In order to complete their life cycles, vertebrates require oxygen and water. However, environments are not always forgiving when it comes to constantly providing these basic needs for vertebrate life. The annual killifish Austrofundulus limnaeus is possibly the most well described extremophile vertebrate and its embryos have been shown to tolerate extremes in oxygen, salinity, and water availability. This phenotype is likely a result of the annual killifish life history, which includes periods of temporary habitat desiccation and oxygen deprivation, and requires the production of stress-tolerant embryos that depress metabolism in a state of suspended animation, known as diapause. Over the past several decades, the basic morphology and physiology of annual killifish development has become better characterized. However, there are still basic cellular processes that remain to be described in annual killifish such as A. limnaeus. Specifically, changes in DNA structure, expression, and copy number are known to have profound impacts on the phenotype and survival of an organism. Little is known as to how A. limnaeus maintains genome integrity during cell stress, nor how the A. limnaeus nuclear and mitochondrial genomes may have evolved under the unpredictable conditions in which A. limnaeus thrive. Early annual killifish embryonic development is also characterized by a complete dispersion and subsequent reaggregation of embryonic blastomeres prior to formation of the embryonic axis. This unusual period of early development may provide a functional adaptation that allows annual killifish embryos to survive these extreme conditions. The overall goals of this project were to (1) characterize the ability of A. limnaeus to tolerate and repair DNA damage through enzymatic and developmental mechanisms, (2) to determine possible consequences of mitochondrial DNA sequence and copy number on the metabolism of A. limnaeus, and (3) to establish a draft genome of A. limnaeus for future comparative genome studies. The results of this project show that embryos of A. limnaeus have an impressive ability to survive and reverse high doses of DNA damage induced by ultraviolet-C (UV-C) radiation, especially when allowed to recover under photoreactivating light. Surprisingly, embryos that survived irradiation during blastomere dispersion phases were able to develop normally. Characterization of gene expression during embryonic development for genes important for axis formation and cellular differentiation suggests that A. limnaeus embryos may delay axis formation until several days after epiboly is complete, thus allowing time for cells that become damaged to be replaced by surrounding pluripotent cells. This outcome would represent first case of a developmental buffering stage in a vertebrate. A. limnaeus embryos are also unique in their mitochondrial response to anoxia. Whereas in other species the amount of mitochondrial DNA (mtDNA) copy number fluctuates following extremes in oxygen availability, A. limnaeus embryonic mtDNA remains stable. Additionally, characterization of the fully sequenced A. limnaeus mitochondrial genome reveals possible evolutionary adaptations that may have facilitated dormancy and anoxia tolerance when compared to other species within the Order Cyprinodontiformes. The final chapter of this project characterizes the draft genome of A. limnaeus and I provide evidence suggesting that epigenetic DNA methylation that may be involved in regulating diapause.

Small molecule screening is commonly used to discover lead compounds for drug development, but it can also be a powerful way to identify chemical probes for studying biological mechanisms. Our lab uses small molecules to study the mechanisms by which the protozoan parasite Toxoplasma gondii infects and replicates within its hosts. In this work, we employed a fluorescence-based assay to screen the Medicines for Malaria Venture (MMV) Open Access Malaria box for compounds that affect T. gondii growth. The box contains 400 previously identified small-molecule inhibitors of the related parasite, Plasmodium falciparum. We identified 79 hits, including a 2,4-diaminoquinazoline (MMV006169; IC50=1.15µM) that strongly inhibits T. gondii intracellular replication and invasion with no evidence of toxicity to mammalian cells. Extensive structure-activity relationship analyses with T. gondii identified a number of analogs with changed potency and altered effects on replication and invasion. These structure-activity analyses provided the information necessary to synthesize a bivalent chemical inducer of dimerization (CID) containing MMV006169 for use in yeast three-hybrid experiments. Yeast growth competition assays showed that this CID is capable of entering the yeast nucleus, as required for yeast three-hybrid screening. Yeast three-hybrid was used in a targeted format to test the hypothesis that MMV006169 works by inhibiting parasite CDC48, an ATPase involved in trafficking and the degradation of misfolded proteins. Large-scale cDNA library screening by yeast three-hybrid suggests that the compound may instead be working through inhibition of a host cell target. This work has provided insight into how MMV006169 affects the parasite's lytic cycle and generated a testable hypothesis for the biologically relevant target of the compound.

Phylogeographic studies have relied on surveying neutral genetic variation in natural populations as a way of gaining better insights into the evolutionary processes shaping present day population demography. Recent emphasis on understanding putative adaptive variation have brought to light the role of epigenetic variation in influencing phenotypes and the mechanisms underlying local adaptation. While much is known about how methylation acts at specific loci to influence known phenotypes, there is little information on the spatial genetic structure of genome-wide patterns of methylation and the extent to which it can extend our understanding of both neutral and putatively adaptive processes. This research examines spatial genetic structure using paired nucleotide and methylation genetic markers in the Sonoran bark beetle, Araptus attenuatus, for which we have a considerable knowledge about its neutral demographic history, demography, and factors influencing ongoing genetic connectivity. Using the msAFLP approach, we attained 703 genetic markers. Of those, 297 were polymorphic in both nucleotide (SEQ) and methylation (METH) were assayed from 20 populations collected throughout the species range. Of the paired SEQ and METH locis, the METH were both more frequent (16% vs. 7%), maintained more diversity (Shannon IMeth = 0.361 vs. ISeq=0.272), and had more among-population genetic structure (ΦST; Meth = 0.035 vs. ΦST; Seq= 0.008) than their paired SEQ loci. Interpopulation genetic distance in both SEQ and METH markers were highly correlated, with 16% of the METH loci having sufficient signal to reconstruct phylogeographic history. Allele frequency variation at five loci (two SEQ and three METH) showed significant relationships with at-site bioclimatic variables suggesting the need for subsequent analysis addressing non-neutral evolution. These results suggest that methylation can be as informative as nucleotide variation when examining spatial genetic structure for phylogeography, connectivity, and, identifying putatively adaptive genetic variance.

Soil and root microbial communities have been studied for decades, and the incorporation of high-throughput techniques and analysis has allowed the identification of endophytic/non-culturable organisms. This has helped characterize and establish the core microbiome of many model plant species which include underground and aboveground organs. Unfortunately, the information obtained from some of these model plants is not always transferable to other agronomic species. In this project, we decided to study the microbiome of the Nicotiana genus because of its importance in plant physiological and plant-microbe interactions studies. The data obtained was used as baseline information that allowed us to better understand the effect of microbial inoculums on the assembly of the microbiome of the plant. We analyzed 16s rRNA amplicons to survey the microbiome in different plant organs and rhizosphere from four different species. Bacterial strains evaluated were screened for a consistent reduction or improvement in plant growth. Four bacterial strains were tested and used as seed inoculum (Lf-Lysinobacillus fusisormis, Ms –Micrococcus sp., Bs–Bacillus sp., Bc–Bacillus cereus). Bs and Bc inoculants caused plant growth promotion, and in contrast Ms caused retarded growth, while Lf acted as a neutral or non-inducing phenotype strain. Data supported that microbial inoculum used as seed treatment caused systemic changes in the host plant microbiome. Functionality of the inoculum was studied and the response in plant growth was linked to hormonal changes (evaluated in the plant and in the bacterial strains). Gene expression analysis using a genome-scale approach revealed that genes that could possibly be involved in stress response are down-regulated for Bc and Bs treatments and up-regulated for Ms. Flexibility variability of the inoculum was also evaluated to have a better understanding of the main factors involved in the promotion or suppression of growth, and possibly its effect in following generations. In summary, the findings of this project support that the plant functional microbiome responds to exogenous stimulation from abiotic and biotic factors by adapting endogenous hormone responses.

In the United States, metastatic breast cancer kills approximately 40,000 women and 400 men annually, and approximately 200,000 new cases of breast cancer are diagnosed each year. Worldwide, breast cancer is the leading cause of cancer deaths among women. Despite advances in the detection and treatment of metastatic breast cancer, mortality rates from this disease remain high because the fact is that once metastatic, it is virtually incurable. It is widely accepted that a major reason breast cancer continues to exhibit recurrence after remission is that current therapies are insufficient for targeting and eliminating therapy-resistant cancer cells. Emerging research has demonstrated that these therapy-resistant cells possess stem cell-like properties and are therefore commonly referred to as breast cancer stem cells (BCSCs). A major hallmark of BCSCs is the cell surface expression of CD44 and lack of expression of CD24, the so-called CD24-/CD44+ phenotype. Research indicates that this dangerous and rare subpopulation of BCSCs may be responsible for cancer onset, recurrence, and ultimately metastasis that leads to death. Two different model systems were utilized in this research. The first was the MCF7 cell line, a luminal A tumor subtype representative of a mildly invasive breast ductal carcinoma with an ER+/PR+/-/HER2- immunoprofile. The second was the MCF10A breast cancer progression model, which consists of three cell lines: MCF10A, MCF10AT1, and MCF10CA1a. In this system, spontaneously immortalized, non-malignant MCF10A cells were transfected with constitutively active H-Ras to form pre-malignant MCF10AT1 cells, which were then subcutaneously injected into mice and allowed to metastasize in order to form the oncogenic MCF10ACA1a cell line. This thesis presents evidence of a CD24low/-/CD44+ BCSC subpopulation within the MCF10A breast cancer progression model system. Findings indicate that RUNX1 and RUNX2 expression levels are involved in maintaining the BCSC phenotype. Across two different model systems, qRT-PCR analysis revealed that decreased levels of RUNX1 expression and increased levels of RUNX2 expression are essential for the maintenance of the BCSC subpopulation. It was also shown that low expression levels of RUNX1 and high expression levels of RUNX2 are present in CD24low/-/CD44+ BCSCs as compared to CD24+/CD44+ non-BCSCs. Furthermore, shRNA knockdown of RUNX1 was shown to enhance tumorigenicity, while shRNA knockdown of RUNX2 repressed tumorigenicity in BCSCs, as measured by the tumorsphere-formation assay. This research lays the groundwork for future investigations into the roles of RUNX1 and RUNX2 in regulating stemness in breast cancer.

Psychiatric genetics is a basic science field that has potential for practical application and effective translation. To date, translational frameworks utilized by this field have been linear (e.g., sequential) in nature, focusing on molecular genetic information. It is proposed that non-linear (e.g., socio-ecological) frameworks are a better way to immediately translate non-molecular genetic information. This dissertation explored the translation of psychiatric genetic information in two ways. First, a survey was sent to academic stakeholders to assess the state of the science regarding the translation of genetic information to the clinical care of mental health disorders. Findings from this indicate a translation-genetic competence gap whereby genetic knowledge reinforces linear frameworks and genetic competence is needed to achieve effective translation in this content area. Second, a new risk factor model for social anxiety was created that incorporated genetic, environmental, and neurophysiological risk factors (behavioral inhibition, parental bonding, emotion reactivity). Findings indicate that genetic etiology is more informative knowledge that can influence risk factor models and possibly prevention and intervention efforts for social anxiety. Overall this dissertation paves the way for examining the translational capacity of psychiatric genetics in a clinical setting. It constitutes the first examination of barriers to and a potential solution for the most effective translation of psychiatric genetic information.

Angiotensinogen is the only known precursor in the renin-angiotensin system, a hormonal system best known as an essential regulator of blood pressure and fluid homeostasis. Angiotensinogen is sequentially cleaved by renin and angiotensin- converting enzyme to generate angiotensin II. As the major effector peptide, angiotensin II mainly function through angiotensin type 1 receptor. Angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and more recently renin inhibitors are widely known as the 3 classic renin-angiotensin system inhibitory drugs against hypertension and atherosclerosis. Here, we developed an array of regents to explore the effects of angiotensinogen inhibition. First, we demonstrated that genetic deficiency of angiotensinogen not only protected against hypercholesterolemia- induced atherosclerosis but also prevented diet-induced obesity. Then we found weekly intraperitoneal injection of antisense oligonucleotides against angiotensinogen remarkably surpressed body weight gain in mice fed a western diet, which was absent from classic renin-angiotensin system inhibition. The suppressed body weight gain was attributable to diminished body fat mass gain and enhanced energy expenditure. More excitingly, angiotensinogen antisense oligonucleotides regressed body weight gain on obese mice. Together, our findings revealed a unique feature of angiotensinogen inhibition beyond classic renin angiotensin inhibition and demonstrated therapeutic potentials of angiotensinogen antisense oligonucleotides against hypertension, atherosclerosis, and obesity. We also developed an in vivo system to explore the functional consequences of disrupting a conserved Cys18-Cys137 disulfide bridge in angiotensinogen. The formation of this disulfide bridge could trigger conformational changes in angiotensinogen, thereby facilitating renin cleavage of angiotensinogen. It was predicted that the redox-sensitive disulfide bridge might change the efficiency of angiotensinogen/renin reaction to release angiotensin II, thus modulate angiotensin II-dependent functions. We determined effects of the presence and absence of the disulfide bridge on angiotensin II concentrations and responses in mice expressing either native angiotensinogen or Cys18Ser, Cys137Ser mutated angiotensinogen in liver via adeno-associated viral vectors. Contrary to the prediction, disruption of Cys18-Cys137 disulfide bridge in angiotensinogen had no discernible effects on angiotensin II production and angiotensin II-dependent functions in mice.

Brain disorders present a significant burden on affected individuals, their families and society at large. Existing diagnostic tests suffer from a lack of genetic biomarkers, particularly for substance use disorders, such as alcohol dependence (AD). Numerous studies have demonstrated that AD has a genetic heritability of 40-60%. The existing genetics literature of AD has primarily focused on linkage analyses in small family cohorts and more recently on genome-wide association analyses (GWAS) in large case-control cohorts, fueled by rapid advances in next generation sequencing (NGS). Numerous AD-associated genomic variations are present at a common frequency in the general population, making these variants of public health significance. However, known AD-associated variants explain only a fraction of the expected heritability. In this dissertation, we demonstrate that systems biology applications that integrate evolutionary genomics, rare variants and structural variation can dissect the genetic architecture of AD and elucidate its heritability. We identified several complex human diseases, including AD and other brain disorders, as potential targets of natural selection forces in diverse world populations. Further evidence of natural selection forces affecting AD was revealed when we identified an association between eye color, a trait under strong selection, and AD. These findings provide strong support for conducting GWAS on brain disorder phenotypes. However, with the ever-increasing abundance of rare genomic variants and large cohorts of multi-ethnic samples, population stratification becomes a serious confounding factor for GWAS. To address this problem, we designed a novel approach to identify ancestry informative single nucleotide polymorphisms (SNPs) for population stratification adjustment in association analyses. Furthermore, to leverage untyped variants from genotyping arrays – particularly rare variants – for GWAS and meta-analysis through rapid imputation, we designed a tool that converts genotype definitions across various array platforms. To further elucidate the genetic heritability of brain disorders, we designed approaches aimed at identifying Copy Number Variations (CNVs) and viral insertions into the human genome. We conducted the first CNV-based whole genome meta-analysis for AD. We also designed an integrated approach to estimate the sensitivity of NGS-based methods of viral insertion detection. For the first time in the literature, we identified herpesvirus in NGS data from an Alzheimer’s disease brain sample. The work in this dissertation represents a three-faceted advance in our understanding of brain disease etiology: 1) evolutionary genomic insights, 2) novel resources and tools to leverage rare variants, and 3) the discovery of disease-associated structural genomic aberrations. Our findings have broad implications on the genetics of complex human disease and hold promise for delivering clinically useful knowledge and resources.

This study aims to trace points of intersection between the too often divorced disciplines of literature, continental philosophy, and the hard sciences in Angela Carter’s “The Lady of the House of Love.” In short, this thesis will not only explore how such conversations surface within the short story, but will also serve as an explication of Maurice Merleau-Ponty’s philosophy of body and space, and the theory of epigenetics. Through these explications, the thesis itself will also gear one discipline towards the other as both theories intimately bind the environment with the body, and the body with the environment. Thus, the body and the environment are not separate and passive, but active and intertwined in a manner much like the aforementioned disciplines I posit are. Therefore, the goal of this thesis is to first postulate that such conversations between literature, philosophy, and science are already occurring, and as such, stress that such conversations need further discussion and exploration.

Nonalcoholic steatohepatitis (NASH) is a severe liver disorder characterized by triglyceride accumulation, severe inflammation, and fibrosis. With the recent increase in prevalence, NASH is now the leading cause of liver transplantation, with no approved therapeutics available. Despite years of research, the exact molecular mechanism of NASH progression is not well understood, but fat accumulation is believed to be the primary driver of the disease. Therefore, diacylglycerol O-acyltransferase 2 (DGAT2), a key enzyme in triglyceride synthesis, has been explored as a NASH target. RNAi-based therapeutics is revolutionizing the treatment of liver diseases, with recent chemical advances supporting long term gene silencing with single subcutaneous administration. Here we identified a hyper-functional, fully chemically stabilized GalNAc conjugated siRNA targeting DGAT2 (Dgat2-1473) that upon injection elicits up to three months of DGAT2 silencing (>80-90%, p<0.0001) in wild-type and NSG-PiZ “humanized” mice. Using an obesity-driven mouse model of NASH (ob/ob-GAN), Dgat2-1473 administration prevents and reverses triglyceride accumulation (> 50%, p:0.0008), resulting in significant improvement of the fatty liver phenotype. However, surprisingly, the reduction in liver fat didn’t translate into a similar impact on inflammation and fibrosis. Thus, while Dgat2-1473 is a practical, long-lasting silencing agent for potential therapeutic attenuation of liver steatosis, combinatorial targeting of a second pathway may be necessary for therapeutic efficacy against NASH.