Molecular Biology Commons^™

Elucidating The Molecular Parameters And Mechanism Of Homology Searching During Meiosis In Neurospora Crassa, Nicholas Adam Rhoades

Theses and Dissertations

Integration of foreign DNA into a host genome is often detrimental to the host organism, as the DNA is often of viral or transposon origin. Many organisms have established DNA surveillance and genome integrity mechanisms to defend against these harmful DNA insertions. Neurospora crassa, a filamentous fungus belonging to the Ascomycota group, has several DNA defense mechanisms to combat foreign DNA integration to its genome. One such mechanism is Meiotic Silencing of Unpaired DNA (MSUD). During meiosis, this remarkable system can detect unpaired genes on homologous chromosomes during sexual reproduction and silence their expression throughout meiosis. In order for the …

Identification Of Novel Nuclear Proteins Required For Meiotic Silencing By Unpaired Dna In Neurospora Crassa, Dilini Ralalage

Theses and Dissertations

A fundamental step that occurs during sexual reproduction is meiosis, which is a specialized type of cell division. During meiosis, pairs of chromosomes exchange genetic information via recombination. At this point, the genome is particularly susceptible to viruses and other foreign genetic invasions. Therefore, it is important to protect the genome to prevent the transmission of foreign genetic materials to the offspring. There are several mechanisms work together to protect host genome from foreign genetic materials. These are known as “genome defense mechanisms”.

The fungus Neurospora crassa is one of the best organisms for genome defense studies due to the …

Altered Na, K- Atpase Isoform Expression In Artemia Franciscana In Response To Hypersaline Environments, Jessica Drenth

Theses and Dissertations

The Na,K-ATPase (NKA) is an essential membrane pump that helps to establish cell ion gradients, and regulate intracellular salt in many organisms. One such species, Artemia franciscana (brine shrimp), extreme halophiles which live in hypersaline environments, expresses 2 distinct α-catalytic subunits of the NKA. One of these subunits, α2-(KK), has two key lysine substitutions located within the cation binding sites. Prior work has demonstrated this specific subunit may be directly involved in brine shrimp adaptation to their extreme environments. However, the precise molecular and physiological effects of α2-(KK) have not been entirely elucidated. I determined through immunohistochemistry that my initial …

Construction And Comparison Of The Recombinant Strains Of Synechocystis Sp. Pcc 6803 Able To Grow On Wood Sugars, Saurabh Jayant Ranade

Theses and Dissertations

The wood sugars present in the lignocellulosic biomass can be used to support microbial growth to synthesize a number of value added products. The objective of this research was to metabolically engineer model cyanobacterium Synechocystis to enable it to harvest carbon and energy stored in the wood sugars in addition to the light energy. In the xylose-specific work, we generated five Synechocystis strains that heterologously expressed XylAB enzymes, which mediate xylose catabolism, either in combination with or without one of four xylose transporters, namely XylE, XylFGH, GalP, or Glf. In terms of dry biomass accumulation and xylose consumption, the strains …

Nitrosative Stress Sensing In Porphyromonas Gingivalis: Structure And Function Of The Heme Binding Transcriptional Regulator Hcpr, Benjamin R. Belvin

Theses and Dissertations

Porphyromonas gingivalis, a Gram negative anaerobe implicated in the progression of periodontal disease, is capable of surviving and causing infection despite high levels of reactive nitrogen species found in the oral cavity due to its efficient nitrosative stress response. HcpR is an important sensor-regulator that plays a vital step in the initiation of the nitrosative stress response in many Gram negative anaerobic bacteria. We employ a combination of X-ray crystallography, SAXS, resonance Raman spectroscopy, UV-Vis spectroscopy, and molecular biology techniques to better understand this key regulator. Knockout of the hcpR gene in W83 P. gingivalis results in the inability of …

Characterization Of Staphylococcal Nuclease And Tudor Domain Containing Protein 1 (Snd1) As A Molecular Target In Hepatocellular Carcinoma And Non-Alcoholic Steatohepatitis, Nidhi H. Jariwala

Theses and Dissertations

CHARACTERIZATION OF STAPHYLOCOCCAL NUCLEASE AND TUDOR DOMAIN CONTAINING PROTEIN 1 (SND1) AS A MOLECULAR TARGET IN HEPATOCELLULAR CARCINOMA AND NON-ALCOHOLIC STEATOHEPATITIS

Nidhi Jariwala, PhD

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Integrative Life Sciences

Virginia Commonwealth University, 2017

Devanand Sarkar, M.B.B.S., PhD.

Associate Professor, Department of Human and Molecular Genetics

Virginia Commonwealth University

Richmond, Virginia

SND1, a subunit of the miRNA regulatory complex RISC, has been implicated as an oncogene in hepatocellular carcinoma (HCC). Oncoprotein SND1 regulates gene expression at a post-transcriptional level in multiple cancers including hepatocellular carcinoma (HCC). …

Sh3 And Multiple Ankyrin Repeat Domain 3 (Shank3) Affects The Expression Of Hyperpolarization-Activated Cyclic Nucleotide-Gated (Hcn) Channels In Mouse Models Of Autism, Nikhil N. Shah

Theses and Dissertations

SH3 and multiple ankyrin repeat domains 3 (SHANK3) is a multidomain scaffold protein that is highly augmented in the postsynaptic density (PSD) of excitatory glutamatergic synapses within the central and peripheral nervous systems. SHANK3 links neurotransmitter receptors, ion channels, and other critical membrane proteins to intracellular cytoskeleton and signal transduction pathways. Mutations in SHANK3 are linked with a number neuropsychiatric disorders including autism spectrum disorders (ASDs). Intellectual disability, impaired memory and learning, and epilepsy are some of the deficits commonly associated with ASDs that result from mutations in SHANK3. Interestingly, these symptoms show some clinical overlap with presentations of human …

Epigenetic Editing To Validate Findings From Methylome-Wide Association Studies Of Neuropsychiatric Disorders, Robin F. Chan

Theses and Dissertations

DNA methylation is necessary for learning, memory consolidation and has been implicated in a number of neuropsychiatric disorders. Obtaining high quality and comprehensive data for the three common forms of methylation in brain is challenging for methylome-wide association studies (MWAS). To address this we optimized a panel of enrichment methods for screening the brain methylome. Results show that these enrichment techniques approach the coverage and fidelity of the current gold standard bisulfite based techniques. Our MBD-based method can also be used with low amounts of genomic material from limited human biomaterials. Psychiatric disorders have high prevalence and are often chronic …

Relb Acts As A Molecular Switch To Drive Chronic Inflammation In Glioblastoma Multiforme (Gbm)., Michael R. Waters

Theses and Dissertations

Inflammation is a homeostatic response to tissue injury or infection, which is normally short- lived and quickly resolves to limit tissue damage. In contrast, chronic inflammation has been linked to a variety of human diseases, including cancers such as glioblastoma multiforme (GBM). GBMs are very aggressive tumors with very low patient survival rates, which have not improved in several decades. GBM tumors are characterized by necrosis and profound inflammation; with cytokines secreted by both GBM cells and the tumor microenvironment. The mechanisms by which chronic inflammation develops and persists in GBM regardless of multiple anti-inflammatory feedback loops remain elusive. This …

Biochemical Analysis Of Putative Single-Stranded Nucleic Acid Binding Proteins In Porphyromonas Gingivalis, Steve H. Kokorelis

Theses and Dissertations

Proteins that bind to both DNA and RNA embody the ability to perform multiple functions by a single gene product. These nucleic acid binding proteins in prokaryotes can play a vital role in many cellular processes, including replication, transcription, gene expression, recombination, and repair, to name a few. Nucleic acid binding proteins have unique functional characteristics that stem from their structural attributes that have evolved in a widely-conserved manner. In Escherichia coli (E. coli), the highly-conserved histone-like protein, HU, which predominates as a heterodimer of HUα and HUβ, has been found to bind to both dsDNA and ssDNA. …

Membrane Biophysics Of Listeria Monocytogenes: Analysis Of An Alternate Pathway Of Branched-Chain Fatty Acid Biosynthesis And Elasticity Of Fatty Acid Utilization, Laura E. Kuczek

Theses and Dissertations

The membrane homeostasis of the psychrotolerant foodborne pathogen Listeria monocytogenes involves maintaining fluidity in a range of different conditions by incorporation of specific branched-chain fatty acids (BCFAs). BCFAs are synthesized through a well-characterized pathway from branched-chain amino acids into short branched-chain acyl-CoAs followed by elongation by the FAS II system. Branched-chain alpha-keto acid dehydrogenase (Bkd) is a major player in this pathway. MOR401 is a transposon insertion mutant deficient in Bkd and has decreased membrane BFCAs. Low levels of BCFAs in L. monocytogenes are linked to diminished growth, less resistance to antimicrobials, and a severe reduction in virulence. Rescued growth …

Alternative Pathway For Provision Of Acyl Coa Precursors For Fatty Acid Biosynthesis: Purification And Kinetic Characterization Of Phosphotransbutyrylase And Butyrate Kinase From Listeria Monocytogenes, Sirisha Sirobhushanam

Theses and Dissertations

Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a disease characterized by gastroenteritis, meningitis, spontaneous miscarriages and high mortality rate among infected individuals. L. monocytogenes is a major concern in the food industry, due to its ability to grow at refrigeration temperatures and the zero tolerance policy of the FDA, resulting in expensive food recalls. Growth at low temperatures is aided in part by the high membrane content of branched-chain fatty acids (BCFAs) which imparts greater fluidity to the membrane. Mutants of L. monocytogenes impaired in BCFA biosynthesis display diminished growth at normal and low temperatures, exhibit lower tolerance …

Investigating The Roles And Interactions Of Sad-6 Within The Parameters Of Meiotic Silencing By Unpaired Dna ( Msud )., Zachary J. Smith

Theses and Dissertations

Meiotic silencing by unpaired DNA (MSUD) is a process observed in the model organism Neurospora crassa. During this process unpaired DNA between homologous chromosomes is detected and silenced, resulting in the suppression of unpaired genes. The effects of MSUD can be seen using phenotypic markers such as the Roundspore gene and evidence supports the existence of a physical search for unpaired DNA. However, the mechanism for detecting unpaired DNA remains uncertain. Previously, we have shown evidence that a Rad54-like protein, SAD-6 is required for the efficient completion of MSUD and may be necessary for the detection of unpaired DNA. Currently, …

Investigation Of Anaplasma Phagocytophilum And Anaplasma Marginale Adhesin-Host Cell Interactions, Kathryn S. Hebert

Theses and Dissertations

Anaplasma phagocytophilum and A. marginale are the etiologic agents of bovine anaplasmosis and human granulocytic anaplasmosis, respectively. As obligate intracellular pathogens, binding and entry of host cells is a prerequisite for survival. The molecular events associated with these processes are poorly understood. Identifying the adhesins mediating binding, delineating their key functional domains, and determining the molecular determinants to which they bind not only benefits better understanding of Anaplasma spp. pathobiology, but could also benefit the development of novel approaches for protecting against infection. We previously demonstrated that A. phagocytophilum outer membrane protein A (ApOmpA) is critical for bacterial binding and …

Investigating The Mechanism Of The Compartmentalized Cbp (Creb-Binding Protein) Ubiquitin Ligase Activities, Oluwatoyin E. Akande

Theses and Dissertations

CBP (CREB Binding Protein) is global transcriptional co-activator and histone acetyltransferase. CBP is involved in the modulation of the transcription of many genes via histone acetylation at the promoter regions of the target genes. Also, non-histone proteins and transcription factors can be acetylated by CBP to promote their transcriptional activation. In addition to its transcription co-activator role, CBP is involved in many other pathological and physiological cellular processes such as cell growth and differentiation, cell transformation and development, response to stress, cell cycle regulation and apoptosis.

CBP and its paralogue p300, play double-edged roles in the regulation of p53, a …

Conditional Cardiac-Specific Akap13 Knockout Induces Sex Dependent Biventricular Dilated Cardiomyopathy With Sarcomeric And Mitochondrial Defects, Kimberlyn M. Baig-Ward

Theses and Dissertations

Heart disease is a complex and heterogeneous disease. Notably, studies have demonstrated gender differences in the expression and types of cardiovascular disease, such as dilated cardiomyopathy (DCM), a major underlying cause of heart failure. Previously we showed that loss of A-Kinase Anchoring Protein 13 (Akap13), a unique proto-oncogene and estrogen receptor modulator, resulted in enlarged embryonic hearts, defective cardiac sarcomere formation, and embryonic lethality in mice. Data have also shown cAMP-dependent Protein Kinase A (PKA) to be involved in DCM pathophysiology. Given the established role of AKAP13 in cell signaling, its ability to bind and modulate ligand-activated nuclear hormone …

Role Of Vav2 In Podocyte Inflammasome Activation And Glomerular Injury During Hyperhomocysteinemia, Sabena Conley

Theses and Dissertations

Hyperhomocysteinemia (hHcys) is a widely known pathogenic factor in the progression of end-stage renal disease (ESRD) and it is also associated with an increased risk for injurious cardiovascular pathologies during ESRD. HHcys is linked to the formation and activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, characterized as a critical early mechanism initiating the inflammatory response. NADPH oxidase (NOX)-derived reactive oxygen species (ROS) mediate the activation of the NLRP3 inflammasome in podocytes in response to elevated levels of homocysteine (Hcys) in vitro and in vivo. However, it remains unknown how NLRP3 inflammasome activation is triggered by NOX. The …

Atm Phosphorylates Subunit A Of Pp2a Resulting In Its Nuclear Export And Spatiotemporal Regulation Of The Dna Damage Response, Amrita D. Sule

Theses and Dissertations

Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and major regulator of the DNA damage response (DDR). One critical ATM target is protein phosphatase 2A (PP2A) known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylation of many proteins during the recovery from the DDR while returning the cell to normalcy. Interestingly, ATM and PP2A are known to form an auto-regulatory yin-yang kinase-phosphatase relationship. Herein, we show that the phosphorylation of the PP2A-Aα structural subunit at S401 by ATM results in nuclear export, which regulates the DDR at multiple levels and affects genomic …

Biophysical Properties Of Cellular Membranes In Gram-Positive Bacterial Pathogens And Their Impact On Major Physiological Attributes And Virulence Determinants, Suranjana Sen

Theses and Dissertations

The cytoplasmic membrane of bacterial cells, forming an essential barrier from the surroundings, is a critical component of cellular physiology ensuring proper survival and maintenance of major cellular functions. The integrity of the membrane is an important feature that plays an essential role in the transport of solutes and nutrients through active and passive pathways, functions of membrane-associated proteins, electron transport and ATP synthesis, maintaining turgor pressure and combating environmental stresses, and thus is a crucial factor of a majority of cellular adaptations. The various biophysical properties affecting the integrity of this membrane are mainly determined by the composition and …

Genomic Deoxyuracil: Targeting, Regulation And Repair, Nate Warren Holton

Theses and Dissertations

Maintaining genomic integrity is critical to avoid sequence mutations and genetic disease. However the immune system requires genomic rearrangements in order to produce novel antibodies and illicit a full immunological response. The expression of DNA modifying enzymes is essential for antibody production but the off target effects are not well understood. The work presented here was performed to elucidate molecular mechanisms responsible for the regulation and targeting of DNA editing enzymes, as well as characterize repair of these lesions at off target loci.

Cell Wall Mutants In Arabidopsis Thaliana, Christy Jane Moore

Theses and Dissertations

Plant cell walls are versatile structures, playing important roles in communication, defense, organization and support. The importance of each of these functions varies by cell type, with specialized cells often utilizing one or two functions more than others. Trichomes, or leaf hairs, and hypocotyl cells for instance, exhibit distinct cell wall characteristics. Trichomes have developed very thick cell walls with several raised structures, known as papillae, on their surfaces. It is believed that these cells function in defense against predators, making it difficult to crawl on the leaf surface, and in protection against ultra violet radiation, through refraction of light …

G-Quadruplex Dna Structures And Site Specific Genetic Instability, Jonathan David Williams

Theses and Dissertations

Repetitive DNA comprises a majority of the human genome yet functions and overall impacts on site-specific genetic instability are not fully defined. Repetitive G-rich sequences have the propensity to form G-quadruplex (G4), which are stable non-B form DNA structures. G4 structures are conspicuously found at regions of site-specific instability. Even so, human genomic loci capable of forming this structure and their connection to DNA rearrangements are just beginning to be elucidated. My dissertation focuses on G4 structures and their capacity to promote site-specific changes in the human genome, particularly at oncogenes. I identified and investigated new biologically relevant G4 loci …

Elucidation Of A Novel Pathway In Staphylococcus Aureus: The Essential Site-Specific Processing Of Ribosomal Protein L27, Erin A. Wall

Theses and Dissertations

Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center where it interacts with both A-site and P-site tRNAs as well as with 23S rRNA. We observed that L27 in S. aureus and other Firmicutes is encoded with a short N-terminal extension that is not present in most Gram-negative organisms, and is absent from mature ribosomes. The extension contains a conserved cleavage motif; nine N-terminal amino acids are …

Mysteries Of The Trypanosomatid Maxicircles: Characterization Of The Maxicircle Genomes And The Evolution Of Rna Editing In The Order Kinetoplastida, Preethi Ranganathan Iyengar

Theses and Dissertations

The trypanosomatid protists belonging to Order Kinetoplastida are some of the most successful parasites ever known to mankind. Their extreme physiological diversity and adaptability to different environmental conditions and host systems make them some of the most widespread parasites, causing deadly diseases in humans and other vertebrates.

This project focuses on their unique mitochondrion, called the kinetoplast, and more specifically involves the characterization of a part of their mitochondrial DNA (also called kinetoplast DNA or kDNA), the maxicircles, which are functional homologs of eukaryotic mitochondrial DNA in the kinetoplastid protists. We have sequenced and characterized the maxicircle genomes of 20 …

Characterization Of A Novel Protease In Staphylococcus Aureus, Adam L. Johnson

Theses and Dissertations

A newly discovered cysteine protease, Prp, has been shown to perform an essential, site-specific cleavage of ribosomal protein L27 in Staphylococcus aureus. In Firmicutes and related bacteria, ribosomal protein L27 is encoded with a conserved N-terminal extension that must be removed to expose residues critical for ribosome function. Uncleavable and pre-cleaved variants were unable to complement an L27 deletion in S. aureus, indicating that this N-terminal processing event is essential and likely plays an important regulatory role. The gene encoding the responsible protease (prp) has been shown to be essential, and is found in all organisms …

Ex Vivo Dna Cloning, Adam B. Fisher

Theses and Dissertations

Genetic engineering of microbes has developed rapidly along with our ability to synthesize DNA de novo. Yet, even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. While technological advances have resulted in powerful techniques for in vitro and in vivo assembly of DNA, each suffers inherent disadvantages. Here, an ex vivo DNA cloning suite using crude cellular lysates derived from E. coli is demonstrated to amplify and assemble DNA containing small sequence homologies. Further, the advantages of an ex vivo approach are …

Functional Characterization Of Rai1 In Zebrafish, Joshua S. Beach

Theses and Dissertations

Smith-Magenis Syndrome (SMS; OMIM #182290) is a multiple congenital abnormality and intellectual disability (ID) disorder caused by either an interstitial deletion of the 17p11.2 region containing the retinoic acid induced-1 (RAI1) gene or a mutation of the RAI1 gene. Individuals diagnosed with SMS typically present characteristics such as ID, self-injurious behavior, sleep disturbance, ocular and otolaryngological abnormalities, craniofacial and skeletal abnormalities, neurological and behavioral abnormalities, as well as other systemic defects and manifestations. Previous work by Vyas in 2009 showed temporal expression of rai1 in zebrafish embryos as early as 9 hpf. We hypothesize that there is maternal …

Rheb Dynamics On Lysosomal Membranes Determines Mtorc1 Activity After Loss Of P53 Or Activation Of Ampk, Catherine M. Bell

Theses and Dissertations

The tumor suppressor TP53 is the most frequently altered gene in human cancers. The growth-promoting complex, mTORC1 plays a part of the oncogenic profile caused by dysfunctional p53. mTORC1 sits downstream of AMPK and other crucial tumor suppressors/oncogenes, PTEN, LKB1, and Akt. The antifolate pemetrexed was found by this laboratory to activate AMPK via the inhibition of the enzyme AICART in de novo purine synthesis. This work presents a mechanism of mTORC1 activation with p53 loss, as well as of mTORC1 inhibition by pemetrexed-induced AMPK. We have found that mTORC1 activity was substantially upregulated by the loss …

Dysregulation Of Micrornas In Blood As Biomarkers For Diagnosing Prostate Cancer, Rhonda W. Daniel

Theses and Dissertations

Prostate cancer is the most common noncutaneous cancer among men, yet current diagnostic methods are insufficient and more reliable diagnostic markers need to be developed. The answer that can bridge this gap and enable more efficient diagnoses may lie in microRNAs. These small, single stranded RNA molecules impact protein expression at the translational level and regulate important cellular pathways. Dysregulation of these small RNA molecules can have tumorigenic effects on cells and lead to many types of cancers.

Currently the Prostate-Stimulating Antigen (PSA) is used as a diagnostic marker for prostate cancer. However, many factors can elevate PSA levels such …

Hif-Independent Responses In Hypoxia, Divya Padmanabha

Theses and Dissertations

The adaptive response to hypoxia is accompanied by widespread transcriptional changes that allow for prolonged survival in low oxygen. Many of these changes are directly regulated by the conserved hypoxia-inducible factor-1 (HIF-1) complex; however, even in its absence, many oxygen-sensitive transcripts in Caenorhabditis elegans are appropriately regulated in hypoxia. To identify mediators of these non-HIF-dependent responses, I established a hif-1 mutant reporter line that expresses GFP in hypoxia or when worms are treated with the hypoxia mimetic cobalt chloride (cobalt chloride). The reporter is selective and HIF-independent, in that it remains insensitive to a number of cellular stresses, but is …