Streptococcus thermophilus est une bactérie lactique utilisée dans l’industrie laitière pour la fabrication de yaourts et de divers fromages. S. thermophilus se développe en chaîne de cellules. Le mécanisme de la séparation des cellules n’est pas connu chez S. thermophilus. Un mutant de S. thermophilus présentant des chaînes de cellules extrêmement longues a été caractérisé. Le gène identifié est nommé cse pour cell separation. La particularité de cse est qu’il résulte d’un réassortiment de modules. En effet, l’analyse a montré que son extrémité 5’ est homologue à celle de sip de S. salivarius, tandis que son extrémité 3’ est homologue à celle de pcsB de S. thermophilus. Le gène cse spécifique de S. thermophilus code une protéine modulaire. A son extrémité N-terminale, Cse possède un peptide signal et un domaine de liaison à la paroi, LysM. Et à son extrémité C-terminale, Cse possède un domaine CHAP, présent dans les hydrolases du peptidoglycane. Dans cette étude, la localisation de Cse à la surface des cellules de S. thermophilus a été réalisée par microscopie électronique à transmission et immunofluorescence à l’aide d’anticorps dirigés contre cette protéine. Cse est localisée spécifiquement aux septa matures de S. thermophilus. De plus, l’activité de Cse a été démontré par zymogramme et présente une activité lytique qui est conférée par son domaine CHAP. L’analyse par RP-HPLC des muropeptides de la paroi de S. thermophilus après digestion avec le domaine CHAP a révélé que Cse est une hydrolase du peptidoglycane et plus précisément une endopeptidase. L’ensemble de ces résultats montre que Cse est l’enzyme majeure de la séparation cellulaire de S. thermophilus. / Streptococcus thermophilus is a lactic bacteria used a stater of fermentation in dairy factories for the production of yogurt and many cheeses. S. thermophilus grows as chains of ovoid cells. However, the genetic basis of S. thermophilus cell separation is still unknown. A S. thermophilus mutant displaying extremely long chains of cells was characterized and demonstrated to be impaired a gene that we named cse for cell separation. The originality of this gene is that cse creation resulted from domain shuffling. Indeed, the analysis has revealed that its 5’extremity has homology with that of sip from S. salivarius, while its 3’extremity shares homology with pcsB from S. thermophilus.The cse gene specific from S. thermophilus, encodes a modular protein. The N-terminal end of Cse contains a secretion signal and cell-wall binding LysM domain. Its C-terminal end includes a CHAP domain found in bacterial cell-wall hydrolases. In this study, the localization of Cse on S. thermophilus cell surface has been undertaken by immunogold electron and immunofluorescence microscopies using of antibodies raised against this protein. Immunolocalization shows that the presence of the Cse protein at mature septa. Moreover, the CHAP domain of Cse exhibits a lytic activity on the cell wall of S. thermophilus that has been demonstrated by zymogram. Additionally, RP-HPLC analysis of muropeptides released from S. thermophilus after digestion with the CHAP domain shows that Cse is a cell wall hydrolase that can function as an endopeptidase. Alltogether, these results suggest that Cse is a major cell wall hydrolase involved in daughter cell separation of S. thermophilus.

L'Epoxide Hydrolase soluble (sEH) est une enzyme exprimée dans les vaisseaux. Elle possède un domaine hydrolase à l'extrémité COOH-term (sEH-H) qui métabolise des facteurs vasodilatateurs et anti-inflammatoires comme les acides époxyeicosatriénoïques (EETs). Elle possède également un domaine phosphatase à l'extrémité NH2-term (sEH-P) dont le rôle biologique n'est pas totalement élucidé. Afin de comprendre le rôle de sEH-H et sEH-P dans la calcification vasculaire, des anneaux aortiques de rats et des cellules musculaires lisses vasculaires humaines (CMLVh) ont été exposés à des conditions procalcifiantes pendant 7 et 14 jours respectivement. Le N-acétyl-S-farnesyl-L-cystéine (AFC), un inhibiteur de sEH-P, et le trans-4-(4-(3-adamantan-1-yl-ureido)-)cyclohexyloxy) acide benzoïque (t-AUCB), un inhibiteur de sEH-H, ont été utilisés entre 0,1 et 10 μM. En condition procalcifiante, l'AFC réduit de façon dose-dépendante la calcification vasculaire. Au contraire, le t-AUCB augmente de façon dose-dépendante la minéralisation au sein des anneaux aortiques. Une augmentation de l'activité TNAP a été observée dans les surnageants de culture des anneaux aortiques avec le t-AUCB. Sur les anneaux désendothélialisés ou sur les cultures de CMLVh, les inhibiteurs n'ont pas d'effet sur la calcification, soulignant le rôle crucial joué par les facteurs endothéliaux métabolisés par la sEH. L'ensemble de nos résultats montrent que l'inhibition pharmacologique de la sEH-H augmente la calcification vasculaire in vitro en augmentant la biodisponibilité des EETS. Au contraire, l'inhibition de la sEH-P protège contre la calcification vasculaire à travers un mécanisme dépendant de l'endothélium / Expressed in the vasculature, soluble epoxide hydrolase (sEH) exhibits a COOH-terminal hydrolase domain metabolizing endothelial vasodilator and anti-inflammatory factors like epoxyeicosatrienoic acids (EETs) and, a NH2-terminal phosphatase domain whose biological role remains unclear. To assess the role of sEH phosphatase and hydrolase domains in vascular calcification, rat aortic rings and hVSMCs were exposed to procalcifying culture media for 7 and 14 days, respectively. N-acetyl-S-farnesyl-L-cysteine (AFC), an inhibitor of the phosphatase domain, and trans-4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxy)-benzoic acid (t-AUCB), a hydrolase domain inhibitor, were used at concentrations ranging from 0.1 to 10 μM. Under procalcifying culture condition, AFC significantly and dose-dependently reduces aortic calcification. Conversely, addition of t-AUCB results in a significant and dose-dependent increase in aortic calcification in rats, without modification of tissue viability. A concomitant increase in TNAP activity was observed in supernatants of aortic rings cultured in the presence of t-AUCB. On de-endothelialized aortic rings or hVSMCs cultures, both inhibitors had no significant effect on the calcification process, pointing out the crucial role played by endothelial factors metabolized by sEH in the control of this biomineralization process. Together, our data demonstrates that pharmacological inhibition of sEH hydrolase increases vascular calcification in vitro by majoring the bioavailability of endothelium- derived EETs. Contrarily, the inhibition of sEH phosphatase is protective against vascular calcification through an endothelium-dependent mechanism.

The research in this dissertation documents connections between the primary amino acid sequence of proteins, the dynamics of proteins, and their catalytic function. This research project studied two proteins called protein-tyrosine phosphatases (PTPs): the human enzyme PTP1B, and the bacterial enzyme YopH. PTP1B is a human enzyme that down regulates the insulin receptor on the outer cellular membrane, and causes the insulin receptor to be less responsive to insulin. A deeper knowledge of how PTP1B is different from other human PTPs might be useful in designing drugs to increase insulin sensitivity in diabetics. Yersinia Pestis is the bacteria that caused the Black Plague, and YopH is an essential for virulence factor that helps Yersinia Pestis to escape the human immune response. Using these proteins, the primary sequence of amino acids in a small but critical loop region was altered and the effect on the catalytic efficiency was measured. This research shows how some residues are key to the catalytic efficiency of PTPs while others could be changed with little to no effect on the catalytic efficiency. A deeper understanding of the difference between key residues and structural residues may allow future scientists to create designer enzymes and perhaps design pharmaceuticals that mediate enzyme activity by affecting their protein dynamics.

Thesis (M.Sc. (Pharmaceutical Chemistry)--Potchefstroom University for Christian Higher Education, 2002.

Listeria monocytogenes is the etiological agent of a life-threatening, opportunistic infection caused by the ingestion of contaminated foods. Although L. monocytogenes is divided into 13 serotypes, 98% of human illness is caused by serotype 1/2a, 1/2b and 4b strains, with serotype 4b accounting for almost all the major outbreaks of human listeriosis. The principle objective of this work was to develop surface-binding monoclonal antibodies (MAbs) highly specific for serotype 4b, as well as characterize their antigen targets to aid in the detection and isolation of serotype 4b strains using an antibody based procedure. To create such antibodies, mice were immunized with formalin killed whole cells of L. monocytogenes serotype 4b strain LI0521. A total of 15 MAbs reactive to serotype 4b isolates were shown to recognize a ~77 kDa surface antigen subsequently identified by mass spectrometry as surface associated autolysin, IspC. Epitope mapping experiments further revealed that each of the 15 MAbs bound to the cell wall binding GW domain of IspC and can be essentially divided into 4 major groups based on epitope localization. ELISA analysis of the reactivity of each of the MAbs with various L. monocytogenes serotypes indicated that several MAbs were 100% specific for serotype 4b isolates. Surface plasmon resonance experiments showed that the affinity constants for each of these MAbs fell within the range of 1.0 x 10-7 to 6.4 x 10-9 M. To determine whether IspC, shown to be well conserved among various serotype 4b strains, is a useful diagnostic marker with antibody-based methods, the expression of IspC was assessed in L. monocytogenes cultured under normal and stress conditions. A functional promoter directing the transcription of ispC gene was identified immediately upstream of the ispC open reading frame by constructing the promoterless lacZ gene fusion with the putative ispC promoter region and by 5'RACE analysis. Data obtained with the lacZ reporter gene system and immunofluorescent microscopy revealed that IspC is expressed on the cell surface under all growth conditions tested (temperature, osmotic stress, pH, ethanol, oxidative stress, anaerobic conditions, carbon source and enrichment media) that allow for cellular division, although the level of ispC gene expression varies. In addition, a significant effort were put into elucidating the hydrolytic bond specificity of IspC by HPLC and mass spectrometry analysis of muropeptides released from IspC-mediated hydrolysis of L. monocytogenes peptidoglycan (PG). The results demonstrated that IspC functions as an N-acetylglucosaminidase capable of cleaving the β-1,4-glycosidic bond of the PG glycan strand. Furthermore, IspC was more efficient at hydrolysing fully Nacetylated PG from a PG deacetylase gene (pgdA) deletion mutant of L. monocytogenes than partially de-N-acetylated wild-type PG, indicating that modification of PG by de-Nacetylation of GlcNAc residues renders PG resistant to IspC hydrolysis. In conclusion, the surface autolysin IspC with the N-acetylglucosaminidase activity is a novel diagnostic marker for the 4b serotype strains, which can be explored , in conjunction with specific MAbs developed here, for detection and isolation of L. monocytogenes serotype 4b strains directly from food, environmental and clinical samples with the need for minimal or no culture enrichment.

No description available.

Stereochemistry and chirality are arguably two of the most important subjects pertaining to the development of new pharmaceutical drugs. Since enantiomers have the potential to encompass different pharmacological effects in biological systems, both enantiomers have to be tested for pharmacological activity. Not only has obtaining these single enantiomers become crucial, but formulation of the pure enantiomer of a drug also has the potential to contain advantages for both pharmaceutical formulation and therapeutic effect. Epoxide hydrolase is an enzyme commonly found in nature that catalyses the hydrolysis of epoxides, resulting in the formation of the corresponding vicinal diol. Over the last few years a large amount of research has been completed on these enzymes from sources such as mammals, insects, bacteria and fungi. Micro-organisms especially have enjoyed ample attention because of their abundant supply. Recently it was found that certain yeasts contain this enzyme and have the ability to enantioselectively catalyse certain hydrolysis reactions. Styrene oxides are terminal epoxides that are, due to the reactivity of the epoxide ring, useful synthons in the organic synthesis of pharmaceutical products. The first objective of this project was to synthesize three nitro derivatives of styrene oxide namely para-, meta-, and ortho-nitrostyrene oxide. Al three products were obtained from the corresponding nitrophenacyl bromide in yields of 52%, 90% and 57% respectively. The second objective was lo find a suitable yeast slrain containing the epoxide hydrolase enzyme to enantioselectively hydrolyse the synthesised products and unsubstituted styrene oxide. A screening was completed during which 410 yeast strains from more than 44 genera were tested. Epoxide hydrolase activity was found to be widespread throughout the screened yeast domain, while the genera Candida, Debaryomyces, Pichia, Rhodosporidium, Rhodotorula and Trichosporon specifically were very successful in catalysing the hydrolysis of the substrates. Rhodosporidium toruloides UOFS Y-0471 and Rhodotorula glutinis UOFS Y-0653 were chosen for further studies because of their superior enantioselectivity. The final objective was to optimise these reactions in terms of pH, temperature and substrate concentration. It was found that a pH value of 7.2 and a temperature of 45’C yielded optimal enzyme activity. Increased temperatures (45’C), however, lead to a decrease in enantioselectivity and, in the case of R. toruloides together with the substrate puranitrostyrene oxide, reversed enantioselectivity. Lower temperatures (15’C) increased enantioselectivity, resulting in a remarkable improvement from a 10% yield of the single enantiomer (45’C) to a 35% yield. Surprisingly this temperature decrease had a very small affect upon the reaction time. / Thesis (M.Sc. (Pharmaceutical Chemistry)--Potchefstroom University for Christian Higher Education, 2002.

Listeria monocytogenes is the etiological agent of a life-threatening, opportunistic infection caused by the ingestion of contaminated foods. Although L. monocytogenes is divided into 13 serotypes, 98% of human illness is caused by serotype 1/2a, 1/2b and 4b strains, with serotype 4b accounting for almost all the major outbreaks of human listeriosis. The principle objective of this work was to develop surface-binding monoclonal antibodies (MAbs) highly specific for serotype 4b, as well as characterize their antigen targets to aid in the detection and isolation of serotype 4b strains using an antibody based procedure. To create such antibodies, mice were immunized with formalin killed whole cells of L. monocytogenes serotype 4b strain LI0521. A total of 15 MAbs reactive to serotype 4b isolates were shown to recognize a ~77 kDa surface antigen subsequently identified by mass spectrometry as surface associated autolysin, IspC. Epitope mapping experiments further revealed that each of the 15 MAbs bound to the cell wall binding GW domain of IspC and can be essentially divided into 4 major groups based on epitope localization. ELISA analysis of the reactivity of each of the MAbs with various L. monocytogenes serotypes indicated that several MAbs were 100% specific for serotype 4b isolates. Surface plasmon resonance experiments showed that the affinity constants for each of these MAbs fell within the range of 1.0 x 10-7 to 6.4 x 10-9 M. To determine whether IspC, shown to be well conserved among various serotype 4b strains, is a useful diagnostic marker with antibody-based methods, the expression of IspC was assessed in L. monocytogenes cultured under normal and stress conditions. A functional promoter directing the transcription of ispC gene was identified immediately upstream of the ispC open reading frame by constructing the promoterless lacZ gene fusion with the putative ispC promoter region and by 5'RACE analysis. Data obtained with the lacZ reporter gene system and immunofluorescent microscopy revealed that IspC is expressed on the cell surface under all growth conditions tested (temperature, osmotic stress, pH, ethanol, oxidative stress, anaerobic conditions, carbon source and enrichment media) that allow for cellular division, although the level of ispC gene expression varies. In addition, a significant effort were put into elucidating the hydrolytic bond specificity of IspC by HPLC and mass spectrometry analysis of muropeptides released from IspC-mediated hydrolysis of L. monocytogenes peptidoglycan (PG). The results demonstrated that IspC functions as an N-acetylglucosaminidase capable of cleaving the β-1,4-glycosidic bond of the PG glycan strand. Furthermore, IspC was more efficient at hydrolysing fully Nacetylated PG from a PG deacetylase gene (pgdA) deletion mutant of L. monocytogenes than partially de-N-acetylated wild-type PG, indicating that modification of PG by de-Nacetylation of GlcNAc residues renders PG resistant to IspC hydrolysis. In conclusion, the surface autolysin IspC with the N-acetylglucosaminidase activity is a novel diagnostic marker for the 4b serotype strains, which can be explored , in conjunction with specific MAbs developed here, for detection and isolation of L. monocytogenes serotype 4b strains directly from food, environmental and clinical samples with the need for minimal or no culture enrichment.

Thèse (M.Sc.)--Université Laval, 2004. / Bibliogr.: f. 62-68. Publié aussi en version électronique.

Thèse (M.Sc.) -- Université Laval, 1997. / Les chapitres 3 à 5, rédigés par l'auteur et collab. en anglais, avec résumés en français, ont été soumis ou publiés dans différents titres de périodiques. Bibliogr. Publ. aussi en version électronique.