<p> Recent advances in the study of archaic hominin DNA have resulted in the rapid development and application of new methods designed to test our relationship to our nearest relatives. These methods have been applied with archaic samples in contexts with ghost admixture, sparse sampling, ascertainment bias, and poorly understood historical events. They have also been applied to modern samples with complex relationships which will exacerbate the same problems. Here, we introduce a new method for estimating the admixture fraction, and test it and several previous methods to determine their sensitivity to the above problems. Finally, we apply these methods to Single Nucleotide Polymorphism (SNP) microarray and whole genome data, and compare our estimates to those published previously.</p>

At present, the genus Clivia consists of six species, including Clivia nobilis Lindl., C. miniata (Lindl.) Regel, C. gardenii Hook., C. caulescens RA Dyer, C. mirabilis Rourke and C. robusta Murray, Ran, De Lange, Hemmet, Truter & Swanevelder. Many of the species and cultivars are extensively grown worldwide, making this group of considerable horticultural importance. This study mostly focused on Clivia caulescens with a natural habitat on the escarpment from Limpopo to Swaziland through Mpumalanga. The overlapping distribution between C. miniata and C. caulescens resulted in the formation of a natural hybrid between these species at the Bearded Man Mountain. The occurrences of natural hybrids between the various species are rarely recorded. In an attempt to find out if genetic erosion is currently a threat to the various C. caulescens populations and Bearded Man Mountain clivias, this study was conducted to establish if genetic variation is present. Genetic variation refers to the variation in the genetic material of a population, and includes the nuclear, mitochondrial, ribosomal DNA as well as the DNA of other organelles. The relative genetic diversity among individuals or populations can be determined using morphological and molecular markers. Five chloroplast DNA regions, i.e. atpH-I, matK, rpoB, rpoC and trnL-F, were used in an attempt to study the molecular diversity of C. caulecsens. This study concentrated on Single Nucleotide Polymorphisms (SNPs) from these regions and microsatellites to study genetic variation. The aim of this study was to determine the genetic variation between and within the different populations of C. caulescens, to determine whether gene flow occur between the different populations and to determine which of the DNA regions included in the study can contribute to the identification of plants from a specific geographical area. Regarding the study of Clivias situated at the Bearded Man Mountain, the main objectives were to estimate genetic diversity and determine the genetic relationship among the different species of Clivia (C. miniata, C. caulescens and C. xnimbicola) from this area. Of the initial five regions that were sequenced, trnL-F amplification failed repeatedly, and this region was therefore excluded from all analyses. The other four regions showed variation between the different populations of C. caulescens and for the Bearded Man Mountain clivias, except the rpoC1 region. When the results of the phylogenetics and statistical analysis (genetic distances) were combined, it was detected that most Bearded Man Mountain specimens and Godâs Window specimens clustered together in the cladograms and in the mean distances tables. Intraspecific variation was present in all the regions and combined dataset. All attempts during this study to amplify STRs and test allelic diversity in 13 microsatellite loci for 20 specimens failed. Cross-species amplification was not as effective as hoped. Microsatellitesâ species-specific nature could have a negative effect on obtaining results, although other researchers (as mentioned in the introduction of Chapter 4) could employ cross species markers successfully. Glen & Schabble (2005) reported that a given pair of microsatellite primers rarely works across broad taxonomic groups, so primers are usually developed anew for each species. The next step would therefore be to attempt the designing of specific primers for C. caulescens.

<p> The past decade of genetics research has been defined by the discovery of the profound effects non-coding genetic variation can have on the phenotypes that distinguish humans from each other and from our close evolutionary relatives. The full implications of this new understanding are largely unexplored, however, as modern ethics restricts experimentation in humans and most primates, rendering data from dynamic processes almost non-existent. The study of regulatory molecular dynamics has been changed entirely by the availability of protocols to generate iPSCs and differentiate them into adult cell types. The molecular basis of disease mechanisms, drug response, and developmental processes can now be studied in the relevant tissue, presenting an overwhelming spectrum of possible applications. Of particular interest to comparative biologists, long-standing questions about the relative conservation of early developmental states can now, for the first time, be ethically explored in closely related primates. In this dissertation, we first discuss evidence that iPSCs can faithfully model genetic variation, even when sourced from highly dysregulated cells. We then use an iPSC-based model to study the temporal profile of conservation between humans and chimpanzees during early endoderm development and identify patterns of divergence over developmental stages.</p>

<p>In the past decade, there have been a series of breakthroughs in human genetics. The advent of next-generation sequencing (NGS) has made it possible, for the first time, to sequence an entire human genome inexpensively and efficiently. The affordability and ease of NGS has led to an explosion of data. Now, the largest hurdle in human genetics has shifted from technology-based limitations of sequencing to developing a framework interpreting the large amount of data that has been generated. Specifically, in medical genetics, the key challenge is recognizing which of a given patient's many mutations may be contributing to disease.</p><p>The most successful methodologies for this problem rely on conservation. However, conservation cannot capture human-specific intolerance to variation. Other methodologies rely on biochemical annotations, which can indicate a genomic region's functional role, but do not directly assess its intolerance to variation in the context of disease. Therefore, despite these available methodologies, detecting causal variation still remains incredibly challenging.</p><p>In my thesis, I describe three methodologies, based on population genetics and standing human variation, which can help identify the regions of the genome that are most likely to cause disease when mutated. The first, subRVIS, focuses on sub-regions within genes. The second, ncRVIS, focuses on the regulatory regions of genes. The third, Orion, tackles the daunting problem of interpreting and prioritizing variants across the entire genome.</p><p>In Chapter 1, we will review some of the history that has brought us to this point and some of the methodologies currently in use for detecting disease-causing variants.</p><p>In Chapter 2, we describe subRVIS, a methodology that divides the gene into sub-regions based on sequence homology to known protein domains, and then ranks those sub-regions based on their tolerance to functional variation. We show that this ranking is associated with the sub-region's likelihood of carrying a previously known pathogenic mutation. Further, we demonstrate that the biological division into domains adds significant information in comparison to dividing the gene into random regions matched in size. This methodology is useful in localizing where pathogenic mutations are most likely to fall within genes.</p><p>Chapter 3 describes a methodology to rank genes based on the likelihood that mutations falling in their regulatory regions are pathogenic. We demonstrate that this ranking is associated with whether or not a gene is sensitive to changes in its dosage. This methodology is useful in assessing the pathogenicity of mutations occurring in known regulatory regions that have been associated with genes.</p><p>In Chapter 4 we tackle one of the most intimidating and challenging problems in the field of medical genetics: detecting intolerance to variation across the entire human genome. Using a sliding window, we generate a score per base to highlight the regions of the genome that are intolerant to variation, with higher scores corresponding to more intolerant sequence. We term this approach Orion. We demonstrate that exons and DNase hypersensitive sites are enriched for higher Orion scores. This methodology will transform the way whole-genome sequence data are interpreted, by giving researchers the ability to assess the pathogenicity of variants in regions of the genome that are not yet fully understood.</p><p>We have developed methodologies to tackle the key problem of detecting disease-causing variation in patients' sequence data. In an era overwhelmed by NGS data, these methodologies bring us closer to understanding the genetics of disease.</p> / Dissertation

<p><italic>Drosophila</italic> larvae respond to potentially tissue-damaging stimuli with nocifensive escape locomotion (NEL). NEL is a stereotyped withdrawal behavior in which the larva rotates along its long-body axis in a "corkscrew" pattern distinct from normal locomotion and is triggered by noxious heat (>39°C) or noxious mechanical (>30mN) stimuli. The Class IV multidendritic (md) neurons are the polymodal nociceptors responsible for triggering NEL. Recent evidence suggests that the <italic>pickpocket</italic> (<italic>ppk</italic>) gene, a degenerin/epithelial sodium channel (DEG/ENaC) subunit, is involved in the mechanotransduction of these neurons. While removal of this ion channel diminishes the larval response to noxious mechanical force, it does not completely abolish it, which leads us to hypothesize that additional mechanical nociception channels have yet to be identified. To identify these channels, we carried out a tissue specific RNAi screen and knocked down all known and predicted ion channels in the larval Class IV md nociceptors. Knockdown animals for each channel subunit were tested for behavioral responses to noxious mechanical force or noxious heat. Taking this approach, two novel channel genes were specifically required for mechanical responses - we named them <italic>stallone</italic> and <italic>balboa</italic>. Remarkably, both genes are predicted to encode DEG/ENaC subunits and neither is required for optogenetic NEL responses, suggesting that both genes are required near the transduction step, perhaps forming a multimeric channel with PPK. Therefore, my research focused on demonstrating that these two DEG/ENaC genes, <italic>stallone</italic> and <italic>balboa</italic>, are required for mechanical nociception responses in <italic>Drosophila</italic> larvae. </p><p>To extend the RNAi results from the previous mechanical nociception screen and confirm <italic>stallone's</italic> role in detecting harsh touch responses, I first generated and rigorously analyzed the mechanical nociception phenotype of a <italic> stallone</italic> mutant. Interestingly, <italic>stallone</italic> may have a general mechanosensory function as gentle touch responses were mildly reduced in the knockout mutant. Additionally the mutant allele was used to confirm that thermal nociception responses were normal and mechanical nociception behavioral defects were not a consequence of morphological defects in the nociceptors. </p><p>Next I focused my efforts on the <italic>balboa</italic> gene as this locus encodes a DEG/ENaC ion channel subunit similar in amino acid sequence to PPK. Converging lines of evidence strongly support the hypothesis that Balboa and PPK physically interact and likely form a functional ion channel <italic> in vivo </italic>. First, both genes are functionally required for mechanical nociception. Second, both genes show very specific expression in the Class IV nociceptor neurons. Third, <italic>ppk</italic> expression causes a dramatic redistribution of Balboa::GFP fluorescence in the multidendritic neurons. Fourth, Balboa::GFP is altered by PPK knockdown and PPK::Venus is dramatically reduced by Balboa knockdown. Fifth, GFP is reconstituted by co-expression of <italic>balboa::CGFP</italic> and <italic>NGFP::ppk</italic> within neurons of the larval peripheral nervous system.</p><p>In summary, two DEG/ENaC genes, <italic>stallone</italic> and <italic>balboa</italic>, were discovered in an unbiased forward, genetic screen to identify genes required for mechanical nociception in <italic>Drosophila melanogaster </italic>. I demonstrated that <italic>stallone</italic> and <italic>balboa</italic> are specifically required for mechanical nociception behaviors documenting the first functional role of these two genes. The body of work outlined in this doctoral thesis has contributed to the overall field of mechanosensation by uncovering two novel genes encoding DEG/ENaCs that are functionally required for animal nociception responses to noxious mechanical force and may shed light on multimeric pain-sensing complexes.</p> / Dissertation

A self-administered thirteen page Hearing Loss Questionnaire (HLQ) was designed in order to systematically collect medical and family history data on deaf children and their families. Data were collected from over 400 families with one or more children enrolled in September 1979 at the Maryland School for the Deaf (MSD). Almost 70% of the parents provided pedigree and family history information by completing the detailed HLQ. Computer analyses of the collected data allowed a thorough examination of almost 200 medical and family history variables, providing useful reference data on the MSD probands. Parental responses to a four-step rating scale of proband hearing ability were compared with actual audiometric data, allowing comparison with similar data from previous studies of hearing populations. Family history data on the non-respondents were available from school records, providing a unique opportunity to assess the potential response bias in questionnaire studies of genetic disease. Segregation analysis was performed on the informative sibships ascertained by incomplete truncate selection. The pooled estimate of the ascertainment probability, π, was 0.488, with no significant evidence of heterogeneity among the respondents and non-respondents. The hypothesis of fully penetrant dominant inheritance (H0:p=0.50) was accepted in the Deaf by Hearing matings. However, the maximum likelihood estimate of the segregation ratio (p=0.257) was consistent with reduced penetrance in these families, as it also was in the Deaf by Deaf matings (p=0.31). There were no significant differences in the maximum likelihood estimates of p or of the proportion of sporadic cases, x, between respondents and nonrespondents in the Hearing by Hearing matings. Among the non-consanguineous Hearing by Hearing matings with no family history of hearing loss, the maximum likelihood estimate of x was 0.81. The removal of 46 sibships with probands born during the 1964-65 rubella epidemic reduced x to 0.71, indicating the potential value of segregation analysis for monitoring the secular trends in sporadic vs. genetic deafness. Among Hearing by Hearing matings with a family history of early onset hearing loss, a recessive hypothesis with no sporadic cases (H0:p=0.25, x=O.OO) fit the data well. However, the same hypothesis was rejected among the Hearing by Hearing matings with a family history of "presbycusis", where x=0.59. Thus, although a family history of early onset hearing loss appears to be a much more reliable index of a genetic etiology that does a family history of "presbycusis", the results of this study suggest that the latter may also be a positive risk factor. The HLQ data implied that both parents and doctors may underestimate the extent to which genetic factors contribute to childhood hearing loss, even in the presence of a positive family history. Genetic factors were estimated to account for approximately 35% of the deafness in the MSD population. In the group with genetic deafness, the estimated proportions of recessive, dominant, and X-linked deafness were 57%, 39.%, and 5% respectively. Comparison of the estimates in the respondent vs. the non-respondent groups revealed remarkable similarity between the two groups, indicating that the use of the HLQ did not further confound existing biases. This study has demonstrated the value and utility of using self-administered questionnaires in genetic research. Indeed, the HLQ may serve as a useful prototype for future large scale population based studies of deafness in man.

<p>A blood draw is one of the most readily accessible, commonly practiced medical procedures with biomarker utility. In particular, transcriptome signatures of blood cells provide valuable insights into the developmental history and adaptations of these circulating cells. The majority of cells within whole blood consist of erythrocytes, or red blood cells (RBCs) that are primarily responsible for the transport of gases throughout the bloodstream. Terminally differentiated and anucleate, RBCs were once thought to lack most RNAs. However, erythrocytes have been recently shown to contain select microRNAs that lend insight into erythrocyte pathophysiology. Erythrocyte RNAs possess the ability to both distinguish between erythroid disease subphenotypes, and provide insight into mechanisms contributing to these differences. We aim to further dissect the events controlling erythroid differentiation and pathophysiology with these readily-accessible genetic materials.</p><p>However, the complete repertoire of either small or large erythrocyte RNAs has not been determined. Based on this knowledge gap, my dissertation has two goals: 1) to define the comprehensive erythrocyte transcriptome, and 2) to utilize an in vitro erythroid differentiation model to elucidate the functions of these newly identified erythrocyte RNAs during development.</p><p>Using high-throughput sequencing, we show an extensive, diverse repertoire of both small-sized (short, 18-24 nt) and large-sized (long, >200 nt) RNA species in mature erythrocytes. Though many erythrocyte RNAs have known functions in erythroid cells, we describe several RNAs with unknown functions; these RNAs provide a wealth of genetic loci for further inquiry.</p><p> </p><p>Additionally, several newly described, primate-specific RNAs were identified within the miR-144/-451 locus formerly involved in erythroid development. I performed a functional investigation of a previously uncharacterized microRNA, miR-4732-3p, within this locus. My study demonstrates that this microRNA is upregulated during erythroid development, represses SMAD2/4-dependent TGF-&#946; signaling, and promotes proliferation during erythropoiesis. Thus, miR-4732-3p emphasizes the importance of balanced TGF-&#946; signaling during primate erythropoiesis, and represents a key modulator with potential therapeutic utility.</p><p>These two studies highlight the advantage of venipuncture to provide a catalog of erythrocyte RNAs, both for an in vitro and in vivo understanding of erythrocyte biology. This in vitro approach functions as a continuous window into the erythrocyte development program, whereas this in vivo approach provides a snapshot of the mature erythrocyte population state in real-time. The integration of these two approaches provides a broad perspective covering the lifetime of erythroid cells.</p> / Dissertation

<p>Lymphomas comprise a diverse group of malignancies derived from immune cells. High throughput sequencing has recently emerged as a powerful and versatile method for analysis of the cancer genome and transcriptome. As these data continue to emerge, the crucial work lies in sorting through the wealth of information to hone in on the critical aspects that will give us a better understanding of biology and new insight for how to treat disease. Finding the important signals within these large data sets is one of the major challenges of next generation sequencing.</p><p>In this dissertation, I have developed several complementary strategies to describe the genetic underpinnings of lymphomas. I begin with developing a better method for RNA sequencing that enables strand-specific total RNA sequencing and alternative splicing profiling in the same analysis. I then combine this RNA sequencing technique with whole exome sequencing to better understand the global landscape of aberrations in these diseases. Finally, I use traditional cell and molecular biology techniques to define the consequences of major genetic alterations in lymphoma.</p><p>Through this analysis, I find recurrent silencing mutations in the G alpha binding protein GNA13 and associated focal adhesion proteins. I aim to describe how loss-of-function mutations in GNA13 can be oncogenic in the context of germinal center B cell biology. Using in vitro techniques including liquid chromatography-mass spectrometry and knockdown and overexpression of genes in B cell lymphoma cell lines, I determine protein binding partners and downstream effectors of GNA13. I also develop a transgenic mouse model to study the role of GNA13 in the germinal center in vivo to determine effects of GNA13 deletion on germinal center structure and cell migration.</p><p>Thus, I have developed complementary approaches that span the spectrum from discovery to context-dependent gene models that afford a better understanding of the biological function of aberrant events and ultimately result in a better understanding of disease.</p> / Dissertation

<p>My dissertation work integrates comparative transcriptomics and functional analyses to investigate gene expression changes underlying two significant aspects of sea urchin evolution and development: the dramatic developmental changes associated with an ecologically significant shift in life history strategy and the development of the unusual radial body plan of adult sea urchins. </p><p>In Chapter 2, I investigate evolutionary changes in gene expression underlying the switch from feeding (planktotrophic) to nonfeeding (lecithotrophic) development in sea urchins. In order to identify these changes, I used Illumina RNA-seq to measure expression dynamics across 7 developmental stages in three sea urchin species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph Heliocidaris tuberculata, and an outgroup planktotroph Lytechinus variegatus. My analyses draw on a well-characterized developmental gene regulatory network (GRN) in sea urchins to understand how the ancestral planktotrophic developmental program was altered during the evolution of lecithotrophic development. My results suggest that changes in gene expression profiles occurred more frequently across the transcriptome during the evolution of lecithotrophy than during the persistence of planktotrophy. These changes were even more pronounced within the GRN than across the transcriptome as a whole, and occurred in each network territory (skeletogenic, endomesoderm and ectoderm). I found evidence for both conservation and divergence of regulatory interactions in the network, as well as significant changes in the expression of genes with known roles in larval skeletogenesis, which is dramatically altered in lecithotrophs. I further explored network dynamics between species using coexpression analyses, which allowed me to identify novel players likely involved in sea urchin neurogenesis and endoderm patterning. </p><p>In Chapter 3, I investigate developmental changes in gene expression underlying radial body plan development and metamorphosis in H. erythrogramma. Using Illumina RNA-seq, I measured gene expression profiles across larval, metamorphic, and post-metamorphic life cycle phases. My results present a high-resolution view of gene expression dynamics during the complex transition from pre- to post-metamorphic development and suggest that distinct sets of regulatory and effector proteins are used during different life history phases. </p><p>Collectively, my investigations provide an important foundation for future, empirical studies to investigate the functional role of gene expression change in the evolution of developmental differences between species and also for the generation of the unusual radial body plan of sea urchins.</p> / Dissertation

BACKGROUND: Telomeres are specialized heterochromatic structures found at the ends of chromosomes that serve to protect the integrity of the genome. Theoretically, telomeres, and thereby chromosomes, should be constantly shortening however that is not the case. Telomere length homeostasis is maintained via the activity of the enzyme Telomerase as well as a complex of 6 proteins, called the shelterin complex. Decreased telomere length is associated with a variety of neuropsychological diseases such as Alzheimer’s disease, and chronic depression. OBJECTIVE: The primary objective was to understand the effect of alcohol exposure on telomere dynamics, specifically on telomere length as well as the expression of the shelterin complex genes. This was accomplished using human embryonic stem cells (hESCs) as models. This study was exploratory with the goal of increasing our knowledge of genes involved in telomere maintenance. METHODS: hESCs were cultured in cell culture media containing 25 mM, 50 mM, or 100 mM of ethanol. After a 3-, 7-, or 14-day ethanol exposure plus a 24-hour withdrawal, hESCs were collected and genomic DNAs and total RNAs was extracted. Ethanol-induced telomere length changes were examined by quantitative polymerase chain reactions (qPCR).Ethanol-induced shelterin complex gene expression changes were examined by reverse-transcription and quantitative polymerase chain reactions (RT-qPCRs). RESULTS: Extended alcohol exposure exerted a deleterious effect on telomeres, causing them to diminish in length. The effects of alcohol on the expression of shelterin complex genes were varied, ranging from consistent upregulation or downregulation to a combination of both. The concentration of alcohol was inversely correlated with the expression of shelterin complex genes, and the correlation was influenced by the duration of ethanol exposure. CONCLUSION: Chronic exposure to alcohol resulted in the shortening of telomeres. The effect ethanol was both time- and concentration-dependent, with telomere length having a negative relationship with the concentration of ethanol as well as the duration of exposure. Additionally, the shelterin subunit genes had their expression levels altered by ethanol in a manner which was consistent with what has been observed in prior studies as well as what aligns with their respective functions.