Medical Molecular Biology Commons^™

Metabotropic Glutamate Receptor 5 In The Medial Prefrontal Cortex: Role In Cocaine Sensitization And Addiction, Kristin Marie Timmer

Theses and Dissertations (ETD)

Cocaine sensitization is associated with cocaine-induced hyperexcitability of pyramidal projection neurons within the medial prefrontal cortex (mPFC). Such hyperexcitability presumably results in increased glutamatergic input to reward-affiliated brain regions such as the ventral tegemental area (VTA) and nucleus accumbens (NAc), consequently facilitating drugseeking behavior. Metabotropic glutamate receptor 5 (mGluR5) has been implicated in cocaine addiction and demonstrated to increase neuronal excitability, therefore, the aim of the present study was to investigate the effect of intra-mPFC mGluR5 manipulation on behavioral and neurochemical sensitization and drug-seeking. Bilateral cannulae were implanted into the mPFC of male Sprague-Dawley rats and mGluR5 antagonist MTEP (15 …

The Molecular Basis Of Fitness And Transmissibility Of Neuraminidase Inhibitor Resistant Influenza A Viruses, Susu Duan

Theses and Dissertations (ETD)

Neuraminidase (NA) inhibitors including oral oseltamivir and inhaled zanamivir are among the first line of defense against influenza virus infection. Development of resistance to NA inhibitors is a huge drawback for limited options for the control of influenza. During the first decade of NA inhibitor use, the detection rates of resistance to both NA inhibitors had remained low in circulating influenza viruses. However, the 2008~2009 season was marked by a radical increase of prevalence of oseltamvir resistance from <1% to >90% in worldwide surveillance in less than a year. The resistance was solely linked to NA H275Y variants of seasonal H1N1 viruses, …

Hp Turns 17: T Helper 17 Cell Response During Hypersensitivity Pneumonitis (Hp) And Factors Controlling It, Hossam Abdelsamed

Theses and Dissertations (ETD)

Hypersensitivity Pneumonitis (HP) is an interstitial lung disease caused by repeated inhalation of a wide range of environmental antigens. It is characterized by alveolitis, granuloma formation, and fibrosis. Since HP is a T cell-mediated disease, it is important to determine the type of T cell response associated with granuloma formation and the factors that control this response. We hypothesized that HP is associated with a predominant Th17 cell response where both T-bet and TLRs 2 and 9 are controlling T cell response during HP. The results demonstrated a predominant Th17 response associated with granuloma formation in the lungs of C57BL/6J …

Cns Penetration Of Tyrosine Kinase Inhibitors In Mouse Models, Mohamed Elmeliegy

Theses and Dissertations (ETD)

For the past three decades, advances in the treatment of central nervous system (CNS) tumors such as malignant glioma have only been modest. One particular challenge facing treatment of brain tumors is the delivery of therapeutically effective concentrations of anti-cancer agents to the target site in the brain. The sanctuary of the brain is protected by several barrier systems such as the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). These barriers restrict the passage of anti-cancer drugs into the brain via several protective mechanisms.

In the present study, we used cerebral microdialysis sampling, a technique for sampling unbound …

Testing Whether Mrp4 (A Camp Efflux Pump) And The Beta 2 Andrenergic Receptor (An Upstream Regulator Of Camp Signaling Pathways, Praveen Kumar Potukuchi

Theses and Dissertations (ETD)

Background and Aim: MRP4/ABCC4 is an ABC transporter that can efflux the second-messenger, cAMP, from cells. MRP4 has a PDZ interacting motif at its carboxy terminal end through which it binds to scaffolding proteins NHERF1 and PDZK1. Previous studies have shown that PDZK1 serves as a scaffold physically coupling MRP4 with the cystic fibrosis transmembrane conductance regulator (CFTR). This protein complex functionally couples cAMP regulation of CFTR function with MRP4 cAMP transporter activity [Li, C., et al., Spatiotemporal coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia. Cell, 2007. 131(5): p. 940-51]. We hypothesized that the …

Characterization Of The Sigma Factor Proteins And The Dna Binding Protein Euo Of Chlamydia, Cory L. Blackwell

Theses and Dissertations (ETD)

Chlamydia spp. are prokaryotic obligate intracellular pathogens with a unique, biphasic developmental cycle in which an infectious, extracellular form termed the elementary body (EB) interconverts with a metabolically active intracellular reticulate body (RB) within host cells. Subsets of genes are differentially expressed during the developmental cycle, and these genes are believed to be responsible for the transitions between the EB and RB forms. The goal of these studies was to explore two potential mechanisms that may function in regulating developmental cycle stage‑specific gene expression in chlamydiae: a cascade of sigma factor expression and the binding of the early stage protein …

The Biochemical Pathway Leading To Lpa Generation Upon Blood Coagulation, Alyssa Lynn Jefferson Bolen

Theses and Dissertations (ETD)

Platelet activation initiates an upsurge in 18:2 and 20:4 lysophosphatidic acid (LPA) production. The biochemical pathway responsible for LPA production during blood clotting is not fully understood. We have purified a phospholipase A₁ (PLA₁) from thrombin-activated human platelets using sequential chromatographic steps followed by fluorophosphonate‑biotin affinity labeling and proteomics. We identified acyl‑protein thioesterase 1 (aka. lysophospholipase A1, accession code O75608) as a novel PLA₁. Addition of this recombinant PLA₁ significantly increased the production of sn‑2‑esterified polyunsaturated LPCs and the corresponding LPAs in plasma. We next examined the regioisomeric preference of lysophospholipase …

Glycan Shielding Of The Influenza Virus Hemagglutinin Elicits Evasion Of The Adaptive Immune Response And T-Cell-Driven Pathology, Keith C. Wanzeck

Theses and Dissertations (ETD)

Three separate influenza pandemics have emerged in the human population since 1918, each characterized by viruses that lack N-linked glycosylation sites on the globular head of the hemagglutinin protein. In contrast, recent non-pandemic isolates have acquired such sites. Here we constructed isogenic viruses containing differing numbers of additional N-linked glycosylation sites to assess the impact on the host immune response. These studies show that mice infected with a glycosylated virus remain susceptible to challenge with a non-glycosylated virus, glycosylated viruses elicit an inferior immune response, and in this context T-cell pathology and death may occur. We conclude from these data …

Discovery And Validation Of New Regulatory Rna Elements In Chlamydia Trachomatis, Yasser Mohammed Elsayed Metwally Abdelrahman

Theses and Dissertations (ETD)

Chlamydia trachomatis is an obligate intracellular bacterium that exhibits a unique biphasic developmental cycle that can be disrupted by growth in the presence of IFN-g and b-lactams, giving rise to an abnormal growth state termed persistence. Relatively little is known about the regulatory mechanisms that control temporal gene expression during the developmental cycle or the control of persistence and reactivation. Here we have examined the expression of a newly defined family of non-coding RNAs (ncRNAs) that are differentially expressed during the developmental cycle and the induction of persistence and reactivation (Using IFNγ and Carbenicillin). Non-coding RNAs were initially identified using …

Generation And Characterization Of A Knock-In Allele Of Eklf: Probing The In Vivo Role Of The Chromatin Remodeling Domain In Definitive Hematopoietic Cells, Valerie Malyvanh Jansen

Theses and Dissertations (ETD)

The zinc finger-encoding transacting factor EKLF, or erythroid Krüppel-like factor, binds key regulatory elements of many erythroid-specific genes, and is essential for definitive erythropoiesis. Mice lacking this factor die of anemia by E15.5 of gestation, failing to activate β-globin gene transcription, and demonstrating a block in the erythroid differentiation program at the primitive erythroblast stage. In contrast, megakaryocytic progenitors are amplified in EKLF-null embryos, with increased Fli-1 gene expression, a marker of early megakaryocytic differentiation. These observations are consistent with the idea that EKLF modulates the megakaryocytic-erythroid (M-E) differentiation switch.

Our laboratory has previously demonstrated that an amino terminal sequence …

Studies To Characterize The Requirements For The Binding And Release Of Erdj3, A Mammalian Er Dnaj Homolog, From Substrates, Yi Jin

Theses and Dissertations (ETD)

Heat shock protein 70s (Hsp70s) and their DnaJ co-factors exist in all organisms and in all eukaryotic organelles. These highly conserved chaperone pairs facilitate a large number of cellular processes. ERdj3 was identified as a soluble, lumenal DnaJ family member that binds to unassembled immunoglobulin heavy chains (HC) along with the BiP chaperone complex in the endoplasmic reticulum of mammalian cells. Here we demonstrate that ERdj3 binds directly to two unfolded substrates: immunoglobulin γHC and denatured firefly luciferase. Using mutagenesis studies on ERdj3 in both in vivo and in vitro binding assays, we defined ERdj3’s critical amino acids in domain …

The Tetraspanin Cd9 Localizes To Platelet-Platelet Contacts And Regulates Thrombus Stability, Sarah Kathleen Hill

Theses and Dissertations (ETD)

CD9, a member of the tetraspanin family, is highly expressed on platelets (50,000-80,000 copies per platelet). Tetraspanins have been implicated in modulation of integrin function, and it is hypothesized that CD9 will modulate GPIIb-IIIa, the major platelet integrin.

The association between CD9 and GPIIb-IIIa was analyzed using immunoprecipitations and confocal microscopy. These two proteins were found to associate with each other, particularly at areas of platelet-platelet contact and at the periphery. Confocal analysis revealed CD9 localization was most intense at platelet-platelet contact, as well as in platelet filopodia and lamellipodia, but there is a lack of CD9 at areas of …

The Structural And Functional Study Of Git1 Paxillin Binding Domain, Ziwei Zhang

Theses and Dissertations (ETD)

The G protein coupled receptor (GPCR)-kinase (GRK) interacting protein 1 (GIT1) is a multidomain protein that plays an important role in cell adhesion, motility, cytoskeletal remodeling, and membrane trafficking. GIT1 mediates the localization of p21-activated kinase (PAK) and PAK-interactive exchange factor (PIX) to focal adhesions, and its activation is regulated by the interaction between its C terminal paxillin-binding domain (PBD) and the LD motifs of paxillin.

In this dissertation, we determined the solution structure of rat GIT1 PBD by nuclear magnetic resonance (NMR) spectroscopy. The PBD folds into a four-helix bundle, which is structurally similar to the focal adhesion targeting …

Molecular Mechanism Of Tetraspanin Cd9 Mediated Cell Motility, Jayaprakash Kotha

Theses and Dissertations (ETD)

CD9, a member of the tetraspanin superfamily of proteins participates in the regulation of cell adhesive functions such as cell migration. The mechanisms underlying CD9 mediated cell migration are not known. In the current study, we investigated the molecular basis for the CD9 promoted cell migration. Our findings show that the phosphatidylinositol-3 kinase (PI-3K) inhibitors, wortamannin and LY294002 inhibited CD9 promoted cell motility in Chinese hamster ovary (CHO) cells. In contrast, inhibitors targeting protein kinase C or mitogen-activated protein kinase had no effect on CD9 driven CHO cell motility. Furthermore, inhibition of PI-3K activity in CHO cells by dominant/negative PI-3K …