Biology Research Projects 2024

Sinthia Ahammed

Advisor: Alison Weber

Investigating Neural Responses from Manduca sexta Wing Nerve During Flight to Determine Sensory Encoding of Natural Behavior

Flying insects exhibit a remarkable capacity to recover from perturbations affecting their flight, demonstrating an ability to detect disturbances and initiate rapid feedback to maintain flight stability. A substantial number of mechanosensory receptors known as campaniform sensilla have been identified in the wings of flying insects, such as Manduca sexta, which  play a crucial role in encoding sensory information during destabilizing flight dynamics. However, there is a notable gap in the literature regarding the neural mechanisms governing insect flight during natural behavior. To address this gap, this study employs extracellular electrophysiology techniques to obtain neural recordings from the wing nerve of M. sexta with the aim of investigating the functionality of these small neural circuits, particularly in generating rapid reflexes during natural behaviors. Furthermore, this research seeks to elucidate the distinct neural signals which are produced in response to various types and magnitudes of perturbations. The insights gained from this research hold the potential to inform the development of advanced sensor systems in drones and airborne technology.

Ahado Ali

Advisor: Tamara Davis

Changes in Methylation Patterns impact Gene Expression

Background/ Importance: Genomic imprinting is the process by which only one copy of a gene, either maternally or paternally inherited, is expressed while the other remains silenced. DNA Methylation is an epigenetic mark that helps differentiate the two parental alleles and determine which copy gets expressed; this is done by covalently attaching a methyl group to the cytosines present in cytosine:guanine dinucleotides on one parental allele, resulting in a differentially methylated region (DMR), which often leads to the silencing of a gene. Once established, DNA methyltransferase 1 (Dnmt1) maintains methylation at these regions. The proper function of Dnmt1 and maintenance of differential DNA methylation are essential to the expression of imprinted genes and the normal development of mammals. The improper maintenance can result in developmental disorders such as Beckwith-Wiedemann and Angelman syndrome.

Question/ Hypothesis: A mutation in the intrinsically disordered domain of Dnmt1 (P-allele mutation) results in a loss of global DNA methylation during embryogenesis and perinatal death in homozygous mutant mice. Previous research has shown that homozygous P-allele mice experience decreased percent methylation at secondary DMRs compared to wildtype mice. This variability in percent methylation between wildtype and P-allele mice prompted the lab to investigate how changes in methylation influenced by the Dnmt1 P-allele mutation impacts the expression of imprinted genes. Because the P-allele mutation decreases methylation globally and at secondary DMRs, and methylation is associated with gene silencing, we hypothesize that mutant embryos with less methylation will express higher levels of imprinted genes.

Methods:  I will investigate this using techniques such as Quantitative Reverse Transcription PCR, with RNA derived from 18.5 dpc wildtype and P-allele mouse embryo brains to measure and compare gene expression levels in each of the samples. Preliminary data collected from two genes (H19 and Dlk1) normalized to housekeeping genes (CycloA and GAPDH) showed increased expression in P-allele mice as expected. More experiments will be conducted at different loci and in biological replicates to ensure the data is consistent and reliable.

Future Direction: Future steps for this project consist of analyzing two additional biological replicates from 18.5 dpc embryos and studying different developmental stages, such as 12.5 dpc, to determine whether expression levels remain consistent across development. Additionally, various tissue samples, such as the liver, can be investigated to assess whether changes in expression are global or tissue-specific. In the far future, the protein products of some of the imprinted loci can be explored to determine the mutation's impact on protein concentration.

Leila Byerly

Advisor: Adam Williamson

Building a CLN Knockout Cell Line Library for Functional Studies of the Batten Disease Proteins

Batten Disease is a debilitating neurodegenerative disease which often appears in children and can lead to severe symptoms including blindness, seizures, and early mortality. Batten Disease is a class of lysosomal storage disorders generally thought to result from inefficient turnover of intracellular debris. The disease is monogenic, associated with mutations in 13 mapped ceroid lipofuscinosis (CLN) genes with different, mostly unknown, molecular mechanisms of action which lead to similar symptoms in patients. To better understand the role and function of these genes and proteins in contributing to Batten Disease symptoms, we aim to uncover their relationship to phagocytosis and disruption of anti-inflammatory debris clearance. I will be conducting gene knockout studies using CRISPR-Cas9 to impede the functional version of the gene and mimic disease. I will then observe the effects of this model of disease on cellular processes through a series of various functional assays including live-cell imaging to compare knockout cells to wild-type controls in terms of their phagocytic and lysosomal function as well as other activities. These studies could be foundational to providing us with more information on how to treat each genetic cause according to its unique mechanism of disease; mutations that cause similar cellular dysfunctions may be treatable by similar regimens, whereas for those that disrupt the cell differently would require specialized approaches. We hope to clarify the relationship between these CLN proteins and the process of cell debris clearance, allowing us to move forward in understanding how these mutations lead to Batten Disease phenotypes and contribute new information that hastens development of new treatments for Batten Disease.

Jack Carlson

Advisor: Thomas Mozdzer

Measuring potential greenhouse gas production rates across genotype in Phragmites Australis rhizosphere samples exposed to global change factors

The common reed Phragmites australis is a globally distributed ecosystem engineer in coastal wetlands. Due to its cosmopolitan distribution and genetic diversity Phragmites has been used as a model organism to study the possible impacts of global change factors such as elevated CO2 and N on wetland ecosystems. Previous research has found that soil microbial communities vary significantly in Phragmites at the lineage level, with some preliminary data suggesting genotypic differences across populations. The bacterial associations in the plant rhizosphere may contribute to a genotype鈥檚 capacity to alter nutrient cycling and nutrient availability. While treatment level variation in soil microbial communities has been found, little is known about the level of variation in soil microbial communities within a population when sampled at high sampling rates (>3 samples per population), or in exposure to experimental treatments of elevated CO2 or N. To address this gap in knowledge I will collect soil samples associated with unique Phragmites australis genotypes from a long-term global change experiment at the Smithsonian Global Change Research Wetland, Edgewater MD, USA. I will develop a method to measure potential rates of methanogenesis and CO2 emissions. I hypothesize that microbial rates will vary by genotype and by environmental treatment. This research will provide a deeper understanding of the factors that affect greenhouse gas cycling and the potential impacts of near future global change. Methods developed in this study can be used for future research on Phragmites F1 genotypes from quantitative genetic experiment growing in the common garden at 黑料社区.

Cynthia Clark

Advisor: Alison Weber

Analyzing Neural Activity Behind Proprioceptive Responses in Moths.

When insects are in flight, they use mechanosensory receptors on their wings, called campaniform sensilla (CS), to detect deformations of their wings due to slight changes in air currents, or perturbations, such as a sudden gust of wind or a collision. The CS enable them to respond to the perturbation by adjusting the position of their wings and abdomen, which allows them to remain stable. One insect in particular that uses CS is Manduca sexta, or the tobacco hawkmoth. Since M. sexta beat their wings about 25 times per second, we will use high-speed videography to record the moths in flight, and then use the software Anipose in DeepLabCut to track the motions of the wing during individual wingbeats. In addition, we will use electrophysiology to study the neural responses elicited in CS on the moth鈥檚 forewings (Pratt et al., 2017). Using a suction electrode, we will take extracellular recordings from the forewing nerve, which is comprised of the sensory neurons innervating the wing. Simultaneously, we will manually move the moth鈥檚 wing in the configurations recorded by the video, to identify which neurons are firing when the moth makes particular movements. So much of how action potentials are generated, processed, and responded to is unknown, even though everything that every animal, from the tiniest fly to humans, does is controlled by them. With these experiments, we hope to take a tiny step forward in understanding more about how they work. The next step in this research will be to determine a way to record the electrical signals of these neurons while the moth is naturally generating its own movements. Our current experiments will inform our understanding of neural responses during flight, but future work is needed to also understand how the system works in nature.

Jessica Cramer

Advisor: Gregory Davis

Identifying the Maternal Signal Responsible for Inducing Asexual Reproductive Fate:

The Davis Lab is investigating the pea aphid, Acyrthosiphon pisum, which exhibits a reproductive polyphenism: individuals of the same genotype develop into asexual and sexual forms depending on environmental conditions. During long summer days, aphids reproduce asexually via live birth (viviparously). As winter approaches, they give birth to future sexually reproducing offspring, male and female. Sexual females lay eggs (oviparous) that withstand the winter frost. During spring, these eggs hatch into asexually reproducing aphids. This switch is likely mediated by an unknown maternal factor dubbed 鈥渧irginoparin鈥 that鈥檚 delivered to embryonic progeny.

One candidate is juvenile hormone (JH), which is important for insect development. We have previously found JH to be sufficient to specify asexual fate in sexually fated oocytes when topically applied to their mothers. Building off a previous student鈥檚 work, which identified the gene Krh1 as a downstream target of JH signaling, and my own work, which demonstrated that Kr-h1 paralog 1 is upregulated when aphids are treated with JH/JH analogs, I hope to further confirm JH鈥檚 role in specifying asexual fate by looking for differential gene expression of JH between sexually and asexually fated embryos, using this Kr-h1 assay.

We are also investigating an alternative pathway as another virginoparin candidate. We plan to interfere with it to test the loss of function phenotype by using RNAi or ReMOT Control, a novel technique making use of vitellogenesis, allowing for the delivery of molecules from the mother鈥檚 hemolymph to developing oocytes鈥 yolk. 

Sarabjit Dhillon

Advisor: Adam Williamson

Designing Phagocytes that Target Correlates of Neurodegenerative Disease

Pathogenic and neurotoxic debris buildup contribute to brain decline in neurodegenerative diseases. Although current treatments for these diseases focus on symptom management, there are no medications that significantly slow disease progression. In this project, I will engineer anti-inflammatory phagocytic immune responses which promote cell ingestion of disease-causing material in the central nervous system and study if this proposed debris clearance method could be used to approach forming treatments for various neurodegenerative diseases.

I will build upon lab-synthesized engulfment receptors, Chimeric Antigen Receptors (CARs), which direct immune cells to ingest targets of therapeutic relevance through phagocytosis, the process by which the cells鈥 cytoskeletons reform to surround and engulf targets. In the Williamson group, there is a focus on programming phagocytosis for specific targets of interest through the development of Chimeric Antigen Receptors for Phagocytosis (CAR-Ps), which expand the reprogramming properties of CARs to macrophage lineages. My project aims to develop two novel CAR-Ps that effectively drive this process and express in cells using Gibson assembly and lentiviral transduction.

With better treatment options for neurodegenerative diseases needed, and the strain on medical resources to keep up with neurology demands in coming years, I hope to use my work to present new protective immune response strategies that may not only relieve symptoms of neurodegenerative decline, but also inhibit the diseases from progressing.

Hope Hsu

Advisor: Tamara Davis

Analysis of 5hmC levels in 5 dpp BL/6 brain DNA 

Within mammals lie imprinted genes. Usually, mammals have two copies, or alleles, of each gene, one from each parent, and both alleles have the possibility of being expressed. However, imprinted genes silence one parent's allele and express the other. This process is very important because incorrect gene expression can cause problems like cancers and developmental disorders. We can identify which copies are silenced or expressed because one of the alleles is tagged with a chemical modification called methylation. One allele is methylated while the other is not, resulting in areas called differently methylated regions (DMRs). DMRs are categorized in two ways based on the time of methylation acquisition and other characteristics. Methylation at primary DMRs is inherited from the parents at fertilization. Methylation at secondary DMRs is gained during embryogenesis. Primary DMRs are very symmetrical and have 90-100% of the methylated allele methylated while the unmethylated allele is only 0-10% methylated. Secondary DMRs have more variable methylation patterns and high levels of methylation asymmetry on the complementary DNA strands. 

During replication, the maintenance methyltransferase Dnmt1 modifies the complementary strand of DNA to mirror the template strand, therefore, the high levels of asymmetry or hemimethylation that we found are highly unexpected. We aim, through this analysis, to discover why secondary DMRs have significantly more hemimethylation than primary DMRs. We believe that this discrepancy is due to the oxidation of the methyl group at these sites. Oxidation could either prevent methyltransferase activity, or it could be recognized as damage and be replaced with an unmethylated nucleotide. We will use glucosylation of oxidized cytosine followed by methylation-sensitive restriction digestion and quantitative PCR to determine the amount of 5-hydroxymethylcytosine at secondary DMRs as compared to primary DMRs to test this hypothesis.

Kuankuan Hu

Advisor: B谩rbara Bitarello

A Simulation Study for Detecting Balancing Selection under Diverse Mechanisms

Natural selection drives evolutionary changes that are reflected by different effects on the genetic structure of a population. Under circumstances where higher mean population fitness is associated with higher polymorphism, balancing selection (BLS) occurs and maintains the genetic diversity at the targeted loci, resulting in signatures such as excessive local heterozygosity, an excess of intermediate-frequency alleles, or excessive polymorphism-to-divergence ratios. The diversity in its mechanisms 鈥  e.g., heterozygote advantage, negative frequency dependent selection, antagonistic selection, and fluctuating selection across time and/or space 鈥 and timescales contributes to the complexity in understanding the BLS. Classic signatures of BLS do not differ distinctively between mechanisms but vary significantly in response to the timespan that it has persisted.

Research shows that BLS balancing selection is more prevalent in the human genome than previously thought, as insight made possible by the development of methods tailored to detect BLS in the past decade. These methods have different requirements and optimal conditions for maximum statistical power. As a complement to review existing BLS testing methods, quantifying the power of the tests in a more uniform environment allows fair comparisons between methods and assists future decisions on selecting test(s) for given scenarios. Through forward evolution simulation by SLiM, different settings of a genome under BLS can be generated to test the statistical power of BLS detection methods. At the same time, this project will include modeling study of sexual antagonism, a BLS-maintaining mechanism that has not been extensively applied in models, in order to compare the power and limitations of current methods in various settings. With the results from valid statistical tools, future research can aspire to bring insights to research questions such as the strength and prevalence of BLS due to different mechanisms, the conditions for populational fitness benefits to overwhelm the costs under BLS, and stability of long-term BLS equilibria.

Fiona McHale

Advisor: Adam Williamson

Through Thick and Thin: The Effect of Microenvironment and Pathogen Consistency on Phagocytotic Decisions

Phagocytosis, the clearing of debris from organisms by engulfment, is a billion-year-old immunological process dating back to before the evolution of tyrosine phosphorylation based cellular signaling. The majority of research into cues for phagocytosis has investigated the role of this biochemical chemical signaling. However, as phagocytosis is older than this form of signaling, there is a likelihood that the physical cues from a phagocyte鈥檚 microenvironment play a role in its activation.

            A majority of previous research into phagocytosis has been conducted on stiff surfaces using glass beads to model pathogens. These materials do not accurately mimic the microenvironment that phagocytes operate in. Pathogens and tissue microenvironments can vary significantly between soft and stiff within an organism. As phagocytes are migratory, understanding their function in different environments is necessary.

How macrophages accomplish their varied tasks in physically distinct environments is a focus of my project. The material of the substrate I am using can be manipulated to change its consistency from soft to stiff. By using confocal microscopy, I will be able to precisely assess how the consistency of model pathogens affects which ones get digested and how the hardness of substrate can influence these choices. These findings will provide an important understanding of how to accurately research phagocytosis in lab environments and inform how immune system functioning is affected by an organism's biological history.

Asal Mogharehdehkordy

Advisor: Alison Weber

Analyzing Flight Dynamics in Response to Perturbations in Manduca sexta

In order to perform precise movements in response to the changing environment, animals need sensory feedback. Sensory feedback from the wings of flying insects is fundamental for producing stabilizing behaviors in flight. Mechanosensory neurons, which are responsible for sensory feedback, are scattered over the surface of insect wings and play a crucial role in detecting wing bending. Our work focuses on studying the behavior of the hawkmoth Manduca sexta in response to destabilizing perturbations to explain how a small and scattered population of mechanosensory receptors called Campaniform Sensilla effectively encodes the perturbations and enables stable flight. My summer research goal is to understand how moths respond to perturbations behaviorally. To achieve this goal, I will capture some videos using a high-speed camera to track and quantify moths鈥 behavioral responses to the imposed perturbations while the moth is held in a stationary position. Various destabilizing perturbations will be tested on the moth, including wind gusts of different magnitudes and collisions with different locations on the wings. Then we will analyze these videos with a software called DeepLabCut. It is expected that the moth will show compensatory and consistent reflexes to the perturbations. Via extracellular electrophysiology, we will then record neural responses from these mechanosensory nuerons while the moth is experiencing perturbations in order to identify which neural circuits are responsible for the observed behaviors. This research not only helps us deepen our understandings of sensory-motor integration in biological systems but also has the potential to contribute to the development of advanced robotic systems that benefit from rapid sensory feedback.

Max Monks

Advisor: Thomas Mozdzer

Investigating variation in Phragmites australis fecundity by genotype and exposure to global change

Coastal wetlands are vital to the protection of our coastlines from natural disasters and erosion. The common reed, Phragmites australis, is a marsh plant that is considered to be a model organism for studying plant invasions and responses to global change (Meyerson et al 2016, Eller et al 2019).  Previous studies have demonstrated rapid evolution of Phragmites in response to elevated CO2 and N conditions, but little is known about the effects of global change exposure on reproductive fitness. Research in our lab has preliminarily demonstrated that the number of seeds produced in response to global change increases with N enrichment. While this demonstrates a treatment effect on seed production, it is unknown if this difference is solely due to global change factors or if reproductive output is influenced by genotype as well.

This study will determine if plant fecundity is impacted by genotype (G) as well as by exposure to elevated CO2 and N (E).  We aim to determine if the response is driven by G, E, or a combination of those factors.

Phragmites australis seeds were collected in 2022 and 2023 on previously genotyped ramets collected from a long-term global change experiment conducted at the Smithsonian Global Change Research Wetland (GCReW) in Edgewater, MD. These plants are grown in conditions with elevated CO2, elevated N, both, or neither. Seeds will be collected from panicles and germinated in order to determine percent germination and death rates of seedlings at the G and E scale. This will allow me to determine if genotype and maternal treatment conditions affect the percent of seeds that germinate, and percent death of the seedlings. I hypothesize that fecundity will vary at the genotype level but that maternal plant treatment will have no effect on percent germination. This study will continue after the summer, observing growth rates of the viable seedlings.

Kira Morozova

Advisor: B谩rbara Bitarello

Exploring models of balancing selection: mechanisms and timescales

Balancing selection (BLS) maintains adaptive polymorphisms in populations. It increases genetic diversity, ensuring that alleles do not go extinct. BLS can occur via several mechanisms: heterozygote advantage, negative frequency dependent selection, antagonistic selection (including sexually antagonistic selection), and selection that changes across time or space in a panmictic population. This research project will focus on exploring existing models pertaining to heterozygote advantage and sexually antagonistic selection.

Heterozygote advantage (HA) describes a situation in which the heterozygous genotype is more fit for survival than the homozygous recessive or homozygous dominant genotypes. Theory predicts the conditions under which a stable equilibrium can be achieved under a simple model of HA but, to our knowledge, the following remains unaddressed: what are the predictions for maintenance of genetic variation when there are differences in the relative fitness between males and females under HA? Should further investigation reveal that similar questions have been investigated in the past, we will focus on analyzing the techniques used in the relevant literature. Otherwise, we will use previous models used to make predictions about BLS, such as the one presented in Kidwell et al. 1977 or Fisher鈥檚 geometric model, as a basis for further manipulation during our inquiry.

The term sexual antagonism describes a case when males and females of a given genotype express a trait at the same rate, but the fitness of one sex is decreased in comparison to the other. Sexually antagonistic selection involves a trade-off between relative fitnesses of the sexes. Trade-offs like this can be one of the driving causes of BLS. Our exploration of this topic will include an assessment of how sexual antagonism models previously published in the literature can be implemented using forward population genetics simulators such as SLiM. Based on an evaluation of the results of the simulations, we will consider whether, and how, these models can be improved upon.

Anna Skiba

Advisor: Thomas Mozdzer

Heritable trait variation in seedling success and relative growth rates in Phragmites Australis

Coastal ecosystems provide many valuable environmental services such as acting as buffers against storms, protecting shorelines, and serving as the greatest carbon sinks on the planet. The globally distributed common reed, Phragmites australis, acts as an ecosystem engineer, as well as an invasive species in coastal ecosystems. Previous research has demonstrated evolutionary responses to near future conditions of elevated CO2 and Nitrogen. The C-EVO at 黑料社区 has been investigating how rapid evolution in response to future global change factors influences carbon cycling and carbon storage. A key measure of evolutionary fitness is success in reproduction and survival of offspring. Little is known about how exposure to near future global change influences the germination and survival of Phragmites seeds. We collected seeds from genetically identified ramets of Phragmites australis from a long-term global change experiment at the Smithsonian Global Change Research Wetland, Edgewater, MD, USA. The seeds will be germinated to evaluate the influence of genotype and exposure to elevated CO2 and N on growth and mortality rate. This research will provide insight into if genotypic variation and exposure to near future global change factors affect the success of Phragmites seedlings, how those possible effects can influence future genetic diversity of Phragmites australis populations, and what that could mean for carbon cycling and carbon storage in the ecosystem.

Jo Smith

Advisor: Thomas Mozdzer

Heritable Trait Variation in Leaf Toughness and Herbivory among Phragmites australis Genotypes from Two Populations

Salt marsh ecosystems provide many vital ecosystem services, but their existence is threatened by accelerating global change factors including rising concentrations of CO2, nutrient pollution, and human development. The globally distributed common reed, Phragmites australis, is considered a model organism for studying invasive species and plant physiology. Recent research from our lab group has demonstrated that exposure to both near future levels of CO2 and nutrient enrichment have reduced intraspecific levels of genetic diversity and altered plant traits suggesting that populations are rapidly evolving. Global change factors can also act as a selective agent on organisms, resulting in evolutionary responses in heritable trait variation in plant traits such as leaf toughness, a proxy for herbivore resistance. To evaluate the heritability of plant traits, unique genotypes of P. australis were collected from two populations in MD, USA, the Smithsonian Global Research Wetland (Edgewater, MD) as well as Parker鈥檚 Creek (Prince Frederick, MD) and were grown in a common garden at 黑料社区. Common garden experiments allow researchers to evaluate traits by removing confounding environmental factors. Previous research has found that genotypes from SERC plants have tougher leaves, suggesting greater resistance to herbivory.  Unfortunately, not at genotypes were able to be measured last year.  This summer, I will measure leaf toughness on genotypes from both populations that had not fully developed last growing season. Data analysis comparing leaf toughness between individual genotypes and among the two populations will be evaluated against the future F1 leaf toughness traits to see what aspect of herbivory defense such as leaf toughness is a heritable trait response or an example of phenotypic plasticity in this species. This research aims to provide insights into which traits are heritable, to provide further insight to the evolutionary ecology of salt marshes.

Sarah Stanger

Advisor: Thomas Mozdzer

Linking Genotype to Trait Variation in Phragmites australis under Global Change Conditions

Coastal ecosystems provide many ecosystems services including carbon sequestration, nutrient removal, and aid in the protection of coastal environments from storms and rising sea levels. Given their position at the land-sea interface, these systems are also very sensitive to global change factors including increasing concentrations of carbon dioxide (CO2) in the atmosphere and nutrient enrichment. The common reed, Phragmites australis subs. australis is a foundation species and a model organism for studying plant invasions and physiological responses to global change. There is growing evidence that shifts in genetic variation within a population exposed to global change, or rapid evolution, might influence the ecosystem response. In order to differentiate phenotypic plasticity from genotypic responses, I will combine several unique data sets to explicitly link genotypic data with previously measured plant trait data from a long-term experiment at the Smithsonian Global Change Research Wetland, Edgewater, MD, USA.  An open top chamber experiment, simulating near future global change, was initiated in 2011 in a fully factorial design with two levels of CO2 (ambient or +340 ppm) and two levels of nitrogen concentrations (ambient or 25 g N m-2yr).  Annual measurement of plant traits (plant height, stem diameter, biomass, herbivory, relative growth rates, photosynthetic rates, leaf area, specific leaf area, and flower presence) will be linked to plant genotypes from a previously published study from our group. By combining these disparate data streams, I will be able to differentiate between genetic variation (G), phenotypic plasticity to exposure (E), as well as GxE interactions. This research aims to bridge the fields of ecosystem ecology and evolutionary ecology to create better predictions of the response of foundation species to near future global change.

Sarah Stephens

Advisor: Adam Williamson

Investigating intracellular phagocytic signaling pathways using an in vitro recapitulation of the Draper and Shark system

The Williamson lab focuses on the process of phagocytosis, an ancient mechanism of clearing dead cells and debris. In the species Drosophila, phagocytic cells express the receptor Draper, which provides the 鈥渆at me鈥 signal from the target to the cell via an intracellular signaling pathway. After the Draper receptor has come into contact with its target and then been activated by phosphorylation by the protein Src42a, it recruits a kinase, Shark. The protein Shark is the next step in initiating phagocytosis and is crucial for facilitating engulfment. While its abilities have been compared to similar proteins in T cells, the complete structure and binding capabilities of Shark have remained unknown. To further elucidate this signaling pathway, I will create an in vitro recapitulation of this system with the ability to quantify the interaction. These experiments will utilize a variety of methods to obtain pure protein stocks and reconstitute the system including affinity purification and size exclusion chromatography. Then, using these proteins, I will conduct in vitro binding assays. This immune pathway is incredibly essential and well-conserved throughout phyla, yet understudied; my work will help build understanding of these key immunological proteins.

Hannah Tobin

Advisor: Tamara Davis

Analysis of DNA methylation in wildtype and DNA methyltransferase mutant mice

For some mammalian genes, expression is based on the parent of origin. Either the mother鈥檚 copy is always expressed or the father鈥檚 copy is always expressed, while the other allele is silenced. Differential DNA methylation, where a methyl group is added to cytosine, is generally used to mark the DNA of a specific parent, so the mother鈥檚 and father鈥檚 DNA can be distinguished and their expression regulated accordingly. This is referred to as genomic imprinting. Misregulation of imprinted genes is responsible for human imprinting disorders, or diseases inherited in a parent-specific manner; this can be caused by genetic alterations or epigenetic defects that cause changes in which parent鈥檚 copy of a gene is expressed. Prader-Willi syndrome, Angelman鈥檚 syndrome, and Beckwith-Wiedemann syndrome are examples of human imprinting disorders. Better understanding the mechanism by which DNA methylation is acquired and maintained might lead to the future development of therapies for such diseases.

I am analyzing the consequences of a DNA methyltransferase mutation in mouse liver DNA. P allele mice have a lethal mutation in the Dnmt1 gene which is responsible for maintaining DNA methylation, reducing methylation levels and preventing the mice from surviving after birth. This summer, I will amplify the differentially methylated regions associated with imprinted loci from liver DNA derived from different developmental stages of wildtype and homozygous P allele mice. Then I will compare the liver methylation levels to levels of methylation in the brain, which were previously determined in the Davis lab, to determine whether activity of mutant methyltransferase is different in different tissue types. This research will lead to a better understanding of the mechanisms of gene imprinting and ultimately how variation in methylation impacts expression.