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Bacteriophages cohabit with their bacterial hosts and shape microbial communities. To initiate infection, phages use bacterial components as receptors to recognize and attach to hosts. Flagellotropic phages utilize bacterial flagella as receptors. Studies focused on uncovering mechanistic details of flagellotropic phage infection are lacking. As the number of phage-based applications grows, it is important to understand these details to predict the potential outcomes of phage therapy. To this end, we studied two flagellotropic phages: Agrobacterium phage 7-7-1 and bacteriophage χ. Phage 7-7-1 infects Agrobacterium spp., while bacteriophage χ infects Salmonella and Escherichia coli. Chapter 1 consists of a literature review. Chapter 2 addresses factors underlying phage-bacteria coexistence. We document the emergence of a sector-shaped lysis pattern following co-inoculation of phage χ and one of its Salmonella hosts on swim plates. We propose that this pattern serves as a reporter for balanced phage-bacteria coexistence. Using a combined experimental and mathematical modelling approach, we discovered that variations to intrinsic factors (i.e., bacterial motility, phage adsorption) skews the pattern towards either bacterial or phage predominance. Thus, this computational model may be used to predict phage therapy application outcomes. Chapter 3 details the identification of cell surface receptors essential for phage 7-7-1 infection using a transposon mutagenesis approach. We identified three Agrobacterium sp. H13-3 genes involved in phage 7-7-1 infection. Using mass spectrometry and other analyses, we determined that the LPS profiles of strains lacking these genes varied compared to the wild type. Thus, LPS is a secondary cell surface receptor for phage 7-7-1. Chapter 4 focuses on the discovery of phage encoded receptor binding proteins (RBPs) in Agrobacterium phage 7-7-1. Using an RBP screen, we discovered three candidate RBPs. We learned that our top candidate, Gp4, inhibits the growth of Agrobacterium sp. H13-3 cells in a motility and glycan dependent manner. Because of its bacteriostatic activities, this protein is a promising candidate for therapeutic use. Overall, the described works contribute to a deepened understanding of flagellotropic phage infection and the factors influencing their coexistence with motile bacteria. These works will contribute towards the development of phage therapies using whole phage or their components. / Doctor of Philosophy / Bacteriophages, or phages for short, are the natural killers of bacteria. Like antibiotics, they can also be used as medicines to treat bacterial infections. Their attack on bacteria begins by recognizing specific parts of the bacterial cell and attaching to them. These parts are called receptors. To use phages as medicines it is important to understand how they recognize and kill bacteria. This information is helpful when deciding which phage should be given to treat a bacterial infection and to predict the outcomes of these treatments. In this work, we focused on two phages to answer different questions. Both phages use long helical thread-like structures, called flagella, as receptors. Flagella help the bacteria to move through the environment and reach new areas with more nutrients. One of these flagella-dependent phages, called phage 7-7-1, infects plant pathogens that cause tumor-like growth in plants. We found that this phage uses two very different host cell components during infection and identified one of the phage proteins that interacts with these receptors. This protein prevents the growth of the plant pathogen, which makes it a promising candidate for therapeutic use. We also investigated how another bacterial virus, bacteriophage χ, is spread throughout the environment and co-exists with its motile bacterial host. We built a computational model that can predict how altering different variables affects phage-bacteria coexistence. With additional research, this model will be a useful tool for predicting the outcomes following phage treatment.

Orientador: José Luiz Morais Vasconcelos / Banca: Ciro Moraes Barros / Banca: José Buratini Junior / Banca: Mario Binelli / Banca: Paula de Carvalho Papa / Resumo: O objetivo desse experimento foi avaliar a histomorfometria uterina, a expressão gênica endometrial de ER, PR, OTR, COX-1 e COX-2, e a expressão protéica de ER e PR (porcentagem de núcleos positivos e intensidade de coloração dos núcleos positivos, em unidades arbitrárias [UA], à imunohistoquímica) em vacas em anestro ao longo do desenvolvimento da onda folicular. Vacas Nelore primíparas em anestro pós-parto foram avaliadas diariamente por ultrassonografia ovariana, visando acompanhar o desenvolvimento folicular, e submetidas à histerectomia quando o folículo dominante atingiu o diâmetro de 7,0 mm (FP; n = 4), 8,5 mm (FM; n = 4) ou 10,0 mm (FG; n = 4). Um grupo adicional (PRO; n = 4) consistiu em vacas submetidas a remoção de bezerros (RB; 48 h) quando o folículo dominante atingiu o diâmetro de 9,5 mm e histerectomizadas ao final da RB. Os dados foram analisados pelo PROC GLM do programa SAS. A concentração sérica de estradiol no dia da histerectomia foi maior nas vacas do grupo PRO em relação aos demais grupos (FP: 0,7 pg/mL; FM: 0,8 pg/mL; FG: 1,1 pg/mL; PRO: 2,9 pg/mL; P < 0,05). Não houve efeito de grupo (P > 0,1) nas variáveis histomorfométricas e na expressão gênica endometrial de ER, PR, OTR, COX-1 e COX-2. As vacas do grupo PRO apresentaram maior intensidade de coloração dos núcleos positivos para ER nas células epiteliais glandulares superficiais (FP: 75,9 UA; FM: 75,2 UA; FG: 71,5 UA; PRO: 88,5 UA), intensidade de coloração dos núcleos positivos para PR nas células epiteliais glandulares profundas (FP: 70,2 UA; FM: 73,1 UA; FG: 69,2 UA; PRO: 85,7 UA) e porcentagem de núcleos positivos para PR no estroma subepitelial superficial (FP: 58,2%; FM: 58,0%; FG: 57,3%; PRO: 63,4) em relação às vacas dos demais grupos (P < 0,05). As vacas dos grupos FG e PRO apresentaram maior porcentagem de núcleos positivos para ER no estroma... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to evaluate the uterine histomorphometry, gene expression of ER, PR, OTR, COX-1 and COX-2, and protein expressions of ER and PR (percentage of positive nuclei and staining intensity of positive nuclei, in arbitrary units [AU], at immunohistochemistry) in anestrous cows throughout the development of a follicular wave. Primiparous postpartum anestrous Nelore cows were evaluated daily by ovarian ultrasonography and submitted to hysterectomy when the dominant follicle reached the diameter of 7.0 mm (FP; n = 4), 8.5 mm (FM; n = 4), or 10.0 mm (FG; n = 4). An additional group (PRO; n = 4) consisted in cows submitted to temporary weaning (TW; 48 h) when the dominant follicle reached 9.5 mm and hysterectomized at the end of TW. Data were analyzed by PROC GLM of SAS. Serum concentration of estradiol at the day of hysterectomy was greater in cows from PRO group than in cows from the other groups (FP: 0.7 pg/mL; FM: 0.8 pg/mL; FG: 1.1 pg/mL; PRO: 2.9 pg/mL; P < 0.05). There were no effects of group (P > 0.1) on histomorphometrical variables and on endometrial gene expression of ER, PR, OTR, COX-1, and COX-2. Cows from PRO group had greater staining intensity of ER-positive nuclei in shallow glandular epithelium (FP: 75.9 AU; FM: 75.2 AU; FG: 71.5 AU; PRO: 88.5 AU), staining intensity of PR-positive nuclei in deep glandular epithelium (FP: 70.2 AU; FM: 73.1 AU; FG: 69.2 AU; PRO: 85.7 AU), and greater percentage of PR-positive nuclei in shallow subepithelial stroma (FP: 58.2%; FM: 58.0%; FG: 57.3%; PRO: 63.4%) than cows from the other groups (P < 0.05). Cows from FG and PRO groups had a greater percentage of ER-positive nuclei in deep subepithelial stroma than cows from the other groups (FP: 76.0%; FM: 76.4%; FG: 83.6%; PRO: 86.1%; P < 0.05). In the other endometrial areas, the protein expressions of ER and PR were similar among groups. We concluded that in anestrous... (Complete abstract click electronic access below) / Doutor

生長滯後是包括炎症性腸病在內的炎症疾病引起的併發症。實驗表明，炎症使肝臟對生長激素(GH)的作用變得不敏感或引起生長激素抵抗。生長激素抵抗會引起胰島素生長因子-1 (IGF-I)的表達下降，並且會啟動一系列的代謝反應。多年來的研究證明炎症因子白介素-6 (IL-6)，腫瘤壞死因子 -α (TNF-α)和白介素-1β(IL-1β)參與肝臟生長激素抵抗的病理過程。然而這些炎症因子調控生長激素通路的具體機理尚不清楚。通過用人肝癌細胞系Huh-7和慢性炎症及急性炎症兩種老鼠模型，我們發現: 1) TNF-α和IL-1β抑制生長激素受體(GHR)的表達; 2) IL-6誘導細胞因子信號轉導抑制因子-3 (SOCS3)的高表達; 3) IL-6-SOCS3途徑對GH-IGF-I信號通路的抑制作用依賴于GHR的表達量，當TNF-α及IL-1β升高而使GHR的表達量下降後，IL-6就不再對GH-IGF-I信號通路有抑制作用。以上結果表明IL-6， TNF-α和IL-1β抑制肝臟生長激素信號通路的機制是不一樣的，這些結果或許對臨床上治療青少年中炎症引起的生長激素抵抗疾病有一定的指導意義。 / 成纖維細胞生長因子(FGF) 通過結合和啟動成纖維細胞生長因子受體(FGFR)而參與許多生理過程。FGF19屬於FGF15/19亞家族，這個亞家族還包括FGF21和FGF23。FGF19調節肝臟中膽汁酸的穩態及蛋白和糖原的合成。FGF19通過與FGFR4及共受體β-klotho結合來啟動信號通路。研究表明，TNF-α通過抑制共受體β-klotho的表達來抑制脂肪細胞中的FGF21信號通路。然而IL-6，TNF-α和IL-1β在調節肝臟FGF19信號通路中的作用尚不清楚。我們的體外細胞和體內動物實驗結果表明，IL-1β通過JNK和NF-κB通路抑制肝臟中β-klotho的表達。IL-6與TNF-α不調節Huh-7細胞中β-klotho的表達。 / 綜上所述，IL-6，TNF-α及IL-1β在肝臟生長激素及FGF19通路中起不同的調節作用。 / Growth failure is a major complication of inflammatory diseases including inflammatory bowel disease. Evidence suggests that during inflammation, the liver becomes resistant to growth hormone (GH) actions, leading to downregulation of the anabolic gene IGF-I and the activation of catabolic processes. Decades of studies demonstrated that pro-inflammatory cytokines IL-6, TNF-α and IL-1β are involved in the pathogenesis of hepatic GH resistance. However, the exact mechanisms used by these individual cytokines to regulate GH signaling are not defined. Using Huh-7 human hepatoma cells and mouse models of chronic and acute inflammation, we show that TNF-α and IL-1β but not IL-6 inhibited hepatic GH receptor (GHR) expression, and that IL-6 but not TNF-α and IL-1β stimulated expression of suppressor of cytokine signaling-3 (SOCS3). TNF-α/IL-1β and IL-6 acted primarily at GHR and SOCS3 respectively to inhibit the GH-IGF-I pathway. While TNF-α/IL-1β exerted a tonic inhibition on hepatic GH signaling, IL-6 activity is dependent on the active GH pathway. IL-6 lost its inhibition on the GH-IGF-I pathway when GHR expression was blocked as the inflammation progressed. These results reveal previously undefined distinct mechanisms used by TNF-α/IL-1β and IL-6 to inhibit the hepatic GH pathway. Our results may provide a new guidance for clinical practice in treating pediatric infammation-induced GH resistance. / Fibroblast growth factors (FGFs) play critical roles in many physiological processes by binding to and activating FGF receptor (FGFR) family. FGF19 belongs to FGF15/19 subfamily of FGFs that includes FGF15/19, FGF21 and FGF23. FGF19 has been shown to regulate bile acid homeostasis, and protein and glycogen synthesis in the liver. FGF19 binds FGFR4 and the co-receptor β-klotho to initiate signaling. Studies have shown that proinflammatory cytokines such as TNF-α can impair FGF21 signaling in adipose cells by repressing the expression of β-klotho. However, little is known about the effects of IL-6, TNF-α and IL-1β on regulating hepatic FGF19 signaling. In the present study, we found that IL-1β inhibited β-klotho expression both in vitro and in vivo, and this inhibition required JNK and NF-κB pathways. IL-6 and TNF-α did not inhibit β-klotho expression in Huh-7 cells. / Taken together, our results demonstrate that IL-6, TNF-α and IL-1β play different roles in regulating the GH and FGF-19 pathways in the liver. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhao, Yueshui. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 147-182). / Abstracts also in Chinese.

Szeto Yi Ki. / Thesis submitted in: December 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 83-113). / Abstracts in English and Chinese. / Acknowledgement --- p.iv / Abstract --- p.v / List of Abbreviations --- p.ix / Chapter CHATPER ONE --- INTRODUCTION / Chapter 1.1 --- Heroin --- p.1 / Chapter 1.1.1 --- Historical Background --- p.2 / Chapter 1.1.2 --- Manufacturing of Heroin --- p.5 / Chapter 1.1.3 --- Route of Administration and Absorption Rate --- p.6 / Chapter 1.1.4 --- Metabolism of Heroin --- p.8 / Chapter 1.1.5 --- Physical and Psychological Effects of Heroin --- p.9 / Chapter 1.2 --- Opioid Receptors --- p.10 / Chapter 1.2.1 --- Mu Opioid Receptors (MOR) --- p.11 / Chapter 1.2.2 --- Kappa Opioid Receptors (KOR) --- p.14 / Chapter 1.2.3 --- Delta Opioid Receptors (DOR) --- p.15 / Chapter 1.3 --- Dopamine Receptors --- p.17 / Chapter 1.4 --- Dopamine Transporter (DAT) --- p.19 / Chapter 1.5 --- Gamma-Aminobutyric Acid (GABA) Receptors --- p.21 / Chapter 1.6 --- Mesocorticolimbic Pathway --- p.22 / Chapter 1.6.1 --- Neural Substrates of Drug Reinforcement --- p.25 / Chapter 1.6.2 --- Molecular and Cellular Basis of Addiction --- p.26 / Chapter 1.6.3 --- Intracellular Substrates of Relapse --- p.29 / Chapter 1.7 --- Environmental Factors in Drug Addiction --- p.30 / Chapter 1.8 --- Genetic Factors in Drug Addiction --- p.32 / Chapter 1.9 --- Aim of Project --- p.35 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- Recruitment of Subjects 、 --- p.39 / Chapter 2.1.1 --- Heroin-dependent Subjects --- p.39 / Chapter 2.1.1.1 --- Phenotype Assessment --- p.39 / Chapter 2.1.1.2 --- Establishment of Socio-demographic Data --- p.40 / Chapter 2.1.2 --- Control Subjects --- p.42 / Chapter 2.2 --- DNA Extraction --- p.42 / Chapter 2.3 --- Genotyping --- p.43 / Chapter 2.3.1 --- A118G Polymorphism in Exon 1 of the Human MOR (hMOR) Gene --- p.43 / Chapter 2.3.2 --- C1031G Polymorphism in Intron 2 of the hMOR Gene --- p.45 / Chapter 2.3.3 --- T921C Polymorphism in Exon 3 of the Human DOR (hDOR) Gene --- p.46 / Chapter 2.3.4 --- 3'VNTR Polymorphism of the DAT Gene --- p.47 / Chapter 2.3.5 --- TaqI A Polymorphism of the DRD2 Gene --- p.48 / Chapter 2.3.6 --- NciI Polymorphism of the GABRG2 Gene --- p.48 / Chapter 2.4 --- DNA Sequencing --- p.49 / Chapter 2.5 --- Statistical Analysis --- p.50 / Chapter CHAPTER THREE --- RESULTS / Chapter 3.1 --- Socio-demographic Data --- p.52 / Chapter 3.1.1 --- Age of the Control and Heroin-dependent Subjects --- p.52 / Chapter 3.1.2 --- Education Standard of the Heroin-dependent Subjects --- p.52 / Chapter 3.1.3 --- Years of Heroin Use --- p.53 / Chapter 3.2 --- Addition Severity Index (ASI) --- p.53 / Chapter 3.2.1 --- ASI-Medical --- p.53 / Chapter 3.2.2 --- ASI-Employment --- p.54 / Chapter 3.2.3 --- ASI-Drug --- p.54 / Chapter 3.2.4 --- ASI-Legal --- p.54 / Chapter 3.2.5 --- ASI-Family/Social Relationships --- p.55 / Chapter 3.2.6 --- ASI-Psychiatry --- p.55 / Chapter 3.2.7 --- Correlation Among the Factors of ASI --- p.55 / Chapter 3.3 --- A118G Polymorphism in Exon 1 of the Human Mu Opioid Receptor (hMOR) Gene --- p.56 / Chapter 3.4 --- C1031G Polymorphism in Intron 2 of the hMOR Gene --- p.58 / Chapter 3.5 --- T921C Polymorphism in Exon 3 of the Human Delta Opioid Receptor (hDOR) Gene --- p.59 / Chapter 3.6 --- Interaction Between Genotypes --- p.60 / Chapter 3.6.1 --- Combined Genotypes of A118G and C1031G Polymorphisms of the hMOR Gene --- p.60 / Chapter 3.6.2 --- Combined Genotypes of A118G Polymorphism of the hMOR Gene and T921C Polymorphism of the hDOR Gene --- p.61 / Chapter 3.6.3 --- Combined Genotypes of C1031G Polymorphism of the hMOR Gene and T921C Polymorphism of the hDOR Gene --- p.61 / Chapter 3.7 --- Correlation Between Allelic Frequencies and Factors of the ASI --- p.62 / Chapter 3.8 --- 3'VNTR Polymorphism of DAT Gene --- p.62 / Chapter 3.9 --- TαqI A Polymorphism of DRD2 Gene --- p.63 / Chapter 3.10 --- NciI Polymorphism of GABRG2 Gene --- p.64 / Chapter CHAPTER FOUR --- DISCUSSION & CONCLUSION --- p.66 / REFERENCES --- p.83 / APPENDIX I The Addiction Severity Index / APPENDIX II Table of Severity Ratings / APPENDIX III Allelic Frequency of A118G Polymorphism in Different Populations / APPENDIX IV Details Information About the Single Nucleotide Polymorphisms In Present Study