Biochemistry, Biophysics, and Structural Biology Commons^™

Examination Of The Neurochemical Mechanisms That Mediate Nicotine Withdrawal In Adolescent And Adult Rats, Luis Alberto Natividad

Open Access Theses & Dissertations

Introduction: The mechanisms that mediate nicotine withdrawal are presently unclear and age group differences in the neurochemical effects of withdrawal have been largely unexplored. Previous studies in our laboratory demonstrated that adult rats display a decrease in extracellular levels of dopamine in the nucleus accumbens (NAcc) during nicotine withdrawal and this decrease is reduced in adolescent rats (Natividad et al., 2010). The goal of this dissertation was to examine whether these age group differences in dopamine during withdrawal are mediated via excitatory and inhibitory mechanisms that modulate dopamine in the cell body region of the ventral tegmental area (VTA). Methods: …

Arachidonic Acid Signaling In Invasive And Non-Invasive Breast Cancer Cells, Debarshi Roy

Open Access Theses & Dissertations

Breast cancer is the second largest cause of cancer-related deaths in women all over the world. Epidemiological studies suggest that the consumption of high-fat diets can promote the incidence of breast cancers in both developed and developing countries. In particular, the lipid-rich diet contains arachidonic acid (AA, a C20:4 polyunsaturated fatty acid), which has been shown to be associated with tumor formation in breast tissues. Nevertheless, the actual mechanism by which AA induces the metastatic transformation and malignancy is not well understood. The goal of my dissertation, therefore, is to identify the molecules and unravel the pathways that participate in …

Use Of Methionine Sulfoximine To Dissect The Role Of Glutamine Synthetase And Glutamine In Progression Of Acute Liver Failure, Amruta Anil Jambekar

Wayne State University Dissertations

Methionine sulfoximine (MSO) is a modified amino acid and a well characterized irreversible inhibitor of glutamine synthetase (GS) enzyme. Glutamine is synthesized by GS enzyme and it is the most abundant amino acid in the body. Glutamine is required by immune cells for generation, propagation and maintenance of an immune response.

To induce acute liver failure (ALF) in mice, animals were given intraperitoneal injections of E. coli lipopolysaccharides (LPS) and sugar D-galactosamine (D-GalN). When these animals were pretreated with MSO, 80% of the animals were completely rescued from liver failure. Moreover, when we characterized the immune response generated during ALF …

The Role Of Cad,Flash And Fam129b In Cancer Cell Survial And Apoptosis, Song Chen

Wayne State University Dissertations

Apoptosis is a normal process in the human body. However, apoptosis is desregulated in cancer cells. Most cancer cells gain resistance to apoptosis, leading to uncontrolled proliferation. In this dissertation, we identified three proteins, associated with apoptosis pathway. 1) CAD, a large multifunctional complex that is invariably elevated in tumor cells, 2) FLASH, a large protein with multiple growth related functions and 3) FAM129B. We demonstrate that CAD could interact with FLASH by using yeast two hybrid, co-immunopreciptation and fluorescence microscopy. In addition, functional analysis using siRNA technology further indicated that CAD could co-operate with FLASH and play roles in …

Identifying Sm22 As A Key Player In Arterial Diseases, Jianbin Shen

Wayne State University Dissertations

Background : Expression of vascular smooth muscle cell (VSMC) cytoskeleton markers including SM22 is down-regulated in arterial diseases including atherosclerosis where inflammation and osteochondrogenesis are present. However, the role of this downregulation in arterial pathogenesis is unknown. Hypothesis : Downregulation of SM22 may actively contribute to arterial pathogenesis. Methods : Five Sm22 knockout (Sm22^-/-) mice and their wild type littermates were subjected to carotid artery denudation, an artery injury model. Analyses were conducted on carotid arteries 2 weeks after injury. Primary VSMCs were isolated from mouse aortas and investigated individually at passage 2 to 4. Sm22 knockdown was …

Identification Of Transcriptional Mechanisms Downstream Of Nf1 Gene Defeciency In Malignant Peripheral Nerve Sheath Tumors, Daochun Sun

Wayne State University Dissertations

Malignant peripheral nerve sheath tumor (MPNST) is a type of soft tissue sarcoma that occurs in carriers of mutations in the neurofibromatosis type I gene (Nf1) as well as sporadically. Plexiform neurofibromas in NF1 patients have a significant risk of developing into MPNSTs leading to increased morbidity and mortality from this syndrome. Surgery is the primary intervention but it is not always effective due to the tendency of MPNSTs to infiltrate the surrounding tissue or grow in an inoperable location. Neurofibromin, the protein coded by the Nf1 gene, functions as a GTPase activating protein (GAP) whose mutation leads to constitutive …

Understanding The Gender-Based Mechanism Of Mso In Als Mice: A Metabolic Characterization Of The Sod1-G93a Mouse Model, Monica Ann Bame

Wayne State University Dissertations

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease characterized by motor neuron death and a corresponding loss of neuromuscular connections resulting in muscle atrophy. Patients become paralyzed shortly after symptom onset and typically die within one to five years of pulmonary complications. ALS is a relatively rare disease, with an overall incidence of approximately 2 in 100,000 people per year and a prevalence of about 5 in 100,000 people. It is typically associated with increasing age and has a slight male prevalence, with a male to female ratio of approximately 3:2. ALS is classified as either familial (the less …

Identification Of Cellular Functions Of Cardiolipin As Physiological Modifiers Of Barth Syndrome, Amit Shridhar Joshi

Wayne State University Dissertations

Cardiolipin (CL) is an anionic phospholipid synthesized in the mitochondrial inner membrane. Perturbation of CL metabolism leads to Barth syndrome (BTHS), a life threatening genetic disorder. I utilized genetic, biochemical and cell biological approaches in yeast to elucidate the cellular functions of CL. Understanding the functions of CL is expected to shed light on the pathology and possible treatments for BTHS.

BTHS is caused by mutations in TAZ1, which encodes a CL remodeling enzyme called tafazzin. BTHS patients exhibit a wide range of clinical presentations, indicating that physiological modifiers influence the BTHS phenotype. A targeted synthetic lethality screen was performed …

Hdm2 Small-Molecule Inhibitors For Therapeutic Intervention In B-Cell Lymphoma, Angela Sosin

Wayne State University Dissertations

Lymphomas frequently retain wild-type (wt) p53 function but overexpress HDM2, compromising p53 activity. Therefore, lymphoma is a suitable model for studying therapeutic value of disrupting HDM2-p53 association by small-molecule inhibitors (SMIs). HDM2 SMIs have been developed and are currently under various stages of preclinical and clinical investigation. This study examined various molecular mechanisms associated and biological effects of two different classes of HDM2 SMIs: the spiro-oxindoles (MI-219) and cis-imidazoline (Nutlin-3) in lymphoma cell lines and patient-derived B-lymphoma cells. Surprisingly, results revealed significant quantitative and qualitative differences between these two agents. At the molecular level, effect of Nutlin-3 was generally more …

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 …

Cdk1 And Plk1 Mediate A Clasp2 Phospho-Switch That Stabilizes Kinetochore–Microtubule Attachments, Ana R. R. Maia, Zaira Garcia, Lilian Kabeche, Marin Barisic

Dartmouth Scholarship

Accurate chromosome segregation during mitosis relies on a dynamic kinetochore (KT)-microtubule (MT) interface that switches from a labile to a stable condition in response to correct MT attachments. This transition is essential to satisfy the spindle-assembly checkpoint (SAC) and couple MT-generated force with chromosome movements, but the underlying regulatory mechanism remains unclear. In this study, we show that during mitosis the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of KT-MT attachments. CLASP2 S1234 was phosphorylated by Cdk1, which primed CLASP2 for association with Plk1. Plk1 recruitment to KTs …

The Binding Properties And Functional Consequences Of Ryr2-Cam Interaction, Yi Yang

Dissertations

The aim of my dissertation is to understand the regulation of RyR2. The whole dissertation is composed of two parts. The first part focused on RyR2-CaM interaction. The second focused on synthetic RyR2 domain peptide (DPc10), which worked as a powerful molecular tool for RyR2 functional and structural studies.

CaM has been long identified as an important cardiac RyR regulator. Broad studies suggest CaM is a critical RyR2 stabilizer and CaM-RyR2 interaction is a critical molecular substrate for arrhythmias and HF pathogenesis, but the in situ binding properties for CaM-RyR2 are still unknown. Here we, Using FRET detection and permeabilized …

Myricetin Inhibits Islet Amyloid Polypeptide (Iapp) Aggregation And Rescues Living Mammalian Cells From Iapp Toxicity, Casey Zelus, Ayano Fox, Anastasia Calciano, Bianca S. Faridian, Luiza A. Nogaj, David A. Moffet

Chemistry and Biochemistry Faculty Works

The aggregation of the amyloidogenic polypeptide IAPP (Islet Amyloid Polypeptide, amylin) is believed to play a direct role in the death of pancreatic β-islet cells in type II diabetes. Preventing the initial aggregation event of IAPP is one strategy for slowing, and possibly preventing, the progression of this disease. Here, we investigate myricetin’s potential as an inhibitor of IAPP aggregation. We show that myricetin prevented thioflavin T binding in a concentration dependent manner. Atomic force microscopy revealed that myricetin prevented fiber formation under rigorous conditions conducive to forming IAPP aggregates. Using an IAPP-EGFP (Enhanced Green Fluorescent Protein) protein construct, we …

Structural Basis Of Substrate Recognition In Thimet Oligopeptidase And Development Of Nanoparticles For Therapeutic Enzyme Delivery, Jonathan Mark Wagner

Theses and Dissertations--Molecular and Cellular Biochemistry

Neuropeptidases are responsible for degradation of signaling peptides in the central nervous system and periphery. Some neuropeptidases have also been shown to play a role as part of the cell’s hydrolytic machinery responsible for breaking down proteins and peptides into amino acids, and these enzymes therefore influence small peptide availability for antigen presentation. A better understanding of how neuropeptidases recognize their substrates could lead to therapeutics that modulate the activity of these important enzymes. Alternatively, re-engineering these enzymes to selectively hydrolyze undesirable peptides could make them attractive as therapeutics themselves. A key question in understanding the activity of these enzymes …

Role Of Lysophosphatidic Acid In Regulation Of Cancer Cell Metabolism, Abir Mukherjee

Theses and Dissertations

The simplest phospholipid, lysophosphatidic acid (LPA), is a heat stable component of serum known for its proliferative and migratory activities in cancer cells. Strong evidence suggests that LPA production and expression of its receptors are dysregulated in multiple human malignancies. The mechanism behind LPA-mediated tumor cell growth and oncogenesis remains poorly understood. In this thesis project I used ovarian and other cancer cells as a model system to examine the hypothesis that LPA present in the tumor microenvironment is a pathophysiological determinant of hyperactive de novo lipogenesis and aerobic glycolysis, two hallmarks of cancer cells. We demonstrated that LPA induced …

Lipase-Kinase Associations Involving Pld2, Jak3 And Fes That Underlie Cancer Cell Proliferation And Invasion, Qing Ye

Browse all Theses and Dissertations

Phospholipase D (PLD) is an enzyme that breaks down phospholipids in the cell membrane. It has been suggested that PLD may play a role during cell proliferation and cell invasion of cancer cells. The objective of this thesis was to define new molecular signaling pathways in which PLD2 might be involved in terms of cell proliferation (first part) and cell invasion (second part). To this, I compared molecular and biochemical aspects between untransformed cell lines with highly invasive, transformed breast cancer cells. In the first part, I investigated the interaction of two tyrosine kinases with PLD2 and the effect of …

Crosstalk Between Lysophospatidic Acid (Lpa) And Transforming Growth Factor Beta (Tgfβ) In Breast And Ovarian Cancer Cells, Jinhua Wu

Theses and Dissertations

Lysophosphatidic acid (LPA) and transforming growth factor beta (TGFβ) are platelet-derived intercellular mediators of cell proliferation and motility. LPA is a general growth, survival and motility-stimulating factor in mammalian cells. TGFβ prevents proliferation of normal epithelial cells. However, the growth-inhibitory effect of TGFβ is lost or reduced in most malignant cells. Instead, TGFβ promotes migration and invasion of advanced cancer cells. Since LPA and TGFβ are both present in the blood and tumor microenvironments, we were interested in signal integration and functional outcomes in malignant epithelial cells in an LPA and TGFβ co-stimulatory context. In a subset of breast and …

Exploration Of Ypel3 Response To Hormones And Ability To Induce Senescence, Joseph E. Rotsinger

Browse all Theses and Dissertations

p53 activation through different cellular senescence pathways can trigger cell cycle arrest via regulation of p53 target genes. One such target gene is YPEL3 which is expressed upon binding of tumor suppressor protein p53 at its p53 binding sites (Kelley, 2010). The ability of p53 to induce YPEL3 gene expression led to the discovery that YPEL3 is one of several p53 target genes which induce cellular senescence (Kelley, 2010). Additionally YPEL3 can be regulated independently of p53 by estrogen signaling through estrogen receptor α (Tuttle, 2011). The loss of estrogen receptor α or removal of estrogen induces YPEL3 gene expression …

Study Of Microrna-34a Mediated Post Transcriptional Regulation Of Mdm4, Pooja P. Mandke

Browse all Theses and Dissertations

MDM4 is an important negative regulator of the tumor suppressor p53. In normal unstressed cells the activity of p53 is kept under control by MDM4 and its homologue MDM2. MDM4 is said to possess oncogenic potential based on the evidence of its overexpression in many cancers. Until recently it was believed MDM4 is constitutively transcribed; however a decrease in full length MDM4 in response to genotoxic stress was observed paving way for exploring the mechanism responsible for this.

It was observed miR-34a a member of the miR34 family which is a direct transcriptional targets of p53 could have a potential …

The Role Of Syntaxin And Tomosyn In Platelet Secretion, Shaojing Ye

Theses and Dissertations--Molecular and Cellular Biochemistry

Platelet secretion is important for hemostasis and thrombosis. The components released are also involved in atherosclerosis, inflammation, angiogenesis, and tumor growth. Though the exact mechanism(s) of platelet secretion is still elusive, accumulating evidence demonstrates that SNAREs (Soluble N-ethylmaleimide Sensitive Factor Associated Receptor) and their regulatory partners are critical for platelet exocytosis. Formation of a trans-bilayer complex composed of one v-SNARE (i.e. VAMPs) and two t-SNAREs (i.e. syntaxin and SNAP-25-type) is minimally required for membrane fusion. Regulatory proteins control the rate and specificity of the complex assembly. VAMP-8 and SNAP-23 (a SNAP-25-type t-SNARE) are clearly important; however, the …