H2S é um gasotransmissor com atua regulando processos fisiológicos e fisiopatológicos. É sintetizado principalmente pelas enzimas cistationinagama-liase (CSE) e cistationina beta-sintase (CBS), ambas dependentes do co-fator 5-fosfato de pirodoxal, e que utilizam o L-cisteína como substrato. Essas enzimas estão presentes no fígado. O camundongo knockout para CBS (CBS KO) apresenta fenótipo de homocistinúria, ou seja, além da homocisteinemia apresenta estresse oxidativo, fibrose e esteatose hepática. Ademais, há estudos in vitro sugerindo um papel desse gás reduzindo a captação de glicose hepática. A hipótese desse projeto é que haja redução precoce da produção de H2S pelo fígado de camundongos alimentados com dieta hiperlipídica (DHL) e esse seja um evento anterior à instalação do fígado gorduroso ou DGHNA associada à obesidade induzida pela DHL, DIO. Para isso, avaliamos a evolução ponderal, consumo alimentar e calórico diários, a tolerância a glicose e a insulina (GTT, ITT e Kitt), a morfohistologia hepática com corantes HE e picrocirius red, expressão proteica e gênica das enzimas CBS e CSE, a quantificação do conteúdo de triglicérides, a deteção de F4/80 e a atividade de enzimas antioxidantes (catalase, GPx e arginase) em amostras de fígado de camundongos machos C57Bl6 alimentados com a DHL por 5 períodos distintos (10, 20, 40, 60 e 80 dias de dieta). Detectamos aumento da massa corporal final a partir de 20 dias de DHL. O consumo alimentar diário foi menor nos animais com a DHL, porém com maior consumo calório do que os respectivos controles. A glicemia basal foi maior nos animais com DHL desde os 10 dias, com exceção o grupo com 40 dias. Houve maior intolerância à glicose desde 10 dias de DHL seguida de resistência à insulina a partir de 20 dias de DHL. Foi detectada presença de vacúolos intracelular nos hepatócitos a partir de 20 dias de DHL, no entanto, a presença de hepatócitos comprometidos foi de aproximadamente 2% aos 20 dias da DHL, cerca de 5% aos 40 dias da DHL e aproximadamente 60% com 60 e 80 dias da DHL. Aos 80 dias de DHL foi detectada balonização de hepatócitos, presença de infiltrado inflamatório identificado pelo aumento da marcação com F4/80, maior detecção de fibras colágenas nos espaços vasculares e aumento da atividade da enzima sérica AST. Houve redução para aproximadamente 50% da produção do H2S nas amostras de fígado dos animais a partir de 40 dias da DHL, porém sem relação clara com as modificações detectadas na expressão das enzimas CBS e CSE. Diante desses dados, concluímos que a DHL leva à intolerância à glicose, seguida de aumento da massa corporal e resistência ao efeito hipoglicemiante da insulina e que o desenvolvimento de fígado gorduroso é um evento mais tardio. Vimos que o espectro da DGHNA desenvolvida apresenta características de esteato-hepatite incipiente, com balonização dos hepatócitos, infiltrado inflamatório presente, discreto aumento de deposição de fibras colágenas e aumento da enzima hepática circulante AST. A redução da produção do gás apareceu após a instalação da resistência à insulina, mas anterior ao estabelecimento da DGHNA. Portanto, concluímos que a alteração na produção hepática do H2S pode ser um evento precoce associado ao desenvolvimento da DGHNA e que merece ser investigado seu potencial papel na progressão de estatose a EHNA com fibrose e cirrose. No entanto, será importante buscar um outro modelo animal para estudo dessa evolução. / Hydrogen sulfide (H2S) is a gaseous signaling molecule that regulates a variety of physiological and pathophysiological processes. It is primarily synthesized by cystathionine-gamma-lyase (CSE) and cystathionine beta-synthase (CBS), which are expressed in the liver, require 5\'-pyrodoxal phosphate as a co-factor, and use L-cysteine as a substrate. Interestingly, CBS knockout (CBS KO) mice present homocystinuria and associated homocysteinemia, as well as oxidative stress, fibrosis and hepatic steatosis. Furthermore, in vitro studies suggest that this gas reduces hepatic glucose uptake. It was hypothesized that mice fed a high fat diet (HFD) would display an early reduction H2S production by the liver and that this event would occur prior to the establishment of fatty liver or NAFLD, which is associated with diet induced obesity (DIO). Towards the goal of gaining a better understanding of H2S production during the onset and progression of DIO, male C57B16 mice were fed a HFD for 10, 20, 40, 60 or 80 days. During the experimental period, weight evolution, daily food and caloric intake, as well as glucose and insulin tolerance (GTT, ITT and Kitt) were evalauted. At the end of each time period hepatocyte morphology was assessed by HE and picrocirius red staining, CBS and CSE gene and protein expression were evaluated, triglyceride content was quantified, F4/80 antigen was detected and antioxidant enzyme (catalase, GPx and arginase) activites were determined. The results showed that mice fed a HFD for 20 days or longer presented an increase in final body weight. All of the HFD groups presented reduced daily food intake, but higher caloric intake, when compared to their respective controls. All of the animals fed a HFD had increased basal glycemia levels, with the exception of the group fed the diet for 40 days. Reduced glucose tolerance and insulin resistance were observed after consuming the HFD for 10 and 20 days, respectively. Intracellular vacuoles were detected in the hepatocytes after 20 days of the HFD. The amount of compromised hepatocytes increased from 2% after 20 days of the HFD, to about 5% after 40 days, to approximately 60% after 60 and 80 days. After consuming the HFD for 80 days, there was an observed ballooning of hepatocytes, increased F4/80 detection indicating the presence of inflammatory infiltrates, increased collagen fiber detection in the vascular spaces, and increased serum AST activity. There was a ~50% reduction in H2S production in the liver samples from animals fed a HFD for 40 days; however, there was no clear relationship with the alterations detected in CBS and CSE gene or protein expression. Based on the results, it was concluded that an HFD promotes glucose intolerance, followed by increased body mass and insulin resistance and that the development of fatty liver occurs later. Additionally, the spectrum of the developed NAFLD presented incipient steatohepatitis characteristics, such as: hepatocyte ballooning, inflammatory infiltrate, a slight increase in collagen fiber deposition, and an increase in circulating liver AST levels. The reduction in H2S gas production appeared after the installation of insulin resistance, but prior to the establishment of NAFLD. Therefore, we conclude that alterations in hepatic H2S production may be an early event associated with the onset of NAFLD and that its potential role in the progression of steatosis and NASH with fibrosis and cirrhosis deserves to be investigated. However, it will be important to evaluate this evolution in other animal models.

L’une des particularités fondamentales caractérisant les cellules végétales des cellules animales est la présence de la paroi cellulaire entourant le protoplaste. La paroi cellulaire joue un rôle primordial dans (1) la protection du protoplaste, (2) est impliquée dans les mécanismes de filtration et (3) est le lieu de maintes réactions biochimiques nécessaires à la régulation du métabolisme et des propriétés mécaniques de la cellule. Les propriétés locales d’élasticité, d’extensibilité, de plasticité et de dureté des composants pariétaux déterminent la géométrie et la forme des cellules lors des processus de différentiation et de morphogenèse. Le but de ma thèse est de comprendre les rôles que jouent les différents composants pariétaux dans le modelage de la géométrie et le contrôle de la croissance des cellules végétales. Pour atteindre cet objectif, le modèle cellulaire sur lequel je me suis basé est le tube pollinique ou gamétophyte mâle. Le tube pollinique est une protubérance cellulaire qui se forme à partir du grain de pollen à la suite de son contact avec le stigmate. Sa fonction est la livraison des cellules spermatiques à l’ovaire pour effectuer la double fécondation. Le tube pollinique est une cellule à croissance apicale, caractérisée par la simple composition de sa paroi et par sa vitesse de croissance qui est la plus rapide du règne végétal. Ces propriétés uniques font du tube pollinique le modèle idéal pour l’étude des effets à courts termes du stress sur la croissance et le métabolisme cellulaire ainsi que sur les propriétés mécaniques de la paroi. La paroi du tube pollinique est composée de trois composantes polysaccharidiques : pectines, cellulose et callose et d’une multitude de protéines. Pour comprendre les effets que jouent ces différents composants dans la régulation de la croissance du tube pollinique, j’ai étudié les effets de mutations, de traitements enzymatiques, de l’hyper-gravité et de la gravité omni-directionnelle sur la paroi du tube pollinique. En utilisant des méthodes de modélisation mathématiques combinées à de la biologie moléculaire et de la microscopie à fluorescence et électronique à haute résolution, j’ai montré que (1) la régulation de la chimie des pectines est primordiale pour le contrôle du taux de croissance et de la forme du tube et que (2) la cellulose détermine le diamètre du tube pollinique en partie sub-apicale. De plus, j’ai examiné le rôle d’un groupe d’enzymes digestives de pectines exprimées durant le développement du tube pollinique : les pectate lyases. J’ai montré que ces enzymes sont requises lors de l’initiation de la germination du pollen. J’ai notamment directement prouvé que les pectate lyases sont sécrétées par le tube pollinique dans le but de faciliter sa pénétration au travers du style. / One of the most important features characterizing plant cells and differentiating them from animal cells is the cell wall that surrounds them. The cell wall plays a critical role in providing protection to the protoplast; it acts as a filtering mechanism and is the location of many biochemical reactions implicated in the regulation of the cell metabolism and the mechanical properties of the cell. The local stiffness, extensibility, plasticity and elasticity of the different cell wall components determine the shape and geometry of the cell during differentiation and morphogenesis. The goal of my thesis is to understand the role played by the different cell wall components in shaping the plant cell and controlling its growth behaviour. To achieve this goal, I studied the pollen tube, or male gametophyte, as a cellular model system. The pollen tube is a cellular protuberance formed by the pollen grain upon its contact with the stigma. Its main purpose is to deliver the sperm cells to the female gametophyte to ensure double fertilization. The pollen tube is a tip-growing cell characterized by its simple cell wall composition and by the fact that it is the fastest growing cell of the plant kingdom. This makes it the ideal model to study the effects of drugs, mutations or stresses on cellular growth behaviour, metabolism and cell wall mechanics. The pollen tube cell wall consists mainly of proteins and three major polysaccharidic components: pectins, cellulose and callose. To understand the role played by these components in regulating pollen tube growth, I investigated the effects of mutations, enzymatic treatments, hyper-gravity and omni-directional gravity on the pollen tube cell wall. Using mathematical modeling combined with molecular biology and high-resolution electron and fluorescent microscopy I was able to show that the regulation of pectin chemistry is required for the regulation of the growth rate and pollen tube shape and that cellulose is crucial for determining the pollen tube diameter in the sup-apical region. Moreover, I investigated the role of the pectate lyases, a group of pectin digesting enzymes expressed during pollen tube development, and I showed that this enzyme activity is required for the initiation of pollen germination. More importantly, I directly showed for the first time that the pollen tube secretes cell wall loosening enzymes to facilitate its penetration through the style.

Submitted by GILMAR JOSÉ PICOLI JUNIOR null (gilmarpicoli@yahoo.com.br) on 2016-03-01T21:07:14Z No. of bitstreams: 1 Tese versão definitiva.pdf: 3101518 bytes, checksum: 929d40efab9e6e768e639133eb923580 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-04T12:31:13Z (GMT) No. of bitstreams: 1 picolijunior_gj_dr_bot.pdf: 3101518 bytes, checksum: 929d40efab9e6e768e639133eb923580 (MD5) / Made available in DSpace on 2016-03-04T12:31:13Z (GMT). No. of bitstreams: 1 picolijunior_gj_dr_bot.pdf: 3101518 bytes, checksum: 929d40efab9e6e768e639133eb923580 (MD5) Previous issue date: 2016-01-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / No Brasil, o azevém (Lolium multiflorum) foi identificado como resistente ao glyphosate se tornando um grande problema em determinadas lavouras. Dessa forma, entender o comportamento a nível bioquímico e fisiológico desta planta daninha são ferramentas que auxiliam num manejo eficiente. Com isso, o objetivo deste trabalho foi comparar o perfil bioquímico e fisiológico de populações de azevém suscetíveis e resistentes ao herbicida glyphosate aplicação do mesmo. Foram realizados quatro estudos em casa-de-vegetação com delineamento experimental inteiramente casualizados com quatro repetições sendo semeadas três populações de azevém (Lolium multiflorum) consideradas como suscetível (S), com suspeita de resistência (R1) e resistente (R2) ao herbicida glyphosate. No primeiro estudo foi obtido o controle aos 21 dias após a aplicação (DAA) e quantificada a massa seca aos 28 DAA das três populações. Os tratamentos foram constituídos da aplicação do herbicida glyphosate composto pelas doses: 0, 135, 270, 540, 1080, 2160, 4320, 8640 g e.a. ha-1. O segundo estudo teve como objetivo determinar a atividade da enzima fenilalanina amônia liase (PAL) nas diferentes populações as 12, 24, 48 e 72 horas após a aplicação (HAA). Os tratamentos foram compostos de duas doses (720 g e.a. ha-1 e 1080 g e.a. ha-1) mais uma testemunha sem aplicação. No terceiro estudo foram realizadas avaliações da fotossíntese nas três populações ao 1, 3, 7 e 28 DAA. As variáveis analisadas foram: taxa de assimilação líquida de CO2, condutância estomática, concentração interna de CO2, transpiração, eficiência do uso da água e eficiência instantânea de carboxilação. Os tratamentos foram compostos de duas doses (720 g e.a. ha-1 e 1080 g e.a. ha-1) mais uma testemunha sem aplicação. O quarto estudo teve o objetivo de quantificar compostos alterados da rota do ácido chiquímico. Para isso, foram utilizados os mesmos tratamentos do primeiro estudo e realizadas coletas das folhas aos 5, 11 e 28 DAA. Os compostos analisados foram: glyphosate, AMPA (ácido aminometilfosfônico), ácido chiquímico, ácido quínico, shiquimato-3-fosfato, os aminoácidos aromáticos fenilalanina, tirosina e triptofano, ácido ferúlico, ácido coumárico e ácido cafeico. Na população considerada resistente, a atividade da enzima fenilalanina amônia liase manteve-se alta após a aplicação do glyphosate. Todas as variáveis fisiológicas foram afetadas após a aplicação do glyphosate nas três populações, porém, R2 foi capaz de se recuperar apresentando valores semelhantes à testemunha. Os níveis de ácido chiquímico e quínico apresentaram padrões semelhantes onde houve aumento para as populações suscetíveis com o aumento da dose do herbicida enquanto que para a resistente os valores se mantiveram semelhantes. Ocorreu aumento dos níveis de shiquimato-3-fosfato para a população R2 se mantendo constante para as suscetíveis. Houve redução dos aminoácidos aromáticos com a aplicação do glyphosate para as populações suscetíveis. / In Brazil, ryegrass (Lolium multiflorum) was identified as resistant to glyphosate becoming a major problem in certain crops. Thus, understanding the behavior of the biochemical and physiological level of this weed are tools that help in efficient management. Thus, the aim of this study was to compare the biochemical and physiological profile of ryegrass populations susceptible and resistant to glyphosate after spray it. Four studies were carried out in greenhouse with experimental design completely randomized with four replications being seeded three populations of ryegrass (Lolium multiflorum) considered as susceptible (S), suspected of having resistance (R1) and resistant (R2) to the herbicide glyphosate. In the first study was measured the control at 21 days after application (DAA) and at 28 DAA, the dry mass the three populations. The treatments consisted of application of the glyphosate composed of doses: 0, 135, 270, 540, 1080, 2160, 4320, 8640 g a.i. ha-1. The second study aimed to determine the phenylalanine ammonia lyase (PAL) activity in different populations at 12, 24, 48 and 72 hours after application (HAA). The treatments consisted of two doses (720 g a.i. ha-1 and 1080 g a.i. ha-1) plus a control without application. In the third study were carried out photosynthesis assessments at three populations at 1, 3, 7 and 28 DAA. The variables analyzed were: CO2 net assimilation rate, stomatal conductance, CO2 internal concentration, transpiration, water use efficiency and instantaneous carboxylation efficiency. The treatments consisted of two doses (720 g a.i. ha-1 and 1080 g a.i. ha-1) plus a control without application. The fourth study aimed to quantify altered compounds of the shikimic acid pathway. For this, the same treatments of the first experiment were used and made collections of leaves at 5, 11, 28 DAA. The compounds analyzed were: glyphosate, AMPA (aminomethylphosphonic acid), shikimic acid, quinic acid, shikimate 3-phosphate, the aromatic amino acids phenylalanine, tyrosine and tryptophan, ferulic acid, coumaric acid and caffeic acid. The phenylalanine ammonia lyase enzyme was not influenced by glyphosate in resitant population. All physiological variables were affected after the application of glyphosate at the three populations, but R2 was able to recover with values similar to the control. The shikimic and quinic acid levels showed similar patterns where, there was an increase for susceptible populations with increasing doses of the herbicide while in resistant, the values remained similar. There was increase in levels of shikimate-3-phosphate to the R2 population, remaining constant for susceptible. There was a reduction of the aromatic amino acids with the application of glyphosate for the susceptible populations.

Made available in DSpace on 2014-12-17T14:03:41Z (GMT). No. of bitstreams: 1 TaffarelMT_DISSERT.pdf: 2213102 bytes, checksum: 779f70cd3a9747e4f3a386630bd3d1cc (MD5) Previous issue date: 2013-04-09 / Funda??o de Apoio ? Pesquisa do Estado do Rio Grande do Norte / Oilseeds are a high-value natural resource, due to its use as a substitute for petroleum. However, the storage time can reduce seed viability and oil quality. Therefore, scientific efforts have been made to provide a increment of storage time, germination rates and plant establishment of high-value oilseeds. The seedling establishment depends of the plant pass over the functional transition stage, characterized by a metabolic change from heterotrophic condition to autotrophic one. The storage oil mobilization is performed by β-oxidation process and the glyoxylate cycle. Also, the functional transition involves acclimation to photosynthetic condition, which generally includes the participation of antioxidant system and the reactive oxygen species, the latter are produced in various reactions of primary and secondary metabolism. In the present study, Catalase was inhibited during the functional transition of sunflower and safflower, after were performed many analyzes to elucidate the effects caused on the SOD and APX antioxidant systems. Also, were checked the changes in expression pattern of the glyoxylate cycle enzymes markers, ICL and MLS. It was observed that after CAT inhibition, the SOD and APX antioxidant systems allow the seedling establishment. Besides, was verified that both oilseeds can be accelerate the reverse mobilization and the photosynthetic establishment when Catalase activity has dramatically decreased / As sementes oleaginosas armazenam ?leos, um recurso natural de alto valor econ?mico devido a sua aplicabilidade como substituto aos derivados do petr?leo. Por?m, o tempo de armazenamento pode inviabilizar a semente provocando a redu??o da qualidade do ?leo e perda da viabilidade da semente. Por este motivo, esfor?os cient?ficos t?m sido realizados para proporcionar um maior tempo de armazenamento e incrementar taxas de germina??o e estabelecimento destas plantas com alto valor econ?mico. O estabelecimento da pl?ntula depende da capacidade do vegetal transpassar a etapa de transi??o funcional, caracterizada pela mudan?a do estado heterotr?fico para o autotr?fico. O consumo das reservas de ?leo depende das vias de β-oxida??o e do ciclo do glioxilato. No entanto, o processo de transi??o envolve a aclimata??o do vegetal ? condi??o de organismo fotossintetizante, que em geral conta com a participa??o fundamental do sistema antioxidante do vegetal devido ? produ??o de esp?cies reativas de oxig?nio em v?rias rea??es do metabolismo prim?rio e secund?rio. Neste estudo, a Catalase foi inibida durante a transi??o funcional de girassol e c?rtamo para analisar os efeitos provocados nos sistemas antioxidantes de APX e da SOD e verificar as modifica??es no padr?o de express?o das enzimas marcadoras do ciclo do glioxilato, ICL e MLS. Constatou-se que diante da inibi??o da Catalase o sistema antioxidante da APX e da SOD permitem o estabelecimento da pl?ntula. Verificou-se tamb?m que as duas oleaginoasas parecem acelerar a mobiliza??o de reservas e o estabelecimento fotossint?tico quando a Catalase tem a atividade drasticamente reduzida

Made available in DSpace on 2014-07-29T15:16:29Z (GMT). No. of bitstreams: 1 Renata Silva do Prado.pdf: 1314483 bytes, checksum: 0832c7a22af0d5f60e10293a7d47b2d0 (MD5) Previous issue date: 2010-11-20 / The fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomicosys (PCM), the most prevalent human systemic mycosis in Latin America. The toxicity of drugs and the appearance of resistant strains have imposition the search for new therapeutic approaches. Plants with reputed antimicrobial uses represent a rich source for the screening of potential antifungal compounds. In this work, we investigated the inhibitory action of argentilactone, extracted from the essential oil of Hyptis ovalifolia, and its analogues on P. brasiliensis yeast cells, during the differentiation from mycelium to yeast, on native and recombinant PbICL. Sensitivity tests on plates, which assessed the activity of argentilactone on yeast cells of P. brasiliensis showed both argentilactone as reduced argentilactone interfered in fungus growth, affecting the cells in a dose-dependent way. The experiments evaluating the minimum inhibitory concentration (MIC), which evaluated the influence of the compounds during the transition from mycelium to yeast in P. brasiliensis showed that argentilactone and reduced argentilactone interferes in this process, also in a dose-dependent way. A specific activity of isocitrate lyase of the fungus, both recombinant as native, as measured by tests of enzyme activity was also affected by the compounds. The type of inhibition promoted by argentilactone and reduced argentilactone was defined as inhibition of mixed type through tests of enzyme kinetics. Different carbon sources (acetate and glucose) have directly influence on the processs of inhibition. The analogues epoxy argentilactone and diol argentilactone not inhibit the growth and differentiation of P. brasiliensis. In addition, it not inhibited the enzyme isocitrate lyase native or recombinant. / O fungo Paracoccidioides brasiliensis é o agente etiológico da paracoccidioidomicose (PCM), micose humana sistêmica de maior prevalência na América Latina. Devido à toxicidade dos antifúngicos existentes, e ao aparecimento de isolados resistentes, estudos em busca de novas terapias tem adquirido relevância. Plantas com propriedades terapêuticas representam uma fonte em potencial para descoberta de novos antifúngicos. Neste trabalho, foi investigada a influência de argentilactona, extraída do óleo essencial de Hyptis ovalifolia, e seus análogos obtidos sinteticamente sobre células leveduriformes de P. brasiliensis, durante o processo de diferenciação de micélio para levedura e sobre a enzima isocitrato liase recombinate (PbICLr) e nativa. Os testes de sensibilidade em placas, que avaliaram a atividade de argentilactona sobre células leveduriformes de P. brasiliensis, mostraram que tanto argentilactona como argentilactona reduzida interferiram no crescimento do fungo, afetando as células de modo dose-dependente. Os experimentos de avaliação de concentração inibitória mínima (CIM), que avaliaram a influência dos compostos durante a transição de micélio para levedura em P. brasiliensis mostraram que argentilactona e argentilactona reduzida interferem nesse processo, também de modo dose-dependente. A atividade específica da isocitrato liase do fungo, tanto recombinante como nativa, mensurada por testes de atividade enzimática, também foi afetada pelos compostos. O tipo de inibição promovida pela argentilactona e argentilactona reduzida foi definido como inibição do tipo mista, através de testes de cinética enzimática. Diferentes fontes de carbono (acetato e glicose) influenciaram diretamente o processo de inibição. Os análogos epoxi argentilactona e diol argentilactona não inibiram o crescimento de células leveduriformes e o processo de diferenciação de P. brasiliensis. Em adição, também não inibiram a atividade da enzima isocitrato liase recombinante e nativa.

Salicylic acid (SA) is an important plant hormone with a wide range of effects on plant growth and metabolism. Plants lacking SA exhibit enhanced susceptibility to pathogens. SA plays important signaling roles in resistance against biotrophic and hemi- biotrophic phytopathogens. It is synthesized in plastids along two pathways, one involving phenylalanine ammonia lyase (PAL) and the other isochorismate synthase (ICS). In Arabidopsis , during immune response most SA is synthesized through the ICS-dependent pathway, but clearly an ICS-independent pathway also exists. Several SA effector proteins have been identified and characterized which mediate downstream SA signaling. This includes SABP, a catalase, SABP2, a methyl salicylate esterase, SABP3, a carbonic anhydrase, NPR1 (nonexpressor of pathogenesis-related 1), NPR3 (a NPR1 paralog), and NPR4 (another NPR1 paralog). NPR3 and NPR4 regulate the turnover of NPR1, a process which plays a key role in activating defense gene expression. The role of SA in abiotic stress signaling is gradually becoming clearer. Various components of SA signaling in biotic stress also appear to impact abiotic stress signaling.

Indiana University-Purdue University Indianapolis (IUPUI) / Spore photoproduct lyase (SPL) is a radical SAM (S-adenosylmethionine) enzyme that is responsible for the repair of the DNA UV damage product 5-thyminyl-5,6-dihydrothymine (also called spore photoproduct, SP) in the early germination phase of bacterial endospores. SPL initiates the SP repair process using 5'-dA• (5'-deoxyadenosyl radical) generated by SAM cleavage to abstract the H6proR atom which results in a thymine allylic radical. These studies provide strong evidence that the TpT radical likely receives an H atom from an intrinsic H atom donor, C141 in B. subtilis SPL. I have shown that C141 can be alkylated in native SPL by iodoacetamide treatment indicating that it is accessible to the TpT radical. Activity studies demonstrate a 3-fold slower repair rate of SP by C141A which produces TpTSO2 - and TpT simultaneously with no lag phase observed for TpTSO2- formation. Additionally, formation of both products shows a Dvmax kinetic isotope effect (KIE) of 1.7 ± 0.2 which is smaller than the DVmax KIE of 2.8 ± 0.3 for the WT SPL reaction. Removal of the intrinsic H atom donor by this single mutation disrupts the rate-limiting process in the enzyme catalysis. Moreover, C141A exhibits ~0.4 turnover compared to the > 5 turnovers in the WT SPL reaction. In Y97 and Y99 studies, structural and biochemical data suggest that these two tyrosine residues are also crucial in enzyme catalysis. It is suggested that Y99 in B. subtilis SPL uses a novel hydrogen atom transfer pathway utilizing a pair of cysteinetyrosine residues to regenerate SAM. The second tyrosine, Y97, structurally assists in SAM binding and may also contribute to SAM regeneration by interacting with radical intermediates to lower the energy barrier for the second H-abstraction step.

No description available.

A hiperplasia adrenal congênita (HAC) por deficiência no P450c17 é uma doença de herança autossômica recessiva raramente relatada em pacientes 46, XX. Nosso objetivo foi o de caracterizar o fenótipo e genótipo desta doença rara revendo os dados clínicos, laboratoriais, genéticos, e da função ovariana de pacientes 46,XX de uma coorte brasileira avaliada no HCFMUSP e dos casos já publicados na literatura. Foram avaliados retrospectivamente os dados de 18 pacientes brasileiras pertencentes a 12 famílias avaliadas no HCFMUSP, e revisados os dados de literatura de pacientes de 10 coortes com deficiência da atividade P450c17 (14 pacientes, 6 delas com defeitos no CYP17A1 e 8 com defeitos no POR). Fenótipo: A maioria das pacientes apresentou-se com amenorreia primária (74 %) e 90 % das pacientes não desenvolveram pubarca; 69,5 % das pacientes apresentavam hipertensão arterial no momento do diagnóstico. Observamos uma alta incidência de distúrbios psiquiátricos (76%), como depressão, ansiedade e síndrome do pânico, em nossa coorte, mas não foram encontrados relatos na literatura. O ultrassom mostrou um aumento de pelo menos um dos ovários em 87 % dos pacientes antes do tratamento e macrocistos ovarianos em 65%, 6 pacientes (26%) referiram terem sido submetidas a cirurgia anterior para tratamento de torção ou ruptura de ovário. O tratamento com dexametasona, estrogênio e progesterona resultou em redução efetiva do volume ovariano. Todos os pacientes apresentaram níveis basais elevados de progesterona e LH e redução dos níveis de androgênios. Não observamos correlação entre os níveis de LH, da relação LH/FSH e de progesterona com o volume ovariano nos dois defeitos. Genótipo: O estudo molecular revelou que 17 pacientes de nossa coorte apresentavam mutações inativadoras no gene CYP17A1 e 1 paciente no gene POR. Duas novas mutações foram identificadas no gene CYP17A1, a p.R362H no éxon 6 e a p.G478S no éxon 8. A mutação mais prevalente no CYP17A1 foi a p.W406R identificada em 41 % das nossas famílias. Algumas das mutações no CYP17A1 foram encontradas apenas na coorte brasileira, mas a mutação p.A287P encontrada no gene POR em uma das nossas pacientes é a mais prevalente na literatura. Em relação à etnia, houve um predomínio dos defeitos moleculares no CYP17A1 em pacientes chinesas e brasileiras, enquanto que os defeitos no gene POR foram mais relatados nas pacientes europeias e norte - americanas. Em conclusão, a análise retrospectiva do fenótipo, genótipo e morfologia ovariana de trinta e duas pacientes 46,XX portadoras de deficiência da atividade P450c17, nos permitiu destacar os seguintes aspectos: a importância da dosagem da progesterona basal para este diagnóstico, a alta prevalência de aumento dos ovários com a presença de macrocistos ovarianos com risco de torção, e de transtornos psiquiátricos identificados em nossa coorte. Acreditamos que esta revisão possa contribuir para o diagnóstico mais precoce desta rara doença congênita / Congenital adrenal hyperplasia due to P450c17 deficiency is rarely reported on 46,XX patients. Our aim was to characterize the phenotype and genotype of this rare disorder reviewing the clinical, laboratory, genetic and ovarian imaging of 46,XX patients of our cohort and of other already reported cases. We retrospectively reviewed 32 patients with deficiency of P450c17 activity: 18 Brazilian patients belonging to 12 families and 10 cohorts already published (fourteen 46,XX patients, 6 of them with CYP17A1 defects and 8 with POR defects). Phenotype: most patients had primary amenorrhea (74%) and 90% of the patients did not develop pubarche; 69.5% of the patients had blood hypertension at diagnosis. We observed a high incidence of psychiatric disorders such as depression, anxiety and panic (76%) in our cohort but no reports were found in the literature. Ultrasound showed an increase of at least one of the ovaries in 87% of the patients before treatment and ovarian macrocysts in 65% of them; 6 patients (26%) had had previous surgery for twisting or ovarian rupture. Treatment with dexamethasone, estrogen and progesterone resulted in ovarian volume reduction. All patients showed elevated basal progesterone and LH and levels, and decreased androgen levels. There was no correlation between the levels of LH and progesterone and of LH/FSH ratio and the ovarian volume in both defects. Genotype: the molecular study showed that 17 patients from our cohort had inactivating mutation in the CYP17A1 gene and 1 in POR gene. Two novel mutations were identified in the CYP17A1 gene, the p.R362H in exon 6 and p.G478S in exon 8. The most prevalent mutation in the CYP17A1 was the p.W406R, identified in 41% of our families. Some of the CYP17A1 mutations were found only in the Brazilian cohort, but the mutation p.A287P found in the POR gene is the most prevalent in the literature. Regarding the ethnicity of the defects, there was a predominance of Chinese and Brazilian patients with defects in the CYP17A1 whereas defects in POR were most reported in European and North-American subjects.Conclusion: In this data review of thirty-two 46,XX patients with dysfunction of P450c17 activity we characterized the phenotype and genotype of this rare disorder and emphasize: the importance of basal progesterone measurement for this diagnosis, the high prevalence of ovarian macrocysts with risk of twisting, and the psychiatric disorders. We believe that this review may contribute to the early diagnosis of this disorder

Recent findings have shown an inverse association between the circulating levels of pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0) with the risk of pathological development in type 2 diabetes, cardio vascular disease and neurological disorders. From previously published research, it has been said that both these odd chain fatty acids are biomarkers of their dietary intake and are significantly correlated to dietary ruminant fat intake. However, there are profound studies that show the contrary where they do not display this biomarker correlation. Additionally, several astute studies have suggested or shown odd chain fatty acid endogenous biosynthesis, most often suggested via alpha oxidation; the cleavage of a single carbon unit from a fatty acid chain within the peroxisomes. To better understand the correlations and interactions between these two fatty acids with pathological development, the origin of these odd chain fatty acids needed to be determined, along with confirming their association with the disease aetiology. To minimise animal & human experimentation we made use of existing sample sets made available through institutional collaborations, which produced both animal and human interventional study samples suitable for odd chain fatty acid investigations. These sample collaborations allowed us to comprehensively investigate all plausible contributory sources of these odd chain fatty acids; including from the intestinal microbiota, from dietary contributions, and derived from novel endogenous biosynthesis. The investigations included two intestinal germ-free studies, two ruminant fat diet studies, two dietary fat studies and an ethanol intake study. Endogenous biosynthesis was assessed through: a stearic acid infusion, phytol supplementation, and an Hacl1 knockout mouse model. A human dietary intervention study was used to translate the results. Finally, a study comparing circulating baseline C15:0 and C17:0 levels with the development of glucose intolerance. We found that the circulating C15:0 and C17:0 levels were not significantly influenced by the presence or absence of intestinal microbiota. The circulating C15:0 levels were significantly and linearly increased when the C15:0 dietary composition increased; however, there was no significant correlation in the circulating C17:0 levels with intake. Circulating levels of C15:0 were affected by the dietary composition and factors affecting the dietary intake, e.g. total fat intake and ethanol, whereas circulating C17:0 levels were found to be independent of these variables. In our studies, the circulating C15:0 levels were not significantly affected by any expected variations in alpha oxidation caused by pathway substrate inhibition or gene knockout. However, C17:0 was significantly related, demonstrating it is substantially endogenously biosynthesised. Furthermore, we found that the circulating C15:0 levels, when independent of any dietary variations, did not correlate with the progression of glucose intolerance when induced, but the circulating C17:0 levels did significantly relate and linearly correlated with the development of glucose intolerance. To summarise, the circulating C15:0 and C17:0 levels were independently derived; the C15:0 levels substantially correlated with its dietary intake, whilst the C17:0 levels proved to be separately derived from its endogenous biosynthesis via alpha oxidation of stearic acid. C15:0 was found to be minimally endogenously biosynthesised via a single cycle of beta oxidation of C17:0 in the peroxisomes, however, this did not significantly contribute to the circulating levels of C15:0. Additionally, only the baseline levels of C17:0 significantly correlated with the development of glucose intolerance. These findings highlight the considerable differences between both of these odd chain fatty acids that were once thought to be homogeneous and similarly derived. On the contrary, they display profound dietary, metabolic, and pathological differences.