No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Elimination of bacteria and active infection within an infected root canal system is one of the primary objectives of nonsurgical root canal treatment. One of the measures of successful root canal treatment is subsequent bone healing of periapical lesions caused by previous infection. A previous study by Yumoto et al. showed that electromagnetic wave stimulation can increase proliferation of osteoblastic cells with no cytotoxicity, and it can also up-regulate growth factors such as vascular endothelial growth factor and platelet-derived growth factor.18 They also showed increased proliferation of an immortalized osteoblastic MC3T3-E1 cell line 3 days following electromagnetic stimulation (EMS).18 Previously, Pauly et al. found increased alkaline phosphatase (ALP) activity with 10 mA EMS application to primary murine calvaria-derived osteoblastic cells with 5 pulses at 1 second per pulse, but no significant differences were found for MTS proliferation nor mineral deposition compared to a negative control group.82 Optimization of the different variables including post-treatment incubation time, current delivery, and number of pulses per treatment may be necessary to improve osteogenic activity. The use of mesenchymal stem cells from murine bone marrow may also offer a physiologically relevant model for osteoblastic regeneration of periapical lesions. Objectives: The goal of this study was to investigate and optimize the effects of electromagnetic wave stimulation (EMS) on murine bone marrow mesenchymal stem cells (MSCs) by evaluating the proliferation and differentiation of the cells after exposure to different EMS treatment regimens. Materials and Methods: 5 x104 stromal osteoblasts (SOBs) were cultured in 24-well plates in α-MEM containing 10% fetal bovine serum. Cells were then subjected to pulsed EMS treatments of 1 mA, 10 mA, and 50 mA. EMS was generated using an electromagnetic apical treatment (EMAT) device created by J. Morita MFG Corp. Proliferation was assessed via MTS assay 1 days after treatment. For osteogenic differentiation, ascorbic acid and β-glycerol phosphate were added to the culture media, and SOBs were cultured for 14 days. Afterwards, alkaline phosphatase (ALP) activity and Alizarin-red S mineral deposition were quantified as measures of osteoblast activity. Cells grown in osteogenic media without EMS treatment served as the negative control. Results: Although MSC proliferation was unaffected by different EMS treatment regimens, 50 mA EMS resulted in a decrease in ALP activity and mineral deposition by osteoblasts. Conclusions: Our findings suggest bone healing by EMS may involve a different cellular mechanism, that is not reproduced in vitro in our studies. Utilizing different amperage and EMS regimens may improve osteogenic differentiation.

A fototerapia com laser em baixa intensidade (FTLBI) é capaz de aumentar o metabolismo celular, o que poderia influenciar na diferenciação ósseo/odontogênica das células-tronco da polpa dentária humada (hDPSCs). O PDGF e o BMP-2 são fatores de crescimento envolvidos na dentinogênese e na reparação tecidual. O PDGF tem papel importante durante o desenvolvimento embrionário, na proliferação e migração celular e na angiogênese, enquanto o BMP-2 está fortemente associado à diferenciação celular em tecidos mineralizados, como o osso e a dentina. Sendo assim, o objetivo do estudo foi analisar os efeitos da FTLBI e dos fatores de crescimento (PDGF-BB ou BMP-2), isolados ou em associação, na diferenciação ósseo/odontogênica das hDPSCs. Para o estudo hDPSCs foram cultivadas em meio regular (G1) e irradiadas (G2), meio mineralizante (G3) e irradiadas (G4), meio mineralizante contendo PDGF-BB (G5) e irradiadas (G6), meio mineralizante contendo BMP-2 (G7) e irradiadas (G8). Para os grupos irradiados, a FTLBI foi realizada no modo pontual e em contato, com um laser de diodo semi-condutor, com área de feixe de 0,028cm2 e comprimento de onda 660nm (InGaAlP-vermelho), utilizando-se os seguintes parâmetros: potência de 20mW, densidade de energia de 5J/cm2, tempo de irradiação de 7 segundos por ponto e 0,14J de energia por ponto. A expressão dos genes relacionados à diferenciação ósseo/odontogênica (DSPP, DMP-1 e OCN) através do PCR quantitativo em tempo real (qRT-PCR), a atividade da fosfatase alcalina e os depósitos de cálcio foram analisados em 3, 7 e 14 dias. Os dados obtidos foram comparados pelo teste ANOVA complementado pelo teste de Tukey (p<0,05). As culturas tratadas com meio mineralizante contendo BMP-2 e irradiadas (G8) foram as que mostraram os maiores índices de diferenciação ósseo/odontogênica nos testes realizados. As expressões de DSPP, OCN e DMP-1, ao menos em 14 dias, foram significantemente maiores no G8 que nos demais grupos experimentais, exceto os grupos G3 e G7. Estes grupos apresentaram expressões de DSPP e OCN semelhantes às do G8 em 14 dias. A maior atividade de ALP foi observada no G8 em 3 dias e a menor no mesmo grupo aos 14 dias. A maior quantidade de depósitos de cálcio também foi encontrada no G8 em 14 dias. A associação de FTLBI e BMP-2 se mostrou capaz de induzir a diferenciação ósseo/odontogênica em células-tronco de polpa dentária humana de forma mais marcante que as demais terapias isoladas ou associadas estudadas. Portanto, o uso de uma terapia associando FTLBI e BMP-2 poderia ser de relevância para o restabelecimento da fisiologia pulpar quando aplicada em casos de exposição deste tecido, uma vez que poderia favorecer a diferenciação das células indiferenciadas da polpa dentária. / Laser phototherapy (LPT) is able to increase cellular metabolism, which in turn could influence the odontogenic differentiation of dental pulp stem cells (hDPSCs). PDGF and BMP-2 are growth factors involved in dentinogenesis and tissue repair. PDGF plays a role in embryonic development, cell proliferation, cell migration, and angiogenesis, whereas BMP-2 is strongly associated with cell differentiation in mineralized tissues such as bone and dentin. The aim of this study was to analyze the effects of LPT and the growth factors PDGF-BB and BMP-2 combined or not on the odontogenic differentiation of hDPSCs. These cells were grown in regular medium (G1) and irradiated (G2), mineralizing medium (G3) and irradiated (G4), mineralizing medium containing PDGF-BB (G5) and irradiated (G6), mineralizing medium containing BMP-2 (G7) and irradiated (G8). For irradiated groups, LPT was performed in punctual and contact mode with a semiconductor diode laser, with a beam spot area of 0.028 cm2 and wavelength of 660nm (InGaAlP-visible red), using the following parameters: power of 20mW, energy density of 5J/cm2 and irradiation time of 7 seconds per point (0,14 J per point). Differentiation was assessed by the following analysis: expression of genes related to odontogenic differentiation (DSPP, DMP-1 and OCN) using quantitative real time PCR (qRT-PCR); alkaline phosphatase activity and calcium deposition using alizarin red staining in 3, 7 and 14 days. Data were compared by ANOVA and Tukey´s test (p<0.05). The cultures treated with mineralizing medium containing BMP-2 and irradiated (G8) showed the highest rate of odontogenic differentiation. The expressions of DSPP, DMP-1 and OCN genes, at least in 14 days, were significantly higher in G8 compared to all other groups, except for the groups G3 and G7. These groups showed similar expressions of DSPP and OCN than G8 in 14 days. G8 showed the highest ALP activity in 3 days and the lowest in 14 days compared to all other groups. The largest amount of calcium deposits was observed in G8 in 14 days. The most striking feature on induction of odontogenic differentiation of hDPSCs was observed when LPT was applied in association with BMP-2. Therefore, the use of a combined LPT and BMP-2 therapy could be of relevance for the re-establishment of pulp physiology when applied in cases of dental pulp exposure by promoting the differentiation of hDPSCs.

A alveolite é uma complicação pós-operatória de carácter inflamatório que acomete alvéolos de dentes recém-extraídos. A incidência dessa complicação varia de 1 a 4% e pode chegar a 30%. O objetivo deste estudo foi analisar os mecanismos biológicos envolvidos no processo de reparo de alvéolos intencionalmente infectados, em ratos; comparar diferentes modalidades de tratamento e correlacionar os resultados encontrados através de duas análises (microscópica e molecular). Foram utilizados 84 ratos, divididos nos grupos: I: alvéolo não infectado; II: alvéolo infectado sem nenhum tratamento; III: alvéolo infectado tratado com irrigação de solução de iodeto de sódio a 2% e peróxido de hidrogênio a 3% na proporção de 1:1; e IV: alvéolo infectado submetido à curetagem, irrigação com soro fisiológico e preenchimento com uma pasta à base de metronidazol. Os animais foram eutanasiados aos 6, 15 e 28 dias pós-operatório. Foi realizada a análise quantitativa da expressão de genes envolvidos no processo de reparo [colágeno tipo I (COL-I), fator de crescimento do endotélio vascular (VEGF), osteocalcina (OCN), fosfatase alcalina (ALP), runt-related transcription factor 2 (RUNX2) e fator de necrose tumoral alfa (TNF-\'alfa\')], através da RealTimePCR, correlacionando sua expressão com as características microscópicas observadas qualitativa e quantitativamente. Com base nos resultados da análise microscópica e molecular, podemos concluir que os marcadores RUNX2, OCN e TNF-\'alfa\' podem ser usados como indicadores para avaliar a neoformação óssea e a quantidade de infiltrado inflamatório em alveolite. Os marcadores ALP e VEGF não representaram adequadamente o que se observou microscopicamente. Embora o tratamento da alveolite com a pasta à base de metronidazol promova maior densidade de neoformação óssea aos 28 dias, não há diferenças entre os tratamentos. / Dry socket is an inflammatory postoperative complication that undertakes sockets of recently extracted teeth. The incidence of such complication varies from 1 to 4% and might reach up to 30%. The objective of this study was to analyze the biological mechanisms involved in the repair process of intentionally infected sockets in mice; compare different treatment conditions and correlate the results of two different analysis (microscopic and molecular). 84 mice were used in this study, divided according the following groups: I: uninfected socket; II: infected socket without any treatment; III: infected socket treated with irrigation of 2% sodium iodide and 3% hydrogen peroxide solution at 1:1 proportion; and IV: infected socket submitted to curettage, physiological saline solution irrigation and fulfillment with metronidazole base paste. The animals were killed at a postoperative period of 6, 15 and 28 days. A quantitative analysis was performed using a RealTimePCR to evaluate the genes expression involved [Collagen Type I (COL-I), vascular endothelial growth factor (VEGF), osteocalcin (OCN), alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2) and tumor necrosis factor-alpha (TNF-\'alpha\')], in the repair process, correlating its expression with the microscopic characteristics observed in both qualitative and quantitative manner. Based in the results of the microscopic and molecular analysis, it can be concluded that the RUNX2, OCN and TNF-\'alpha\' markers can be used as indicators to evaluate the dry socket bone neoformation and inflammatory infiltrate quantity. The ALP and VEGF markers did not represented appropriately what was observed microscopically. Although the dry socket treatment with metronidazole base paste promotes an increase in the bone neoformation density at 28 days, no difference was found among the treatments.

A alveolite é uma complicação pós-operatória de carácter inflamatório que acomete alvéolos de dentes recém-extraídos. A incidência dessa complicação varia de 1 a 4% e pode chegar a 30%. O objetivo deste estudo foi analisar os mecanismos biológicos envolvidos no processo de reparo de alvéolos intencionalmente infectados, em ratos; comparar diferentes modalidades de tratamento e correlacionar os resultados encontrados através de duas análises (microscópica e molecular). Foram utilizados 84 ratos, divididos nos grupos: I: alvéolo não infectado; II: alvéolo infectado sem nenhum tratamento; III: alvéolo infectado tratado com irrigação de solução de iodeto de sódio a 2% e peróxido de hidrogênio a 3% na proporção de 1:1; e IV: alvéolo infectado submetido à curetagem, irrigação com soro fisiológico e preenchimento com uma pasta à base de metronidazol. Os animais foram eutanasiados aos 6, 15 e 28 dias pós-operatório. Foi realizada a análise quantitativa da expressão de genes envolvidos no processo de reparo [colágeno tipo I (COL-I), fator de crescimento do endotélio vascular (VEGF), osteocalcina (OCN), fosfatase alcalina (ALP), runt-related transcription factor 2 (RUNX2) e fator de necrose tumoral alfa (TNF-\'alfa\')], através da RealTimePCR, correlacionando sua expressão com as características microscópicas observadas qualitativa e quantitativamente. Com base nos resultados da análise microscópica e molecular, podemos concluir que os marcadores RUNX2, OCN e TNF-\'alfa\' podem ser usados como indicadores para avaliar a neoformação óssea e a quantidade de infiltrado inflamatório em alveolite. Os marcadores ALP e VEGF não representaram adequadamente o que se observou microscopicamente. Embora o tratamento da alveolite com a pasta à base de metronidazol promova maior densidade de neoformação óssea aos 28 dias, não há diferenças entre os tratamentos. / Dry socket is an inflammatory postoperative complication that undertakes sockets of recently extracted teeth. The incidence of such complication varies from 1 to 4% and might reach up to 30%. The objective of this study was to analyze the biological mechanisms involved in the repair process of intentionally infected sockets in mice; compare different treatment conditions and correlate the results of two different analysis (microscopic and molecular). 84 mice were used in this study, divided according the following groups: I: uninfected socket; II: infected socket without any treatment; III: infected socket treated with irrigation of 2% sodium iodide and 3% hydrogen peroxide solution at 1:1 proportion; and IV: infected socket submitted to curettage, physiological saline solution irrigation and fulfillment with metronidazole base paste. The animals were killed at a postoperative period of 6, 15 and 28 days. A quantitative analysis was performed using a RealTimePCR to evaluate the genes expression involved [Collagen Type I (COL-I), vascular endothelial growth factor (VEGF), osteocalcin (OCN), alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2) and tumor necrosis factor-alpha (TNF-\'alpha\')], in the repair process, correlating its expression with the microscopic characteristics observed in both qualitative and quantitative manner. Based in the results of the microscopic and molecular analysis, it can be concluded that the RUNX2, OCN and TNF-\'alpha\' markers can be used as indicators to evaluate the dry socket bone neoformation and inflammatory infiltrate quantity. The ALP and VEGF markers did not represented appropriately what was observed microscopically. Although the dry socket treatment with metronidazole base paste promotes an increase in the bone neoformation density at 28 days, no difference was found among the treatments.

A fototerapia com laser em baixa intensidade (FTLBI) é capaz de aumentar o metabolismo celular, o que poderia influenciar na diferenciação ósseo/odontogênica das células-tronco da polpa dentária humada (hDPSCs). O PDGF e o BMP-2 são fatores de crescimento envolvidos na dentinogênese e na reparação tecidual. O PDGF tem papel importante durante o desenvolvimento embrionário, na proliferação e migração celular e na angiogênese, enquanto o BMP-2 está fortemente associado à diferenciação celular em tecidos mineralizados, como o osso e a dentina. Sendo assim, o objetivo do estudo foi analisar os efeitos da FTLBI e dos fatores de crescimento (PDGF-BB ou BMP-2), isolados ou em associação, na diferenciação ósseo/odontogênica das hDPSCs. Para o estudo hDPSCs foram cultivadas em meio regular (G1) e irradiadas (G2), meio mineralizante (G3) e irradiadas (G4), meio mineralizante contendo PDGF-BB (G5) e irradiadas (G6), meio mineralizante contendo BMP-2 (G7) e irradiadas (G8). Para os grupos irradiados, a FTLBI foi realizada no modo pontual e em contato, com um laser de diodo semi-condutor, com área de feixe de 0,028cm2 e comprimento de onda 660nm (InGaAlP-vermelho), utilizando-se os seguintes parâmetros: potência de 20mW, densidade de energia de 5J/cm2, tempo de irradiação de 7 segundos por ponto e 0,14J de energia por ponto. A expressão dos genes relacionados à diferenciação ósseo/odontogênica (DSPP, DMP-1 e OCN) através do PCR quantitativo em tempo real (qRT-PCR), a atividade da fosfatase alcalina e os depósitos de cálcio foram analisados em 3, 7 e 14 dias. Os dados obtidos foram comparados pelo teste ANOVA complementado pelo teste de Tukey (p<0,05). As culturas tratadas com meio mineralizante contendo BMP-2 e irradiadas (G8) foram as que mostraram os maiores índices de diferenciação ósseo/odontogênica nos testes realizados. As expressões de DSPP, OCN e DMP-1, ao menos em 14 dias, foram significantemente maiores no G8 que nos demais grupos experimentais, exceto os grupos G3 e G7. Estes grupos apresentaram expressões de DSPP e OCN semelhantes às do G8 em 14 dias. A maior atividade de ALP foi observada no G8 em 3 dias e a menor no mesmo grupo aos 14 dias. A maior quantidade de depósitos de cálcio também foi encontrada no G8 em 14 dias. A associação de FTLBI e BMP-2 se mostrou capaz de induzir a diferenciação ósseo/odontogênica em células-tronco de polpa dentária humana de forma mais marcante que as demais terapias isoladas ou associadas estudadas. Portanto, o uso de uma terapia associando FTLBI e BMP-2 poderia ser de relevância para o restabelecimento da fisiologia pulpar quando aplicada em casos de exposição deste tecido, uma vez que poderia favorecer a diferenciação das células indiferenciadas da polpa dentária. / Laser phototherapy (LPT) is able to increase cellular metabolism, which in turn could influence the odontogenic differentiation of dental pulp stem cells (hDPSCs). PDGF and BMP-2 are growth factors involved in dentinogenesis and tissue repair. PDGF plays a role in embryonic development, cell proliferation, cell migration, and angiogenesis, whereas BMP-2 is strongly associated with cell differentiation in mineralized tissues such as bone and dentin. The aim of this study was to analyze the effects of LPT and the growth factors PDGF-BB and BMP-2 combined or not on the odontogenic differentiation of hDPSCs. These cells were grown in regular medium (G1) and irradiated (G2), mineralizing medium (G3) and irradiated (G4), mineralizing medium containing PDGF-BB (G5) and irradiated (G6), mineralizing medium containing BMP-2 (G7) and irradiated (G8). For irradiated groups, LPT was performed in punctual and contact mode with a semiconductor diode laser, with a beam spot area of 0.028 cm2 and wavelength of 660nm (InGaAlP-visible red), using the following parameters: power of 20mW, energy density of 5J/cm2 and irradiation time of 7 seconds per point (0,14 J per point). Differentiation was assessed by the following analysis: expression of genes related to odontogenic differentiation (DSPP, DMP-1 and OCN) using quantitative real time PCR (qRT-PCR); alkaline phosphatase activity and calcium deposition using alizarin red staining in 3, 7 and 14 days. Data were compared by ANOVA and Tukey´s test (p<0.05). The cultures treated with mineralizing medium containing BMP-2 and irradiated (G8) showed the highest rate of odontogenic differentiation. The expressions of DSPP, DMP-1 and OCN genes, at least in 14 days, were significantly higher in G8 compared to all other groups, except for the groups G3 and G7. These groups showed similar expressions of DSPP and OCN than G8 in 14 days. G8 showed the highest ALP activity in 3 days and the lowest in 14 days compared to all other groups. The largest amount of calcium deposits was observed in G8 in 14 days. The most striking feature on induction of odontogenic differentiation of hDPSCs was observed when LPT was applied in association with BMP-2. Therefore, the use of a combined LPT and BMP-2 therapy could be of relevance for the re-establishment of pulp physiology when applied in cases of dental pulp exposure by promoting the differentiation of hDPSCs.

Caractériser la fonction des protéines est crucial pour notre compréhension des mécanismes moléculaires de la vie, des maladies, et aussi pour inspirer de nouvelles applications en bionanotechnologie. Pour y arriver, il est nécessaire de caractériser la structure et la dynamique de chaque état occupé par la protéine durant sa fonction. La caractérisation expérimentale des états transitoires des protéines représente encore un défi majeur parce que les techniques à haute résolution structurelle, telles que la spectroscopie RMN et la cristallographie aux rayons X, peuvent difficilement être appliquées à l’étude des états de courte durée. De plus, les techniques à haute résolution temporelle, telles que la spectroscopie de fluorescence, nécessitent généralement une chimie complexe pour introduire des fluorophores à des endroits spécifiques dans la protéine. Dans cette thèse nous introduisons l’utilisation des nanoantennes fluorescentes en tant que nouvelle stratégie pour détecter et signaler les changements de conformation des protéines via des interactions non covalentes entre des fluorophores spécifiques et la surface de la protéine. En utilisant des expériences et des simulations moléculaires, nous démontrons que des fluorophores chimiquement divers peuvent se lier et être utilisés pour sonder différentes régions d’une enzyme modèle, la phosphatase alcaline (PA). Ces nanoantennes peuvent être fixées directement aux protéines ou utilisées à l'aide du système de fixation simple et modulaire, le complexe biotine-streptavidine (SA), qui permet un criblage rapide et efficace de la nanoantenne optimale tant dans sa composition que sa longueur. Dans le cas de la PA, nous montrons que nos nanoantennes permettent la détection et la caractérisation des conformations distinctes incluant les changements conformationnels nanoscopiques produisant durant la catalyse du substrat. Nous démontrons également que les signaux fluorescents émis par la nanoantenne peuvent également permettre de caractériser la cinétique enzymatique d’une protéine en une seule expérience tout en incluant la détermination des paramètres « Michaelis-Menten » de ses substrats et inhibiteurs. Nous avons également exploré l'universalité de la stratégie ces nanoantennes fluorescentes en utilisant une autre protéine modèle, la Protéine G et son interaction avec les anticorps, et avons démontré son utilité pour mettre au point un essai permettant de détecter les anticorps. Ces nanoantennes simples et faciles à utiliser peuvent être appliquées pour détecter et analyser les changements conformationnels de toutes tailles et nos résultats suggèrent qu'elles pourraient être utilisées pour caractériser n’importe quel type de fonction. / The characterisation of protein function is crucial to understanding the molecular mechanisms of life and disease, and inspires new applications in bionanotechnology. To do so, it is necessary to characterise the structure and dynamics of each state that proteins adopt during their function. Experimental study of protein transient states, however, remains a major challenge because high-structural-resolution techniques, including NMR spectroscopy and X-ray crystallography, can often not be directly applied to study short-lived protein states. On the other hand, high-temporal-resolution techniques, such as fluorescence spectroscopy, typically require complicated site-specific labelling chemistry. This thesis introduces the use of fluorescent nanoantennas as a new strategy for sensing and reporting on protein conformational changes through noncovalent dye-protein interactions driven by a high local concentration. Using experiments and molecular simulations, we first demonstrate that chemically diverse dyes can bind and be used to probe different regions of a model enzyme, intestinal alkaline phosphatase (AP). These nanoantennas can be attached directly to proteins or employed using the simple and modular biotin-streptavidin (SA) attachment system, which enables rapid and efficient screening for high sensitivity by tuning their length and composition. We show that these nanoantennas enable the detection and characterisation of distinct conformational changes of AP, including nanoscale conformational changes that occur during substrate catalysis. We also show that the fluorescent signal emitted by the nanoantenna enables complete characterisation of enzyme kinetics in one experiment, including determination of Michaelis-Menten parameters of substrates and inhibitors of AP. We then explored the universality of the nanoantenna strategy by using a different model protein system. Protein G was shown to interact with antibodies, using a rapid screening strategy for antibody detection. These effective and easy-to-use nanoantennas could potentially be employed to monitor various conformational changes, and our results offer potential for characterising various protein functions.

No description available.

Heteropolymeric O antigen (O-Ag)-capped lipopolysaccharide is the principal constituent of the Gram-negative bacterial cell surface. It is assembled via the integral inner membrane (IM) Wzx/Wzy-dependent pathway. In Pseudomonas aeruginosa, Wzx translocates lipid-linked anionic O-Ag subunits from the cytoplasmic to the periplasmic leaflets of the IM, where Wzy polymerizes the subunits to lengths regulated by Wzz1/2. The Wzx and Wzy IM topologies were mapped using random C-terminal-truncation fusions to PhoALacZα, which displays PhoA/LacZ activity dependent upon its subcellular localization. Twelve transmembrane segments (TMS) containing charged residues were identified for Wzx. Fourteen TMS, two sizeable cytoplasmic loops (CL), and two large periplasmic loops (PL3 and PL5 of comparable size) were characterized for Wzy. Despite Wzy PL3–PL5 sequence homology, these loops were distinguished by respective cationic and anionic charge properties. Site-directed mutagenesis identified functionally-essential Arg residues in both loops. These results led to the proposition of a “catch-and-release” mechanism for Wzy function. The abovementioned Arg residues and intra-Wzy PL3–PL5 sequence homology were conserved among phylogenetically diverse Wzy homologues, indicating widespread potential for the proposed mechanism. Unexpectedly, Wzy CL6 mutations disrupted Wzz1-mediated regulation of shorter O-Ag chains, providing the first evidence for direct Wzy–Wzz interaction. Mutagenesis studies identified functionally-important charged and aromatic TMS residues localized to either the interior vestibule or TMS bundles in a 3D homology model constructed for Wzx. Substrate-binding or energy-coupling roles were proposed for these residues, respectively. The Wzx interior was found to be cationic, consistent with translocation of anionic O-Ag subunits. To test these hypotheses, Wzx was overexpressed, purified, and reconstituted in proteoliposomes loaded with I−. Common transport coupling ions were introduced to “open” the protein and allow detection of I− flux via reconstituted Wzx. Extraliposomal changes in H+ induced I− flux, while Na+ addition had no effect, suggesting H+-dependent Wzx gating. Putative energy-coupling residue mutants demonstrated defective H+-dependent halide flux. Wzx also mediated H+ uptake as detected through fluorescence shifts from proteoliposomes loaded with pH-sensitive dye. Consequently, Wzx was proposed to function via H+-coupled antiport. In summary, this research has contributed structural and functional knowledge leading to novel mechanistic understandings for O-Ag biosynthesis in bacteria. / Bookmarks within the document have been provided for ease of access to a particular section in the body of the thesis. Each entry in the Table of Contents, List of Tables, and List of Figures has been "linked" to its respective position and as such can be clicked for direct access to the entry. Similarly, each in-text Figure or Table reference has been "linked" to its respective figure/table for direct access to the entry. / 1.) Canadian Institutes of Health Research (CIHR) Frederick Banting and Charles Best Canada Graduate Scholarship doctoral award, 2.) CIHR Michael Smith Foreign Study Award, 3.) Cystic Fibrosis Canada (CFC) doctoral studentship, 4.) University of Guelph Dean's Tri-Council Scholarship, 5.) Ontario Graduate Scholarship in Science and Technology, 6.) Operating grants to Dr. Joseph S. Lam from CIHR (MOP-14687) and CFC