Family 6 Glycosyltransferases (GT6s) are involved in the biosynthesis of complex glycans and can be found in all vertebrates, cyanophages, and some bacteria and unicellular eukaryotes. Understanding variations within family 6 GTs is important because of the roles of their products in cellular recognition, intercellular interactions, pathogenicity, and immunity and is likewise important for understanding the evolution of GTs. PaGT6 (from Parchlamydia acanthamoebae) and α3GT (from Bos taurus) both require a divalent metal ion for catalysis which binds to a DXD motif. In BoGT6a from Bacteroides ovatus a NXN motif replaces DXD, and activity is metal-independent. However, mutating the NXN motif in BoGT6a to DXD did not introduce metal-dependency, indicating that metal-dependency is linked to additional differences. Calorimetric studies have shown that the presence of a divalent metal ion enhances UDP and donor substrate binding to PaGT6 and causes an increase in the entropy of the interaction. Protein modelling of PaGT6 has revealed that the presence of Mn2+ allows a hydrogen bond to form between Asp 97 and UDP-GalNAc, causing the donor substrate to bend and form hydrogen bonds with His 119, Asn 229, Lys 228, and Arg 234. These interactions do not occur in the absence of Mn2+. Investigation of acceptor substrate binding revealed that the presence of UDP enhances acceptor substrate binding to BoGT6a and PaGT6. Calorimetric titrations of BoGT6a with 2-fucosyllactose in the absence and presence of UDP showed that UDP increases the affinity of 2-fucosyllactose 16-fold with little effect on ΔH. Measurements of ΔCp for 2-fucosyllactose binding indicate that there is not a hydrophobic effect for the binding of 2-fucosyllactose. The preferred acceptor substrate for the bovine and Bacteroides GT6 has a β-1,4 linked galactose, but P. acanthamoebae GT6 prefers an acceptor substrate with a β-1,3 linked galactose. The N-terminus of the catalytic domain of bacterial GT6s is truncated by 47 residues relative to the catalytic domain of bovine α3GT. Removal of this region from α3GT results in an unfolded protein, indicating that although this region is not directly involved in substrate binding, it forms interactions necessary for the stability of the catalytic domain. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Thèses (M.A.)--Université de Sherbrooke (Canada), 1997. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Se estudió la presencia de Amebas de Vida Libre (AVL) (Acanthamoeba spp. y Naegleria spp.), y bacterias del orden Chlamydiales como agentes infecciosos en bovinos de la provincia de La Pampa, Argentina. Se obtuvo desarrollo de AVL en el 83,07 % de las muestras de agua destinada a consumo bovino (N=65). Se identificó por cultivo y técnicas moleculares la presencia de Acanthamoeba spp. en el 24,07 %. Se identificaron los genotipos T4, T5 y T15 del género Acanthamoeba. No se encontró asociación entre queratitis con la presencia de AVL. No se detectaron AVL del género Naegleria. Se estudiaron 709 muestras bovinas, para detectar la presencia de bacterias del orden Chlamydiales, y 656 sueros bovinos para estimar la prevalencia de infección por estas bacterias. Se detectó ADN de familia Chlamydiaceae en el 4,78 % de las muestras de órganos parafinados provenientes de pérdidas reproductivas y de Chlamydia abortus en el 1,99 % (N=251). Se determinó una prevalencia de anticuerpos séricos contra Chlamydia abortus del 25,00 % sobre 128 lotes bovinos, con una seroprevalencia individual (N=656) del 8,07 %. No se encontraron diferencias significativas entre categoría. Se detectó un 6 % de ADN del orden Chlamydiales y 2 % de la familia Chlamydiaceae sobre 50 muestras oculares de terneros estudiadas. Se identificó por secuenciación la presencia de Uncultured Chlamydiales. Se detectó ADN del orden Chlamydiales en el 30,76 % de las 104 muestras de cérvix estudiadas y el 3,84 % correspondió a la familia Chlamydiaceae. No se encontraron diferencias significativas entre las prevalencias de hembras con y sin problemas reproductivos a nivel de orden, pero si a nivel de familia. Se detectó en un 8% (1 muestra) de lavado de útero de vacas donadoras de embriones, ADN a nivel de orden Chlamydiales y de familia Chlamydiaceae sobre 12 muestras estudiadas. Se detectó ADN del orden Chlamydiales en el 30,00 % de las muestras de semen de toros de cabañas estudiadas y de familia Chlamydiaceae en el 6,00 % (N=50). Se detectó ADN del orden Chlamydiales en el 23,33% de las muestras de esmegma prepucial de toros provenientes de cabañas y servicio natural estudiadas (N=150). La familia Chlamydiaceae fue identificada en el 4,66% de ellas. Se encontraron diferencias significativas entre toros en servicio natural y de cabaña solo a nivel de orden. Se detectó ADN del orden Chlamydiales en el 4,76% de muestras de órganos formolados de fetos abortados y ADN de la familia Chlamydiaceae en el 2,38% de ellas (N=42). Se detectó en 50 muestras de órganos de fetos no abortados ADN del orden Chlamydiales en el 20,00 % y 10,00 % de la familia Chlamydiaceae. Se identificó por secuenciación la presencia de una clamidia ambiental (Neochlamydia spp.) en un cultivo de Acanthamoeba spp. (genotipo T5), probablemente como endosimbionte. Los resultados obtenidos indican la necesidad de profundizar el estudio de las AVL y las clamidias en ambientes ganaderos como agentes infecciosos con potenciales características zoonóticas. / The aim of this study was to evaluate the presence of free-living amoebae (FLA) (Acanthamoeba spp. and Naegleria spp.) and bacteria of the order Chlamydiales as infectious agents in cattle in the province of La Pampa, Argentina. FLA was isolated from 83.07% of the watering trough samples. The identification by Polymerase chain reaction was performed with genus-specific primers and followed by direct sequencing. The sequencing revealed the presence of genotypes T4, T5, and T15 of the genus Acanthamoeba in the samples studied. No association was found between keratitis and the presence of FLA. No FLA of the genus Naegleria was detected. A total of 709 bovine samples collected from paraffined organs of reproductive loss, eye, cervix, washing of uterus from cow donating embryos, semen, preputial smegma, formalin-fixed organs from aborted fetuses and organs from unborn were studied to detect the presence of bacteria of the order Chlamydiales.To assess the prevalence of infection by these bacteria serum samples from 656 cattle were analyzed for the presence of specific IgG antibodies. DNA of the family Chlamydiaceae was detected in 4.78 % of sample paraffin organs proceeding of reproductive loss and Chlamydia abortus in 1.99 % (N=251). Antibodies to Chlamydia abortus were found in 8.07% sera based on ELISA result (N= 656). The overall herd prevalence of anti-Chlamydia abortus antibodies in 128 bovine lots was 25.00%. No significant differences were found between categories. Six percent of DNA bacteria of the order Chlamydiales and 2 % of the family Chlamydiaceae were detected on 50 eye samples of calves studied. The presence of Uncultured Chlamydiales was identified by sequencing. DNA of the order Chlamydiales and the family Chlamydiaceae was detected in 30.76% and 3.84 % of the cervix samples studied (N=104). No significant differences were detected between the prevalence of females with and without reproductive problems at the order level, but yes at the family level. One of 12 samples (8.00%) of washing of uterus of cow donating embryos, DNA was detected at order and family level. DNA of the order Chlamydiales and the family Chlamydiaceae was detected in 30.00% and in 6.00% of the semen samples studied (N=50). The order Chlamydiales DNA was detected in 23.33% of the preputial smegma sample of bulls from breeders and natural service studied (N=150). DNA of the family Chlamydiaceae was identified in 4.66% of them. Significant differences were found between bulls in natural service and breeders only at the order level. DNA of the order Chlamydiales and the family Chlamydiaceae was detected in 4.76 % and 2.38 % of formalin-fixed organ samples of aborted fetuses (N=42). In unborn organ samples, DNA of the order Chlamydiales was detected in 20.00 %. The family Chlamydiaceae DNA was detected in 10% (N=50). The presence of Chlamydia-like organism in an Acanthamoeba spp. (genotype T5) was identified by sequencing, probably as an endosymbiont. The results obtained indicate the need to carry out more studies of FLA and chlamydia in cattle ranch environments as infectious agents with potential zoonotic characteristics.