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La dégradation des fibres alimentaires est une fonction essentielle des écosystèmes digestifs microbiens. Chez le ruminant, elle est assurée par des bactéries, champignons et protozoaires capables de produire de nombreuses enzymes nécessaires à l’hydrolyse des polysaccharides de paroi végétale. Parmi les facteurs susceptibles d’influencer l’efficacité de dégradation des fibres, qui est une composante importante de la productivité et de la santé animales, des additifs tels que des levures probiotiques apparaissent comme un levier intéressant. Afin d’approfondir les connaissances sur les facteurs de modulation de l’activité fibrolytique, une biopuce ADN fonctionnelle, outil moléculaire haut-débit, ciblant les gènes codant les enzymes clés de la dégradation de la cellulose et des xylanes dans les écosystèmes digestifs a été développée. Aussi, une méthode efficace dédiée à des échantillons ruminaux pour la soustraction des ARNr à partir des ARN totaux a été mise au point afin d’accroitre la sensibilité de l’outil. La biopuce fonctionnelle a été validée sur échantillons de complexité croissante et démontre d’excellents caractères de spécificité et de sensibilité tout en étant exploratoire et quantitative. Des régulations différentielles de l’arsenal des gènes de la fibrolyse de la bactérie du rumen Fibrobacter succinogenes ont pu être montrées. De même, les résultats sur échantillons de rumen suggèrent un rôle des microorganismes eucaryotes dans la fibrolyse pouvant être plus important qu’initialement envisagé. Cette approche métatranscriptomique dirigée pourra in fine continuer d’être appliquée dans l’étude de l’impact de facteurs biotiques et abiotiques sur la fonction fibrolytique microbienne chez les animaux d’élevage. / Dietary fibre degradation is an essential function of microbial digestive ecosystems. In ruminants, this function is ensured by bacteria, fungi and protozoa, producing a large array of enzymes able to degrade plant cell wall polysaccharides. Among factors likely to influence the efficiency of fibre degradation, which is an important component in animal productivity and health, dietary additives such as probiotic yeasts appear as an interesting tool. To provide more insight on factors modulating fibrolytic activity, we designed a functional DNA microarray targeting genes coding for key enzymes involved in cellulose and xylan degradation by digestive microbiota. Also, an efficient method dedicated to rumen samples for removing microorganisms’ rRNA from total RNA samples was developed to increase the sensitivity of the tool. The DNA microarray was validated using targets of increasing complexity and demonstrated sensitivity and specificity as well as explorative and quantitative potential. Differential expression of genes involved in fibrolysis was evidenced in the rumen bacterium Fibrobacter succinogenes. Moreover, results on rumen samples suggest a more important role of eucaryotes in fibre degradation than previously thought. This targeted metatranscriptomic approach will be further applied to the study of the impact of biotic and abiotic factors on the microbial mechanisms of fibre degradation in livestock.

<p>Enzymes are useful tools as specific analytical reagents. Two different analysis methods were developed for use in the separate fields of protein science and organic synthesis. Both methods rely on the substrate specificity of enzymes. Enzyme catalysis and substrate specificity is described and put in context with each of the two developed methods.</p><p>In <strong>paper I </strong>a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage.</p><p>In paper<strong> II </strong>and <strong>III</strong>, three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase,<em> Candida antarctica</em> lipase<strong> </strong>B<strong> </strong>and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD⁺. The conversion and enantiomeric excess of the sample could be calculated from the relative differences in absorbance.</p>

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-27T15:29:57Z No. of bitstreams: 1 Tereza Cristina de Assis.pdf: 813581 bytes, checksum: 8b96b058018eb3373d6a748191e4e45c (MD5) / Made available in DSpace on 2017-03-27T15:29:57Z (GMT). No. of bitstreams: 1 Tereza Cristina de Assis.pdf: 813581 bytes, checksum: 8b96b058018eb3373d6a748191e4e45c (MD5) Previous issue date: 2006-02-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The expressive increase in production of tropical flowers in Pernambuco indicates the State rising interest in ornamentals. This study had the objectives of: 1) screening plant parasitic nematodes in Zingiberales (Etlingera elatior, Zingiber spectabilis, Alpinia purpurata, Musa coccinea, Tapeinoquilos ananassae e Heliconia spp.) producing areas of Pernambuco, 2) predicting number of samples for nematodes monitoring at different acceptable error levels, 3) studying the effect of resistance abiotic inductors on nematode comunity associated with plant roots and soil, 4) evaluating enzymes activity in E. elatior, and 5) evaluating enzymes production in A. purpurata infected with Meloidogyne incognita. Samples (soil and roots of nematode parasited plants) were collected in producing areas from 2002 to 2004. Populational density of plant parasitic nematodes were evaluated and used to predict number of samples for monitoring. The study of induction resistance was carried out in one and two years old plants of a commercial area, fromSeptember to December in 2005. Acibenzolar-S-methyl (ASM), potassium silicate and neem cake were used as inductors, being analyzed free living and plant parasitic nematodes population density, area under nematode population density curve (AUNPDC), reproduction factor (RF), diameter of plant stem, β-1,3-glucanase and peroxidase activity. Enzymatic activity of esterase, phosphatase and peroxydase was evaluated in A. purpurata infected with M. incognita. Pratylenhus sp., Rotylenchulus sp., Meloidogyne spp., Helicotylenchus sp. and Criconemella sp. were frequents in most of samples. Symptoms of root-knot nematodes were verified in 100 % of the growing areas, being levels of Meloidogyne spp. considered high, up to 375 nematodes per 300 cm3 soil and 6090 nematodes per 20 g of roots, considering different areas and hosts. Among Meloidogyne species, M. incognita, M. arenaria and M. javanica were detected. Data analysis indicatedthat at least 11, 20, 10, 18 and 10 samples per area are recommended for monitoring the genus Pratylenchus, Rotylenchulus, Meloidogyne, Helicotylenchus and Criconemella, respectively, at 20 % error. The associations M. arenaria with E. elatior, Rotylenchulus sp. with A. purpurata, Heliconia spp. and M. coccinia, and Criconemella sp. with A. purpurata consisted of the first occurrence report in Pernambuco, Brazil. ASM was the best resistance inductor, reducing M. incognita population density, AUNPDC and FR with results similar to the nematicide control, but with no influence on free living nematodes in soil. Fitotoxic symptoms were not verified in any treatment. The action of ASM seems to be more related with peroxidases production than the β-1,3-glucanases activity in the plant. Among all of the inductors evaluated, ASM was the most effective inducing resistance against M. incognita in E. elatior. Enzymatic production was higher in A. purpurataparasited than in the controls. The acid phosphatase activity was high in contrast with the low esterase and peroxydase activity expressed in parasited plants. / O aumento expressivo na área plantada com ornamentais tropicais em Pernambuco reflete o crescente interesse pela floricultura no Estado. Este estudo teve por objetivos: 1) realizar levantamento de fitonematóides em áreas produtoras de Zingiberales em Pernambuco, 2) estimar o número de amostras recomendado para monitoramento, 3) avaliar os efeitos de indutores abióticos de resistência sobre a nematofauna associada à rizosfera, 4) avaliar a atividade de enzimas em plantas de bastão do imperador (Etlingera elatior), 5) analisar a produção de enzimas em plantas de alpínia (Alpinia purpurata) sob parasitismo de Meloidogyne incognita. No período de 2002 a 2004, foram visitadas 10 áreas produtoras de Zingiberales ornamentais (Etlingera elatior, Zingiber spectabilis, Alpinia purpurata, Musa coccinea, Tapeinochilos ananassae e Heliconia spp.), no estado de Pernambuco. Em cada propriedade, foram coletadas raízes de plantas parasitadas por fitonematóides e solo da rizosfera. Após processamento das amostras, foram determinadas as densidades populacionais dos gênerosde nematóides presentes e estimado o número recomendado de amostras para monitoramento, considerando diferentes níveis de erro aceitável. O estudo com indutores foi desenvolvido em plantio comercial, com plantas cultivadas por um e dois anos, no período de setembro a dezembro de 2005. Acibenzolar-S-metil (ASM), silicato de potássio e torta de nim foram utilizados como indutores. Foram analisadas a densidade populacional de nematóides parasitos e de vida livre, curva de densidade populacional dos nematóides, área abaixo da curva da densidade populacional(AACDP), fator de reprodução (FR), diâmetro do pecíolo e atividade das enzimas β-1,3-glucanase e peroxidase. No estudo de enzimas em plantas de alpínia, sob parasitismo de M. incognita foram determinadas as atividades da esterase, fosfatase ácida e peroxidase. No levantamento, constatou-se ocorrência freqüente dos gêneros: Pratylenhus, Rotylenchulus,Meloidogyne, Helicotylenchus e Criconemella. Sintomas da meloidoginose foram constatados em 100 % das áreas de plantio, com os níveis populacionais médios de Meloidogyne spp. elevados, alcançando 375 espécimes/300 cm3 de solo e 6090 espécimes/20 g de raízes, considerando diferentes hospedeiros e áreas. Entre as espécies de Meloidogyne foram detectadas M. incognita, M. arenaria e M. javanica. As estimativas do número de amostras indicaram que pelo menos 11, 20, 10, 18 e 10 amostras por área são recomendadas para monitoramento dos gêneros Pratylenchus, Rotylenchulus, Meloidogyne, Helicotylenchus e Criconemella, respectivamente, considerando um nível de erro de 20 %. As associações M. arenaria com E. elatior, Rotylenchulus sp. com A. purpurata, Heliconia spp. e M. coccinia, e Criconemella sp. com A. purpurata constituíram os primeiros relatos da ocorrência em Pernambuco. ASM reduziu a densidade populacional, AACDP e FR de M. incognita, apresentando resultados semelhantes aos obtidos pelo nematicida, destacando-se dos demais tratamentos, sem no entanto influenciar negativamente as populações de outros nematóides presentes no solo. Não foram verificados sintomas de fitotoxidez em nenhum dos tratamentos realizados. A ação do ASM mostrou-se mais relacionada com a produção de peroxidases do que com a atividade de β-1,3-glucanases em plantas de bastão do imperador. A produção de enzimas foi maior em A. purpurata parasitada do que na testemunha. A maior atividade foi verificada pela enzima fosfatase ácida, enquanto esterase e peroxidase expressaram fraca atividade em plantas parasitadas.

Enzymes are useful tools as specific analytical reagents. Two different analysis methods were developed for use in the separate fields of protein science and organic synthesis. Both methods rely on the substrate specificity of enzymes. Enzyme catalysis and substrate specificity is described and put in context with each of the two developed methods. In paper I a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage. In paper II and III, three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The conversion and enantiomeric excess of the sample could be calculated from the relative differences in absorbance. / QC 20101108

<p>U okviru ove doktorske disertacije ispitan je hemijski sastav i biolo&scaron;ke aktivnosti metanolnih i vodenih ekstrakata odabranih samoniklih vrsta tribusa Urticeae, rod&nbsp; Urtica:&nbsp; U. <em>dioica</em>&nbsp; subsp.&nbsp; <em>dioica</em>&nbsp; var.&nbsp;<em> pubescens</em>, U.<em>&nbsp; dioica&nbsp;</em> subsp.&nbsp; <em>dioica&nbsp; </em>var.&nbsp; <em>dioica</em>&nbsp; i&nbsp; U. <em>kioviensis</em>&nbsp; i tribusa Parietarieae, rod&nbsp; Parietaria:<em>&nbsp; P. officinalis</em>,&nbsp; <em>P.</em><br /><em>lusitanica</em> L. subsp<em>. lusitanica</em>,<em> P. judaica</em> L. subsp. <em>judaica</em> i<em> P. serbica</em>. Cilj rada bio je da se odredi sadrţaj biolo&scaron;ki aktivnih jedinjenja u ovim, do sada veoma malo ispitanim vrstama famijije Urticaceae, i utvrdi njihov potencijal primene kao pomoćnih lekovitih sredstava i dodataka ishrani.<br />Hemijski sastav ekstrakata ispitivanih vrsta određen je primenom: tečnohromatografskih tehnika (LC-DAD-MS i LC-MS-MS) za kvalitativnu analizu metanolnih ekstrakata, dok je za kvantitativnu analizu odabranih fenolnih jedinjenja primenjena LC-MS-MS tehnika. Spektrofotometrijskim metodama je određen sadržaj<br />ukupnih fenolnih komponenti i flavonoida. Ispitivanja biolo&scaron;kih aktivnosti ekstrakata obuhvatila su: određivanje antioksidantne i antiinflamatorne aktivnosti kao i sposobnost ekstrakata da inhibiraju acetilholinesterazu.&nbsp; Određen je uticaj odabranih metanolnih ekstrakata na imuni odgovor i proliferaciju intestinalnih ćelijskih linija pacova (IEC18) i ĉoveka (Caco2).<br />Dobijeni rezultati ukazuju da odabrane vrste tribusa Urticeae i Parietarieae, odnosno rodova&nbsp; Urtica&nbsp; i Parietaria&nbsp; predstavljaju bogate izvore biolo&scaron;ki aktivnih jedinjenja koja ispoljavaju raznovrsne biolo&scaron;ke aktivnosti. Sa hemotaksonomskog aspekta izdvajaju se sledeća jedinjenja kao potencijalni taksonomski markeri: vi&scaron;i sadržaj 5-O-kafeoilhinske kiseline u ekstraktima herbi vrsta roda&nbsp; Urtica, i visok sadrţaj<br />epikatehina u ekstraktima korena vrsta roda&nbsp; Parietaria. Ekstrakt herbe vrste&nbsp; U. kioviensis&nbsp; se od ostalih izdvaja po tome &scaron;to ne sadrži rutin a sadrži&nbsp; C-glikozide, u najvećoj meri viteksin. Od svih ispitivanih ekstrakata, ekstrakti korena&nbsp; Parietaria&nbsp; vrsta su ispoljili najbolji antioksidantni potencijal u većini izvr&scaron;enih testova. Najsnažniji antiinflamatorni potencijal je ispoljio ekstrakt korena vrste&nbsp; P. officinalis&nbsp; a prate ga ekstrakti korena vrsta roda&nbsp; Urtica.&nbsp; Veoma dobar antiinflamatorni potencijal su ispoljili infuzi herbi vrste U.<em> dioica</em>&nbsp; (čajevi od koprive). Svi ispitani metanolni ekstrakti su ispoljili odličnu inhibiciju enzima acetilholinesteraze a kao najbolji se izdvajaju ekstrakti korena&nbsp; Parietaria&nbsp; vrsta i vrste&nbsp; U.<em> kioviensis</em>. Povećanu sekreciju citokina rat MCP1 i GRO&alpha;&nbsp; izazivaju ekstrakti korena vrsta&nbsp; P. officinalis&nbsp; i&nbsp; P. judaica&nbsp; u<br />bazalnim uslovima i uslovima LPS-stimulisane inlamacije, dok ekstrakti vrste&nbsp; U. dioica&nbsp; povećavaju bazalnu a smanjuju LPS-stimulisanu sekreciju. Stimulaciju sekrecije ova dva citokina, ispitivani ekstrakti vr&scaron;e interakcijom sa adapternim proteinom MyD88 (ali ne intereaguju sa TLR4 receptorom) i NF -&kappa;B signalnim<br />putem. Ekstrakt korena vrste&nbsp; P. <em>officinalis</em>&nbsp; povećava LPS-om indukovanu ekspresiju enzima COX-2 u IEC18 ćelijama, dok je ekstrakt korena vrste&nbsp; U.<em> dioica</em>&nbsp; smanjuje. Efekat epitelizacije ili zarastanja rane na monosloju IEC18 ćelija ispoljavaju ekstrakti herbe i korena vrste&nbsp; P. <em>officinalis</em>. Ispitivani ekstrakti ne menjaju značajno seksreciju citokina hMCP1 i IL-8 u Caco2 ćelijama niti ispoljavaju značajan uticaj na &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; njihovu proliferaciju.</p> / <p>Within this doctoral thesis the chemical composition and biological activity of methanol and aqueous extracts of the selected plant species belonging to the Urticeae and Parietarieae tribe, more specifically to the&nbsp; Urtica&nbsp; and&nbsp; Parietaria&nbsp; genuses was evaluated (Urtica:&nbsp; U.<em> dioica</em>&nbsp; subsp.<em> dioica</em>&nbsp; var.&nbsp; <em>pubescens</em>,&nbsp; U.&nbsp; <em>dioica</em>&nbsp; subsp.&nbsp;<em> dioica</em>&nbsp; var.&nbsp; <em>dioica</em>&nbsp; and&nbsp; U.<em> kioviensis</em>;&nbsp; Parietaria:&nbsp; P<em>. officinalis</em>,&nbsp; P.<em> lusitanica</em>&nbsp; subsp.&nbsp; <em>lusitanica,</em>&nbsp; P. <em>judaica&nbsp;</em> subsp.<em>&nbsp; judaica&nbsp;</em> and<em>&nbsp; P. serbica</em>). The principal aim was to determine the content of biologically active&nbsp; compounds in this, poorly<br />examined species of the Urticaceae family, and determine their potential as additional remedy and dietary supplements.<br />Qualitative analysis of methanol extracts was performed by LC-DAD-MS i LC-MS/MS analysis, and LC-MS/MS for quantitative analysis of selected phenolic compounds. Total phenolics and flavonoids were determined spectrophotometrically. In order to assess the biological potential, the antioxidant and anti-inflammatory activities of the extracts were studied as well as&nbsp; their ability to inhibit acetylcholinesterase. The immuno-modulatory effects of the selected methanol extract on the immune response and proliferation of intestinal epithelial cells (IEC18 and Caco2)<br />was determined.<br />The obtained results suggest that the examined species of the Urticeae and Parietarieae tribe (genuses&nbsp; Urtica&nbsp; and&nbsp; Parietaria) are abundant with the biologically active compounds that express a broad spectrum of biological activities. As a potential chemotaxonomic markers stand out the following&nbsp; compounds: 5-O-caffeoilquinic acid (highly abundant in the herb extracts of the&nbsp; Urtica&nbsp; spp.) and epicatechin (highly abundant in the root extracts of the&nbsp; Parietaria&nbsp; spp.).&nbsp; U.<br />kioviensis&nbsp; herb extracts differs from the rest by high content of vitexin and total lack of rutin. The best antioxidant potential have exhibited the root extracts of the&nbsp; Parietaria&nbsp; species. The strongest anti-inflammatory potential had the root extract of the&nbsp; P. officinalis, followed by root extracts of the&nbsp; Urtica&nbsp; spp. Excellent anti-inflammatory activity have exhibited the aqueous&nbsp; extracts of&nbsp; U.<em> dioica</em>&nbsp; herbs&nbsp; &ndash;&nbsp; stinging nettle teas. All tested methanol extracts have inhibited enzyme acetylcholinesterase, the best inhibitors being root extracts of&nbsp; U.<em> kioviensis&nbsp;</em> and Parietaria&nbsp; species. Root extracts of&nbsp; P<em>. officinalis</em>&nbsp; and&nbsp; P.<em> judaica</em>&nbsp; have increased the basal and LPS-stimulated secretion of rat MCP1 and GRO&alpha;, while&nbsp; U. <em>dioica&nbsp;</em> extracts increased the basal but decreased the LPS-stimulated secretion. The examined extracts interact with the MyD88 (but not the TLR4) and NF-&kappa;B signaling pathway. The root extract of&nbsp; P<em>. officinalis&nbsp; </em>increase LPS-stimulated expression of COX-2 in IEC18 cells, while the root extract of&nbsp; U<em>. dioica</em>&nbsp; decreases it.<br />The herb and root extract of P. <em>officinalis</em>&nbsp; exhibit the wound healing effect. Investigated extracts do not significantly alter the secretion of hMCP1 and IL-8 in Caco2 cells and exhibit no significant effect to their proliferation.</p> / null