Thesis (M.E.)--University of Canterbury, 2006. / Typescript (photocopy). "March 2006." Includes bibliographical references (p. 145-151). Also available via the World Wide Web.

The calcium-sensing receptor (CaR) is a G-protein coupled receptor that controls mammalian extracellular calcium (Ca2+o) homeostasis. CaR downstream signalling involves intracellular calcium (Ca2+i) mobilisation which can be negatively modulated by protein kinase C (PKC)-mediated phosphorylation of CaR residue Thr-888 (CaRT888). The nature of this regulation was investigated here using siRNA-based knockdown of individual PKC isotypes. Knocking down PKCα expression increased CaR-induced Ca2+i mobilisation in CaR-HEK cells, significantly lowering the EC50 for Ca2+o relative to control siRNA-transfected cells. In accordance, PKCα knockdown also decreased CaRT888 phosphorylation which also permitted the triggering of Ca2+i mobilisation in CaR-HEK cells at sub-threshold Ca2+o concentrations. Interestingly, PKCε knockdown attenuated CaR-induced Ca2+i mobilisation in CaR-HEK cells, significantly increasing the EC50 for Ca2+o. However, this knockdown was also also found to inhibit CaRT888 phosphorylation and this is the first time that CaRT888 phosphorylation has been shown to be dissociate from Ca2+i mobilisation. The results show the complexity of the interactions that potentially underlie the CaR’s pleiotropic signalling and provides novel targets for examining signal bias. Classically an increase in cAMP is known to trigger PTH seceretion. The observation in this study shows that raising intracellular cAMP levels with forskolin also decreased CaRT888 phosphorylation permitting increased Ca2+i mobilisation. This suggests that cAMP may stimulate the phosphatase (most likely protein phosphatase 2A (PP2A)). Nevertheless, knocking down Gα12, which has been shown to activate PP2A, resulted in increased CaRT888 phosphorylation and lower Ca2+i mobilisation (increased EC50 for Ca2+o). This suggests the possibility of CaR as a cAMP sensor that can detect an increase in intracellular cAMP in order to stop PTH serection. Three novel CaR effectors, P70 ribosamal protein S6 kinase, insulin-like growth factor receptor-1 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, were identified in CaR-HEK cells. It was shown that a) high Ca2+o stimulated the activation of these effectors and b) each effector was inhibited by knockdown of PKCα and Gα12, which further confirmed the association of these signals with CaR. These data show that CaR also plays an important role outside Ca2+o homeostasis, such as growth and inflammation. Finally, five CaR mutations associated with autosomal dominant hypocalcaemia (ADH) were found to increase Ca2+o-induced Ca2+i mobilisation, as well as ERK and p38MAPK activation, when transfected stably in HEK-293 cells. Cotreatment with the calcilytic NPSP795 inhibited ERK and p38MAPK phosphorylation in all 5 gain-of-function mutants and in the wild type CaR cells, with IC50s for the compound in the nanomolar range. These data highlight the potential utility of CaR negative allosteric modulators in the treatment of gain-of-function CaR mutations. Together these data enhance our understanding of CaRT888 phosphorylation and CaR signalling.

Made available in DSpace on 2014-08-20T13:25:41Z (GMT). No. of bitstreams: 1 Tese_Juliana_Severo_Castelo_Branco.pdf: 7339681 bytes, checksum: 5c86995be26b0fd8b4d1fda6177461ec (MD5) Previous issue date: 2008-10-01 / Among the cereals, wheat (Triticum aestivum L.) is the most important crop reaching the third largest grain production in the world. Wheat is cultivated under many different environmental conditions, presenting high grain yield capacity, nutritional quality and high degree of adaptability. In the State of Rio Grande do Sul, Brazil, approximately 5.4 million hectares are considered hydromorphic soils and are mostly used for rice farming and cattle grazing, due to its low drainage capability. Annually, only one million hectares are cultivated with rice, leaving large amounts of land without cropping or cultivated with forage crops. Therefore, there is a strong challenge for breeding programs to develop genotypes capable of tolerating this environment, with a satisfactory cost/benefit ratio for farmers. In order to suppress the energy deficit provoked by hypoxic conditions, several species redirect their metabolic pathways in order to garantee the extra ATP production resulting from an increase in fermentation rates. In those species, a group of proteins that cathalyse glycolisis and phosphate sugar metabolism such as the enzyme alcohol dehydrogenase (ADH), is selectively synthesized. The objective of this work was to verify the possibility of selecting wheat genotypes tolerant to flooding stress, in the greenhouse, by using different periods of flooding conditions alterned with draining periods. Also, to estimate the degree of conservation of the ADH1 gene in some plant species. The results indicate that it is possible to detect genetic variability for the character flooding tolerance in greenhouse. The condition of eight days of flooding revealed that the genotypes BRS 120 and BRS 208 were the most tolerant and the genotypes CD 111 and BRS 194 were the most sensitive. The molecular phylogenetic analysis obtained for the alcohol dehydrogenase (ADH1), indicates that Poaceae members present a high degree of conservation. The closest species were Saccharum oficinarum and Zea mays, presenting higher nucleotide identity. The model species (Oryza sativa) presented a higher dissimilarity regarding the remaining species of the Poaceae, suggesting that this enzyme has acquired novel functions and or mutations that were selected under the flooding condition. / Entre os cereais, o trigo (Triticum aestivum L.) é a mais importante cultura, alcançando a terceira maior produção de grãos no mundo sobrepujado apenas pelo milho e arroz. O trigo é cultivado sob diversas condições ambientais, apresentando alta capacidade de produção de grãos, qualidade nutricional e alto grau de adaptabilidade. No Estado do Rio Grande do Sul, aproximadamente 5,4 milhões de hectares são considerados solos hidromórficos e são principalmente utilizados para cultivo de arroz e pastagem, devido a sua baixa capacidade de drenagem. Anualmente, apenas um milhão de hectares está sendo utilizado para o cultivo de arroz, deixando grandes quantidades de terras para o cultivo de culturas forrageiras. Portanto, existe um forte desafio para programas de melhoramento no desenvolvimento de genótipos capazes de tolerar esse ambiente, com uma boa relação custo/benefício para os agricultores. A fim de suprimir o déficit energético provocado pelas condições de hipoxia, várias espécies redirecionam suas vias metabólicas, a fim de garantir a produção extra ATP resultantes do aumento das taxas de fermentação. Nessas espécies, um grupo de proteínas que catalisam a glicólise e metabolismo de açúcares, como a enzima álcool desidrogenase (ADH), é sintetizada seletivamente. O objetivo deste trabalho foi verificar a possibilidade de seleção de genótipos de trigo tolerantes ao estresse por alagamento, em casa de vegetação, utilizando diferentes períodos de alagamento intercalados com drenagem. Além disso, estimar o grau de conservação do gene ADH1 em algumas espécies de plantas. Os resultados indicam que é possível a detecção de variabilidade genética para o caráter tolerância ao alagamento em casa de vegetação. A condição de oito dias de alagamento revelou que os genótipos BRS 120 e BRS 208 foram as mais tolerantes e os genótipos CD 111 e BRS 194 foram as mais sensíveis. A análise filogenética molecular obtida para o álcool desidrogenase (ADH1), indica que os membros Poaceae apresentaram um elevado grau de conservação. As espécies mais próximas foram Saccharum oficinarum e Zea mays, apresentando maior identidade de nucleotídeos. A espécie modelo para plantas monocotiledôneas (Oryza sativa) apresenta uma maior dissimilaridade quanto ao restante das espécies de Poaceae, sugerindo que esta enzima tenha adquirido níveis de funções ou mutações que foram selecionadas sob a condição alagamento.

Indiana University-Purdue University Indianapolis (IUPUI) / Alcohol dehydrogenase (ADH) genes encode proteins that metabolize ethanol to acetaldehyde. Humans have seven ADH genes in a cluster. The hypothesis of this study was that by controlling the levels of ADH enzymes, cis-regulatory regions could affect the risk for alcoholism. The goal was thus to identify distal regulatory regions of ADHs. To achieve this, sequence conservation across 220 kb of the ADH cluster was examined. An enhancer (4E) was identified upstream of ADH4. In HepG2 human hepatoma cells, 4E increased the activity of an ADH4 basal promoter by 50-fold. 4E was cell specific, as no enhancer activity was detected in a human lung cell line, H1299. The enhancer activity was located in a 565 bp region (4E3). Four FOXA and one HNF-1A protein binding sites were shown to be functional in the 4E3 region. To test if this region could affect the risk for alcoholism, the effect of variations in 4E3 on enhancer activity was tested. Two variations had a significant effect on enhancer activity, decreasing the activity to 0.6-fold. A third variation had a small but significant effect. The effect of variations in the ADH1B proximal promoter was also tested. At SNP rs1229982, the C allele had 30% lower activity than the A allele. In addition to studying the regulatory regions of ADH genes, the effects of alcohol on liver-derived cells (HepG2) were also explored. Liver is the primary site of alcohol metabolism, and is highly vulnerable to injuries due to chronic alcohol abuse. To identify the effects of long term ethanol exposure on global gene expression and alternative splicing, HepG2 cells were cultured in 75 mM ethanol for nine days. Global gene expression changes and alternative splicing were measured using Affymetrix GeneChip® Human Exon 1.0 ST Arrays. At the level of gene expression, genes involved in stress response pathways, metabolic pathways (including carbohydrate and lipid metabolism) and chromatin regulation were affected. Alcohol effects were also observed on alternative transcript isoforms of some genes.

Strongyloidiasis stercoralis hyperinfection presenting as vasculitic-like skin lesions is rare. An autoinfection cycle allows intestinal strongyloidiasis, usually a benign infection, to persist for many decades. We report a woman with disseminated S stercoralis infection presenting as nonpalpable purpuric skin rash and syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Upon admission, she was treated with corticosteroids for her vasculitic skin lesions, which then worsened her status. When the diagnosis was recognized, steroids were stopped, thiabendazole treatment was instituted, and she gradually recovered. Serious or fatal infection can occur in patients with strongyloidiasis who were treated with immunosuppressive drugs. Stool specimen screening and/or serological tests for S stercoralis infection in patients who require immunosuppressive therapy helps to prevent complications before embarking on such treatment. Unexplained hyponatremia, severe hypoalbuminemia without proteinuria, and unusual skin rashes, especially over the lower aspect of the abdomen and upper aspects of the thighs, in persons living in areas endemic to S stercoralis should raise suspicion of S stercoralis infection.

Many different chemicals are used in the everyday life, like detergents and pharmaceuticals. However, their production has a big impact on health and environment as much of the raw materials are not renewable and the standard ways of production in many cases includes toxic and environmentally hazardous components. As the population and as the life standard increases all over the planet, the demand for different important chemicals, like pharmaceuticals, will increase. A way to handle this is to apply the concept of Green chemistry, where biocatalysis, in the form of enzymes, is a very good alternative. Enzymes do not normally function in industrial processes and needs modifications through protein engineering to cope in such conditions. To be able to efficiently improve an enzyme, there is a need to understand the mechanism and characteristics of that enzyme. Acyloins (α-hydroxy ketones) are important building blocks in the synthesis of pharmaceuticals. In this thesis, the enzyme alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber has been in focus, as it has been shown to display a wide substrate scope, also accepting aryl-substituted alcohols. The aim has been to study the usefulness of ADH-A as a biocatalyst towards production of acyloins and its activity with aryl-substituted vicinal diols and to study substrate-, regio-, and enantioselectivity of this enzyme. This thesis is based on four different papers where the focus of the first has been to biochemically characterize ADH-A and determine its mechanism, kinetics and its substrate-, regio-, and enantioselectivity. The second and third paper aims towards deeper understanding of some aspects of selectivity of ADH-A. Non-productive binding and its importance for enantioselectivity is studied in the second paper by evolving ADH-A towards increased activity with the least favored enantiomer through protein engineering. In the third paper, regioselectivity is in focus, where an evolved variant displaying reversed regioselectivity is studied. In the fourth and last paper ADH-A is studied towards the possibility to increase its activity towards aryl-substituted vicinal diols, with R-1-phenyl ethane-1,2-diol as the model substrate, and the possibility to link ADH-A with an epoxide hydrolase to produce acyloins from racemic epoxides.

Indiana University-Purdue University Indianapolis (IUPUI) / Strawberry (Fragaria spp.) is an agricultural crop grown often in temperate regions that has high variability in its susceptibility to freezing injury. To breed cultivars for frost and freezing tolerance, identification of molecular markers associated with low temperature tolerance is advantageous. In this work, I investigated a high-throughput method for protein assays and western blotting. Success in streamlining these processes saves an immense amount of time and allows for the processing of more samples and obtaining larger datasets. Thirty-three octoploid varieties were tested for their accumulation of five different potential biomarkers in response to cold exposure. It was found that total dehydrin content, has the strongest potential to be reliable biomarkers for breeding programs. Previous work identified seven putative dehydrins in Fragaria, where two were purified and positively identified by mass spectrometry and determined to be COR47-like (SKn) and XERO2-like (YnSKn). This work demonstrated that cold tolerance positively correlated with dehydrin protein expression levels. To understand the cold-regulated expression of dehydrins as a function of cold exposure time, the levels of transcripts and corresponding proteins were examined in strongly cold tolerant (Alta) and lesser cold tolerant (FDP817, NCGR1363) Fragaria diploid genotypes. The COR47-like (SKn) and XERO2-like (YnSKn) dehydrins both had higher transcript accumulation and protein levels in the more cold tolerant line in comparison to the two less cold tolerant lines. Lack of correlation between transcript and resulting COR47 protein level in Alta were observed at several different timepoints, where protein accumulation preceded an increase in RNA. This trend was not seen with XERO2. This initiated an investigation to discover at what level COR47 is being regulated. First, the COR47 coding region was sequenced for all the genotypes to confirm against the predicted sequence. In addition, since two isoforms of the COR47 gene exist, and could possibly explain the discrepancy in transcript counts, primers were designed for both isoforms and RT-qPCR was performed to examine the transcripts of COR47 more closely. Through examination of the non-congruence of COR47 transcripts and protein, it was found that transcriptional mechanisms of regulation are not involved, and that post transcriptional and post-RNA splicing mechanisms are likely to be responsible for the observed trend in Alta. Conclusions from this work demonstrate that dehydrin transcripts and dehydrin protein accumulations are strong potential biomarkers for identifying low temperature tolerance in diploid strawberry.

Une attention croissante est portée aux cascades multi enzymatiques pour l’élaboration de procédés de synthèse plus efficaces. Cependant, le contrôle de l’expression hétérologue de plusieurs gènes dans un même hôte s’avère difficile et peut mener à un déséquilibre du flux réactionnel. Pour exploiter au mieux les avantages d’une cascade in vivo, il est nécessaire d’ajuster les activités de chaque étape, et de construire des catalyseurs cellulaires capables de programmer la stœchiométrie des enzymes. Nous avons développé dans ce projet une approche originale pour moduler le ratio de deux enzymes in cellulo en jouant sur le nombre de copies de plasmides par cellule (PCN). Nous avons choisi comme modèle un système autosuffisant associant une Alcool Déshydrogénase (ADH) et une Baeyer-Villiger MonoOxygenase (BVMO), NADP(H)-dépendantes. Plusieurs plasmides recombinants portant les deux gènes ont été conçus et combinés dans E. coli. Les souches de co-expression construites ont été comparées en termes de PCN, de production d’enzymes et d’activité. Nous avons montré l’importance d’un choix judicieux de la combinaison de plasmides ainsi que l’existence d’une corrélation entre ratios d’enzymes et activité. Nos biocatalyseurs s’étendent sur une gamme allant du système inactif à un système affichant un TTN d’environ 6000. Ce système a permis la synthèse de lactones d’intérêt industriel, la dihydrocoumarine et la caprolactone, à partir d’indanol et de cyclohexanol. Enfin, sur ce modèle de combinaison de plasmides, trois nouveaux biocatalyseurs cellulaires, associant l’ADH à diverses BVMOs, ont été créés afin d’élargir la gamme d’esters et de lactones synthétisables à partir d’alcools. / Growing attention is paid to multienzymatic cascades to develop more efficient synthetic processes. However, in in cellulo process, the control of the simultaneous heterologous expression of several genes in the same host is often difficult and can lead to imbalances in the reaction flow. To exploit the benefits of cascades, activities of each step have to be adjusted and thus, cellular biocatalysts capable of programming enzymes stoichiometry have to be constructed. In this work, to modulate the stoichiometry of two enzymes in vivo, we developed an original approach based on the copy number per cell of plasmids (PCN) used as vectors. The PCN is regulated in bacteria by three main mechanisms leading, according to the replicon, to low, medium or high PCN. As proof of concept, we chose a self-sufficient system combining an Alcohol Dehydrogenase (ADH) and a Baeyer-Villiger MonoOxygenase (BVMO), both NADP(H)-dependent. Several recombinant plasmids harboring both genes were designed and combined in E. coli. Coexpression strains constructed were compared in terms of PCN, enzyme production and activity. We showed the importance of a judicious choice of plasmids combination and the existence of a correlation between enzymes ratios and activity. Our biocatalysts ranged from an inactive system to a system with a TTN of about 6000. This system allowed the synthesis of lactones of industrial interest, dihydrocoumarin and caprolactone, via double oxidation of indanol and cyclohexanol. Finally, based on this plasmids combination model, three new cellular biocatalysts combining ADH with various BVMOs were designed to broaden the range of esters and lactones synthesizable from alcohols.

La consommation de poisson et de mammifères marins représente une source importante d’acides gras oméga-3 connus pour leurs effets bénéfiques sur le développement des fonctions cérébrales et notamment, sur le développement du système visuel. Afin de tester l’hypothèse selon laquelle l’exposition prénatale aux acides gras oméga-3 a des effets bénéfiques à long terme, nous avons examiné les fonctions visuelles chez des enfants Inuits d’âge scolaire exposés à de grandes quantités d’oméga-3 durant la période de gestation. Des enfants Inuits (n = 136; moyenne d’âge = 11.3 ans) du nord du Québec (Nunavik) ont participé à cette étude. Un protocole de potentiels évoqués visuels (PEVs) utilisant des stimuli en couleur et en mouvement a été employé afin d’appréhender les réponses parvo- et magnocellulaires respectivement. Les concentrations d’acide docosahexaénoïque (ADH) ont été mesurées à la naissance à partir du sang de cordon ombilical et au moment du testing, reflétant ainsi les expositions pré- et post-natales. Les relations entre les niveaux sanguins d’ADH et les PEVs ont été examinées à l’aide d’analyses de régression multiples, en tenant compte des contaminants environnementaux et d’autres variables potentiellement confondantes. Aucune association significative n’a été trouvée en ce qui concerne les stimuli de mouvement. Cependant, après ajustement pour les covariables, les concentrations d’ADH à la naissance étaient associées à une latence plus courte des composantes N1 et P1 des PEVs couleur. Notre étude démontre, pour la première fois, des effets bénéfiques de l’exposition prénatale à l’ADH sur le système parvocellulaire à l’âge scolaire. / Fish and sea mammals consumption is an important source of omega-3 fatty acids, known for their beneficial effects on human brain development. Several lines of evidence indicate that omega-3 fatty acids are beneficial especially for the development of the visual system. However, the long-term effect of prenatal exposure to omega-3 fatty acids on human visual development is unknown. This question was addressed using visual evoked potentials (PEVs) to study a cohort of school-age Inuit children (n = 136; mean age = 11.3 years old) from Arctic Quebec (Nunavik) who received high levels of omega-3 intake during gestation. PEV protocols using color and motion-onset stimuli were used to assess the parvocellular and magnocellular responses, respectively. Concentrations of the omega-3 fatty acid DHA were measured at birth in the umbilical cord and at the time of testing, reflecting pre- and post-natal exposure, respectively. Relations between omega-3 and VEPs were assessed by multivariate regression analyses, taking into account environmental contaminants and other potential confounding variables. No significant associations were found with motion-onset VEPs. However, after adjustment for covariables, cord blood concentrations of DHA were associated with a shorter latency of the N1 and P1 components of the color VEPs. Our study suggests beneficial effects of DHA on the visual parvocellular system at school age. This is the first study supporting the longlasting beneficial effects of prenatal exposure to DHA.

La consommation de poisson et de mammifères marins représente une source importante d’acides gras oméga-3 connus pour leurs effets bénéfiques sur le développement des fonctions cérébrales et notamment, sur le développement du système visuel. Afin de tester l’hypothèse selon laquelle l’exposition prénatale aux acides gras oméga-3 a des effets bénéfiques à long terme, nous avons examiné les fonctions visuelles chez des enfants Inuits d’âge scolaire exposés à de grandes quantités d’oméga-3 durant la période de gestation. Des enfants Inuits (n = 136; moyenne d’âge = 11.3 ans) du nord du Québec (Nunavik) ont participé à cette étude. Un protocole de potentiels évoqués visuels (PEVs) utilisant des stimuli en couleur et en mouvement a été employé afin d’appréhender les réponses parvo- et magnocellulaires respectivement. Les concentrations d’acide docosahexaénoïque (ADH) ont été mesurées à la naissance à partir du sang de cordon ombilical et au moment du testing, reflétant ainsi les expositions pré- et post-natales. Les relations entre les niveaux sanguins d’ADH et les PEVs ont été examinées à l’aide d’analyses de régression multiples, en tenant compte des contaminants environnementaux et d’autres variables potentiellement confondantes. Aucune association significative n’a été trouvée en ce qui concerne les stimuli de mouvement. Cependant, après ajustement pour les covariables, les concentrations d’ADH à la naissance étaient associées à une latence plus courte des composantes N1 et P1 des PEVs couleur. Notre étude démontre, pour la première fois, des effets bénéfiques de l’exposition prénatale à l’ADH sur le système parvocellulaire à l’âge scolaire. / Fish and sea mammals consumption is an important source of omega-3 fatty acids, known for their beneficial effects on human brain development. Several lines of evidence indicate that omega-3 fatty acids are beneficial especially for the development of the visual system. However, the long-term effect of prenatal exposure to omega-3 fatty acids on human visual development is unknown. This question was addressed using visual evoked potentials (PEVs) to study a cohort of school-age Inuit children (n = 136; mean age = 11.3 years old) from Arctic Quebec (Nunavik) who received high levels of omega-3 intake during gestation. PEV protocols using color and motion-onset stimuli were used to assess the parvocellular and magnocellular responses, respectively. Concentrations of the omega-3 fatty acid DHA were measured at birth in the umbilical cord and at the time of testing, reflecting pre- and post-natal exposure, respectively. Relations between omega-3 and VEPs were assessed by multivariate regression analyses, taking into account environmental contaminants and other potential confounding variables. No significant associations were found with motion-onset VEPs. However, after adjustment for covariables, cord blood concentrations of DHA were associated with a shorter latency of the N1 and P1 components of the color VEPs. Our study suggests beneficial effects of DHA on the visual parvocellular system at school age. This is the first study supporting the longlasting beneficial effects of prenatal exposure to DHA.