Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências Médicas, Programa de Pós-Graduação em Ciências Médicas, 2015. / A Síndrome de Robinow se caracteriza por dismorfias faciais associadas a encurtamento mesomélico e genitália hipoplásica. Defeitos de segmentação costovertebral que incluem fusões de costelas também podem ser observados e permitem distinguir os pacientes com a forma autossômica recessiva da síndrome daqueles com a forma autossômica dominante. A forma recessiva é causada por mutações no gene ROR2 (related orphan receptor 2), sendo que apenas dezenove diferentes mutações foram descritas em pacientes com a síndrome. O objetivo desse trabalho foi identificar novas mutações no gene ROR2 em pacientes com a Síndrome de Robinow autossômica recessiva. Foram selecionados onze pacientes originários do Brasil, Estados Unidos, Índia e Turquia com características clínicas da forma recessiva da síndrome. O sequenciamento foi realizado a partir de amostras de sangue e saliva por sequenciamento Sanger, ou por sequenciamento de nova geração na plataforma Ion PGM. Em um paciente a mutação foi detectada por MLPA. Identificamos 13 diferentes mutações sendo que 10 ainda não haviam sido descritas na literatura. A comparação dos dados clínicos dos pacientes estudados com as frequências dos sinais clínicos proposta por Mazzeu e cols. (2007) revelou frequência semelhante para a maioria dos sinais clínicos com exceção da fusão de costelas, ausente no paciente 5, a língua bífida presente em todos os 11 pacientes, o lábio superior fino que teve uma frequência de 72% e anteriormente era de 26% e a má-oclusão dentária que teve uma frequência de 60% enquanto que a frequência descrita anteriormente era de 93%. Assim, a descrição clínica detalhada de pacientes com diagnóstico confirmado molecularmente contribuiu para a definição das frequências dos principais sinais clínicos nessa forma da síndrome. Observamos também uma maior gravidade das manifestações esqueléticas nos pacientes com mutações que alteram o quadro de leitura mostrando assim que pode haver correlação entre o tipo de mutação e o fenótipo dos afetados. O conjunto de novas mutações detectadas no trabalho colaboram para um melhor entendimento da fisiopatologia da síndrome. / Robinow syndrome is characterized by facial dysmorphisms, mesomelic limb shortening and hypoplastic genitalia. Costovertebral segmentation defects including rib fusions may be present and allow the distinction between the autosomal recessive and the autosomal dominant forms. Recessive form is caused by mutations in ROR2 gene (related orphan receptor 2) and only 19 different mutations have been described in the literature in patients with the syndrome. The main goal of this work was to identify new mutations in ROR2 in patients with RRS. Eleven patients with clinical signs of RRS from Brazil, United States, India and Turkey have been selected. Sequencing was performed from either blood or saliva samples by Sanger sequencing or Next generation sequencing using Ion PGM platform. In one patient the mutation was detected by MLPA. We identified 13 different mutations, 10 not previously described in the literature. Comparison of clinical data from our patients and the frequencies of clinical signs proposed by Mazzeu e cols., (2007) revealed similar frequencies for most clinical signs except for rib fusions, absent in patient 5; bifid tongue, present in all 11 patients; thin upper lip that had a frequency of 72% compared to 26% in the previous report and dental malocclusion that showed a frequency of 60% and the previous frequency was 93%. Therefore, a detailed clinical description of patients with a molecularly confirmed diagnosis contributed to the definition of frequencies of the main clinical signs of the syndrome. We also observed a more severe skeletal phenotype in patients with frameshift mutations showing that genotype and phenotype may correlate. The group of new mutations detected in the present work contribute to a better understanding of the physiopathology of the syndrome.

No description available.

No description available.

Lichens produce a diverse array of bioactive secondary metabolites, many of which are unique to the organisms. Their potential applications, however, are limited by their finite sources and the slow-growing nature of the organisms in both laboratory and environmental conditions. This thesis set out to investigate polyketide synthase genes in lichens, with the ultimate goal of providing a sustainable source of lichen natural products to support these applications. To expand the diversity of PKS genes that could be detected in lichens, new degenerate primers targeting ketoacylsynthase (KS) domains of specific clades of PKS genes have been developed and tested on various lichen samples. Using these primers, 19 KS domains from various lichens were obtained. Phylogenetic analysis of the KS domains was used to infer the function of the PKS genes based on the predicted PKS domain architecture and chemical analysis by TLC and/or HPLC. KS domains from PKS clades not previously known in lichens were identified; this included the clade III NR (non-reducing)-PKSs, PR (partially reducing)-PKSs and HR (highly reducing)-PKSs. The discovery of clade III NR-PKSs with C-methyltransferase (CMeT) domain and their wide occurrence in lichens was especially significant. Based on the KS domain phylogenetic analysis and compounds detected in the individual lichens, the clade III NR-PKSs were hypothesized to be involved in the biosynthesis of β-orsellinic acid and methylphloroacetopheno ne - the monoaromatic precursors for many lichen coupled phenolic compounds, such as β-orcinol depsides/depsidones and usnic acid. A strategy has been developed to isolate clade III NR-PKSs directly from environmental lichen DNA using clade III NR-type KS amplified from the degenerate primers (NR3KS-F/R) as homologous probes. Another pair of degenerate primers specific to the CMeT domain of NR-PKSs has also been developed to facilitate the cloning and probing of new clade III NR-PKS genes in lichens. A clade III NR-PKS gene (xsepks1) from X. semiviridis was cloned successfully. This is the first report of the isolation of a full-length PKS gene from environmental lichen DNA. The domain architecture of xsepks1 is KS-AT-ACP-CMeT, as expected for a clade III NR-PKS, suggesting that the newly developed clade-specific primers are useful for cloning new clade III NR-PKS genes and that KS domain phylogenetic analysis can predict the functional domains in PKSs. Attempts were made to characterize the function of xsepks1 by heterologous expression in Aspergillus species. Both A. nidulans (transformed with 5´partial xsepks1 including native promoter) and A. oryzae (transformed with full-length xsepks1 under the regulation of starch-inducible amyB promoter) were tested as potential hosts for the expression of lichen PKS genes. Transcriptional analysis showed that A. nidulans could potentially utilize the lichen PKS gene promoter and both fungal hosts could splice the introns of a lichen PKS gene. Several compounds unique to the A. oryzae transformants carrying xsepks1 were detected, but they could not be reproduced in subsequent fermentations even though the gene was transcribed into mRNA. None of the expected products (β-orsellinic acid, methylphloroacetophenone or similar methylated monoaromatic compounds) was detected in A. oryzae transformants, and the function of xsepks1 remains to be determined. The other clade III NR-PKS genes detected in X. semiviridis cou ld also be responsible for the biosynthesis of β-orsellinic acid or methylphloroacetophenone, as precursors of the major secondary metabolites detected in X. semiviridis (i.e. fumarprotocetraric acid, succinprotocetraric acid and usnic acid). Overall, the work in this thesis demonstrated the prospect of using a molecular approach to access the lichen biosynthetic potential without going through the cumbersome culturing stage.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

There are a vast number of transcription factors that regulate plant growth and development. The NAC gene superfamily is one of the largest families of transcription factors in the plant kingdom. NAC gene expression profiles using Affymetrix ATH1 gene chips were obtained for different plant organs: heart embryo, mature embryo, leaf, root and flower. NAC gene expression profiles proved to be very complex, except for one NAC gene detected only in floral tissue, At1g61110. At1g61110 was shown to be specifically expressed in the anther tapetum of Arabidospis; therefore, its name was changed to TAPNAC. TAPNAC became the focus of our studies. We identified a tapnac T-DNA knockout (KO) line, SALK_069450. A molecular phenotype was observed. Several oligopeptide, sugar and metal transporters were differentially expressed. Coincidentally, a wheat NAC gene, named TaNAM-B1 for its high sequence similarity to ATNAM, TAPNAC and At3g15510 was found to be involved in nutrient remobilization. PHOSPHOLIPASE Dα1 (PLDα1) was also found to be down-regulated in the tapnac KO. PLDα1 is an enzyme which hydrolyzes phospholipids that are part of tapetal cell membranes and tapetal lipid bodies. Once these tapetal cell structures are disrupted, the secretion of the compounds that form part of the pollen coat (i.e. proteins, flavonoids and lipids) into the anther locule is facilitated. Promoter deletion analysis using a GUS reporter and later GUS immuno-localization confirmed the findings of Wellmer and others. TAPNAC is a tapetal specific gene. The cis-regulatory sequence that enhances tapetal expression in the TAPNAC promoter was identified. The consensus motif TCGTGT increased tapetal expression of a GUS reporter gene, only when flanked by the TAPNAC minimal promoter region (-217 bp to +51 bp). In summary, TAPNAC transcription factor has been characterized and data indicates that it could play a role in nutrient remobilization from the tapetum to the pollen grains, particularly during late floral stages. Also, important information on tapetal specifcation cis-regulatory sequences was discovered. The consensus motif TCGTGT, present in TAPNAC promoter, was shown to enhance tapetal expression of a GUS reporter gene.

Seed vigor, although not well understood, is a key critical component for yield and is in part due to a well establishment and vigorous stand of canola (<i>Brassica napus</i>) seedling under less than ideal conditions in Western Canada. My objective was to determine what constitutes vigor by studying the response of a black seed line and a yellow seed line imbibed at 8 ºC in either water, saline or osmotic solutions, abscisic acid (ABA), ABA biosynthesis inhibitor, gibberellin (GA4+7), inhibitor of GA biosynthesis and a germination promoter, fusicoccin. Also tested was the effect of seed coat (testa) on seed germination rate and percent germination. Previous studies have established that seed vigor is in part hormonal controlled and genetically controlled. In our study, gene expression was investigated by using transcriptome analysis and hormonal analysis was used to quantitate the changes in hormones and their metabolites during germination. <p> Both the black and the yellow canola seed lines were very sensitive to increasing concentrations of saline and osmotic solutions; however, at the same osmotic potential, osmotic solutions were more inhibitory. The yellow seed line was more sensitive to these conditions than the black seed line. As expected, ABA delayed seed germination, whereas GA4+7 enhanced seed germination and GA4+7 partially overcame the inhibitory effect of ABA. The seed coat was a major factor affecting the germination rate of the yellow seed line; however, GA4+7 overcame the inhibitory effect of the seed coat, whereas ABA exacerbated it. Fusicoccin was more stimulatory to germination than GA4+7; however, unlike GA4+7, it was unable to overcome the inhibitory effect of paclobutrazol, a GA biosynthesis inhibitor. Fluridone, an ABA biosynthesis inhibitor, was unable to overcome the inhibitory effects of a saline solution suggesting that the inhibitory effect was not due to elevated ABA levels. Ethylene, a stimulator of germination, did not appear to be involved in the germination of these two lines. Controlled deterioration at 35 ºC, 85% RH was either partially or completely overcome by exogenous GA4+7. This study demonstrates that the role of hormones, salinity and seed coat on the germination of canola seed under low temperature environmental conditions. <p>During germination, ABA declined while GA4 increased. Higher ABA was found in un-germinated seeds compared to germinated seeds. GA4+7 was lower in seeds imbibed in the saline solution compared to seeds imbibed in water. Un-germinated seeds imbibed in ABA had lower GA4+7 compared to un-germinated seeds imbibed in water; however, the contents of GA4+7 were similar for germinated seeds imbibed in either water or ABA. Phaseic acid (PA) and dihydrophaseic acid (DPA) increased in seeds imbibed in either water, the saline solution or ABA, while they decreased in seeds imbibed in GA4+7. In addition, we found that ABA inhibited GA4 biosynthesis, whereas, GA had no effect on ABA biosynthesis, but altered the ABA catabolic pathway. <p> Gene expression profiles revealed that there are significant differences between un-germinated and germinated seeds. Seeds imbibed in water, GA4+7, a saline solution or ABA had different gene profiles. LEA genes, hormone-related genes, hydrolase-related genes and specific seed germination-related genes were identified and their expression profiles were finely associated with seed germination performance.

Seed vigor, although not well understood, is a key critical component for yield and is in part due to a well establishment and vigorous stand of canola (<i>Brassica napus</i>) seedling under less than ideal conditions in Western Canada. My objective was to determine what constitutes vigor by studying the response of a black seed line and a yellow seed line imbibed at 8 ºC in either water, saline or osmotic solutions, abscisic acid (ABA), ABA biosynthesis inhibitor, gibberellin (GA4+7), inhibitor of GA biosynthesis and a germination promoter, fusicoccin. Also tested was the effect of seed coat (testa) on seed germination rate and percent germination. Previous studies have established that seed vigor is in part hormonal controlled and genetically controlled. In our study, gene expression was investigated by using transcriptome analysis and hormonal analysis was used to quantitate the changes in hormones and their metabolites during germination. <p> Both the black and the yellow canola seed lines were very sensitive to increasing concentrations of saline and osmotic solutions; however, at the same osmotic potential, osmotic solutions were more inhibitory. The yellow seed line was more sensitive to these conditions than the black seed line. As expected, ABA delayed seed germination, whereas GA4+7 enhanced seed germination and GA4+7 partially overcame the inhibitory effect of ABA. The seed coat was a major factor affecting the germination rate of the yellow seed line; however, GA4+7 overcame the inhibitory effect of the seed coat, whereas ABA exacerbated it. Fusicoccin was more stimulatory to germination than GA4+7; however, unlike GA4+7, it was unable to overcome the inhibitory effect of paclobutrazol, a GA biosynthesis inhibitor. Fluridone, an ABA biosynthesis inhibitor, was unable to overcome the inhibitory effects of a saline solution suggesting that the inhibitory effect was not due to elevated ABA levels. Ethylene, a stimulator of germination, did not appear to be involved in the germination of these two lines. Controlled deterioration at 35 ºC, 85% RH was either partially or completely overcome by exogenous GA4+7. This study demonstrates that the role of hormones, salinity and seed coat on the germination of canola seed under low temperature environmental conditions. <p>During germination, ABA declined while GA4 increased. Higher ABA was found in un-germinated seeds compared to germinated seeds. GA4+7 was lower in seeds imbibed in the saline solution compared to seeds imbibed in water. Un-germinated seeds imbibed in ABA had lower GA4+7 compared to un-germinated seeds imbibed in water; however, the contents of GA4+7 were similar for germinated seeds imbibed in either water or ABA. Phaseic acid (PA) and dihydrophaseic acid (DPA) increased in seeds imbibed in either water, the saline solution or ABA, while they decreased in seeds imbibed in GA4+7. In addition, we found that ABA inhibited GA4 biosynthesis, whereas, GA had no effect on ABA biosynthesis, but altered the ABA catabolic pathway. <p> Gene expression profiles revealed that there are significant differences between un-germinated and germinated seeds. Seeds imbibed in water, GA4+7, a saline solution or ABA had different gene profiles. LEA genes, hormone-related genes, hydrolase-related genes and specific seed germination-related genes were identified and their expression profiles were finely associated with seed germination performance.

There are a vast number of transcription factors that regulate plant growth and development. The NAC gene superfamily is one of the largest families of transcription factors in the plant kingdom. NAC gene expression profiles using Affymetrix ATH1 gene chips were obtained for different plant organs: heart embryo, mature embryo, leaf, root and flower. NAC gene expression profiles proved to be very complex, except for one NAC gene detected only in floral tissue, At1g61110. At1g61110 was shown to be specifically expressed in the anther tapetum of Arabidospis; therefore, its name was changed to TAPNAC. TAPNAC became the focus of our studies. We identified a tapnac T-DNA knockout (KO) line, SALK_069450. A molecular phenotype was observed. Several oligopeptide, sugar and metal transporters were differentially expressed. Coincidentally, a wheat NAC gene, named TaNAM-B1 for its high sequence similarity to ATNAM, TAPNAC and At3g15510 was found to be involved in nutrient remobilization. PHOSPHOLIPASE Dα1 (PLDα1) was also found to be down-regulated in the tapnac KO. PLDα1 is an enzyme which hydrolyzes phospholipids that are part of tapetal cell membranes and tapetal lipid bodies. Once these tapetal cell structures are disrupted, the secretion of the compounds that form part of the pollen coat (i.e. proteins, flavonoids and lipids) into the anther locule is facilitated. Promoter deletion analysis using a GUS reporter and later GUS immuno-localization confirmed the findings of Wellmer and others. TAPNAC is a tapetal specific gene. The cis-regulatory sequence that enhances tapetal expression in the TAPNAC promoter was identified. The consensus motif TCGTGT increased tapetal expression of a GUS reporter gene, only when flanked by the TAPNAC minimal promoter region (-217 bp to +51 bp). In summary, TAPNAC transcription factor has been characterized and data indicates that it could play a role in nutrient remobilization from the tapetum to the pollen grains, particularly during late floral stages. Also, important information on tapetal specifcation cis-regulatory sequences was discovered. The consensus motif TCGTGT, present in TAPNAC promoter, was shown to enhance tapetal expression of a GUS reporter gene.