Wang, Yejun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 97-105). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

This thesis studies two particular bacterial outer membrane proteins called OmpC and Wzi, focusing on their expression, purification, crystallization and X-ray structure determination. A series of four naturally occurring OmpC mutants were isolated from a single patient with an E. coli infection of liver cysts. The isolated E. coli strains progressively exhibited increasing breadth of antibiotic resistance in which OmpC was predicted to take a partial role. We carried out an assay in which a strain of E. coli lacking OmpC was used to express the first (antibiotic sensitive) and the last (antibiotic resistant) of the clinical OmpC mutants and drug permeation assessed. Single channel conductance measurements were carried out and the X-ray structures for all the isolates were determined. Protein stability was assessed. With these data we propose that changes in the transverse electric field, not the pore size, underlie the clinically observed resistance to the antibiotics. This is the first demonstration of this strategy for antibiotic resistance. Wzi is a novel outer membrane protein involved in the biosynthesis and translocation mechanism of the K30 antigen from E. coli. The mechanism is a complicated process that requires several proteins including outer and inner membrane proteins. The protein Wzi was expressed, purified and crystallized. Initial crystals were tested and diffracted to 15Å. After optimization, a crystal diffracting to 2.4Å has been obtained.

Orientador: Janete Apparecida Desidério / Banca: Odair Aparecido Fernandes / Banca: Agda Paula Facincani / Banca: Paula Cristina Brunini Crialesi Legori / Banca: Juliana Regina Rossi / Resumo: As proteínas Vip3Aa e Cry1Ia apresentam potencial no controle de lepidópteros-praga e surgem como alternativa promissora no manejo da resistência de pragas as proteínas Cry1A, que tem sido altamente utilizada na formulação de bioinseticidas comerciais à base de Bacillus thuringiensis (Bt) e em plantas transgênicas. Assim sendo, o presente trabalho teve como objetivo a clonagem e expressão das proteínas Vip3Aa42, Vip3Aa43 e Cry1Ia10 em Escherichia coli, a fim de se analisar a correlação entre a união aos receptores por meio de análises de competição entre as diferentes toxinas Vip3Aa e a toxina Cry1Ia10, e a toxicidade a lepidópteros-praga, inferindo-se quais as combinações que poderiam ser utilizadas na produção de plantas transgênicas, contendo múltiplos genes, as quais vêm sendo empregadas para contornar a evolução da resistência dos insetos às toxinas Bt. Para tanto, os genes vip3Aa e cry1Ia10 foram clonados no vetor pET SUMO, expressos em E. coli e a toxicidade das proteínas foram testadas em bioensaios com lagartas neonatas de Spodoptera frugiperda, Anticarsia gemmatalis e Heliothis virescens. As BBMVs ("Brush Border Membrane Vesicles") foram preparadas a partir dos intestinos das três espécies e ensaios de competição homóloga e heteróloga foram realizados. As proteínas Vip3Aa42 e Vip3Aa43 apresentaram toxicidade para S. frugiperda e A. gemmatalis. Já a proteína Cry1Ia10 apresentou toxicidade apenas para A. gemmatalis e, as proteínas não se mostraram tóxicas para H. virescens. Os ensaios de ligação às BBMVs demonstraram que as proteínas Vip3Aa42, Vip3Aa43 e Cry1Ia10 se unem aos receptores presentes no intestino médio de forma efetiva nas três espécies e que, portanto, houve correlação entre a toxicidade e a união aos receptores para as populações de S. frugiperda e A. gemmatalis, porém para H. virescens não houve relação entre a toxicidade e a união aos receptores. Sendo assim ... / Abstract: Vip3Aa and Cry1Ia proteins have potential in control of Lepidopteran pest and emerge as a promising alternative in the pest resistance management the Cry1A proteins, which has been highly used in the formulation of commercial insecticides based on Bacillus thuringiensis (Bt) and in transgenic plants. Therefore, this study aimed to cloning and expression of Vip3Aa42, Vip3Aa43 and Cry1Ia10 proteins in Escherichia coli, in order to analyze the correlation between the binding to receptors through competition assays between the different Vip3Aa toxins and toxin Cry1Ia10, and toxicity to lepidopteran pests inferring up which combinations that could be used to produce transgenic plants containing multiple genes, which have been used to circumvent the development of insects resistance to Bt toxins. Therefore, vip3Aa and cry1Ia10 genes were cloned into the pET SUMO vector, expressed in E. coli and toxicity of proteins were tested in bioassays with neonate larvae of Spodoptera frugiperda, Anticarsia gemmatalis and Heliothis virescens. The BBMVs (Brush Border Membrane Vesicles) were prepared from the gut of the three species, and homologous and heterologous competition assays were performed. The Vip3Aa42 and Vip3Aa43 proteins were toxic to S. frugiperda and A. gemmatalis. Already Cry1Ia10 protein showed toxicity only for A. gemmatalis and proteins were not toxic to H. virescens. Binding assays to BBMVs demonstrated that Vip3Aa42, Vip3Aa43 and Cry1Ia10 proteins binding effectively to receptors present in the midgut in three species and, therefore, was correlation between toxicity and the binding to receptors for the populations of S. frugiperda and A. gemmatalis, but for H. virescens there was no relationship between toxicity and the binding to receptors. Thus, the combination of Cry1Ia10 and Vip3Aa42 or Vip3Aa43 proteins is indicated for the production of biological insecticidal, as well as for the production of transgenic plants to circumvent ... / Doutor

Diseases caused by pathogenic bacteria continue to be major health concerns. For example, it is estimated that in the year 2000 typhoid fever caused over 21,000,000 illnesses and ~200,000 deaths (Crump et al., 2004). The disease is caused by S. typhi, a closely-related serotype of S. typhiumurium, the salmonella strain in which BipA was first identified. The CDC estimated that in 2013, multidrug resistant bacteria caused over 2 million infections in the United States, ending in more than 23,000 deaths (CDC, 2013). This number is set to rise as more bacteria become resilient to the collection of conventional antibiotics. The increasing number of multidrug resistant bacterial strains necessitates the development of new antimicrobial drugs. BipA is an attractive target for drug research. As mentioned in Section 2.5.2, BipA is ubiquitous in eubacteria and lower eukaryotes such as protozoa, but is absent from higher-order eukaryotes such as humans. Because the protein is essential for bacterial survival, BipA presents a major vulnerability of pathogenic bacteria. A drug targeting the protein itself or its interactions to the ribosome will disable only the bacteria, but have no effect on the eukaryotic host. A comprehensive model of BipA bound to the 70S ribosome will provide unparalleled insight into BipA's binding site and its mechanism. Toward this goal, cryo-EM techniques were employed to visualize the binding site of BipA on the 70S ribosome, characterize its interactions with the ribosome, and elucidate its mechanism on the ribosome. An X-ray structure of isolated BipA-GMPPNP was elucidated, by collaborators, and used for further molecular modeling of the protein to reveal possible atomic interactions between BipA and 70S ribosome. Additional biochemical studies were performed to fully characterize the specific ribosomal complex that optimizes binding of the factor. Together, the cryo-EM reconstruction, the BipA X-ray structure, the subsequent molecular modeling, and the additional biochemical studies provide a comprehensive model for BipA binding. Over the last years, the introduction of new automated algorithms for particle selection (AutoPicker) and classification (RELION) for the cryo-EM technique has revolutionized the workflow of the entire imaging and reconstruction process. The BipA dataset was primed to be used as a test bed for these algorithms and classification technique, respectively. Using old and new techniques to process the dataset allows a discussion of how the single particle reconstruction process can be vastly improved, with greater automation and efficiency.

Thesis (Ph.D.) -- McMaster University, 2006. / Supervisor: David W. Andrews. Includes bibliographical references (leaves 110-117).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 135-141).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 78-98).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 88-102).

Thesis (Ph. D.)--University of Oregon, 2003. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 101-114). Also available for download via the World Wide Web; free to University of Oregon users.