Cell and Developmental Biology Commons^™

Identification Of Proteins At Active, Stalled, And Collapsed Replication Forks Using Isolation Of Proteins On Nascent Dna (Ipond) Coupled With Mass Spectrometry, Bianca M. Sirbu, W. Hayes Mcdonald, Huzefa Dungrawala, Akosua Badu-Nkansah, Gina M. Kavanaugh, Yaoyi Chen, David L. Tabb, David Cortez

Molecular Biosciences Faculty Publications

Both DNA and chromatin need to be duplicated during each cell division cycle. Replication happens in the context of defects in the DNA template and other forms of replication stress that present challenges to both genetic and epigenetic inheritance. The replication machinery is highly regulated by replication stress responses to accomplish this goal. To identify important replication and stress response proteins, we combined isolation of proteins on nascent DNA (iPOND) with quantitative mass spectrometry. We identified 290 proteins enriched on newly replicated DNA at active, stalled, and collapsed replication forks. Approximately 16% of these proteins are known replication or DNA …

Functional And Mutational Analysis Of Human Recq-Like Dna Helicases In Saccharomyces Cerevisiae, Hamed Mirzaei-Souderjani

USF Tampa Graduate Theses and Dissertations

RecQ-like proteins are a family of DNA helicases that are evolutionary conserved from prokaryotes to eukaryotes. A large amount of experimental evidence suggests these proteins have a major role in the maintenance of genome stability. In humans five RecQ like helicase have been identified (RecQL1, BLM, WRN, RecQL4, and RecQL5), three of which are associated with rare genetic disorders with sever chromosomal and developmental abnormalities, and an elevated predisposition to cancer. Among the disease associated RecQ-like helicases, BLM and WRN have been subject to extensive research, while our collective knowledge about the function of RecQL4 is still very limited. Similarly, …

Sirt1 Regulation Of The Heat Shock Response In An Hsf1-Dependent Manner And The Impact Of Caloric Restriction, Rachel Rene Raynes

USF Tampa Graduate Theses and Dissertations

The heat shock response (HSR) is the cell's molecular reaction to protein damaging stress and is critical in the management of denatured proteins. Activation of HSF1, the master transcriptional regulator of the HSR, results in the induction of molecular chaperones called heat shock proteins (HSPs). Transcription of hsp genes is promoted by the hyperphosphorylation of HSF1, while the attenuation of the HSR is regulated by a dual mechanism involving negative feedback inhibition from HSPs and acetylation at a critical lysine residue within the DNA binding domain of HSF1, which results in a loss of affinity for DNA. SIRT1 is a …

Regulation Of The Tumor Suppresser P53 And Survivin By Ras And Ral Gtpases:Implications For Malignant Transformation, Awet G. Tecleab

USF Tampa Graduate Theses and Dissertations

Abstract

Although the critical role of the small GTPases Ras and Ral in oncogenesis has been well documented, much remains to be investigated about the molecular mechanism by which these GTPases regulate malignant transformation. The work under this thesis made two major contributions to this field. The first is the discovery that K-Ras, RalA and/or RalB are required for the maintenance of the high levels of the anti-apoptotic protein survivin in some human cancer cells, and the second is the demonstration that down regulation of K-Ras, RalA and/or RalB, but not Raf-1 or Akt1/2, stabilizes the tumor suppressor p53 and …

Epigenetic Modifiers To Augment The Immunogenicity Of Chronic Lymphocytic Leukemia, Jason A. Dubovsky

USF Tampa Graduate Theses and Dissertations

Cancer cells employ a litany of immunosuppressive and immunevasive strategies to avoid detection and elimination by the various arms of the innate and adaptive immune system. Many hematologic and solid tumors progressively develop a specialized microenvironment which promotes tissue restructuring inflammation while masking the immune signature of the tumor cells themselves. Chronic lymphocytic leukemia, a malignancy of mature B lymphocytes must constantly balance on the precipice of immune recognition. A mature antigen presenting cell themselves, CLL clonal growth is dependent on the very interactions which, if effective, could potentially lead to their demise. To circumvent this, CLL clones set up …

Exploring Potential Risk Factors Of Fetal Origins Of Diabetes: Maternal Stressors During Pregnancy And Birth Outcomes Among Women In A Hospital In The Municipality Of Caguas, Puerto Rico, Juan Pablo Arroyo

USF Tampa Graduate Theses and Dissertations

Puerto Rico has the highest prevalence of type 2 diabetes, low birth-weight, and the second highest prevalence of preterm-birth in all the U.S. and its non-incorporated territories. These conditions are related. Birth-weight at both ends of the spectrum and preterm-birth are associated with an increased risk for developing type 2 diabetes and immune-inflammatory dysregulations. Maternal psychosocial stressors during pregnancy have also been recognized as potential risk factors for type 2 diabetes, and have been consistently associated with preterm-birth and low birth-weight across populations. Current evidence points toward epigenetic fetal metabolic-programming as the mechanism that underlies the increased risk for the …

Phosphorylation Of Histone Deacetylase 6 Within Its C-Terminal Region By Extracellular Signal Regulated Kinase 1, Kendra Allana Williams

USF Tampa Graduate Theses and Dissertations

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Drug Resistance Mechanisms To Gamma-Secretase Inhibitors In Human Colon Cancer Cells, Cindy R. Timme

USF Tampa Graduate Theses and Dissertations

Colorectal cancer is the third leading cause of cancer-related mortality. Much progress has been achieved in combating this disease with surgical resection and chemotherapy in combination with targeted drugs. However, most metastatic patients develop drug resistance so new modalities of treatment are needed.

Notch signaling plays a vital role in intestinal homeostasis, self-renewal, and cell fate decisions during post-development and is activated in colorectal adenocarcinomas. Under debate is its role in carcinomas and metastatic disease. In theory, blocking Notch activation using gamma-secretase inhibitors (GSIs) may show efficacy alone or in combination with chemotherapy in the treatment of colon cancer.

In …

The Role Of Argininosuccinate Synthase Serine 328 Phosphorylation In Nitric Oxide Production, Ricci Haines

USF Tampa Graduate Theses and Dissertations

Until recently, the main mechanism of argininosuccinate synthase (AS) regulation was described to exist mainly at the level of transcription. Transcriptional regulation of AS has been shown to be coordinate with eNOS in response to shear stress, hypoxia, tumor necrosis factor á (TNF-á), and PPAR ã agonist troglitizone. However, it is now understood that one level of NO regulation is cellular control of arginine availability to eNOS via post-translational modifications of AS such as phosphorylation. The purpose of this investigation was to determine under what conditions AS is phosphorylated at S328, identify the pathway that AS phosphorylation at S328 plays …

Identification Of Amino Acids Essential For Estrone-3-Sulfate Transport Within Transmembrane Domain 2 Of Organic Anion Transporting Polypeptide 1b1, Nan Li, Weifang Hong, Hong Huang, Hanping Lu, Guangyun Lin, Mei Hong

School of Information Faculty Publications

As an important structure in membrane proteins, transmembrane domains have been found to be crucial for properly targeting the protein to cell membrane as well as carrying out transport functions in transporters. Computer analysis of OATP sequences revealed transmembrane domain 2 (TM2) is among those transmembrane domains that have high amino acid identities within different family members. In the present study, we identify four amino acids (Asp70, Phe73, Glu74, and Gly76) that are essential for the transport function of OATP1B1, an OATP member that is specifically expressed in the human liver. A substitution of these four amino acids with alanine …

The Role Of Tumor Suppressors, Ship And Rb, In Immune Suppressive Cells, Michelle Marie Collazo Ruiz

USF Tampa Graduate Theses and Dissertations

Regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC) have been extensively studied in the past 30-40 years. Their potent suppressive capacity shown in several pathological and clinical settings, such as cancer and transplantation, has made it evident that better understanding their development and function is critical.

Specifically, Tregs play a pivotal role in preventing autoimmunity, graft-versus-host disease (GvHD), and organ graft rejection. We previously demonstrated that germline or induced SH2 domain-containing inositol 5-phosphatase (SHIP) deficiency in the host abrogates GvHD. Here we show that SHIP-deficiency promotes an increase of FoxP3+ cells in both the CD4+CD25+ and the CD4+CD25- T …

Cell Wall Antibiotics Provoke Accumulation Of Anchored Mcherry In The Cross Wall Of Staphylococcus Aureus, Wenqi Yu, Friedrich Götz

Molecular Biosciences Faculty Publications

A fluorescence microscopy method to directly follow the localization of defined proteins in Staphylococcus was hampered by the unstable fluorescence of fluorescent proteins. Here, we constructed plasmid (pCX) encoded red fluorescence (RF) mCherry (mCh) hybrids, namely mCh-cyto (no signal peptide and no sorting sequence), mCh-sec (with signal peptide), and mCh-cw (with signal peptide and cell wall sorting sequence). The S. aureus clones targeted mCh-fusion proteins into the cytosol, the supernatant and the cell envelope respectively; in all cases mCherry exhibited bright fluorescence. In staphylococci two types of signal peptides (SP) can be distinguished: the +YSIRK motif SPlip and the −YSIRK …

Discovery Of Marinopyrrole A (Maritoclax) As A Selective Mcl-1 Antagonist That Overcomes Abt-737 Resistance By Binding To And Targeting Mcl-1 For Proteasomal Degradation*, Kenichiro Doi, Rongshi Li, Shen-Shu Sung, Hongwei Wu, Yan Liu, Wanda Manieri, Gowdahalli Krishnegowda, Andy Awwad, Alden Dewey, Xin Liu, Shantu Amin, Chunwei Cheng, Yong Qin, Ernst Schonbrunn, Gary W. Daughdrill, Thomas P. Loughran Jr., Said M. Sebti, Hong-Gang Wang

Molecular Biosciences Faculty Publications

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-XL and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-XL with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified …

An Ims/Atp Assay For The Detection Of Mycobacterium Tuberculosis In Urine, Dawn M. Hunter, Daniel V. Lim

Molecular Biosciences Faculty Publications

Background. Although sputum smears are the gold standard for diagnosis of tuberculosis, sensitivity in HIV/TB coinfection cases is low, indicating a need for alternative methods. Urine is being increasingly evaluated. Materials and Methods. A novel method for detecting Mycobacterium tuberculosis (MTB) in synthetic urine using a combined IMS/ATP assay was evaluated. Preliminary work established standard ATP conditions and the sensitivity and specificity of the MTB antibody. Eighty-four blinded samples in four replicate assays were evaluated for the presence of MTB using labeled immunomagnetic beads for capture. Beads were separated, washed, and resuspended in broth and added to a …

Identification Of New Cell Size Control Genes In S. Cerevisiae, Huzefa Dungrawala, Hui Hua, Jill Wright, Lesley Abraham, Thivakorn Kasemsri, Anthony Mcdowell, Jessica Stilwell, Brandt L. Schneider

Molecular Biosciences Faculty Publications

Cell size homeostasis is a conserved attribute in many eukaryotic species involving a tight regulation between the processes of growth and proliferation. In budding yeast S. cerevisiae, growth to a “critical cell size” must be achieved before a cell can progress past START and commit to cell division. Numerous studies have shown that progression past START is actively regulated by cell size control genes, many of which have implications in cell cycle control and cancer. Two initial screens identified genes that strongly modulate cell size in yeast. Since a second generation yeast gene knockout collection has been generated, we screened …

The Two-Component Signal Transduction System Arlrs Regulates Staphylococcus Epidermidis Biofilm Formation In An Ica-Dependent Manner, Yang Wu, Jiaxue Wang, Tao Xu, Jingran Liu, Wenqi Yu, Qiang Lou, Tao Zhu, Nianan He, Haijing Ben, Jian Hu, Friedrich Götz, Di Qu

Molecular Biosciences Faculty Publications

Due to its ability to form biofilms on medical devices, Staphylococcus epidermidis has emerged as a major pathogen of nosocomial infections. In this study, we investigated the role of the two-component signal transduction system ArlRS in regulating S. epidermidis biofilm formation. An ArlRS-deficient mutant, WW06, was constructed using S. epidermidis strain 1457 as a parental strain. Although the growth curve of WW06 was similar to that of SE1457, the mutant strain was unable to form biofilms in vitro. In a rabbit subcutaneous infection model, sterile disks made of polymeric materials were implanted subcutaneously followed with inoculation of WW06 or SE1457. …

Wipi-1 Positive Autophagosome-Like Vesicles Entrap Pathogenic Staphylococcus Aureus For Lysosomal Degradation, Mario Mauthe, Wenqi Yu, Oleg Krut, Martin Krönke, Friedrich Götz, Horst Robenek, Tassula Proikas-Cezanne

Molecular Biosciences Faculty Publications

Invading pathogens provoke the autophagic machinery and, in a process termed xenophagy, the host cell survives because autophagy is employed as a safeguard for pathogens that escaped phagosomes. However, some pathogens can manipulate the autophagic pathway and replicate within the niche of generated autophagosome-like vesicles. By automated fluorescence-based high content analyses, we demonstrate that Staphylococcus aureus strains (USA300, HG001, SA113) stimulate autophagy and become entrapped in intracellular PtdIns(3)P-enriched vesicles that are decorated with human WIPI-1, an essential PtdIns(3)P effector of canonical autophagy and membrane protein of both phagophores and autophagosomes. Further, agr-positive S. aureus (USA300, HG001) strains were more efficiently …

Comparative Developmental Transcriptomics Of Echinoderms, Roy Vaughn

USF Tampa Graduate Theses and Dissertations

The gastrula stage represents the point in development at which the three primary germ layers diverge. At this point the gene regulatory networks that specify the germ layers are established and the genes that define the differentiated states of the tissues have begun to be activated. These networks have been well characterized in sea urchins, but not in other echinoderms. Embryos of the brittle star Ophiocoma wendtii share a number of developmental features with sea urchin embryos, including the ingression of mesenchyme cells that give rise to an embryonic skeleton. Notable differences are that no micromeres are formed during cleavage …

Lenalidomide Targets The T-Cell Co-Stimulatory Pathway To Mediate Immune Modulation, Jessica Marie Mcdaniel

USF Tampa Graduate Theses and Dissertations

T-cells are lymphocytes that make up part of the adaptive arm of the immune system, and are essential for efficient protection from and eradication of viruses and pathogens. T-cells not only play an important role in protection from external agents, but also regulate and prevent activation towards self-peptides and detect and remove erratically growing cells. Alterations in T-cell activation and suppression contribute to auto-immunity, immunocompromised disorders, and cancer progression.

The immune system, and T-cells in particular, provides daily surveillance, recognition and destruction of aberrant cells. Although the immune system is proficient at suppressing malignant progression, tumor cells acquire various methods …

Identification Of Novel Stat3 Target Genes Associated With Oncogenesis, Rachel Haviland

USF Tampa Graduate Theses and Dissertations

Cytokine and growth factor signaling pathways involving STAT3 are frequently constitutively activated in many human primary tumors, and are known for the transcriptional role they play in controlling cell growth and cell cycle progression. However, the extent of STAT3's reach on transcriptional control of the genome as a whole remains an important question. We predicted that this persistent STAT3 signaling affects a wide variety of cellular functions, many of which still remain to be characterized.

We took a broad approach to identify novel STAT3 regulated genes by examining changes in the genome-wide gene expression profile by microarray, using cells expressing …

Disorder Predictors Also Predict Backbone Dynamics For A Family Of Disordered Proteins, Gary W. Daughdrill, Wade M. Borcherds, Hongwei Wu

Molecular Biosciences Faculty Publications

Several algorithms have been developed that use amino acid sequences to predict whether or not a protein or a region of a protein is disordered. These algorithms make accurate predictions for disordered regions that are 30 amino acids or longer, but it is unclear whether the predictions can be directly related to the backbone dynamics of individual amino acid residues. The nuclear Overhauser effect between the amide nitrogen and hydrogen (NHNOE) provides an unambiguous measure of backbone dynamics at single residue resolution and is an excellent tool for characterizing the dynamic behavior of disordered proteins. In this report, we show …

Regulation Of Cell Adhesion Strength By Spatial Organization Of Focal Adhesions, Kranthi Kumar Elineni

USF Tampa Graduate Theses and Dissertations

Cell adhesion to extracellular matrix (ECM) is critical to various cellular processes like cell spreading, migration, growth and apoptosis. At the tissue level, cell adhesion is important in the pathological and physiological processes that regulate the tissue morphogenesis. Cell adhesion to the ECM is primarily mediated by the integrin family of receptors. The receptors that are recruited to the surface are reinforced by structural and signaling proteins at the adhesive sites forming focal adhesions that connect the cytoskeleton to further stabilize the adhesions. The functional roles of these focal adhesions extend beyond stabilizing adhesions and transduce mechanical signals at the …

From Dna To Protein: A Study Of Genomic Instability Candidate Genes During Zebrafish Development, Kristine Griffett

USF Tampa Graduate Theses and Dissertations

The zebrafish, Danio rerio, is a type of freshwater minnow often used to model human diseases including cancer, anxiety and aging diseases. The overall biology of zebrafish is strikingly similar to that of humans, allowing these fish to be used for drug discovery and toxicology studies for preclinical trials. In this study, zebrafish embryos were used to identify and characterize several candidate genes within two known regions of genomic instability on chromosome 18 and chromosome 4. This fish that were used in this study had been previously classified as genomic instability (gin) mutants due to increased incidence of somatic mutation …

Inhibition Of P53 Dna Binding Function By The Mdm2 Acidic Domain, Brittany Lynne Cross

USF Tampa Graduate Theses and Dissertations

MDM2 regulates p53 predominantly by promoting p53 ubiquitination. However, ubiquitination-independent mechanisms of MDM2 have also been implicated. Here we show that MDM2 inhibits p53 DNA binding activity in vitro and in vivo. MDM2 binding promotes p53 to adopt a mutant-like conformation, losing reactivity to antibody Pab1620, while exposing the Pab240 epitope. The acidic domain of MDM2 is required to induce p53 conformational change and inhibit p53 DNA binding. ARF binding to the MDM2 acidic domain restores p53 wild type conformation and rescues DNA binding activity. Furthermore, histone methyl transferase SUV39H1 binding to the MDM2 acidic domain also restores p53 wild …

Role Of Protein Kinase C-Iota In Glioblastoma, Shraddha R. Desai

USF Tampa Graduate Theses and Dissertations

The focus of this research was to investigate the role of protein kinase C-iota (PKC-é) in the regulation of Bad function, a pro-apoptotic member of the Bcl-2 family and Cdk7 function, a master cell cycle regulator in glioblastoma.

The results were obtained from the human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-é co-localized and directly associated with Bad as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-é directly phosphorylated Bad at phospho specific residues, S112, S136 and S155 which in turn induced inactivation of Bad and disruption of …

Using A Focus Measure To Automate The Location Of Biological Tissue Surfaces In Brightfield Microscopy, Daniel Toby Elozory

USF Tampa Graduate Theses and Dissertations

The study of microstructures in brightfield microscopy using unbiased stereology plays a large and growing role in bioscience research. Stereology enables objective quantitative analysis of biological structures within a tissue sample. A first step in the stereology process is to calculate the thickness of a tissue sample by locating the top and bottom surfaces of the sample. The aim of this project is to fully automate this location process by using the relative optical focus measure as an indicator of tissue surface boundary.

The current method for identification of focus bounding planes requires a trained user to manually select the …

Role Of Protein Kinase C-Iota In Neuroblastoma And The Effect Of Ica-1, A Novel Protein Kinase C-Iota Inhibitor On The Proliferation And Apoptosis Of Neuroblastoma Cells, Prajit P. Pillai

USF Tampa Graduate Theses and Dissertations

Protein Kinase C-iota (PKC-é), an atypical protein kinase C isoform manifests its potential as an oncogene by targeting various aspects of cancer cells such as growth, invasion and survival. PKC-é confers resistance to drug-induced apoptosis in cancer cells. The acquisition of drug resistance is a major obstacle to good prognosis in neuroblastoma. The focus of the dissertation was three-fold: First to study the role of PKC-é in the proliferation of neuroblastoma. Secondly, to identify the efficacy of [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) as a novel PKC-é inhibitor in neuroblastoma cell proliferation and apoptosis. Finally, to analyze whether PKC-é could self-regulate …

The Roles Of Elevated Bcl-2 In Ovarian Cancer, Nicole Shree Anderson

USF Tampa Graduate Theses and Dissertations

Ovarian cancer (OC) is the second most common gynecologic cancer; however it is responsible for the most gynecologic cancer-related deaths. Apoptosis evasion is an important mechanism in OC tumorigenesis, and the prototypic anti-apoptotic protein, B-cell lymphoma 2 (Bcl-2), is often overexpressed in OC tumors. Gaining a better understanding of the mechanism(s) behind Bcl-2 overexpression and potential extra-anti-apoptotic functions of Bcl-2 could elucidate the importance of elevated Bcl-2 in OC. In the current study, I show through immunohistochemical analysis of normal, benign, and OC tissue sections, that both epithelial and stromal Bcl-2 expression decreases with OC progression. However, the number of …

Molecular Mechanism Of Agc Kinases In Human Malignant, Shaokun Shu

USF Tampa Graduate Theses and Dissertations

The maintenance of normal cell function and tissue homeostasis is dependent on the precise regulation of multiple signaling pathways that control cellular decisions to either proliferate, differentiate, arrest cell growth, or initiate programmed cell death (apoptosis). Cancer arises when clones of mutated cells escape this balance and proliferate inappropriately without compensatory apoptosis. Deregulated cell growth occurs as a result of perturbed signal transduction that modulates or alters cellular behavior or function to keep the critical balance between the rate of cell-cycle progression (cell division) and cell growth (cell mass) on one hand, and programmed cell death (apoptosis, autophagy) on the …

The Human In 3d: Advanced Morphometric Analysis Of High-Resolution Anatomically Accurate Computed Models, Summer J. Decker

USF Tampa Graduate Theses and Dissertations

Computed virtual models of anatomical structures are proving to be of increasing value in clinical medicine, education and research. With a variety of fields focused on craniofacial and pelvic anatomy there is a need for accurate anatomical models. Recent technological advancements in computer and medical imaging technologies have provided the tools necessary to develop three-dimensional (3D) functional models of human anatomy for use in medicine (surgical planning and education), forensics and engineering (computer-aided design (CAD) and finite element analysis).

Traditionally caliper methodologies are used in the quantitative analysis of human anatomy. In order for experts in anatomy and morphometrics to …