Gene expression is regulated by local genetic sequence, and researchers have identified thousands of common genetic variants in the human population that associate with altered gene expression. These expression quantitative trait loci (eQTLs) often co-localize with genome wide association study (GWAS) loci, suggesting that they may hold the key to understanding genetic effects on human phenotype and cause disease. eQTLs are enriched in cis-regulatory elements, suggesting that many affect gene expression via non-coding mechanisms. However, many of the discovered loci lie in noncoding regions of the genome for which we lack understanding, and determining their mechanisms of action remains a challenge. To complicate matters further, genetic variants may have varied effects in different tissues or under different environmental conditions. The research presented here uses statistical methods to investigate genetic variants’ mechanisms of actions and context specificity. In Chapter 1, we introduce eQTLs and discuss challenges associated with their discovery and analysis. In Chapter 2, we investigate cross-tissue eQTL and gene expression patterns, including for GWAS genes. We find that eQTL effects show increasing, decreasing, and non-monotonic relationships with gene expression levels across tissues, and we observe higher eQTL effects and eGene expression for GWAS genes in disease-relevant tissues. In Chapter 3, we use the natural variation of transcription factor activity among tissues and between individuals to elucidate mechanisms of action of eQTL regulatory variants and understand context specificity of eQTL effects. We discover thousands of potential transcription factor mechanisms of eQTL effects, and we investigate the transcription factors’ roles with orthogonal datasets and experimental approaches. Finally, in Chapter 4, we focus on a locus implicated in coronary artery disease risk and unravel the likely causal variants and functional mechanisms of the locus’s effects on gene expression and disease. We confirm the locus’s colocalization with an eQTL for the LIPA gene, and using statistical, functional, and experimental approaches, we highlight two potential causal variants in partial linkage disequilibrium. Taken together, this work develops a framework for understanding eQTL context variability and highlights the complex genetic and environmental contributions to gene regulation. It provides a deeper understanding of gene regulation and of genetic and environmental contributions to complex traits and disease, enabling future research surrounding the context variability of genetic effects on gene expression and disease.

Due to their underlying genetic complexity, chromosomal disorders such as Down syndrome (DS), which is caused by trisomy 21, have long been understudied and continue to lack effective treatments. With over 200 genes on the extra chromosome, even the specific cell pathologies and pathways impacted in DS are not known, and it has not been considered a viable target for the burgeoning field of gene therapy. Recently, our lab demonstrated that the natural mechanism of dosage compensation can be harnessed to silence the trisomic chromosome in pluripotent cells. Using an inducible XIST transgene allows us to study the effects of trisomy in a tightly controlled system by comparing the same cells with either two or three active copies of chromosome 21. In addition, it raises the prospect that insertion of a single gene into a trisomic chromosome could potentially be developed in the future for “chromosome therapy”. This thesis aims to utilize this inducible system for dosage compensation to study the neurodevelopmental effects of trisomy 21 in vitro, and to answer basic epigenetic questions critical to the viability of chromosome silencing as a therapeutic approach. Foremost, for XIST to have any prospect as a therapeutic, and to strengthen its experimental utility, it must be able to initiate chromosome silencing beyond its natural context of pluripotency. Here I demonstrate that, contrary to the current literature, XIST is capable of initiating chromosome silencing in differentiated cells and producing fully dosage compensated DS neurons. Additionally, I show that silencing of the trisomic chromosome in neural stem cells enhances their terminal differentiation to neurons, and transcriptome analysis provides evidence of a specific pathway involved. Separate experiments utilize novel three-dimensional organoid technology and transcriptome analysis to model DS neurodevelopment in relation to isogenic euploid cells. Overall, this work demonstrates that dosage compensation provides a powerful experimental tool to examine early DS neurodevelopment, and establishes that XIST function does not require pluripotency, thereby overcoming a perceived obstacle to the potential of XIST as a therapeutic strategy for trisomy.

The overall goal of this project was to develop, optimize, and test an ultrasound-responsive targeted nanobubble for delivering osteoporosis-related silencing genes such as Cathepsin K small interfering RNA (CTSK siRNA) for osteoporosis treatment. The nanobubbles were synthesized using an in situ sonochemical method. The nanobubble (NB) is composed of a gas core made from perfluorocarbon, stabilized with albumin, encapsulated with CTSK siRNA, and embedded with alendronate (AL) for bone targeting (CTSK siRNA-NB-AL). Following its development, the responsiveness of CTSK siRNA-NB-AL to a therapeutic ultrasound probe was examined. The results of biocompatibility tests with human bone marrow-derived mesenchymal stem cells proved no significant cell death (p > 0.05). When the CTSK siRNA-NB-AL was supplemented with human osteoclast precursors, they suppressed osteoclastogenesis. Thus, this project establishes the potential of nanotechnology and ultrasound to deliver genes into the osteoclasts. This research also presents a novel ultrasound responsive and targeted nanobubble platform that can be used as a gene, drug, and/or oxygen delivery system for various diseases including cancer, neurodegenerative diseases, or bone disorders.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder involving a mutation in the CF transmembrane conductance regulator protein (CFTR), which causes dysfunctional transport of chloride ions across cell membranes. CF affects multiple body systems and a few of its symptoms include chronic cough, difficulty breathing, obstructive airway disease, bacterial pulmonary infections, maldigestion, malabsorption, pancreatitis, and male infertility. Until recently, treatment options have been limited to alleviating symptoms, but a new classification of drugs, CFTR modulators, provide an opportunity to slow the progression of the disease and improve clinical outcomes. The effect of CFTR modulators may be attributed to the reduction of persistently colonizing bacteria in CF lungs. Though, the effects of modulators on microbial communities colonizing the CF lung remains unknown, specifically with common respiratory pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. Particularly, previous CF studies have been limited in scope due to focusing on only one type of modulator and by using low-yield sequencing techniques. To address this gap, we seek to study the changes in CF respiratory pathogens of patients initiating CFTR modulator therapy at Nemours Hospital using long-read metagenomic sequencing (Oxford Nanopore) of longitudinally collected respiratory samples. We have optimized a protocol for host DNA depletion and microbial metagenomic sequencing to characterize the respiratory microbiome. This study focuses on utilizing these sequencing data to compare the microbiome among two healthy controls to pre-CFTR-treatment microbial communities of two recruited pediatric CF patients.

Aging of the brain is the leading risk factor for neurodegenerative diseases and brain cancers and has deleterious effects on brain functions. It follows that attempts to reverse the aging process may be therapeutically valuable. Neural stem cells (NSC) have been shown to play a critical role in maintaining brain functions, and their number is severely decreased with age. The development of senescence-like characteristics and declining functions in NSCs have been proposed to be responsible for brain aging and tumorigenesis. MS-818 is a pyrrolopyrimidine that has been shown to increase the NSC population and reverse the decline of behavioral function in aged rodent models. While MS-818 has demonstrated such benefits, the mechanism by which it affects particular pathways of biological age in NSCs is not yet known. Understanding how MS-818 relates to the molecular mechanisms underlying cellular aging may help accelerate the development of anti-aging therapies for neurodegenerative diseases and cancer. This study attempts to elucidate the mechanism of action of MS-818 on NSCs using an in vitro accelerated-aging model produced by Hydroxyurea (HU) treatment. Our analysis of NSC population size post-MS-818 exposure supports the idea that MS-818 treatment can increase NSC proliferation. qPCR analysis of aging-related genes revealed HU treatment produced a trend of increased p16 and Il-6 and decreased Lamin B1 relative expression, supporting the notion that HU treatment can induce senescence in NSCs. MS-818 treatment alone also produced notable trends for targets including BRCA1. In addition, MS-818 treatment post-HU exposure appeared to influence the relative expression of targets, including PGC1a and Lamin B1. Such MS-818 treatment produced similarly noteworthy trends for the expression of genes including PGC1a, Lamin B1, BRCA1, RPTOR, and Il-6, whether in media containing 2.5% or 7.5% serum. These results indicate that MS-818 may have influenced some aging-related pathways.

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No description available.

<p>Chondrosarcoma is a unique type of bone cancer in that it does not respond to chemotherapy or radiation therapy, and therefore many affected patients die from metastatic disease. Metastasis has been correlated to upregulation of the matrix metalloproteinase (MMP) family of proteases, which can degrade a wide range of extracellular components. MMP2 is an enzyme secreted from the cell and degrades extracellular gelatin, as denatured collagen. ETV5 is a transcription factor which has shown to be overexpressed in multiple types of invasive tumours. The regulatory relationship between ETV5 and MMP2 has not been studied in chondrosarcoma. We hypothesized that ETV5 regulates MMP2 in human chondrosarcoma with the protease acting as a downstream effector. We have determined that both ETV5 and MMP2 are upregulated in the KC human chondrosarcoma cell line. Gene knockdown of ETV5 in KC cells resulted in a reduction in MMP2 mRNA expression as well as decreased protein production, and significantly decreased MMP2 gelatinase activity. When ETV5 is overexpressed, MMP2 expression is upregulated at the RNA and protein levels. Data from our bone resorption studies revealed that when a matrix metalloproteinase 2 inhibitor is added to the KC cell growth media, collagen released from bone chips decreased significantly. Upon histological examination, bone chips incubated with KC cells and the MMP2 inhibitor showed less bone resorption. Analyses of patient cell lines show similar expression profiles of ETV5 and MMP2 as the KC cell line. ETV5 knockdown (KD) in the patient cell lines also showed a decrease in MMP2 expression, similar to the KC cells, suggesting that the ETV5-MMP2 pathway has clinical importance. This data advocates that ETV5 has a significant role in regulating MMP2 expression and activity, and bone resorption in human chondrosarcoma, and thus may be a targetable effector of the metastatic cascade in this cancer.</p> / Master of Science (MSc)

This thesis presents genetic studies aiming at enlarging our knowledge regarding the genetic factors underlying two immune-mediated diseases, hypothyroidism and autoimmune Addison’s disease (AAD), in dogs and humans, respectively. Genetic and environmental factors are indicated to contribute to canine hypothyroidism, which can be considered a model for human Hashimoto’s thyroiditis (HT). In Paper I we performed the first genome-wide association (GWA) study of this disease in three high-risk dog breeds (Gordon Setter, Hovawart and Rhodesian Ridgeback). Using an integrated GWA and meta-analysis strategy, we identified a novel hypothyroidism risk haplotype located on chromosome 12 being shared by the three breeds. The identified haplotype, harboring three genes previously not associated with hypothyroidism, is independent of the dog leukocyte antigen region and significantly enriched across the affected dogs. In Paper II we performed a GWA study in another high-risk breed (Giant Schnauzer) and detected an associated locus located on chromosome 11 and conferring protection to hypothyroidism. After whole genome resequencing of a subset of samples with key haplotypes, we fine mapped the region of association that was subsequently screened for the presence of structural variants. We detected a putative copy number variant overlapping with the upstream region of the IFNA7 gene, which is located in a region of high genomic complexity. Remarkably, perturbed activities of type I Interferons have been extensively associated with HT and general autoimmunity. In Paper III we performed the first large-scale genetic study of human AAD, a rare autoimmune disorder characterized by dysfunction and ultimately destruction of the adrenal cortex. We resequenced 1853 immune-related genes comprising of their coding sequences, untranslated regions, as well as conserved intronic and intergenic regions in extensively characterized AAD patients and control samples, all collected in Sweden. We identified BACH2 gene as a novel risk locus associated with AAD, and we showed its independent association with isolated AAD. In addition, we confirmed the previously established AAD association with the human leukocyte antigen complex. The results of these studies will hopefully help increasing the understanding of such diseases in dogs and humans, eventually promoting their well-being.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase and the limiting factor for the enzyme activity. The expression of TERT and telomerase activity is increased in atherosclerotic plaques. However, the role of TERT dysregulation during atherosclerosis formation remains unknown. The work herein first identified a multi-tiered regulation of TERT expression in smooth muscle cells (SMC) through histone deacetylase (HDAC) inhibition. HDAC inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Furthermore, during vascular remodeling in vivo, TERT protein expression in the neointima is prevented by HDAC inhibition. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition. TERT is highly expressed in replicating SMC of atherosclerotic and neointimal lesions. Using a model of guidewire-induced arterial injury, neointima formation was reduced in TERT-deficient mice. Studies in SMC isolated from TERT-deficient and TERT overexpressing mice with normal telomere length established that TERT is necessary and sufficient for cell proliferation. TERT deficiency did not induce a senescent phenotype but resulted in G1 arrest albeit hyperphosphorylation of the retinoblastoma protein. This proliferative arrest was associated with stable silencing of the E2F1-dependent S-phase gene expression program which could not be reversed by ectopic overexpression of E2F1. Chromatin immunoprecipitation and accessibility assays revealed that TERT was recruited to E2F1 target sites to increase chromatin accessibility for E2F1 by facilitating the acquisition of permissive histone modifications. These data indicate a mitogenic effect of TERT on SMC growth and neointima formation through epigenetic regulation of proliferative gene expression. Furthermore, TERT expression is induced in activated macrophages during experimental and human atherosclerosis formation. To investigate the role for TERT in lesional macrophages and the subsequent effect on atherosclerosis formation, TERT-deficient mice were crossbred with LDL-receptor-deficient (LDLr-/-) mice to generate first generation G1TERT-/-LDLr-/- offsprings, which were then further intercrossed to obtain third generation G3TERT-/-LDLr-/- mice. G1TERT-/-LDLr-/- mice revealed no telomere shortening while severe telomere attrition was evident in G3TERT-/-LDLr-/- mice. When fed an atherogenic diet, G1TERT-/-LDLr-/- and G3TERT-/-LDLr-/- mice were both protected from atherosclerosis formation compared to their wild-type controls, indicating that genetic TERT-deletion prevents atherosclerosis, and formation of the disease is not affected by telomere attrition. Similarly, atherosclerosis development was decreased in chimeric LDLr-/- mice with TERT deletion in hematopoietic stem cells after bone marrow transplantation. TERT deficiency reduced macrophage accumulation in atherosclerotic lesions and altered chemokine expression, including CXC1/2/3, CCL3, CCL5, CCL21, CCR7, IL-6, and IL-1α. In isolated macrophages, gene ontology (GO) enrichment analysis of silenced inflammatory genes indicated that TERT positively regulates signal transducer and activator of transcription (STAT) cascade, which was confirmed by the decreased tyrosine phosphorylation of STAT3 protein resulting from TERT deletion. These findings indicate genetic TERT deficiency decreases atherosclerosis formation by silencing inflammatory chemokine transcription through inactivation of the STAT3 signaling pathway in activated macrophages. In conclusion, the dysregulation of TERT expression within atherosclerotic plaques plays a causative role for vascular remodeling, including injury-induced neointima formation and hypercholesterolemia-induced atherosclerosis, through inducing SMC proliferation and a pro-inflammatory phenotype in infiltrating macrophages. These findings unveil a mechanism of TERT exacerbating the pathological vascular remodeling, which may provide a novel therapeutic target to combating vascular diseases.