Autism spectrum disorder (ASD) is a highly variable neurodevelopmental disorder. The main hallmarks of individuals with ASD are social communication impairments and repetitive sensory-motor behaviors, and they may present with additional comorbidities such as intellectual disability, epilepsy, anxiety, and/or attention-deficit/hyperactivity disorder. The underlying cause of ASD is similarly heterogeneous. More than a thousand associated genetic variants including chromosomal abnormalities and de novo rare genetic variants have been identified to be associated with ASD. However, there is a general lack of understanding of how genetic variants contribute to the fetal development of ASD. One interesting idea between the biological mechanism and ASD centers around immune associations and neurodevelopment. Many studies have found that a subset of genes are connected to immune pathways that converge on associated mechanisms of ASD. This work interrogates this idea by examining single cell expression of highly associated ASD genes in non-diseased human fetal thymus and brain. The high resolution of single cell expression highlights potential associations between specific cell types or pathways to ASD at a time point that is critical for neurodevelopment. Additionally, gene expression analysis has largely been focused on the brain, and this work investigates the thymus, a transient organ responsible for T cell development and a central component of the immune system. By analyzing highly associated ASD genes in non-diseased tissues of the fetal brain and thymus, the finding that a subset of genes are enriched in thymus tissue substantiates the reason for further interrogation of the possible associations of the thymus and ASD. This analysis also offers a baseline to compare to upon similar analyses of affected tissues of fetal brain and thymus from individuals with ASD.

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The structure and function of the eye is conserved across vertebrates. Mutagenesis screens of zebrafish have led to the discovery of many genes essential for retinal development and function. In one such screen, a recessive allele on the zvm9 locus was identified due to the lack of an optokinetic reflex in larvae of otherwise normal appearance. Preliminary histology indicated a lack of lamination in the zvm9 mutant retina and immunohistochemistry confirmed phenotypic defects in all layers of the retina. These included thin or absent plexiform layers, disorganization of cells within laminae, and the loss of specific retinal cell types. Additionally, cell death in both the retina and forebrain was identified by TUNEL. The presence of ectopic mitoses during retinal development and the later observation of ectopic cones suggests that the zvm9 product is a crucial component of a complex regulating cell-cell junctions or apical-basal polarity. The notable absence of synaptic structures in cone photoreceptors is consistent with my hypothesis that this complex is also required for proper cell-cell interactions. / A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science. / Fall Semester, 2014. / October 30, 2014. / Lamination, Retina, Synapse, Zebrafish / Includes bibliographical references. / James Fadool, Professor Directing Thesis; Cathy Levenson, Committee Member; Laura Keller, Committee Member; Hong-Guo Yu, Committee Member.

INTRODUCTION: Adaptation to environmental changes is a foundational characteristic of all life. Along with temperature, oxygen concentration and many other variables, relative pH is a key factor that cells must regulate in order to maintain viability and functionality. Studies have shown upregulated gene expression of acid tolerance response genes in bacteria when exposed to high acidity. Research has also linked significant pH changes in extracellular environment to different diseases and the cells subsequent responses. Acidosis is a hallmark of both in tumors of various cancers and in inflammation. Previous studies have found solid tumors maintain acidic extracellular pH due to increased respiration associated with glycolysis due to lack of proper vascularization which decreases oxygen saturation in the tumor. Other studies have demonstrated that acidic pH promotes expression of pro-apoptotic and pro-inflammatory cytokines. Many studies have also focused on pH as it pertains to specific pathologies associated with disease conditions. Despite the limited progress, research on the effects of the acidic environment on broader gene expression patterns corresponding to critical signaling pathways that are targeted for various treatment strategies remain unexplored. OBJECTIVE: To evaluate the effect of microenvironmental acidity on regulation of gene expression related to regulating transcription in epithelials, human embryonic kidney cells and human fibroblasts. METHODS: Human embryonic kidney (HEK293), immortal breast MCF10A, fibroblast (HDFa) and cancerous (HCT116) cells were grown in stock medium. Cells were cultured in 1mL wells under control, 12.5 µL of 0.2M HCl/mL, 25 µL of 0.2M HCl/mL and 37.5 µL of 0.2M HCl/mL of culture. Cells were collected after 3 days under experimental conditions. RNA and DNA were collected and purified usingDirect-Zol DNA/RNA Miniprep Kit. For RT-qPCR analysis, we synthesized cDNA from eluted RNA via SuperScript™ IV First-Strand Synthesis System, while concentrations of DNA, RNA and cDNA of each sample was determined via Nanodrop U-Vis spectrophotometer. Expression levels of neurotrophic tyrosine receptor kinase 2 (NTRK2), DNA (cytosine-5)-methyltransferase 1 & 3α (DNMT1, 3A) and Transforming Growth Factor Beta 2 & 1 (TGFB2, 1) were determined by RT-PCR analysis using Bio Rad CFX-Manager Program. The expression levels of sirtuin-silent-mating type information regulation 2 homolog-1 (SIRT1) was also tested for but only in the cancer cell line. Note that the selection of this group of genes was based on our recent studies that provided evidence for the expression of genes such as TGFB2, NTRK2 and DNMT3A exhibiting inverse correlation to pH in an expression microarray analysis of human post-mortem brain samples. RESULTS: Decreasing environmental pH did not directly indicate increased cellular replication as DNA and RNA concentrations in collected samples had no significant changes as extracellular pH increased. Strong association between decreased extracellular pH and increased gene expression was not unilateral across all tested genes and cell types. Increased DMNT1 expression directly correlated w/ decreased pH in all tested cell types. DMNT3A and TGFB1 increased in HDFa and HEK293; TGFB2 expression increased while TGFB1 expression exhibited no change as pH decreased in MCF10A cells. SIRT1 expression remained unchanged under any acid condition in the HCT116 cell line. Our data showed a strong correlation between decreased pH and increased NTRK2 expression in HEK293 and HDFa cells. Furthermore, we found no evidence for altered expression of these genes as well as SIRT1 in the cancer cell line. DISCUSSION: The increased expression of cytokines such as TGFB1/2 and DNMTs affecting transcription indicates that non-cancerous cells have an overall increased gene expression in response to acidic pH. Research procedures should be revisited for optimization using more normal and cancer cell lines to eliminate factors potentially responsible for the inconsistencies in altered gene expression patterns in order to establish their importance in disease pathogenesis and for exploring novel therapeutic remedies.

Gene therapy holds a significant promise of bettering the treatment modality. Gene therapy offers the potential of lifetime cure. Suffering patients become asymptomatic with the respective disorder when gene therapy is administered. However, there exists an underlying issue on the matter concerning long term effects and the high cost associated with gene therapy. This study is meant to overview the benefits and safety associated with gene therapy and compare it with the standard ways of treating patients suffering from genetic disorders. The study will offer a comprehensive resource on the available evidence of future and ongoing trials and research.

archives@tulane.edu / For many long-lived, outcrossing trees, a variety of ecological and evolutionary processes impact a species’ ability to respond to environmental change by shaping patterns of genetic diversity and variation. For example, because many plant species rely on animals to disperse their pollen and seeds, the factors that determine the movement of animal mutualists influences the distribution of genetic diversity. Furthermore, the environment often changes across the range of many plant species and exerts differing selective pressures that impact variation in allele frequencies among populations. Despite the importance of understanding the processes that impact a species’ evolutionary potential, the mechanisms that drive these relationships remain unclear. In this dissertation I used the tropical palm Oenocarpus bataua to explore how the distribution of resources, the amount of suitable habitat, and climate influence micro-evolutionary outcomes and patterns of genetic diversity and variation across the species’ range in Ecuador. I found that substantial temporal variation in the density of concurrently flowering conspecific trees influenced pollination parameters, such as genetic diversity, and that the relationship between density and pollination outcomes is scale dependent. Next, I found that that surrounding forest cover at the landscape scale had a similar significant positive association with measures of genetic diversity, suggesting that habitat loss equally dampens the genetic outcomes of both pollen and seed dispersal.Finally, I detected signatures of selection in O. bataua and found that that regional differences in climate influence population- specific responses in genetic and phenotypic variation. This work illustrates how a variety of interacting ecological and evolutionary processes mechanistically shape patterns of genetic diversity and variation within and among populations. / 1 / Zoe Diaz-Martin

Circulating tumor cells are cells that break away from a primary malignant site and circulate in the blood stream. From there, they can relocate to another part of the body, and cause metastasis. They can be used as biomarkers for cancer prognosis, and may also be used for cancer diagnosis in the presence of comorbid conditions. Micro RNAs (miRNA) are non-coding small RNAs found in humans that can degrade or upregulate protein transcription. miRNA is dependent on the Argonaute2 protein for incorporation into an RNA-induced silencing complex. miRNA utilizes exosomes to move outside the cytosol while being protected from extracellular enzymes such as RNases. miR-24 is implicated in many cancers, including breast cancer, and has the ability to upregulate the expression of its neighboring genes. As it has been shown that it can use the 3’untranslated regions on mRNA to influence expression of nearby targets, it also may influence its own transcription. The aim of this study was to evaluate the capability of transfected miR-24 to translocate into the nucleus of MCF-7 cells. It also evaluated the ability of transfected mirR-24 to integrate into exosomes, and whether this exosomal miRNA could similarly integrate into the nucleus in newly cultured cells. Results showed that transfected miR-24 does translocate into the nucleus of MCF-7 cells. It also showed that transfected miR-24 readily incorporates into exosomes. Because of procedural difficulties and time constraints, transfections with exosomal miR-24 have not yet been completed, but are a key next step in illustrating the ability of miR-24 to influence genetic expression on a broad spectrum. As miR-24 is upregulated in many disease states, including in many forms of breast cancer, the use of exosomes may outline a novel method for miRNA to integrate into malignant cells, and modulate protein transcription.

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