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Les fonctions organiques du corps humain sont liées à des constants biologiques. Variations de ces constantes induisent divers états pathologiques. Parmi ces constantes, le pH constitue le cœur de ces travaux de thèse. Chez les êtres vivants, les fonctions biologiques dépendent de constant acides ou alcalines. En fait, l’action d’une protéine dépend du pH du milieu environnant. Une valeur inadéquate du pH rend les protéines inactives ce qui est délétère pour l’organisme. Il existe donc un besoin pour des capteurs de pH qui puissant être utilisés dans le corps humain pour des applications cliniques (échelle macroscopique), sur des cellules en culture pour des recherches en biologie (échelle mesoscopique) et pour étudier les échanges ioniques au niveau des membranes cellulaires pour des travaux plus fondamentaux (échelle microscopique). Parmi le large éventail de technologies potentiellement candidates pour ces applications, la mesure de pH par fibre optique exploitant la fluorescence permet d’être adaptée aux trois échelles dimensionnelles susnommées. Ce manuscrit de thèse adresse des contraintes par l’étude de capteurs fluorescents à fibre optique utilisant deux types d’indicateur de pH: les SNARF et la fluorescéine. En parallèle de ces développements expérimentaux, des descriptions mathématiques des propriétés de fluorescence de ces deux indicateurs sont proposées. Ces descriptions permettent de progresser vers une mesure du pH sans calibration / Organic functions of the human body are linked to biological constants. Variations of these constants induce pathological troubles. Among these constants, the pH is the central subject of this PhD work. In living beings, biological functions are related to either acid or alkaline constants. Indeed, the action of a protein depends on the surrounding pH. An inadequate value of the pH makes the proteins non active which is deleterious for the organism. There exist a need for pH sensors which can be used in the human body for clinical applications (macroscopic scale), on cells in culture for biology researches (mesoscopic scale) or at a cell membrane level for more fundamental studies (microscopic scale). Among the wide range of technologies potentially useful for these applications, fiber optic fluorescence pH sensing offers the possibility to be adapted to the three above mentioned dimensional scales. This PhD dissertation addresses these constraints by studying fluorescence fiber optics pH sensors using two kind of pH indicators: SNARF and fluorescein. Together with these experimental studies, mathematical descriptions of the fluorescence properties of these indicators are proposed. They allow progressing towards calibration free pH sensing.

Artificial insemination using cooled, transported semen has become a popular practice in the equine industry. However, equine sperm are assumed to show a decline in their fertilizing ability after 24 to 48 hours of cooled storage. Two measures that are commonly used to estimate the fertility of an ejaculate are sperm motility and sperm membrane integrity (SMI). Recently, it has been suggested that SMI may have a better correlation with fertility of an inseminate than sperm motility. The effect of cooled-storage on sperm quality over an extended time period was evaluated to illustrate changes in sperm characteristics that might be related to an ejaculate’s fertility. Semen was stored at 4°C in INRA 96 extender containing 10% seminal plasma for a period of 10 days. Data were collected daily on sperm motion characteristics, SMI, mitochondrial membrane potential, and DNA quality. To measure daily changes in SMI in stallion sperm, two fluorescent vital-staining protocols used in flow cytometric analysis were compared – a combination of SNARF-1, Yo-Pro-1, and Ethidium Homodimer 1 (SYE) and a combination of lectin from Pisum sativum and propidium iodide (PSA/PI). We hypothesized that the SYE protocol adapted for use with stallion sperm could detect more subtle, and perhaps earlier, damage to the sperm plasma membrane than the PSA/PI protocol. A combination of SYBR 14, propidium iodide, and JC-1 (SYPIJC) was used to measure mitochondrial membrane potential, as well as SMI. A computer-assisted sperm motion analysis (CASA) instrument was used to evaluate sperm motion characteristics; the sperm chromatin structure assay (SCSA) was used to measure the degree of DNA fragmentation. In this study, with the exception of sperm motility, the measures of sperm quality retained values consistent with “viability” after 10 days of cooled-storage. This suggests that the fertility of some stallions may last considerably longer than previously assumed, which could ultimately alter the time-table used for artificial insemination using cooled, transported semen.

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