Ces travaux de recherche ont porté sur la thématique de concentration de traces d'analytes dans un échantillon grâce à l'électropréconcentration dans un dispositif fluidique contenant une nanofente. La technique étudiée est très récente, car mise en évidence il y a quelques années. De nombreux paramètres sont impliqués dans l'électropréconcentration, dont une grande majorité sont difficilement maîtrisables, rendant l'interprétation des résultats très délicate. Le Chapitre 1 présente le contexte sociétal et économique qui justifient les efforts de recherche pour les dispositifs fluidiques intégrant des structures nanométriques. Les techniques concurrentes permettent de fixer les limites actuelles. Enfin, la théorie et l'état de l'art de l'électropréconcentration fixe le point de départ de ces trois années. Le Chapitre 2 présente le procédé de fabrication initial. Les optimisations testées et leurs résultats sont décrits mais n'ont pas permis d'obtenir des résultats satisfaisants. Une partie importante de ce travail de thèse a donc servi au développement d'une solution alternative qui a permis de continuer les expériences fluidiques. Le procédé innovant développé a mené à une demande de brevet. Le Chapitre 3 présente le banc expérimental optimisé. Le protocole, très important pour ces expériences, a été étudié afin d'obtenir des résultats fluidiques expérimentaux fiables. Le Chapitre 4 décrit les expériences fluidiques réalisées au cours de ces trois ans. Les expériences originales de transport ionique à travers la nanofente grâce à une surpression seule, un champ électrique seul puis grâce à la combinaison d'un champ électrique et hydrodynamique y sont présentées.

Salmonella outbreaks associated with orange juices have been reported in the past. Though there have been studies on the internalization of Salmonella into oranges there is inadequate information on the ability of this pathogen to survive on orange surfaces, become internalized, and survive the low pH internal conditions. The objective of this work was to study the survival of Salmonella gaminara and Salmonella agona on oranges obtained from the field and retail outlets and investigate their attachment and internalization potential. These studies showed that oranges obtained from both the field and retail outlets harbored relatively high concentrations of aerobic heterotrophic bacterial populations. There were significant differences in the survival of Salmonella agona and Salmonella gaminara at 4??C, room temperature (25??C) and 37??C. Survival was highest at 37??C and lowest at 4??C for both Salmonella gaminara and Salmonella agona. Salmonella agona and Salmonella gaminara showed significant differences in recovery when the cells were treated with pH 4.0, 7.0 and 9.5 buffers. The internalization studies suggest that a negative temperature differential favors the internalization of Salmonella cells into the fruit. Significant differences in the internalization of Salmonella into field and market oranges were observed with more internalization in the field oranges as compared to the market oranges. These results suggest that to prevent Salmonella contamination of orange juices adequate pre-harvest protection against pathogen contamination and post-harvest cleaning and disinfection strategies need to be employed.

Specialized cells of the innate immune system, such as macrophages, employ lysosomal enzymes, together with cationic peptides and reactive oxygen intermediates, to eliminate invading microorganisms ensnared within phagosomes. The effectiveness of this impressive armamentarium is potentiated by the acid pH generated by the vacuolar-type ATPase (V-ATPase). The determinants of the luminal pH of phagosomes and of the lysosomes they fuse with are not completely understood, but the V-ATPase is known to be electrogenic and net accumulation of protons requires charge compensation. For this reason, counter-ion pathways are thought to serve a central role in the control of acidification. It has generally been assumed that a parallel anion influx accompanies proton pumping to dissipate the voltage that tends to build up. In fact, impaired chloride channel activity in cystic fibrosis has been proposed to underlie the defective phagolysosome acidification and microbial killing reported in lung macrophages. In the first part of this thesis, I devised methods to dialyze the lumen of lysosomes in intact cells, while monitoring lysosomal pH, in order to assess the individual contribution of counter-ions to acidification. Surprisingly, anions were found to be completely dispensable for proton pumping, whereas the presence of permeant cations in the lysosomal lumen was essential. Accordingly, defects in lysosomal anion permeability cannot explain the impaired microbicidal capacity of phagocytes in cystic fibrosis. Even though counter-ion permeation pathways exist, dissipation of the electrical contribution of the V-ATPase may not be complete. If present, a transmembrane potential would alter the rate and extent of proton accumulation in phagosomes and lysosomes. However, no estimates of the voltage across the phagosomes were available. To overcome this deficiency, in the second part of this thesis, I describe a noninvasive procedure to estimate the voltage across the phagosome using fluorescence resonance energy transfer. This novel approach, in combination with organellar pH measurements, demonstrated that proton pumping is not limited by counter-ion permeability.

Helicobacter pylori (H. pylori) is a gram-negative pathogenic bacterium that infects half of the world’s population. It resides in the human stomach, where it survives extremely acidic conditions. Efficient colonization by H. pylori requires urease and hydrogenase enzymes, both of which utilize nickel as a cofactor. The intracellular level of nickel in H. pylori is strictly maintained by a nickel-responsive transcription factor, HpNikR, which acts as a master regulator to control both activation of the urease genes and repression of a variety of other genes including its own. In addition to its role in nickel homeostasis, HpNikR has been implicated in the adaptive response of H. pylori to acidic environmental conditions. In this work, two representative genes, ureA and nikR, are confirmed to be regulated by HpNikR directly in response to changes in nickel concentration, with HpNikR binding to the promoter region of each gene. The binding sequences on the two promoters are distinct from each other and no consensus sequence could be identified from them. The binding affinity of HpNikR to the ureA promoter is much tighter than to the nikR promoter. Another signal that can activate the DNA-binding activity of HpNikR is a change in pH. Once HpNikR is activated by proton binding, it binds to the ureA promoter independently of nickel concentration, but still binds to the nikR promoter in a nickel-dependent manner. Several amino acids at the N-terminus of HpNikR, including Asp7, Asp8 and Lys6, are critical for the specific response of HpNikR to pH changes. In addition, the binding of HpNikR to distinct DNA sequences induces different degrees of DNA bending, which provides another possible means of gene regulation by HpNikR. The ability of HpNikR to differentially regulate distinct genes in response to several signals allows a graduated level of control of gene expression by HpNikR. In vivo studies to evaluate the physiological relevance of the above in vitro results have been initiated. Given the relatively low abundance of transcription factors in H. pylori, information about the effects of nickel and pH on HpNikR in vivo is important for understanding the multifaceted roles of HpNikR in fine-tuning the physiology of this organism. Ultimately this study may provide us with a better idea of how to control H. pylori-caused diseases.

Inflammation is an ubiquitous response of the body to cellular damage and injury. It not only leads to increased production of inflammatory molecules such as prostaglandins, bradykinins, histamine, and nerve growth factor (NGF) that sensitize the neurons, but is also associated with acidosis due to a local physiological decrease in extracellular pH, to as low as 5.3. The addition of protons to the sensitized area results in increased membrane conductance and depolarization in sensory neurons involving the acid sensing ion channels (ASICs), the capsaicin sensitive transient receptor potential vanilloid type 1 receptor (TRPV1), and blockade of background potassium conductance. Collectively, this combination heightens the pain state. As expression and activation of the NGF receptor tropomyosin-related kinase A (TrkA) are critically linked to inflammation-associated nociceptor sensitization, this led us to hypothesize that decreased pH may contribute to this process by depolarizing the neuron and mobilizing more TrkA to the cell membrane. This hypothesis is premised by previous experiments in other neuronal populations demonstrating that increased neuronal activity mobilizes more of the neurotrophin receptor TrkB to the membrane. We explored this question utilizing an in vitro model of acidosis akin to that observed with inflammation. Primary sensory neurons were grown on coverslips at a physiological pH of 7.4. The pH of the media was decreased to an acidic pH of 6.5 in half of the cultures for 30 minutes, followed by fixation under permeablizing or non-permeablizing conditions. Immunocytochemical analysis revealed a significant increase in the mobilization of TrkA to the plasma membrane in response to acidosis (confirmed using a biotinylation assay) and an enhanced level of TrkA activation in response to brief NGF challenge. This rapid mobilization of TrkA was attenuated with the addition of proton-sensitive channel blockers capsazepine and amiloride, for the TRPV and ASIC channels respectively. Unexpectedly, the amount of activated TrkA was also increased at pH 6.5 in the absence of NGF challenge. Taken together, the data suggests that sensory neurons can be rapidly sensitized to NGF in response to a decrease in pH and as such likely plays a role in the sensitization and hyperalgesia associated with an inflammatory state.

Specialized cells of the innate immune system, such as macrophages, employ lysosomal enzymes, together with cationic peptides and reactive oxygen intermediates, to eliminate invading microorganisms ensnared within phagosomes. The effectiveness of this impressive armamentarium is potentiated by the acid pH generated by the vacuolar-type ATPase (V-ATPase). The determinants of the luminal pH of phagosomes and of the lysosomes they fuse with are not completely understood, but the V-ATPase is known to be electrogenic and net accumulation of protons requires charge compensation. For this reason, counter-ion pathways are thought to serve a central role in the control of acidification. It has generally been assumed that a parallel anion influx accompanies proton pumping to dissipate the voltage that tends to build up. In fact, impaired chloride channel activity in cystic fibrosis has been proposed to underlie the defective phagolysosome acidification and microbial killing reported in lung macrophages. In the first part of this thesis, I devised methods to dialyze the lumen of lysosomes in intact cells, while monitoring lysosomal pH, in order to assess the individual contribution of counter-ions to acidification. Surprisingly, anions were found to be completely dispensable for proton pumping, whereas the presence of permeant cations in the lysosomal lumen was essential. Accordingly, defects in lysosomal anion permeability cannot explain the impaired microbicidal capacity of phagocytes in cystic fibrosis. Even though counter-ion permeation pathways exist, dissipation of the electrical contribution of the V-ATPase may not be complete. If present, a transmembrane potential would alter the rate and extent of proton accumulation in phagosomes and lysosomes. However, no estimates of the voltage across the phagosomes were available. To overcome this deficiency, in the second part of this thesis, I describe a noninvasive procedure to estimate the voltage across the phagosome using fluorescence resonance energy transfer. This novel approach, in combination with organellar pH measurements, demonstrated that proton pumping is not limited by counter-ion permeability.

Helicobacter pylori (H. pylori) is a gram-negative pathogenic bacterium that infects half of the world’s population. It resides in the human stomach, where it survives extremely acidic conditions. Efficient colonization by H. pylori requires urease and hydrogenase enzymes, both of which utilize nickel as a cofactor. The intracellular level of nickel in H. pylori is strictly maintained by a nickel-responsive transcription factor, HpNikR, which acts as a master regulator to control both activation of the urease genes and repression of a variety of other genes including its own. In addition to its role in nickel homeostasis, HpNikR has been implicated in the adaptive response of H. pylori to acidic environmental conditions. In this work, two representative genes, ureA and nikR, are confirmed to be regulated by HpNikR directly in response to changes in nickel concentration, with HpNikR binding to the promoter region of each gene. The binding sequences on the two promoters are distinct from each other and no consensus sequence could be identified from them. The binding affinity of HpNikR to the ureA promoter is much tighter than to the nikR promoter. Another signal that can activate the DNA-binding activity of HpNikR is a change in pH. Once HpNikR is activated by proton binding, it binds to the ureA promoter independently of nickel concentration, but still binds to the nikR promoter in a nickel-dependent manner. Several amino acids at the N-terminus of HpNikR, including Asp7, Asp8 and Lys6, are critical for the specific response of HpNikR to pH changes. In addition, the binding of HpNikR to distinct DNA sequences induces different degrees of DNA bending, which provides another possible means of gene regulation by HpNikR. The ability of HpNikR to differentially regulate distinct genes in response to several signals allows a graduated level of control of gene expression by HpNikR. In vivo studies to evaluate the physiological relevance of the above in vitro results have been initiated. Given the relatively low abundance of transcription factors in H. pylori, information about the effects of nickel and pH on HpNikR in vivo is important for understanding the multifaceted roles of HpNikR in fine-tuning the physiology of this organism. Ultimately this study may provide us with a better idea of how to control H. pylori-caused diseases.

El objetivo de la presente tesis fue estudiar el efecto de algunos factores relacionados con el comportamiento, y el uso de bicarbonato sódico, sobre los procesos digestivos que afectan la función ruminal en terneros de cebo intensivo alimentados con pienso y paja ad libitum. En el primer experimento, 4 terneras Frisonas fistuladas en el rumen fueron asignadas a un Cuadrado Latino 4 × 4 para determinar el efecto del aumento en la concentración de bicarbonato sódico (0, 1,25, 2,50 y 5 % de la MS del pienso) sobre la ingestión, el consumo de agua, la fermentación ruminal, y los comportamientos de masticación e ingestión. La ingestión de pienso disminuyó mientras que la de paja aumentó linealmente con el aumento de bicarbonato. El consumo de agua aumentó linealmente sólo cuando se expresó en L/kg de MS. La media y el valor mínimo del pH diario del líquido ruminal no fueron afectados. Sin embargo, el pH ruminal aumentó linealmente a las 2 y 4 h post-alimentación, y el número de horas con pH ruminal por debajo de 5,8 mostró altos valores en el tratamiento sin bicarbonato. La proporción molar de propionato en rumen disminuyó linealmente, mientras que las proporciones de acetato, butirato y ácidos grasos volátiles no-ramificados aumentaron linealmente con el nivel de bicarbonato. La frecuencia de las comidas y la velocidad de ingestión disminuyeron linealmente. El tiempo comiendo por unidad de ingestión (min/kg MS) aumentó linealmente con el nivel de tampón. En el segundo experimento, se utilizaron 72 terneras Frisonas distribuidas en un diseño factorial con 3 tratamientos y 3 bloques con similar peso vivo para estudiar el efecto de la competencia social sobre la producción, el comportamiento y el bienestar durante las 4 semanas siguientes a la llegada al cebadero. Los tratamientos consistieron en aumentos en el nivel de competencia social a través del uso de 1, 2 y 4 espacios en el comedero de pienso por corral (8 terneras/corral). Hubo una disminución lineal en la ingestión de pienso y en el crecimiento durante la semana 1 después de la llegada al cebadero pero el efecto fue cuadrático durante las semana 3 y 4, con valores bajos cuando la competencia fue más alta. El consumo de paja y la variabilidad del crecimiento entre terneras dentro de cada corral tendieron a aumentar linealmente con el aumento en la competencia. El aumento de competencia por el pienso aumentó los efectos de la dominancia sobre el crecimiento, siendo su relación negativa en la primera pero positiva en la tercera semana. Los mismos animales con el mismo diseño experimental se utilizaron para estudiar el efecto de la competencia sobre la fermentación ruminal, la producción y el bienestar durante 6 períodos experimentales de 28 días cada uno. La ingestión de pienso disminuyó linealmente pero esto no afectó a ningún parámetro productivo. Hubo un alto porcentaje de hígados con abscesos en el nivel más alto de competencia. El tiempo diario comiendo pienso disminuyó, mientras que su velocidad de ingestión, el tiempo diario de pie y el número de agresiones aumentaron con la competencia. La corticosterona en heces mostró altos valores sólo en animales dominantes del nivel más alto de competencia. La concentración de haptoglobina en sangre aumentó linealmente, particularmente en las terneras mas subordinadas. El aumento en la competencia redujo el pH ruminal sólo en los primeros 2 períodos, y la proporción de terneras que mostraron un pH ruminal menor a 5,6 y la concentración de ácido láctico aumentaron linealmente. Esta tesis muestra que el comportamiento de la alimentación de los terneros en cebo intensivo permite entender mejor el funcionamiento ruminal y procurar así su bienestar. / The objective of the present thesis was to study the effects of some behavioral factors, and the use of sodium bicarbonate, on the digestion processes affecting ruminal function of beef cattle fed concentrate and straw ad libitum. In the first experiment, four ruminally fistulated Holstein heifers were used in a 4 x 4 Latin square design to determine the effect of increasing levels of sodium bicarbonate (0, 1.25, 2.50 and 5 %, on concentrate DM basis) on intake, water consumption, ruminal fermentation, and chewing and feed intake behaviors. Concentrate and barley straw were fed once daily at 0830 and ad libitum. Concentrate decreased and straw DMI increased linearly with buffer level. Water consumption increased linearly when expressed in L/kg of DMI but not in L/d. Daily mean and lowest ruminal pH were not affected by treatments. However, ruminal fluid pH increased linearly at 2 and 4 h after feeding and the number of hours in which ruminal pH was below 5.8 was greatest when no buffer was added. Daily average molar proportion of propionate decreased linearly but acetate, butyrate, and branched-chain VFA increased linearly as the level of bicarbonate increased. Meal frequency and eating rate decreased linearly. Time spent eating per unit of DMI increased linearly with buffer level. In the second experiment, 72 Friesian calves were distributed in a factorial design with 3 treatments and 3 blocks of similar fasted BW to study the effect of increasing competition on performance, behavior, and welfare indicators throughout the 4 wk after arrival. Treatments consisted of increasing levels of social competition with the use of 1, 2 or 4 concentrate feeding places/pen (8 calves/pen). Concentrate and straw were fed ad libitum at 0830 and in individual feeders. Increasing competition resulted in a linear decrease of concentrate DMI and ADG during wk 1 after arrival but the response was quadratic in wk 3 and 4, being lowest at the greatest competition. Straw intake and the within-pen SD of ADG tended to increase linearly with competition during the 4-wk receiving period. Increasing competition at the concentrate feeders reduced lying time, increased standing time, and changed the diurnal feeding pattern (concentrate eating time decreased but straw eating time increased during peak feeding times). Increasing social competition at the concentrate feeders accentuated the effects of dominance rank on ADG, with their relationship being negative at wk 1 but positive at wk 3. The same 72 heifers with the same experimental setup were used to study the effect of increasing competition on performance, behavior, welfare, and ruminal fermentation of feedlot heifers. After the 4-wk adaptation period, DMI and ADG were measured, and blood and rumen samples were taken during 6, 28-d experimental periods. Fecal corticosterone and behavior were measured at periods 1, 3 and 6. Concentrate intake decreased linearly with competition but final BW, ADG, and G:F ratio were not affected by treatments. The proportion of abscessed livers responded quadratically with high proportion at the greatest competition. Concentrate eating time decreased, and eating rate, standing time, and aggressions increased linearly with competition for concentrate. The pen-average fecal corticosterone was not affected by treatments but that of dominants responded quadratically, being greatest with the greatest competition. Serum haptoglobin concentration increased linearly with competition, particularly within the most subordinate heifers. Increased competition reduced ruminal pH only in periods 1 and 2 but tended to increase the proportion of heifers with ruminal pH below 5.6 and increased ruminal lactate. The present thesis shows that feeding behavior in beef cattle allows better understand ruminal function and to assess welfare as well.

Inflammation is an ubiquitous response of the body to cellular damage and injury. It not only leads to increased production of inflammatory molecules such as prostaglandins, bradykinins, histamine, and nerve growth factor (NGF) that sensitize the neurons, but is also associated with acidosis due to a local physiological decrease in extracellular pH, to as low as 5.3. The addition of protons to the sensitized area results in increased membrane conductance and depolarization in sensory neurons involving the acid sensing ion channels (ASICs), the capsaicin sensitive transient receptor potential vanilloid type 1 receptor (TRPV1), and blockade of background potassium conductance. Collectively, this combination heightens the pain state. As expression and activation of the NGF receptor tropomyosin-related kinase A (TrkA) are critically linked to inflammation-associated nociceptor sensitization, this led us to hypothesize that decreased pH may contribute to this process by depolarizing the neuron and mobilizing more TrkA to the cell membrane. This hypothesis is premised by previous experiments in other neuronal populations demonstrating that increased neuronal activity mobilizes more of the neurotrophin receptor TrkB to the membrane. We explored this question utilizing an in vitro model of acidosis akin to that observed with inflammation. Primary sensory neurons were grown on coverslips at a physiological pH of 7.4. The pH of the media was decreased to an acidic pH of 6.5 in half of the cultures for 30 minutes, followed by fixation under permeablizing or non-permeablizing conditions. Immunocytochemical analysis revealed a significant increase in the mobilization of TrkA to the plasma membrane in response to acidosis (confirmed using a biotinylation assay) and an enhanced level of TrkA activation in response to brief NGF challenge. This rapid mobilization of TrkA was attenuated with the addition of proton-sensitive channel blockers capsazepine and amiloride, for the TRPV and ASIC channels respectively. Unexpectedly, the amount of activated TrkA was also increased at pH 6.5 in the absence of NGF challenge. Taken together, the data suggests that sensory neurons can be rapidly sensitized to NGF in response to a decrease in pH and as such likely plays a role in the sensitization and hyperalgesia associated with an inflammatory state.

The sperm in the seminal fluid is affected by various environmental factors, where one is the pH-value in the environment. An extremely low or high pH-value has been observed in other studies to be lethal to sperm cells, thus effecting the sperms capability to perform a conception. Addition of nutrion, such as glucose and fructose, also has been a factor contributing to the survival of the sperm.In this essay we found that the pH-value did not affect the lifespan of the sperm. Mainly due to the pH-value in the seminal fluid and the buffer capacity of the same, the different pH-solutions we used showed no significant effect on the sperm’s lifespan. Addition of nutrion (glucose or fructose) did however affect the time the sperm survived, adding hours to the lifespan. The best media for survival was the IVFTM nutrion solution used to perform so called ”swim up” tests, where the sperms get ”washed” and used without the seminal fluid’s content. The IVFTM improved the motility of the sperms and also made them longer living.