Microbiology Commons^™

Oxygen Sensing By The Aer And Tsr Receptors Of Escherichia Coli, Jessica Claire Edwards

Loma Linda University Electronic Theses, Dissertations & Projects

Escherichia coli senses the oxygen concentration of its environment via the Aer and Tsr receptors, and swims to an oxygen concentration that provides optimal energy generation. Aer and Tsr do not sense oxygen per se but, sense the internal energy state via the electron transport system (ETS) in a previously unidentified mechanism. Proton motive force (PMF) and electron flow were proposed as possible sensory signals but, distinguishing between the two was not possible, previously, in live, motile cells. I differentiated PMF from electron flow by creating a series of ETS mutant strains that express ternary combinations of i) aerotaxis receptors, …

Enterotoxin B Subunit Lectins As Adjuvants For Improvement Of Mucosal Vaccine, Nak-Won Choi

Loma Linda University Electronic Theses, Dissertations & Projects

In comparison with whole organism vaccines, subunit vaccines may be safer for immunization but may lack sufficient immunogenicity to provide complete immunity to the pathogen. To resolve this problem, bacterial and plant enterotoxin B subunit adjuvants containing a variety of receptor-binding properties were used to enhance the immunogenicity of rotavirus subunit vaccines. Enterotoxin B subunit adjuvants were employed to enhance protection against virus infection. Pentameric cholera toxin B subunit (CTB), shiga toxin-1 B subunit (STB) and monomeric ricin toxin B subunit (RTB) molecules were genetically linked to a 90 amino acid peptide from the simian rotavirus (SA11) nonstructural protein NSP4 …

The Potential Role Of Ledgf/P75 In Prostate Cancer, Tracy Ruth Daniels

Loma Linda University Electronic Theses, Dissertations & Projects

The serum autoantibody repertoire from cancer patients is currently being exploited for the identification of tumor associated antigens (TAA) and the design of TAA arrays. Such arrays would facilitate autoantibody profiling and potentially aid in the serological diagnosis and prognosis of tumors. The goal of this study was to identify and characterize prostate-specific TAA. We observed that while the general frequency and titers of autoantibodies in PCa patients were relatively similar to those in matched controls, significant differences could be detected between the two groups in the autoantibody response to the lens epithelial-derived growth factor p75 (LEDGF/p75). LEDGF/p75 is a …

Mechanisms Of Oxidative Stress Resistance In Porphyromonas Gingivalis W83, Neal Antonio Johnson

Loma Linda University Electronic Theses, Dissertations & Projects

Porphyromonas gingivalisis one of the most important bacterial etiological agents involved in adult periodontitis. It possesses several virulence factors which aid in its persistence in the periodontal pocket leading to the development of some systemic illnesses. As an anaerobe, P. gingivalismust employ mechanisms to avoid oxidative stress generated by reactive oxygen species during phagocytosis, or the occasional exposure to air. Presently, only three proteins with antioxidant enzymatic function have been identified as playing a role in oxidative stress resistance in P. gingivalis. In this study, attention was focused on the alkyl hydroperoxidase subunit C (ahpC)and bacterioferritin comigratory …

Detection Of Bacteria To Dental Tissue By Low-Light Imaging, G-Hong Robert Hsu

Loma Linda University Electronic Theses, Dissertations & Projects

Bacteria have been shown to play an important role in the development of pulp and periradicular disease. The source of the infective bacteria is most often the oral cavity. Previous studies have detected bacteria in teeth that are structurally intact that seemingly have no communication intraorally. Anachoresis has been proposed as a mechanism for localizing circulating bacteria to such teeth. In several previous bacteriologic anachoresis studies, bacteria have been found to localize in inflamed dental tissues. The criticism most commonly associated with these investigations has been the potential contribution of contaminant bacteria resulting in false positive findings.

Low-light image analysis …

The Biological Burden Of Antibiotic Resistance In Staphylococcus Aureus, Natasha Samantha Dean

Loma Linda University Electronic Theses, Dissertations & Projects

Antibiotic resistance is a major problem. Resistance is often mediated by genes on bacterial plasmids and dissemination of resistance is commonly achieved by plasmid transfer. The mechanisms of resistance that are presently understood suggest that resistance is energetically costly. If this is generally true, resistance would reduce the fitness of cells in the absence of antibiotics. We examined the hypothesis that resistance reduces the fitness of microorganism and requires continuous selection to be maintained. To test this hypothesis, we measured the effects of plasmid-borne resistance genes on cell growth by comparing the growth rates of Staphylococcus aureus with and without …

A Novel Method To Detect Bacterial Restriction-Modification (R-M) Systems, Julie Kay Armantrout Kasarjian

Loma Linda University Electronic Theses, Dissertations & Projects

Analysis of recently sequenced microbial genomes has revealed many DNA sequences that code for previously unknown restriction endonucleases and their corresponding methyltransferases. These findings show that numerous restriction enzymes abundant in bacteria have yet to be discovered. Traditionally, restriction enzymes have been discovered by the classical restriction and modification (R-M) phenomena of bacteriophages (type I and III enzymes), or by direct enzyme assays (type II enzymes). To avoid the limitations of these traditional approaches, a quantitative R-M test based on plasmid transformation efficiency (Plasmid R-M Test) was established using DNA fragments derived from the E. coli bacteriophage lambda. This test …

Potential Mechanisms Explaining The Antitumor Effect Of Total-Body Irradiation, Glen Michael Miller

Loma Linda University Electronic Theses, Dissertations & Projects

While numerous reports have documented that radiation exposure increases the risk for malignancy and suppresses immune mechanisms, increasing evidence has suggested that low-dose total-body irradiation (TBI) may alter leukocyte composition and function leading to heightened immune responsiveness and long-term remission of certain cancers. Having observed that moderate-dose TBI produces an antitumor effect in the Lewis lung carcinoma (EEC) model, the major goal of this study was to determine whether changes in tumor growth could be correlated with radiation-induced alterations of immune system parameters. The governing hypothesis was that selective immune augmentation, i.e. upregulation of specific leukocyte subsets, is primarily responsible …

Mechanisms Of Autoantigen Cleavage During Cell Death, Xiwei Wu

Loma Linda University Electronic Theses, Dissertations & Projects

Specific intracellular autoantigens targeted by autoantibodies in systemic autoimmune and chronic inflammatory diseases undergo posttranslational modifications, such as cleavage, during cell death that could potentially enhance their immunogenicity. In light of the increasing interest in the immunological consequences of defective clearance of apoptotic cells, we explored whether autoantigens cleaved during apoptosis undergo an additional wave of proteolysis as apoptosis progresses to secondary necrosis in the absence of phagocytosis. Jurkat T cells treated with different apoptosis inducers underwent a rapid apoptosis that gradually progressed to secondary necrosis. During the initial apoptotic stages, several nuclear autoantigens, including PARP, Topo I, and LEDGF/p75 …

Hamp Domain And Signaling Mechanism Of The Aer Protein, Qinhong Ma

Loma Linda University Electronic Theses, Dissertations & Projects

Escherichia coli utilizes the Aer protein to sense changes in the intracellular energy level and guide the bacteria to a niche where the cellular energy level is optimal. Aer contains a PAS sensory domain and an FI fragment in the N terminus and a chemotaxis signaling domain in the C terminus, separated by a putative transmembrane region. In this dissertation, I report that the linker region of Aer (residues 205-265), which joins the transmembrane region to the signaling domain, is a conserved signal transduction module in numerous methyl-accepting chemoreceptors, histidine kinases, adenylyl cyclases, and other sensory proteins in all three …

Development Of A Model For Infective Endocarditis Via Intravenous Injection And Tooth Infection Of Rats Using Pathogenic Bacteria, Shahrokh Shabahang

Loma Linda University Electronic Theses, Dissertations & Projects

Historically, focal areas of infection have been linked to a number of systemic ailments. During the early part of the 20th century, patients who were suffering from inexplicable diseases and who were suspected of having infected teeth were referred to the dentist for multiple extraction of their dentition. The discovery of antibiotics provided a false sense of security that the war against microorganisms is no longer a concern for mankind. However, more recently, it has become common belief that increasing numbers of systemic diseases are linked to chronic infections. Recovery of bacteria from representative tissues has led investigators to believe …

A Novel Strategy Utilizing Co-Expression Of Murine Il-12 And Antisense Tgf-Pl Against H238 Tumor Formation, Craig A. Seheult

Loma Linda University Electronic Theses, Dissertations & Projects

Cytokines have a profound effect on immune modulation, thus playing a significant role in cancer gene therapy. A variety of human carcinomas secrete transforming growth factor-beta (TGF-β), a cytokine with potent immunosuppressive properties. Suppressing TGF-β could be a key to successfully combating malignancies, such as gliomas, mammary, and colon cancers, that over-express TGF-β. Conversely, interleukin-12 (IL-12) is a potent immunostimulatory cytokine capable of regulating T and NK cell-mediated cytotoxic responses (Trinchieri, 1994) during an anti-tumor response. Activation of the immune system with such immunostimulatory cytokines renders it inert to the suppressive effects of TGF-β. To better understand these processes our …

Effects Of Liposome-Encapsulated Tnf-Alpha And Radiation Against Colon Cancer, Dong Won Kim

Loma Linda University Electronic Theses, Dissertations & Projects

Previous studies have shown that tumor necrosis factor-α (TNF-α) can augment the antitumor effects of radiation, inhibiting tumor progression to a greater extent than either modality alone. However, nonspecific toxicities associated with TNF-α have limited its use as an adjuvant. This limitation is potentially resolved with the encapsulation of TNF-α in sterically stabilized, PEGylated liposomes (STEALTH®). Thus, the overall hypothesis of the research was that the combination of radiation with STEALTH® liposome encapsulated TNF-α (SL-TNF-α) will have greater efficacy and fewer dose-limiting side effects in the LS174T human colorectal tumor xenograft model compared to the combination of free TNF-α and …

Identification Of The Polar Flagellum Genes In Azospirillum Brasilense, Enid Thanna Mckinley

Loma Linda University Electronic Theses, Dissertations & Projects

Azospirillum brasilenser, a nitrogen fixing rhizobacterium, can colonize plant roots and increase the yield of economically important cereals, such as rice and grasses. This organism has a mixed pattern of flagellation: a single polar flagellum for motility in liquid medium and lateral flagella which might be involved in motility across the root surface and or attachment to the root hairs of plants. Flagella dictate A. brasilense chemotactic ability and motility, which plays an important role in colonization of plant roots. However, nothing is known about the genes that code for the polar flagellum. To study the polar flagellum, different mutants …

Il-2 And Antisense Tgf-B1 Gene Therapy Of The H238 Tumor And Analysis Of Tgf-B Receptors, Azim Moiz Mohamedali

Loma Linda University Electronic Theses, Dissertations & Projects

Transforming growth factor-beta (TGF-p) belongs to a family of immunosuppressive cytokines, capable of regulating macrophages, T, B and NK cells. We have detected the TGF-p transcript being synthesized by H238 cells. This is herpes simplex-Type2 transformed murine fibrosarcoma cell line. The amount of TGF-p formed/106 cells was >2000pg/ml. Previous attempts to control H238 tumorigenicity had sub-optimal efficacies. In addition, studies had shown that the FI238 cell line was not responsive to TGF-p. This project was an attempt to develop an improved modality to reduce H238 tumorigenicity and investigate potential abnormalities in expression of TGF-p receptors in the H238 cell line. …

Vaccinia Virus-Mediated Glioma Gene Therapy Using P53, Il-2 And Il-12, Bing Chen

Loma Linda University Electronic Theses, Dissertations & Projects

Malignant brain tumors are generally lethal soon after their diagnosis, and the five-year survival rate for the patient is low in spite of neurosurgery, radiation therapy, and/or chemotherapy. Gene therapy has the potential to improve patient survival and quality of life by delivering specific combinations of therapeutic genes directly to the tumor for localized treatment.

We have based our cancer gene therapy for glioma on a highly attenuated variant of the Lister strain of vaccinia virus (VV) which has been proven to be a safe and effective vaccine vector. Other advantages include a wide host range, a strong promoter system, …

Connexin-32 Contributes To Radiation Resistance In Thyroid Follicles, Da-Thao Dinh Tran

Loma Linda University Electronic Theses, Dissertations & Projects

The objective of this study was to examine the role of gap junctions in the radiation resistance of thyroid follicles. The question of whether gap junctional intercellular communication plays an important role in the “contact effect”, wherein cells in direct apposition are more resistant to the damaging effects of radiation, has been debated for years. We used the Fischer rat thyroid cell line (FRTL-5) to carry out our experiments. This thesis is divided into two parts: 1) characterization of gap junctional properties in FRTL-5 cells and, 2) exposure of these cells to radiation.

We found that, unlike primary thyroid cells, …

Function And Properties Of Groe Chaperonins In Bacterial And Mammalian Cells, Gregory M. Nelson

Loma Linda University Electronic Theses, Dissertations & Projects

Molecular chaperones play an integral role in the folding of most polypeptides in vivo, and protect proteins against aggregation when a cell is under stress. The GroESL proteins of Escherichia coli are the best characterized of the ringed chaperones, or chaperonins. Chaperonins of the eukaryotic cytoplasm interact with a limited number of polypeptides, whereas GroEL is promiscuous as it binds and mediates the folding of many polypeptides. This feature makes GroEL an attractive protein for investigating various aspects of protein folding in eukaryotic cells because its substrate interaction is diverse. In this work we have expressed the groES and …

Identification And Characterization Of Aer, An Energy Sensor In Escherichia Coli, Anuradha Rebbapragada

Loma Linda University Electronic Theses, Dissertations & Projects

This study investigated the mechanism of signal transduction by Aer, the aerotaxis transducer in E.coli. A computerized search of GenBank revealed an open reading frame at 69.1 min on the E.co//chromosome that had a C-terminal fragment with 96.7% identity to the highly conserved domain of Tsr. The open reading frame was renamed aer for aerotaxis and energy responses. When the aer gene was inactivated, approximately 50% of aerotaxis responses were eliminated. When both aer and fsr were inactivated, aerotaxis, redox taxis and glycerol taxis were completely abolished. Expressing Aer in aer fsr cells restored aerotaxis responses and the duration …

Intracellular Traits Of Chimeric Trnalys3-Ribozymes And Their Inhibition Of Hiv, Zongli Chang

Loma Linda University Electronic Theses, Dissertations & Projects

Host tRNALYs3is the primer recruited by HIV-1 for initiating reverse transcription. When ribozymes targeted upstream of the primer binding site of the HIV-1 genome are tethered to the 3' end of the tRNALYs3, it is expected that the tRNA will carry the ribozymes into the virions. We made constructs that express chimeric tRNALYs3- ribozymes from internal tRNA promoters. These chimeras were also adjoined downstream of a U6 promoter, resulting in a hybrid U6/tRNA expression cassette with extragenic U6 and intragenic tRNA promoters. Juxtaposition of the tRNA and U6 promoters significantly enhanced expression levels compared to the tRNA promoter alone. A …

A Preclinical Model Of Ionizing Radiation And Tnf-Alpha Gene Therapy For Cancer, Jun Li

Loma Linda University Electronic Theses, Dissertations & Projects

Long term control of high-grade brain tumors is rarely achieved with current therapeutic regimens. This study sought to determine if pretreatment with plasmid DNA expressing Tumor Necrosis Factor-α (TNF-α) could enhance the effect of ionizing radiation in an aggressive, rapidly growing glioma tumor (C6) model. Plasmid DNA is an attractive vector system for delivery of foreign genes into eukaryotic cells due to its advantages including low immunogenicity in vivo, lesser toxicity and relatively low cost. We have constructed a plasmid-based mammalian expression vector (pGL1-TNF) to secrete human TNF-α from cancer cells. Plasmid DNA was successfully transfected into C6 cells …

Functional Analysis Of Human Igfbp-6 In Human Osteoblasts, Tao Yan

Loma Linda University Electronic Theses, Dissertations & Projects

Insulin-like growth factor binding proteins (IGFBPs) are a family of six secreted proteins that bind to and modulate the functions of insulin-like growth factors (IGF-I and IGF-II). As a relatively new member of the IGF system, IGFBP-6 was found to be produced by human osteoblasts and exert significant biological effects on human osteoblasts.

Our previous studies indicated that all-trans retinoic acid (ATRA) caused more than a10 fold increase in IGFBP-6 production and a50-60% reduction of ALP activity in human osteoblasts. Based on these findings, we proposed the hypothesis that ATRA induced human osteoblast differentiation was mediated at least in part …

Use Of Bioinformatics In The Analysis Of Chemotaxis Proteins, Sean Andrew Bulloch

Loma Linda University Electronic Theses, Dissertations & Projects

Bacterial chemotaxis is one of the best-known signal transduction systems. Levels of attractants and repellents are sensed in the surrounding environment by various chemoreceptors. The signal is passed to the excitation pathway via the transfer of a phosphoryl group from the receptor-associated histidine kinase CheA to the response regulator CheY. Phospho-CheY binds to the flagellar motor switching the direction of rotation of the flagella and thus allowing the cell to move towards or away from the attractant or repellent. The phosphatase CheZ removes the phosphoryl group from phospho-CheY restoring default flagellar rotation. Adaptation to stimuli occurs via addition of methyl …

Antitumor Effect Of Il-2, P53, And Bax Naked Gene Transfer In C6 Glioma Cells, Peyman Haghighat

Loma Linda University Electronic Theses, Dissertations & Projects

[Abstract not included]

Acute Effects Of Whole-Body Proton Irradiation On The Immune System Of The C57bl/6 Mouse, Eric H. Kajioka

Loma Linda University Electronic Theses, Dissertations & Projects

The acute effects of proton whole-body irradiation (WBI) on leukocytes, lymphocytes, and hematological parameters in the spleen and blood of C57BI/6 mice were examined and compared to the effects of photon (gamma) WBI derived from a 60cobalt (60Co) source. Adult, female C57BL/6 mice were exposed to a single dose (3 Gy, 0.4 Gy/min dose rate) of either proton WBI at the Bragg peak, proton WBI at the entry plateau, or Co WBI, and sacrifice intervals were at 1,4, 7, and 10 days post- WBI. Flow cytometry analysis of the spleen and peripheral blood showed depression in cell counts for all …

Dna-Dna Checkerboard Hybridization To Identify Bacteria In Human Plaque Samples, Jasan Lee Zimmerman

Loma Linda University Electronic Theses, Dissertations & Projects

Previous research has shown that using DNA probes to identify the presence of specific bacteria in human plaque samples is superior to the commonly used anaerobic cultural procedures. Some periodontal pathogens are either uncultivable or very difficult to grow, such as Prevotella intermedia, P. nigrescens, and Eubacterium brachy. Digoxygenin probes were prepared for 15 oral bacteria. These probes were used with a checkerboard DNA-DNA hybridization scheme to analyze whether human plaque samples contained any of the 15 bacterial species. Our results have shown that this method is successful in identifying bacterial DNA present in the subgingival patient plaque …

Engineering Secreted Proteins For Gene Transfer And Dna Vaccination, Jingxue Liu

Loma Linda University Electronic Theses, Dissertations & Projects

In recent years gene therapy has become a promising way of alleviating incurable human ailments, its concept emerging as the ultimate therapy for many infectious and genetic diseases. Two important aspects of the development of successful gene therapy protocols are the ability to monitor gene transfer readily, and the establishment of new protocols for treating specific diseases. In this work, Renilla luciferase and human glutamic acid decarboxylase (GAD) 65 were engineered for secretion to address some aspects of these issues.

Secreted reporter proteins are promising tools to study gene transfer and expression in a non-destructive manner, and bioluminescent proteins are …

Analysis Of Protein And Rna Interactions Of The Hiv-1 Rev Protein, John Francis

Loma Linda University Electronic Theses, Dissertations & Projects

The HIV-1 Rev protein enters the nucleus via it’s nuclear localization sequence/RNA binding domain and interacts with a ≈234 nt region of viral RNA, termed the Rev Response Element (RRE), which is located in the env region of unspliced and singly-spliced HIV-1 mRNA. The Rev nuclear export sequence (NES) then mediates translocation of the viral transcript to the cytoplasm, allowing for translation of viral structural genes and active virion formation. We report identification of two yeast nucleopore proteins, NUP49 and NUP100, that have been found to interact with Rev in vivo using the yeast two-hybrid system. NUP49 and NUP100 contain …

Igf-Ii Expression In Human Tumor Tissues And Human Igf-Ii Ribozyme Action, Zhaodong Xu

Loma Linda University Electronic Theses, Dissertations & Projects

Insulin-like growth factors (IGFs) are potent mitogens for a variety of cancer cells in vitro. In breast, prostate and neuroblastoma cancer cells, it has been suggested that IGF-II plays a paracrine/autocrine role. However, information on cell-type -specific IGF-II expression in vivo is limited. In situ hybridization and immunohistochemistry were carried out to determine the cell type expressing IGF-II in different tumor tissues. Both IGF-II mRNA and protein were localized to malignant cells, and expression in the stroma was minimal in all the tumors. The data are consistent with the hypothesis that cancer cell growth is regulated by IGF-II, and therefore …

Plant-Delivered Cholera Toxin B Subunit As An Immunomodulatory Molecule, Takeshi Arakawa

Loma Linda University Electronic Theses, Dissertations & Projects

The B subunit of cholera toxin (CTB) functions as an effective mucosal carrier molecule of chemically or genetically conjugated antigens. Orally delivered CTB or CTB fusion protein binds to G_M1-ganglioside receptor molecules located on cells in the gutassociated lymphoid tissues, the site of immunological induction. We have generated transgenic plants synthesizing CTB conjugated with vaccine antigens for food plant-based prevention of cholera and rotavirus toxin-induced diarrhea and for autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM).

Mice fed with transgenic potato tubers synthesizing 0.3% of CTB as total tuber protein generated serum and mucosal antibodies with toxin neutralizing …