In the post-genomic era, one of the most important goals for the community of plant biologists is to take full advantage of the knowledge generated by the Arabidopsis thaliana genome project, and to employ state-of-the-art functional genomics techniques to assign function to each gene. This will be achieved through a complete understanding of what all cellular components do, and how they interact with one another to produce a phenotype. Among the proteins encoded by the Arabidopsis genome are 24 related carboxyl methyltransferases that belong to the SABATH family. Several of the SABATH methyltransferases convert plant hormones, like jasmonic acid, indole-3-acetic acid, salicylic acid, gibberellins, and other plant constituents into methyl esters, thereby regulating the biological activity of these molecules and, consequently, myriad important physiological processes. Our research aims to decipher the function of proteins belonging to the SABATH family by applying a combination of genomics tools, including genome-wide expression analysis and gas-chromatography coupled with mass spectrometry-based metabolite profiling. Our results, combined with available biochemical information, provide a better understanding of the physiological role of SABATH methyltransferases, further insights into secondary plant metabolism and deeper knowledge of the consequences of modulating the expression of SABATH methyltransferases, both at the genome-wide expression and metabolite levels. / Ph. D.

The bacterial pathogen Vibrio cholerae is responsible for ongoing cholera outbreaks in Haiti and elsewhere. Association of V. cholerae with the human host is responsible for fatal disease, but the bacteria also reside as natural inhabitants of aquatic environments, commonly attaching as biofilms to chitinous surfaces of copepods and crabs. Prior studies in V. cholerae demonstrated that competence for genetic transformation, a mechanism of horizontal gene transfer (HGT), requires the TfoX regulator protein that is triggered by chitin, and the HapR transcription factor that is made in response to quorum sensing (QS) signals produced by V. cholerae and Vibrios. To define regulatory components connecting extracellular signals to natural competence, I first demonstrated that QS molecules produced by Vibrios within multi-species chitinous biofilms are required for DNA uptake by V. cholerae, confirming the critical role of QS signals in HGT. Second, I identified by transposon-mutagenesis a new positive regulator of competence, CytR (cytidine repressor), only studied prior in E. coli as a regulator of nucleoside scavenging. Specific mutations in V. cholerae CytR impaired expression of competence genes and halted DNA uptake; and the addition of exogenous cytidine had similar affects as predicted in E. coli. V. cholerae and other competent Vibrios encode TfoX, HapR, and CytR, although none of these regulators directly controls genes coding for the DNA uptake apparatus. Thus, these results have uncovered a regulatory network, likely used by many Vibrios, that contains additional factors linking several extracellular chemical molecules (cytidine, chitin, and QS signals) to DNA uptake. My study has begun to define a molecular mechanism by which both environment and genetics contribute to genome evolution for this important marine pathogen.

Genetic studies have sought to identify subtypes or endophenotypes of schizophrenia in an effort to improve the reliability of findings. A number of chromosomal regions or genes have now been shown to have had replicated linkage to schizophrenia susceptibility. Molecules involved in neurodevelopment or neurotransmitter function are coded by many of the genes that have been implicated in schizophrenia. Studies of neurotransmitter function have identified, among others, a possible role for GABA, glutamate and dopamine in animal models of schizophrenia. GABA neurons that co-express the calcium binding protein parvalbumin have been implicated as have glutamatergic metabotropic receptors and dopamine D3 receptors. Stress influences glutamate and dopamine providing another environmental factor that may interact with the influence of genes on neurotransmitter function. Neurotransmitter interactions include influences on signaling molecules and these too have been implicated in forms of learning thought to be affected in schizophrenia. Results continue to unravel the interplay of genes and environment in the etiology of schizophrenia and other psychotic disorders.

P2X7 receptors are members of the ATP-gated cationic channel family with a preferential localization at the microglial cells, the resident macrophages of the brain. However, these receptors are also present at neuroglia (astrocytes, oligodendrocytes) although at a considerably lower density. They mediate necrosis/apoptosis by the release of pro-inflammatory cytokines/chemokines, reactive oxygen species (ROS) as well as the excitotoxic (glio)transmitters glutamate and ATP. Besides mediating cell damage i.e., superimposed upon chronic neurodegenerative processes in Alzheimer’s Disease, Parkinson’s Disease, multiple sclerosis, and amyotrophic lateral sclerosis, they may also participate in neuroglial signaling to neurons under conditions of high ATP concentrations during any other form of neuroinflammation/neurodegeneration. It is a pertinent open question whether P2X7Rs are localized on neurons, or whether only neuroglia/microglia possess this receptor-type causing indirect effects by releasing the above-mentioned signaling molecules. We suggest as based on molecular biology and functional evidence that neurons are devoid of P2X7Rs although the existence of neuronal P2X7Rs cannot be excluded with absolute certainty.

As plantas se defendem do ataque de patógenos através de um sistema imune composto por duas fases. A primeira delas é mediada por receptores localizados na membrana celular ou intracelularmente, os quais são conhecidos como receptores de reconhecimento padrão (do inglês, pattern recognition receptors - PRR). Esses receptores reconhecem moléculas derivadas de microrganismos, as quais são conservadas evolutivamente e são chamadas de padrões moleculares associados a patógenos (do inglês, pathogen-associated molecular patterns - PAMPs). Esse reconhecimento dispara uma resposta de defesa conhecida como PTI (do inglês, PAMP-triggerd immunity - PTI). Alguns patógenos foram aptos a sintetizar moléculas capazes de suprimir a PTI e essas moléculas são denominadas de efetores. A resposta que ocorre devido à ação dos efetores é chamada de susceptibilidade disparada por efetores (do inglês, effector-triggered susceptibility - ETS). Entretanto, plantas resistentes podem reconhecer os efetores através de proteínas de resistência localizadas intracelularmente, ativando a imunidade disparada por efetores (do inglês, effector-triggeredimmunity - ETI). De modo geral, as respostas advindas da PTI e da ETI são similares, mas a segunda é ativada mais rapidamente e é mediada por um único gene de resistência R. Por essa razão, a ETI é conhecida como uma resposta à doença qualitativa e as plantas não desenvolvem sintomas, caracterizando a interação incompatível. Por outro lado, a PTI é mediada por diversos genes e as respostas de defesa são tardias, possibilitando a disseminação do patógeno pelas células da planta e a ocorrência da doença, o que caracteriza a interação compatível. Nas respostas de defesa, moléculas como o óxido nítrico, as poliaminas e o ácido salicílico participam do processo de sinalização. O sistema antioxidante da planta é ativado de modo a mitigar os efeitos das espécies reativas de oxigênio e o metabolismo da planta é alterado. Dessa maneira, o estudo das respostas de defesa contra patógenos, pode ser uma ferramenta útil para estabelecer controles efetivos para as doenças de plantas / Plants defend themselves from pathogen attack through an active immunity system composed by two phases. The first is mediated by cell surface and intracellular pattern recognition receptors (PRR), which recognizes conserved molecules derived from microbes known as pathogen-associated molecular patterns (PAMPs). This recognition triggers a defense response called PAMP-triggered immunity (PTI). Throughout evolution, pathogens were able to synthesize molecules capable of suppressing PTI. These molecules are named effectors and they are responsible for effector-triggered susceptibility (ETS). However, resistant plants can recognize effectors by intracellular resistance (R) proteins, initiating effector-triggered immunity (ETI). In general, responses derived from PTI and ETI are the same, but the latter is activated faster and is mediated by a single R gene. For this reason, ETI-response is also known as qualitative disease response (QDR) and plants do not develop disease symptoms, characterizing the incompatible interaction. On the other hand, PTI is mediated by several genes and the defense response is delayed, enabling the pathogen to spread out and to cause disease. This interaction is known as compatible. In defense responses, molecules like nitric oxide, polyamines and salicylic acid can participate in signaling process. The antioxidant system can be activated to quench the ROS effects and the plant metabolism is altered. In this sense, studying defense responses against pathogens can help to develop tools to establish effective control methods for plant disease

Patients with type II diabetes present alterations in glucose homeostasis due to insufficient amount of insulin (β-cell dysfunction) and inability to properly use the insulin that is secreted (insulin resistance). Combined genetical and environmental factors are believed to be responsible for these dysfunctions and the resulting impairment in glucose homeostasis. The pancreatic gland is composed of exocrine and endocrine tissues. The endocrine part of the organ couples glucose sensing to insulin release. Within this endocrine gland, also known as islets of Langerhans, the insulin secreting β-cell is the main player and therefore highly important for proper glucose metabolism. In this thesis, mice were developed in order to assess the role of BMP signaling molecule and Arsenite induced ATPase-1 (Asna1) for pancreas development and β-cell function. The mature β-cell responds to elevated glucose levels by secreting insulin in a tightly controlled manner. This physiological response of the β-cell to elevated blood glucose levels is critical for maintenance of normoglycaemia and impaired Glucose stimulated insulin secretion (GSIS) is a prominent feature of overt type 2 diabetes. Thus, the identification of signals and pathways that ensure and stimulate GSIS in β-cells is of great clinical interest. Here we show (Paper I) that BMPRIA and its high affinity ligand BMP4 are expressed in fetal and adult islets. We also provide evidence that BMPRIA signaling in adult β-cell is required for GSIS, and that both transgenic expression of Bmp4 in β-cells or systemic administration of BMP4 protein to mice enhances GSIS. Thus, BMP4-BMPRIA signaling in β-cells positively regulates the genetic machinery that ensures GSIS. Arsenite induced ATPase (Asna1), the homologue of the bacterial ArsA ATPase, is expressed in insulin producing cells of both mammals and the nematode Caenorhabditis elegans (C.elegans). Asna1 has been proposed to act as an evolutionary conserved regulator of insulin/insulin like factor signaling. In C.elegans, asna-1 has been shown to regulate growth in a non-cell autonomous and IGF-receptor dependent manner. Here we show that transgenic expression of ASNA1 in β-cells of mice leads to enhanced Aktactivity and β-cell hyperplasia (manuscript). ASNA1 transgenic mice develop, however, diabetes due to impaired insulin secretion. The expression of genes involved in secretion stimulus coupling and insulin exocytosis is perturbed in islets of these mice. These data suggest that activation of ASNA1, here mimicked by enhanced expression, positively influences β-cell mass but negatively affects insulin secretion.

As plantas se defendem do ataque de patógenos através de um sistema imune composto por duas fases. A primeira delas é mediada por receptores localizados na membrana celular ou intracelularmente, os quais são conhecidos como receptores de reconhecimento padrão (do inglês, pattern recognition receptors - PRR). Esses receptores reconhecem moléculas derivadas de microrganismos, as quais são conservadas evolutivamente e são chamadas de padrões moleculares associados a patógenos (do inglês, pathogen-associated molecular patterns - PAMPs). Esse reconhecimento dispara uma resposta de defesa conhecida como PTI (do inglês, PAMP-triggerd immunity - PTI). Alguns patógenos foram aptos a sintetizar moléculas capazes de suprimir a PTI e essas moléculas são denominadas de efetores. A resposta que ocorre devido à ação dos efetores é chamada de susceptibilidade disparada por efetores (do inglês, effector-triggered susceptibility - ETS). Entretanto, plantas resistentes podem reconhecer os efetores através de proteínas de resistência localizadas intracelularmente, ativando a imunidade disparada por efetores (do inglês, effector-triggeredimmunity - ETI). De modo geral, as respostas advindas da PTI e da ETI são similares, mas a segunda é ativada mais rapidamente e é mediada por um único gene de resistência R. Por essa razão, a ETI é conhecida como uma resposta à doença qualitativa e as plantas não desenvolvem sintomas, caracterizando a interação incompatível. Por outro lado, a PTI é mediada por diversos genes e as respostas de defesa são tardias, possibilitando a disseminação do patógeno pelas células da planta e a ocorrência da doença, o que caracteriza a interação compatível. Nas respostas de defesa, moléculas como o óxido nítrico, as poliaminas e o ácido salicílico participam do processo de sinalização. O sistema antioxidante da planta é ativado de modo a mitigar os efeitos das espécies reativas de oxigênio e o metabolismo da planta é alterado. Dessa maneira, o estudo das respostas de defesa contra patógenos, pode ser uma ferramenta útil para estabelecer controles efetivos para as doenças de plantas / Plants defend themselves from pathogen attack through an active immunity system composed by two phases. The first is mediated by cell surface and intracellular pattern recognition receptors (PRR), which recognizes conserved molecules derived from microbes known as pathogen-associated molecular patterns (PAMPs). This recognition triggers a defense response called PAMP-triggered immunity (PTI). Throughout evolution, pathogens were able to synthesize molecules capable of suppressing PTI. These molecules are named effectors and they are responsible for effector-triggered susceptibility (ETS). However, resistant plants can recognize effectors by intracellular resistance (R) proteins, initiating effector-triggered immunity (ETI). In general, responses derived from PTI and ETI are the same, but the latter is activated faster and is mediated by a single R gene. For this reason, ETI-response is also known as qualitative disease response (QDR) and plants do not develop disease symptoms, characterizing the incompatible interaction. On the other hand, PTI is mediated by several genes and the defense response is delayed, enabling the pathogen to spread out and to cause disease. This interaction is known as compatible. In defense responses, molecules like nitric oxide, polyamines and salicylic acid can participate in signaling process. The antioxidant system can be activated to quench the ROS effects and the plant metabolism is altered. In this sense, studying defense responses against pathogens can help to develop tools to establish effective control methods for plant disease

Yra žinoma, jog nervinės ląstelės gali regeneruoti savo aksonus po periferinės, o tam tikrais atvejais ir po centrinės nervų sistemos pažeidimų. Tačiau aksonų augimas yra sudėtingas, o jo reguliacija turi kritinę įtaką tiek neuronų vystymęsi, tiek regeneracijoje. Vekiami aplinkinių ląstelių, išskiriamų pritraukiančiųjų ir atstumiančiųjų molekulių, aksonai augdami nuolat keičia augimo kryptis iki kol pasiekia galutinius taikinius. Dorsalinių ragų ganglijų (DRG) aksonų augimas priklauso nuo semaforinų klasės molekulių. Sekretuojantys, ar su membrana surišti semaforinai dalyvauja įvairiuose biologiniuose procesuose, tokiuose, kaip centrinės ir periferinės nervų sistemos (CNS ir PNS) vystymęsi ir regeneracijoje, širdies ir kraujagyslių vystymęsi ir imuninės sistemos funkcijose. DRG aksonų vystymasis ir išlikimas smarkiai priklauso nuo nervų augimo faktoriaus (NGF). Darbo tikslas buvo įvertinti NGF koncentracijos įtaką DRG aksonų augimo atsakams į semaforiną 3A. 15 parų pelių embrionų DRG buvo preparuojami iš C57/Bl linijos pelių embrionų. DRG neuronų auginimui naudoti sterilūs dengiamieji stikleliai buvo padengiami poli-L-lizino 0,01 mg/ml ir laminino 0,01 mg/ml tirpalu, pagamintu GBSS terpėje. Aksonų augimo kūgelių vertinimas buvo atliekamas praėjus 60 minučių, o aksonų ilgio vertinimas - praėjus 16 valandų po DRG pasodinimo. Tam, kad nustayti DRG apoptozės lygį, DRG neuronuose priklausomai nuo NGF koncentracijos buvo įvertinta Bcl-2, Bax, c-jun genų raiška, naudojant RT - PGR... [toliau žr. visą tekstą] / It is known that nerve cells can regenerate their axons after damage to peripheral and in some cases central nervous system (PNS and CNS). However, axon growth over longer distances, especially in central nervous system, is complicated. Regulation of axon growth is a critical event both in neuronal development and regeneration. To reach their proper targets, axons rely upon the actions of attractive and repulsive guidance molecules. It is known that growth of dorsal root ganglion (DRG) axons depend on guidance molecules of semaphorin class. Secreted and membrane bound semaphorins participate in diverse biological processes, including development and regeneration of central and peripheral nervous system, cardiovascular development, and immune system functioning. In addition to regulation of DRG axon growth by semaphorin class molecules, DRG axon growth and survival is strongly dependent on nerve growth factor (NGF). The aim of this study was to evaluate responses of DRG axons to semaphorin 3A in dependence of NGF concentration. DRG were dissected from C57/Bl mice E15 embryos in dissection HBSS/glucose medium. DRG were plated on cover slips coated with laminin and poly-L-lysine and grown in Neurobasal medium supplemented with 2% of B27 supplement. To evaluate collapse rate the morphology of axons growth cones were evaluated after 60 minutes and axons length were evaluated 16 hours after DRG plating. To evaluate DRG survival and level of apoptosis in dependence of NGF... [to full text]