Submitted by João Arthur Martins (joao.arthur@ufpe.br) on 2015-03-10T19:07:24Z No. of bitstreams: 2 Dissertaçao Hesdras Viana.pdf: 2998238 bytes, checksum: de42b675472ac4632a3a3c04688a77d5 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Approved for entry into archive by Daniella Sodre (daniella.sodre@ufpe.br) on 2015-03-10T19:43:06Z (GMT) No. of bitstreams: 2 Dissertaçao Hesdras Viana.pdf: 2998238 bytes, checksum: de42b675472ac4632a3a3c04688a77d5 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-10T19:43:06Z (GMT). No. of bitstreams: 2 Dissertaçao Hesdras Viana.pdf: 2998238 bytes, checksum: de42b675472ac4632a3a3c04688a77d5 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013-02-26 / Extrair características da fala é uma etapa fundamental para os sistemas de reconhecimento de voz. É através dos descritores que extraímos a energia do sinal, a frequência fundamental (pitch) e a estrutura dos formantes que serão utilizados como identificadores para cada palavra pronunciada. Descritores como MFCC (Mel-Frequency Cepstral Coefficient), RASTA-PLP (RelAtive SpecTrAl - Perceptual Linear Predictive) e PNCC (Power Normalized Cepstral Coefficient) são muitos utilizados no estado da arte na área de reconhecimento de voz, porém, essas técnicas não conseguem apresentar bons resultados quando expostos a amostras com presença de ruído, variabilidade de locutor e fala contínua. O objetivo deste trabalho é desenvolver um descritor para a fala que seja invariante ao ruído, ambiente e locução. Para isso, fizemos um estudo dos descritores de voz mais utilizados na literatura, identificando as vantagens e desvantagens, expondo a situações variadas. Para avaliação das técnicas, utilizamos a base NOIZEUS (Noisy Speech Corpus) e dois classificadores: HMM (Hidden Markov Models) e SVM (Support Vector Machine). Essa base tem como característica a presença de ruído variando de 0dB, 5dB, 10dB e 15dB, gravada em diversos ambientes. A utilização dos classificadores serviu para validar os descritores de voz. O descritor proposto, chamado de MINERS (Model Invariant to Noise and Environment and Robust for Speech), apresentou melhores resultados entre todos os descritores avaliados (MFCC, MFCC combinado com Wavelet Denoising, RASTAPLP e PNCC). A abordagem que obteve maior sucesso foi a utilização do MINERS com o classificador SVM.

No description available.

This work concerns new borders, heuristics, algoritms and mathematic models of pallet loading problem (PLP). We try to describe these computational methods and find out if we can use them in real. We maximalize number of boxes placed on rectangular pallets in a particular warehouse by using chosen heuristics. Every box has a rectangular form with the same lenght and width and is fully placed on the pallet. We can rotate with the box by 90% degree until it is fixed as we want and its side lies parallelly with side of the pallet. All instances are setted in model (X, Y, a, b), where X is lenght, Y width of the pallet, a lenght and b width of the box.

Orientadores: Áureo Tatsumi Yamada, Michael Joseph Soares / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T04:12:34Z (GMT). No. of bitstreams: 1 Lippe_ElianaMaraOliveira_D.pdf: 1508385 bytes, checksum: f80492236b91aa11f6167dbfd5369199 (MD5) Previous issue date: 2011 / Resumo: Um processo de remodelação progressiva da vascularização uterina precede a placentação em humanos e roedores para prover o suprimento sanguíneo adequado na interface materno-fetal, tendo as células NK um papel modulador através da produção de citocinas como o IFN'gama', fatores de crescimento como o VEGF e radicais livres como o óxido nítrico (NO). A regulação das células uNK nesta atividade tem sido atribuída à influência de fatores exógenos como a hipóxia e parácrinos como a prolactin-like protein-A (PLP-A). Contudo, como estes mecanismos são integrados no controle da angiogênese e vasculogênese da interface materno-fetal envolvendo as células uNK não são plenamente compreendidos. No presente trabalho, foram investigadas experimentalmente a resposta das células uNK relacionadas com os mecanismos de regulação dos fatores angiogênicos sob influência da hipóxia no período pré-placentário. Para tanto, foram avaliados camundongos CD1 e geneticamente modificados IL15-/-, PLPA-/- e IL15-/-/PLPA-/- gestantes no 8° dia de gestação (dg), mantidos em hipóxia (42 0Torr - 11%O2) durante 48 horas. Em comparação com os animais mantidos em normóxia (760Torr - 21%O2) a quantidade de sítios anormais apresentou incremento estatisticamente significativo sob hipóxia nos animais CD1, porém este índice era substancialmente maior nos animais depletados dos genes IL15 e PLPA. As amostras dos sítios uterinos de desenvolvimento embrionários coletados foram processados para obtenção de criocortes destinados às reações citoquímicas, imunocitoquímicas e hibridização in situ, e homogeneizados teciduais para extração do RNAm ou proteínas. Conforme esperado, os resultados da citoquímica com lectina DBA e imunocitoquímica de perforina comprovam a ausência de células uNK nos animais IL15-/- e IL15-/-/PLPA-/-, enquanto nos animais PLPA-/-, a incidência das células uNK perforina positivas não difere dos CD1 em normóxia ou hipóxia. A concentração protéica de VEGF e dos genes das isoformas VEGFA, VEGFB e VEGFC, assim como do IFN? e das isoformas iNOS e eNOS, o do TNF? e seus receptores TNFR1 e TNFR2 não apresentaram variações em suas concentrações ou níveis de expressões com padrões definidas de regulação negativa ou positiva entre os animais avaliados. Contudo a imunocitoquímica demonstrou redução de marcação de células endoteliais endoglinpositivas no endométrio e aumento no potencial invasivo de células trofoblásticas TROMA-I positivas dos animais IL15-/-. O conjunto destes resultados confirma que a ausência das células uNK afeta a vascularização normal do endométrio, a qual pode resultar em perdas gestacionais e induz a hipertrofia placentária, sem a participação direta da atividade citotóxica destas células. Comprovam também que os mecanismos de controle da expressão de fatores angiogênicos no útero gestante são multifatoriais, não sendo dependente de uma via única como o da PLPA/VEGF, ou exclusivamente das células uNK como fontes de fatores que modulam a angiogênese na interface maternofetal do útero gestante / Abstract: A gradual process of remodeling of uterine vasculature precedes placentation in humans and rodents to provide adequate blood supply in maternal-fetal interface, where NK cells producing cytokines like growth factor (VEGF) and IFN'gama' and free radical as nitric oxide (NO). Regulation of this uNK cells activity seems to be under influence of exogenous and endogenous factors such as hypoxia as paracrine effects of prolactin-like protein-A (PLPA). Nevertheless, how these mechanisms are integrated in the control of maternal-fetal interface angiogenesis and vasculogenesis involving uNK cells are not fully understood. In this study, we investigated experimentally the uNK cells response related to the mechanisms of regulation of angiogenic factor under hypoxia influence in pre-placental period. Thus, we evaluated genetically modified mice and CD1-IL15-/-, PLPA-/- and IL15-/- /PLPA-/-pregnant on the 8th day of gestation (dg), maintained in hypoxia (420Torr-11%O2) for 48h. When compared to the control animals in normoxia (760Torr-21%O2) the amount of abnormal embryo developing sites showed statistically significant increase under hypoxia in CD1 animals, but this rate was substantially higher in the PLPA and IL15 gene depleted animals. Uterine samples were processed to obtain cryosection for cytochemical, immunocytochemical and in situ hybridization reactions and extraction of mRNA or protein for PCR or ELISA reactions, respectively. As expected, the results of cytochemistry and immunocitochemistry with DBA lectin and perforin prove the absence of uNK cells in IL15-/- and IL15-/-/PLPA-/-animals, while in PLPA-/-animals, the incidence of uNK cells perforinpositive did not differ from CD1 animlas in normoxia or hypoxia. The concentration of protein VEGF and the gene isoforms expression of VEGF (A, B and C), as well as, IFN?, iNOS and eNOS, TNF? and their receptors TNFR1 and TNFR2 did not show constancy in the pattern of variations. However, the immunocytochemistry showed reduced staining of endoglin-positive endothelial cells in the endometrium and increase in invasive potential of trophoblast cells by TROMA-I positive in IL15-/- animals. This set of results confirms that the absence of uNK cells affects the normal vasculature of the endometrium which may increased intrauterine growth restriction (IUGR) or pregnancy failure rates and induces placental hypertrophy. This abnormality at the maternal-fetal interface does not seems to be involves direct participation of cytotoxic activity of uNK cells. Furthemore, the control mechanisms of angiogenic factors expression in the pregnant uterus are multifactorial rather than dependent of a single pathway like the PLP-A/VEGF, or limited to uNK cells as a source of factors that modulate angiogenesis in the maternal-fetal interface of pregnant uterus / Doutorado / Histologia / Doutor em Biologia Celular e Estrutural

The thesis describes structural and functional studies of two PLP-dependent enzymes, diaminopropionate (DAP) ammonia lyase (DAPAL) and N-acetylornithine aminotransferase (AcOAT). The main objective of this work was to understand the structural features that control and impart specificity for PLP-dependent catalysis. DAPAL is a prokaryotic enzyme that catalyzes the degradation of D and L forms of DAP to pyruvate and ammonia. The first crystal structure of DAPAL was determined from Escherichia coli (EcDAPAL) in holo and apo forms, and in complex with various ligands. The structure with a transient reaction intermediate (aminoacrylate-PLP azomethine) bound at the active site was obtained from crystals soaked with substrate, DL-DAP. Apo and holo structures revealed that the region around the active site undergoes transition from disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. Based on the crystal structures and biochemical studies, as well as studies on active site mutant enzymes, a two base mechanism of catalysis involving Asp120 and Lys77 is suggested. AcOAT is an enzyme of arginine biosynthesis pathway that catalyses the reversible conversion of N-acetylglutamate semialdehyde and glutamate to N-acetyl ornithine and α-ketoglutarate. It belongs to subgroup III of fold type I PLP dependent enzymes. Many clinically important aminotransferases belong to the same subgroup and share many structural similarities. We have carried out extensive comparative analysis of these enzymes to identify the unique features important for substrate specificity. Crystal structures of AcOAT from Salmonella typhimurium were determined in presence of two ligands, canaline and gabaculine, which are known to act as general inhibitors for most of the enzymes of this class. There structures provided important insights into the mode of binding of the substrates. The structures illustrated the switching of conformation of an active site glutamate side chain on binding of the two substrates. In addition to that, structural transitions involving three loop regions near the active site were observed in different ligand bound structures. Kinetics of single turnover fast reactions and multiple turnover steady state reactions indicated that N-AcOAT dimer might follow a mechanism involving sequential half site reactivity for efficient catalysis. The changes observed in loop conformation that resulted in asymmetric forms of the dimer enzyme might form the structural basis for half site reactivity. Single site mutants were designed to understand the significance of these structural transitions and the specific role of active site residues in determining substrate specificity and catalysis. Biochemical characterization of wild type and mutant enzymes by steady state and fast kinetic studies, along with their crystal structures provided detailed insights into subtlety of active site features that manifest substrate specificity and catalytic activity. The thesis also describes the investigations on fold type II enzymes directed towards analyses of polypeptide folds of these enzymes, features of their active sites, nature of interactions between the cofactor and the polypeptide, oligomeric structure, catalytic activities with various ligands, origin of specificity and plausible regulation of activity. Analysis of the available crystal structures of fold type II enzymes revealed five different classes. The dimeric interfaces found in these enzymes vary across the classes and probably have functional significance. Contributions made towards structural and functional studies of three other PLP-dependent enzymes, serine hydoxymethyltransferase (SHMT), D-serine deaminase (DSD) and D-cysteine desulfhydrase (DCyD) are described in an appendix.

A produção animal atualmente conta com diferentes biotecnologias para aumentar sua produtividade, incluindo técnicas aplicadas na reprodução. A inseminação artificial, por exemplo, vem sendo aplicada há muitos anos e de maneira extensiva. Esta tem como objetivo o aproveitamento de apenas um ejaculado em diversas prenhezes viabilizando a realização de testes de progênies e um grande aproveitamento de touros melhoradores. Atualmente essa técnica pode contar com um material ainda mais específico, o sêmen sexado que possui maior porcentagem (acima de 87%) de espermatozóides portadores de cromossomo X ou Y resultando numa progênie com o sexo predeterminado. A pré-seleção do sexo vem sendo estudada desde final dos anos 70, uma das linhas de pesquisa resultaram no desenvolvimento de um equipamento, o citômetro de fluxo, em que a célula espermática tem seu conteúdo de DNA estimado e, na seqüência é orientado para separação. Atualmente essa técnica é utilizada para produção de sêmen sexado comercialmente, anteriormente à comercialização do produto, a confirmação da taxa de separação dos cromossomos X e Y é realizada pelo próprio equipamento, o que acrescenta um custo operacional e pode introduzir um viés na confirmação da taxa de sexagem. Sendo assim o objetivo desse trabalho foi elaborar um protocolo de confirmação da taxa de sexagem mediante PCR em tempo real, utilizando sonda TaqMan® em regiões dos genes X-específico (PLP) e Y-específico (SRY). Foram analisadas duas sub-espécies de bovinos (Bos primigenius taurus e Bos primigenius indicus), sendo 21 doses de sêmen sexado X, 21 doses de sêmen sexado Y e 42 doses de sêmen não sexado (controle). A extração do DNA foi desenvolvida com a utilização de di-etiltreitol (DTT) e isotiocianato de guanidina. As amostras foram, em seguida, submetidas à técnica de PCR em tempo real aplicando sondas específicas. Foi possível amplificar os fragmentos específicos dos cromossomos X e Y separadamente e em conjunto e estimar a proporção relativa entre ambos. Todavia a repetibilidade do teste e sua acurácia não permitem recomendá-lo da maneira que se encontra para a aplicação na prática. / There are different biotechnologies to apply to increase the animal production nowadays, reproduction techniques are including. The artificial insemination (AI), for example, is a technique very used during many years ago and it is used at extensive production. The aim of this technique is to increase the numbers of pregnancies with just one ejaculation what will propose easier testing of offspring and better use of genetic selected bulls. At present the AI can associate a more specific material, the sexed semen, that contains a higher percentage (more than 87%) of X- or Y- bearing spermatozoa. Its application will result in a offspring with the desired sex. The sorting sex process has been studied since the 1970\'s, since then, some researchers have determined the cytometric sexing flow, a \"promising technique\" that allows the separation of the X-bearing chromosome sperm from the Y-bearing chromosome sperm based on the estimation of the DNA content in each measured cell. The cytometric flow is the unique commercial technique applied for mammalian species in many countries. Before the commercialization the bovine sexed semen has its percentage of the desired sex confirmed by using the same technique that sorted at the beginning of the process. This point can improve the cost and benefits the sorting technique. This way, the objective of this study was to determine a protocol of sex proportion confirmation by real time PCR using TaqMan® probe at X-specific gene (PLP) and Y-specific gene (SRY). Two subspecies of cattle semen were analyzed (Bos primigenius taurus and Bos primigenius indicus). Twenty-one sexed semen for female, other twenty-one sexed semen for male and forty-two non sexed semen were used in this research. The DNA extraction used DTT and guanidine isothiocyanate, and then the obtained samples were used to the real time PCR applying specific probes. This study allows the amplification in different reactions of specific products of X-chromosome bearing and Y-chromosome bearing. Putting the results together, it was possible to determine the relative proportion between the products. But the repeatability and accuracy of the test don\'t permit use this technique in this way of practice.

Pyridoxal 5’-phosphate (PLP), the active form of vitamin B6, is a cofactor for many enzymes involved in the metabolism of amino acids, amino acid derived metabolites and some amino sugars. PLP is one of the most versatile cofactors and the PLP-dependent enzymes catalyze a variety of reactions including transamination, decarboxylation, inter-conversion of L-and D-amino acids and removal or replacement of chemical groups bound at β or γ carbon of amino acids. The thesis describes the structural studies carried out on three PLP-dependent enzymes; N-acetylornithine aminotransferase (AcOAT), serine hydroxymethyltransferase (SHMT) and diaminopropionate ammonia lyase (DAPAL). Chapter 1 of the thesis begins with a brief introduction to PLP-dependent enzymes and their classification. This is followed by a review of structures of enzymes belonging to the subgroup II aminotransferases. The last section of chapter I contains a detailed description of the structures available till date for SHMT from various sources and the mutational studies carried out on SHMT. All the common experimental procedures and computational methods used for the current investigations are described in chapter II, as most of these are applicable to all structure determinations and analyses. The experimental procedures described include cloning, overexpression, purification, crystallization, and X-ray diffraction data collection. Computational methods include details of various programs used during data processing, structure determination, refinement, model building, structure validation and analysis. AcOAT is one of the key enzymes in arginine and lysine metabolism. AcOAT belongs to the fold type I (αfamily) subgroup II family of PLP dependent enzymes. Both S. typhimurium and E. coli have two genes each, one involved in the biosynthesis of arginine and another in the biodegradation of arginine. Biosynthetic AcOAT catalyzes the conversion of N-acetylglutamate semialdehyde to N-acetylornithine (AcOrn) in the presence of L-glutamate and the conversion of N-succinyl-L-2-amino-6-oxopimelate to N-succinyl-L,L-diaminopimelate in lysine biosynthesis. Meso-DAP and lysine, the products of lysine biosynthesis pathway, are known to function as cross-linking moieties in the peptidoglycan component of bacterial cell wall. Therefore N-acetylornithine aminotransferase could serve as a target for designing antibacterials. Chapter III gives the details of the work carried out on AcOAT. Two genes each from S. typhimurium and E. coli coding for biosynthetic and biodegradative AcOAT were cloned in E. coli, overexpressed and purified by Ni-NTA affinity chromatography. Of the four enzymes, biosynthetic AcOAT from S. typhimurium (sArgD) crystallized in the unliganded form and in the presence of the inhibitor gabaculine or one of the substrates L-glutamate, diffracted to a maximum resolution of 1.90 Å and contained a dimer in the asymmetric unit. The structure was determined by the molecular replacement method using human ornithine aminotransferase (hOAT) as the starting model. The structure of unliganded sAcOAT showed significant electron density for PLP in only one of the subunits (subunit A). The asymmetry in PLP binding could be attributed to the ordering of the loop Lαk-βm in only one subunit. The Km and kcat/Km values determined with the purified sArgD suggested that the enzyme could accept both acetylornithine (AcOrn) and ornithine (Orn) as the substrates and had much higher affinity for AcOrn than for Orn. However, OAT accepts only Orn as the substrate. Comparison of the structurte of sArgD with T. thermophilus AcOAT and hOAT suggested that the higher specificity of sArgD towards AcOrn may not be due to specific differences in the active site residues but could result from minor conformational changes in some of them. sArgD was inhibited by gabaculine with an inhibition constant (Ki) of 7 µM and a second order rate constant (k2) of 0.16 mM-1s-1. The crystal structure of sArgD obtained in the presence of gabaculine and the spectral studies of sArgD with gabaculine suggested that the enzyme might have a low affinity for the PLP-gabaculine complex. Biosynthetic AcOAT from E. coli (eArgD) crystallized in the presence of gabaculine in hanging drop vapor diffusion method and diffracted X-rays only to a resolution of 3.5 Å. Two data sets were collected for the eArgD crystals. One of the data sets belonged to P1 (data 1) and the other to P321 space group (data 2) with a solvent content of ~70%. Data 1 was twinned and the unit cell was unusually large and could accommodate ~24 molecules in the asymmetric unit where as data 2 had four molecules in the asymmetric unit. Biodegradataive AcOAT from E. coli also crystallized in presence of gabaculine in hanging drop vapor diffusion method and suffered from low diffraction quality, where as that from S. typhimurium did not yield crystals. In chapter IV, X-ray crystallographic studies on various site specific mutants of SHMT from Bacillus stereotherophilus (bs) and a detailed comparison of structural data with the biochemical results in relation to mechanism of catalysis are presented. SHMT is a member of the α-class of PLP-dependent enzymes and catalyzes the reversible conversion of L-Ser and THF to glycine and 5,10-methylene THF. 5,10-methylene THF serves as a major source of one-carbon units in the biosynthesis of nucleotides and a few amino acids. SHMT also catalyses the cleavage of β-hydroxy amino acids like L-allo-threonine, transamination, racemization and decarboxylation reactions. SHMT shows increased activity along with enhanced nucleotide synthesis and therefore is a potential target for cancer chemotherapy. The availability of structural and biochemical data on SHMT from different sources ranging from human to E. coli enabled the identification of active site residues and a more critical examination of the role of these residues in the different steps of catalysis. The important mutants studied in the present investigation are E53Q, Y51F, Y61F, Y61A, Y60A, N341A and F351G of bsSHMT. The crystal structures of all these mutants are solved in the presence of various ligands, which gave many interesting results. E53, one of the active residues, interacts with the side chain hydroxyl group of serine bound to PLP in the wild type serine complex and N10 and formyl oxygen in the wild type glycine-FTHF complex. In E53Q glycine and serine complexes, glycine carboxyl and serine side chain were in two conformations, respectively, the new conformation being stabilized by their interaction with the mutated residue Q53. The structure of E53Q-Gly complex obtained in the presence and absence of 5-formyl THF(FTHF) showed an interesting case of enzyme memory in which the final conformational state depends on the way it was obtained and suggested that E53 is crucial for FTHF/THF binding. Though the spectrum showed that FTHF binds to the mutant initially, no density was observed for FTHF in the final structure. FTHF is believed to dissociate from the active site with prolonged incubation leaving behind a few significant conformational changes. Y51, one of the highly conserved tyrosines in SHMT, has hydrogen bonding interactions with the phosphate group of PLP and the active site lysine (K226) in bsSHMT. Mutation of Y51 to F resulted in significant changes at the active site. In all the structures of Y51F complexes, the phosphate group is in two conformations and F51 has moved away from the phosphate and in turn changed the position of Y61, another tyrosine in the active site. The residue Y61 is hydrogen bonded to R357 in the internal aldimine complex of bsSHMT. Addition of glycine/serine to bsSHMT resulted in the conformational change of Y61 away from R357 and towards E53, allowing the added glycine/serine to interact with R357. Mutation of Y61 to A did not bring significant structural changes. Structures of Y51F and Y61A mutants complexed with L-allo-Thr (cleaved to Gly by the wild type enzyme) showed that L-allo-Thr was not cleaved to glycine and acetaldehyde and confirmed the biochemical observation that these two residues are essential even for the THF-independent reaction. Residues Y60 and N341 are also highly conserved residues among SHMTs. Y60 stacks over PABA ring of FTHF in the wild type glycine-FTHF ternary complex. N341 has strong hydrogen bonding interactions with N1 and N8 atoms of the pteridine ring of FTHF. Mutation of either Y60 or N341 to A destroys the binding ability of FTHF/THF to the enzyme according to the biochemical and structural observations. The residue F351 exhibits different conformations in the two subunits of wild type glycine-FTHF ternary complex and is thought to be an important residue in determining the asymmetric binding of FTHF. Mutation of F351 to G did not affect the catalytic activity. Surprisingly, in the crystal structure obtained in the presence of L-allo-Thr, the ligand did not get cleaved to glycine, though in solution, the mutant is as active as the wild type enzyme. Chapter V describes the preliminary structural studies carried out on DAPAL from E. coli and S. typhimurium. DAPAL catalyzes the α, βelimination of both L-and D-diaminopropionate (DAP). DAP is the immediate precursor of two neurotoxins 3oxalyl and 2,3-dioxalyl DAP present in Lathyrus sativus, a grain legume rich in proteins and capable of growing well in drought conditions. The presence of these two neurotoxins precludes its use as a source of protein rich food. This enzyme is present only in bacteria and few species of actinomycetes. Unlike many other PLP-dependent enzymes, DAPAL does not catalyze any side reaction and is the only enzyme known to remove an amino group from the βcarbon of the substrate. The enzymes from E. coli (eDAPAL) and S. typhimurium (sDAPAL) produced diffraction quality crystals. However, crystals of sDAPAL did not survive heavy atom soaking and eDAPAL crystals suffered from poor reproducibility and severe non-isomorphism making it difficult to obtain suitable heavy atom derivatives for structure determination. Production of selenomethionine labelled proteins for these enzymes was initiated and thin crystals were obtained for eDAPAL. Improvement of the quality of these crystals is necessary in order to solve the structure of DAPAL by MAD method.

A produção animal atualmente conta com diferentes biotecnologias para aumentar sua produtividade, incluindo técnicas aplicadas na reprodução. A inseminação artificial, por exemplo, vem sendo aplicada há muitos anos e de maneira extensiva. Esta tem como objetivo o aproveitamento de apenas um ejaculado em diversas prenhezes viabilizando a realização de testes de progênies e um grande aproveitamento de touros melhoradores. Atualmente essa técnica pode contar com um material ainda mais específico, o sêmen sexado que possui maior porcentagem (acima de 87%) de espermatozóides portadores de cromossomo X ou Y resultando numa progênie com o sexo predeterminado. A pré-seleção do sexo vem sendo estudada desde final dos anos 70, uma das linhas de pesquisa resultaram no desenvolvimento de um equipamento, o citômetro de fluxo, em que a célula espermática tem seu conteúdo de DNA estimado e, na seqüência é orientado para separação. Atualmente essa técnica é utilizada para produção de sêmen sexado comercialmente, anteriormente à comercialização do produto, a confirmação da taxa de separação dos cromossomos X e Y é realizada pelo próprio equipamento, o que acrescenta um custo operacional e pode introduzir um viés na confirmação da taxa de sexagem. Sendo assim o objetivo desse trabalho foi elaborar um protocolo de confirmação da taxa de sexagem mediante PCR em tempo real, utilizando sonda TaqMan® em regiões dos genes X-específico (PLP) e Y-específico (SRY). Foram analisadas duas sub-espécies de bovinos (Bos primigenius taurus e Bos primigenius indicus), sendo 21 doses de sêmen sexado X, 21 doses de sêmen sexado Y e 42 doses de sêmen não sexado (controle). A extração do DNA foi desenvolvida com a utilização de di-etiltreitol (DTT) e isotiocianato de guanidina. As amostras foram, em seguida, submetidas à técnica de PCR em tempo real aplicando sondas específicas. Foi possível amplificar os fragmentos específicos dos cromossomos X e Y separadamente e em conjunto e estimar a proporção relativa entre ambos. Todavia a repetibilidade do teste e sua acurácia não permitem recomendá-lo da maneira que se encontra para a aplicação na prática. / There are different biotechnologies to apply to increase the animal production nowadays, reproduction techniques are including. The artificial insemination (AI), for example, is a technique very used during many years ago and it is used at extensive production. The aim of this technique is to increase the numbers of pregnancies with just one ejaculation what will propose easier testing of offspring and better use of genetic selected bulls. At present the AI can associate a more specific material, the sexed semen, that contains a higher percentage (more than 87%) of X- or Y- bearing spermatozoa. Its application will result in a offspring with the desired sex. The sorting sex process has been studied since the 1970\'s, since then, some researchers have determined the cytometric sexing flow, a \"promising technique\" that allows the separation of the X-bearing chromosome sperm from the Y-bearing chromosome sperm based on the estimation of the DNA content in each measured cell. The cytometric flow is the unique commercial technique applied for mammalian species in many countries. Before the commercialization the bovine sexed semen has its percentage of the desired sex confirmed by using the same technique that sorted at the beginning of the process. This point can improve the cost and benefits the sorting technique. This way, the objective of this study was to determine a protocol of sex proportion confirmation by real time PCR using TaqMan® probe at X-specific gene (PLP) and Y-specific gene (SRY). Two subspecies of cattle semen were analyzed (Bos primigenius taurus and Bos primigenius indicus). Twenty-one sexed semen for female, other twenty-one sexed semen for male and forty-two non sexed semen were used in this research. The DNA extraction used DTT and guanidine isothiocyanate, and then the obtained samples were used to the real time PCR applying specific probes. This study allows the amplification in different reactions of specific products of X-chromosome bearing and Y-chromosome bearing. Putting the results together, it was possible to determine the relative proportion between the products. But the repeatability and accuracy of the test don\'t permit use this technique in this way of practice.

No description available.

Enzymatic fluorination of natural products is extremely rare. Of the 4000 halogenated natural products identified, only 13 possess a fluorine atom. The C-F bond forming enzyme from the soil bacterium, Streptomyces cattleya, remains the only native enzyme to be identified that is capable of such biochemistry. It generates 5’-fluoro-5-deoxyadenosine (5‘-FDA) from S-adenosyl-L-methionine (SAM) and F-. The “fluorinase” is the first committed step toward the biosynthesis of the two fluorometabolites, 4-fluorothreonine and fluoroacetate, via the common intermediate, fluoroacetaldehyde (FAld). The enzymatic steps responsible for the conversion of 5’-FDA to the fluorometabolites remained to be fully characterised when this project began. Previously, a purine nucleoside phosphorylase was identified that was capable of generating 5-fluorodeoxyribose-1-phosphate (5-FDRP) from 5’-FDA. 5-FDRP is subsequently isomerised to 5-fluorodeoxyribulose-1-phosphate (5-FDRulP) by an aldose-ketose isomerase enzyme. Chapter 2 describes the identification of the isomerase gene from the genomic DNA of S. cattleya and the corresponding protein product was capable of generating 5-FDRulP from 5-FDRP. The next intermediate, FAld, is generated from 5-FDRulP by a fuculose aldolase. Attempts to identify the aldolase gene from S. cattleya were unsuccessful, however a putative fuculose aldolase from Streptomyces coelicolor was isolated that could generate FAld from 5-FDRulP, which is described in Chapter 3. Following the identification and over expression of a PLP-dependant transaldolase, which generates 4-fluorothreonine (4-FT) from FAld and L-threonine in S. cattleya, Chapter 4 details the successful in vitro reconstitution of fluorometabolite biosynthesis using five over- expressed enzymes. In Chapter 5, attempts to develop a novel assay for fluorinase activity was explored. The colorimetric detection of L-methionine produced by the fluorinase in a coupled L-amino acid oxidase and horseradish peroxidase assay, leading to the oxidation of a dye substance. This was carried out with interest in developing a high-throughput assay for fluorinase mutants, generated by random mutagenesis, in order to identify those with increased activity. In the event, it proved unsuccessful.