Cell and Developmental Biology Commons^™

Dynamic Remodeling Of The Stressed Heart: Role Of Protein Degradation Pathways, Deborah Vela

Dissertations and Theses (Open Access)

The heart is a remarkable organ. In order to maintain its function, it remodels in response to a variety of environmental stresses, including pressure overload, volume overload, mechanical or pharmacological unloading and hormonal or metabolic disturbances. All these responses are linked to the inherent capacity of the heart to rebuild itself. Particularly, cardiac pressure overload activates signaling pathways of both protein synthesis and degradation. While much is known about regulators of protein synthesis, little is known about regulators of protein degradation in hypertrophy. The ubiquitin-proteasome system (UPS) selectively degrades unused and abnormal intracellular proteins. I speculated that the UPS may …

Mcam/Muc18 Regulates Melanoma Progression By Modulating The Expression Of Id-1, Maya Zigler

Dissertations and Theses (Open Access)

The acquisition of the metastatic melanoma phenotype is associated with increased expression of the melanoma cell adhesion molecule MCAM/MUC18 (CD146). However, the mechanism by which MUC18 contributes to melanoma metastasis remains unclear. Herein, we stably silenced MUC18 expression utilizing lentivirus-incorporated small hairpin RNA, in two metastatic melanoma cell lines, A375SM and C8161, and conducted cDNA microarray analysis. We identified and validated that the transcriptional regulator, Inhibitor of DNA Binding-1 (Id-1), previously shown to function as an oncogene in several malignancies, was downregulated by 5.6-fold following MUC18 silencing. Additionally, we found that MUC18 regulated Id-1 expression at the transcriptional level via …

The Role Of Tyrosine Phosphorylation In The Functions Of The Tumor Suppressor Gprc5a, Xiaofeng Lin

Dissertations and Theses (Open Access)

The retinoic acid inducible G protein coupled receptor family C group 5 type A (GPRC5A) is expressed preferentially in normal lung tissue but its expression is suppressed in the majority of human non-small cell lung cancer cell lines and tissues. This differential expression has led to the idea that GPRC5A is a potential tumor suppressor. This notion was supported by the finding that mice with a deletion of the Gprc5a gene develop spontaneous lung tumors. However, there are various tumor cell lines and tissue samples, including lung, that exhibit higher GPRC5A expression than normal tissues and some reports by other …

Genetic Analysis Of The Function Of The Drosophila Doublesex-Related Factor Dmrt93b, Diana O'Day

Dissertations and Theses (Open Access)

DMRT (Doublesex and Mab-3 related transcription factor) proteins generally associated with sexual differentiation in many organisms share a common DNA binding domain and are often expressed in reproductive tissues. Aside from doublesex, which is a central factor in the regulation of sex determination, Drosophila possesses three different dmrt genes that are of unknown function. Because the association with sexual differentiation and reproduction is not universal and some DMRT proteins have been found to play other developmental roles we chose to further characterize one of these Drosophila genes. We carried out genetic analysis of dmrt93B, which was previously found to be …

Defining The Role Of Il-15 Trans-Presentation By Distinct Cell-Types During The Development And Homeostasis Of Natural Killer And Invariant Natural Killer T Cells, Eliseo F. Castillo

Dissertations and Theses (Open Access)

The immuno-regulatory functions displayed by NK and iNKT cells have highlighted their importance as key lymphocytes involved in innate and adaptive immunity. Therefore, understanding the dynamics influencing the generation of NK and iNKT cells is extremely important. IL-15 has been shown to provide a critical signal throughout the development and homeostasis of NK and iNKT cells; however, the cellular source of IL-15 has remained unclear. In this investigation, I provide evidence that the cell-type providing IL-15 to NK and iNKT cells via trans-presentation is determined by the tissue site and the maturation status of NK and iNKT cells. For NK …

Loss Of Gprc5a Enhances Survival In Normal And Malignant Lung Epithelial Cells By Eliciting Persistent Stat3 Activation Induced By Autocrine Lif, Yulong Chen

Dissertations and Theses (Open Access)

Signal transduction and activator of transcription 3 (Stat3) is activated by cytokines and growth factors in many cancers. Persistent activation of Stat3 plays important role in cell growth, survival, and transformation through regulating its targeted genes.

Previously, we found that mice with a deletion of the G protein-coupled receptor, family C, group 5, member a (Gprc5a) gene develop lung tumors indicating that Gprc5a is a tumor suppressor. In the present study, we examined he mechanism of Gprc5a-mediated tumor suppression. We found that epithelial cells from Gprc5a knockout mouse lung (Gprc5a^-/- cells) survive better in vitro in medium deprived …

Immune Recognition Of Self Nucleic Acids Driven By Endogenous Antimicrobial Peptides: Role In Autoimmunity, Dipyaman Ganguly

Dissertations and Theses (Open Access)

Innate immune recognition of extracellular host-derived self-DNA and self-RNA is prevented by endosomal seclusion of the Toll-like receptors (TLRs) in the dendritic cells (DCs). However, in psoriasis plasmacytoid dendritic cells have been found to be able to sense self-DNA molecules in complex with the endogenous cationic antimicrobial peptide LL37, which are internalized into the endosomal compartments and thus can access TLR9. We investigated whether this endogenous peptide can also interact with extracellular self-RNA and lead to DC activation. We found that LL37 binds self-RNA as well as self-DNA going into an electrostatic interaction; forms micro-aggregates of nano-scale particles protected from …

Artemis Interacts With The Cul4a Ubiquitin E3 Ligase Complex And Regulates The Cell Cycle Progression, Yiyi Yan

Dissertations and Theses (Open Access)

Artemis, a member of the SNM1 gene family, is one of the six known components of the non-homologous end joining pathway. It is a multifunctional phospho-protein that has been shown to be modified by the phosphatidylinositol 3-kinases (PIKs) DNA-PKcs, ATM and ATR in response to a variety of cellular stresses. Artemis has important roles in V(D)J recombination, DNA double strand breaks repair and damage-induced cell-cycle checkpoint regulation. The detailed mechanism by which Artemis mediates its functions in these cellular pathways needs to be further elucidated. My work presented here demonstrates a new function for Artemis in cell cycle regulation as …

Nherf1 – New Modifier Of Colorectal Cancer Progression, Yuho Hayashi

Dissertations and Theses (Open Access)

Colorectal cancer (CRC) develops from multiple progressive modifications of normal intestinal epithelium into adenocarcinoma. Loss of cell polarity has been implicated as an early event in this process, but the molecular players involved are not well known. NHERF1 (Na+/H+ Exchanger Regulatory Factor 1) is an adaptor protein with apical membrane localization in polarized epithelia. In this study, we tested our hypothesis that NHERF1 plays a role in CRC. We examined surgical CRC resection specimens for changes in NHERF1 expression, and modeled these changes in two- and three-dimensional (2D and 3D) Caco-2 CRC cell systems. NHERF1 had significant alterations from normal …

E2f1 And Tumor Suppression: The Role Of P21, Mirnas, And The Dna Damage Response, Regina L. Weaks

Dissertations and Theses (Open Access)

E2F1 is a multi-faceted protein that has roles in a number of important cellular processes including cell cycle regulation, apoptosis, proliferation, and the DNA damage response (DDR). Moreover, E2F1 has opposing roles in tumor development, acting as either a tumor suppressor or an oncogene depending on the context. In human cancer, E2F1 is often deregulated through aberrations in the Rb-p16INK4a-cyclin D1 pathway. In these studies we examined three mechanisms by which E2F1 might mediate its tumor suppressive properties: p21-induced senescence, miRNAs, and the DNA damage response. We found that E2F1 acts as a tumor suppressor in response to ras activation …

Role And Regulation Of Epha2 In Pancreatic Cancer, Pavel A. Levin

Dissertations and Theses (Open Access)

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cancer cause of death in the US. Gemcitabine is the first-line therapy for this disease, but unfortunately it shows only very modest benefit. The focus of the current study was to investigate the role and regulation of EphA2, a receptor tyrosine kinase expressed in PDAC, to further understand this disease and identify new therapeutic targets.

The role of EphA2 was determined in PDAC by siRNA mediated silencing. In combination with gemcitabine, silencing of EphA2 caused a dramatic increase in apoptosis even in highly resistant cells in vitro. Furthermore, EphA2 silencing was found …

Delta Like Ligand 4 Is A Critical Regulator Of Bone Marrow Cell Differentiation Into Pericytes/Vascular Smooth Muscle Cells And Is Essential For The Vasculogenesis That Supports The Growth Of Ewing’S Sarcoma, Keri L. Stewart

Dissertations and Theses (Open Access)

We have previously shown that vasculogenesis, the process by which bone marrow-derived cells are recruited to the tumor and organized to form a blood vessel network de novo, is essential for the growth of Ewing’s sarcoma. We further demonstrated that these bone marrow cells differentiate into pericytes/vascular smooth muscle cells(vSMC) and contribute to the formation of the functional vascular network. The molecular mechanisms that control bone marrow cell differentiation into pericytes/vSMC in Ewing’s sarcoma are poorly understood. Here, we demonstrate that the Notch ligand Delta like ligand 4 (DLL4) plays a critical role in this process. DLL4 is essential for …

Validation Of The Activation Of Aurora B Kinase By Caenorhabditis Elegans To Usled-Like Kinase And The Identification Of Cyclin B3 As A Phospho-Specific Tlk-1 Interactor, Gary Michael Deyter

Dissertations and Theses (Open Access)

A hallmark of tumorigenesis and certain birth defect syndromes is the loss of ploidy that can result from incorrect chromosome segregation. Chromosomes that are not partitioned properly during mitosis are often fragmented, changing the genetic makeup of daughter cells. Inheriting extrachromosomal fragments that contain cell survival genes or losing chromosomal loci that encode tumor suppressors can promote tumor development. Thus, it is essential to elucidate molecular mechanisms required for correct chromosome segregation. Chromosomes are connected to mitotic spindle microtubules by way of a proteinacous, chromosome-bound organelle called the kinetochore. Two decades of research have confirmed that the conserved Aurora B/AIR-2 …

Xenoestrogen-Specific Mechanisms Of Developmental Reprogramming Correlate With Gene Expression And Tumor Development, Kristen L. Greathouse

Dissertations and Theses (Open Access)

Environmental exposures during sensitive windows of development can reprogram normal physiological responses and alter disease susceptibility later in life in a process known as developmental reprogramming. We have shown that neonatal exposure to the xenoestrogen diethylstilbestrol (DES) can developmentally reprogram the reproductive tract in genetically susceptible Eker rats giving rise to complete penetrance of uterine leiomyoma. Based on this, we hypothesized that xenoestrogens, including genistein (GEN) and bisphenol A (BPA), reprogram estrogen-responsive gene expression in the myometrium and promote the development of uterine leiomyoma. We proposed the mechanism that is responsible for the developmental reprogramming of gene expression was through …

Myogenin Modulates Exercise Endurance By Altering Skeletal Muscle Metabolism, James Eric Meadows

Dissertations and Theses (Open Access)

The function of myogenic regulatory factors (MRFs) during adult life is not well understood. The requirement of one of these MRFs, myogenin (Myog), during embryonic muscle development suggests an equally important role in adult muscle. In this study, we have determined the function of myogenin during adult life using a conditional allele of Myog. In contrast to embryonic development, myogenin is not required for adult viability, and Myog-deleted mice exhibited no remarkable phenotypic changes during sedentary life. Remarkably, sedentary Myog-deleted mice demonstrated enhanced exercise endurance during involuntary treadmill running. Altered blood glucose and lactate levels in sedentary Myog-deleted mice after …

Smooth Muscle Hyperplasia Due To Acta2/Myh11 Mutations: Identification Of Novel Pathology And Pathways Leading To Aneurysms And Diverse Vascular Occlusive Diseases, Christina L. Papke

Dissertations and Theses (Open Access)

Missense mutations in smooth muscle cell (SMC) specific ACTA2 (á-actin) and MYH11 (â-myosin heavy chain) cause diffuse and diverse vascular diseases, including thoracic aortic aneurysms and dissections (TAAD) and early onset coronary artery disease and stroke. The mechanism by which these mutations lead to dilatation of some arteries but occlusion of others is unknown. We hypothesized that the mutations act through two distinct mechanisms to cause varied vascular diseases: a loss of function, leading to decreased SMC contraction and aneurysms, and a gain of function, leading to increased SMC proliferation and occlusive disease. To test this hypothesis, ACTA2 mutant SMCs …

The Consequences Of Disrupting The Mdm2-P53 Balance In Hematopoiesis, Hussein A. Abbas

Dissertations and Theses (Open Access)

The bone marrow accommodates hematopoietic stem cells and progenitors. These cells provide an indispensible resource for replenishing the blood constituents throughout an organism’s life. A tissue with such a high turn-over rate mandates intact cycling checkpoint and apoptotic pathways to avoid inappropriate cell proliferation and ultimately the development of leukemias. p53, a major tumor suppressor, is a transcription factor that regulates cell cycle, and induces apoptosis and senescence. Mice inheriting a hypomorphic p53 allele in the absence of Mdm2, a p53 inhibitor, have elevated p53 cell cycle activity and die by postnatal day 13 due to hematopoietic failure. Hematopoiesis progresses …

New Target Genes For Tumor Suppressors P53 And P73 In Regenerating Liver, Svitlana M. Kurinna

Dissertations and Theses (Open Access)

The p53-family of proteins regulates expression of target genes during tissue development and differentiation. Within the p53-family, p53 and p73 have hepatic-specific functions in development and tumor suppression. Despite a growing list of p53/p73 target genes, very few of these have been studied in vivo, and the knowledge regarding functions of p53 and p73 in normal tissues remains limited. p53+/-p73+/- mice develop hepatocellular carcinoma (HCC), whereas overexpression of p53 in human HCC leads to tumor regression. However, the mechanism of p53/p73 function in liver remains poorly characterized. Here, the model of mouse liver regeneration is used to identify new target …

Survival Prediction For Brain Tumor Patients Using Gene Expression Data, Vinicius Bonato

Dissertations and Theses (Open Access)

Brain tumor is one of the most aggressive types of cancer in humans, with an estimated median survival time of 12 months and only 4% of the patients surviving more than 5 years after disease diagnosis. Until recently, brain tumor prognosis has been based only on clinical information such as tumor grade and patient age, but there are reports indicating that molecular profiling of gliomas can reveal subgroups of patients with distinct survival rates. We hypothesize that coupling molecular profiling of brain tumors with clinical information might improve predictions of patient survival time and, consequently, better guide future treatment decisions. …

Mechanism-Based Strategies To Enhance The Actions Of A, Fabiola C. Gomez

Dissertations and Theses (Open Access)

Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the functional stability of client oncoproteins, such as STAT3, Raf-1 and Akt, which play a role in the survival of malignant cells. The chaperone function of HSP90 is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins. However, treatment with 17-AAG results in the elevation of the levels of antiapoptotic proteins HSP70 and HSP27, which may lead to cell death resistance. The increase in HSP70 and HSP27 protein levels is due …

Cip4 And Src In Promoting The Migration And Invasion Of Breast Cancers, Christina S. Pichot

Dissertations and Theses (Open Access)

Cellular invasion represents a critical early step in the metastatic cascade, and many proteins have been identified as part of an “invasive signature.” The non-receptor tyrosine kinase Src is commonly upregulated in breast cancers, often in conjunction with overexpression of EGFR. Signaling from this pathway stimulates cell proliferation, migration, and invasion and frequently involves proteins that regulate the cytoskeleton. My data demonstrates that inhibition of Src, using the small-molecule inhibitor dasatinib, impairs cellular migration and invasion. Furthermore, Src inhibition sensitizes the cells to the effects of the chemotherapeutic doxorubicin resulting in dramatic, synergistic inhibition of proliferation with combination treatments. The …

Inhibition Of Deubiquitinase Activity And Ubiquitination Of Jak2 Blocks Cytokine Signaling And Induces Tumor Cell Apoptosis, Vaibhav Kapuria

Dissertations and Theses (Open Access)

The Jak-stat pathway is critical for cellular proliferation and is commonly found to be deregulated in many solid tumors as well as hematological malignancies. Such findings have spurred the development of novel therapeutic agents that specifically inhibit Jak2 kinase, thereby suppressing tumor cell growth. Tyrphostin AG490, the first described Jak2 inhibitor, displays poor pharmacology and requires high concentrations for anti-tumor activities. Our research group screened a small library of AG490 structural analogues and identified WP1130 as a potent inhibitor of Jak2 signaling. However, unlike AG490, WP1130 did not directly inhibit Jak2 kinase activity. Our results show that WP1130 induces rapid …

The Ubiquitin Ligase Ube4b Is Required For Efficient Epidermal Growth Factor Receptor Degradation, Natalie Sirisaengtaksin

Dissertations and Theses (Open Access)

The length of time that integral membrane proteins reside on the plasma membrane is regulated by endocytosis, a process that can inactivate these proteins by removing them from the membrane and may ultimately result in their degradation. Proteins are internalized and pass through multiple distinct intracellular compartments where targeting decisions determine their fate. Membrane proteins initially enter early endosomes, and subsequently late endosomes/multivesicular bodies (MVBs), before being degraded in the lysosome. The MVB is a subset of late endosomes characterized by the appearance of small vesicles in its luminal compartment. These vesicles contain cargo proteins sorted from the limiting membrane …

Cellular And Developmental Functions Of The Xenopus Arvcf-Catenin:Kazrin Complex, Kyucheol Cho

Dissertations and Theses (Open Access)

Xenopus ARVCF (xARVCF), a member of p120-catenin subfamily, binds cadherin cytoplasmic domains to enhance cadherin metabolic stability, or when dissociated, modulates Rho-family GTPases. We previously found that xARVCF binds directly to Xenopus KazrinA (xKazrinA), a widely expressed, conserved protein that bears little homology to established protein families. xKazrinA is also known to influence keratinocyte proliferation-differentiation and cytoskeletal activity. In my study, I first evaluated the expression pattern of endogenous Kazrin RNA and protein in Xenopus embryogenesis as well as in adult tissues. We then collaboratively predicted the helical structure of Kazrin’s coiled-coil domain, and I obtained evidence of Kazrin’s dimerization/oligomerization. …

Identification Of A Conserved Cluster In The Rh Domain Of Grk Critical For Activation By Gpcrs, Faiza Baameur

Dissertations and Theses (Open Access)

One of the most critical aspects of G Protein Coupled Receptors (GPCRs) regulation is their rapid and acute desensitization following agonist stimulation. Phosphorylation of these receptors by GPCR kinases (GRK) is a major mechanism of desensitization. Considerable evidence from studies of rhodopsin kinase and GRK2 suggests there is an allosteric docking site for the receptor distinct from the GRK catalytic site. While the agonist-activated GPCR appears crucial for GRK activation, the molecular details of this interaction remain unclear. Recent studies suggested an important role for the N- and C-termini and domains in the small lobe of the kinase domain in …