Genetics and Genomics Commons^™

Chaperoning Ef Hands That Shape Calcium Response: Ncald, Hpca And S100b, Jingyi Zhang

Graduate School of Biomedical Sciences Theses and Dissertations

All organisms have an internal clock with a defined period between repetitions of activities. The period for circadian clock in human is 24.5 hours, while in mouse and rat, it is 23.5 hours. However, all organisms are forced to be in synchronization with their environment. A major environmental force that resets the internal clock to 24 hours is light. This phenomenon is defined as “light entrainment” or “phase-setting”. It is unclear how this entrainment process occurs. Studies from this laboratory indicate a role for two neuronal calcium sensor proteins: Neurocalcin  (NCALD) and S100B. For these two genes, mRNA as …

Characterization Of E-Cadherin Regulation In Response To Zeb1 Inhibition In Endometrial Cancer Cell Lines, Chidozie Paul Chukwu

Graduate School of Biomedical Sciences Theses and Dissertations

Epithelial to mesenchymal transition (EMT) is the process in which cells lose their epithelial structure during gastrulation. This process also affects the migration and movement of tumor cells and promotes invasion and metastases of endometrial carcinomas. Down-regulation of E-cadherin (CDH1) by transcription factors is the key target of EMT modulators and is achieved mainly by ZEB1 (zinc finger E-box binding homeobox 1). Current research looking at restoration of E-cadherin expression in vitro involves the use of small molecules such as histone deacetylase (HDAC) inhibitors and DNA methyltransferase inhibitors. Trichostatin A (TSA) and small interfering ribonucleic acid (siRNA) are tools that …

The Role Of The Expansion Segment 7 Of 25s Rrna During Oxidative Stress In Saccharomyces Cerevisiae, Ethan Gardner

Graduate School of Biomedical Sciences Theses and Dissertations

Translation is an essential process for protein expression in both eukaryotes and prokaryotes. Like any cellular process, translational factors are prone to damage when the cell is under stress. One common stressor that nearly all cells may experience is abnormal levels of reactive oxygen species (ROS). Damage caused by ROS has been associated with disease ranging from neurodegenerative impairments, to the aging process of cells. These oxygen radicals are capable of damaging a litany of molecules including nucleic acids, and molecular factors involved in translation. It has been shown that tRNA can be cleaved upon ROS-induced stress and these fragments …

Association Of Ifih1 And Pro-Inflammatory Mediators: Potential New Clues In Sle-Associated Pathogenesis, Melissa E. Munroe, Nathan Pezant, Michael A. Brown, Dustin A. Fife, Joel M. Guthridge, Jennifer A. Kelly, Graham Wiley, Patrick M. Gaffney, Judith A. James, Courtney G. Montgomery

College of Science & Mathematics Departmental Research

Antiviral defenses are inappropriately activated in systemic lupus erythematosus (SLE) and association between SLE and the antiviral helicase gene, IFIH1, is well established. We sought to extend the previously reported association of pathogenic soluble mediators and autoantibodies with mouse Mda5 to its human ortholog, IFIH1. To better understand the role this gene plays in human lupus, we assessed association of IFIH1 variants with soluble mediators and autoantibodies in 357 European-American SLE patients, first-degree relatives, and unrelated, unaffected healthy controls. Association between each of 135 genotyped SNPs in IFIH1 and four lupus-associated plasma mediators, IL-6, TNF-α, IFN-β, and IP-10, …

Time-Course Analysis Of Gene Expression During The Saccharomyces Cerevisiae Hypoxic Response, Nasrine Bendjilali, Samuel Macleon, Gurmannat Kalra, Stephen D. Willis, A.K.M. Nawshad Hossian, Erica Avery, Olivia Wojtowicz, Mark J. Hickman

College of Science & Mathematics Departmental Research

Many cells experience hypoxia, or low oxygen, and respond by dramatically altering gene expression. In the yeast Saccharomyces cerevisiae, genes that respond are required for many oxygen-dependent cellular processes, such as respiration, biosynthesis, and redox regulation. To more fully characterize the global response to hypoxia, we exposed yeast to hypoxic conditions, extracted RNA at different times, and performed RNA sequencing (RNA-seq) analysis. Time-course statistical analysis revealed hundreds of genes that changed expression by up to 550-fold. The genes responded with varying kinetics suggesting that multiple regulatory pathways are involved. We identified most known oxygen-regulated genes and also uncovered new …

Punctuated Evolution Within A Eurythermic Genus (Mesenchytraeus) Of Segmented Worms: Genetic Modification Of The Glacier Ice Worm F1f0 Atp Synthase, Shirley A. Lang

Graduate School of Biomedical Sciences Theses and Dissertations

Segmented worms (Annelida) are among the most successful animal inhabitants of extreme environments worldwide. An unusual group of Mesenchytraeus worms endemic to the Pacific Northwest of North America occupy geographically proximal ecozones ranging from low elevation temperate rainforests to high altitude glaciers. Along this altitudinal transect, Mesenchytraeus representatives from disparate habitat types were collected and subjected to deep mitochondrial and nuclear phylogenetic analyses. Evidence presented here employing modern bioinformatic analyses (i.e., maximum likelihood, Bayesian inference, multi-species coalescent) supports a Mesenchytraeus “explosion” in the upper Miocene (5-10 million years ago) that gave rise to ice, snow and terrestrial worms, derived from …

Replication-Transciption Switch In Human Mitochondria, Karen Agaronyan

Graduate School of Biomedical Sciences Theses and Dissertations

Coordinated replication and expression of mitochondrial genome is critical for metabolically active cells during various stages of development. However, it is not known whether replication and transcription can occur simultaneously without interfering with each other and whether mtDNA copy number can be regulated by the transcription machinery. Human mitochondrial RNA polymerase (mtRNAP) is a central enzyme involved in gene expression in mitochondria. It generates genome-size polycistronic transcripts and also makes replication primers at two origins of replication. MtRNAP is distantly related to phage T7 RNAP. While T7 RNAP is optimized to produce large amounts of transcripts to overcompete the bacterial …

De Novo Deciphering Three-Dimensional Chromatin Interaction And Topological Domains By Wavelet Transformation Of Epigenetic Profiles., Yong Chen, Yunfei Wang, Zhenyu Xuan, Min Chen, Michael Q Zhang

College of Science & Mathematics Departmental Research

Defining chromatin interaction frequencies and topological domains is a great challenge for the annotations of genome structures. Although the chromosome conformation capture (3C) and its derivative methods have been developed for exploring the global interactome, they are limited by high experimental complexity and costs. Here we describe a novel computational method, called CITD, for de novo prediction of the chromatin interaction map by integrating histone modification data. We used the public epigenomic data from human fibroblast IMR90 cell and embryonic stem cell (H1) to develop and test CITD, which can not only successfully reconstruct the chromatin interaction frequencies discovered by …

Assembly And Structure Of Human Mitochondrial Transcription Initiation Complex, Yaroslav Morozov

Graduate School of Biomedical Sciences Theses and Dissertations

Mitochondria play a key role in energy metabolism in nearly all eukaryotic cells. Apart from that, they participate in many other biochemical pathways, such as amino acid and nucleotide metabolism, and biosynthesis of cholesterol, heme, and iron-sulfur clusters. Mitochondria are also involved in cell signaling, apoptosis and regulation of cell cycle. Abnormalities of mitochondrial morphology and function are often associated with tumor progression, neurodegeneration and aging.

Mitochondria are unique organelles in mammalian cells because they have their own genomic DNA, transcription and translation systems. Various mutations and defects in mitochondrial gene expression lead to severe diseases that include various mitochondrial …

Climp: Clustering Motifs Via Maximal Cliques With Parallel Computing Design., Shaoqiang Zhang, Yong Chen

College of Science & Mathematics Departmental Research

A set of conserved binding sites recognized by a transcription factor is called a motif, which can be found by many applications of comparative genomics for identifying over-represented segments. Moreover, when numerous putative motifs are predicted from a collection of genome-wide data, their similarity data can be represented as a large graph, where these motifs are connected to one another. However, an efficient clustering algorithm is desired for clustering the motifs that belong to the same groups and separating the motifs that belong to different groups, or even deleting an amount of spurious ones. In this work, a new motif …

Investigating Evolutionary Dynamics Of Rha1 Operons, Yong Chen, Dandan Geng, Kristina Ehrhardt, Shaoqiang Zhang

College of Science & Mathematics Departmental Research

Grouping genes as operons is an important genomic feature of prokaryotic organisms. The comprehensive understanding of the operon organizations would be helpful to decipher transcriptional mechanisms, cellular pathways, and the evolutionary landscape of prokaryotic genomes. Although thousands of prokaryotes have been sequenced, genome-wide investigation of the evolutionary dynamics (division and recombination) of operons among these genomes remains unexplored. Here, we systematically analyzed the operon dynamics of Rhodococcus jostii RHA1 (RHA1), an oleaginous bacterium with high potential applications in biofuel, by comparing 340 prokaryotic genomes that were carefully selected from different genera. Interestingly, 99% of RHA1 operons were observed to exhibit …

Functional Analysis Of Rpl22: Coordinating Differentiation Signals And Developmental Gene Translation To Promote Cell Fate Decisions, Stephen Kim

Graduate School of Biomedical Sciences Theses and Dissertations

The execution of meiosis and spore formation in S. cerevisiae requires a transient transcription program that is initiated by Ime I-dependent destruction of the repressor Ume6. IMEJ transcription is tightly contro1led by both cell type and nutritional cues but how its translation is regulated has been unknown. This study demonstrates that Rpl22A and Rpl22B, eukaryotic-specific ribosomal protein paralogs of the 60S large subunit, are required for efficient Ime I translation. While IME1 mRNA is induced normally in the absence of both para logs following transfer to sporulation medium, Ume6 remains stabilized, downstream early meiotic gene remain repressed, and development is …

The Development Of A Platform For Expression And Purification Of The Mammalian Transmembrane Transporter Abca4, Amaryllis Ortiz

Graduate School of Biomedical Sciences Theses and Dissertations

Purpose- ABCA4 is an ATP Binding Cassette (ABC) protein localized at cone and rod photoreceptor outer segments of the retina. Though mutations in ABCA4 have been found to cause a broad spectrum of disorders, ABCA4's high molecular weight (~210 kDa) and biochemical transmembrane properties have made ABCA4 functional studies unsuccessful. Identifying how ABCA4 functions might have significant clinical impact and open unanticipated avenues for therapeutic interventions. Thus, the objective of this study was to develop a new expression system able to produce the full-length, correctly folded and fully functional ABCA4 protein.

Methods- the ABCA4 gene was amplified from the PCMV6-ABCA4 …

Hippocalcin Response To Calcium: Do Conserved Tryptophans – W30 Or W103 – Matter?, Sunkesula K. Sagar

Graduate School of Biomedical Sciences Theses and Dissertations

Changes in intracellular calcium levels play a very important role in cell signaling, in turn, affecting neuronal functions such as memory, learning and cell death. A class of proteins called Neuronal Calcium Sensor (NCS) proteins serves to modulate the functioning of the neuronal cells in response to changes in calcium levels, and prevent neuronal apoptosis. Structurally, all NCS proteins have 4 calcium-binding EF hand motifs, although EF1 does not bind to calcium in many members. All NCS proteins have an acyl modification at the N- terminus – where a myristoyl group is added post-translationally. Hippocalcin (HPCA) is an NCS protein, …

Conformational Dynamics Of Dnaa Protein Of Bacillus Anthracis In The Initiation Of Dna Replication: A Fluorescence Resonance Energy Transfer Study, Meera J. Patel

Graduate School of Biomedical Sciences Theses and Dissertations

A precursor of the origin recognition complex proteins in eukaryotes, the DnaA protein, a single-subunit protein found in all prokaryotes, p1ays a significant role in the initiation of DNA replication. The DnaA protein in Gram-negative Escherichia coli (DnaA_EC) identifies, binds and activates specific DNA sequences at the origin of replication, oriC, initiating the duplication of the prokaryotic genome. Using DnaA_EC as a guide, we selected the full-length form of DnaA from Gram-positive Bacillus anthracis (DnaA_BA) as our model for studying. We have cloned, mutated, expressed and purified large quantities of DnaA_BA protein due to …

Structural And Functional Interactions Between Bro1 Domain Of Human Alix Protein And Nucleocapsid Packaging Rna Complex From Hiv, Scott Gross

Graduate School of Biomedical Sciences Theses and Dissertations

A virus is only as powerful as its ability to spread. Enveloped retroviruses, namely HIV-1, use exocytosis pathways that normal host cells use to release particles from the plasma membrane. The main pathways of interest in this study are the Endosomal Sorting Complex Required for Transport (ESCRT) and adjacent ALIX pathways. The ESCRT pathway is especially important for degradation of receptor/cargo complexes that form Multi-Vesicular Bodies (MVBs). Currently, there is no known therapy that targets this endosomal pathway, which would prevent the spread of the virus to other cells. The virus has adapted to jump from pathway to pathway when …

Age-Dependent Breakdown Of The Blood Brain Barrier And Associated Changes In S100b Ko Mice, Eric V. Brown

Graduate School of Biomedical Sciences Theses and Dissertations

Autoantibodies play an important role in many autoimmune diseases. Recent research has shown that breakdown of the blood brain barrier (BBB) occurs concomitant to generation of brain reactive autoantibodies in many neurodegenerative diseases, which serve as biomarkers and drivers of pathology. SI00B, a calcium binding protein found most highly expressed in astrocytes which ensheathe the BBB, has many functions in neural development and signaling. Currently literature indicates that S100B KO mice develop normally, with no phenotypic abnormalities. Here, it is demonstrated that S100B KO mice seem to develop a chronic BBB breakdown similar to that seen in human neurodegenerative diseases. …

Evolution Of Sperm Activation In Nematodes, Qing Wei

Graduate School of Biomedical Sciences Theses and Dissertations

Three hermaphroditic species in the nematode genus Caenorhabditis - C. elegans, C. briggsae and C. tropicalis evolved independently from a male/female ancestor. Thus, the hermaphroditic reproductive system provides an ideal subject for studying the origin of new complex traits. Previous studies implied that this transition required two steps: a mutation that caused XY animals to make sperm, and a mutation that allowed XX spermatids to activate and fertilize oocytes. In this dissertation, I show how sperm activation is regulated in different hermaphroditic species.

First, I used TALENs to create genetic tools for C. briggsae and C. tropicalis. These tools were …

Genome Engineering To Create Dominant Alleles In Caenorhabditis Elegans Using Crispr-Cas9 Technology, Abrar Sulaimani

Graduate School of Biomedical Sciences Theses and Dissertations

Many investigators have being using CRISPR-Cas9 as a method of genome engineering because it is easy, accurate and fast. This technique has been used to modify the genomes of a wide variety of organisms, including the nematode Caenorhabditis elegans (C. elegans). The short life cycle and ease of introducing exogenous plasmids make C. elegans an ideal system for advancing this technique. My thesis had two aims that focused on developing methods to create dominant alleles in C. elegans. Genetic modifications like precise deletion and insertions into a locus of chromosome are technically challenging. Additionally, although there are several ways of …

Meiotic Checkpoint Activation Promotes Anaphase Promoting Complex/Cyclosome Substrate Specificity And Ume6/Sin3/Rpd3-Independent Reduction In Emg Expression, Rebecca Lewandowski

Graduate School of Biomedical Sciences Theses and Dissertations

Complex differentiation pathways, such as yeast meiosis and sporulation, are initiated in response to cell type and external stimuli and are driven by changes in tightly regulated and temporally expressed transcriptional programs. These programs are monitored by checkpoint mechanisms that couple meiotic progression to transcriptional activity to ensure each stage is successfully completed prior to progression into the next phase. This work investigated transcriptional regulation controlling meiotic progression when cells sense an insult to genetic integrity. These studies revealed that the early meiotic gene (EMG) repressor Ume6p is degraded during meiotic checkpoint activation triggered by the deoxyribonucleotide reductase inhibitor and …

Integrated Transcriptome Analysis Reveals Mirna-Mrna Crosstalk In Laryngeal Squamous Cell Carcinoma., Yang Zhang, Yong Chen, Jinhai Yu, Guiming Liu, Zhigang Huang

College of Science & Mathematics Departmental Research

Next generation sequencing (NGS) has proven to be a powerful tool in delineating myriads of molecular subtypes of cancer, as well as in revealing accumulation of genomic mutations throughout cancer progression. Whole genome microRNA (miRNA) and mRNA expression profiles were obtained from patients with laryngeal squamous cell carcinoma (LSCC) using deep sequencing technology, and were analyzed by utilizing integrative computational approaches. A large number of protein-coding and non-coding genes were detected to be differentially expressed, indicating a functional switch in LSCC cells. A total of 127 mutated genes were detected to be significantly associated with ectoderm and epidermis development. Eleven …

Nack Is An Integral Component Of The Notch Transcriptional Activation Complex And Is Critical For Development And Tumorigenesis, Kelly L Weaver, Marie-Clotilde Alves-Guerra, Ke Jin, Zhiqiang Wang, Xiaoqing Han, Prathibha Ranganathan, Xiaoxia Zhu, Thiago Dasilva, Wei Liu, Francesca Ratti, Renee M Demarest, Cristos Tzimas, Meghan Rice, Rodrigo Vasquez-Del Carpio, Nadia Dahmane, David J Robbins, Anthony J Capobianco

Rowan-Virtua School of Osteopathic Medicine Departmental Research

The Notch signaling pathway governs many distinct cellular processes by regulating transcriptional programs. The transcriptional response initiated by Notch is highly cell context dependent, indicating that multiple factors influence Notch target gene selection and activity. However, the mechanism by which Notch drives target gene transcription is not well understood. Herein, we identify and characterize a novel Notch-interacting protein, Notch activation complex kinase (NACK), which acts as a Notch transcriptional coactivator. We show that NACK associates with the Notch transcriptional activation complex on DNA, mediates Notch transcriptional activity, and is required for Notch-mediated tumorigenesis. We demonstrate that Notch1 and NACK are …

Regulation Of Strand-Specific Processing In Human Pre-Mir-371 And Its Mouse Homologs, Jianting Shi

Graduate School of Biomedical Sciences Theses and Dissertations

MicroRNAs (miRNAs) are small noncoding RNAs, known to regulate genes by inducing target mRNA degradation or translational repression. This regulatory function has been shown to assist developmental timing control and increasingly more evidence reveals some of the miRNAs may have tissue/cell type-specific function, showing the potential to assist cell differentiation. Mouse miR-290-295 cluster is embryonic stem cell (ESCs)-specific miRNA cluster and human miR-371-373 cluster has been verified to be its functional homolog. We have previously demonstrated that human pre-miR-371 is processed into multiple miRNA species, which represent the seeds of the mouse of miR-290-5p, miR-292-5p, miR-292-3p-iso2 and miR-293-3p. Deep sequencing …

The Development Of A Comprehensive Antifungal Susceptibility Testing Assay For Vulvovaginal Candidiasis Therapy, David Hardaker

Graduate School of Biomedical Sciences Theses and Dissertations

Vulvovaginal candidiasis is the most common fungal infection of the female urogenital tract, commonly caused by Candida albicans and C. glabrata. However, treatment can be difficult when caused by non-albicans strains due to resistance to the oral antifungal drug fluconazole. The mechanism through which strains of Candida, particularly C. glabrata, develop resistance to different antifungal classes has not been completely characterized. The ergosterol biosynthesis pathway, an important component of the cell membrane, is the major target of antifungals such as fluconazole, an inhibitor of the ERG 11 gene. Current research shows that an upregulation of specific genes in C. glabrata …

Structural And Functional Analysis Of Harv1 Lipid Binding, Jamie Francisco

Graduate School of Biomedical Sciences Theses and Dissertations

Cardiovascular disease (CVD) has the highest mortality rate worldwide and is considered epidemic in the United States. Statins as the primary and almost sole method of treatment for cholesterol-related CVD, but they have limitations. Proper sterol transport is needed to maintain normal blood lipid levels. Studies have shown that misregulation of lipid trafficking aids in the progression of CVD and in the activation of the unfolded protein response (UPR). Misregulation of lipid and sterol homeostasis also results in the loss of lipid rafts, known cellular signaling hubs and major components of ER associated membrane contact sites. Arv1 is a highly …

Prioritizing Protein Complexes Implicated In Human Diseases By Network Optimization., Yong Chen, Thibault Jacquemin, Shuyan Zhang, Rui Jiang

College of Science & Mathematics Departmental Research

BACKGROUND: The detection of associations between protein complexes and human inherited diseases is of great importance in understanding mechanisms of diseases. Dysfunctions of a protein complex are usually defined by its member disturbance and consequently result in certain diseases. Although individual disease proteins have been widely predicted, computational methods are still absent for systematically investigating disease-related protein complexes.

RESULTS: We propose a method, MAXCOM, for the prioritization of candidate protein complexes. MAXCOM performs a maximum information flow algorithm to optimize relationships between a query disease and candidate protein complexes through a heterogeneous network that is constructed by combining protein-protein interactions …

Integrated Omics Study Delineates The Dynamics Of Lipid Droplets In Rhodococcus Opacus Pd630., Yong Chen, Yunfeng Ding, Li Yang, Jinhai Yu, Guiming Liu, Xumin Wang, Shuyan Zhang, Dan Yu, Lai Song, Hangxiao Zhang, Congyan Zhang, Linhe Huo, Chaoxing Huo, Yang Wang, Yalan Du, Huina Zhang, Peng Zhang, Huimin Na, Shimeng Xu, Yaxin Zhu, Zhensheng Xie, Tong He, Yue Zhang, Guoliang Wang, Zhonghua Fan, Fuquan Yang, Honglei Liu, Xiaowo Wang, Xuegong Zhang, Michael Q Zhang, Yanda Li, Alexander Steinbüchel, Toyoshi Fujimoto, Simon Cichello, Jun Yu, Pingsheng Liu

College of Science & Mathematics Departmental Research

Rhodococcus opacus strain PD630 (R. opacus PD630), is an oleaginous bacterium, and also is one of few prokaryotic organisms that contain lipid droplets (LDs). LD is an important organelle for lipid storage but also intercellular communication regarding energy metabolism, and yet is a poorly understood cellular organelle. To understand the dynamics of LD using a simple model organism, we conducted a series of comprehensive omics studies of R. opacus PD630 including complete genome, transcriptome and proteome analysis. The genome of R. opacus PD630 encodes 8947 genes that are significantly enriched in the lipid transport, synthesis and metabolic, indicating a super …

Identifying Potential Cancer Driver Genes By Genomic Data Integration., Yong Chen, Jingjing Hao, Wei Jiang, Tong He, Xuegong Zhang, Tao Jiang, Rui Jiang

College of Science & Mathematics Departmental Research

Cancer is a genomic disease associated with a plethora of gene mutations resulting in a loss of control over vital cellular functions. Among these mutated genes, driver genes are defined as being causally linked to oncogenesis, while passenger genes are thought to be irrelevant for cancer development. With increasing numbers of large-scale genomic datasets available, integrating these genomic data to identify driver genes from aberration regions of cancer genomes becomes an important goal of cancer genome analysis and investigations into mechanisms responsible for cancer development. A computational method, MAXDRIVER, is proposed here to identify potential driver genes on the basis …

Integrating Human Omics Data To Prioritize Candidate Genes., Yong Chen, Xuebing Wu, Rui Jiang

College of Science & Mathematics Departmental Research

BACKGROUND: The identification of genes involved in human complex diseases remains a great challenge in computational systems biology. Although methods have been developed to use disease phenotypic similarities with a protein-protein interaction network for the prioritization of candidate genes, other valuable omics data sources have been largely overlooked in these methods.

METHODS: With this understanding, we proposed a method called BRIDGE to prioritize candidate genes by integrating disease phenotypic similarities with such omics data as protein-protein interactions, gene sequence similarities, gene expression patterns, gene ontology annotations, and gene pathway memberships. BRIDGE utilizes a multiple regression model with lasso penalty to …

Bacterial Transcription Factors Grea, Greb And Dksa: Characterization Of Their Interaction With Rna Polymerase And Molecular Mechanism Of Action, Andrey Parshin

Graduate School of Biomedical Sciences Theses and Dissertations

The activity of RNA polymerase (RNAP), the key enzyme of transcription process, is regulated by a large number of transcription factors acting at each step of the transcription cycle. Our research project is focused on studying the structure and function of bacterial transcript cleavage factors GreA, GreB and the stringent response regulator DksA. Gre factors bind in the secondary channel of RNAP in backtracked (inactive) ternary elongation complexes and stimulate the enzyme's intrinsic RNase activity, which is required for suppression of transcription pause and arrest, enhancement of transcription fidelity, and efficient promoter escape. DksA also acts through the RNAP secondary …