MS, University of Cincinnati, 2022, Medicine: Genetic Counseling

Genome-wide assessment of copy number variants (CNVs) by chromosomal microarray (CMA) is a first-line test offered to individuals with developmental delay, multiple congenital anomalies, autism spectrum disorder, and other indications. Exome reanalysis studies have demonstrated that reanalysis of genetic tests over time can yield additional diagnoses due to updates in genetic databases and the literature. Compared to exome reanalysis studies, there is limited data regarding the yield of microarray reanalysis. We conducted a manual reanalysis of 154 non-diagnostic patient SNP microarrays five years after they were initially performed in 2016. We found that 25/339 (7.4%) CNVs changed in classification after reanalysis with 24/154 (15.6%) patients having at least one variant that changed in classification. Of those variants that changed, 15/25 changed from benign to uncertain significance. However, only 2/154 patients (1.3%) had a diagnostic change, with a variant upgraded to likely pathogenic or pathogenic. The most common reasons variants changed in classification were changes in CNV classification standards, in particular the decoupling of patient phenotype to variant classification and new scoring guidelines. Overall, we demonstrate that while variants can be classified differently over time, SNP microarray reanalysis yields lower diagnostic rates compared to previously published exome reanalysis studies, if done within a relatively recent time-period of initial analysis (5-6 years).

Committee: Melanie Myers Ph.D. (Committee Member); Alyxis Coyan M.S. (Committee Member); Qiaoning Guan (Committee Member); Wenying Zhang M.D. Ph.D. M.B.A. (Committee Member); Teresa Smolarek Ph.D. (Committee Member); Elizabeth Ulm M.S. (Committee Member); Jie Liu (Committee Member) Subjects: Genetics

PhD, University of Cincinnati, 2007, Medicine : Biostatistics (Environmental Health)

DNA microarrays are a revolutionary technology able to measure the expression levels of thousands of genes simultaneously, providing a snapshot in time of a tissue or cell culture's transcriptome. Although microarrays have been in existence for several years now, research is yet ongoing for how to best analyze the data, at least partly due to the combination of small sample sizes (few replicates) with large numbers of genes. Several challenges remain in maximizing the amount of biological information attainable from a microarray experiment. The key components of microarray analysis where these challenges lie are experimental design, preprocessing, statistical inference, identifying expression patterns, and understanding biological relevance. In this dissertation we aim to improve the analysis and interpretation of microarray data by concentrating on two key steps in microarray analysis: obtaining accurate estimates of significance when testing for differentially expressed genes, and identifying key biological functions and cellular pathways affected by the experimental conditions. We identify opportunities to enhance analytical techniques, and demonstrate that these enhancements significantly improve the functional interpretation of microarray results. We develop three related Bayesian statistical models to improve the estimates of significance by exploiting the information available from all genes, and functionally relating the gene variances to their expression levels. These novel methodologies are compared to previously proposed methods both in simulations and with real-world experimental data performed on multiple microarray platforms. In addition, we introduce a logistic regression method for identifying key biological categories and molecular pathways and compared this method with the commonly used Fisher's exact test and other relevant previously developed methods. We make our statistical methods available to the biomedical research community through the use (open full item for complete abstract)

Committee: Dr. Mario Medvedovic (Advisor) Subjects:

Doctor of Philosophy, University of Akron, 2022, Integrated Bioscience

Cadherins are cell-adhesion molecules that play important roles in animal development, maintenance and/or regeneration of adult animal tissues. In order to understand cadherins' functions in adult vertebrate visual structures, one must study their distribution in those structures. First, I examined expression of cadherin-6, cadherin-7, protocadherin-17 and protocadherin-19 in the visual structures of normal adult zebrafish using RNA in situ hybridization, followed by studying expression of two Kruppel-like transcription factors (klf6a and klf7), that are known markers for regenerating adult zebrafish retinas and optic nerves, in normal adult zebrafish brain, normal and regenerating adult zebrafish retinas. Then, I investigated expression of these cadherins in regenerating adult zebrafish retinas using both RNA in situ hybridization and quantitative PCR. Finally, as the first step in elucidating molecular mechanisms underlying protocadherin-17 (one of the cadherins that I studied) function in zebrafish visual system development, I used DNA microarray analysis to study gene expression of zebrafish embryos with their protocadherin-17 expression blocked by morpholino antisense oligonucleotides (these embryos are called protocadherin- 17 morphants). The major findings include: 1) cadherin-6, cadherin-7, protocadherin-17 and protocadherin-19 were differently expressed in the retina and major visual structures of normal adult zebrafish brain. 2) klf6a and klf7 showed similar expression patterns in most visual structures in the adult fish brain, and in regenerating retinas, but klf6a appeared to be a superior regeneration marker based on RNA in situ hybridization. 3) These four cadherin molecules showed distinct expression patterns in the regenerating zebrafish retinas. 4) Several genes involved in vision and/or visual development were significantly downregulated in the protocadherin-17 morphants compared to control embryos. My results suggest that (open full item for complete abstract)

Committee: Qin Liu (Advisor); Richard Londraville (Committee Member); Rolando Ramirez (Committee Member); Brian Bagatto (Committee Member); Zhong-Hui Duan (Committee Member) Subjects: Anatomy and Physiology; Bioinformatics; Biology; Developmental Biology; Molecular Biology

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2022, Biomedical Sciences (Bioinformatics and Proteomics/Genomics)

Suicide is defined as the act of self-injurious behavior that results in an outcome of death. Nearly 800,000 people are estimated to die by suicide each year. Approximately 90% of those that die by suicide are suspected to have a comorbid neuropsychiatric condition, such as major depressive disorder (MDD), schizophrenia (SCZ), or bipolar disorder (BP). Given this clear correlation, studies of suicide have been largely conducted in the context of gene transcription associated with neuropsychiatric disorders. Such approaches, like RNAseq and microarray technologies, have been invaluable in assessing gene expression changes in the brain in subjects diagnosed with a neuropsychiatric disorder, offering significant insight into the biological processes that are perturbed in these disorders and therefore associated with an outcome of suicide. In this analysis, we utilized public postmortem brain tissue datasets from frontal cortical regions (i.e. DLPFC, frontal cortex, prefrontal cortex) to identify transcriptional gene signature specific to suicide across multiple neuropsychiatric disorders (MDD, SCZ, BP). Three RNAseq (150 total subjects) and four microarray (322 total subjects) datasets were collected from 4 NCBI GEO to construct a meta-analysis of various neuropsychiatric disorders. We assessed differentially expressed genes (DEGs) between suicide and natural death groups. We further explored DEG results using full transcriptome (GSEA) and targeted transcriptome (EnrichR) pathway analysis. As chronic psychological stress is associated with an increase in suicidal risk, we identified DEGs from mouse models of chronic stress. We explored these DEGs using similar pathway analyses as human data for comparison. Results in human data show dysfunction in mitochondrial pathways, purine metabolism and purinergic signaling pathways, as well as neuroinflammation associated pathways which have prior implications in suicide risk and underlying pathology of MDD, SCZ, and BP. We ad (open full item for complete abstract)

Committee: Robert Smith (Committee Co-Chair); Cheryl McCullumsmith (Committee Member); Rammohan Shukla (Committee Member); Sinead O'Donovan (Committee Co-Chair) Subjects: Bioinformatics; Biology; Biomedical Research; Neurosciences

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2021, Biomedical Sciences (Bioinformatics and Proteomics/Genomics)

Systemic Sclerosis (SSc) is a complex autoimmune disease of unknown etiology characterized by dysregulation of the immune system, a characteristic vasculopathy, and severe and often progressive fibrotic process that widely varies in extent. Its pathogenesis mechanism is unclear and complex. Discovery of validated biomarkers is crucial for the diagnosis, disease classification, and evaluation of organ involvement and therapeutic response. The objective of this study was to identify a biomarker for SSc using the publicly available microarray profiles of skin, lungs and blood from SSc patients and healthy controls. We sought common gene expression patterns associated with SSc in skin, lungs, and blood. All data were downloaded from the Gene Expression Omnibus (GEO) database. The top 1000 differentially expressed genes (DEGs) for each dataset, and shared genes were identified. Functional annotation of DEGs using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed. The construction and analysis of the protein‑protein interaction (PPI) network and identification and analysis of hub genes was carried out for each tissue type. A total of 29 and 134 significant DEGs were shared among skin datasets and lungs datasets respectively. Analyzing datasets from blood, a total of 39 DEGs were found to be shared between GSE22367 and GSE19617. Four statistically significant genes (CCL2, COL18A1, COL4A1, and COL5A2) from skin, five statistically significant genes (PECAM1, JUN, CDH5, IL7R, and WT1) from lungs, and three (MYLIP, UBE2D1, and WSB1) from blood were identified as hub genes with potential clinical implication for diagnosis or treatment of SSc. In a nutshell, this study identified a number of significant DEGs and a total of 12 hub genes that may, as a group, be useful as a biomarker for early detection and treatment of SSc, in particular CCL2, JUN, CDH5, and IL7R genes. Further studies are suggested to confirm and validate the fin (open full item for complete abstract)

Committee: Sadik Khuder (Committee Chair); Robert Blumenthal (Committee Member); Nezam Altorok (Committee Member) Subjects: Bioinformatics

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2020, Biomedical Sciences (Bioinformatics and Proteomics/Genomics)

Dilated cardiomyopathy (DCM) belongs to the heterogeneous group of heart muscle disorders called cardiomyopathies, characterize by left ventricular dilatation and reduce myocardial muscle contractility that leads to a reduced ejection fraction of the heart. There are several causes of DCM; genetic, toxic, metabolic, endocrine, infiltrative, and idiopathic disorders. Forty causative genes encoding for a variety of proteins have been identified up to date. The objective of this study is to identify potential biomarkers related to the disease process of DCM. Microarray and RNA-Seq profiles of cardiac tissue were used to identify differentially expressed genes (DEGs). Four Microarray datasets (GSE 3585, GSE3586, GSE9800, and GSE42955), and three RNA-Seq datasets were selected (GSE55296, GSE65466, and GSE71613) from the Gene Expression Omnibus (GEO) database. Weighted gene co-expression network analysis (WGCNA) was used to explore the hub genes involved in the disease process of DCM. A total of 51 modules for microarray datasets and 15 modules for RNA-Seq datasets were identified using correlation network analysis. Four common statistically- significant genes were identified among these modules, including AP3M2, ECM2, ERBB2, and ZNF83. AP3M2 gene, which involves protein trafficking to lysosomes and specialized organelles, ECM2 gene, which affects in extracellular matrix protein function, ERBB2 gene, which involves in Erb-B2 Receptor Tyrosine Kinase 2 signaling pathway, and ZNF83 gene which involves in transcriptional regulation in the cell. A total of 9 hub genes that were differentially over-expressed significantly in cardiac tissue from RNA-Seq datasets, including EIF4GA which is related to viral myocarditis, HACD1, MYOM3, PTPN4, and NRBP1 are associated with muscular disorders, CELSR which play an essential role for planar cell polarity, SLC27A6 which is transporter involve in LCFA uptake process, SCMH1 involves in negative regulation of gene expression, and DC (open full item for complete abstract)

Committee: Sadik Khuder (Committee Chair); Robert Blumenthal (Committee Member); Jiang Tian (Committee Member) Subjects: Bioinformatics; Biomedical Research; Genetics

Master of Sciences, Case Western Reserve University, 2020, EECS - Computer and Information Sciences

Microarray and RNA-seq are two transcriptomics data types. Comparison between microarray and RNA-seq was popular in recent research fields. But few works analyzed both data types on the same sample groups and fewer have discussed whether different analytical methods or statistical models will impact on the results. To get an insight of what role analytical method and statistical model play, and also to eliminate variances that might be caused by data types, we applied microarray and RNA-seq data into 5 analytical methods across 4 statistical models to contrast the similarities and differences. In this thesis, we processed and transformed raw microarray and RNA-seq data to fit in 5 different analytical methods across 4 statistical models. We did differential expression analysis on both data for all methods and evaluated the results. Both data types showed high consistency in those two methods applying the same model. All statistical models gave similar detected differentially expressed genes, the extent of similarity varied basing on specific pairs chosen to be compared. Among all analytical methods, NBPSeq and NOIseq are the most consistent for microarray while DESeq and baySeq are the most consistent for RNA-seq. Between data types, RNA-seq gives more detected differentially expressed genes. Overall, statistical models and data types both impact greatly on differential analysis results while analytical method seems trivial.

Committee: Fredrick Schumacher (Committee Chair); Jing Li (Committee Member); Mehmet Koyuturk (Committee Member); Satya Sahoo (Committee Member) Subjects: Bioinformatics

Doctor of Engineering, Cleveland State University, 2018, Washkewicz College of Engineering

Hepatocellular carcinoma (HCC) is an invasive and aggressive cancer of the liver that arises due to chronic cirrhosis. Research into understanding HCC has focused on two-dimensional (2D) and three-dimensional (3D) technologies to simulate the liver microenvironment and use animal models to model how HCC affects the rest of the body. 3D hydrogel models are desired because they can mimic the transport behavior observed in vivo by structurally mimicking the extracellular matrix (ECM) without the ethical concerns of animal models. However, hydrogels can be toxic to cells and require optimal procedures for appropriate handling. In this study, we created 3D models of liver diseases on high-throughput platforms. First, we optimized hydrogel attachment on micropillar chips by coating them with 0.01 w/v % PMA-OD in ethanol. Next, we optimized the protocol for encapsulation of viable Hep3B cells PuraMatrix peptide hydrogel, using a higher seeding density (6 * 106 cells/mL) and two post-print media washes. Then, we established the ability to transduce adenoviruses in situ in encapsulated cells and successfully demonstrated their dose-response behavior towards six compounds. In the second part, we scaled up to using the microwell chip platform and optimized the polymerization of oxidized methacrylated alginate (OMA) for Hep3B encapsulation. First, we plasma-treated microwell chips for 15 minutes at high RF to minimize bubbles. Then, we optimized micro-scale photopolymerization conditions at 45 % methacrylated OMA (OMA-45) and 2 w/v % OMA with 0.05 w/v % PI and reflective background under either low intensity, long duration (2.5 mW/cm2 for 2 minutes) or high intensity, short duration (4.0 mW/cm2, 30 seconds) light by testing cell viability at these conditions. Third, we used these OMA conditions to develop a high-throughput, real-time 3D cell migration assay on a newly engineered 384-pillar plate with sidewalls. We first developed a set of a protocols where out-of-focus cells ar (open full item for complete abstract)

Committee: Moo-Yeal Lee Ph.D. (Committee Chair); Joah Belovich Ph.D. (Committee Member); Nolan Holland Ph.D. (Committee Member); Chandra Kothapalli Ph.D. (Committee Member); Xue-Long Sun Ph.D. (Committee Chair) Subjects: Biomedical Engineering; Biomedical Research; Chemical Engineering; Materials Science; Nanoscience

Doctor of Philosophy, The Ohio State University, 2017, Biomedical Sciences

It is estimated that greater than 40,000 women in the United States will die from breast cancer this year and over 250,000 will be newly diagnosed. Greater than 70% of these cases are attributable to environmental factors, some of which are suspected to be a consequence of extensive human exposure to estrogenic compounds, collectively known as endocrine disrupting compounds (EDCs). Significant evidence from animal models suggests that chronic and/ or early-life exposure to known EDCs, such as bisphenol A (BPA) and genistein (GEN) are linked to increased risk and mediators of epithelial transformation to impart later-life development of breast cancer. However, the consequences of vast human exposures are largely unknown, immeasurable and the molecular mechanisms are largely undefined. Herein, we have utilized two distinct models to address undefined facets of the EDC mechanism. We address molecular mechanisms of chronic exposures that are representative of observed human exposures and examine windows of differential in utero BPA exposure to narrow possible developmental mechanisms at time of exposure that may propagate EDC-mediated alterations to later-life. We emphasize the importance of understanding the transcriptional alterations and epigenetic landscape of adult mammary component cell types, given the primary focus of current work to be on embryonic manifestation of disease and susceptibility. Our analyses demonstrate sustained reprogramming of the estrogen response beyond cessation of chronic exposures. Further we attribute the well-characterized BPA in utero phenotype to iii a period in mammary gland development corresponding to discrete tissue compartment interactions, corroborating the vital paracrine signaling that occurs at this in utero time of exposure. Our analyses are the first of its kind to map transcriptional and epigenetic alterations following in utero BPA exposure in the adult mammary gland. Taken together, our findings can inform analysis of h (open full item for complete abstract)

Committee: Craig Burd (Advisor); Helen Chamberlin (Committee Member); Ruth Keri (Committee Member); Thomas Ludwig (Committee Member); Mark Parthun (Committee Member) Subjects: Biology; Biomedical Research; Developmental Biology; Endocrinology; Environmental Health; Epidemiology; Experiments; Food Science; Health; Histology; Molecular Biology; Morphology; Plastics; Public Health; Toxicology

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2017, Biomedical Sciences (Bioinformatics and Proteomics/Genomics)

Macrophages are critical cells in the immune system implicated in various diseases with a chronic inflammatory component, such as atherosclerosis. The dominant macrophage types, M1 and M2, play key roles in progression and regression of atherosclerotic plaques. In this study, we profiled the transcriptomes of these macrophage subsets using RNA-Seq and microarray to examine molecular signatures and distinctive pathways for each subset. RNA-Seq analysis revealed a total of 2,127 differentially expressed genes (including coding and non-coding transcripts) between the M1 and M2 subsets. Validation by qRT-PCR of 10 top differentially expressed genes showed that 8 of the upregulated and 9 of the downregulated genes followed the expected trend based on the RNA-Seq analysis. Subsequently, pathway analysis of the upregulated and downregulated gene sets showed M1 and M2 macrophages to be enriched in pathways such as the Th1 and glioma signaling respectively. Microarray analysis revealed a total of 163 differentially expressed circRNAs between the two macrophage subsets and predictions of circRNA/miRNA interactions. From these circRNA/miRNA interactions, a number of the differentially expressed circRNAs were predicted to target athero-relevant miRNAs. As examples, circRNA_41878 (gene Ankrd42) was predicted to target miR-382-5p while circRNA_19794 (gene Lmbrd1) was predicted to target miR-124-3p, miRNAs of recognized effects in atherosclerosis. Overall, this work provides insight into distinctive molecular signatures and pathways enriched in bone-marrow derived M1 and M2 macrophages, and reveals the presence and contribution of non-protein-coding RNAs to their transcriptomes. In addition, our circRNA results provide the basis for future studies on the function of circRNAs in the context of macrophage functions in atherosclerosis.

Committee: Guillermo Vazquez (Committee Chair); Bina Joe (Committee Member); Beata Lecka-Czernik (Committee Member); Sivarajan Kumarasamy (Committee Member) Subjects: Bioinformatics; Biomedical Research

Master of Science, The Ohio State University, 2017, Public Health

Colon cancers are second only to lung cancers in the number of cancer deaths in the United States per annum. Common treatment regimens are surgical resection, optionally followed by adjuvant chemotherapy. Successful outcomes are measured by lack of progression to advanced stages at the primary tumor site and the absence of recurrence elsewhere in the body. Stage 3 cancer patients relapse at 50%, while stage 2 cancer patients relapse 25-40%. The most common site of distant metastasis is the liver; about 11% of patients who relapse survive 5 years. Unfortunately, there are no clear means to predict which patients will relapse following treatment. Subtyping of colon cancers is detailed in literature, although no clear translation to predicting clinical outcome has occurred. A 2014 meta-analysis by the Agency for Healthcare Research and Quality showed existing commercial means of predicting relapse provided dubious benefits to patients. This thesis details a method to create a predictive model of relapse in colon cancer patients that is an improvement over existing standards of care using gene expression patterns in specific stages coordinated with histopathological subtypes to be examined in vitro. We conducted a retrospective analysis of mRNA expression in colon cancer patients at the time of treatment, integrating genomic data from microarray and RNA-seq platforms with matching clinical data. The main focus of this research was genes with bimodal gene expression due to the ability of bimodal genes to fall along tumor subtypes with unique biological, clinical, and prognostic characteristics. Our results successfully identified bimodal genes through a novel ensemble testing system that recognizes clusters of gene expression values that decompose a single Gaussian distribution into two component Gaussian distributions. The utility and efficacy of the method was demonstrated with known bimodal gene markers in breast cancer patients as a positive contro (open full item for complete abstract)

Committee: Kun Huang PhD (Advisor); Randall Harris MD,PhD (Committee Member); James Chen MD (Committee Member); Joanna Groden PhD (Committee Member) Subjects: Bioinformatics; Biostatistics; Medicine; Public Health

Doctor of Philosophy, The Ohio State University, 2017, Pharmaceutical Sciences

Circadian rhythms are defined as oscillations in biological processes with a period similar to the 24 hour period of the earth's rotation. In mammals, circadian rhythms are controlled by the suprachiasmatic nucleus (SCN), a hypothalamic structure working as a central pacemaker that drives the circadian rhythms that are expressed in other brain regions, as well as throughout the rest of the organism. The manifestation of circadian rhythms is structured by the presence in nearly all cells of a molecular clock, a transcriptional and translational feedback loop that directs the timing of numerous cellular processes. Although endogenous circadian rhythms are self-sustained, entrainment of the SCN circadian clock is induced by diverse environmental factors in order to maintain a synchrony with external geophysical cycles, with light being recognized as one of the most robust entrainment signals. Here, I first present a study aimed at describing genome-wide expression changes in the SCN triggered by light inputs in the SCN (Chapter2). In this respect, previous studies have shown that the effects of light in the SCN are time-of-day specific, resulting from differential responses in specific neuronal signaling pathways. In particular, activation of the mitogen-activated protein kinases/ extracellular signal–regulated kinases (MAPK/ERK) signaling pathway in the SCN couples photic stimuli with circadian entrainment through changes in gene expression. Using microarray analysis and pharmacological interventions, I describe here the number and nature of transcripts induced by light at multiple points of the circadian cycle, with a particular emphasis on the subgroup of light-induced genes whose expression is modulated by the activation of the MAPK/ERK signaling pathway. This examination is followed by studies aimed to determine the influence of circadian rhythms on the function of the hippocampus. First, I assess the role of circadian rhythms in the formation and retrieval of co (open full item for complete abstract)

Committee: Kari Hoyt PhD (Advisor); Karl Obrietan PhD (Other) Subjects: Neurobiology; Neurosciences; Pharmacology; Pharmacy Sciences

Master of Science, University of Akron, 2017, Biology

Mammalian leptin (LEP) is a pleiotropic peptide hormone best characterized for its roles related to obesity and diabetes. However, the molecular function of the leptin signal transduction pathway in non-mammals is less clear. Comparative studies that address leptin signaling in non-model organisms are integral components of the leptin phylogenetic history, and there is little evidence addressing the functional disparities between the teleost leptin paralogues and mammalian leptins. To demarcate genes and biochemical pathways regulated by leptin signaling in developing zebrafish, microarray gene expression data were generated with total RNA isolated at 48 hours post fertilization from leptin-a morpholino oligonucleotide “knockdown”, recombinant leptin-a “rescue”, and wild type embryos. Expression estimates were computed for 26,046 genes across 16 microarray samples. Differentially expressed genes (DEG), (KEGG) pathways, and Gene Ontologies (GO) were evaluated for three contrasts (Morphant:Control, Rescue:Morphant, Rescue:Control). Signaling pathways that respond to leptin-a knockdown and rescue are analogous to gene targets of the mammalian LEP system (“GnRH”, “MAPK”, “Adipocytokine”, “Phosphatidylinositol”, “mTOR”, “ErbB”, “FoxO”, and “Notch”). A subset of differentially expressed transcription factors in leptin-a morphants are homologous to putative regulators of LEP expression in mammals (cebpb, creb5, fosl1a, mybl1, pax5, pou3f1, pparg, stat1a). “Neuroactive ligand-receptor interaction” as well as cAMP-responsive hormone signaling pathways responded to leptin-a. Consistent with leptin-a as an endocrine regulator, agouti-related peptide-2 (agrp2), cocaine-and-amphetamine-related-transcript (LOC557301), gonadotropin-releasing hormone 2 (gnrh2), and melanocortin receptor 5a (mc5ra) were dysregulated in rescue embryos. Further, “Notch signaling” and “Spinal cord/CNS development” were enriched in morphants whereas rescue arrays were comparable to wild type e (open full item for complete abstract)

Committee: Richard Londraville (Advisor) Subjects: Bioinformatics; Biology; Comparative; Developmental Biology; Endocrinology

MS, University of Cincinnati, 2017, Medicine: Genetic Counseling

Chromosomal microarrays have been used for over a decade to improve clinical diagnostic rates by identifying copy number variations (CNVs) across the genome. CNVs in noncoding regions of the genome are difficult to interpret because less is understood about the etiology of disease in these cases. Topologically-associating domains (TADs) describe the higher order chromatin structure that creates a landscape for enhancer-promoter interaction. The human genome has roughly two thousand such regions marked by high levels of chromatin interaction within these regions and separated by genomic boundaries, across which interaction occurs much less frequently. These domains and boundaries may provide the underlying genomic infrastructure for gene regulation and long range activation or repression of gene expression. Disruption of the boundary regions has been associated with human disease in two types of limb abnormalities and in other small disease specific populations. To date only one study has estimated the impact of TAD boundary deletions in an affected population and that study focused on large, known pathogenic deletions that were likely to disrupt multiple boundaries. In this study, a large scale, quantitative, comparative case-control study design was used to examine the presence of deletions across TAD boundaries by comparing patients with clinical indications for microarray testing to population-based controls. 5033 patients with a broad variety of clinical indications for microarray were compared with 312 controls to assess overall deletion burden, enrichment of topologically-associated domain boundary region disruption, and phenotypic trends among patients. A nonparametric t-test was used to assess the difference in the proportion of deletions which span an entire TAD boundary in cases compared to controls. No significant difference was found between cases and controls when both heterozygous and homozygous deletions were compared together. However, cases had sign (open full item for complete abstract)

Committee: Kristen Sund Ph.D. (Committee Chair); Iouri Chepelev Ph.D. (Committee Member); Lisa Dyer Ph.D. (Committee Member); Lisa Martin Ph.D. (Committee Member); Auvo Reponen Ph.D. (Committee Member); Teresa Smolarek Ph.D. (Committee Member) Subjects: Health Sciences

MS, University of Cincinnati, 2017, Medicine: Genetic Counseling

Chromosomal microarray analysis (CMA) is currently the first-tier diagnostic test recommended for children with undiagnosed intellectual disabilities. Long ordered by geneticists, CMAs are now increasingly being ordered by non-geneticist providers. This study explores parents' understanding and emotions during the process of ordering CMA by geneticists and non-geneticists, and parents' subsequent ability to derive utility from results. Twenty-two parents of children who underwent CMA testing participated in semi-structured interviews oriented around the domains of parental understanding, perceived utility, and affective response. Transcribed interviews were analyzed using inductive and deductive coding. Themes were explored for patterns, similarities, and differences between geneticist versus non-geneticist providers, as well as among those who received positive, negative, or variant of uncertain clinical significance (VUCS) results. We found that all parents of children with VUCS or pathogenic results received post-test counseling, but no parents of children with normal CMA results received post-test counseling. Nearly all parents reported that written resources and advice about online research was valuable, and this support was more likely to have been offered by genetics providers such as geneticists and genetic counselors rather than non-geneticist providers. Parents' perceived utility of CMA results included having an answer or name for their child's condition, providing information about reproductive risks and options, shaping expectations for the future, guiding their child's medical management, and helping parents explain the condition to others. Emotions expressed by parents in relation to genetic testing included anxiety, frustration, guilt, relief, and hope. Nearly all parents reported anxiety while waiting for test results. Frustration was mostly expressed in relation to communicating with providers, particularly in trying to obtain their child's tes (open full item for complete abstract)

Committee: Melanie Myers Ph.D. (Committee Chair); Kyle Brothers M.D. Ph.D. (Committee Member); Abigail Masunga M.S. (Committee Member) Subjects: Genetics

Master of Science, The Ohio State University, 2016, Public Health

Spatial patterns of gene expression in brain tissue have been observed in both human and more thoroughly in mouse. Because of the increased complexity and data availability, these spatial gene expression patterns in human have not been biologically explained in the same way that they have in murine models. In mice they can be explained by the spatial distribution of cell types. Using this insight, we address the human biology behind these spatial transcription changes by informing the human model with a-priori mouse transcriptional profiles. We apply clustered single cell RNA sequencing (scRNA-Seq) derived expression profiles across transcriptomics platforms to show: i) ordinary least squares regression can accurately predict the cell types within a tissue sample (validated through biomarkers and training/test data), ii) the distribution of cell types in the human brain vary between different locations in the brain, and iii) the distribution of human and mouse neural densities correlate between human and mouse. We developed a methodology to deconvolute cell types within heterogeneous tissue samples that can be applied to disparate data and achieve meaningful results. We then map these results to the large Allen Brain microarray database to evaluate changes in tissue between brain location.

Committee: Kun Huang PhD (Advisor); Po-Yin Yen PhD (Advisor); Raghu Machiraju PhD (Committee Member); Albert Lai PhD (Committee Member) Subjects: Biomedical Research

Master of Science, The Ohio State University, 2016, Dentistry

Objectives: Bone marrow mesenchymal stem cells (BM-MSCs) from limb bones have demonstrated promises in regenerating craniofacial bones; yet little is known about the potential of BM-MSCs from craniofacial bones. This study compared BM-MSCs isolated from limb and craniofacial bones in a commonly used preclinical large animal model, the pig. Methods: Bone marrow was aspirated from the tibia and mandible (symphysis) of 4-month-old pigs (n=4). Subsequently, BM-MSCs were isolated, expanded and confirmed by flow cytometry. To assess cell proliferation, cell doubling times were calculated from serial cell number counts over 2 weeks. Total mRNA was extracted from freshly isolated BM-MSCs and analyzed for gene expression using an Affymetrix GeneChip porcine genome array, followed by real-time RT-PCR for validation. Osteogenic capacity was assessed by quantifying alkaline phosphatase activity. Using temperature-responsive culturing plates, the abilities of BM-MSCs to form multi-layer cell sheets (for future in-vivo transplantation), along with cell viability and morphology, were evaluated by fluorescent labeling and histological staining. Results: BM-MSCs from both locations expressed MSC markers but not hematopoietic markers. Mandibular BM-MSCs proliferated significantly faster than tibial BM-MSCs (median cell doubling times: 2.25 vs. 2.80 days, Mann-Whitney U test, p<0.01). Without any osteogenic induction, mandibular BM-MSC alkaline phosphatase activities were 3.3-fold (factorial ANOVA, p<0.001) to those of tibial BM-MSCs. Microarray analysis on one hand confirmed that overall mandibular and tibial BM-MSC gene expressions are highly correlated with each other and genes related to osteogenesis and angiogenesis were strongly expressed, and on the other hand revealed that there were several dozen genes which were indeed differentially expressed between mandibular and tibial BM-MSCs. They include cranial neural crest-related genes nestin (1.23-fold) and BMP-4 (1.79-f (open full item for complete abstract)

Committee: Zongyang Sun DDS/PhD/MS/MSD (Advisor) Subjects: Dentistry

Doctor of Philosophy, Case Western Reserve University, 2015, Neurosciences

Following spinal cord injury (SCI), immune-mediated secondary processes exacerbate the extent of permanent neurological deficits. We develop here a contusion model of SCI to investigate the capacity of adult bone marrow-derived stem cells, termed multipotent adult progenitor cells (MAPCs), to alter inflammation and promote recovery following SCI. MAPCs were derived from human or rat donors and delivered either locally to the lesion or systemically via intravenous injection immediately following injury. Rats receiving intravenous human MAPCs displayed greater recovery in locomotion than animals treated with rat MAPCs or MAPCs of either origin delivered to the lesion. Delaying intravenous administration of human MAPCs by one day further improved locomotor recovery and significantly enhanced lower urinary tract recovery, re-establishing coordination between the detrusor and external urethral sphincter muscles. Intravenous MAPCs altered the immune response in the spinal cord and periphery, however biodistribution studies revealed that no MAPCs were found in the cord and instead preferentially homed to the spleen. Nonetheless, in the injured cord of treated animals we observed a decrease in macrophage number and activation, which was associated with significant sparing of neural tissue. In vitro, we show that human MAPCs have the ability to modulate macrophage activation, and prior exposure of macrophages to MAPC secreted factors can reduce macrophage-mediated axonal dieback of dystrophic axons. Our results demonstrate that a single intravenous dose of human MAPCs one day following severe contusive SCI attenuates immune mediated secondary spinal cord injury, resulting in significant neural tissue sparing and substantial improvements in locomotor and lower urinary tract function. This preclinical study provides strong support for the use of MAPC therapy in acute human contusive SCI.

Committee: Jerry Silver (Advisor); Richard Zigmond (Committee Chair); Robert Miller (Committee Member); Thomas Dick (Committee Member); Yu-Shang Lee (Committee Member) Subjects: Biology; Biomedical Engineering; Biomedical Research; Immunology; Neurobiology; Neurosciences

Doctor of Philosophy, The Ohio State University, 2015, Animal Sciences

Study of cell cycle activity is critical to understand development of adipose tissue, because the switch on and off of cell cycle activity is tightly related to hyperplasia and hypotrophy of the fat cells. Three cell cycle inhibitors - CCNG2, CDKN2C and PMP22, and three cell cycle activators for adipose tissue - CCNA1, CCND3 and ANAPC5, were selected as important cell cycle regulators for adipogenesis based on analyses of Gene Expression Omnibus (GEO) DataSets (GDSs) in the website of the National Center for Biotechnology Information (NCBI). The three important cell cycle inhibitors showed higher expression in fat cells than in preadipocytes and increased exprerssion during primary cell culture and development of adipose tissue in quantitative real-time PCR (qPCR) or western blotting. The results suggest that expression of these cell cycle inhibitors generally increases with the differentiation of fat cells and the growth of pig adipose tissue. In broiler chickens, expression of CCNG2, CDKN2C, CCND3 and ANAPC5 reaches a peak at post-hatch day 5 and decreases in the later stages, suggesting that day 5 may be an important time with active proliferation and differentiation of preadipocytes. The result of PCNA immunostaining suggests that proliferation activity of fat cells keeps decreasing from embryo day 15 to post-hatch day 33. The result of H&E staining suggests that fat cell size keeps increasing until post-hatch day 11. Therefore, both hypertrophic and hyperplastic growth is active before post-hatch day 5. From post-hatch day 5 to day 11, as proliferation activity decreases and most preadipocytes become differentiated, hypertrophic growth gradually becomes dominant. In the third experiment, some tissue specific secretory factors in mouse were selected based on GEO data analysis and bioinformatic tools. The tissue specific expressions of the selected genes were confirmed using semi-quantitative PCR. Secretion of five novel genes were then validated thro (open full item for complete abstract)

Committee: Michael Davis E. (Advisor); Kichoon Lee (Committee Member); Macdonald Wick (Committee Member); Harald Vaessin (Committee Member) Subjects: Animal Sciences; Animals; Bioinformatics; Biology

Doctor of Philosophy, University of Akron, 2014, Integrated Bioscience

In this dissertation, I tested the hypothesis that leptin A in zebrafish (D. rerio) plays a similar role to mammalian leptin in regulating metabolic rate and immune function, whereas leptins's effects on the zebrafish transcriptome may be distinct. Leptin is now identified in all major vertebrate lineages, but its role in controlling food intake, development, metabolic rate, and fat storage is best studied in mammals. In that group, leptin has pleiotropic effects including those on angiogenesis, bone formation, reproductive status, immune function, and energy expenditure. A homozygous mutation (ob-/ ob-) for leptin is the most common model for leptin study in mammals. The use of leptin-null mutants in non-mammal models is not common. We recently developed a leptin knockdown model in zebrafish and applied a comparative approach to studying some well-characterized mammalian leptin functions in this new system. I tested the impact of leptin expression on metabolism in the developing zebrafish embryo. Leptin knockdown reduced oxygen consumption most prominently during early development (24-48 hours post fertilization, hpf) whereas carbonic acid production was most significantly attenuated later in development (48-72 hpf). Cardiac output was significantly reduced in embryos with reduced leptin expression (leptin morphants); all of these effects could be rescued by co-injection of recombinant fish leptin. The second part of my research focused on the innate immune response. When presented with a bacterial challenge, leptin morphants had reduced macrophage respiratory burst activity and bacterial load clearance was unaffected 12 hours post infection (hpi). By 36 hpi, leptin morphants had significantly increased bacterial burden and reduced survival compared to control embryos. I then focused on the transcriptomic effects of reduced leptin A expression in the developing zebrafish embryo. Microarray analysis identified sensory and development pathways as the most signific (open full item for complete abstract)

Committee: Richard Londraville Dr (Advisor); Qin Liu Dr (Committee Member); Zhong-Hui Duan Dr (Committee Member); Ahmed Mustafa Dr (Committee Member); Brian Bagatto Dr (Committee Member) Subjects: Bioinformatics; Molecular Biology