Thèse d'exercice : Médecine. Médecine générale : Université de Nantes : 2004. / Bibliogr. f. 101-109 [97 réf.].

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 29-33)f.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Thesis (Ph. D.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 227-228).

Thesis (M. Med. Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 23-28).

Thesis (M.Med.Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 29-33). Also available in print.

Thesis (M.Med.Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 23-28). Also available in print.

Vibrio cholerae, the etiological agent of the severe diarrheal disease cholera, is an enteric pathogen that can be found in aquatic ecosystems when not colonizing the human gastrointestinal tract. Under adverse environmental conditions, V. cholerae is capable of entering dormant states that increase its survival during these ecological fluctuations. In these states, V. cholerae slows its metabolic activity and exhibits drastically altered gene expression and morphology. Stressors that lead to entry into these states vary from nutrient limitation, suboptimal pH, or antimicrobials. Cells in these dormant states are highly resistant to antimicrobials and cannot be detected using standard microbiological techniques which poses major public health challenges such as food or water contamination. In V. cholerae, proteolysis of virulence regulator ToxR has been identified to be required for entry into a dormant state called viable but nonculturable (VBNC) under nutrient limitation and alkaline pH mediated by the sigma-E stress response. However, the mechanisms that lead to the initiation of this cascade remain unknown. The stringent response is another mechanism involved in mediating bacterial survival during late stationary phase. The stringent response involves the alarmone (p)ppGpp, which acts at the level of transcription to inhibit cellular processes that consume significant resources and activate genes responsible for biosynthetic processes. RelA is one enzyme responsible for the synthesis of (p)ppGpp, which in turn activates transcription of RpoE, suggesting a potential connection with ToxR proteolysis. Therefore, the aim of this study is to define the role of RelA in dormancy and ToxR proteolysis in V. cholerae. Our results show that RelA alone is not sufficient to control dormancy and ToxR proteolysis in V. cholerae. Nonetheless, another regulator (SpoT) is also associated with (p)ppGpp synthesis, indicating that other stringent response-associated mechanisms might be involved in ToxR proteolysis.

Globally, tuberculosis, a disease caused by the species of Mycobacterium tuberculosis (Mtb) complex, stands as a leading cause of death from a single infectious agent. Even though antituberculous drugs are available, treatment is challenging due to antibiotic resistance associated with point mutations in the bacterial genome. Resistance to the first-line antibiotics – rifampin and isoniazid – results in multidrug-resistant tuberculosis (MDR) requiring a more complicated treatment regimen. Timely and accurate identification of drug-resistant TB cases can help prescribe the most effective treatment and prevent the spread of infection. This research aims to develop an assay to discern multi-drug resistant forms of tuberculosis using a molecular assay based on split deoxyribozyme hybridization probes. For the probe design, a catalytic core of an RNA-cleaving deoxyribozyme is split into two parts, with each part elongated with a target-recognizing fragment ("arm"). In the presence of a fully complementary nucleic acid target, but not the one containing point mutations, the catalytic core of the deoxyribozyme can be re-formed due to the assembling of the target-probe complex, which recognizes and allows cleavage of a fluorophore- and quencher-labeled signal reporter, thereby ensuring increase in fluorescence in a target-dependent manner. The target-binding arms of the probes were optimized in terms of the signal-to-background ratio and selectivity of target recognition using synthetic targets corresponding to the fragments of the katG and rpoB genes with point-mutation sites implicated in the resistance to isoniazid and rifampin, respectively. The optimized probe sequences were used to interrogate the targets obtained by amplifying the correspondent fragments of the Mtb genes using Linear- After-The-Exponential (LATE) PCR, which allows efficient synthesis of a single-stranded amplicon. The signal triggered by cognate targets can be read using a portable fluorometer, which eliminates the need to use a sophisticated real-time PCR instrument for the assay. The success of the split deoxyribozyme assay can establish an affordable and user-friendly molecular diagnostic assay where a sample can be amplified and analyzed in a single tube.