Cell and Developmental Biology Commons^™

The Ras Effector Nore1a Forms A Tumor Suppressor Complex With Brca1., Nicholas C Nelson

Electronic Theses and Dissertations

Ras proteins function as molecular signaling switches that can stimulate multiple mitogenic pathways in response to extracellular signaling. Oncogenic activation of Ras by structural mutation is a highly transforming event in ~1/3 of human cancers. However, aberrant Ras activation can also promote oncogene-induced senescence. This Ras-induced irreversible growth arrest is a physiological process that acts as a barrier to malignancy. The mechanisms by which Ras drives senescence and how this process is bypassed during Ras-driven transformation remains poorly understood.

Although mutations in the RAS gene are extremely rare in human breast cancer, the Ras signaling pathway is constitutively activated in …

Regulation Of The Retinoblastoma Tumor Suppressor By The Novel Ras Effector Nore1a., Thibaut François Barnoud

Electronic Theses and Dissertations

Ras is the most frequently mutated oncogene in human cancers. It acts as a critical branch point in signal transduction, regulating numerous downstream effectors involved in cell growth and differentiation. While Ras can activate many growth promoting pathways, it can paradoxically regulate growth inhibitory pathways leading to apoptosis and cell cycle arrest. One of the ways Ras can inhibit the growth of cells is via a family of effectors called the RASSF proteins. RASSF5 (NORE1A) is a tumor suppressor that is frequently inactivated in human tumors by epigenetic mechanisms. NORE1A binds directly to Ras and promotes Ras-induced senescence. We have …

Regulation Of Synaptogenesis By The Mirna Pathway And Fmr/P Bodies, Jacqueline Rochelle Furlong

Electronic Theses and Dissertations

Post-transcriptional regulation of mRNA is facilitated by different mechanisms, such as microRNA (miRNA) induced gene silencing or fragile X mental retardation protein (FMRP) mediated repression either independent of or acting through cytoplasmic RNA Processing bodies (P bodies). DPTP99A, Lar, and Wg have known functions during synaptogenesis and may be targets of miR-8. Here, we provide evidence that miR-8 regulates DPTP99A in vitro. Non-endogenous miR-8 expressed using an UAS driver regulates Lar. Endogenous miR-8 may regulate DPTP99A in vivo. Here we show that FMRP is capable of colocalizing with the P body components: DCP1, HPat, and Me31B, but not …

Rab8 Directs Tubulation And Furrow Ingression During Epithelial Formation In Drosophila Melanogaster, Lauren Mackenzie Mavor

Electronic Theses and Dissertations

One of the most fundamental changes in cell morphology is the formation of a plasma membrane furrow. The Drosophila embryo undergoes several cycles of rapid furrow ingression during early development, which culminates in the formation of an epithelial sheet. Previous studies have demonstrated the requirement for intracellular trafficking pathways in furrow ingression; however, the pathways that link compartmental behaviors with cortical furrow ingression events have remained unclear. This research shows that Rab8, a small GTPase associated with late exocytic trafficking events, demonstrates striking dynamic behaviors in vivo; transitioning from punctate structures to a stable association with the cortex during …

Evaluating The Effects Of Aroclor 1260 In Non-Alcoholic Fatty Liver Disease : The Role Of Xenobiotic Receptors., Banrida Wahlang

Electronic Theses and Dissertations

Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of US adults and dose-dependently associated with nonalcoholic fatty liver disease (NAFLD) in epidemiologic studies. PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), liver-X-receptor and farnesoid-X-receptor. This study evaluated the hepatic effects of the PCB mixture, Aroclor 1260, whose composition mimics human bioaccumulation patterns, in a mouse model of diet-induced obesity. Male C57Bl/6J mice were fed a control or 42% high fat diet (HFD) …

The Role Of Stem Cells In Adipose Tissue Remodeling., Candice Reshay Holden 1988-

Electronic Theses and Dissertations

The work presented in this dissertation outlines the role of stem cells in the remodeling of adipose tissue under conditions of nutrient excess. Obesity-induced variations in adipose tissue stem cell distribution were uncovered by analysis of the stromal vascular fraction isolated from mice fed a high fat diet for several weeks. Bone marrow cell transplantation was used to determine the derivation of progenitor cells found in adipose tissue; and supplementation of depleted progenitor cell populations achieved via direct cell transplantation, helped to determine the contribution of these progenitor cells to the remodeling process. The dissertation is presented in five chapters …

Rala And Rab8 Regulate Drosophila Embryogenesis, Zhongyuan Zuo

Electronic Theses and Dissertations

Pseudocleavage furrow dynamics initiate at cycle 10 to help with mitotic divisions that lack cytokinesis. Permanent furrows then form at the beginning of cellularization (cycle 14). Cytoskeletal networks such as F-actin and non-muscle Myosin II have been demonstrated to be required for furrow formation and provide anchor points for chromosomal segregation. However, what drives the onset of furrow formation and membrane addition remains largely unclear. In my thesis, I have explored RalA and Rab8 function during furrow formation at metaphase and cellularization stages. These furrows are critical for correct chromosomal segregation, while disruption of furrow formation results in severe developmental …

New Insights Into The Roles Of Human Dna Damage Checkpoint Protein Atr In The Regulation Of Nucleotide Excision Repair And Dna Damage-Induced Cell Death, Zhengke Li

Electronic Theses and Dissertations

Integrity of the human genome is frequently threatened by endogenous and exogenous DNA damaging reagents that may lead to genome instability and cancer. Cells have evolved multiple mechanisms to repair DNA damage or to eliminate the damaged cells beyond repair and to prevent diverse diseases. Among these are ataxia telangiectasia and Rad3-related (ATR)-mediated DNA damage checkpoint and nucleotide excision repair (NER) that are the major pathways by which cells handle ultraviolet C (UV-C)- or other exogenous genotoxin-induced bulky DNA damage. However, it is unclear how these 2 pathways may be coordinated. In this study we show that ATR physically interacts …

Investigating Potential Bioactive Compounds From Rhodococcus And Their Effects On Mcf7 Breast Cancer Cells, Megan N. Crabtree

Electronic Theses and Dissertations

Many drugs used in the treatment of various cancers are derived from or influenced by compounds from nature. The soil bacterium Rhodococcus is of interest because of its identified secondary metabolic pathways and the production of novel natural antibiotics from several strains. In this study, a solid agar extraction method was used to collect compounds from strains of Rhodococcus. These bacterial compound extracts were then tested using a MTT assay in order to evaluate their effectiveness in augmenting MCF7 breast cancer cell death. The results of two way ANOVA analyses revealed 18 compound extracts from 15 strains of Rhodococcus that …

A Role For Dynamin-Dependent Endocytosis During Drosophila Gastrulation, Marissa Kay Kuhl

Electronic Theses and Dissertations

Gastrulation, a process conserved among many higher organisms, is the directed migration of cells into layers that will establish various tissues targeted to become anatomical structures. This process is accomplished through another conserved morphogenetic event, known as cell intercalation. Early in development, this movement of cells within an organized tissue leads to unique cellular arrangements where neighboring cells contract their shared interfaces in order to meet at a shared vertex. In this thesis, I present work that demonstrates a requirement for Dynamin-dependent endocytosis during these contraction events. Using quantitative analysis, I have identified varied cell behaviors during experiments which knockdown …

Estradiol Modulation Of Calcium Dynamics In Pituitary Mmq Lactotroph Cells, Monika Pauckova

Electronic Theses and Dissertations

Pituitary lactotrophs are excitable cells that exhibit spontaneous, calcium influx triggering prolactin (PRL) secretion to stimulate lactation. Lactotrophs express estrogen receptors (ER) and are a well established estrogen-responsive cell system. 17B-estradiol (E2) is known to directly affect lactotrophs by increasing PRL transcription and biosynthesis, intracellular Ca2+ concentration ([Ca2+]i) and PRL secretion. This study demonstrates that the MMQ clonal cell line, isolated from the 7315a rat pituitary tumor, is a model lactotroph cell line that is E2-responsive. Spontaneous and evoked Ca2+ transients were especially sensitive to L-type channel block, but not affected by block of omega-conotoxin-GVIA-sensitive CaV channels or TTX-sensitive voltage-gated …

Flow Cytometric Analysis Of Crayfish Hemocytes., Sarah Kathryn Allen

Electronic Theses and Dissertations

Crayfish exhibit innate immune responses via hemocytes and their products. There are 3 hemocyte populations: hyaline cells, granular cells, and semigranular cells. Hemocytes from laboratory housed, untreated crayfish (normal crayfish) have been quantified on the basis of cell type, cell size, and cell granularity using Flow Cytometry. These data present the first overall picture of normal hemocytes from Red Swamp Crayfish with regard to cell type, cell size, and cell granularity and will serve as a baseline for all future studies in our lab. Experiments using crayfish injected with Pseudomonas aeruginosa, Staphylococcus aureus, or crayfish saline alone showed …

Mosaic Analysis With Double Markers (Madm) As A Method To Map Cell Fates In Adult Mouse Taste Buds., Preston D. Moore

Electronic Theses and Dissertations

Taste buds are chemosensory endorgans embedded in the oral epithelium composed of cells that undergo continuous replacement. Mature taste cells live on average 10-14 days and are replaced by new cells when they die. However, the mechanism by which taste cells are produced and integrated into the taste bud as mature taste cells remains unknown. Previous studies approached this issue from either cell cycle gene expression properties or lineage tracing of precursor cells. In our study, we apply a new fate mapping technique that combines these two ideas. This technique, Mosaic Analysis with Double Markers, allows for simultaneous gene knockout …

Neuronal Calcium Regulation And Cellular Stress In A Malignant Hyperthermia Disease Model, Jason Santiago

Electronic Theses and Dissertations

Calcium ions (Ca2+) are essential signaling molecules and must be carefully regulated to preserve specificity in function. One indispensible Ca2+ signaling protein is the ryanodine receptor (RyR) calcium release channel. RyR1 is essential for muscle contraction, and RyR1mutations can lead to severe muscle disorders such as malignant hyperthermia (MH). Recently, a mouse model of human MH (Y522S-RyR1) has been developed. In skeletal muscle, Y522S-RyR1 exhibits an increased sensitivity to activation resulting in Ca2+ leak, mitochondrial disorganization, and cellular stress. Although RyR1 expression is limited in the brain, the cerebellum may be particularly vulnerable to this disease because RyR1 is highly …

Prostasin Is Expressed In Benign Prostatic Hyperplasia And Regulates Cell Proliferation And Invasion Via Inos, Icam-1, And Cycli, Meghan Hatfield

Electronic Theses and Dissertations

Prostasin is expressed in normal prostate epithelial cells but down-regulated in prostate cancers, while prostasin re-expression in invasive prostate cancer cells reduced invasion. We examined prostasin expression and function in benign prostatic hyperplasia (BPH). We evaluated prostasin expression in 12 BPH specimens by immunohistochemistry, and evaluated the impact of prostasin silencing by siRNA on the expression of the inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), and cyclin D1, as well as on cell proliferation and invasion, using the BPH-1 human prostate epithelial cell line model. Prostasin expression was localized in the glands of BPH tissues by immunohistochemistry, in …

Role Of Transient Receptor Potential Canonical-6 (Trpc6) Channel In Metastasis Of Glioblastoma Multiforme, Rajarajeshwari Venkataraman

Electronic Theses and Dissertations

Glioblastoma multiforme (GBM) is one of the extremely fatal brain tumors. The main reason that makes it so lethal is its capability to invade and spread to other parts of CNS producing secondary tumors. Among other factors hypoxia, reduced oxygen availability, is linked to higher metastatic potential of cancers. Hypoxia causes numerous changes in genome and proteome of the cell. These changes help a normal cell to adapt to nutritional deficiency, but the same changes can increase the malignancy and metastasis in tumor cells. Extensive research by a number of curious scientists reveal that various pathways involving numerous proteins cross-talk …

Purinergic Receptors In The Circumvallate Papillae Of Rats, Alana Marselle Montoya

Electronic Theses and Dissertations

P2X2 receptors and P2Y4 receptors are ATP-activated cell surface receptors that gate movement of K+, Na+, and Ca2+. We used immunocytochemistry for P2X2 and P2Y4 receptors as well as taste cell type markers to learn if P2X2 immunoreactivity (IR) is present in nerve processes in close apposition to Type II and/or Type III taste cells. We also tested to see if P2Y4 IR is present in Type II and/or Type III taste cells. Our results indicate that P2X2 is present only in intragemmal nerve processes. P2X2-LIR nerve processes form close contacts with Type II and Type III taste cells. P2Y4 …

Vitamin E (Tocotrienols) And Prostate Cancer: A Proteomics Approach., Christian Mbangha Muenyi

Electronic Theses and Dissertations

Proteomics is the large scale study of proteins in cells or organisms. The purpose of this study was to characterize the proteomic alterations occurring in a prostate cancer (LNCaP) cell line after treatment with delta-tocotrienol (a form of vitamin E not very prevalent from most dietary sources). We found that both gamma- and delta-tocotrienols induced time and concentration dependent growth inhibition and programmed cell dead (apoptosis) in LNCaP cells. Secondly, we used two-dimensional gel electrophoresis (2-DE) to characterize changes in protein expression levels associated with this treatment. Our results show that a specific set of proteins are regulated at both …

Mechanism Of Action And Regulation Of Membrane Serine Protease Prostasin In The Prostate And Prostate Cancer, Mengqian Chen

Electronic Theses and Dissertations

The glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin (PRSS8) is expressed at the apical membrane surface of epithelial cells and acts as a suppressor of tumor invasion when re-expressed in highly invasive human prostate and breast cancer cell lines. To better understand the molecular mechanisms underlying the anti-invasion phenotype associated with prostasin re-expression in prostate cancer cells, we expressed wild-type human prostasin or a serine active-site mutant prostasin in the PC-3 human prostate carcinoma cells. Molecular changes were measured at the mRNA and the protein levels. The expression of several invasion-promoting molecules is regulated by prostasin re-expression, mediated by a protein-level down-regulation …

Lim Kinase 1 Modulates Expression Of Matrix Metalloproteinases And Associates With Gamma-Tubulin: Dual Role In Invasion And Mito, Tenekua Tapia

Electronic Theses and Dissertations

LIM kinase 1 (LIMK1) is a unique dual specificity serine/threonine kinase containing two N-terminal LIM domains in tandem, a PDZ domain and a C-terminal catalytic domain. LIMK1 is involved in modulation of actin cytoskeleton through inactivating phosphorylation of the ADF (actin depolymerization factor) family protein cofilin. Recent studies have shown that LIMK1 is upregulated in breast and prostate cancer cells and tissues, promotes metastasis in animals and induces acquisition of an invasive phenotype when ectopically expressed in benign prostate epithelial (BPH) cells. Furthermore, overexpression of LIMK1 was associated with altered sub cellular localization of the membrane type 1 matrix metalloprotease …

Effects Of Oxidized Low Density Lipoprotein On Nitric Oxide Production In Macrophages, Annong Huang

Electronic Theses and Dissertations

The effects of oxidatively modified low density lipoprotein (oxLDL) on atherogenesis may be partly mediated by alterations in nitric oxide (NO) production by macrophages. A major goal of this study was to identify the lipid components in oxLDL modulating NO production. The effect of a water soluble antioxidants (N-acetylcysteine) and lipid soluble antioxidant (alpha-tocopherol) on NO production in macrophages was also determined. A second goal was to determine if the effects of oxLDL occurred at the transcriptional level. Human LDL was oxidized using an azo-initiator 2,2$\sp\prime$-azobis (2-amidinopropane) HCI (ABAP). OxLDL markedly decreased the production of NO in LPS stimulated RAW264.7 …

Comparison Of Beta-Adrenoceptor Coupled Camp Production In Cultured Human Mononuclear Leukocytes And Myometrial Cells, Yu-Li Liu

Electronic Theses and Dissertations

$\beta\sb2$-Adrenoceptor ($\beta\sb2$-AR) agonists, such as terbutaline, are used as tocolytic agents in the treatment of preterm labor. $\beta$-Adrenoceptor stimulation relaxes myometrium through specific receptors coupled through Gs to adenylyl cyclase (AC) that catalyzes the conversion of ATP to cAMP. The purpose of this study was to compare $\beta$-adrenoceptors and cAMP production in cultured human leukocytes and myometrial cells, and to determine the importance of $\beta$-adrenoceptors and cAMP production in isoproterenol-induced myometrial relaxation. $\sp{125}$I-iodopindolol was used to assess $\beta$-adrenoceptor affinity and number cAMP levels were analyzed before and after stimulation by isoproterenol, AlF$\sb4\sp-$, forskolin, and PGE$\sb1$. Isometric recording was used to …

Endogenous Alkylglycerol Functions As A Mediator Of Protein Kinase C Activity And Cell Proliferation, Fritz G. Buchanan

Electronic Theses and Dissertations

To explore the possibility that 1-O-alkyl-sn-glycerol (alkylglycerol) may serve a regulatory role in the control of cell proliferation or PKC activity, we examined the ability of alkylglycerol to influence PKC activity and subcellular distribution as well as the ability of alkylglycerol to effect cell proliferation. MDCK cells grown to confluence show a loss of PKC activity associated with the membrane, as reported in fibroblasts. Preconfluent cultures of MDCK cells have a high level of PKC activity associated with the membrane. However, treatment of preconfluent cultures with alkylglycerol causes a reduction of PKC activity. A similar inhibition was observed with alkylglycerol …

Microcirculation: Electrophysiological Basis For The Response Of Endothelial Cells To Inflammatory Mediators-Bradykinin, Kai Miao

Electronic Theses and Dissertations

Using conventional microelectrodes, I studied the electrical basis for determining the resting V$\sb{\rm m}$ in intact EC's from hamsters. The resting V$\sb{\rm m}$ were found to be $-$40 mV for aortic EC's and $-$43 mV for vena caval EC's. The contributions of ions to the resting V$\sb{\rm m}$ of aortic EC's were compared in terms of the transference number (t$\sb{\rm ion}$). To develop a technique for in situ monitoring changes in V$\sb{\rm m}$ of postcapillary venular EC's in the hamster mesentery, a voltage-sensitive fluorescent probe, bisoxonol, was used to load the cells and the fluorescence signals were analyzed under an …

Unusual Structure Of A Human Middle Repetitive Dna, Duminda D. Ratnasinghe

Electronic Theses and Dissertations

The L2Hs sequences are a polymorphic, interspersed, middle repetitive DNA family unique to human genomes. Genomic fingerprinting indicates that these DNAs vary from one individual to another and between tissues of the same individual. Sequence analysis reveals that they are AT-rich (76%) and contain many unusual sequence arrangements (palindromes, inverted and direct repeats). These sequence properties confer on the L2Hs elements the potential to fold into non-B-form structures, a characteristic of recombination hot spots. To test this hypothesis carbodiimide, osmium tetroxide and S$\sb1$ nuclease were used as single-strand specific probes to study a recombinant plasmid, pN6.4.39, containing a single L2Hs …

Changes In Intracellular Chloride During Osmotic Stress And L-Alanine Uptake In Mouse Hepatocytes, Kening Wang

Electronic Theses and Dissertations

A stable intracellular ionic environment is necessary for hepatocytes to function normally. Thus, during hypotonic shock or L-alanine uptake, hepatocytes swell and then exhibit a regulatory volume decrease (RVD), which comprises an increase in K$\sp+$ conductance (G$\sb{\rm K}$), an increased K$\sp+$ efflux, and a hyperpolarization of transmembrane potential (V$\sb{\rm m}$). Since hepatocyte intracellular Cl$\sp-$ has been demonstrated to distribute passively with V$\sb{\rm m}$, this study is designed to test the hypothesis that the hypotonic shock- or L-alanine uptake-induced hyperpolarization of V$\sb{\rm m}$ might provide an electromotive force for the efflux of hepatocyte intracellular Cl$\sp-$, which in turn would contribute osmotically …

Molecular And Cellular Analysis Of Chlamydia Trachomatis: Persistence And Reactivation, Kimberly R. Tau

Electronic Theses and Dissertations

Chlamydia trachomatis (CT) is the most prevalent sexually-transmitted infection in the United States. It has been suggested that CT infections can become latent. This has not been substantiated. CT persistence was examined at the molecular and cellular level in vitro and in vivo. Penicillin treatment of CT in vitro results in abnormal inclusions and reduced recovery of infectious CT. Penicillin did not inhibit initial stages of infection, but did downregulate CT rRNA levels after 25 hours post-inoculation (p.i.). DNA amplification was employed to differentiate between a resolved infection and a persistent one. Utilizing a primer pair that amplified a 144 …

Characterization Of Two Temperature-Sensitive Mutants Of Escherichia Coli Exhibiting An Altered L22 Ribosomal Protein, Bonnie A. Burnette-Vick

Electronic Theses and Dissertations

Analysis of E. coli strains SK1047 and SK1048 have shown them to be temperature-sensitive, protein-synthesis deficient. An alteration in ribosomal protein L22 was detected in both strains using two dimensional gel electrophoresis. Protein L22 was purified from both strains by reversed phase high performance liquid chromatography and from two dimensional electrophoretic gels. Purified ribosomal protein L22 was labeled by reductive methylation and used in 23S RNA binding assays with and without ribosomal protein L4. At the permissive temperature, protein L22 from SK1047 bound less efficiently than the control while protein L22 from SK1048 bound as efficiently as the control. At …

Mouse Mast Cell Proteases: Induction, Molecular Cloning, And Characterization, Wei Chu

Electronic Theses and Dissertations

Tryptase, a mast cell-specific serine protease with trypsin-like specificity, has been identified in a mouse mast cell line (ABFTL-6) based on it's enzymatic activity, inhibition properties, and cross-reactivity to a human mast cell tryptase antibody. The effects of fibroblast-conditioned medium and sodium butyrate on ABFTL-6 mast cell differentiation and tryptase expression have been examined. ABFTL-6 mouse mast cells undergo phenotypic changes upon culturing in media supplemented with fibroblast-conditioned media at 50% or 1 mM sodium butyrate. The induced cells increased in size, had larger and more metachromatic cytoplasmic granules, and increased their total cellular protein about four-fold. Tryptase activity increased …

Electrophysiology, Cell Calcium, And Mechanisms Of Hepatocyte Volume Regulation, Walid E. Khalbuss

Electronic Theses and Dissertations

The electrophysiologic technique (Reuss, L., Proc. Natl. Acad. Sci. USA 82:6014, 1985) was modified to measure changes in steady-state hepatocyte volume during osmotic stress. Hepatocytes in mouse liver slices were loaded with tetramethylammonium ion (TMA$\sp{+}$) during transient exposure of cells to nystatin. Intracellular TMA$\sp{+}$ activity (a$\sp{\rm i}\sb{\rm TMA}$) was measured with TMA$\sp{+}$-sensitive, double-barreled microelectrodes. Loading hepatocytes with TMA$\sp{+}$ did not change their membrane potential (V$\sb{\rm m}$), and under steady-state conditions a$\sp{\rm i}\sb{\rm TMA}$ remained constant over 4 min in single impalements. Hyperosmotic solutions (50, 100, & 150 mM sucrose added to media) and hyposmotic solutions (sucrose in media reduced by …