Science Explorations, Vol. 10 | 2020–2021

SCIENCE EXPLORATIONS Saint Ann’s School VOLUME 10

2020 – 2021

Table of Contents Investigating the lifelong behavioral effects of bright light stress on Drosophila melanogaster and their offspring Johanna M. (Mentor:Jessica Zayas) ….…………………………….………………………………………………...…2 Examining Epigenetics through the Lens of Development and Aging Cuatro V. (Mentor: Nicholas Harbison) .…………..……………………………………………………………….8 “I can tell you’re smiling from your eyes”: How the pandemic is affecting development and social interactions among preschoolers and kindergarteners Lily S.-K. (Mentor: Virtue Sankoh) ………………………………………..……………………………………….14 Modeling mTORC1 and its inhibitors for the treatment of tuberous sclerosis complex Una R. (Mentor: Daniel Radoff) ……………………………...….…………………….………….…..………. 25 Constructing an Electromagnetic Railgun Henry K. (Mentor: Justin Pelzer)……………………………………………………………………………………28 An Exploration of Nuclear Magnetic Resonance and Dynamic Nuclear Polarization William T. (Mentor: Nicholas Harbison)…………………..……………………..……………….……………….38

1

Investigating the lifelong behavioral effects of bright light stress on Drosophila melanogaster and their offspring Johanna M.

Abstract Despite common conception, DNA is not the only factor that determines human traits; rather, the epigenome also affects how our genes manifest. The epigenome is determined by an individual’s lifetime experiences and facilitates a conversation between her environment and her DNA. In recent years, trauma and the possibility of its intergenerational effects has been a subject of interest within the field of genetics, particularly historical events like the Holocaust and famine. In this study, the impact of stress induced anxiety on an individual and that individual’s offspring was explored using Drosophila melanogaster, the common fruit fly or vinegar fly. In order to test the effects of stress on individuals and their heritability, the flies were stressed by a 1500 lumen flashlight in the larval stage and, using the Caltech software Ctrax, were tested as adults for wall following behavior (WAFO) and locomotive changes, known signs of stress in Drosophila. Previous studies1 have shown that stressed flies avoid central areas (centrophobia) within an enclosure and prefer to stay near walls of an enclosure as compared to control flies. This study aims to measure these effects and determine their potency in subsequent generations by breeding affected flies and testing for the effects of stress on their offspring.

Introduction In recent years, epigenetics has become a topic of discussion among scientists and non-scientists alike. While both epigenetics and genetics contribute to an individual’s phenotype (appearance), epigenetics differs from genetics in a few key ways. While genetics is concerned with issues like the structure, function, and alteration of genes due to events like genetic mutation, epigenetics zooms out further and looks at inheritable changes caused by modifications to gene expression. In short, genetics addresses the inner-workings of DNA and its sequence, while epigenetics concerns itself with what lies on top of the DNA, regulation of DNA and gene expression.

The study of epigenetics, which means “on top of the genome (DNA)” attempts to answer questions like: how can identical twins end up being physiologically different? Am I more likely to be depressed if my dad was? My mother’s father survived a famine; does his trauma affect me chemically? Many of these questions can be answered by looking closely at gene expression, which is the toggling on and off of certain genes. Genes can be turned on/off due to a process called methylation, where semi-permanent chemical tags (methyl 1

Khaira, Harjit Khaira, et al. Measuring the effects of larval life stress on adult anxiety behavior in Drosophila melanogaster. Camden, NJ, Rutgers Camden-The Journal of Biological Science, 6 May 2019. The Journal of Biological Sciences, jbs.camden.rutgers.edu/sites/jbs/files// Measuring%20the%20effects%20of%20larval%20life%20stress%20on%20adult%20anxiety%20beha vior%20in%20Drosophila%20melanogaster.pdf. Accessed 27 May 2021.

2

groups) are attached to DNA thereby affecting transcription and, ultimately, expression. The changes in methyl groups cause DNA to coil more tightly or loosely around proteins, such as histones, making it easier or more difficult to transcribe genes.

Expressivity is the culprit for many of the genetic phenomena that we observe. For example, if DNA is methylated and wrapped more closely around a histone, certain segments of that DNA might become inaccessible, resulting in the underproduction of proteins that might be coded for in that specific “hidden” gene. This could result in major changes in the body like, in the case of Holocaust offspring in a study by Rachel Yehuda, lower levels of stress hormones like cortisol in the blood.2 My interest in the field of epigenetics was piqued by the work of Yehuda, a leader in the field of traumatic stress studies who has devoted a number of years to studying Holocaust surviviors, their children, and the intergenerational effects of traumatic events of similar magnitude. Through this work she addresses the role of epigenetics in psychological disorders like PTSD and anxiety by looking closely at gene regulation, methylation, and the behavioral outcomes of these genetic effects.3 In my own investigation of epigenetics and its generational potency, I was particularly interested in the impact of stress induced anxiety upon an individual and that individual’s offspring.

Anxiety is the body’s response to traumatic circumstances. The onset of anxiety can result in changes in an individual’s mental and physical health. In my research, I tested the hypothesis that when larvae of D. melanogaster are exposed to stress, they will display ambulatory differences due to increased levels of anxiety as adults caused by stress induced DNA methylation. It has been established that D. melanogaster become stressed when exposed to bright-light4 and observations of their motor activity as adults reveal more physical signs of anxiety in stressed flies than in flies that were not subjected to bright light stress. This outcome supports the assumption that anxiety can have extreme effects on individuals later in life and, potentially, their offspring.

Procedure Drosophila melanogaster was used to study transgenerational epigenetics because of its ability to reproduce rapidly and show the effects of light-induced trauma suggesting the occurrence of DNA modifications in a relatively short period of time. The Oregon-R wild type strain of D. melanogaster used was supplied by Carolina Biological. The flies were kept in culture vials at approximately 20 degrees Celsius in Formula 4-24 Instant Drosophila Medium. The larvae present in the media were tested upon and observed after being allowed to mature to adulthood. Specifically, larvae in the third instar of development (the stage of 2

Yehuda, Rachel. Holocaust Exposure Induced Intergenerational Effects on FKBP5 Methylation.

Biological Psychiatry, 21 Sept. 2016. Biological Psychiatry Journal, www.biologicalpsychiatryjournal.com / article/S0006-3223(15)00652-6/pdf. Accessed 27 May 2021.

3 Van Derveer, Dr. Will, and Keith Kurlander, narrators. "What We Know About The Transmission of Intergenerational Trauma – Dr. Rachel Yehuda – HPP 59." Higher Practice Podcast, Integrative Psychiatry Institute file, episode 59, Dr. Will Van Derveer, Keith Kurlander.

4 Khaira, Harjit Khaira, et al. Measuring the effects of larval life stress on adult anxiety behavior in Drosophila melanogaster.

3

larval growth approximately two to three days after hatching from the egg) were used as subjects for this experiment. This stage is most readily identified by the size of the larvae, which are the biggest present in the media before pupation. Another method of recognizing the 3rd instar is identification of spiracle shape; larvae in the 3rd instar have branched spiracles as opposed to clubbed spiracles that are present in the 2nd instar. Larvae in the third instar were then separated from the others in the vial using a sorting brush (a plastic paintbrush). After sorting, the larvae were placed in petri dishes for experiments. The larvae were then exposed to one kind of stressor: bright light exposure. After being exposed to this stimulus, flies were expected to exhibit decreased locomotor activity. The bright light used was the Letion Rechargeable LED Flashlight with a wattage of 25 W (1500 lumens). The flies were then subject to three rounds of light stimulus every 24 hours until pupation; the length of each exposure was ten minutes with ten minute breaks between during which the larvae were exposed to no light. The mature flies were then placed in transparent boxes with dimensions 2 x 1 x 1 inches. Fly locomotion was video recorded on an iPhone. The software Ctrax was then utilized to track the path and wall-following behavior of each fly. The Tracking Wizard was used to determine the velocity, amount of movement, and wall following behavior (WAFO). D. melanogaster are centrophobic and tend to remain along the walls of their enclosure, particularly when feeling stressed.5 Thus, the stressed flies’ time spent near a wall was compared to that of the control group and was used as an indicator of epigenetic changes. In tandem with increased centrophobia, change in locomotive action was studied as an indicator of stress. Three minute videos were taken to analyze this and any movement was categorized as a response. According to previous studies, unstressed flies show more movement than flies that were exposed to stress inducing stimuli.

Results To test if bright light caused a locomotive reaction in Drosophila, the amount of time the flies spent walking during the duration of a three minute video was analyzed. The results show that the six flies in the stress group walked around their enclosure less than the control group, even stopping completely for long periods of time (decreased locomotor activity).

Additionally, the results of the WAFO (wall following) measurements were consistent with this outcome. The same graphs and videos, at a duration of three minutes, were used for WAFO analysis; the graphs show the flies’ movement in their chambers. The control flies tracked through the center of their enclosure, even flying at some points, while the stressed flies remained mostly along the walls of the enclosure and did not fly.

5

Khaira, Harjit Khaira, et al. Measuring the effects of larval life stress on adult anxiety behavior in Drosophila melanogaster.

4

Figure 1 Shows the movement of the stressed fly in its container in X and Y coordinates over three minutes. Black lines indicate the boundaries of the flies’ enclosure.This data was obtained from Ctrax’s Analysis tab under the heading “Plot Trajectories.”

Figure 2 Shows the movement of the control fly in its container in X and Y coordinates over three minutes.Black lines indicate the boundaries of the flies’ enclosure. This data was obtained from Ctrax’s Analysis tab under the heading “Plot Trajectories.”

Discussion The results of this study indicate that stress in early life can instigate behavioral alterations in adulthood due to the onset of anxiety. It has been confirmed that bright light exposure can induce higher levels of anxiety in adult Drosophila, which is exemplified through their wall following (WAFO) activity and locomotive tendencies. Decrease in locomotive behavior has been identified as an indicator of anxiety in Drosophila that have been exposed to heat shock. Decreased motor activity was also the result of this study of bright light effects on Drosophila indicating that bright light can induce the same anxiety related responses in the flies . The WAFO results supported this data: the control flies showed less WAFO activity while the stressed flies exhibited greater WAFO activity. Previous studies have established that stressed flies are centrophobic; these data show that light-stressed flies are also centrophobic.

5

The results of this study work in tandem with the above conclusion about the flies’ decreased locomotor activity to indicate that the flies had been substantially stressed. According to a Rutgers University study, Drosophila melanogaster has been used in many experiments due to its “genetic similarity with humans”6 Therefore, it can be logically concluded that the flies’ larval stage is “analogous to infancy in humans.”7 Having demonstrated that exposing Drosophila to stress in their larval stage results in their having consistently higher levels of stress as adults, it is reasonable to assume that exposing humans to stress during pregnancy might result in their offspring displaying behavioral changes in adulthood. While in this study stress was indicated by locomotive changes, in humans, these behaviors might manifest as physiological or psychological differences relating to organ health8 and mental health. For example, Rachel Yehuda’s Holocaust offspring exhibited higher levels of PTSD, akin to that of their parents, than children whose biological parents did not survive the traumatic events of the Holocaust.9 Whether this result in humans is due to nature (epigenetic changes) or nurture (behavioral tendencies inherited from parents) is still unclear. However, the results of this study can be used to support the former, given that parental behavior has little bearing on offspring’s behavior in Drosophila, especially because larvae were separated from their adult parents.

To further investigate these issues regarding behavioral responses to stress and stress inducing stimulus in Drosophila, future research can alter aspects of the flies’ environment as an added variable. Also, time exposed to stimuli can be varied and observed as a factor that might contribute to the severity of stress in the flies. Further, the offspring of the stressed Drosophila (potentially multiple generations) can be observed to indicate the intergenerational potency of light induced stress and address the issue of intergenerational trauma.

6

Khaira, Harjit Khaira, et al. Measuring the effects of larval life stress on adult anxiety behavior in Drosophila melanogaster.

7 ibid 8 Pembrey, Marcus. Human transgenerational responses to early-life experience: potential impact on development, health and biomedical research. J Med Genet, 25 July 2014. NCBI, www.ncbi.nlm.nih.gov/pmc/articles/PMC4157403/. Accessed 27 May 2021.

9 Transgenerational transmission of cortisol and PTSD risk. Prog Brain Res., 2008. National Library of Medicine, pubmed.ncbi.nlm.nih.gov/18037011/. Accessed 27 May 2021.

6

BIBLIOGRAPHY: links https://ed.ted.com/lessons/how-the-choices-you-make-can-affect-your-genes-carlos-guerrero-bosag na#digdeeper https://www.nature.com/news/epigenetics-the-sins-of-the-father-1.14816 https://www.nature.com/articles/43657 https://onbeing.org/programs/rachel-yehuda-how-trauma-and-resilience-cross-generations-nov201 7/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020004/ https://youtu.be/zV9sya4F5KQ https://www.biologicalpsychiatryjournal.com/article/S0006-3223(15)00652-6/fulltext https://en.wikipedia.org/wiki/Hsp90 https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/fkbp5 https://medlineplus.gov/genetics/understanding/genomicresearch/snp/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4084612/ https://flexikon.doccheck.com/en/Ultrashort_feedback https://www.sciencedaily.com/releases/2010/09/100928193450.htm https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020004/ http://symposium.cshlp.org/content/69/155.full.pdf+html https://www.biologicalpsychiatryjournal.com/article/S0006-3223(15)00652-6/pdf https://en.wikipedia.org/wiki/FKBP5 https://en.wikipedia.org/wiki/Glucocorticoid_receptor https://www.research.va.gov/currents/1016-3.cfm https://www.mountsinai.org/about/newsroom/2017/on-being-pbs-how-trauma-and-resilience-croS s-generations-rachel-yehuda-phd https://psychiatryinstitute.com/podcast/transmission-intergenerational-trauma-yehuda

7

Examining Epigenetics through the Lens of Development and Aging Cuatro V.

Abstract In high school biology, students often learn why Lamarckian Inheritance, the theory that offspring inherit physical characteristics acquired by parents,1 is flawed. However, the recent field of epigenetics may suggest that Lamarck was partially correct.2 The epigenome is another dimension of genetic influence apart from the nucleotide sequence. While many scientists once believed the epigenome disappeared in embryos, today, it is known that it is not entirely rebuilt from scratch.3 In this project, the correlation between an organism’s behavior and epigenetic traces is analyzed. Specifically, how prenatal and postnatal experiences and a parent’s epigenome can have significant influences on offspring. In a study on mice, researchers found an alteration in offsprings’ epigenome at a glucocorticoid receptor gene promoter in the hippocampus caused by increased pup licking and grooming and arched-back nursing by rat mothers.4 There were significant disparities in DNA methylation between offspring with mothers who consistently groomed and nursed and offspring with mothers who did not do so. While these differences were in the first week of life, the mice would see them persist into adulthood with altered histone acetylation. This study proved a direct link between behavioral programming and the epigenome. In this project, the question is raised as to how this link might be present in humans. For instance, like the stress response in mice, adverse childhood experiences (ACEs) in humans may have lasting effects on the genome.5 They can be correlated to a higher risk of cardiovascular disease,6 autoimmune disease,7 obesity, and type-two diabetes, among other disadvantageous conditions.8 Epigenetics may hold many answers to the questions scientists 1

Britannica, The Editors of Encyclopaedia. "Lamarckism". Encyclopedia Britannica, 11 Oct. 2017, https://www.britannica.com/science/Lamarckism. Accessed 26 May 2021.

2 Jablonka, Eva, and Marion J. Lamb. Epigenetic Inheritance and Evolution the Lamarckian Dimension. Oxford Univ.

Pr, 2005.

3 The Epigenome Learns From Its Experiences, www.learn.genetics.utah.edu/content/epigenetics/memory/.

4 Weaver IC;Cervoni N;Champagne FA;D'Alessio AC;Sharma S;Seckl JR;Dymov S;Szyf M;Meaney MJ; “Epigenetic Programming by Maternal Behavior.” Nature Neuroscience, U.S. National Library of Medicine, www.pubmed.ncbi.nlm.nih.gov/15220929/.

5 Lang, Jason, et al. “Adverse Childhood Experiences, Epigenetics and Telomere Length Variation in Childhood and beyond: a Systematic Review of the Literature.” European Child & Adolescent Psychiatry, Springer Berlin Heidelberg, 9 Apr. 2019, link.springer.com/article/10.1007/s00787-019-01329-1.

6 Murphy MO, Cohn DM, Loria AS. Developmental origins of cardiovascular disease: impact of early life stress in humans and rodents.

7 Dube SR, Fairweather D, Pearson WS, Felitti VJ, Anda RF, Croft JB. Cumulative childhood stress and autoimmune diseases in adults.

8 Jiang, Shui et al. “Epigenetic Modifications in Stress Response Genes Associated With Childhood Trauma.” Frontiers in psychiatry vol. 10 808. 8 Nov. 2019, doi:10.3389/fpsyt.2019.00808

8

have been asking for decades. While it is vital to understand the nucleotide sequence in this novel genomic era, it may be equally crucial to explore the mechanisms of epigenetics.

Introduction Initially, the term “epigenetics” was used to refer to the unexplored processes by which fertilized eggs became complex organisms.9 However, in 1942 developmental biologist Conrad H. Waddington redefined epigenetics as the dynamic interactions between an organism's environment and phenotypic expression.10 The Greek prefix “epi” means “on” or “over.”11 Epigenetics, in this way, examines the additional instructions lying “over” DNA, impacting the way genes are expressed. Although little was known about the hereditary role of genes at the time, Waddington’s refinement as well as work from other scientists, such as Ernst Hadorn, would lay the foundation for the field of modern epigenetics.

DNA methylation was among the first identified epigenetic mechanisms and occurs when a methyl group (CH₃) is added to a DNA molecule, typically acting to repress gene transcription (Figure 1).12 This chemical addition is known as an ‘epigenetic mark,’ and was first described in 1948 by Rollin Hotchkiss when he detected 5-methylcytosine using paper chromatography in a calf thymus preparation.13 DNA methylation was later linked to a biological function in 1969, when John S. Griffith and Henry R. Mahler hypothesized that it was associated with better cognitive memory.14 By the mid-1970s, numerous groups had confirmed DNA methylation was involved in turning genes “on” and “off” and in cell differentiation.15 Today it is known that about 45% of the mammalian genome is methylated to silence viral and transposable elements, the main role being to suppress harmful genetic components; however, many environmental and behavioral changes can cause potentially detrimental epigenetic changes.16

9

Felsenfeld, Gary. “A brief history of epigenetics.” Cold Spring Harbor perspectives in biology vol. 6,1 a018200. 1 Jan. 2014, doi:10.1101/cshperspect.a018200 10 Tronick, Ed, and Richard G Hunter. “Waddington, Dynamic Systems, and Epigenetics.” Frontiers in behavioral neuroscience vol. 10 107. 10 Jun. 2016, doi:10.3389/fnbeh.2016.00107 11 “Epigenetics: Fundamentals, History, and Examples.” What Is Epigenetics?, www.whatisepigenetics.com/fundamentals/.

12 “Epigenetics Studies Affect of Chemical Tags Linked to DNA.” WhatisBiotechnology.org, www.whatisbiotechnology.org/index.php/science/summary/epigenetics/.

13 Moore, Lisa D, et al. “DNA Methylation and Its Basic Function.” Nature News, Nature Publishing Group, 11 July 2012, www.nature.com/articles/npp2012112.

14 Griffith JS, Mahler HR. DNA ticketing theory of memory. Nature. 1969 Aug 9;223(5206):580-2. doi: 10.1038/223580a0. PMID: 5799529.

15 Holliday, R, and J. Pugh. “DNA Modification Mechanisms and Gene Activity during Development.” Science, vol.

187, no. 4173, 1975, pp. 226–232., doi:10.1126/science.1111098.

16 Schulz WA, Steinhoff C, Florl AR (2006). Methylation of endogenous human retroelements in health and disease.

Curr Top Microbiol Immunol

9

Figure 117 Only adenine and cytosine are known to undergo natural enzymatic DNA methylation.18 There are three classes of methyltransferase enzymes that recognize, establish and modify DNA methylation: readers, writers, and erasers. Readers influence gene expression by recognizing and binding to methyl groups. Writers act as the catalyst for methyl group addition onto a DNA nucleotide, whereas erasers act to remove this group.

The development of DNA cloning and sequencing techniques, microarray technology, and improved antibody staining, further enhanced epigenomic understanding, setting the field up to experience an unprecedented increase in research in the 2000s.19 The emergence of evidence that epigenetic mechanisms are correlated to pathologies had fueled this sudden interest and further clarified specific epigenetic mechanisms such as histone modification of the chromatin.

Chromatin is not a fixed structure, it is constantly shifting in response to external cues to accommodate and carry out a variety of DNA functions. While not changing the nucleotide sequence, histone modification acts as one of these cues to regulate biological processes by manipulating the expression of DNA.20 During developmental periods, histone modification and DNA methylation have influence on one another.21 Unique histone methylation patterns during early embryonic systems ensure that each tissue type is being expressed by the 17

Bearer, Elaine L, and Brianna S Mulligan. “Epigenetic Changes Associated with Early Life Experiences: Saliva, A Biospecimen for DNA Methylation Signatures.” Current genomics vol. 19,8 (2018): 676-698. doi:10.2174/1389202919666180307150508 18 Iyer, Lakshminarayan M et al. “Adenine methylation in eukaryotes: Apprehending the complex evolutionary history and functional potential of an epigenetic modification.” BioEssays : news and reviews in molecular, cellular and developmental biology vol. 38,1 (2016): 27-40. doi:10.1002/bies.201500104 19 “Epigenetics Studies Affect of Chemical Tags Linked to DNA.” WhatisBiotechnology.org, www.whatisbiotechnology.org/index.php/science/summary/epigenetics/.

20 Bannister, A., Kouzarides, T. Regulation of chromatin by histone modifications. Cell Res 21, 381–395 (2011). https://doi.org/10.1038/cr.2011.22 21 Cedar, H., Bergman, Y. Linking DNA methylation and histone modification: patterns and paradigms. Nat Rev Genet 10, 295–304 (2009). https://doi.org/10.1038/nrg2540

10

appropriate genes. Studies demonstrate that during embryogenesis, aberrant methylation can cause development defects in organs, body patterning, and skeletal and nervous systems.22 Other epigenetic mechanisms playing a considerable role in the regulation of gene expression are non-coding RNA (ncRNA) sequences, occurring when ncRNA (a transcribed RNA molecule that is not translated into proteins) causes gene silencing. Long non-coding RNAs, microRNAs and short interfering RNAs are the most well understood forms of ncRNA.23 While many researchers originally regarded this process as genomically wasteful, it is now evident that ncRNA plays a vital role in varying phenotypic expression, particularly between human populations.24 Recent exploration into the biological processes guiding development and aging suggest that epigenetic markers play a significant role. Prenatally, epigenetic mechanisms are vital in the differentiation of cells. Such markers continue to be important in postnatal development, with vital implications for the future health of the organism.25 For example, in developing mouse sperm, disruption of histone methylation by exposure to the KDM1A transgene critically impaired survivability and development of offspring for two generations (Figure 2).26 Figure 2

22

Jambhekar, A., Dhall, A. & Shi, Y. Roles and regulation of histone methylation in animal development. Nat Rev Mol Cell Biol 20, 625–641 (2019). https://doi.org/10.1038/s41580-019-0151-1 23 Aboud, Nora M. Al. “Genetics, Epigenetic Mechanism.” StatPearls [Internet]., U.S. National Library of Medicine, 6 Nov. 2020, www.ncbi.nlm.nih.gov/books/NBK532999/.

24 Frías-Lasserre D, Villagra CA. The Importance of ncRNAs as Epigenetic Mechanisms in Phenotypic Variation and Organic Evolution. Front Microbiol. 2017;8:2483.

25 Murgatroyd, Chris, and Dietmar Spengler. “Epigenetics of Early Child Development.” Frontiers, Frontiers, 4 Apr. 2011, www.frontiersin.org/articles/10.3389/fpsyt.2011.00016/full.

26 Siklenka, Keith, et al. “Disruption of Histone Methylation in Developing Sperm Impairs Offspring Health Transgenerationally.” Science, American Association for the Advancement of Science, 6 Nov. 2015, www.sciencemag.org/content/350/6261/aab2006#:~:text=Disruption%20of%20histone%20methylation%20in%20de veloping%20sperm%20by%20exposure%20to,absence%20of%20KDM1A%20germline%20expression.

11

A study in 2003 focused on the epigenetics of the agouti gene (a small 131-amino acid protein secreted by dermal papillae cells) in mice. When activated, this gene causes a yellow coat (Figure 2) and predisposition to certain pathogens such as diabetes and obesity.27 In this experiment, female agouti mice were fed a diet high in methyl groups such as folic acid, B12 vitamin, betaine and choline both before and during pregnancy.28 While the control group’s pups were yellow and obese, when mice with altered diet gave birth, the offspring were thin and brown. The results of this study suggest not only that the diet and environment of parents can have lasting epigenetic effects on children and grandchildren, but also that epigenetic traits are reversible through manipulating such factors.

Figure 329

Also potentially detrimental to aging and developmental processes, adverse childhood experiences (ACEs) can cause abnormal alterations in DNA methylation. Regulated by the methylation of cytosine (5mC), a “conserved transcriptional response to adversity” in leukocytes has been recognized.30 Additionally, ACEs cause a methylation of the cortisol promoter hormone (CRF/CRH), causing immune and cognitive suppression.31 Child maltreatment and suicide has also been associated with GR/NR3C1 protein-coding gene methylation.32 27

Morgan, H., Sutherland, H., Martin, D. et al. Epigenetic inheritance at the agouti locus in the mouse. Nat Genet 23, 314–318 (1999). https://doi.org/10.1038/15490 28 Murphy, Glenn. “Mother's Diet Changes Pups' Colour.” Nature News, Nature Publishing Group, 4 Aug. 2003, www.nature.com/news/2003/030728/full/news030728-12.html.

29 Maternal nutrient supplementation counteracts bisphenol A-induced DNA hypomethylation in early development Dana C. Dolinoy, Dale Huang, Randy L. Jirtle Proceedings of the National Academy of Sciences Aug 2007, 104 (32) 13056-13061; DOI: 10.1073/pnas.0703739104 30 Loneliness, eudaimonia, and the human conserved transcriptional response to adversity. Cole SW, Levine ME, Arevalo JM, Ma J, Weir DR, Crimmins EM Psychoneuroendocrinology. 2015 Dec; 62():11-7.

31 Chen J., Evans A.N., Liu Y., Honda M., Saavedra J.M., Aguilera G. Maternal deprivation in rats is associated with Corticotrophin-releasing Hormone (CRH) promoter hypomethylation and enhances CRH transcriptional responses to stress in adulthood. J. Neuroendocrinol. 2012;24(7):1055–1064.

32 Perroud N., Paoloni-Giacobino A., Prada P., Olie E., Salzmann A., Nicastro R., Guillaume S., Mouthon D., Stouder C., Dieben K., Huguelet P., Courtet P., Malafosse A. Increased methylation of glucocorticoid receptor gene (NR3C1) in adults with a history of childhood maltreatment: A link with the severity and type of trauma. Transl. Psychiatry.

2011;1:e59.

12

Methods Prior to the global pandemic, more epigenetic studies were conducted than ever before.

Using advanced technological methods such as high resolution melt analysis and chromatin immunoprecipitation, researchers have further developed our understanding of the epigenome.33 This section will be used to elucidate future objectives and potential experimental methodology for a long-term study beyond the time and technical constraints of high school.

The proposed study will start by recruiting participants ages 15-45. Each participant will be given an ACEs questionnaire and then divided into two groups: high levels of ACEs and normal levels of ACEs. This questionnaire will be highly specific and the definition of an ACE will be determined by the CDC and American Psychiatric Association. A comparison of each group's histone modifications and telomere lengths of a specific gene in both groups will be conducted. The GR/NR3C1 gene and the serotonin transporter gene (5htt/sert/slc6a4), among others, will be included in this analysis.

Results The results of this experiment will determine the correlation between methylation patterns and ACEs. With the emergence of such findings, the original groups will be followed to measure epigenetic changes and the prevalence of specific pathologies such as cardiovascular disease, type II diabetes, cancer, and high cortisol levels. If these patients reproduce, DNA methylation analysis will be conducted on offspring with familial consent.

Discussion Identifying and locating the differences of particular 5mC sites between the two groups will elucidate the transgenerational effects of ACEs in Homo sapiens. Analyzing both the epigenetics of aging and development, the results of this longitudinal study will determine what specific epigenetic modifications are most prevalent and likely to pass down to offspring.

These findings become particularly relevant in a world where our environment and behavioral practices as a species are changing at unprecedented rates. By determining which epigenetic changes adversely affect the human population at high rates, we can clarify the path forward in prevention.

33

“Epigenetics Analysis.” Bio, www.bio-rad.com/en-us/applications-technologies/epigenetics-analysis?ID=LUSNNRCZF.

13

“I can tell you’re smiling from your eyes”: How the pandemic is affecting development and social interactions among preschoolers and kindergarteners.

Lily S.-K. Abstract: The goal of this project was to delve into how the COVID-19 pandemic is affecting cognitive development, social interactions and overall behavior among preschoolers and kindergarteners. Between the ages of three and five is a critical period of cognitive and social development which I hypothesized would be affected by COVID. I researched the psychological consequences of traumatic events, helping me speculate about the consequences of today's pandemic. I observed at the Saint Ann’s preschool and kindergarten weekly, formulated a teacher survey regarding the effects of COVID, and interviewed fifteen preschoolers and kindergarteners themselves. The results of all this showed that although the pandemic has definitely detrimentally impacted children, causing some to regress in play, develop language more slowly and be overall more anxious and aggressive, there also have been some positive outcomes. Adjusting to the circumstances caused by COVID was tricky, yet they all showed resilience, developing new ways of interacting, reading social-cues, and showing affection. They gained a more mature understanding of the world around them as well. One kindergartener, when asked what he would change about the pandemic, said: “People don't always wear masks and I wanna change that. Like the people in Texas!” However, the amount of joy that resonates as they play with each other is immense and “raise your hand if you love school” is shouted frequently, showing, as one teacher puts it, “that they are rockstars!”

Introduction: How preschoolers and kindergarteners develop cognitively and socially fascinates me. But this year, what captivated me even more was how resilient they were in regards to the COVID-19 pandemic, and their astounding ability to adjust to the environment around them.

Development between the ages of three and five is critical and many skills are established including: socializing, which strengthens negotiation skills and language; and collaboration, which helps evolve moral reasoning. However, almost all of this is learned through being around other children and the pandemic limited kids from socializing. Since development is influenced by one’s environment, not going to in-person school for months drastically altered the “normal” period when children learn these skill sets, and when schools did open up again, they had many restrictions. In an environment with social restrictions--where play can be limited, where people wear masks, where the learning of facial expressions, communication, and language is restricted, and where demonstrating affection and other kinds of physical closeness is discouraged--it is speculated that limitations in the formation of certain areas of the brain can occur, and as a result of this there is an impairment in the acquisition of cognitive, behavioral, social, and communication skills.31

14

I wanted to know exactly how all of this was affecting Saint Ann’s preschoolers and kindergarteners and how they were adapting to it. After spending time observing, interviewing and researching, I came to the conclusion that although the pandemic has many negative effects, there are also some positive outcomes. While there is a lack of development in some areas, a higher anxiety spectrum and for some a regression in play, there is also a tighter community, more creativity, new ways of communicating (through body language and eyes) and less conflict. Unfortunately, there has not been a lot of research done on how epidemics affect children’s development, and therefore there still remains a lot of controversy around the topic, as some believe it will cause many detrimental effects while others think children are “biologically wired to adapt.”

Background ACEs (adverse childhood experiences) are traumatic or stressful events that occur in childhood and the pandemic can be understood as one. ACEs have detrimental effects on brain development and overall health, leading possibly to learning disabilities, depression, anxiety, obesity, and heart conditions.31 Recent neurobiological studies have shown that traumatic experiences in childhood can diminish concentration, memory, and the organizational and language abilities children need to succeed in school. Also, children may have difficulty identifying, expressing, and managing emotions, as well as reasoning and problem solving. Trauma can interfere with the capacity for creative play, which is one of the ways children learn how to cope with the problems of everyday life.14 Studies comparing the symptoms of post-traumatic stress in parents and children confined in pandemic situations with those of families in normal routines indicate that stress levels are four times higher for children who have been in quarantine. However, if the appropriate care and support is given to a child under stress, their body reorganizes itself biochemically and quickly returns to levels of physiological functioning without further damage.31 Nonetheless, there is a lot of controversy around how the trauma of COVID will affect development down the line; some researchers think children are “adaptable” and it won't affect them at all, while others think the trauma of it could lead to severe consequences.

Nadine Burke Harris, a Canadian-American pediatrician and Surgeon General of California, wrote: “Decades of studies tell us that the kind of trauma and stress many children are experiencing during the Covid-19 emergency has the potential to embed itself in children’s DNA, dramatically affecting their brains and other critical body systems and, as a result, their health across a lifetime.”12 By contrast, Dr. Jack Shonkoff, a pediatrician and early-childhood-development expert at Harvard’s Center on the Developing Child, wrote: “Even though this is unusual, most kids will come out of this fine because we’re biologically wired to adapt.”27 Although we are “wired to adapt,” undoubtedly there will be some effects--whether long lasting or not--from the pandemic, especially for families who are not able to provide the support needed for their kids at this time.

School helps children develop cognitively, socially and emotionally, as well as help them master basic foundational academic skills. In the absence of school there have been noticeable delays in speech and language as well as trouble sharing, which leads to problems

15

being in groups. A study executed before the pandemic by cognitive neuroscientist Arjen Stolk found that kids who attended day care were able to interact more effectively with peers than kids who didn’t. In the study, 5-year-olds were asked to use non-verbal communication to guide children of different ages through a video game. Those who had been in day care were better able to tailor their interactions to the children's ages.23 When comparing the 1918 pandemic to today, there are many similarities, one being school closures. During the 1918-1919 influenza pandemic around 675,000 people died in the United States alone and the majority of public schools were closed for weeks to months on end. When influenza struck New York City in the fall of 1918, its school system comprised more than a million children.19 Most of these children stayed at home, not getting the proper education and care needed. The long term physiological effects of people who survived the Spanish flu were severe, as they reported sleep disturbances, depression, mental distraction, dizziness, and difficulties coping in social settings.9 With the restrictions of masks now, the ability to communicate with nonverbal cues is important. It is thought by some researchers that mask wearing might affect development.

Kang Lee, a professor of applied psychology and human development at the University of Toronto who studies the development of facial recognition skills in children, pointed to three potential problems masks might pose. One was that children might encounter difficulty recognizing people, because kids often focus on individual features. Second was that a lot of emotional information is displayed through movement of facial musculature and because that will be obscured by a mask, children may have issues with “emotional recognition and social interaction.” And lastly, children may have problems with speech recognition. Even though it’s thought speech communication takes place through sound, a great deal of information is communicated visually.30 However, not all researchers have the same beliefs regarding masks and development. For instance, a 2012 study found children could recognize facial emotions equally well whether or not a person was wearing a face mask.

Children raised in cultures where face coverings are fairly common don’t fare any worse than other children at recognizing faces or emotions.17 Isolation could lead to the impairment of children’s development. Dr. Barbara Nosal, chief clinical officer at Newport Academy, wrote: “During periods of social development, when a child is deprived of the opportunity to build these skills, it can potentially delay their socio-emotional development.”18 Social isolation damages the nervous system’s support cells, which in turn affects the development of cognitive functioning.16 Previous research has also revealed that socially isolated children tend to have lower subsequent educational attainment, be part of a less advantaged social class in adulthood, and are more likely to be psychologically distressed in adulthood.32 However, this study fails to take into account economic status from the start, as well as the notion that some parents might voluntarily want their kids to be isolated. Play facilitates cognitive development as well, and quarantine limits essential interactive experiences that lead to the development of relationships outside of the family.

16

However, young children can be very flexible and exhibit great strength in difficult times.

There are also some positive aspects to the current pandemic situation. One aspect is that children are spending more time with their parents. A survey in the past year of 1,319 American adults — including 284 fathers — suggested that 68% of fathers feel closer or much closer to their children since the pandemic started. This increased involvement with their fathers can have many benefits for kids, including strengthening cognitive and emotional development.33 Materials and Methods: At the start of the year I went to the preschool and kindergarten weekly to observe the kids play and interact, in addition to getting a sense of how they were adjusting to the school year. Last year I also observed the preschoolers and so I had some basis of comparison. As I was doing this I also read up on articles about the effects of the pandemic on children and tried to find some correlation to past epidemics and their consequences as well. When I first started researching in September there was barely any written data on the topic. However, as the year went on more articles were published that I further examined. After observing for several months, I constructed a list of questions regarding the effects of the pandemic and created a survey from it that I sent to all the teachers at the preschool and kindergarten. The questions consisted of: 1. Do preschoolers/kindergarteners interact with each other differently now that they have to wear masks and are supposed to social distance?

2. How are their friendships affected? For instance, are they forming friendships more slowly this year?

3. Are there kids who talk only with the teachers or play by themselves more than usual?

4. Is it harder for them to learn and remember information with the teacher and themselves wearing masks?

5. Do they have more trouble than usual concentrating?

6. Is not being able to see people's mouths affecting speech and language development?

Are they finding different ways to communicate?

7. They’re supposed to social distance as best as possible, therefore they can't hug/hit each other as much. Is there less conflict between kids because of this? Are there new ways they show affection?

8. How is the pandemic manifested in their play and conversations? For instance, are there new elements in their dramatic play? Could you give some examples?

9. Do you have any other observations regarding the effects of COVID19?

I got thirteen vivid responses for each question, seven from kindergarten teachers and six from preschool teachers. I then analyzed each answer, trying to find some common thread among them all, although they ranged in their responses.

After doing this I went back to the preschool and kindergarten to interview the kids themselves, asking seven preschoolers and nine kindergarteners:

17

1. What do you know about COVID? If I did not know anything about it, how would you describe it to me?

2. Do you think when you wear a mask you play with your friends differently?

3. If you can't see someone smile, can you still tell if they are happy?

4. What do you notice that is different about this year? How do you feel?

5. You're supposed to social distance as best as possible. Is it hard not to hug your friends?

6. If you could change something right now (about COVID) what would you change?

7. You have to follow so many rules (social distancing, wearing a mask). Is it hard to follow all of these rules?

8. Do you know why we wear masks?

I recorded each kid as I asked them these questions so I could remember every detail. I finally compiled all my data from observation, the teachers’ responses, and the kids’ responses to formulate a conclusion on how the pandemic is affecting development and social interactions.

Results: When reviewing the responses of the preschoolers and kindergarteners, it was clear that they have an immense amount of knowledge on the topic of the pandemic. They also have a deeper understanding of sickness and death than in years past. They had a wide range of responses to What do you know about COVID? From the kindergarteners these included: ● “It’s a really bad virus that can kill people.” ● “It’s been going on for like a year now.” ● “I believe it started in China but I don't blame them, China.” ● “If you don’t want to quarantine you have to take your vaccine.”

There were also other elaborate answers mostly consisting of it being “bad” and able to kill you. The students clearly incorporated what the adults in their life were saying about it, including the severity of it. In addition, they all said these quotes without seeming upset. At this age it is hard for kids to internalize real death and disease, since when they do encounter it, it is usually in a make-believe-game, TV-show, or book. COVID has also been going on for so long that they have almost gotten used to it. After all, it has been almost a quarter of their lives.

In response to Do you think when you wear a mask you play with your friends differently? they said things like: ● “It’s harder to understand my friends.” ● “It’s hard because you can't be close to your friends.” ● “I can’t recognize people.” ● One kindergartener even said, “It’s harder for them to hear you so then it’s like one of your friends making all the rules and the other isn't.”

18

This is showing the kind of imbalanced negotiation which can lead to group problems. There are also communication problems between them, which when you’re four or five and don’t have the largest vocabulary to express yourself, makes it that much harder to interact.

In regards to emotional recognition, the question If you can't see someone smile can you still tell if they are happy? was answered by everyone almost the same way; they all said they could still see the person's eyes and therefore could still tell if they were happy or sad.

When asked what they noticed was different about this year, most mentioned physical differences, like wearing masks. For example: ● “We have to wear a mask and do zoom a lot, not in person.” ● “We have to stay six feet apart when we are eating and our masks are off.” ● The question You are supposed to distance. Is it hard not to hug your friends? was answered differently by every student: ● One boy said, “It’s easy to distance.” ● A girl said, “If I haven't seen someone in a while I wanna hug them...but it’s COVID so I can't really do it.”

I did not gather enough data to make a conclusion on this, however, most of the girls I interviewed said it was hard to distance, whereas the three boys I interviewed said it was not that hard.

In response to the question on what they would change about COVID, almost all of them said stop wearing a mask, or as one boy said: “People don't always wear masks and I wanna change that. Like the people in Texas!”

When asked, Is it hard to follow all of these rules? (social distancing, wearing a mask), there were a variety of responses. Some said it was easy to follow while others iterated that it was very difficult and took time to get the hang of it. One kindergartener said: “It’s sorta hard, but it’s kinda just to keep us protected so it's kinda helpful,” exemplifying her understanding of the importance of protection; this kindergartener was sophisticated enough to understand that she had to give up some things, like hugging, in order to stay safe.

The most interesting range of responses was for the question Do you know why we wear masks? All of them had a different “scientific” explanation that they shared. ● One boy said: “Yes, to keep ourselves safe. And also because CoronaVirus is in you but not attacking you. If you sneeze on someone that person would get CoronaVirus.” ● Another girl said: “To protect our mouth and nose because the COVID can go into either one of them. When you open your mouth there is a hole in there and COVID could go in or your nose.”

Overall, their responses were elaborate, showing their ability to adapt and adjust to this new “world” they are living through as well as their awareness of it all. The preschoolers' answers, however, were more straightforward, although still very knowledgeable, while the kindergarteners had a larger vocabulary to express their creative answers. All of the answers

19

quoted above are from the kindergarteners since the preschoolers had responses consisting of: ● “The pandemic is bad.” ● “I don’t like masks.” ● “I want it to end.”

Although interesting, it did not meet the level of eloquence of the kindergarteners. This, however, demonstrates the vast language developmental jump that takes place from preschool to kindergarten. The preschoolers don't know as many details about COVID, and unlike the kindergarteners, they don’t understand that some of the “rules” are for their own good.

The responses to the teacher survey ranged greatly. There were 108 written responses in total, but main themes popped out. With the question, How is the pandemic manifested in their play? some of the responses the teachers wrote included: ● “A child made a drawing of herself at Pierrepont park to describe a moment she was proud of. Everyone in the drawing was wearing masks, but she explained how she had asked two sisters to put on their masks, and she felt really proud of herself for doing so.” ● “They have one game in which our littlest student is always in some state of need (though not necessarily ill) and all the other kids are vying for the chance to care for her.”

Teachers also wrote that in the kids’ dramatic play, phrases exemplifying what COVID has taken away from them, like: “I can’t have a birthday party because of the virus” and "My mom can't bring me to school today because she has COVID,” are stated frequently. In the survey a teacher also said that a group of kids were playing with Magna-Tiles and created a room in the center of the house called the “COVID room” where people went to “die.” When I observed in the beginning of the year, I also noticed more medical play taking place; in the yard at the kindergarten, a little girl was giving a boy CPR and screaming “We have to call an ambulance, you’re gonna die!” This little girl was trying to find a way to help her friend.

Children tend to work through difficult aspects in their life through their play and are evidently doing so now, as they are aware of the dangers of COVID. Another question asked was: Is it harder for them to learn and remember information wearing a mask? Almost all of the teachers said yes, that language, writing and reading are all more difficult to teach and learn as well as that it is difficult to understand the kids. I also asked if it was harder for the children to concentrate. To this a lot of teachers said yes, and that the kids often do not seem to know where a voice is coming from without seeing a mouth moving.

Lastly, related to cognitive development, I asked if speech and language development were affected. Some of the responses were:

20

●

●

●

“Yes. Not being able to see each other's mouths definitely has affected their language development. They seem to physically express how they feel and use that as another form of communication.”

“Yes...I also think they probably have adapted by changing behaviors- getting right in front of a peer while talking, for example. We do use thumbs up a fair amount and they are doing some of that too.”

“Kids are finding more ways to express themselves through creative work/art.”

Clearly there have been difficulties that the children have had to overcome regarding communication, but they also have learned to adapt, finding new ways of expressing themselves, demonstrating their creativity.

The last theme pulled out was about friendship formation and interactions. To the question, Is there less conflict between kids? there were some contradictions in the responses. Most teachers believe that “There's definitely less conflict and more love, more empathy. We have elbow bumps, side hugs, lots of sign language.” However, one kindergarten teacher said that there was more conflict because of the tension around social distancing, as it creates a stressful environment.

To the question, How are their friendships affected? Are they forming friendships more slowly? most teachers said that they were. A few of the responses included: ● “Socializing during the pandemic was/is limited. Not being able to socialize as much during the pandemic has greatly affected them. A lot of children have regressed because of the lack of play.” ● “They are connecting with fewer peers-less opportunities. Friendship groups are smaller. Definitely takes longer to form new friend groups.” ● “It has been harder for everyone to understand each other verbally. However, I think that the kiddos have gotten very good at reading each other’s body language.”

Nevertheless, the slower formation of friendships did not affect the community. In fact, multiple teachers said that living through this time has created one of the tightest communities ever. Most of the teachers also said that there was less conflict among students, which could be one of the causes of the “tightness” seen.

Discussion and Conclusion: Some researchers have said that the pandemic will detrimentally affect kids’ development while others have said that kids will adapt and it won't be a problem. There is no consensus, partly because it is subjective to every child's circumstance, but also because humans can adapt while also suffering consequences. At the Saint Ann’s preschool and kindergarten there were both beneficial and detrimental effects as a result of COVID. Some of the benefits included: ● Listening more carefully to one another. ● The ability to read emotions better (through eyes and body language). ● The ability to express themselves through creative work. ● Less conflict (one of the factors that leads to a tighter community).

21

When the world around you seems to be “falling” apart, having a close community to rely on is crucial, and aspects like less conflict and listening more carefully undoubtedly are some of the causes of the tighter community. There were also two surprising benefits that were found in the survey, one being a teacher wrote that it was easier to teach reading on zoom than in the classroom because they didn’t have to deal with class management. And the other was that for some kids wearing a mask enhanced their language development since they needed to articulate and express themselves more clearly to be understood. However, that definitely did not apply to all of the children, as some had more trouble developing language this year.

Some of the detrimental effects COVID caused included: ● Mask anxiety and parental anxiety being passed down. ● Regressing socially because of the lack of play in quarantine, which could be one of the causes of their difficulty forming friendships. ● Difficulty understanding, hearing others and therefore concentrating. ● Difficulty learning language and reading. ● Lack of negotiation and collaboration skills (which could be a result of the lack of play in quarantine).

It is clear that everyone is struggling through COVID, but in times of hardships people come together. And while we don’t know for sure the long-term effects three, four, and five year olds are going to face, we do know how resilient humans are and that the preschoolers and kindergarteners are, as one teacher writes, “working so hard to open themselves to joy and connection everyday and it’s beautiful to witness.”

Bibliography: 1.

2.

3.

4.

5.

6.

7.

Baltimore, Wesley J. "Helping Our Clients Parse Speech Through Masks During COVID-19."

Leader Pub. Last modified June 5, 2020. Accessed May 26, 2021. https://leader.pubs.asha.org/do/10.1044/leader.MIW.25062020.34/full/.

Bremner, Douglas J. "Traumatic stress: effects on the brain." NCBI. Accessed May 26, 2021. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3181836/#:~:text=Brain%20areas%20imp licated%20in%20the,norepinephrine%20response%20to%20subsequent%20 stressors.

Child Mind Institute. Accessed May 26, 2021. https://childmind.org/.

Child Study Center. Accessed 5/26/2021. https://nyulangone.org/locations/child-study-center.

Collins, Francis. "What We Know About COVID-19's Effects on Child and Maternal Health."

NIH. Last modified July 30, 2020. Accessed May 26, 2021. https://directorsblog.nih.gov/2020/07/30/what-we-know-about-covid-19s-effects-on-childand-maternal-health/.

"Coping with COVID-19." NAEYC. Accessed May 26, 2021. https://www.naeyc.org/resources/topics/covid-19.

"Early childhood development and COVID-19." UNICEF. Accessed May 26, 2021. https://data.unicef.org/topic/early-childhood-development/covid-19/.

22

8. "Effects of the Global Coronavirus Disease-2019 Pandemic on Early Childhood Development: Short- and Long-Term Risks and Mitigating Program and Policy Actions." NCBI. Last modified August 2020. Accessed May 26, 2021. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7234941/.

9. Eghigian, Greg. "The Spanish Flu Pandemic and Mental Health: A Historical Perspective."

Psychiatric Times. Last modified May 28, 2020. Accessed May 26, 2021. https://www.psychiatrictimes.com/view/spanish-flu-pandemic-and-mental-health-historical -perspective.

10. Ehmke, Rachel. "Talking to Kids About the Coronavirus Crisis." Child Mind Institute.

Accessed May 26, 2021. https://childmind.org/article/talking-to-kids-about-the-coronavirus/.

11. Grose, Jessica. "Will the Pandemic Socially Stunt My Kid?" New York Times. Last modified September 30, 2020. Accessed May 26, 2021. https://www.nytimes.com/2020/09/30/parenting/coronavirus-masks-kids-socialization.ht ml?auth=linked-google&referringSource=articleShare.

12. Harris, Nadine Burke. "Children will pay long-term stress-related costs of Covid-19 unless we follow the science." Stat News. Last modified August 4, 2020. Accessed May 26, 2021. https://www.statnews.com/2020/08/04/children-long-term-stress-related-costs-covid-19/.

13. Hawn, Goldie. "Why policymakers and school leaders can't ignore how the pandemic hurts childhood brain development." The Hechinger Report. Last modified August 28, 2020.

Accessed May 26, 2021. https://hechingerreport.org/opinion-why-policymakers-and-school-leaders-cant-ignore-how -the-pandemic-hurts-childhood-brain-development/?utm_source=The+Hechinger+Report& utm_campaign=17f2aea516-weekly_2020_09_01&utm_medium=email&utm_term=0_d3ee 4c3e04-17f2aea516-322608333.

14. "Helping Traumatized Children Learn." Trauma Sensitive Schools. Accessed May 26, 2021. https://traumasensitiveschools.org/trauma-and-learning/the-problem-impact/#:~:text=Tra umatic%20experiences%20can%20impact%20learning,need%20to%20succeed%20in%20sc hool.

15. "How COVID-19 Could Affect Kids' Long-Term Social Development." Healthline. Accessed May 26, 2021. https://www.healthline.com/health-news/social-distancing-effects-on-social-development.

16. "How does social isolation affect a child's mental health and development?" No Isolation. Last modified April 29, 2019. Accessed May 26, 2021.

17. Klass, Perri. "Do Masks Impede Children's Development?" New York Times. Last modified September 14, 2020. Accessed May 26, 2021. https://www.nytimes.com/2020/09/14/well/family/Masks-child-development.html.

18. Lindberg, Sara. "Impact of Isolation on Childhood Development." Very Well Family. Last modified August 2, 2020. Accessed May 26, 2021. https://www.verywellfamily.com/impact-of-isolation-on-childhood-development-4843122.

19. Markel, Howard. "Analysis: Why some schools stayed open during the 1918 flu pandemic."

PBS News. Last modified July 13, 2020. Accessed May 26, 2021. https://www.pbs.org/newshour/health/analysis-why-some-schools-stayed-open-during-the1918-flu-pandemic.

20. The National Child Traumatic Stress Network. Accessed May 26, 2021. https://www.nctsn.org/what-is-child-trauma/trauma-types/complex-trauma/effects#:~:text =Children%20who%20has%20experienced%20complex,depression%2C%20anxiety%2C%20 or%20anger.

23

21. "Policy Brief: The Impact of COVID-19 on children." United Nations. Last modified April 15, 2020. Accessed May 26, 2021. https://www.un.org/sites/un2.un.org/files/policy_brief_on_covid_impact_on_children_16 _april_2020.pdf.

22. "Preschoolers: Tips for Supporting Learning at Home." Child Mind Institute. Accessed May 26, 2021. https://childmind.org/article/preschoolers-tips-for-supporting-learning-at-home/.

23. Preston, Caroline. "'A drastic experiment in progress': How will coronavirus change our kids?" The Hechinger Report. Last modified April 15, 2020. Accessed May 26, 2021. https://hechingerreport.org/a-drastic-experiment-in-progress-how-will-coronavirus-changeour-kids/.

24. Rich, Gina. "During the 1918 flu pandemic, at-home learning meant little schoolwork." The Washington Post. Last modified May 13, 2020. Accessed May 26, 2021. https://www.washingtonpost.com/lifestyle/kidspost/school-during-the-1918-spanish-flu-pa ndemic/2020/05/12/7d606c90-7f32-11ea-9040-68981f488eed_story.html.

25. Sheldon-Dean, Hannah. "Will My Child Bounce Back From the Coronavirus Crisis?" Child Mind Institute. Accessed May 26, 2021.

26. "Social, Emotional, and Mental Well-being of Young Children during COVID-19." CDC. Accessed May 26, 2021. https://www.cdc.gov/coronavirus/2019-ncov/daily-life-coping/parental-resource-kit/early-c hildhood.html.

27. Sohn, Emily. "Worried About Your Kids' Social Skills Post-Lockdown?" New York Times. Last modified June 18, 2020. Accessed May 26, 2021. https://www.nytimes.com/2020/06/18/parenting/kids-social-needs-quarantine.html?action =click&module=RelatedLinks&pgtype=Article.

28. UNICEF. Accessed May 26, 2021. https://www.unicef.org/.

29. "What Is COVID-19? And How Does It Relate to Child Development?" Center on the Developing Child at Harvard University. Accessed May 26, 2021. https://developingchild.harvard.edu/resources/what-is-covid-19-and-how-does-it-relate-tochild-development/.

30. "Will face masks impede a child's development? Here's what the experts say." Advisory Board. Last modified September 17, 2020. Accessed May 26, 2021. https://www.advisory.com/en/daily-briefing/2020/09/17/masks.

31. "The potential impact of the COVID-19 pandemic on child growth and development: a systematic review." NCBI. Last modified September 3, 2020. Accessed May 26, 2021. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510529/.

32. "Social isolation in childhood and adult inflammation: evidence from the National Child Development Study." National Library of Medicine. Accessed May 26, 2021. https://pubmed.ncbi.nlm.nih.gov/25197797/#:~:text=In%20 addition%2C%20 children%20who%20were,be%20obese%20and%20to%20smoke.

33. Weissbourd, Richard. "How the Pandemic is Strengthening Fathers' Relationships with Their Children." Harvard Graduate School of Education. Last modified June 2020. Accessed May 26, 2021. https://static1.squarespace.com/static/5b7c56e255b02c683659fe43/t/5eea124b6fa0cb590b 968a90/1592398414030/How+the+Pandemic+is+Strengthening+Fathers+Relationships+wi th+their+Children+FINAL.pdf.

24

Modeling mTORC1 and its inhibitors for the treatment of tuberous sclerosis complex Una R.

Abstract Tuberous sclerosis complex (also known as tuberous sclerosis or TSC) is a rare genetic disease that is predominantly identified by the growth of benign tumors all over the body.

TSC also causes various neuropsychiatric disorders, as well as pulmonary and cardiovascular pathologies. TSC is caused by mutations in the TSC1-TSC2 complex, a pair of proteins that bind together as a nexus of multiple cell growth pathways. The TSC1-TSC2 complex controls cellular growth in response to factors such as oxygen levels, hormones, and general stress.

Pathogenic mutations are typically ones that occur in the binding interfaces of TSC1 and TSC2, and result in dissociation of the complex. This project attempted to propose or design a drug that could prevent the symptoms of TSC by compensating for failures in the relevant pathways. We focused on a downstream target of TSC1-TSC2, a protein complex called mTORC1. mTORC1 activates cell growth in response to TSC signals via a G protein called Rheb, so our drug needed to block the Rheb binding site to prevent this activation. The precursor to these experiments, however, was finding a way to manipulate our protein structure in silico. We tried many protein modeling softwares, including but not limited to ChimeraX, Phyre2, and Missense3D. Unfortunately, each software was inaccessible in one way or another and prohibited us from proceeding with our drug design. Had we been successful in modeling the protein complex, we would have then designed and modeled a drug to fit in the binding site between Rheb and mTORC1, blocking their interaction and potentially preventing excess cellular growth.

Introduction Tuberous sclerosis complex is a disorder characterized by physical symptoms including but not limited to angiofibroma, cardiac rhabdomyoma, angiomyolipoma, oral fibromas, retinal astrocytic hamartomas, and lymphangioleiomyomatosis. It also manifests in neuropsychiatric disorders such as hyperactivity, aggression, psychiatric conditions, intellectual disability, and problems with communication and social interaction. Diagnosis usually requires some combination of major features, like skin lesions and problems in the central nervous system, as well as minor features, like bone cysts or pits in dental enamel.

Despite this stunning array of symptoms, tuberous sclerosis can be caused by mutations as simple as swapping one single amino acid for another. Most TSC cases result from de novo mutations, meaning that most cases are spontaneous despite TSC being an autosomal dominant disorder.

Tuberous sclerosis complex affects approximately 1 in 10,000 births. Symptoms like hypopigmented macules appear in patients younger than 5 years, and most others worsen with age. TSC patients have a fivefold higher mortality rate than the general population, with

25

the most common direct causes of death being epilepsy, kidney complications, and complications from other infections.

Tuberous sclerosis is caused by mutations in the TSC1 or TSC2 genes, which encode proteins known as either TSC1 and TSC2 or hamartin and tuberin. In healthy cells, TSC1 and TSC2 form a complex (known as the TSC1-TSC2 complex) that helps control cell growth in response to positive or negative factors such as hypoxia, energy stress, Wnt signaling, cytokines, and amino acid levels. The TSC1-TSC2 complex in healthy cells is essentially a suppressor of cell growth, meaning that when activated, it inhibits growth. It does this by inhibiting the GTP-binding protein Rheb (Ras homologue enriched in brain), which in turn prevents the activation of mTORC1 (mammalian target of rapamycin complex 1). mTORC1 is a dimer whose most important element is mTOR, a kinase that catalyzes the addition of phosphate groups to the hydroxyl groups of serine or threonine.

This project aimed to use free online protein modeling software to propose a potential drug that might offer relief to victims of tuberous sclerosis. We first attempted to focus on the TSC1-TSC2 complex, but as structural information about these proteins was lacking, we switched to mTORC1, finding it to be a much more plausible target to simply inhibit. In addition, most pathogenic TSC1 or TSC2 mutations caused the dissociation of helices or the complex altogether, and designing a drug to act as a scaffold was unnecessarily complicated.

Instead, we decided to target mTORC1, itself the target of the TSC1-TSC2 complex. Our hypothetical drug would have needed to inhibit mTORC1 in order to curb excess cell growth.

Methods Electron microscopy images were obtained via rcsb.org (PDB) as follows: the core domain of TSC1 (PDB: 4KK0), the GAP (GTPase-activating protein) domain of TSC2 (6SSH), mTORC1 (6BCX), and mTORC1 activated by RHEB (6BCU). TSC1 and TSC2 mutations and phosphorylation sites were obtained from Huang et al, 2008 and Sudarshan et al, 2019.

Potential platforms for modeling software were discovered from the Wikipedia list of protein-ligand docking software and narrowed down by cost and accessibility. The most promising programs included ChimeraX, Phyre2, and Missense 3D. Results and Discussion Before the project could commence to the drug docking stage, however, the design or price point of each software made protein modeling itself impossible, let alone mutation mapping or drug docking. ChimeraX was able to load the PDB files, but not manipulate the protein structures or even tag specific amino acids with any accuracy. Phyre2 generated a PDB file of TSC2, but when we loaded it into Chimera, it was 2-dimensional. Missense 3D required the input of a PDB file, chain ID, residue number, and wild-type amino acid before selecting a mutant amino acid. However, due to discrepancies between the residue numbers, the program never worked beyond the first field of input. The rest of the software options we tried were even less useful. Had we found success in the technology, we would have started to design a drug by first examining the Rheb-binding site of mTORC1, then selecting a structure based on the layout of residues.

26

References 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

Chen, Y., & Zhou, X. (2020). Research progress of mTOR inhibitors. European Journal of Medicinal Chemistry, 208, 112820. https://doi.org/10.1016/j.ejmech.2020.112820 Deng, L., Chen, L., Zhao, L., Xu, Y., Peng, X., Wang, X., Ding, L., Jin, J., Teng, H., Wang, Y., Pan, W., Yu, F., Liao, L., Li, L., Ge, X., & Wang, P. (2018). Ubiquitination of Rheb governs growth factor-induced mTORC1 activation. Cell Research, 29(2), 136-150. https://doi.org/10.1038/s41422-018-0120-9 Henske, E. P., Jóźwiak, S., Kingswood, J. C., Sampson, J. R., & Thiele, E. A. (2016). Tuberous sclerosis complex. Nature Reviews Disease Primers, 2(1). https://doi.org/10.1038/nrdp.2016.35 Huang, J., & Manning, B. (2008). The TSC1–TSC2 complex: A molecular switchboard controlling cell growth. Biochemical Journal, 412(2), 179-190. https://doi.org/10.1042/BJ20080281 Ittisoponpisan, S., Islam, S. A., Khanna, T., Alhuzimi, E., David, A., & Sternberg, M. J.E. (2019). Can Predicted Protein 3D Structures Provide Reliable Insights into whether Missense Variants Are Disease Associated? Journal of Molecular Biology, 431(11), 2197-2212. https://doi.org/10.1016/j.jmb.2019.04.009 Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., & Sternberg, M. J. E. (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols, 10(6), 845-858. https://doi.org/10.1038/nprot.2015.053 List of protein-ligand docking software. (n.d.). https://en.wikipedia.org/wiki/List_of_protein-ligand_docking_software Rayhan, A., Faller, A., Chevalier, R., Mattice, A., & Karagiannis, J. (2017). Using genetic buffering relationships identified in fission yeast to reveal susceptibilities in cells lacking hamartin or tuberin function. Biology Open. https://doi.org/10.1242/bio.031302 Rosset, C., Netto, C. B. O., & Ashton-Prolla, P. (2017). TSC1 and TSC2 gene mutations and their implications for treatment in Tuberous Sclerosis Complex: A review. Genetics and Molecular Biology, 40(1), 69-79. https://doi.org/10.1590/1678-4685-GMB-2015-0321 Sudarshan, S., Kumar, M., Kaur, P., Kumar, A., G., S., Sapra, S., Gulati, S., Gupta, N., Kabra, M., & Roy Chowdhury, M. (2019). Decoding of novel missense TSC2 gene variants using in-silico methods. BMC Medical Genetics, 20(1). https://doi.org/10.1186/s12881-019-0891-y Sun, W., Zhu, Y. J., Wang, Z., Zhong, Q., Gao, F., Lou, J., Gong, W., & Xu, W. (2013). Crystal structure of the yeast TSC1 core domain and implications for tuberous sclerosis pathological mutations. Nature Communications, 4(1). https://doi.org/10.1038/ncomms3135 UCSF ChimeraX: Structure visualization for researchers, educators, and developers.

Pettersen EF, Goddard TD, Huang CC, Meng EC, Couch GS, Croll TI, Morris JH, Ferrin TE. Protein Sci. 2021 Jan;30(1):70-82.

Yang, H., Jiang, X., Li, B., Yang, H. J., Miller, M., Yang, A., Dhar, A., & Pavletich, N. P. (2017).

Mechanisms of mTORC1 activation by RHEB and inhibition by PRAS40. Nature, 552(7685), 368-373. https://doi.org/10.1038/nature25023 Zöllner, J. P., Franz, D. N., Hertzberg, C., Nabbout, R., Rosenow, F., Sauter, M., Schubert-Bast, S., Wiemer-Kruel, A., & Strzelczyk, A. (2020). A systematic review on the burden of illness in individuals with tuberous sclerosis complex (TSC). Orphanet Journal of Rare Diseases, 15(1). https://doi.org/10.1186/s13023-019-1258-3 Zou, Z., Chen, J., Yang, J., & Bai, X. (2016). Targeted Inhibition of Rictor/mTORC2 in Cancer Treatment: A New Era after Rapamycin. Current Cancer Drug Targets, 16(4), 288-304. https://doi.org/10.2174/1568009616666151113120830

27

Constructing an Electromagnetic Railgun Henry K.

Abstract Railguns are straight out of science fiction. In movies, we see them mounted on starships or defending moon bases. Yet unlike shrink rays or lightsabers, railguns are very much real—just look at the U.S. Navy railgun that can shoot over 120 miles. Given its name, one might assume that a railgun is some type of device that accelerates a projectile, and probably has something to do with rails. That would be correct. Railguns harness electromagnetic forces to accelerate a projectile down parallel lines of a conducting material: in the case of my railgun, copper rods. By passing a huge amount of electrical current down these rails, and through the projectile, a magnetic field, and hence force, is created, hopefully one large enough to move the projectile. Here’s the problem: while the concept and design of a railgun is simple enough, maximizing the current proves troublesome. Railguns need tremendous amounts of power, for which it is essential to optimize the balance between the electrical discharge time and the magnitude of the current, especially to get through the friction barrier. Even the Navy’s railgun wasn’t practical using all that electricity. Since I wanted to construct a railgun, what was I going to do? For one thing, my railgun wasn’t going to shoot 20kg projectiles at Mach 6. To get enough power, I increased the voltage of two 9V batteries through a DC-DC boost converter and used it to charge a capacitor bank. But even with these limitations, a working version isn’t easy. Other factors, such as needing the projectile to enter the rails already with an initial velocity, made it all the more difficult. While my railgun might not look the same as those from sci-fi, the same electromagnetic principles are at work.

Introduction A railgun is essentially a simple circuit with a couple of moving parts, complete with wires, a battery, and a voltage drop across it; the same elements that are behind the circuit of a light bulb or a toaster. The only difference is that, while the energy provided by the battery is released in light with the lightbulb and heat with the toaster, the energy released by the railgun is in motion: electrical energy to kinetic energy. To tell the truth, the differences might be slightly greater than just this. For one, the wires of the railgun are thick copper bars, the projectile itself is conductive, the battery is a capacitor bank charged up with a boost converter, and the circuit breaks as soon as the projectile leaves contact with the rails.

So how does a railgun work? The first thing to understand is that because it is a circuit, electrical current, i.e. the flow of charged particles, moves through the conductive “wires” of the railgun. Another thing to understand is that currents create magnetic fields; the current flowing through the railgun creates a magnetic field. The third thing to understand is that a current within a magnetic field experiences a force, called the Lorentz force; the current within the railgun is within a field that it creates, and so experiences a force from it. With

28

these three facts, the way a railgun works is surprisingly clear. You can model these facts easily with a method called the right hand rules. The magnetic field created due to a current is always perpendicular to the direction of the current. You can see the direction of the field at a certain point by sticking out your right hand, like giving a thumbs up. The direction your thumb points is the direction of the current, and the direction your other fingers curl is the direction of the magnetic field. The magnetic field around a current will always be circling around it, just due to it having to be perpendicular. See Figure 1. The second right hand rule is used to model the direction of the force experienced by a current within a magnetic field.

This force has to be both perpendicular to the direction of the current as well as the direction of the magnetic field. You can see the direction of the field by sticking out your right hand, with your thumb and index finger pointing out, both perpendicular to each other. The direction your palm faces is the direction of the magnetic field, the direction of your index finger is the direction of the current, and the direction of your thumb is the direction of the force. See Figure 2.

Figure 1

Figure 2

Through the right hand rules, we now know the direction of the magnetic field created by a current, as well as the direction of the Lorentz force, but we do not know the magnitude of either. Two equations give us the values. The one that models the created magnetic field is equation 1, and the one that models the force experienced by a current in a magnetic field is equation 2. In these equations, B is the magnetic field, I is the current, r is the distance of the desired point at which you want to know the magnitude of the field from the current carrying wire, F is the Lorentz force, l is the length of the current-carrying wire that experiences the force––in the case of the railgun this is the length of the projectile––, and µ𝑜 and 2π are constants. B is inversely proportional to r, so as r decreases, i.e. the distance between the point and the wire gets smaller, B increases. The Lorentz force is proportional to the current; more current means more force.

29

Equation 1 µ𝐼

𝐵 =

𝑜

2π𝑟

Equation 2

𝐹 = 𝐼𝑙 × 𝐵

A railgun uses these properties to create the force that accelerates the projectile. The rails are parallel to each other and the conductive projectile fits perpendicularly in between them.

When the battery is connected, electrical current flows up one of the rails, across the projectile, and down the other parallel rail, completing the circuit. See Figure 3.

Figure 3

Figure 4

With the first right hand rule, we see that the direction of the magnetic field created by the current flowing down the rails is looping outwards from each rail, with the field facing directly up on the plane in between the rails. See Figure 4. This is the same plane the projectile lies on, and so it is within a field facing up, perpendicular to the direction of the current flowing through it. Now we can use the second right hand rule; the projectile has a current running through it, and because it is in an upwards facing magnetic field created by the rails, it experiences a force facing out of the rails. This is the force that accelerates the projectile.

We now know the direction of this force, but not the magnitude of it. To calculate the acceleration of the projectile, which we would need to do to see how effective the railgun is, we have to know this magnitude, but this is not an easy calculation. Given the inverse r relationship of the magnetic field, and thus force as well, the force upon the projectile will not be constant along the length of it. Finding the average magnetic field along the projectile will give us a way to find it. First we can calculate the magnetic field at some arbitrary point on the projectile, and then integrate across the length of it to find this average. If we call the length of the projectile L then we can say the magnetic field at this point is the magnetic field from the right rail added to the magnetic field from the left rail, with the radii being L r and r respectively. See Figure 5. Then we can just add up the magnetic field from both the

30

sides, and get equation 3. Now once we’ve got this, all we have to do is integrate it along L, or actually L minus a slight amount of space called δ as the projectile will not be infinitely close to the current of the rails, and δ is this negligible distance. If the projectile was indeed touching the current, i.e. 𝑟 = 0, then the force at those contact points would be infinite, so instead we integrate from δto 𝐿 − δ, and get equation 4, where r has been replaced with X. Solving this equation leads to the values of the average magnetic field on the projectile: equation 5. By substituting this average B into equation 2, the Lorentz Force equation, we can find the force on the projectile: equation 6. Then, since force is just mass times acceleration, we can substitute 𝑚𝑎for 𝐹, and find the acceleration of the projectile: equation 7.

Figure 5

Equation 3

Equation 6

Key

Equation 4

Equation 5

Equation 7

The acceleration of the projectile is dependent on the current squared, the mass, and the length of the projectile. More current and less mass optimizes the acceleration. We can see the relationship between the current and acceleration in a graph of the equation: Figure 6.

The y-axis is the acceleration, and the x-axis is the current. Values are set for 𝑚, µ0, 𝐿, 𝑎𝑛𝑑 δ.

The mass is 1 gram, the length is 1cm, andδis 0.1mm. With this, we see how more current means more acceleration, but what about the length of the projectile; how does that relate to the acceleration? The longer the projectile is, the more acceleration it experiences, but this increased acceleration asymptotes if L gets sufficiently large: see figure 7. Here the current has been set at 100A, and the x-axis is the projectile length. This means as long as the

31

projectile surpasses some minimum length, it doesn’t matter how long it is; actually the longer it gets, the more mass it has and thus lower acceleration.

Figure 6 Figure 7

Building The Railgun Knowing the theory behind a railgun was one thing, constructing a working version was another. A railgun is made up of three important parts; a power source to provide the necessary current, the rails to create the magnetic field, and the projectile.

Rails Setup The first of these I attempted to create was the rail setup. For the rails themselves I used ½ inch copper rods; copper has a low resistivity and so the current will not diminish as much and a larger area of the wire means a larger current as well. However, it wasn’t as simple as putting two rails parallel to each other. The same Lorentz Force responsible for accelerating the projectile also accelerates the rails away from each other; the rails repel when current runs through them. This is because they also are in a magnetic field from the other rail, and since current is running through them, they experience a force. Using the right hand rule we see that the direction of this force points away from the other rail. The railgun will rip apart when firing. For this reason I 3D printed a plastic sheath that fit around each rail, and these sheaths fit into a 2in PVC pipe, holding the rails together. See Figure 8 and 9.

Figure 8

Figure 9

32

This wasn’t enough for the completed rail setup; the projectile has to enter the rails with an initial velocity or it will be welded to them when the current is released. When all the current goes through a non-moving projectile at once, the current can melt the projectile and rails together. This required a trigger mechanism to provide the initial push, acting as a kickstart to the projectile.

Initial Trigger Velocity Device Attempt 1: Gauss Rifle A Gauss rifle, Gauss gun, or Gauss cannon, is another type of linear magnetic accelerator, although a much simpler one than the railgun. I decided to use this to provide the trigger velocity because of how it would combine two magnetic accelerators into one, as well as provide an opportunity to research the Gauss rifle. Unlike the railgun, a Gauss rifle doesn’t use electrical energy to create magnetic fields; instead it uses magnets themselves to increase the kinetic energy of the projectile. If one places any metal pellet near a magnet, it will accelerate towards the magnet, gaining kinetic energy. The closer the pellet is to the 2

magnet, the more force exists, in a 1/𝑟 relationship. These properties aren’t unfamiliar, we know them through simply playing with magnets, yet it’s essentially what drives a Gauss rifle. The magnet is an accelerator, just like a railgun, to the pellet. The only difference is that when the pellet slams into the magnet, all the gained kinetic energy is released. The Gauss rifle uses conservation of momentum to sidestep this. If a secondary pellet were initially connected to the magnet, the first pellet's momentum would be transferred through the magnet to the second pellet (P2). The problem here is that the P2, provided that P1 and P2 are the same mass, will only move as far as the P1 went, since P2 experiences the same magnetic force back towards the magnet. Then P2 would accelerate towards the magnet, and the cycle repeats.

Initial position

Final position

While this is better than the previous situation, with only P1, it still isn’t a successful accelerator. A third pellet allows the same momentum transfer, but P3 isn’t constrained by the same magnetic force as P2 was. This way, P3 can continue moving forward, and thus be a projectile.

Initial position

Final position

33

Just by the fact that P3 is farther from the magnet than P2, and so the attractive magnetic force is weaker, can P3 continue moving forward. Adding more pellets continues to reduce this constraining magnetic field, and thus increase the velocity of the final pellet.

Now we can go one step further. Stacking up copies of this magnet-pellet unit, by having P5 be the trigger pellet for a new unit, the velocities just keep adding up.

I used 1.25in neodymium magnets and 0.75in steel ball bearings for the pellets. 3D printed units housed these components, which were made with a stacking connection. As the first pellet needs to be released to accelerate at the magnet, I printed a separate unit for this purpose, in which all that is needed is to raise a tilted platform upon which the first bearing sits, until it is free to roll down the 3D printed unit on its own.

However, my Gauss rifle wasn’t a successful method to provide the initial velocity into the rail tube. The final ball bearing didn’t have enough speed, as the friction within the 3D-printed cases outweighed the boosts each unit gave it. The bearing also tended to hop, and so not fit into the rails.

Attempt 2: Spring piston For this, I tried a more straight-forward approach: a simple plate pulled back by a spring and then released into the projectile, pushing it into the rails.

34

The trigger mechanism provided the initial velocity well and was used for the final version of the railgun.

Initial Position

Final Position

Power Source Finding a power source large enough to provide the necessary current was the hardest part of the project. Any consumer-level battery wouldn't come close to having it, but I didn’t need the constant current the batteries emitted; I only needed current for the short amount of time the projectile accelerated down the rails. Capacitors offered just this. A capacitor is a device that charges up electrical charge, then releases it all at once, giving off a high current for a short time. The higher voltage the capacitor can handle means the higher current it gives off, and the higher capacitance it has, i.e. the more electrical charge it stores, the longer the current is released. Maximizing both of these is needed for the railgun. A high voltage means more acceleration, but a high capacitance means more time it accelerates. The problem is the higher voltage the capacitor can hold, the less capacitance it has. Finding a balance between these is key. We want the strong force of a high voltage capacitor, but only if there is sufficient time for it to accelerate. Getting a high voltage capacitor with a high capacitance is incredibly expensive, but a solution exists for increasing the capacitance: adding capacitors in parallel adds each capacitance together; this is called a capacitor bank. I used 14 180V capacitors, each with 400mf of capacitance, summing to 5600mf.

But now I had to find a way to charge the 180V capacitors, which required finding a power source of 180V. That's greater than the voltage of an electrical outlet. There is an easy trick to get the required voltage: the use of a DC-DC boost converter. This is a device that boosts up the voltage of a DC power source, for me just two 9V batteries. Putting these batteries together in parallel got the voltage up to 18V, then through the converter to 180V. With this I could charge my capacitor bank. Now I had the rails, projectile––just a copper cylinder––and power source. All I had to do now was assemble.

Assembly The first step was connecting the boost converter to the capacitor bank, which I had made by soldering each capacitor to the next. See Figure 10. When batteries were connected to the converter, the capacitors would start to charge. Given that accidentally brushing against the

35

charged capacitor bank would result in serious injury, I boxed this setup in wood, and connected it to an ammeter so I could measure the charge of the capacitors. Then I connected the capacitor bank to the rails, finishing the design of the railgun. See Figure 11.

Figure 10

Figure 11

Results This railgun design didn’t end up successful because the projectile, when shot through the rails, tended to hop. This interrupted the connection between the projectile and the rails, so the projectile only had current running through it at the few points it was actually touching.

The design of the rails had the projectile just sitting on top of the copper rods, only held down to them by gravity, which wasn’t enough to ensure a constant connection. When fired, at each point the projectile hit the rails the capacitor bank discharged some of the stored charge, which is a good sign as it shows that the electrical components of the railgun were working. Taking off the PVC casing, I could clearly see each hop. See Figure 12. After each hop, the current released is lowered, and the amount of energy released in light, sound, and heat is lowered as well.

Figure 12 Initial rails

First Hop

Second Hop

Third Hop

Next, I plan to create a new rail setup in which the projectile is always in contact with the rails, so that it is impossible for it to hop and lose contact with them. I will use rectangular copper bars and a rectangular projectile that fits directly in between them, always touching both. This will have much more friction than the previous design, but will ideally stop the hopping problem. The railgun is an ongoing project, and hopefully I will get a working version soon.

36

Sources

1. Right Hand Rule Diagram. www.pasco.com/media/files/static/guides/right-hand-rule/curl_rhr.png.

2. Right Hand Rule Lorentz Force. upload.wikimedia.org/wikipedia/commons/thumb/3/3d/Right_hand_rule_cross_p roduct_F%3DJ×B.svg/1200px-Right_hand_rule_cross_product_F%3DJ×B.svg.png 3. Railgun Diagram. www.digikey.com/maker-media/5f091812-8045-4318-9a23-d6865608bd52.

4. “US Navy Railgun - Their Most Powerful Cannon.” YouTube, YouTube, 11 May 2018, www.youtube.com/watch?v=58MmOpSm4LY. 5. MstrJames, director. Making a RAILGUN and Then TESTING It! YouTube, YouTube, 1 Mar. 2018, www.youtube.com/watch?v=fKEaDhDTciQ&t=458s.

6. TheLiquider, director. How To Build a Railgun Experiment. YouTube, YouTube, 3 Oct. 2013, www.youtube.com/watch?v=YH08iDj2yic.

37

An Exploration of Nuclear Magnetic Resonance and Dynamic Nuclear Polarization William T. Abstract Spectroscopy, the study of interactions between matter and electromagnetic radiation, is essential to classifying and understanding molecular structures. Nuclear Magnetic Resonance (NMR), one of the most prevalent types of spectroscopy, is a method of identifying monomolec-ular organic compounds, proteins, and complex molecules. It also provides detailed information about the structure, dynamics, reaction state, and chemical environment of molecules. Building upon our initial understanding of quantum physics, we researched the mechanics behind NMR, specifically its applications to solid state. Solid state NMR is considerably more complicated than NMR in solution, and is characterized by the presence of anisotropic interactions which are usually averaged out in solution NMR. Hence, it is necessary to employ various techniques to suppress these interactions and obtain interpretable NMR spectra. One such method is magic-angle spinning (MAS). By spinning the sample at the “magic angle,” 54.74°, the resolution of the spectrum is increased due to orientation-dependent interactions becoming averaged out. Af-ter gaining insight into the physics behind the technique, we examined recent applications and extensions made. Of particular interest was the combination of Dynamic Nuclear Polarization (DNP), a result of transferring spin polarization from electron to nuclei, with MAS, or MAS-DNP. A brief review of quantum mechanical NMR spectroscopy is presented, a summary of the two individual methods, and a discussion of research into their combination.

1

Introduction

Nuclear Magnetic Resonance is an effect in which magnetic fields are used to perturb atomic nuclei, resulting in them producing an electromagnetic signal that can be used to identify and classify molecular structures. It relies on the intrinsic magnetic field of certain nuclei, and occurs near the resonance frequency of that nuclei. Given its usefulness, NMR is widely used in both organic chemistry and physics, and also has applications in the field of medical imaging. However, the technique has its limitations. For example, NMR is most commonly applied to solutions due to the averaging of anisotropic interactions that occurs due to Brownian motion. Obviously, solid state NMR (ssNMR) does not benefit from random motion in all directions, and hence spectra resolution can be very low and difficult to interpret.

Motivated by the difficulty of this field, we examined the intricacies of ssNMR and its methods.

In order to increase the resolution of solid-state NMR experiments, a number of different techniques are employed. Magic-angle spinning (MAS) is one such technique, and stems from the fact that the three main anisotropic interactions are either completely averaged out or noticeably diminished while rapidly spinning the sample at 54.7 , the magic angle.

However, MAS alone is not often enough, and is constantly being improved. For instance, the MAS must occur at a sufficiently fast frequency, specifically at a rate equal to or greater than the dipolar linewidth, else spinning sidebands will become visible. Hence, most developments in MAS are improvements on rotation speed. However, there is also interest in the combination of MAS with other resolution increasing techniques, such as dynamic nuclear polarization (DNP).

Dynamic nuclear polarization is the result of transferring spin polarizations from electrons to nuclei, thereby aligning nuclear spins to the extent that the electrons were aligned. Since electrons

38

are more easily manipulable than nuclei, by leveraging electron alignment via DNP it is possible to boost signal intensities in NMR experiments by one to three orders of magnitude. Owing to the huge gains in signal intensity and resolution by combinging these techniques, we are motivated to investigate the physics behind them, as well as some of the recent applications.

1.1 1.1.1

Nuclear Magnetic Resonance Nuclear Magnetism

Nuclear magnetic resonance relies on a sample’s nuclear spin and its interactions with magnetic fields. Nuclear spin angular momentum is represented by the nuclear spin quantum number, I. The vector quantity corresponding to nuclear spin angular momentum, characterized by I, has magnitude 1

1

|I| = [I · I] 2 = h̄[I(I + 1)] 2

(1)

Quantum mechanics tells us that only one of the three Cartesian components of I can be specified simultaneously with I2 , and so by convention we specify the z-component with Iz = h̄m

(2)

with the magnetic quantum number, m, ranging from m = ( I, I + 1, . . . , I 1, I), giving Iz 2I + 1 possible values.

Nuclei with nonzero spin angular momentum also have corresponding nuclear magnetic moments.

The nuclear magnetic moment is colinear with the vector representing the nuclear spin angular momentum and is given by µ= I (3) µz = Iz = h̄m

(4)

The gyromagnetic ratio, , is a constant for a given nucleus. In the presence of an external magnetic field, nuclear spin states have energies given by E=

µ·B

(5)

with B being the magnetic field vector. A spin then has minimum energy when the projection of µ onto B is maximized. The quantization of nuclear spin results in both the magnetic moment and magnetic energies being quantized, with these quantized energies proportional to the projection of µ along B. In the presence of a static magnetic field, the main action of the spin is to precess around the direction of the field, at its quantized energy. For a static field along the z axis, the convention in NMR, the energies become Em = Iz B0 = mh̄ B0 (6) where B0 is the static magnetic field strength. We now define the bulk magnetic moment, M, and the bulk angular momentum J. These are given by the vector sum of the corresponding quantities for each given nuclei. At thermal equilibrium, owing to Zeeman splitting and statistical mechanics, the transverse components of µ and I sum to zero. This along with small population differences between energies gives rise to a bulk magnetization parallel to the static magnetic field, which is central to NMR spectroscopy.

1.1.2

Quantum Mechanical NMR Spectroscopy

We now present a brief quantum mechanical description of a one-pulse NMR experiment. The initial state of our system is described via the equilibrium density operator. At thermodynamic equilibrium we assume that our nuclear spin states are at equilibrium with the lattice. By extension the values of the probability distribution, P( ) are constrained such that the populations of the stationary

39

states take the form of a Boltzmann distribution. In addition, we require that the density matrix is diagonal at equilibrium due to the fact that the different subensembles are uncorrelated. These requirements lead us to n o eq = e H/kb T /Tr e H/kb T (7) which in the high temperature limit can be approximated to eq

⇡ E/N

H N kb T

(8)

N represents the dimensionality of the Hilbert space. For example, for M spin-1/2 nuclei N = 2M .

However, this term is a constant that doesn’t affect the NMR experiment and is usually not written.

Removing this term and writing in the Zeeman Hamiltonian we obtain eq

=

M X h̄!0 k Ikz N kb T

(9)

k=1

The complex magnetization recorded during the acquisition period is given by ⌦

↵ M + (t) = N h̄ Tr

(t)F+

(10)

N is volume density of the number of spins, and F+ is F+ =

M X

(11)

(Ikx + iIky ).

k=1

For a single spin, we have that angle ↵ applied to Iz is

eq

/ Iz and hence the effect of a pulse with x-phase and a rotation

Rx (↵)Iz Rx 1 (↵) = Iz cos ↵ when ↵ =

⇡ 2

Iy sin ↵

(12)

this becomes

⇡ ⇡ (13) Rx ( )Iz Rx 1 ( ) = Iy 2 2 This is identical to the result we expect via the classical Bloch model. We now consider the evolution of the Iy magnetization under the Zeeman Hamiltonian (in the rotating frame) (14)

Hz = ⌦Iz Following this out, with the derivation omitted here, we obtain (t) = Ix sin ⌦t

Iy cos ⌦t

(15)

Not only does this allow us to calculate the magnetization, it also implies that magnetization with a positive resonance offset in the rotating frame precesses in the clockwise direction, looking along the z axis.

We can now calculate our detectable magnetization for a one-pulse sequence for a single isolated spin: n ⌦ +↵ ⇡ o N h̄2 !0 M (t) = exp i(⌦t ) (16) 8kb T 2

Hence our quantum mechanical formalism once again outputs the form expected using a classical Bloch model in the absence of relaxation. For further explanation on the classical model and the actual experimental procedure behind NMR, refer to the previous paper.

40

Figure 1:

2

, ✓, and

as referenced in (20), (21)

Results

2.1

Solid-State Nuclear Magnetic Resonance

As previously mentioned, ssNMR is considerably more difficult and complex than solution NMR. This is primarily due to three types of anisotropic interactions: 1. Chemical shift anisotropy (CSA) 2. Dipolar coupling 3. Quadrupolar interactions In a sample with little mobility like crystalline powders, glasses, and other solids, anisotropic local fields have substantial influence on spin behavior. This results in very broad spectra, as the full results of the interactions are observed. In order to get suitable spectra MAS, dilution, multiplepulse sequences, and cross polarization are used, among other methods.

A brief explanation of each interaction is presented, followed by an explanation of MAS and its ability to mitigate each.

2.1.1

Chemical Shift Anisotropy

Chemical shift anisotropy is induced by electron clouds surrounding the nucleus, and while we usually expect direction independent chemical shifts, direction dependent chemical shifts cause broad spectra to occur. In exploring CSA, we first begin with equation for Zeeman splitting, E=

h̄)B0

(17)

Taking into account shielding, , we obtain E= Hence for a single spin

1 2

h̄(1

)B0

(18)

nucleus, we can write hIˆ B0 2⇡

Ĥcs =

41

(19)

Figure 2: Two nuclear spins where is now the chemical shift tensor. We can write the magnetization at the nucleus in terms of ✓ and , the angles from the Z axis and X axis respectively. The chemical shift, !cs is then !cs =

!0 (

!cs =

xx

!0

sin2 ✓ cos2

+

yy

sin2 ✓ sin2

1 !0 [3 cos2 ✓ 2

iso

+

zz

cos2 ✓)

1 + ⌘ sin2 ✓ cos 2 ]

(20) (21)

The proof of which is left to the reader.

2.1.2

Dipolar Coupling

All NMR active nuclear spins exhibit a magnetic dipole moment. This generates a magnetic field that interacts with the dipole moments of the other nuclei. ~ ~ Consider two nuclear spins, Iˆ1 and Iˆ2 , separated by a distance r. The interaction between the ~ 2 is two dipoles, µ ~ 1 and µ E=

µ0 [(~ µ1 · µ ~ 2 )r 4⇡

3

3(~ µ1 · ~r)(~ µ2 · ~r)r

5

]

(22)

Assuming the two dipoles have the same orientation, we obtain E= 2.1.3

µ0 (~ µ1 µ ~ 2 )r 4⇡

3

[1

3 cos2 ✓]

(23)

Quadrupolar Interactions

Nuclei with spin quantum number > 12 have a non spherical charge distribution, and hence have an electric quadrupole moment that couples with surrounding electric field gradients.

The interaction of a quadrupole with a field gradient in an arbitrary frame is given by ĤQ =

eQ Iˆ · V · Iˆ 2I(2I 1)h̄

(24)

Writing this in terms of the Cartesian spin operators and changing the reference frame to that of the electric field gradient, we can see that expansion of the Hamiltonian can be done using perturbation theory. Doing so, and expanding in terms of polar coordinates and raising/lowering operators, we find 1 (3 cos2 ✓ 1)(3Iˆz2 Iˆ2 ) + ...

(25) HˆQ = 4I(2I 1) 2 which is sufficient for our needs.

42

Figure 3: Spherical charge distribution vs. non-spherical. Note: Due to the symmetry of the quadrupole, it does not couple with constant fields, hence the field gradient.

Figure 4: NMR spectra of polycrystalline aluminum. The left-hand diagram is the static specimen, while the right-hand diagram is the specimen under magic angle spinning.

2.2

Magic Angle Spinning

In light of these resolution decreasing interactions, it’s necessary to implement techniques that mitigate their anisotropy. MAS NMR is a powerful method that enables incomparable sensitivity and resolution.

The magic angle, 54.7 can be easily seen via the Hamiltonians of the anisotropic interactions,

(1)

(1)

where HCS , HQ , and HD are the Hamiltonians of chemical shift, first-order nuclear quadrupolar, and first-order dipolar interaction as roughly derived in the previous section. Note that all three of these interactions contain 3 cos2 ✓ 1 terms, which is zero when ✓ = 54.74 . Additionally, the rate of MAS mustbe greater or equal to the magnitude of the anisotropic interactions in order to average it to zero. Hence, the orientation dependent interaction averaging in solution is mimicked by MAS, with CAS averaging to only the isotropic portion, QI being partially averaged, and DI being completely averaged out. Experimentally, MAS is usually conducted via an air turbine mechanism, typically spun by air or nitrogen gas, and was first described in 1958 by Edward Raymond Andrew, A. Bradbury, and R. G. Eades.

2.3

Dynamic Nuclear Polarization

DNP is a technique that utilizes electron polarization to provide a boost to NMR signal intensities by several orders of magnitude. DNP consists of transferring spin polarizations from electrons to nuclei, allowing NMR experiments greater control over nuclear spin polarization. First used in the early

43

Figure 5: Nuclear spin energy level diagram for two spin

1 2

nuclei.

1950s at low magnetic fields, DNP has recently become much more applicable due to the progress made in increasingly high-frequency high-power sources.

DNP NMR takes place by way of four methods: the Overhauser Effect, the solid effect, thermalmixing, and the cross effect. In solution-state NMR only the Overhauser effect is applicable, but all can be used for ssNMR. 2.3.1

The Overhauser Effect

The Overhauser effect (OE) utilizes the transfer of one population of spins to another via crossrelaxation. It was first developed by Albert Overhauser in 1953, who proposed that nuclear spin polarizations could be enhanced by the irradiation of electrons in certain metals.

OE and spin-lattice relaxation are very closely related, with OE allowing transitions between normally not allowed energy levels through dipolar relaxation. Expressed in terms of bulk NMR magnetizations, the experimentally observed steady-state OE for a nucleus, I, is ⌘IS =

MIS M0I M0I

(26)

where M0I is the magnetization (resonance intensity) of nucleus I at thermal equilibrium.

In essence, the Overhauser effect is an attempt by the system to remain at equilibrium when nearby spins are perturbed. When a specific population is magnetically excited and its neighbor is at equilibrium, cross relaxation occurs resulting in the intensity of the nucleus at equilibrium changing.

Hence, by saturating the transitions of electron spin with microwave irradiation we can perturb the nuclear spin level populations.

2.3.2

The Solid Effect

The solid effect governs the polarization transfer if the concentration of electron spins is low and the mutual interactions between them can be neglected. Fundamentally, the solid effect (SE) consists of flip-flop transitions between electron and nuclear spins caused by a microwave field.

Most nuclear spins are far away from electron spins, and hence their interactions are negligible.

Nevertheless, we are still capable of transferring spin polarizations back and forth via microwave field. First, the field induces flip-flop transitions between electron spins and nearby nuclear spins.

Next, this polarization is further transported away by means of flip-flop transitions from nuclear spin to nuclear spin. This is generally described as "nuclear spin diffusion." Lastly, due to the amount of nuclear spins we wish to polarize and conservation of energy, it is necessary to repolarize the electrons via spin-lattice relaxation.

44

Figure 6: Overview of the processes involved in the solid effect.

Figure 7: Overview of the processes involved in the solid effect.

In such an interaction, the polarization transfer rate equation has the shape @ (Ps cos ✓ @t 2.3.3

PI ) =

W± (Ps cos ✓

PI ).

(27)

The Cross Effect and Thermal Mixing

The final two mechanisms through which DNP takes place are known as thermal mixing and the cross effect. In both of these mechanisms, the microwave field first reduces the polarization of some electron spins, creating a variety of different spin polarizations. The hyperfine and superhyperfine interactions between electron and nuclear spins then combine to transfer such differences of polarization to the nuclear spins. If the electron spins are in thermal equilibrium this is known as thermal mixing, otherwise it is known as the cross effect.

Although complicated, full solutions are obtainable from such a system. After applying a microwave field for a long time, a stationary value is shown to appear, PI = tanh !0

NZ

(28)

This result is widely applicable, such as for a proton spin polarization in a glass doped with radical TEMPO, a graph of which is shown in Figure 8.

45

Figure 8: Overview of the processes involved in the solid effect.

Figure 9: Overview of the processes involved in the solid effect.

3 3.1

Discussion Combining Techniques

In the last decade, the use of MAS and DNP in tandem (MAS-DNP) has undergone rapid developments. As each technique individually benefits NMR experiments greatly, it’s no surprised that in combination NMR spectra are significantly improved. MAS-DNP offers many advantages, from its wide range of applicability to its very high rate of sensitivity gains. Additionally, it is also applicable to multidimensional experiments, which are typically quite difficult.

The first DNP-NMR experiments using MAS were reported in 1983 by Wind, R.A. It primarily allowed for the acquisition of high-resolution spectra from materials such as coal, diamonds, polymers, and organic conductors. However, experiments were constrained to B0  1.5T .

5T by MIT in the 1990s. Developments with cyclotron resMAS-DNP was enabled for B0 onance masers, and higher-power microwave sources were the main source of these development, enabling more efficient DNP transfer.

Currently, MAS-DNP represents one of the most powerful techniques in NMR experiments. Its wide range of uses and its huge spectra resolution improvements continue to further drive developments in hardware, polarization agent design, sample preparation, as well as theoretical understanding of the mechanics behind NMR.

46

4

References

1. Kaminker, Ilia. “Recent Advances in Magic Angle Spinning[U+2010]Dynamic Nuclear Polarization Methodology.” Israel Journal of Chemistry, vol. 59, no. 11-12, 2019, pp. 990–1000., doi:10.1002/ijch.201900092.

2. Rankin, Andrew G.M., et al. “Recent Developments in MAS DNP-NMR of Materials.” Solid State Nuclear Magnetic Resonance, vol. 101, 2019, pp. 116–143., doi:10.1016/j.ssnmr.2019.05.009.

3.

“Nuclear Overhauser Effect.” Wikiwand, www.wikiwand.com/en/Nuclear_Overhauser_effect.

4.

Cavanagh, J. (2008). Protein NMR spectroscopy: principles and practice. Academic Press.

5. Wenckebach, W. T. “The Solid Effect.” Applied Magnetic Resonance, vol. 34, no. 3-4, 2008, pp. 227–235., doi:10.1007/s00723-008-0121-9.

6. Dynamic Nuclear Polarization, www.sigmaaldrich.com/US/en/technical-documents/technicalarticle/analytical-chemistry/nuclear-magnetic-resonance/dynamic-nuclear-polarization.

7. Bridge12 Technology, Inc. “Dynamic Nuclear Polarization (DNP) Spectroscopy.” Bridge12, www.bridge12.com/learn/dnp-spectroscopy.

8. Libretexts. “Chemical Shift (Shielding).” Chemistry LibreTexts, Libretexts, 15 Aug. 2020, chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_(P _Theory/ NMR_Interactions/Chemical_Shift_(Shielding).

9.

hysic

Straus, "NMR Interactions: Dipolar Coupling." https://groups.chem.ubc.ca/straus/l4.pdf.

10. Libretexts. “Quadrupolar Coupling.” Chemistry LibreTexts, Libretexts, 15 Aug. 2020, chem.libretexts.org/Bookshelv _Theory/ NMR_Interactions/Quadrupolar_Coupling.

es/Ph

11. “Magic Angle Spinning in Solid State N.m.r. Spectroscopy.” Philosophical Transactions of the Royal Society of London. Series A, Mathematical and Physical Sciences, vol. 299, no. 1452, 1981, pp. 505–520., doi:10.1098/rsta.1981.0032.

12. Jiang, Yi Jin, and James K. Harper. “An Explanation of Magic Angle Spinning NMR Experiments in the Time Domain.” Concepts in Magnetic Resonance Part A, vol. 34A, no. 5, 2009, pp. 249–263., doi:10.1002/cmr.a.20144.

13. Schurko, "Introduction to Solid State NMR" http://www.emory.edu/NMR/web_swu/SSNMR_redor/ssnmr_schur k 14. Polenova, Tatyana, et al. “Magic Angle Spinning NMR Spectroscopy: A Versatile Technique for Structural and Dynamic Analysis of Solid-Phase Systems.” Analytical Chemistry, vol. 87, no. 11, 2015, pp. 5458–5469., doi:10.1021/ac504288u.

47

o.