Molecular Biology Commons^™

The Role Of Fruiting Bodies In Protecting A Community Of Myxococcus Xanthus Spores From The Environment, Dave Lall

Wayne State University Dissertations

Environmental microorganisms have evolved a variety of strategies to survive fluctuations in environmental conditions, including the production of biofilms and differentiation into spores. Myxococcus xanthus are ubiquitous soil bacteria that produce starvation-induced multicellular fruiting bodies filled with environmentally resistant spores (a specialized biofilm). Isolated spores have been shown to be more resistant than vegetative cells to heat, ultraviolet radiation, and desiccation. The evolutionary advantage of producing spores inside of fruiting bodies is not clear. Here, we examine a hypothesis that the fruiting body provides additional protection from environmental insults. We developed a high-throughput method to compare the recovery (outgrowth) of …

Microbial Prevalence And Antibiotic Resistance Associated With Urban Food Production Systems, Nirosha Ruwani Amarasekara

Wayne State University Dissertations

Despite the increasing popularity of urban agriculture and farmers’ markets, there is a lack of scientific data on microbial prevalence and antibiotic resistance associated with this unique food production system. The overall goal of this research is to understand antibiotic resistance and microbial prevalence in soil and fresh produce associated with urban agriculture. Specifically, we aim to investigate ARG in association with resistance genes to metals and other compounds in urban agricultural soil as well as antibiotic resistome and Listeria contamination in fresh produce distributed in urban communities.

In the initial phase of this research, soil samples were collected from …

Benzene Exposure In Reproductive Biology: A Comprehensive Analysis Of Placental Response And Molecular Mechanisms In Trophoblast Function, Anthony Maxwell

Wayne State University Dissertations

The increase in environmental challenges and exposure to pollutants like benzene poses pressing concerns for reproductive health. This dissertation investigates the impact of benzene exposure during gestation on placental function, a key determinant for fetal development and pregnancy outcomes. Our work establishes that benzene exposure during pregnancy induces maternal immune activation, leading to increased rates of fetal resorptions, compromised fetal growth, and abnormal placental development. Intriguingly, we identify a sexual dimorphic response in male and female placentas to benzene exposure, attributable to inherent differences between male and female placental biology. On a molecular level, benzene is metabolized into hydroquinone in …

Biological Adaptations Leading To Metastatic Potential In Ovarian Cancer, Hussein Chehade

Wayne State University Dissertations

Ovarian cancer induces most deaths from gynecological cancers. Patients succumb due to heavy metastatic tumor burden, which accounts for 90% of cancer related deaths and is characterized by the dissemination of malignant cells from primary tumor to secondary site(s), partaking in cancer progression. Ovarian cancer progression is an extremely demanding process that requires cancer cells to be able to survive anoikis conditions and be metabolically flexible to survive microenvironmental changes during transit to a secondary site. Mitochondrial Nuclear Retrograde Regulator 1 (MNRR1) is a bi-organellar protein primarily present in the mitochondria by which it can prevent apoptosis and/or enhance mitochondrial …

From Culture To Metagenomics: A Comprehensive Evaluation Of The Female Reproductive Tract Microbiome In Pregnancy, Jonathan Panzer

Wayne State University Dissertations

The advent of Next Generation Sequencing technologies prompted novel investigations of the microbiome. These revealed both increased diversity of known microbiomes such as those of the gut, oral cavity and vagina, but also bacterial DNA in tissues previously considered sterile such as the blood, liver, brain, and placenta. Thus, previous paradigms of sterility, based primarily on culture data, were called into question. However, interpretation of bacterial DNA signals from low-microbial biomass tissues is complex due to the strong potential for contamination and interference from the surrounding environment. Yet, many new methodologies are being developed to help isolate valid bacterial DNA …

Analyzing The Impact Of Zip Subfamily Ii Mutations On The Zinc And Iron Transport Of Zupt From Escherichia Coli, Bradley Vandecar

Wayne State University Theses

The disfunction or misregulation of the ZIP family of metal ion transporters has been linked to a plethora of diseases and cancers in humans. Zn2+ is the primary substrate for ZIPs and is transported through the M1 metal binding site, though this activity is stimulated through the binding of Fe2+ in the M2 metal binding site. The members of ZIP Subfamily II are predicted to have an inactive M2 site due to the lack of Fe2+ transport and from a previously generated homology model. This work utilizes homology modelling, whole cell metal ion uptake assays, and growth studies in minimal …

In Silico Drug Discovery For Sars-Cov-2 Papain-Like Protease, Olivia Haskin

Wayne State University Theses

SARS-CoV-2 Papain-like protease is an essential viral protein for viral replication. In our lab, we hypothesize that successful inhibition of this protease will stop viral replication and allow for clearance of the infection by the host. The work described here aimed to design and execute an in silico drug discovery workflow for successfully selecting PLpro inhibitors from the Mcule purchasable compound database. This work reports such a workflow and nine potential compounds for in vitro testing. This work also describes structural activity relationship analysis of a proprietary set of PLpro inhibitors synthesized by medicinal chemistry collaborators and makes recommendations for …

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 …

Increased Cd11b+ Immune Cells Contribute To Thoracic Aortic Aneurysm Rupture, Lucynda Pham

Wayne State University Theses

A life-threatening clinical manifestation of Marfan Syndrome (MFS) is the development of thoracic aortic aneurysms (TAA) which can lead to dissection and or rupture. MFS is caused by mutations in extracellular matrix (ECM) protein, Fibrillin-1 (FBN1) which forms the structural component of elastic microfibrils. It is unclear how mutations in FBN1 cause aneurysm development and progression. In recent years, inflammation and immune cells are being considered an important biological process that contributes to TAA. However, the exact roles of the immune cells are unknown. Here we find increased CD11b+ cells recruited and infiltrated into the vessel wall and their spatial …

Genetic Analysis Of Vibrio Cholerae During Its Hyperinfectious State In Danio Rerio (Zebrafish), Ravipaul Singh

Wayne State University Theses

ABSTRACTGENETIC ANALYSIS OF VIBRIO CHOLERAE DURING ITS HYPERINFECTIOUS STATE IN DANIO RERIO (ZEBRAFISH) by RAVIPAUL SINGH AUGUST 2022 Advisor: Dr. Jeffrey Withey Major: Biochemistry and Molecular Biology Degree: Master of Science Vibrio cholerae is a gram-negative, comma-shaped, monotrichous bacillus and the causative agent of the diarrheal disease cholera. V. cholerae is a natural resident of aquatic environments such as rivers and estuaries and is a highly motile bacterium. Upon entering the human host, V. cholerae travels to its site of colonization in the small intestine and upregulates its virulence factors. Upon escape from the host, V. cholerae can enter a …

Effect Of Allosteric Site Binding On Smyd2 Conformational Ensemble, Jacob Daniel Sobota

Wayne State University Theses

Molecular dynamics simulation allows for the simulation of proteins with precise control over what ligands are present at what sites. The conformational ensemble of a protein can be obtained from MD simulation with and without binding at an allosteric site and compared between to gain insight at what effects binding at an allosteric site has on a protein’s structure. SMYD2, a critical regulator of cellular processes, was recently found to have a novel secondary binding site with allosteric effects on its activity. The discovery of an allosteric site has implications for drug design in the inhibition of SMYD2 to treat …

Deconstructing Brown Adipocyte Neogenesis In Brown And White Adipose Tissue, Rayanne Burl

Wayne State University Dissertations

Global incidence of Type 2 Diabetes (T2D) has reached epidemic proportions, and increasing evidence indicates that dysfunctional adipose tissue is an important contributor to the pathogenesis of T2D. Expanding brown adipocyte (BA) populations within adipose tissues through adrenergic activation improves energy balance and insulin sensitivity. In order to exploit this remodeling of adipose tissue for therapeutic benefit, we need to understand the mechanisms by which adrenergic signaling expand populations of BAs in vivo. These studies utilized single-cell RNA-sequencing and transgenic mouse models, in combination with single-molecule fluorescence in situ hybridization (smFISH) and immunoistochemical analysis, to study BA neogenesis in vivo. …

Vaginal Microbiota Of Recurrent Bacterial Vaginosis Patients Under Oral Metronidazole Therapy: Prognosis Based On Specific Species Of Gardnerella And Other Bacterial Vaginosis Associated Species In Association With The Vaginal Microbiome, Ashomathi Ashok Sudhamathi Mollin

Wayne State University Theses

Bacterial Vaginosis (BV) is a state of vaginal dysbiosis in which BV-associated species displace the typically dominant species of Lactobacillus. This urogenital disease elevates the risk for complications such as pre-term birth, STDs and HIV. Currently, there is no consensus on the etiology of BV. Standard of care treatment is oral metronidazole; however, therapy has high rates of recurrence. Our previous qPCR study of recurrent BV patients (3+ episodes/year) found strong associations of Gardnerella_Gsp07, G. swidsinksii, and G. leopoldii with refractory and recurrent responses, and a robust recovery of Lactobacillus in remission patients. However, these findings were not applicable across …

Abhd5 Induced Morphological Changes On Model Membrane Systems, Nasser S. Junedi

Honors College Theses

Proper regulation of neutral lipid storage (lipogenesis) and release (lipolysis) are critical molecular processes localized to an organelle called the Lipid Droplet (LD). The LD consists of a core with neutral lipids such as triacylglycerols (TAGs) and sterol esters surrounded by a phospholipid monolayer. Dysregulation of the processes localized to the LD are involved in the pathology of various diseases such as Neutral Lipid Storage Disease, diabetes, stroke and cancer. The non-enzymatic protein ABHD5 (α-β Hydrolase Domain-Containing Protein 5), is thought to play a key role in the process of lipolysis by forming homo-oligomers on the surface of the LD …

Lysine 53 Acetylation Of Cytochrome C In Prostate Cancer: Warburg Metabolism And Evasion Of Apoptosis, Viktoriia Bazylianska, Hasini A. Kalpage, Junmei Wan, Asmita Vaishnav, Gargi Mahapatra, Alice A. Turner, Dipanwita Dutta Chowdhury, Katherine Kim, Paul T. Morse, Icksoo Lee, Joseph S. Brunzelle, Lisa Polin, Prabal Subedi, Elisabeth I. Heath, Izabela Podgorski, Katrin Marcus, Brian Fp Edwards, Maik HüTtemann

Biochemistry and Molecular Biology Faculty Publications

Prostate cancer is the second leading cause of cancer-related death in men. Two classic cancer hallmarks are a metabolic switch from oxidative phosphorylation (OxPhos) to glycolysis, known as the Warburg effect, and resistance to cell death. Cytochrome c (Cytc) is at the intersection of both pathways, as it is essential for electron transport in mitochondrial respiration and a trigger of intrinsic apoptosis when released from the mitochondria. However, its functional role in cancer has never been studied. Our data show that Cytc is acetylated on lysine 53 in both androgen hormone-resistant and -sensitive human prostate cancer xenografts. To characterize the …

Global Coordination Between Gene Expression Processes In C. Crescentus, James Raymond Aretakis

Wayne State University Dissertations

Gene expression is an essential process of all organisms which occurs in two steps, mRNA transcription, and translation and which is shut off by mRNA decay. For a bacterium to grow and divide, these steps need to be tuned to optimize the synthesis rates of thousands of proteins from their genes. Genome-wide experiments were used to determine the absolute transcription rates, mRNA degradation rates, and translation rates on a gene by gene basis in the model bacterium Caulobacter crescentus. This not only provides a system biology view of the organism’s gene expression resource allocation, but also provides a chance to …

Functional Characterization Of Threonine 49 Phosphorylation Of Cytochrome C, Antoine Khobeir

Wayne State University Theses

Cytochrome c (Cytc) is a pivotal multifunctional mitochondrial protein that serves as a single electron carrier between complexes III and IV of the electron transport chain. It has important roles in both cellular respiration and apoptosis. The novel Thr49 (T49) phosphorylation of Cytc likely affects mitochondrial respiration, membrane potential, ROS production, ATP production, and apoptosis. Based on the functional characterization of previously mapped phosphorylation sites (Tyr97, Tyr48, Thr28, Ser47, Thr58) of the lab, we hypothesize that T49 phosphorylation will lead to controlled respiration, optimal intermediate mitochondrial membrane potential, lower ROS production, and inhibition of apoptosis compared to unphosphorylated Cytc. Here …

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 …

Targeted Combination Therapy Of Triple-Negative Breast Cancer, Ketki Bhise

Wayne State University Dissertations

TARGETED COMBINATION THERAPY OF TRIPLE-NEGATIVE BREAST CANCERby KETKI BHISE May 2021 Advisor: Dr. Arun Iyer Major: Pharmaceutical Sciences Degree: Doctor of Philosophy Triple Negative Breast Cancer (TNBC) accounts for 10-20% of the total breast carcinoma cases. There is no FDA-approved targeted therapy for TNBC due to lack estrogen, progesterone and HER-2. We have identified the role of a hypoxia biomarker, carbonic anhydrase IX (CAIX) in proliferation and metastasis of TNBC. Based on this observation, we have developed CAIX-targeted Doxorubicin (Dox) prodrug, abbreviated as DoxAtz. To improve the delivery of DoxAtz to the tumor, we developed long circulating liposomes (LipoDoxAtz) and …

Mnrr1: Understanding The Role Of A Novel Mitochondrial-Nuclear Regulator, Stephanie L. Gladyck

Wayne State University Dissertations

Mitochondria are complex organelles that generate most of the energy required to sustain life and function in metabolic and signaling pathways required to maintain cellular homeostasis. MNRR1 (mitochondrial nuclear retrograde regulator 1 or CHCHD2) is a small, bi-organellar twin CX9C protein that is emerging as an important regulator of mitochondrial function, apoptosis, and cellular stress by participating in mitochondrial-nuclear crosstalk. Our lab has previously shown that in the mitochondria, MNRR1 regulates complex IV (Cytochrome c oxidase or COX) and is able to finetune the oxidase function through phosphorylation status. We have also shown that during stress, mitochondrial MNRR1 levels deplete, …

Mixed-Lineage Kinase-3 (Mlk3) Plays A Negative Modulatory Role In Insulin Secretion From The Pancreatic Β-Cell, Tyler Russeth, Vijayalakshmi Thamilselvan, Anjaneyulu Kowluru

Medical Student Research Symposium

Glucose-stimulated insulin secretion (GSIS) from the pancreatic β-cell in response to elevated levels of glucose is controlled by a variety of signals including intracellular calcium and nucleotides such as cAMP, ATP and GTP. These cellular signals are responsible for activation of specific kinases that mediate phosphorylation of key exocytotic proteins that lead to GSIS. In the context of protein kinases, mixed-lineage kinases (MLKs) have been implicated in an assortment of cellular functions, including cell proliferation and apoptosis. However, very little is known on potential regulatory roles of MLKs in islet β-cell function, including GSIS. The goal of this study is …

Characterization And Assembly Of The Pseudomonas Aeruginosa Aspartate Transcarbamoylase-Pseudo Dihydroorotase Complex, Chandni Patel, Asmita Vaishnav, Brian Fp Edwards, David R. Evans

Biochemistry and Molecular Biology Faculty Publications

Pseudomonas aeruginosa is a virulent pathogen that has become more threatening with the emergence of multidrug resistance. The aspartate transcarbamoylase (ATCase) of this organism is a dodecamer comprised of six 37 kDa catalytic chains and six 45 kDa chains homologous to dihydroorotase (pDHO). The pDHO chain is inactive but is necessary for ATCase activity. A stoichiometric mixture of the subunits associates into a dodecamer with full ATCase activity. Unlike other known ATCases, the P. aeruginosa catalytic chain does not spontaneously assemble into a trimer. Chemical-crosslinking and size-exclusion chro- matography showed that P. aeruginosa ATCase is monomeric which accounts for its …

Use Of Alkoxyamines To Quantify And Map Uracils And To Kill B Cell Cancers, Madusha Lakshani Watuthanthrige Perera

Wayne State University Dissertations

Majority of B-cell lymphoma and leukemia originates in germinal centers and over express AID resulting high levels of uracils and eventually AP sites. Treating cells with a small alkoxyamine AA3 with a terminal alkyne group specifically killed B cell lymphoma cell lines without killing normal human B cells and non-hematological cancers. To study the cytotoxicity of AA3, I designed and synthesized a series of AA3 analogs with and without the alkyne group. Using cytotoxicity assays I demonstrated that the alkyne functionality plays a major role in AA3 cytotoxicity in B-cell cancers and further showed that the effectiveness is modulated by …

Timing And Duration Of Folate Restriction Differentially Impacts Colon Carcinogenesis., Ali M. Fardous

Wayne State University Dissertations

Colorectal cancer (CRC) constitutes a major burden on the healthcare system as the second most commonly diagnosed cancer in the developed world. Dietary folate is considered an important modulator of colorectal cancer. Folate restriction has been implicated in increasing CRC incidence by disrupting nucleotide synthesis, Impacting DNA methylation and inducing genetic instability. Our research shows that the timing and duration of dietary folate restriction can differentially impact Colorectal cancer initiation. Acclimating mice to folate restriction for 8 weeks results in a reduced number of preneoplastic lesions compared to mice placed of folate restriction for 1 week prior to initiating the …

Hormonal Regulation Of Glycine Decarboxylase And Its Metabolic Outcomes, Ruta Milind Jog

Wayne State University Dissertations

The amino acid glycine is involved in generation of multiple critical metabolites including glutathione, heme, and creatinine. Interestingly, in both humans and rodents, circulating glycine levels are significantly reduced in obesity, glucose intolerance, type II diabetes and non-alcoholic fatty liver disease. The glycine cleavage system is the predominant glycine degradation pathway in humans. The rate-limiting enzyme of glycine cleavage system is glycine decarboxylase (GLDC), and loss-of-function mutations of GLDC cause hyperglycinemia. Here, we show that GLDC gene expression is upregulated in livers of mouse models of diabetes and diet-induced obesity as well as in the fasted state in normal animals. …

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 …

Germinal Center B Cell Expression Of Aire Regulates Antibody Diversification And Autoimmunity, Jordan Zheng Zhou

Wayne State University Dissertations

B cells are a unique subset of immune cells that, in response to antigen, diversify their antibody repertoire to generate progressively higher affinity antibodies of different isotypes through the processes of affinity maturation/somatic hypermutation (SHM) and class switch recombination (CSR). One of the major sites in which this diversification occurs is in T cell dependent microanatomical structures known as germinal centers (GC). Here, we find that GC B cells express the protein, autoimmune regulator (Aire) in a CD40 dependent manner. In these cells, Aire interacts with activation induced cytidine deaminase (AID), the protein responsible for initiating the processes of diversification …

Regulation Of Respiration And Apoptosis By Cytochrome C Threonine 58 Phosphorylation, Junmei Wan, Hasini A. Kalpage, Asmita Vaishnav, Jenney Liu, Icksoo Lee, Gargi Mahapatra, Alice A. Turner, Matthew P. Zurek, Qinqin Ji, Carlos T. Moraes, Maurice-Andre Recanati, Lawrence I. Grossman, Arthur R. Salomon, Brian Fp Edwards, Maik HüTtemann

Biochemistry and Molecular Biology Faculty Publications

Cytochrome c (cytc) is a multifunctional protein, acting as an electron carrier in the electron transport chain (ETC), where it shuttles electrons from bc1 complex to cytochrome c oxidase (COX), and as a trigger of type II apoptosis when released from the mitochondria. We previously showed that cytc is regulated in a highly tissue-specific manner: Cytc isolated from heart, liver, and kidney is phosphorylated on Y97, Y48, and T28, respectively. Here, we have analyzed the effect of a new Cytc phosphorylation site, threonine 58, which we mapped in rat kidney Cytc by mass spectrometry. We generated and overexpressed wild-type, phosphomimetic …

Characterization Of Cytosolic Sulfotransferase Expression And Regulation In Human Liver And Intestine, Sarah Talal Dubaisi

Wayne State University Dissertations

SULTs are conjugation enzymes that can modify the activity of a myriad of foreign and endogenous molecules. SULT expression was detected in various human tissues, including liver, small intestine, and colon. There are 13 human SULT genes that are classified into 4 families, SULT1, SULT2, SULT4, and SULT6. In humans, SULT1 and SULT2 families include 11 genes that are further divided into 6 subfamilies. In addition to their role in xenobiotic detoxification and regulation of physiological processes, SULT enzymes were implicated in the bioactivation of procarcinogens. Previous studies detected the expression of most SULT1 and SULT2 enzymes during early development, …