Molecular Biology Commons^™

The Importance Of Protein Context In Spinocerebellar Ataxia Type 3, Sean Luis Johnson

Wayne State University Dissertations

Spinocerebellar Ataxia Type 3 (SCA3) is a member of the family of polyglutamine (polyQ) neurodegenerative disorders that includes Huntington's Disease and several other SCAs. SCA3, the most common dominant ataxia in the world, is caused by polyQ tract expansion in the protein, ataxin-3. How SCA3 occurs and how to treat it remain unresolved issues. The primary culprit of toxicity in all polyQ diseases is the glutamine repeat: its abnormal expansion leads to neuronal dysfunction and death. With that said, there is indisputable evidence that the way polyQ-dependent toxicity presents—areas impacted, cellular processes perturbed—is predicated in large part on regions outside …

Novel Role Of Endoplasmic Reticulum-Associated Degradation In The Regulation Of Ceruloplasmin, Stephen William Hippleheuser

Wayne State University Theses

Ceruloplasmin (Cp) is a secreted ferroxidase produced by the hepatocytes that assists in the transport of iron throughout the human body. In human aceruloplasminemia patients, due to Cp deficiency excess intracellular iron buildup leads to ailments like liver cirrhosis, neurodegeneration, and blindness. We recently found that the biogenesis of Cp in the endoplasmic reticulum (ER) is regulated by a principal ER quality-control process, ER-associated degradation (ERAD). ERAD clears misfolded ER proteins for cytosolic proteasomal degradation, with the Sel-1 suppressor of lin-12-like (Sel1L)-HMG-CoA reductase degradation 1 (Hrd1) protein complex representing the most conserved branch in mammals. Interestingly, we found that Sel1L-Hrd1 …

Termination-Independent Role Of Rat1 In Cotranscriptional Splicing In Budding Yeast, Zuzer Hakimuddin Dhoondia

Wayne State University Dissertations

Rat1 is a 5′→3′ exoribonuclease in budding yeast belonging to the XRN-family of nucleases. It is a highly conserved protein with homologs being present in fission yeast, flies, worms, mice and humans. Rat1 and its homolog in metazoan have been shown to function in multiple facets of RNA metabolism. In this study, we report a novel role of Rat1 in splicing of pre-mRNA in budding yeast. In the absence of the functional Rat1 in the nucleus, an increase in the level of unspliced transcripts was observed in yeast cells. Strand-specific TRO analysis revealed that the accumulation of unspliced transcripts upon …

Novel Insights Into The Critical Role Of Cardiolipin In Cellular Metabolism And Mitochondrial Physiology, Jiajia Ji

Wayne State University Dissertations

Cardiolipin (CL) is the signature phospholipid of mitochondria. CL and its remodeling exert critical roles in biological processes both inside and outside of mitochondria. CL abnormalities have been associated with various mitochondrial disorders and aging. Understanding the role of CL in mitochondrial physiology and cellular metabolism could provide valuable insights into cell biology and human health. Several metabolic alterations have been reported in CL-deficient cells, including accumulated lactate, decreased PDH activity, and decreased TCA cycle function. This dissertation connected these findings by showing abnormal NAD+ metabolism in various models lacking CL. Importantly, it shows that NAD+ supplementation improves mitochondrial function …

Perturbation Of Energy Metabolism At The Center Of The Mechanism Of Action Of Valproate, Michael Ghassan Salsaa

Wayne State University Dissertations

Bipolar disorder (BD) is a common and debilitating psychiatric disorder. Valproic acid (VPA) is one of the major drugs used to treat BD patients. However, it is not universally effective and, in addition, causes severe side effects. Its mechanism of action is not known, which complicates efforts to develop more effective drugs. Studies have established that VPA perturbs metabolism, which is implicated in both the therapeutic mechanism of action of the drug as well as drug toxicity. However, the mechanism whereby VPA causes these perturbations is not understood. To address this knowledge gap, I investigated the acute and chronic effects …

Identification Of Oxygen Optima For Mouse Trophoblast Stem Cells And Human Embryos And The Stress Responses Upon Departing Optima, Yu Yang

Wayne State University Dissertations

Low level of oxygen (O2) occurs physiologically during in vivo embryo development. As developing embryos moving from fallopian tube to uterus, oxygen level gradually decreases to ≤ 5% at the time of blastocyst implantation. Blastocysts are made of two major cell populations, trophoblast cells and inner cell mass, from which trophoblast stem cells (TSCs) and embryonic stem cells (ESCs) are derived respectively. TSCs serve as placental stem cells that later on proliferate and differentiate into placenta. Previous study has shown that 2% O2 is the optimal O2 level for mTSC in vitro growth and potency maintenance, which agrees with their …

Navigating Human Cytomegalovirus (Hcmv) Envelopment And Egress, William Longeway Close

Wayne State University Dissertations

Human cytomegalovirus (HCMV) is a ubiquitous viral pathogen. In individuals with fully functioning and mature immune systems, HCMV is associated with mild symptoms prior to establishing latency. In individuals with naïve or compromised immune systems, HCMV is capable of causing severe organ damage. HCMV is the leading infectious cause of congenital birth defects and a major non-genetic cause of hearing loss. Unfortunately, antiviral treatment options lack diversity due to limited knowledge of virion replication. If HCMV replication were better understood, new antiviral treatments could be developed.

In this work, we describe the development and implementation of new tools to study …

An Analysis Of The Interaction Between Sin3 And Methionine Metabolism In Drosophila, Mengying Liu

Wayne State University Dissertations

Chromatin modification and cellular metabolism are tightly connected. The mechanism for this cross-talk, however, remains incompletely understood. SIN3 controls histone acetylation through association with the histone deacetylase RPD3. In this study, my major goal is to explore the mechanism of how SIN3 regulates cellular metabolism.

Methionine metabolism generates the major methyl donor S-adenosylmethionine (SAM) for histone methylation. In collaboration with others, I report that reduced levels of some enzymes involved in methionine metabolism and histone demethylases lead to lethality, as well as wing development and cell proliferation defects in Drosophila melanogaster. Additionally, disruption of methionine metabolism can directly affect histone …

A Novel Role For Repetitive Sequences In Recognition Of The Drosophila Melanogaster X Chromosome, Sonal Suresh Joshi

Wayne State University Dissertations

In humans and fruit flies, males have one X chromosome while females have two. This imbalance in gene dosage is potentially lethal, and the process of dosage compensation corrects it. The MSL (Male Specific Lethal) complex, which is composed of five proteins and one of two functionally redundant long non-coding roX (RNA on the X) RNAs, brings about dosage compensation in Drosophila melanogaster. In fruit fly dosage compensation, all the genes on the single male X chromosome are upregulated approximately twofold, via chromatin modifications, to equalize gene dosage with the two X chromosomes of females. This process calls for highly …

Genome Wide Analysis Identifies Sphingolipid Metabolism As A New Target Of Valproic Acid, Shyamalagauri Jadhav Jadhav

Wayne State University Dissertations

Bipolar disorder (BD), which is characterized by depression and mania, affects about 1% of the total world population. Current treatments are effective in only 40-60% of cases and cause severe side effects. Valproic acid (VPA), a branched short-chain fatty acid, is one of the most widely used drugs for the treatment of BD. Although many hypotheses have been postulated to explain the molecular mechanism of action of this drug in BD, the therapeutic mechanism is not understood. This knowledge gap has hampered the development of new drugs to treat this disorder. To identify candidate pathways affected by VPA, I performed …

Cardiolipin Is Required For Optimal Acetyl-Coa Metabolism, Vaishnavi Raja

Wayne State University Dissertations

The phospholipid cardiolipin (CL) is crucial for many cellular functions and signaling pathways, both inside and outside of mitochondria. My thesis focuses on the role of CL in energy metabolism. Many reactions of electron transport and oxidative phosphorylation, the transport of metabolites needed for these processes, and the stabilization of electron transport chain supercomplexes, require CL. Recent studies indicate that CL is required for the synthesis of iron-sulfur (Fe-S) co-factors, which are essential for numerous metabolic pathways. Activation of carnitine-acetylcarnitine translocase, which transports acetyl-CoA into the mitochondria, is CL dependent. The presence of substantial amounts of CL in the peroxisomal …

Analyzing The Interactions Of Kdm5/Lid And Sin3 In Drosophila Melanogaster, Ambikai Gajan

Wayne State University Dissertations

SIN3, the scaffold protein of a histone modifying complex is conserved from yeast to mammals. Drosophila SIN3 associates with both a histone deactylase RPD3 and a histone demethylase dKDM5/LID. Immunopurification of dKDM5/LID verifies a previously observed interaction with SIN3 and RPD3. Furthermore, deficiency of dKDM5/LID phenocopies deficiency of SIN3 in many cellular and developmental processes. Knockdown of both Sin3A and lid hinder cell proliferation in Drosophila cultured cells and developing flies. Knockdown of these genes also results in a curved wing phenotype implicating a role in wing development. Analysis of underlying gene expression changes upon decreased expression of SIN3, dKDM5/LID …

Drosophila Cyclin J And The Somatic Pirna Pathway Cooperate To Regulate Germline Stem Cells, Paul Michael Albosta

Wayne State University Dissertations

Cyclin J (CycJ) is a highly conserved cyclin that is uniquely expressed specifically in ovaries in Drosophila. Deletion of the genomic region containing CycJ and adjacent genes resulted in a genetic interaction with neighboring piRNA pathway gene, armitage (armi). Here I assessed oogenesis in CycJ null in the presence or absence of mutations in armi or other piRNA pathway genes. Although CycJ null flies had decreased egg laying and hatching rates, ovaries appeared normal indicating that CycJ is dispensable for oogenesis under normal conditions. Further double mutant analysis of CycJ and neighbor armi, as well as two other piRNA pathway …

A Protective Role Of Autophagy In A Drosophila Model Of Friedreich's Ataxia (Frda), Luan Wang

Wayne State University Dissertations

Friedreich’s ataxia (FRDA) is an inherited autosomal recessive neurodegenerative disease. It affects 1 in every 50,000 people in central Europe and North America. FRDA is caused by deficiency of Frataxin, an essential mitochondrial iron chaperone protein, and the associated oxidative stress damages. Autophagy, a housekeeping process responsible for the bulk degradation and turnover of long half-life proteins and organelles, is featured by the formation of double-membrane vacuoles and lysosomal degradation. Previous researches indicate that Danon’s disease, the inherited neural disorder disease that shares similar symptoms with FRDA, is due to the malfunction of autophagy. Based on this, we raise the …

The Mechanism Of Regulation Of Autosomal Heterochromatic Genes In Drosophila Melanogaster Males By Rox Rna And Msl Proteins, Satya Kiran Koya

Wayne State University Dissertations

In humans and flies, males and females have different set of sex chromosomes contributing to different levels of X-linked gene expression. To equalize X-linked gene dosage between sexes, both humans and flies developed independent strategies which are called dosage compensation. Human females randomly inactivate one of their X chromosome into barr body and Drosophila males up regulate their single X chromosome two fold. Both strategies equalize of X linked gene dose between sexes.

In Drosophila, dosage compensation is brought about by the ribonucleoprotein Male Specific Lethal (MSL) complex that binds hundreds of sites along the X chromosome and modifies …

Characterization And Identification Of Novel Regulators Of The Synthesis Of Phospholipids, Cunqi Ye

Wayne State University Dissertations

Phospholipids are the most abundant lipids in cell membranes. The synthesis of phospholipids is crucial for cellular membrane biogenesis and nearly all aspects of cellular processes. Understanding the regulation of synthesis of phospholipids is beneficial to our fundamental knowledge of cell biology as well as human health.

Regulation of the synthesis of phospholipids is intensively studied in the yeast S. cerevisiae. Most notably, the synthesis of phospholipids is coordinated with the synthesis of inositol, a precursor of inositol-containing lipids, by controlling expression of the genes encoding phospholipid biosynthetic enzymes. In addition to this well-characterized regulatory circuit controlled by the …

Regulation Of Inositol Biosynthesis And Cellular Consequences Of Inositol Depletion: Implications For The Mechanism Of Action Of Valproate, Rania M. Deranieh

Wayne State University Dissertations

Inositol is a six-carbon cyclitol that is ubiquitous in biological systems. It is a precursor for the synthesis of numerous biologically important compounds, including inositol phosphates and phosphoinositides that are essential for cell function and viability. Inositol compounds play a role in membrane formation, gene regulation, signaling, regulation of ion channels, and membrane trafficking. Furthermore, inositol regulates hundreds of genes, including those involved in the biosynthesis of inositol and phospholipids. While transcriptional regulation of inositol biosynthesis has been extensively studied and well characterized, regulation of inositol biosynthesis at the enzymatic level has not been addressed. The current study shows that …

Sex And Heterochromatin: An Investigation Of Sexual Dimorphism In Drosophila Melanogaster, Manasi S. Apte

Wayne State University Dissertations

Over 30% of Drosophila genome is assembled into heterochromatin. Heterochromatin is relatively gene poor, transcriptionally less active and remains condensed during interphase. Previous studies established that roX RNA and some of the Male Specific Lethal (MSL) proteins, all components of the dosage compensation complex, are required for full expression of autosomal heterochromatic genes in male flies but not in females. This was surprising since heterochromatin is generally not thought to be sexually dimorphic. The genetic basis for the regulation of sex-specific heterochromatin was completely unknown.

To determine if roX RNAs localize directly at the heterochromatic regions that they regulate, I …

Characterizing Cyclin J By Identifying Conserved Protein-Protein Interactions, Phillip Jacob Selman

Wayne State University Theses

Cyclins are proteins that bind to Cyclin-dependent kinases, or Cdks, through a conserved domain called the Cyclin Box. Many Cyclins regulate the cell cycle. A few Cyclins impact cellular processes outside of the cell cycle. Also, a few Cyclins have poorly understood functions.

Cyclin J is a member of the Cyclin superfamily of proteins. Cyclin J is conserved among all metazoans, but is presently not well understood. All the research done on Cyclin J has been done in Drosophila.

Its mRNA is present in the early embryo, then disappears, only to reappear in adult females. When probing protein extracts with …

Investigation Of X Chromosome Recognition: The Role Of Small Rna In Drosophila Dosage Compensation, Debashish Unnikrishnan Menon

Wayne State University Dissertations

In humans and flies, females have two X chromosomes but males have one X chromosome and one Y chromosome. This leads to a fatal imbalance in X-linked gene expression in one sex. In mammals and in the fruit fly Drosophila, modulation of X chromosome expression is critical for survival. This process is termed dosage compensation. Flies increase expression from the male X chromosome two-fold. This is achieved by the Male Specific Lethal (MSL) complex, which consists of two large, non-coding RNA on the X transcripts (roX1 and roX2) and five proteins. The roX RNAs have a critical …

Role Of Phosphoylation Of The Mycobacterium Tuberculosis Proteasome In Oxidative Stress Response And Regulation Of Homoserine Kinase (Thrb) In Corynebacterium Glutamicum, Heather Baun

Wayne State University Theses

Part 1:

One-third of the world's population is infected with the latent form of Mycobacterium tuberculosis. M. tuberculosis must be able to control this latent even in response to nitrosative and oxidative stress conferred by the host. It is not well known how M. tuberculosis controls this state.

One possibility for this control is through its eleven eukaryotic-like serine/threonine protein kinases. Our lab has focused research on two eukaryotic-like ser/thr protein kinases, PknA and PknB. It has been found that PknA expression may affect the stability or the assembly of the proteasome complex, which is essential for M. tuberculosis to …

Investigation Of A 16s Rna Central Domain Pseudoknot, Jenna Marie Jasinski-Bolak

Wayne State University Theses

X-ray crystallography of the prokaryotic 30S ribosomal subunit revealed a myriad of complex RNA-RNA, RNA-protein, and protein-protein interactions. Among these are several phylogenetically conserved RNA pseudoknots. Pseudoknots are structurally and functionally diverse RNA secondary structures. They are generally formed by two short complimentary sequences separated by many bases of single stranded regions or loops. These relatively simple folds are often yield complex structures that are key components of functionally important conformational changes in RNA structure. One such pseudoknot is located in the central domain of the 16S rRNA.

The central domain pseudoknot is formed by Watson-Crick base pairing between G570-C866 …

Prevalence And Physiological Significance Of Gene Looping In Saccharomyces Cerevisiae, Banupriya Mukundan

Wayne State University Dissertations

My Ph.D. dissertation work is focused on studying the role of promoter-bound transcription initiation factors involved in gene looping. In this study we showed that the RNAP II subunit Rpb4 has a significant effect on termination of transcription. Gene looping is disrupted in the absence of Rpb4. Rpb4 shows a strong physical interaction with the Mediator subunit Srb5. Mediator subunit Srb5 crosslinked to the 5' and 3' ends of INO1 and CHA1 genes and is required for proper termination of transcription of these genes. Srb5 affected termination of transcription through its interaction with the CF1 complex. Srb5 interaction with the …

Rox1 Function In Dosage Compensation: Structural / Functional Analysis Of A Non-Coding Rna, Ying Kong

Wayne State University Dissertations

roX1 is a long non-coding RNA involved in the chromosome-wide gene regulation that occurs during dosage compensation in Drosophila. Dosage compensation in Drosophila melanogaster occurs by a global two-fold increase of transcription from the single male X chromosome. This essential process compensates for X chromosome monosomy. The male-specific lethal (MSL) complex, containing five proteins, localizes to the male X chromosome and alters chromatin to modify gene expression. roX1 and roX2 RNAs are redundant components of MSL complex that are required for its exclusive X-localization. Recent studies in our lab have revealed a second role of roX RNAs in heterochromatic gene …