Developing Methods for Access to High Quality Genome Sequences from Wild Ape Populations

Modern evolutionary study of wild ape populations requires the collection of genomic DNA from individuals living in their natural habitat. In order to be maximally useful, these samples must be robust enough for the amplification and subsequent assembly of genomic sequences, which are driving much of modern evolutionary research. Additionally, conservation efforts require that these samples be collected with zero intervention on the study species, because all great apes are now critically endangered. Consequently, the conventional method for genomic DNA collection has been extraction from cells present in fecal samples. However, this approach presents multiple difficulties to investigators, including extensive contamination of sequences from gut microbiota and limited storage time. The purpose of this study is to explore alternative procedures of noninvasive DNA collection to overcome these challenges. Specifically, the study looks at DNA extraction from hair follicle cells and from cells present in urine. These sources of genomic information confer a number of advantages over feces, such as smaller volumes of collection, much lower levels of microbial contamination, and relative ease of storage and transport. In this study, a method for isolating genomic DNA from chimpanzee (Pan troglodytes) hair follicle cells is developed and tested for limit of detection using a decreasing number of hairs per extraction. Validation of the method is then established through the determination of the frequency of polymorphisms due genomic amplification error by comparing sequences obtained from three identically handled samples. After laying out the next steps of development for this method, the study also suggests a similar investigation for samples derived from urine. The overall aim of these studies is a future incorporation of these procedures into the suite of DNA collection techniques available to researchers working with natural populations of great apes and other mammals.Honors Thesis submitted in partial fulfillment of the requirements for graduation with Distinction in Biology in Trinity College of Duke Universit

What\u27s in your Cup? Increasing Transparency and Confidence in Alcohol Use Screening and Brief Intervention

19% of Vermonters report drinking alcohol at levels which puts their health at risk, but many healthcare providers do not feel confident in addressing their patients\u27 usage. This can stem from lack of experience with alcohol use, worries about stigma, and time constraints. However, data has shown that even 5-15 minute interventional conversations can significantly reduce a patient\u27s risky drinking. This project aims to provide real-world, practical advice for having conversations around alcohol, and provides a conversion chart converting popular alcoholic beverages into standard drink equivalents.https://scholarworks.uvm.edu/fmclerk/1597/thumbnail.jp

Training: Key in Recognizing Potential Trafficking Victims in a Healthcare Setting

Background • Human Trafficking (HT) is a crime that involves exploiting a person for labor, services, or commercial sex. • HT can happen in any industry, to persons of any gender, age, economic status, religion, and nationality. • In FY 2018, service agencies in the State of Vermont submitted over 180 reports of HT. • HT has a profound negative impact on survivors’ physical and mental health. • 25-88% of HT victims interact with a healthcare professional while they are being exploited. • Providers have cited a lack of confidence and knowledge on HT as a major barrier to proper care of potential victims, stemming from a lack of adequate training. • There is a need to assess healthcare workers’ knowledge in recognizing and providing appropriate care and options to potential victims of HT. Objectives • Assess awareness of University of Vermont Medical Center (UVMMC) and affiliated primary care staff and providers regarding the recent implementation of a UVMMC policy on providing support to victims of HT. • Understand current behaviors of staff and providers when providing care to a patient suspected of being a victim of HT.https://scholarworks.uvm.edu/comphp_gallery/1277/thumbnail.jp

SOLO: a meiotic protein required for centromere cohesion, coorientation, and SMC1 localization in Drosophila melanogaster

Sisters on the loose (SOLO) protein functions as a meiotic cohesin component critical for correct chromosome segregation in Drosophila

The Drosophila Zinc Finger Protein Trade Embargo Is Required for Double Strand Break Formation in Meiosis

Homologous recombination in meiosis is initiated by the programmed induction of double strand breaks (DSBs). Although the Drosophila Spo11 ortholog Mei-W68 is required for the induction of DSBs during meiotic prophase, only one other protein (Mei-P22) has been shown to be required for Mei-W68 to exert this function. We show here that the chromatin-associated protein Trade Embargo (Trem), a C2H2 zinc finger protein, is required to localize Mei-P22 to discrete foci on meiotic chromosomes, and thus to promote the formation of DSBs, making Trem the earliest known function in the process of DSB formation in Drosophila oocytes. We speculate that Trem may act by either directing the binding of Mei-P22 to preferred sites of DSB formation or by altering chromatin structure in a manner that allows Mei-P22 to form foci

A Ubiquitin Ligase Complex Regulates Caspase Activation During Sperm Differentiation in Drosophila

In both insects and mammals, spermatids eliminate their bulk cytoplasm as they undergo terminal differentiation. In Drosophila, this process of dramatic cellular remodeling requires apoptotic proteins, including caspases. To gain further insight into the regulation of caspases, we screened a large collection of sterile male flies for mutants that block effector caspase activation at the onset of spermatid individualization. Here, we describe the identification and characterization of a testis-specific, Cullin-3–dependent ubiquitin ligase complex that is required for caspase activation in spermatids. Mutations in either a testis-specific isoform of Cullin-3 (Cul3Testis), the small RING protein Roc1b, or a Drosophila orthologue of the mammalian BTB-Kelch protein Klhl10 all reduce or eliminate effector caspase activation in spermatids. Importantly, all three genes encode proteins that can physically interact to form a ubiquitin ligase complex. Roc1b binds to the catalytic core of Cullin-3, and Klhl10 binds specifically to a unique testis-specific N-terminal Cullin-3 (TeNC) domain of Cul3Testis that is required for activation of effector caspase in spermatids. Finally, the BIR domain region of the giant inhibitor of apoptosis–like protein dBruce is sufficient to bind to Klhl10, which is consistent with the idea that dBruce is a substrate for the Cullin-3-based E3-ligase complex. These findings reveal a novel role of Cullin-based ubiquitin ligases in caspase regulation

Mutations in the Catalytic Loop HRD Motif Alter the Activity and Function of Drosophila Src64

The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. We have identified mutations in a Drosophila src gene, src64, that alter the three HRD amino acids. We have analyzed the mutants for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. We also describe the phenotypic effects of other mutations in the SH2 and tyrosine kinase domains of src64, and we compare them to the phenotypic effects of the src64 null allele

A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors

Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4 + T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention

Neurod1 Suppresses Hair Cell Differentiation in Ear Ganglia and Regulates Hair Cell Subtype Development in the Cochlea

Background: At least five bHLH genes regulate cell fate determination and differentiation of sensory neurons, hair cells and supporting cells in the mammalian inner ear. Cross-regulation of Atoh1 and Neurog1 results in hair cell changes in Neurog1 null mice although the nature and mechanism of the cross-regulation has not yet been determined. Neurod1, regulated by both Neurog1 and Atoh1, could be the mediator of this cross-regulation. Methodology/Principal Findings: We used Tg(Pax2-Cre) to conditionally delete Neurod1 in the inner ear. Our data demonstrate for the first time that the absence of Neurod1 results in formation of hair cells within the inner ear sensory ganglia. Three cell types, neural crest derived Schwann cells and mesenchyme derived fibroblasts (neither expresses Neurod1) and inner ear derived neurons (which express Neurod1) constitute inner ear ganglia. The most parsimonious explanation is that Neurod1 suppresses the alternative fate of sensory neurons to develop as hair cells. In the absence of Neurod1, Atoh1 is expressed and differentiates cells within the ganglion into hair cells. We followed up on this effect in ganglia by demonstrating that Neurod1 also regulates differentiation of subtypes of hair cells in the organ of Corti. We show that in Neurod1 conditional null mice there is a premature expression of several genes in the apex of the developing cochlea and outer hair cells are transformed into inner hair cells. Conclusions/Significance: Our data suggest that the long noted cross-regulation of Atoh1 expression by Neurog1 migh