Diss. (sammanfattning) Stockholm : Karolinska institutet, 2001. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (p. 98-113).

Chi-shing Lo. / Thesis submitted in: November 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 119-138). / Abstracts in English and Chinese. / Statement --- p.iii / Acknowledgments --- p.iv / Abbreviations --- p.v / Abstract --- p.vii / Abstract (Chinese version) --- p.ix / Table of contents --- p.xi / List of Figure --- p.xvii / List of Table --- p.xix / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- BIOLOGY OF TOXOPLASMA GONDII --- p.1 / Chapter 1.1.1 --- Life cycle of Toxoplasma gondii --- p.2 / Chapter (a) --- Tachyzoite --- p.3 / Chapter (b) --- Bradyzoite --- p.3 / Chapter 1.1.2 --- Genetics of Toxoplasma gondii --- p.4 / Chapter (a) --- Population genetics --- p.4 / Chapter (b) --- Molecular genetics --- p.5 / Chapter (c) --- Genome analysis --- p.7 / Chapter 1.1.3 --- Invasion --- p.8 / Chapter 1.1.4 --- Surface of Toxoplasma gondii --- p.9 / Chapter (a) --- Tachyzoite surface --- p.9 / Chapter (b) --- Bradyzoite surface --- p.11 / Chapter (c) --- Sporozite surface --- p.11 / Chapter (d) --- Glycoprotein antigens --- p.12 / Chapter 1.2 --- TREATMENT OF TOXOPLASMOSIS --- p.13 / Chapter 1.2.1 --- Chemotherapy --- p.13 / Chapter (a) --- Drug against metabolism and protein synthesis on nuclear genome --- p.13 / Chapter (b) --- Drug against other organelles --- p.14 / Chapter (c) --- Drug resistance --- p.15 / Chapter 1.2.2 --- Toxoplasma vaccine --- p.16 / Chapter (a) --- Mutant strains of Toxoplasma gondii as vaccine --- p.17 / Chapter (b) --- Subunit vaccine --- p.19 / Chapter (c) --- P30 as subunit vaccine --- p.20 / Chapter 1.3 --- AIM OF THE STUDY --- p.22 / Chapter Chapter 2 --- : Expression of P30 in Escherichia coli --- p.23 / Chapter 2.1 --- INTRODUCTION --- p.23 / Chapter 2.1.1 --- Why Escherichia coli? --- p.23 / Chapter 2.1.2 --- protein folding --- p.24 / Chapter 2.1.3 --- T7-based gene expression system --- p.25 / Chapter (a) --- Biology of T7 RNA polymerase --- p.26 / Chapter (b) --- pET translational vector --- p.26 / Chapter (c) --- Hislidine-tagged protein --- p.27 / Chapter (d) --- Host strain for expression --- p.28 / Chapter 2.2 --- MATERIALS --- p.29 / Chapter 2.2.1 --- Bactcrial strains --- p.29 / Chapter 2.2.2 --- Mouse strain --- p.29 / Chapter 2.2.3 --- Chemicals --- p.29 / Chapter 2.2.4 --- Nucleic acids --- p.30 / Chapter 2.2.5 --- Kit and reagents --- p.31 / Chapter 2.2.6 --- Antibodies --- p.31 / Chapter 2.2.7 --- Solutions --- p.32 / Chapter 2.2.8 --- Enzymes --- p.33 / Chapter 2.2.9 --- Sequencing primers --- p.33 / Chapter 2.3 --- METHODS --- p.34 / Chapter 2.3.1 --- Modification of P30 gene --- p.34 / Chapter (a) --- Preparation of recombinant plasmids，pBV220-ASP30PI and pBV220- SP30hisAPI --- p.36 / Chapter (b) --- Digestion of pBV220-ASP30PI and pBV220-SP30hisAPI with DraII and EcoRI --- p.37 / Chapter (c) --- Purification of DNA fragments from agarose gel --- p.37 / Chapter (d) --- Ligation of fragments of pBV220-ΔSP30PI and pBV220-SP30hisAPI --- p.38 / Chapter (e) --- Preparation of DH5α competent cells --- p.38 / Chapter (f) --- Transformation of recombinant pBV220-ΔSP30hisAPI --- p.38 / Chapter (g) --- Plasmid preparation of putative pBV220-ΔSP30API --- p.39 / Chapter (h) --- Plasmid preparation of pET-ΔSP30API --- p.39 / Chapter (i) --- Cycle sequencing reaction on putative plasmid pET-ASP30API --- p.40 / Chapter 2.3.2 --- Expression and Purification of his-tag P30 --- p.41 / Chapter (a) --- Expression profile of his-tag P30 production by IPTG induction --- p.41 / Chapter (b) --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.41 / Chapter (c) --- Purification of his-tag P30 --- p.43 / Chapter (d) --- Bradford Protein Microassay (Bio-Rad) --- p.43 / Chapter 2.3.3 --- Characterization of his-tag P30 --- p.44 / Chapter (a) --- Western blot of induced bacterial lysate by monoclonal anti-his-tag antibody --- p.44 / Chapter (b) --- Western blot of his-tag with seropositive sera of mice，rabbit and human --- p.46 / Chapter (c) --- Enterokinase digestion of his-tag P30 --- p.46 / Chapter (d) --- N'terminal amino acid sequencing of pure and enterokinase-cut his-tag --- p.47 / Chapter (e) --- Western blot of T. gondii lysate with antiserum against his-tag P30 --- p.47 / Chapter 2.4 --- RESULTS --- p.49 / Chapter 2.4.1 --- Modification of P30 gene --- p.49 / Chapter 2.4.2 --- "Expression, purification and characteriziation of his-tag P30 in bacteria" --- p.54 / Chapter 2.5 --- DISCUSSIONS --- p.64 / Chapter 2.5.1 --- Modification of P30 gene --- p.64 / Chapter 2.5.2 --- Expression and purification of his-tag P30 --- p.66 / Chapter 2.5.3 --- Characterization of his-tag P30 --- p.67 / Chapter Chapter 3 --- : Expression of P30 in Arabidopsis thalina --- p.69 / Chapter 3.1 --- INTRODUCTION --- p.69 / Chapter 3.1.1 --- Why Arabidopsis thalina? --- p.69 / Chapter 3.1.2 --- In planta transformation --- p.70 / Chapter 3.1.3 --- Transgenic plants as vacine production systems --- p.72 / Chapter (a) --- Stable expression of E. coli heat-liable enterotoxin B subunit and cholera-toxin B subunit --- p.73 / Chapter (b) --- Stable expression of Hepatitis B surface antigen (HBsAg) --- p.74 / Chapter (c) --- Stable expression of Norwalk virus capsid protein --- p.75 / Chapter (d) --- Transient expression by tobacco mosaic virus --- p.75 / Chapter (e) --- Transient expression by Cowpea mosaic virus capsid protein fusion --- p.76 / Chapter 3.2 --- MATERIALS --- p.77 / Chapter 3.2.1 --- Bacterial strains --- p.77 / Chapter 3.2.2 --- Arabidopsis strains --- p.77 / Chapter 3.2.3 --- Chemicals --- p.77 / Chapter 3.2.4 --- Nucleic acids --- p.78 / Chapter 3.2.5 --- Kit and reagents --- p.78 / Chapter 3.2.6 --- Solutions --- p.79 / Chapter 3.2.7 --- Enzymes and buffers --- p.81 / Chapter 3.2.8 --- PCR and Sequencing primers --- p.81 / Chapter 3.3 --- METHODS --- p.82 / Chapter 3.3.1 --- Construction of V7-ASP30API --- p.82 / Chapter 3.3.2 --- Agrobacterium-mediated transformation of Arabidopsis by vacuum infiltration --- p.83 / Chapter (a) --- Preparation of electro-competent Agrobacterium --- p.83 / Chapter (b) --- Transformation of electro-competent Agrobacterium with V7- ASP30API --- p.84 / Chapter (c) --- Plasmid preparation of V7-ASP30API from transformed Agrobacterium --- p.84 / Chapter (d) --- Vacuum infiltration --- p.85 / Chapter 3.3.3 --- Screening of homozygous transgenic plants --- p.86 / Chapter 3.3.4 --- Detecton of transgene P30 in genomic DNA of transgenic plants --- p.87 / Chapter (a) --- Preparation of DIG-labelled probe --- p.87 / Chapter (b) --- Estimation the yield of DIG-labelled probe --- p.88 / Chapter (c) --- Extraction of genomic DNA from transgenic plants --- p.88 / Chapter (d) --- Restriction digestion of genomic DNA with EcoRI and HindIII --- p.89 / Chapter (e) --- DNA transfer from gel to nylon membrane --- p.89 / Chapter (f) --- Detection of hybridized DIG-labelled probe on membrane/ blot --- p.90 / Chapter (g) --- PCR on genomic DNA of transgenic plants with specific primers --- p.91 / Chapter 3.3.5 --- Analysis of transgene RNA expression in transgenic plants --- p.91 / Chapter (a) --- Extraction of total RNA from plants --- p.91 / Chapter (b) --- Northern blot on RNA of F2 transgenic plants --- p.92 / Chapter (c) --- RT-PCR on RNA of F3 transgenic plants --- p.93 / Chapter 3.3.6 --- Detection of his-tag P30 protein in F3 transgenic plants --- p.93 / Chapter 3.4 --- RESULTS --- p.95 / Chapter 3.4.1 --- Construction of V7-ASP30API --- p.95 / Chapter 3.4.2 --- Screening of homozygous transgenic plants --- p.99 / Chapter 3.4.3 --- Molecular analysis of transgene P30 in transgenic plants --- p.101 / Chapter 3.5 --- DISCUSSIONS --- p.108 / Chapter 3.5.1 --- Construction and optimization of expression construct --- p.108 / Chapter 3.5.2 --- Screening and selection of homozyous transgenic plants --- p.109 / Chapter 3.5.3 --- Analysis of transgenic plants --- p.110 / Chapter Chapter 4 : --- General Discussions --- p.112 / Chapter 4.1 --- Significances of studying Toxoplasma gondii --- p.112 / Chapter 4.2 --- Expression of recombinant P30 in prokaryotic systems --- p.113 / Chapter 4.2 --- Expression of recombinant P30 in eukaryotic systems --- p.115 / Reference --- p.119

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 96-101).

Orientador: Tomomasa Yano / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T06:09:15Z (GMT). No. of bitstreams: 1 Cordeiro_MelinaAparecida_M.pdf: 3889960 bytes, checksum: 3e92b071b7e108ebb8bff09fe751b180 (MD5) Previous issue date: 2015 / Resumo: O Resumo poderá ser visualizado no texto completo da tese digital / Abstract: The Abstract is available with the full electronic digital document / Mestrado / Microbiologia / Mestra em Genética e Biologia Molecular

Orientador: Tomomasa Yano / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T23:05:19Z (GMT). No. of bitstreams: 1 Conceicao_RogerioArcuri1983-_D.pdf: 4676987 bytes, checksum: 96e3babbbb52c358f5daca2c8166f056 (MD5) Previous issue date: 2014 / Resumo: Estudos preliminares sobre adesão e invasão mostraram que 100% de 49 amostras de Escherichia coli associada à sepse humana (SEPEC) foram capazes de aderir e invadir células Vero (Rim de macaco verde africano) e Huvec (Veia umbilical humana). Em análise genotípica, foi observada uma alta prevalência dos genes fimH (98,0%) que codificam para a adesina da fimbria de tipo 1 (F1). No entanto, as cepas analisadas aderiram tanto na presença quanto na ausência de ?-D-manopiranosideo, um inibidor da adesão mediada pela F1. Por esse motivo foram analisadas as proteínas de membrana externa de SEPEC, com o objetivo de se avaliar potenciais fatores adesão e invasão dessas cepas. Por técnicas de proteômica, foram identificadas três proteínas de SEPEC com afinidade a glicoproteínas celulares: OmpA (Proteína de membrana A); FimA (Subunidade maior da fimbria do tipo 1) e YdeR (Subunidade menor da fimbria do tipo 9 - F9). Esses dados foram coerentes com a análise genotípica das amostras SEPEC, pois foi observada uma alta porcentagem dos genes que codificam para as três proteínas descritas anteriormente, sendo que 100% das amostras foram ompA+, 98,0% foram fimA+ e 100% foram ydeR+. Os ensaios de adesão e invasão de SEPEC (OmpA+/ F1+) em células Vero e Huvec mostraram que ?-D-manopiranosideo e GlcNAc, quando atuando juntos, agem como potentes inibidores da adesão e invasão, sugerindo que essas moléculas poderiam estar ocupando os sítios de ligação a carboidratos das duas adesinas F1 e/ou OmpA, respectivamente. Para confirmar essa hipótese, mutantes nulos para ?fimA (OmpA+/ F1-) e ?ompA (OmpA-/F1+) foram construídos, e os resultados de adesão e invasão bacteriana foram similares aos obtidos na presença dos inibidores: ?-D-manopiranosideo e GlcNAc. Mutante nulo para a proteína YdeR da fimbria F9 (OmpA+/ F1+), mostrou significativa redução da adesão e invasão bacteriana em células Vero e Huvec (p ? 0,05) somente na presença dos inibidores da adesão mediada por F1 e OmpA: ?-D-manopiranosideo e GlcNAc, respectivamente. Esses dados sugerem que F9 também contribui para a adesão e invasão de SEPEC. Juntos, nossos dados demonstram que os processos de adesão e invasão de SEPEC são dependentes de OmpA, F1 e F9, as quais podem ser complementares, e devem atuar sinergicamente para a adesão e invasão de SEPEC / Abstract: Our preliminary studies about bacterial adhesion and invasion showed 100% of 49 strains of human sepsis-associated Escherichia coli (SEPEC) were able to adhere and invade Vero (African green monkey kidney) and HUVEC (human umbilical vein) cells. In genotypic analysis, high prevalence of fimH (98.0%), gene encoding for the type 1 fimbrial adhesin was observed. However, all the strains analyzed were able to adhere and invade these cellular lineages both in the presence and absence of the type 1 fimbriae adherence-inhibitor ?-D-mannopiranoside. Consequently, the outer membrane proteins of SEPEC were analyzed in order to find potential adhesion and invasion factors expressed by these strains. Through proteomic techniques, three proteins with affinity to cellular glycoproteins were identified: OmpA (Membrane Protein A); FimA (major subunit of type 1 fimbriae - F1) and YdeR (minor subunit of type 9 fimbriae - F9). These data were consistent with the genotypic analysis of SEPEC strains because a high percentage of the genes encoding these three proteins was observed, being that ompA+ (100%) fimA+ (98%) and ydeR+ (100%). Assays of adhesion and invasion of SEPEC (OmpA+ / F1+) in Vero and HUVEC cells showed that ?-D-manopiranosideo and GlcNAc, when acting together, act as potent inhibitors of adhesion and invasion, suggesting that these molecules could be occupying sites of carbohydrate-binding displayed by adhesins from both F1 and/or OmpA, respectively. To confirm this hypothesis, null mutants ?fimA (OmpA+/F1-) and ?ompA (OmpA-/F1+) were constructed, and the phenotypic results of bacterial adhesion and invasion were similar to those obtained in the presence of inhibitors ?-D-mannopiranoside and GlcNAc. The mutant ?ydeR (OmpA+ / F1+), null mutant for YdeR protein of F9 fimbriae, showed a significant reduction in bacterial adhesion and invasion in Vero and HUVEC (p ? 0.05) just in the presence of inhibitors of cell adhesion mediated by F1 and OmpA. These data suggest that F9 also can contribute to the adhesion and invasion of SEPEC in Vero and HUVEC cells. Hold together, our data demonstrated that adhesion and invasion of SEPEC are OmpA, F1 and F9 dependents, which can be complementary and express synergistic activity leading to enhancing the ability of adhesion and invasion in SEPEC strains / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

Enterohemorrhagic Escherichia coli 0157:H7 (EHEC) and Enteropathogenic E. coli (EPEC) are intestinal pathogens that induce characteristic lesions on mammalian cells called actin pedestals. Attachment to host cells by both EPEC and EHEC is an essential step towards colonization and is associated with the formation of highly organized actin cytoskeletal elements termed as attaching and effacing (AE) lesions beneath bound bacteria. The outer membrane protein intimin is required for the formation of these structures and binds its own translocated mammalian cell receptor called Translocated intimin receptor (Tir). These interactions induce a cascade of events that result in actin pedestal formation. In this thesis, we characterized pedestal formation and the requirements of pedestal formation by host adapted and in vitro cultivated EHEC. Our data indicate that growing EHEC in the mammalian host enhances bacterial cell attachment, expression and translocation of virulence effectors and actin signaling, and this enhancement is likely to entail more than one bacterial activity involved in host cell interactions. We also focused on the interaction between the two key bacterial players involved in pedestal formation, intimin and Tir. We randomly mutagenized the Tir-binding domain of intimin and isolated point mutants that disrupted Tir recognition. The ability of intimin mutants to bind to recombinant Tir correlated with their ability to trigger AE lesions on pre-infected mammalian cells. Half of the mutations fell within the previously identified 50 amino acid C-terminal region of intimin, and alanine scanning mutagenesis of this region identified four residues of EHEC intimin that are critical for Tir recognition. In a model of the EHEC intimin-Tir complex that is based on EPEC intimin and Tir, these four amino acids are predicted to be located at the intimin-Tir interface, indicating that these residues play a functional role in intimin recognition by Tir. To identify critical residues involved in intimin recognition and intimin mediated actin signaling, we generated point mutations in the extracellular domain of EHEC Tir. Based on our data, we conclude that Tir-intimin interaction is essential for triggering actin pedestals, and intimin function in the context of Tir signaling can be replaced by proteins that are entirely unrelated to intimin but that bind to Tir. These data are concordant with the model that intimin functions to cluster Tir in the membrane to induce actin assembly. Finally, as a step to study downstream actin signaling processes after Tir translocation, we mapped the domain of Tir involved in host cell signaling. We found that the clustering of a 12 amino acid stretch of C-terminus encompassing the Nck binding sequence of Tir generated actin nucleation indistinguishable from that mediated by the entire C-terminus, and abrogation of Nck binding by mutation of Y474 to Phenylalanine abolished actin assembly. Although these results do not rule out a role for other domains of Tir involved in actin pedestal formation, this suggests that the essential element of Tir consists of the Nck binding domain.