Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "August 2001." Typescript. Vita. Includes bibliographical references (leaves 135-149). Also available on the Internet.

The World Health Organization has identified antimicrobial resistance as one of the top three threats to human health. Gram-negative bacteria such as Escherichia coli are intrinsically more resistant to antimicrobials. There are very few drugs either on the market or in the pharmaceutical pipeline targeting Gram-negative pathogens. Two mechanisms, the protection of the outer membrane and the active efflux by the multidrug transporters, play important roles in conferring multidrug resistance to Gram-negative bacteria. My work focuses on two main directions, each aligning with one of the known multidrug resistance mechanisms. The first direction of my research is in the area of the biogenesis of the bacterial outer membrane. The outer membrane serves as a permeability barrier in Gram-negative bacteria. Antibiotics cross the membrane barrier mainly via diffusion into the lipid bilayer or channels formed by outer membrane proteins. Therefore, bacterial drug resistance is closely correlated with the integrity of the outer membrane, which depends on the correct folding of the outer membrane proteins. The folding of the outer membrane proteins has been studied extensively in dilute buffer solution. However, the cell periplasm, where the folding actually occurs, is a crowded environment. In Chapter 2, effects of the macromolecular crowding on the folding mechanisms of two bacterial outer membrane proteins (OmpA and OmpT) were examined. Our results suggested that the periplasmic domain of OmpA improved the efficiency of the OmpA maturation under the crowding condition, while refolding of OmpT was barely affected by the crowding. The second direction of my research focuses on the major multidrug efflux transporter in Gram-negative bacteria, AcrB. AcrB is an obligate trimer, which exists and functions exclusively in a trimeric state. In Chapter 3, the unfolding of the AcrB trimer was investigated. Our results revealed that sodium dodecyl sulfate induced unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild type AcrB. In Chapter 4, the correlation between the AcrB trimer stability and the transporter activity was studied. A non-linear correlation was observed, in which the threshold trimer stability was required to maintain the efflux activity. Finally, in Chapter 5, the stability of another inner membrane protein, AqpZ, was studied. AqpZ was remarkably stable. Several molecular engineering approaches were tested to improve the thermal stability of the protein.

Syphilis is a global public health concern with 36.4 million cases worldwide and 11 million new infections per year. It is a chronic multistage disease caused by the spirochete bacterium Treponema pallidum and is transmitted by sexual contact, direct contact with lesions or vertically from an infected mother to her fetus. T. pallidum is a highly invasive pathogen that rapidly penetrates tight junctions of endothelial cells and disseminates rapidly via the bloodstream to establish widespread infection. Previous investigations conducted in our laboratory identified the surface-exposed adhesin, pallilysin, as a metalloprotease that degrades the host components laminin (major component of the basement membrane lining blood vessels) and fibrinogen (primary component of the coagulation cascade), as well as fibrin clots (function to entrap bacteria and prevent disseminated infection). Furthermore, pallilysin expressed on the surface of the non-invasive spirochete Treponema phagedenis conferred upon this bacterium the ability to degrade fibrin clots. It was hypothesized that pallilysin is integral to the process of T. pallidum dissemination, and interference with its functioning will prevent spread throughout the host and establishment of chronic infection. To test this hypothesis, a two-pronged approach was undertaken during my thesis research. Bioinformatics analyses were used to trace the evolutionary history of pallilysin in an attempt to gain further insight into its role in the pathogenesis of T. pallidum. The sequence conservation of pallilysin was analyzed in the context of its homologues. The bioinformatics analyses revealed homologues in three spirochete genera, namely Treponema, Spirochaeta, and Borrelia, presented in decreasing order of the degree of sequence conservation. The HEXXH motif, part of the active site of the pallilysin metalloprotease, was fully conserved only in T. pallidum and T. paraluiscuniculi, both of which are systemic pathogens. However, the flanking sequences showed a high degree of conservation, especially in the Treponema and Spirochaeta genera. The minimum laminin-binding region of pallilysin identified previously was partially conserved among the treponema and spirochaeta homologues with the highest degree of conservation observed with the homologues from T. paraluiscuniculi and T. phagedenis, as well as among the homologues from the human oral pathogens. In vitro dissemination studies were performed to investigate the dissemination capacity of T. phagedenis heterologously expressing pallilysin. Human Umbilical Vein Endothelial Cells were seeded and grown to confluence on permeable inserts coated with growth factor-reduced Matrigel to create an artificial endothelial barrier. Wild type T. phagedenis, and T. phagedenis transformed either with the pallilysin open reading frame or its empty shuttle vector, were incubated with the barriers under anaerobic conditions. Dissemination across the barrier was assessed as percent traversal by both dark-field microscopic counts of treponemes and real-time quantitative PCR of genomic DNA extracted from the treponemes. The results were inconclusive. However, a traversal trend suggested heterologous expression of pallilysin may facilitate traversal of T. phagedenis across the artificial endothelial barrier. This study presented the first step towards elucidating the role of pallilysin in endothelial monolayer traversal and provided supporting evidence for the role of pallilysin in the widespread dissemination of T. pallidum in vivo. / Graduate

Chlamydia trachomatis infections are among the most frequently reported causes of human sexually transmitted infection. In Australia, the reported rate of infection in 2004 reached 175 per 100,000 population, the highest rate since surveillance of the condition began in 1991. Severe adverse sequelae that commonly occur following progression of the infection from the lower to the upper genital tract include pelvic inflammatory disease, infertility and ectopic pregnancy. However the frequent prevalance of asymptomatic infection makes diagnosis and treatment often late and therefore ineffective against upper genital tract complications. Hence there is a great need to develop a vaccine to protect against the sexual transmission of C.trachomatis. Despite many years of research investigating potential vaccine strategies to prevent sexually transmitted C.trachomatis infections, there remains no commercially available C.trachomatis vaccine. Early research showed that the use of live, attenuated or inactivated whole Chlamydia as a vaccine was not a viable option due to adverse effects caused by immunopathogenic cellular components. The early human vaccine trials that utilized whole chlamydial cells and resulted in exacerbated disease when immunized individuals were re-exposed to Chlamydia have led to the investigation of chlamydial subunit components as potential vaccine antigens. The most widely investigated vaccine candidate antigen is the major outer membrane protein (MOMP) as it is known to be immunogenic and surface exposed. Much research using this antigen has been undertaken with the antigen being delivered as a protein, peptide or DNA, via many mucosal and systemic routes of immunization, and in combination with various vaccine adjuvants. However, at best only partial protection against a chlamydial genital tract infection has been achieved. Only a few alternative candidate antigens have been investigated as potential vaccine targets to protect against chlamydial infections. These include the outer membrane porin PorB, the large cysteine rich outer membrane protein Omp2 and the heat shock proteins DnaK and GroEL. Although other candidate antigens have been predicted in various models of chlamydial infection (Finco et al., 2005; Stemke-Hale et al., 2005; Li et al., 2006), few have been tested for their protective efficacy. The aim of this study was to use expression library immunization to screen the whole C.muridarum genome for novel vaccine candidates capable of protecting against a chlamydial genital tract infection. C.muridarum was selected as the disease model for chlamydial genital tract infection as it has similarities to C.trachomatis in pathogenesis, immune response to infection and gene content and order. Once protective antigens had been isolated from an expression library, these were screened individually for immunogenicity and protective efficacy in the C.muridarum model of infection. An expression library containing over 21,000 recombinant C.muridarum clones was constructed and divided into pools of clones. DNA was extracted from these pools and used to immunize mice through gene gun technology, delivering 1μg of DNA to the abdomen of mice. Following the immunization regime, mice were challenged intra-vaginally with live C.muridarum as this route of infection best resembles the natural route of infection that is responsible for the sexual transmission of C.trachomatis in humans. Four in vivo screens of the C.muridarum expression library, each time using reduced numbers of clones, resulted in the identification of seven novel vaccine antigens that conferred protection against a genital tract challenge infection in mice. These warrant further investigation as vaccine antigens in the development of a vaccine against C.trachomatis infection. The identified antigens include antigens not conventionally believed to be potential vaccine candidates such as hypothetical proteins and housekeeping genes, including a DNA gyrase subunit, TC0462, and the ATP-dependent Clp protease, ATP-binding subunit ClpC, TC0559. Other antigens identified were more traditional, surface exposed vaccine targets that have not been previously investigated as vaccine targets, including a novel outer membrane protein, TC0512, a polymorphic membrane protein, TC0693, and TC0850, a protein of the type three secretion system, a family of proteins that allow gram-negative bacteria to inject virulence related proteins into the cytoplasm of a host cell. All antigens were shown to be partially protective with the putative outer membrane protein TC0512 showing an overall reduction in chlamydial burden of 55% and other antigens showing overall reductions in chlamydial burden of 26 - 44%. These antigens were also either capable of stimulating an immune response, or predicted to contain epitopes that may stimulate strong immune responses and so warrant further investigation as vaccine antigens to protect against chlamydial genital tract infections. The results of this research demonstrate that it is possible to identify novel vaccine targets through screening an expression library in a disease model. This study has identified several novel vaccine targets that are partially-protective against a C.muridarum infection and that are thought to be capable of stimulating strong immune responses. These antigens have high homology with C.trachomatis sequences, indicating that they have potential as vaccine candidates capable of protecting against the serovars of C.trachomatis that cause sexually transmitted infections in humans. Although the protection observed in this study was only partial, the immunization strategy utilised only fragments of the genes, an immunization mechanism known to elicit Th2 type immune responses, and no adjuvant to enhance the immunogenicity of the antigens. Through different immunization routes and in conjunction with adjuvants that stimulate Th1 type immune responses, complete protection against chlamydial genital tract infections may be achieved.

Diss. (sammanfattning) Umeå : Umeå universitet, 2007. / Härtill 5 uppsatser.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2010. / Title from PDF title page (viewed on June 28, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / "December 2006" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Thesis (M.S.)--Uniformed Services University of the Health Sciences, 2006 / Typescript (photocopy)

Orientador: Marcelo Lancellotti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T23:54:54Z (GMT). No. of bitstreams: 1 IzidoroJunior_MarioServulo_M.pdf: 2227378 bytes, checksum: fe650bf0745300591928bae4fac67e46 (MD5) Previous issue date: 2013 / Resumo: Embora comensal da nasofaringe humana, algumas linhagens de Neisseria meningitidis ocasionalmente ultrapassam a mucosa respiratória e a barreira hematoencefálica causando doenças como meningite e septicemia. Sabe-se também que bactérias em geral possuem taxia por células cancerígenas, isto é, possuem uma predileção por infectar células tumorais. Desta forma, neste trabalho produzimos OMV de diversas linhagens de Neisseria meningitidis e analisamos as principais características físico-químicas destas vesículas e, além disso, também verificamos as principais interações desta micropartícula com a linhagem celular NG97, uma linhagem de glioblastoma, tecido pelo qual tal bactéria é conhecida por infectar em casos patológicos, por testes morfológicos in vitro e também analisamos as citocinas inflamatórias através de quantificação relativa por Real time PCR. O grande achado deste trabalho é que a utilização de ultrafiltração substituindo a ultracentrifugação na produção de OMVs é viável, menos trabalhosa e mais rápida que os métodos descritos na literatura. Além disso, as vesículas extraídas por tal processo apresentam tamanhas e cargas superficiais como os encontrados na literatura sendo que cada cepa produz diferentes quantidades de OMVs para um mesmo tempo. Ainda, observou-se que as OMVs obtidas de linhagens atenuadas para os fatores de virulência causaram menores alterações morfológicas na linhagem NG97 do que as OMVs obtidas a partir de linhagens selvagens. A influência de tais fatores de virulência, que auxiliam na interação das células do parasita com as células do hospedeiro, também foi observada na análise das citocinas inflamatórias produzidas, onde foram constatados que na ausência da pilina (proteína responsável pela aderência da bactéria com células epiteliais e endoteliais) a produção de citocinas foi praticamente nula quando comparada com suas diferentes linhagens onde este fator de virulência é presente. Para um possível uso destas vesículas como sistemas lipossomais de entrega de drogas, acredita-se que quanto mais inócua a vesícula for maior seria a biodisponibilidade da molécula antitumoral a ser entregue, como é o caso da OMV produzida pela cepa C2135 ?pilE que, além disso, possui um tamanho menor de 200nm sendo capaz de penetrar nas junções celulares da massa tumoral / Abstract: Neisseria meningitidis is a human nasopharyngeal commensal, however, some strains occasionally exceed the respiratory mucosa and the blood brain barrier causing diseases such as meningitis and septicemia. It is well established that bacteria has a natural predilection to infect cancer cells. Thus, this study produced OMVs from different strains of Neisseria meningitidis and analyzed physicochemical characteristics of these vesicles such as size and surface charge, and also, the main interactions between this nanoparticle and NG97 cell line by in vitro morphological tests and the analyze of the production of inflammatory cytokines by relative quantification Real time PCR. The major finding of this study is that the use of ultrafiltration substituting ultracentrifugation on OMVs production makes the extraction viable, less laborous and faster than previous methods. Furthermore, the vesicles extracted by this process exhibited similar sizes and surface charges when compared to the literature and each strain produces different amounts of OMVs when comparing the same production time. In this work, we also observed that the OMVs generated by attenuated strains for virulence factors induced less morphological changes in the NG97 cells than OMVs generated from wild type strains. This trend where virulence factors which assist the parasite-host cells interaction was also observed in the analysis of cytokines produced. It was observed in the absence of pilin (protein responsible for adherence of the bacteria to epithelial and endothelial cells) the production of cytokines was much lower when compared with the different strains where this virulence factor is present. For a possible use of these lipossomal vesicles as delivery systems for drugs, it is believed that the more innocuous the greater the bioavailability of the antitumor molecule to be delivered, as in the case of OMV produced by the strain C2135 ?pilE, moreover, having a size around 200 nm fits perfectly between the cell junctions allowing the nanoparticle to reach the tumor mass, but further studies are required to confirm this hypothesis / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Made available in DSpace on 2014-08-20T13:32:50Z (GMT). No. of bitstreams: 1 dissertacao_nubia_seyffert.pdf: 4325642 bytes, checksum: 2836f7bcdef6a962ef1e00c38936c413 (MD5) Previous issue date: 2007-02-05 / Leptospiral immunoglobulin-like (Lig)proteins are exposed on membrane surface of pathogenic Leptospira and may play a role in host cell attachment and invasion during infection.A pair of Ligs,named LigA and LigB,with identical and non-identical polypeptide regions has recently been identified.The two proteins elicit a strong humoral immune response during acute host infection and have been suggested as targets for development of vaccines and diagnostic tests for leptospirosis.This study reports the production and characterization of monoclonal antibodies (Mabs)against recombinant non-identical fragments of LigA (rLigANI)and LigB (rLigBNI).The recombinant fragments were used for mice immunization and screening hybridomas by indirect ELISA.Four Mabs obtained against rLigANI and six Mabs obtained against rLigBNI were isotyped and evaluated regarding their potential for use in studies of immunoprotection and diagnostic test development.The Mabs were of the IgM (1),Ig 2b (3)and Ig 1 (6)isotypes and reacted with the native proteins from a pathogenic strain of Leptospira i terroga s serovar Copenhageni L1 130 in an indirect ELISA and immunofluorescence.Mabs affinity constants felt between 4x10 7 M -1 and 2x10 8 M -1 ,and epitope mapping by additive ELISA has shown that each Mab either react with the same epitope in the recombinant molecule or cause steric hindrance. Tissue sections from kidneys of hamsters experimentally infected with leptospires and probed with the Mabs reacted positively by immunohistochemistry.These findings suggest that the Mabs obtained can be useful for the studies of immunoprotection and development of diagnostic tests. / As proteínas leptospiral immu oglobuli -like (Lig)estão expostas na superfície da membrana das leptospiras patogênicas e podem estar envolvidas no ataque e penetração às células do hospedeiro durante a infecção.Recentemente,foi identificado um par de Ligs,nomeadas LigA e LigB,com regiões polipeptídicas idênticas e não-idênticas.As duas proteínas estimulam uma resposta humoral forte durante a infecção aguda no hospedeiro e têm sido sugeridas como alvos para o desenvolvimento de vacinas e testes diagnósticos para leptospirose.Este estudo relata a produção e caracterização de anticorpos monoclonais (Mabs)contra fragmentos recombinantes não idênticos de LigA (rLigANI)e LigB (rLigBNI).Os fragmentos recombinantes foram utilizados para a imunização dos camundongos e triagem dos hibridomas por ELISA indireto.Quatro Mabs obtidos contra rLigANI e 6 Mabs contra rLigBNI foram isotipados e avaliados quanto ao seu potencial para uso em estudos de imunoproteção e desenvolvimento de testes diagnósticos.Os Mabs foram dos isotipos IgM (1),Ig 2b (3)and Ig 1 (6)e reagiram com as proteínas nativas de Leptospira i terroga s sorovar Copenhageni cepa L1 130 em ELISA indireto e imunofluorescência.A constante de afinidade dos Mabs manteve-se entre 4x10 7 M -1 and 2x10 8 M -1 ,e o mapeamento de epitopos por ELISA de aditividade demonstrou que cada Mab reage com o mesmo epitopo na molécula recombinante ou causa um impedimento espacial (steric hi dra ce ).Finalmente,os Mabs reagiram positivamente em testes imuno-histoquímicos de seções do tecido renal de hamsters experimentalmente infectados com leptospiras.Esses dados sugerem que os Mabs obtidos podem ser usados para estudos de imunoproteção e desenvolvimento de testes de diagnóstico de leptospirose.