No description available.

Thesis (M.S.)--Northern Michigan University, 2009. / Bibliography: leaves 114-116.

No description available.

No description available.

Le diagnostic précoce de l'hypothyroïdie permet d'initier le traitement au plus tôt et ainsi de préserver la santé du patient. Le bénéfice du traitement de l'hypothyroïdie franche a été depuis longtemps établi, mais les critères de prise en charge des patients en hypothyroïdie fruste sont encore difficiles à définir. En effet, les symptômes ne sont pas toujours présents et leur appréciation est subjective. Afin d'établir le diagnostic et la prise en charge, le médecin s'appuie sur le dosage de la thyréostimuline (TSH) dans le sang, qui peut éventuellement être complété par le dosage des hormones thyroïdiennes. Le dosage de la TSH, très sensible, peut présenter sur un même échantillon sanguin d'importantes variations qui rendent d'autant plus difficiles la décision du médecin et le suivi du patient. Le polymorphisme naturel de la TSH peut expliquer en partie ces variations. La TSH appartient en effet à la famille des hormones glycoprotéiques et sa glycosylation peut constituer jusqu'à 30% de son poids. Dans le cas de l'hypothyroïdie en particulier, ces glycanes sont modifiés et présentent une plus grande quantité d'acides sialiques terminaux. Ainsi, certaines variations entre les dosages de la TSH, qui freinent actuellement leur harmonisation, peuvent être dues à des différences de reconnaissance de glycoformes par les anticorps utilisés dans les dosages. Dans ce contexte, l'objectif de de ces travaux était de contribuer à la construction de dosages plus performants que ceux actuellement utilisés dans le diagnostic de l'hypothyroïdie. Un nouveau calibrateur recombinant sialylé plus proche de la TSH circulante dans l'hypothyroïdie a alors été produit. De nouvelles associations d'anticorps monoclonaux ont été utilisées pour construire des dosages. Les nouveaux dosages sélectionnés ont ensuite été calibrés avec la TSH sialylée produite et le calibrateur de référence international. Ils ont alors servi à doser plusieurs séries de sérums de patients. Ces travaux ont donc validé l'utilisation d'un nouveau calibrateur d'origine recombinante pour les dosages de la TSH, ce qui devrait à l'harmonisation des dosages existants. / If iodine deficiency is the first cause of low thyroid hormone levels in the world, there are also other etiologies to thyroid disorders. Diagnosis of those allow an early treatment to preserve patient's health. Although there is a general agreement concerning treatment of overt hypothyroidism, treatment of subclinical hypothyroidism is still under debate. In these cases, symptoms are, by definition, not always present. In order to establish diagnosis, the clinicians rely on the measurement of circulating thyroid stimulating hormone (TSH, potentially completed with thyroid hormones measurement). TSH assays are now very sensitive, but can present important between assays variations. The diagnosis and follow up of the patient are consequently complicated. Natural polymorphism of TSH can explain a part of this variability. TSH belongs to the glycoprotein hormones family and its glycans can count for more than 30% of its weight. In hypothyroidism, these glycans are subject of modulation and present higher levels of terminal sialylation. Variation in immuno-assays can be explained by these modifications of sialylation if recognition by antibodies used in immuno-assays is glycosylation dependent. In this context, the aim of this work was to contribute to the construction of new immuno-assays, more reliable in the early diagnosis of subclinical hypothyroidism. During this thesis a new recombinant standard closer to circulating TSH was produced. The total level of sialylation was higher and better mimic the circulating forms in hypothyroidism. In order to select the best antibodies associations in immuno-assays, new antibodies were obtained and associated with commercially available antibodies. New immuno assays improvement is based on the following two approaches: the first one is the use of a new standard which presents glycoformes closer to the circulating TSH and the second one consists in an appropriate selection of antibodies involved in the assays. The new assays were used to measure TSH concentration in blood samples. These studies associated with validation steps allow us to select four assays and constitute a proof of concept for the use of a new sialylated recombinant standard for TSH assays. This can contribute to the needed harmonization of TSH assays.

Le diagnostic précoce de l'hypothyroïdie permet d'initier le traitement au plus tôt et ainsi de préserver la santé du patient. Le bénéfice du traitement de l'hypothyroïdie franche a été depuis longtemps établi, mais les critères de prise en charge des patients en hypothyroïdie fruste sont encore difficiles à définir. En effet, les symptômes ne sont pas toujours présents et leur appréciation est subjective. Afin d'établir le diagnostic et la prise en charge, le médecin s'appuie sur le dosage de la thyréostimuline (TSH) dans le sang, qui peut éventuellement être complété par le dosage des hormones thyroïdiennes. Le dosage de la TSH, très sensible, peut présenter sur un même échantillon sanguin d'importantes variations qui rendent d'autant plus difficiles la décision du médecin et le suivi du patient. Le polymorphisme naturel de la TSH peut expliquer en partie ces variations. La TSH appartient en effet à la famille des hormones glycoprotéiques et sa glycosylation peut constituer jusqu'à 30% de son poids. Dans le cas de l'hypothyroïdie en particulier, ces glycanes sont modifiés et présentent une plus grande quantité d'acides sialiques terminaux. Ainsi, certaines variations entre les dosages de la TSH, qui freinent actuellement leur harmonisation, peuvent être dues à des différences de reconnaissance de glycoformes par les anticorps utilisés dans les dosages. Dans ce contexte, l'objectif de de ces travaux était de contribuer à la construction de dosages plus performants que ceux actuellement utilisés dans le diagnostic de l'hypothyroïdie. Un nouveau calibrateur recombinant sialylé plus proche de la TSH circulante dans l'hypothyroïdie a alors été produit. De nouvelles associations d'anticorps monoclonaux ont été utilisées pour construire des dosages. Les nouveaux dosages sélectionnés ont ensuite été calibrés avec la TSH sialylée produite et le calibrateur de référence international. Ils ont alors servi à doser plusieurs séries de sérums de patients. Ces travaux ont donc validé l'utilisation d'un nouveau calibrateur d'origine recombinante pour les dosages de la TSH, ce qui devrait à l'harmonisation des dosages existants. / If iodine deficiency is the first cause of low thyroid hormone levels in the world, there are also other etiologies to thyroid disorders. Diagnosis of those allow an early treatment to preserve patient's health. Although there is a general agreement concerning treatment of overt hypothyroidism, treatment of subclinical hypothyroidism is still under debate. In these cases, symptoms are, by definition, not always present. In order to establish diagnosis, the clinicians rely on the measurement of circulating thyroid stimulating hormone (TSH, potentially completed with thyroid hormones measurement). TSH assays are now very sensitive, but can present important between assays variations. The diagnosis and follow up of the patient are consequently complicated. Natural polymorphism of TSH can explain a part of this variability. TSH belongs to the glycoprotein hormones family and its glycans can count for more than 30% of its weight. In hypothyroidism, these glycans are subject of modulation and present higher levels of terminal sialylation. Variation in immuno-assays can be explained by these modifications of sialylation if recognition by antibodies used in immuno-assays is glycosylation dependent. In this context, the aim of this work was to contribute to the construction of new immuno-assays, more reliable in the early diagnosis of subclinical hypothyroidism. During this thesis a new recombinant standard closer to circulating TSH was produced. The total level of sialylation was higher and better mimic the circulating forms in hypothyroidism. In order to select the best antibodies associations in immuno-assays, new antibodies were obtained and associated with commercially available antibodies. New immuno assays improvement is based on the following two approaches: the first one is the use of a new standard which presents glycoformes closer to the circulating TSH and the second one consists in an appropriate selection of antibodies involved in the assays. The new assays were used to measure TSH concentration in blood samples. These studies associated with validation steps allow us to select four assays and constitute a proof of concept for the use of a new sialylated recombinant standard for TSH assays. This can contribute to the needed harmonization of TSH assays.

<p> Diabetes mellitus is a major independent risk factor for cardiovascular disease (CVD) and stroke, however the cellular mechanisms by which diabetes contributes to vascular dysfunction are not fully understood. In recent decades, multiple molecular mechanisms have been implicated in hyperglycemia-associated vascular damage and CVD [1]. It is well established that hyperglycemia promotes intracellular glucose flux through the hexosamine pathway where the rate-limiting enzyme, glutamine:fructose-6-phosphate amidotransferase (GFAT) produces glucosamine-6-phosphate [2,3]. We have shown that elevated levels of intracellular glucosamine cause ER stress and activation of the UPR in multiple cell types [4]. Additionally, we have previously shown that ER stress is associated with lipid accumulation, activation of inflammatory pathways, and is associated with atherosclerotic plaque formation in hyperglycemic mice [ 4,5]. We hypothesize that the accumulation of intracellular glucosamine, observed in conditions of hyperglycemia, promotes atherogenesis via a mechanism that involves the hexosamine pathway, protein glycosylation and ER stress.</p> <p> Using in vitro over-expression studies, we investigated the role of GFAT in hyperglycemia-associated ER stress. We developed methods to increase GFAT expression in both HepG2 cells and HASMC. However, we found that GFAT over-expression is insufficient to induce an ER stress response. Further investigation of this system suggests that the over-expressed GFAT does not increase intracellular glucosamine levels to sufficiently promote ER stress.</p> <p> We have also investigated the role of protein glycosylation in glucosamine-induced ER stress. We have shown that O-linked glycosylation plays a role in ER stress induction. We have also shown that N-linked protein glycosylation is affected by elevated cellular glucosamine levels. Thus, dysregulated glycosylation of newly synthesized proteins may contribute to the accumulation of unfolded protein in the ER and lead to the activation of the UPR.</p> / Thesis / Master of Science (MSc)

Raman spectroscopy is an established analytical technique for determining molecular structure, whose major drawback is lack of sensitivity. Enhanced Raman scattering techniques, such as surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS), utilise nanoscale substrates to enhance the Raman signal through the interaction of surface charges with the incident electromagnetic radiation. Here, nanoparticle-based SERS was used to detect dipicolinic acid (DPA), a biomarker for bacterial spores. Whilst this has been demonstrated previously, the use of a different nanoparticle aggregation mechanism and the inclusion of an internal standard has enabled a SERS detection method to be developed that is quantitative to almost an order of magnitude lower than previously reported. Moreover, for the first time, a nanoparticle-based SERS method was applied to the detection of viable Bacillus spores. Investigations were made into the possibility of SERS enhancement using deep UV laser excitation at 244 nm using a novel boron nitride surface material. This semiconductor has a band gap of comparable magnitude to the laser excitation wavelength and therefore had the potential to impart a SERS enhancement via a chemical enhancement mechanism. Whilst initial results looked promising using Rhodamine 6G as a test analyte, it was not possible to demonstrate reproducibly and no enhancement was observed on other analytes that were tested. TERS was shown to be able to discriminate between glycosylated and non-glycosylated forms of protein molecules, based on the measurement of just a few molecules at a time. This was achieved even without control of the protein interaction with the TERS substrate. The vibrational peak positions in TERS experiments were shown to be highly dependent on the analyte’s orientation relative to the TERS tip, giving variable and complex spectral data. As such, the data processing and analysis methods had to be carefully considered in order to eliminate bias. Lastly, a novel SERS detector for high-performance liquid chromatography (HPLC) was built and tested. It was shown to be able to quantify purine bases from mixtures in tandem with, and in lower amounts than the conventionally used UV absorbance detection, even when the analyte peaks were co-eluting. This quantitative analysis is conducted on-line and in real-time, making it applicable to high throughput applications. Together the four research projects presented in this thesis make a significant contribution to the field of enhanced Raman scattering and promote its sensitivity and reproducibility as a quantitative analytical technique for the trace analysis of biomolecules.

No description available.

The opportunistic bacterial pathogen Pseudomonas aeruginosa uses type IV pili (T4P) for adherence to, and rapid colonization of, surfaces via twitching motility. T4P are formed from thousands of pilin (PilA) subunits. Two groups of P. aeruginosa pilins were described previously (I and II), distinguished by protein length and sequence. PilA_I was glycosylated with an O-antigen subunit through the action of PilO/TfpO, encoded downstream of pilA_I. To determine if additional pilin variants existed, analysis of the pilin locus of >300 P. aeruginosa strains from a variety of environments was conducted. Three additional pilin alleles were discovered, each of which was invariantly associated with a unique, previously unidentified, downstream gene(s): pilA_III+tfpY, pilAIV+tfpW+tfpX, pilA_V+tfpZ. This survey also revealed that strains with group I T4P were more commonly associated with respiratory infections than strains with other pilins, suggesting that glycosylated T4P may confer a colonization advantage in this environment. The newly identified group IV pilin, represented by strain Pa5196, migrated aberrantly through SDS-PA gels, suggesting it was also glycosylated, a hypothesis confirmed by periodic acid-Schiff staining and mass spectrometry (MS) analyses. Disruption of Pa5196 O-antigen biosynthesis did not prevent the production of glycosylated pilins, demonstrating that these pilins were modified in a novel manner, unlike group I pilins. Using MS, nuclear magnetic resonance spectroscopy and site-directed mutagenesis, the Pa5196 pilins were shown to be uniquely modified with homo-oligosaccharides of mycobacterial-like α-1,5-D-arabinofuranose at multiple locations. Residues Thr64 and Thr66, located on the αβ-loop region of the protein, appear to be the preferred, but not exclusive sites of modification, each being modified with up to four D-Araf sugars. This region of the pilin is partially surface-exposed in the pilus, therefore modification of these sites may influence the surface chemistry of the fibre. Residues Ser81, Ser82, Ser85 and Ser89, located in the β-strand region, were also modified, mainly with mono- and disaccharides. Bioinformatic analyses and mutagenesis of TfpW suggest that this novel protein is an arabinosyltransferase necessary for PilA_IV modification. This research has increased our understanding of the complexity of this virulence factor, and may aid in development of new therapeutics for P. aeruginosa and mycobacterial infections.