The highly conserved Notch signaling pathway regulates cell-cell communication pathways, cell fate, cell determination, cell death, embryonic development, and adult tissue pathways in metazoans. The Notch receptors and ligands that bind to Notch are single pass, transmembrane proteins that communicate cell to cell via juxtacrine signaling. There are reports of the divergent function and localization of the Deltalike 3 (Dll3) ligand. In Mus musculus (an eutherin mammal) the DLL3 protein inhibits the Notch signaling pathway and is localized in the Golgi apparatus. In contrast, the DLL3 protein from zebrafish, Danio rerio (a teleost) activates Notch and is located on the cell surface. This study will focus on examining the evolutionary pathway/evolutionary similarities, localization, and function of the A. carolinensis dll3 gene in comparison to other vertebrate species. This is important because there is not much known about the evolutionary divergence of the DLL3 A. carolinensis protein, its function in Notch signaling, and its subcellular localization.
Evolutionary analysis of vertebrate DLL3 protein sequences using phylogenetic trees showed that D. rerio and A. carolinensis are more evolutionarily similar in comparison to M. musculus suggesting that they may have similar intracellular localization. However, immunofluorescence staining experiments showed that the A. carolinensis DLL3 protein co-localized significantly with an endoplasmic reticulum (ER) specific primary antibody. Since this protein is localized in the secretory system, similar to that of M. musculus DLL3, it suggests that its function is to inhibit the Notch signaling pathway. Protein sequence alignments were created that suggested that there is a region in the protein sequences where the lizard and mouse sequence are conserved, while the zebrafish sequence simultaneously varies. This region of the amino acid sequence could be responsible for the difference in localization and function of the protein in these two species.

Hepatocellular carcinoma (HCC) is a type of liver cancer common in Sub-Saharan Africa and South East Asian countries. Each year more than 700,000 new cases and more than 600,000 deaths are recorded worldwide due to HCC. According to the American Cancer Society HCC is ranked the 5th most common cancer worldwide with a male:female susceptibility ratio ranging between 2:1 and 8:1. HCC risk factors include lifestyle behaviors, such as persistent alcohol abuse and smoking, prolonged exposure to aflatoxins, chronic viral hepatitis infections, inherited metabolic diseases and non-alcoholic fatty liver diseases. To understand the genetic effects underlying sex-bias in HCC, it is necessary to include the sex chromosomes in genomics analyses. X and Y chromosomes are often discluded in genomics studies because of the technical and analytical challenges: sequence homology. The purpose of this thesis is to analyze the effects of sex chromosome complement aware read mapping to germline variant calling. 10 male and 10 female tumor adjacent samples from The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA LIHC) cohort were processed using sex-aware and default reference and the concordance of the two approaches was examined. We detected a higher disconcordance of 0.69% on variants called on the X chromosome and a disconcordance of 0.51% on variants called on the Y chromosomes for the reference and alternative alleles respectively compared to autosomes. Variants called on the REF/ALT genotypes had a disconcordances of 1.00%, 1.05%, 1.35% and 12.34% for the autosomes, chromosome 7, the X, and the Y chromosome, respectively. At the end of the project we concluded that the generated datasets showed the effect of sex-aware read mapping on variant calling. Though the data did not show the sites that can be called as variants in one dataset but not in the other, rather the concordance looked at sites that were called as variants in both data sets.

Lactase persistence is the ability of adults to digest lactose in milk (Segurel & Bon, 2017). Mammals are generally distinguished by their mammary glands which gives females the ability to produce milk and feed their newborn children. The new born therefore requires the ability to breakdown the lactose in the milk to ensure its proper digestion (Segurel & Bon, 2017). Generally, humans lose the expression of lactase after weaning, which prevents them being able to breakdown lactose from dairy (Flatz, 1987).
My research is focused on the people of Turkana, a human pastoral population inhabiting Northwest Kenya. The people of Turkana are Nilotic people that are native to the Turkana district. There are currently no conclusive studies done on evidence for genetic lactase persistence in Turkana. Therefore, my research will be on the evolution of lactase persistence in the people of Turkana. The goal of this project is to investigate the evolutionary history of two genes with known involvement in lactase persistence, LCT and MCM6, in the Turkana. Variants in these genes have previously been identified to result in the ability to digest lactose post-weaning age. Furthermore, an additional study found that a closely related population to the Turkana, the Massai, showed stronger signals of recent selection for lactase persistence than Europeans in these genes. My goal is to characterize known variants associated with lactase persistence by calculating their allele frequencies in the Turkana and conduct selection scans to determine if LCT/MCM6 show signatures of positive selection. In doing this, we conducted a pilot study consisting of 10 female Turkana individuals and 10 females from four different populations from the 1000 genomes project namely: the Yoruba in Ibadan, Nigeria (YRI); Luhya in Webuye, Kenya; Utah Residents with Northern and Western European Ancestry (CEU); and the Southern Han Chinese. The allele frequency calculation suggested that the CEU (Utah Residents with Northern and Western European Ancestry) population had a higher lactase persistence associated allele frequency than all the other populations analyzed here, including the Turkana population. Our Tajima’s D calculations and analysis suggested that both the Turkana population and the four haplotype map populations shows signatures of positive selection in the same region. The iHS selection scans we conducted to detect signatures of positive selection on all five populations showed that the Southern Han Chinese (CHS), the LWK (Luhya in Webuye, Kenya) and the YRI (Yoruba in Ibadan, Nigeria) populations had stronger signatures of positive selection than the Turkana population. The LWK (Luhya in Webuye, Kenya) and the YRI (Yoruba in Ibadan, Nigeria) populations showed the strongest signatures of positive selection in this region. This project serves as a first step in the investigation of lactase persistence in the Turkana population and its evolution over time.

Bats (order Chiroptera) are the longest lived mammals for their size, with particularly extreme longevity evolving in the family Vespertilionidae, or vesper bats. Because of this, researchers have proposed using bats to study ageing and cancer suppression. Here, we study gene duplications across mammalian genomes and show that, similar to previous findings in elephants, bats have experienced duplications of the tumor suppressor gene TP53, including five genomic copies in the genome of the little brown bat (Myotis lucifugus) and two copies in Brandt's bat (Myotis brandtii). These species can live 37 and 41 years, respectively, despite having an adult body mass of only ~7 grams. We use evolutionary genetics and next generation sequencing approaches to show that positive selection has acted on the TP53 locus across bats, and two recently duplicated TP53 gene copies in the little brown bat are both highly conserved and expressed, suggesting they are functional. We also report an extraordinary genomic copy number expansion of the tumor suppressor gene FBXO31 in the common ancestor of vesper bats which accelerated in the Myotis lineage, leading to 34\u201457 copies and the expression of 20 functional FBXO31 homologs in Brandt's bat. As FBXO31 directs the degradation of MDM2, which is a negative regulator of TP53, we suggest that increased expression of both FBXO31 and TP53 may be related to an enhanced DNA-damage response to genotoxic stress brought on by long lifespans and rapid metabolic rates in bats.

This project was designed to develop resources to highlight diverse career options for students achieving a degree within the School of Life Sciences. Many students have a very narrow view of what careers their degree prepares them for. In addition, if they have a career in mind, they have difficulty selecting an appropriate degree that will prepare them for their intended career. The goal of this project was to provide a broader view of career options, as well as illustrate the requirements each student would need to meet in order to pursue these careers. This was done by interviewing five career professionals and developing a major map that corresponds to the specific requirements of that career.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and has been shown to have genetic factors that contribute to cancer susceptibility. These genetic factors can be studied using Genome-Wide association studies (GWAS), which allow for the assessment of associations between specific biologic markers. Through GWAS, associations can be analyzed to identify genetic components that contribute to the onset of HCC. This study uses an extended version of Pathways of Distinction analysis (PoDA) to identify the subset of SNPs within the Antigen Presentation and Processing Pathway that distinguish cases from controls. Further analysis was performed to explore SNP-SNP association differences between HCC cases and controls using R-squared values and p-values. Three SNPs show significant inter-SNP associations in both HCC cases and controls. Additionally, 4 SNPs showed significant SNP-SNP associations exclusively in the control data set, possibly suggesting that control pathways have a greater degree of genetic regulation and robustness that is lost in carcinogenesis. This result suggests that these SNP associations may contribute to HCC susceptibility.

Mammary gland development in humans during puberty involves the enlargement of breast tissue, but this is not true in non-human primates. To identify potential causes of this difference, I examined variation in substitution rates across genes related to mammary development. Genes undergoing purifying selection show slower-than-average substitution rates, while genes undergoing positive selection show faster rates. These may be related to the difference between humans and other primates. Three genes were found to be accelerated were FOXF1, IGFBP5, and ATP2B2, but only the latter one was found in humans and it seems unlikely that it would be related to the differences between mammary gland development at puberty between humans and non-human primates.

Unlike the autosomes, recombination on the sex chromosomes is limited to the pseudoautosomal regions (PARs) at each end of the chromosome. PAR1 spans approximately 2.7 Mb from the tip of the proximal arm of each sex chromosome, and a pseudoautosomal boundary between the PAR1 and non-PAR region is thought to have evolved from a Y-specific inversion that suppressed recombination across the boundary. In addition to the two PARs, there is also a human-specific X-transposed region (XTR) that was duplicated from the X to the Y chromosome. Genetic diversity is expected to be higher in recombining than nonrecombining regions, particularly because recombination reduces the effects of linked selection, allowing neutral variation to accumulate. We previously showed that diversity decreases linearly across the previously defined pseudoautosomal boundary (rather than drop suddenly at the boundary), suggesting that the pseudoautosomal boundary may not be as strict as previously thought. In this study, we analyzed data from 1271 genetic females to explore the extent to which the pseudoautosomal boundary varies among human populations (broadly, African, European, South Asian, East Asian, and the Americas). We found that, in all populations, genetic diversity was significantly higher in the PAR1 and XTR than in the non-PAR regions, and that diversity decreased linearly from the PAR1 to finally reach a non-PAR value well past the pseudoautosomal boundary in all populations. However, we also found that the location at which diversity changes from reflecting the higher PAR1 diversity to the lower nonPAR diversity varied by as much as 500 kb among populations. The lack of genetic evidence for a strict pseudoautosomal boundary and the variability in patterns of diversity across the pseudoautosomal boundary are consistent with two potential explanations: (1) the boundary itself may vary across populations, or (2) that population-specific demographic histories have shaped diversity across the pseudoautosomal boundary.

While specific resistance mechanisms to targeted inhibitors in BRAF-mutant cutaneous melanoma have been identified, surprisingly little is known about the rate at which resistance develops under different treatment options. There is increasing evidence that resistance arises from pre-existing clones rather than from de novo mutations, but there remains the need for a better understanding of how different drugs affect the fitness of clones within a tumor population and promote or delay the emergence of resistance. To this end, we have developed an assay that defines the in vitro rate of adaptation by analyzing the progressive change in sensitivity of a melanoma cell line to different treatments. We performed a proof-of-theory experiment based on the hypothesis that drugs that cause cell death (cytotoxic) impose a higher selection pressure for drug-resistant clones than drugs that cause cell-cycle arrest (cytostatic drugs), thereby resulting in a faster rate of adaptation. We tested this hypothesis by continuously treating the BRAFV600E melanoma cell line A375 with the cytotoxic MEK inhibitor E6201 and the cytostatic MEK inhibitor trametinib, both of which are known to be effective in the setting of constitutive oncogenic signaling driven by the BRAF mutation. While the identification of confounding factors prevented the direct comparison between E6201-treated and trametinib-treated cells, we observed that E6201-treated cells demonstrate decreased drug sensitivity compared to vehicle-treated cells as early as 18 days after treatment begins. We were able to quantify this rate of divergence at 2.6% per passage by measuring the increase over time in average viability difference between drug-treated and vehicle-treated cells within a DDR analysis. We argue that this value correlates to the rate of adaptation. Furthermore, this study includes efforts to establish a barcoded cell line to allow for individual clonal tracking and efforts to identify synergistic and antagonist drug combinations for use in future experiments. Ultimately, we describe here a novel system capable of quantifying adaptation rate in cancer cells undergoing treatment, and we anticipate that this assay will prove helpful in identifying treatment options that circumvent or delay resistance through future hypothesis-driven experiments.

There are several challenges to accurately inferring levels of transcription using RNA-sequencing (RNA-seq) data, including detecting and correcting for reference genome mapping bias. One potential confounder of RNA-seq analysis results from the application of a standardized pipeline to samples of different sexes in species with chromosomal sex determination. The homology between the human X and Y chromosomes will routinely cause mismapping to occur, artificially biasing estimates of sex-biased gene transcription. For this reason we tested sex-specific mapping scenarios in humans on RNA-seq samples from the brains of 5 genetic females and 5 genetic males to assess how inferences of differential gene expression patterns change depending on the reference genome. We first applied a mapping protocol where we mapped all individuals to the entire human reference genome (complete), including the X and Y chromosomes, and computed differential expression between the set of genetic male and genetic female samples. We next mapped the genetic female samples (46,XX) to the human reference genome with the Y chromosome removed (Y-excluded) and the genetic male samples (46, XY) to the human reference genome (including the Y chromosome), but with the pseudoautosomal regions of the Y chromosome hard-masked (YPARs-masked) for the two sex-specific mappings. Using the complete and sex-specific mapping protocols, we compared the differential expression measurements of genetic males and genetic females from cuffDiff outputs. The second strategy called 33 additional genes as being differentially expressed between the two sexes when compared to the complete mapping protocol. This research provides a framework for a new standard of reference genome mappings to correct for sex-biased gene expression estimates that can be used in future studies.