Orientador: Marcelo Menossi Teixeira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T15:57:52Z (GMT). No. of bitstreams: 1 Tavares_RafaelGarcia_M.pdf: 4032767 bytes, checksum: ffe11e896bea6da2737a9d3ce4dd94ff (MD5) Previous issue date: 2011 / Resumo: A cultura da cana-de-açúcar (Saccharum spp) está entre as mais importantes do Brasil, tendo o setor sucroalcooleiro grande importância econômica no país devido ao crescente consumo de álcool e açúcar. A fase de maturação no desenvolvimento da cana-de-açúcar é especialmente interessante, pois está diretamente relacionada com o teor de sacarose. O hormônio etileno está envolvido em muitos aspectos do ciclo de vida da planta, incluindo a maturação. Como primeira parte deste projeto, o objetivo foi avaliar a relação entre etileno e maturação de cana-de-açúcar pela caracterização da expressão dos genes que codificam as isoformas da enzima ACC sintase, responsável por uma das vias da biossíntese desse hormônio. Assim, dois genes (Scacs1 e Scacs2) que codificam para a enzima ACC sintase foram identificados no projeto SUCEST e caracterizados. Nossos resultados de localização subcelular confirmaram estudos anteriores sobre a localização citoplasmática das proteínas ACS. Todavia, nós identificamos por análise in silico a presença de um peptídeo sinal na região N-terminal da proteína ScACS2. Ensaios de duplo-híbrido com bibliotecas de cDNAs a partir de tecidos de folha +1 e entrenó 5, nos revelaram novas interações putativas com a proteína ScACS2, sendo uma delas, a proteína denominada sinaptotagmina envolvida no mecanismo de reparo da membrana celular. Ao contrário do que foi observado em Arabidopsis, nossos resultados de heterodimerização não identificaram uma interação entre as isoformas de cana-de-açúcar. Análise de expressão por PCR quantitativo em duas variedades contrastantes ao teor de sacarose, identificaram uma maior expressão nos entrenós madurados quando comparados aos entrenós jovens para os dois genes ACS. Em uma estudo in situ da expressão, o gene Scacs1 apresentou uma expressão nas células do parenquima e nas células da bainha do feixe vascular, entretanto, não mostrou expressão nos feixes vasculares. Por outro lado, o gene Scacs2 apresentou expressão constitutiva nos diferentes tipos celulares e, inclusive, nos feixes vasculares. Os resultados apresentados nesse trabalho, abrem novas perspectivas e perguntas para os diversos mecanismos fisiológicos e celulares realizados pelo hormônio etileno nas plantas. Todavia, uma prática comum antes da colheita da cana é a aplicação de precursores do hormônio etileno, como o etefon (ácido 2-cloroetil fosfônico) para acelerar o amadurecimento final das plantas, inibir o florescimento e consequentemente aumentar o teor de sacarose. Assim, a manipulação da biossíntese desse hormônio tem o potencial para aumentar a produtividade em cana. Desta forma, um segundo objetivo deste projeto foi desenvolver uma estratégia para promover o amadurecimento controlado da cana-de-açúcar no final da safra, usando o etanol em substituição aos análogos químicos do etileno. Para tal, foi avaliado o uso de um promotor ativado por etanol (sistema de expressão alc de Aspergillus nidulans) controlando a expressão do gene que codifica a ACC sintase, enzima chave na biossíntese de etileno. Nossos resultados foram condizentes com o esperado, mostrando claramente um aumento na produção do hormônio etileno quando plantas transgênicas de tabaco foram induzidas por spray foliar com 5% (v/v) de etanol. Esses resultados demonstram a possível utilização de ferramentas biotecnológicas no controle da expressão de genes envolvidos em características agrícolas interessantes, tais como na cultura de cana-de-açúcar. / Abstract: Sugarcane crop (Saccharum spp) is among the most important of Brazil, due to its economic importance in the country with the increasingly consume of its by-products such as the ethanol and sugar. Throughout development of the sugarcane, the maturation phase is especially interesting, whereas it is highly related to sucrose accumulate. The ethylene hormone is involved in several physiological processes of plants, including the maturation. As first part of this project, the goal was evaluated the correlation between ethylene hormone and sugarcane maturation through characterization of the genes expression encoded ACS enzymes, involved in biosynthesis pathway of this hormone. Thus, two genes (Scacs1 and Scacs2), which encoding the ACS enzyme, were identified in SUCEST project and characterized. Our results of subcellular localization confirmed previous studies regarding to cytoplasmic localization of ACS proteins. Nonetheless, we identify through in silico analyses the presence of a signal peptide in the N-terminal region of the ScACS2 protein. Yeast two-hybrid assay using cDNAs libraries from leaf +1 and internode 5 tissues, revealed putative interactions with the ScACS2 protein, being one of them, a protein called plant synaptotagmin involved in the mechanism of cell membrane repair. Different from Arabidopsis, our results about heterodimerization did not identify an interaction between sugarcane ACS isoenzymes. Expression analyses of quantitative PCR in two sugarcane varieties contrasting to Brix content presented a higher expression of ACS genes in mature internodes than young internodes. In situ analyses for Scacs1 gene showed an expression in parenchyma cells and bundles sheath, however, none expression was observed in vascular bundles. On the other hand, the Scacs2 gene presented an ubiquitous expression in the different cell types and, including in vascular bundles. Our results presented in this work, open further perspectives and issues about the underlying mechanisms by ethylene hormone in plants. Nevertheless, a common practice before harvest of sugarcane is the application of precursors of ethylene hormone, such as ethephon (2-chloroethyl- dioxido-oxophosphorane) to accelerate plants ripening, delay opening of the inflorescence and to increase sucrose accumulate. As a result, the manipulation of biosynthetic pathway of this hormone is a potential tool to increase the productivity in sugarcane. Thus, other goal of this work was develop a strategy to promote a controlled ripening of sugarcane at the end of harvest, using the inductor ethanol instead of ethylene precursor. To achieve this goal, an ethanol-inducible system based on alc switch of the ascomycete fungus Aspergillus nidulans were evaluated controlling gene expression of ACS and consequently the production of the ethylene hormone. Our results were consistent with the expected, showing clearly an increase in the production of ethylene hormone when transgenic tobacco plants were induced for the leaf spray with 5% (v/v) ethanol. This result shows the possible utilization of biotechnology tools in the control of gene expression involved in important agricultural traits, such as in the sugarcane culture. / Mestrado / Genetica Vegetal e Melhoramento / Mestre em Genética e Biologia Molecular

L’ACC Oxydase est une enzyme à Fe(II) non-hémique impliquée dans la biosynthèse de l’éthylène chez les plantes. Notre compréhension du mécanisme ainsi le rôle des différents cofacteurs nécessite l’obtention des données structurales. Une structure cristallographique a été publiée montrant la partie C-terminale (C-term) éloignée du site actif. Ce n’est pas la conformation active car la partie C-term est essentielle à l’activité. Un modèle structural a été construit dans lequel la partie C-term est tournée vers le site actif. Différentes conformations semblent donc possibles. Le marquage de spin couplé à la spectroscopie RPE est une technique puissante pour sonder la dynamique structurale des protéines. Elle implique la liaison de nitroxydes sur des cystéines. Il est possible d’analyser la mobilité des sondes pour obtenir des informations sur leur environnement local. Par l’utilisation de techniques de RPE avancées, des mesures de distances entre deux sondes sont possibles. Des mutants portant une ou deux cystéines ont été conçus. La dynamique des mutants marqués a été étudiée in vitro par RPE. Par RPE impulsionnelle, des distances ont été mesurées pour l’ACCO en présence de différentes combinaisons de cofacteurs. Les distances expérimentales ont été comparées à celles prédites à partir des structures cristallographiques et du modèle structural et aussi à celles obtenues par des calculs de dynamique moléculaire. Pour cibler d’autres positions sur l’ACCO, l’introduction d’un acide aminé non naturel a été réalisée avec succès permettant d’obtenir de premières données structurales. Des données structurales préliminaires par RPE in cell sont également présentées / ACC Oxidase is a nonheme iron(II) containing enzyme involved in the biosynthesis of ethylene in plants. ACCO reaction mechanism and the role of the various cofactors are not well understood and structural and dynamic data are still required. A crystallographic structure has been reported showing the C-terminal part (C-term) away from the active site. This is not the active conformation as it has been shown that the C-term is essential. Later, a structural model has been proposed in which the C-term is folded towards the active site. Different conformations can be hypothesized. A technique well suited to monitor protein dynamics is site-directed spin labeling followed by EPR spectroscopy. It relies on the insertion of a nitroxide derivative on cysteines. Using this approach, it is possible to analyze the mobility of the label in order to obtain information on its local environment. Moreover using advanced EPR techniques, it is possible to acquire interspin distances between two incorporated probes. Mutants bearing one or two cysteines at desirable positions were designed. The dynamics of labeled mutants were studied in vitro using continuous wave EPR. By pulsed EPR, distances were recorded for ACCO in presence of different combinations of cofactors. The experimental distances were compared to the predicted ones obtained from the crystallographic and model structures, and also to the calculated ones obtained by molecular dynamic simulations. A successful introduction of an unnatural amino acid onto the sequence of ACCO was performed, allowing to obtain earliest results. The achievement of preliminary structural data by in cell EPR are also presented

Indiana University-Purdue University Indianapolis (IUPUI) / In automotive industry, driver assistance and active safety features are main areas of research. This thesis concentrates on designing one of the famous ADAS system feature called Adaptive cruise control. Feature development and analysis of various functionalities involved in the system control are done using Petri Nets. A background on the past and current ACC research is noted and taken as motivation. The idea is to implement the adaptive cruise control system in Petri net and analyze how to provide fault tolerance to the system. The system can be evaluated for various cases. The ACC technology implemented in di erent cars were compared and discussed. The interaction of the ACC module with other modules in the car is explained. The cruise system's algorithm in Petri net is used as the basis for developing Adaptive Cruise Control system's algorithm. The ACC system model is designed using Petri nets and various Petri net functionalities like place invariant, transition invariant and reachability tree of the model are analyzed. The results are veri ed using Matlab. Controllers are introduced for ideal cases and are implemented in Petri nets. Then the error cases are considered and fault tolerance techniques are carried out on the model to identify the fault places.

The anterior cingulate cortex (ACC) can influence emotional and motivational states in primates by its dense connections with many neocortical and subcortical regions. Pyramidal neurons serve as the basic building blocks of these neocortical circuits, which have been extensively studied in other brain regions, but their morphological and electrophysiological properties in the primate ACC are not well understood. In this study, we used whole-cell patch clamp and high-resolution laser scanning confocal microscopy to reveal the general electrophysiological properties and detailed morphological features of layer 2 and 3 pyramidal neurons in ACC (area 24/32) of the rhesus monkey. Neurons from both layers had similar passive membrane properties and action potential properties. Morphologically, dendrites of layer 3 ACC neurons were more complex than those of layer 2 neurons, by having dendrites with longer total dendritic lengths, more branch points and dendritic segments, spanning larger convex hull volumes. This difference in total dendritic morphology was mainly due to the apical dendrites. In contrast, the basal dendrites displayed mostly similar features between the two groups of neurons. However, while apical dendrites extend to the same layer (layer 1), the basal dendrites of layer 3 extended into deeper layers than layer 2 because of the difference in soma-pia distance. Thus, basal dendrites of the two groups of neurons receive different laminar inputs. Analysis of spines showed that more spines were found in neurons of layer 3 apical dendritic arbors than layer 2 neurons. However, the apical spine densities were similar between neurons in the two layers. Thus, while higher spine number suggests that layer 3 neurons receive more excitatory input than layer 2 neurons, the similar spine density suggests similar spatial and temporal summation of these inputs. The combined effects of increased number of excitatory input and higher dendritic complexity in layer 3 than in layer 2 ACC neurons suggest the additional information received by layer 3 neurons, especially in the apical dendrites, might undergo more complex integration.

<p>The synthesis and breakdown of sugars is one of the most important functions in Nature. Consequently, sugar structures are used both as energy storage and as building blocks to stabilise and protect the cell. The formation of these structures is performed by glycosyltransferases (GT), an enzyme group structurally conserved within all kingdoms. Until now, only two different folds have been discovered by crystallisation of GTs, i.e. GT-A and GT-B. A third fold family has however been proposed by fold predictions. In this thesis, a multivariate data analysis was successfully used in classifying and predicting both fold and reaction mechanism (inverting or retaining) of GTs. This method was also used to obtain information about the separating parameters for the reaction mechanism classification. This information could be traced back to the amino acid sequence. The method could as well be used to analyse and identify the properties of membrane binding regions of GTs, and subsequently distinguish soluble from membrane-associated enzymes. Most functionally characterised enzymes only use one substrate, synthesising one product. <i>Mycoplasma pneumoniae</i>, a common human pathogen with a small genome has only three proposed GTs. The bacterium was, however expected to have a greater number of GTs, due to its ability to make both glycolipids and capsule. Here we have determined the function of one of these enzymes, MPN483 and discovered its ability to both use different acceptors, and make elongated glycolipids with up to three galactose residues, with both DAG and ceramide as the base. Many of the synthesised glycolipids were also found to be immunogenic, hence showing their biological importance. The properties of lipids are known to be important for the function of a biological membrane. We have here shown that not only the charge but also the shape of the lipids are important for several protein mediated membrane processes in <i>Echerichia coli</i>, such as the function of the LacY.</p>

<p><em>Doktrin för luftoperationer</em> fastställdes som styrande dokument för alla luftoperationer 2005. Har då doktrinen blivit det styrande dokument i Flygvapnet som den är tänkt att vara?</p><p>I denna uppsats avser jag undersöka till vilken grad <em>Doktrin för luftoperationer</em> är implementerad i flygvapnets ledning och om doktrinen används som styrande dokument i utformningen av ordrar, direktiv och TOEM.</p><p>Jag har genom en kvalitativ textanalys granskat två centrala dokument ur olika perspektiv, för att utröna <em>till vilken grad</em> doktrinen används som styrande dokument. Jag har även intervjuat utvalda delar i flygvapnets ledning för att få ledningens uppfattningar av graden av implementering. Jag har i uppsatsen använt mig av en tregradig skala: <em>Inte alls</em>, <em>Till del</em> samt <em>Fullständigt</em> implementerad.</p><p>Genom dessa analyser har jag kunnat dra slutsatsen att <em>Doktrin för luftoperationer</em> endast <em>till del</em> är implementerad i flygvapnets ledning.</p> / <p>The Swedish doctrine for air operations was released and became effective in 2005. The doctrine was supposed to be a ruling document but is that the fact?</p><p>The aim of this study is to investigate at what level the doctrine has been implemented in the leadership of the Swedish Air Force and if the doctrine is in use as a ruling document when writing orders, directive and tactical, organisational, economical objectives (TOEM) for the sub-units.</p><p>The scientific method of this investigation is a qualitative text analysis where I analysed two vital documents in order to measure the level of implementation. For that purpose I have used a three grade scale: <em>not</em> implemented, <em>partly</em> and finally <em>fully</em> implemented.</p><p>The conclusion I draw after examining the subject is that the Swedish doctrine for air operations is only <em>partly</em> implemented in the Air Force.</p>

The synthesis and breakdown of sugars is one of the most important functions in Nature. Consequently, sugar structures are used both as energy storage and as building blocks to stabilise and protect the cell. The formation of these structures is performed by glycosyltransferases (GT), an enzyme group structurally conserved within all kingdoms. Until now, only two different folds have been discovered by crystallisation of GTs, i.e. GT-A and GT-B. A third fold family has however been proposed by fold predictions. In this thesis, a multivariate data analysis was successfully used in classifying and predicting both fold and reaction mechanism (inverting or retaining) of GTs. This method was also used to obtain information about the separating parameters for the reaction mechanism classification. This information could be traced back to the amino acid sequence. The method could as well be used to analyse and identify the properties of membrane binding regions of GTs, and subsequently distinguish soluble from membrane-associated enzymes. Most functionally characterised enzymes only use one substrate, synthesising one product. Mycoplasma pneumoniae, a common human pathogen with a small genome has only three proposed GTs. The bacterium was, however expected to have a greater number of GTs, due to its ability to make both glycolipids and capsule. Here we have determined the function of one of these enzymes, MPN483 and discovered its ability to both use different acceptors, and make elongated glycolipids with up to three galactose residues, with both DAG and ceramide as the base. Many of the synthesised glycolipids were also found to be immunogenic, hence showing their biological importance. The properties of lipids are known to be important for the function of a biological membrane. We have here shown that not only the charge but also the shape of the lipids are important for several protein mediated membrane processes in Echerichia coli, such as the function of the LacY.

<p>The direct addition of enolizable aldehydes and sulfonyl imines to &alpha;-halo thioesters to produce &beta;-hydroxy/amino thioesters enabled by reductive soft enolization is reported. The transformation is operationally simple and efficient and has the unusual feature of giving high <italic>syn</italic>-selectivity, which is the opposite of that produced for the aldol addition with (thio)esters under conventional conditions. This method is tolerant to aldehydes and imines that not only contain acidic &alpha;-protons, but also towards electrophiles containing other acidic protons and base-sensitive functional groups. Moreover, excellent diastereoselectivity is achieved when a chiral non-racemic &alpha;-hydroxy aldehyde derivative is used. Using MgI₂ and Ph₃P, this method gives a wide range of aldol and Mannich products in good yields with high <italic>syn</italic>-diastereoselectivity. The products obtained from the reductive aldol and Mannich reactions are synthetically important intermediates in both polyketide and &beta;-lactam synthesis, respectively, and can be readily derivatized to form many carbonyl derivatives through known manipulation of the thioester moiety.</p><p><p>Also, herein the asymmetric synthesis of (+)-mefloquine, a potent anti-malarial compound, is described. The synthesis is based on a key enantioselective Darzens reaction between a chiral &alpha;-chloro-N-amino cyclic carbamate (ACC) hydrazone and a quinoline-based aldehyde. This is a novel methodology developed by our lab, which gives a highly enantioenriched epoxide that can be further functionalized to give both enantiomers of mefloquine.</p> / Dissertation

Doktrin för luftoperationer fastställdes som styrande dokument för alla luftoperationer 2005. Har då doktrinen blivit det styrande dokument i Flygvapnet som den är tänkt att vara? I denna uppsats avser jag undersöka till vilken grad Doktrin för luftoperationer är implementerad i flygvapnets ledning och om doktrinen används som styrande dokument i utformningen av ordrar, direktiv och TOEM. Jag har genom en kvalitativ textanalys granskat två centrala dokument ur olika perspektiv, för att utröna till vilken grad doktrinen används som styrande dokument. Jag har även intervjuat utvalda delar i flygvapnets ledning för att få ledningens uppfattningar av graden av implementering. Jag har i uppsatsen använt mig av en tregradig skala: Inte alls, Till del samt Fullständigt implementerad. Genom dessa analyser har jag kunnat dra slutsatsen att Doktrin för luftoperationer endast till del är implementerad i flygvapnets ledning. / The Swedish doctrine for air operations was released and became effective in 2005. The doctrine was supposed to be a ruling document but is that the fact? The aim of this study is to investigate at what level the doctrine has been implemented in the leadership of the Swedish Air Force and if the doctrine is in use as a ruling document when writing orders, directive and tactical, organisational, economical objectives (TOEM) for the sub-units. The scientific method of this investigation is a qualitative text analysis where I analysed two vital documents in order to measure the level of implementation. For that purpose I have used a three grade scale: not implemented, partly and finally fully implemented. The conclusion I draw after examining the subject is that the Swedish doctrine for air operations is only partly implemented in the Air Force.

Emotion processing involves specialised brain regions allowing for effective evaluation of the social environment and for the acquisition of social skills that emerge over childhood. In humans, an important aspect of normal development is the ability to understand the facial expressions of others that signal the nature and safety of the environment. Existing functional data, however, have not characterised the developmental trajectories associated with the differing neural and cognitive-behavioural development. The current thesis investigates the functional specialisation and development of the spatial and temporal patterns in neural activities during implicit processing of facial emotions from early childhood through adulthood. The first study identified brain regions engaged in implicit processing of emotional expressions using a simple emotion-processing paradigm (target detection task) with fourteen healthy adults using magnetoencephalography (MEG) recordings. Participants responded to a non-face target (a scrambled pattern) while ignoring the emotional face presented in a different hemifield. Results showed ACC and right-lateralised amygdala activations in early latencies in response to the unattended emotional faces related to rapid and implicit attention to the task-irrelevant facial emotions, specifically during the processing of the fearful emotion. Based on the findings in the first study, the second study investigated the developmental patterns and age-related differences in brain activities associated with the rapid and automatic processing of the emotional expressions in MEG with twelve children 7 – 10 years old, twelve adolescents 12 – 15 years old and twelve young adults (mean age 24.4 years) using the same paradigm. The results showed that emotion processing developed early in childhood in the amygdalae, whereas the processing of fear had later maturation engaging the ACC. The results further demonstrated an age-correlated increase in development in ACC activity and an age-related laterality shift in the amygdalae related to fear processing. The present thesis provides new evidence contributing to the understanding of the protracted but differing normal development in the emotional brain over the childhood into adulthood, and offers critical insights into understanding possible dysfunctions of these brain regions during development.