Phenobarbital (PB) is one of the most important antiseizure drugs (ASDs) to treat canine idiopathic epilepsy (IE). The effect of PB on the taxonomic changes in gastrointestinal microbiota (GIM) and their functions is less known, which may explain parts of its pharmacokinetic and pharmacodynamic properties, especially its antiseizure effect and drug responsiveness or drug resistance as well as its effect on behavioral comorbidities. Fecal samples of 12 dogs with IE were collected prior to the initiation of PB treatment and 90 days after oral PB treatment. The fecal samples were analyzed using shallow DNA shotgun sequencing, real-time polymerase chain reaction (qPCR)-based dysbiosis index (DI), and quantification of short-chain fatty acids (SCFAs). Behavioral comorbidities were evaluated using standardized online questionnaires, namely, a canine behavioral assessment and research questionnaire (cBARQ), canine cognitive dysfunction rating scale (CCDR), and an attention deficit hyperactivity disorder (ADHD) questionnaire. The results revealed no significant changes in alpha and beta diversity or in the DI, whereas only the abundance of Clostridiales was significantly decreased after PB treatment. Fecal SCFA measurement showed a significant increase in total fecal SCFA concentration and the concentrations of propionate and butyrate, while acetate concentrations revealed an upward trend after 90 days of treatment. In addition, the PB-Responder (PB-R) group had significantly higher butyrate levels compared to the PB-Non-Responder (PB-NR) group. Metagenomics of functional pathway genes demonstrated a significant increase in genes in trehalose biosynthesis, ribosomal synthesis, and gluconeogenesis, but a decrease in V-ATPase-related oxidative phosphorylation. For behavioral assessment, cBARQ analysis showed improvement in stranger-directed fear, non-social fear, and trainability, while there were no differences in ADHD-like behavior and canine cognitive dysfunction (CCD) scores after 90 days of PB treatment. While only very minor shifts in bacterial taxonomy were detected, the higher SCFA concentrations after PB treatment could be one of the key differences between PB-R and PB-NR. These results suggest functional changes in GIM in canine IE treatment.

Efterfrågan på energi ökar snabbt, och denna trend förväntas bestå. Det råder dock ingen tvekan om att världen för närvarande befinner sig i en energikris, som är en katastrof både ekonomiskt och miljömässigt. Följaktligen är sökandet efter alternativa energikällor för att ersätta fossila bränslen absolut nödvändigt. Biobränslen som härrör från biomassa ökar i popularitet på grund av deras höga tillgänglighet och förmåga att minska utsläppen av växthusgaser. Butylbutyrat är ett potentiellt substitut för diesel- och flygmotorbränsle, vilket gör det till en viktig energikälla för både land- och lufttransporter. I den här studien undersöks enzymatisk katalys för produktion av butylbutyrat genom förestring av butanol och smörsyra med hjälp av olika membran. Användningen av enzymer som katalysatorer erbjuder många fördelar jämfört med traditionella kemiska katalysatorer, såsom ökad selektivitet, mildare reaktionsförhållanden och miljövänliga processer. Estrarnas byggstenar, n-butanol och smörsyra, är viktiga kemikalier och kan produceras i biotekniska processer med hjälp av förnybara källor som lignocellulosabaserad biomassa, vilket bidrar till övergången från en petroleumbaserad till en hållbar energiförsörjning. Produktionen av butanol och smörsyra i bioreaktorer försvåras av de typiska hindren för biobränsleproduktion, såsom produkthämning, höga bearbetningskostnader i efterföljande led och låga utbyten. NanoLodge-projektet bygger på tre reaktorer, en för produktion av butanol och en för smörsyra, i två separata men sammankopplade kontinuerliga Clostridium fermenteringar, och ett tredje reaktionssystem för enzymatisk förestring. Alla tre processerna i en, med dedikerade membran som separerar de olika reaktiva systemen, förväntas öka utbytet och samtidigt skapa en mycket integrerad anläggning som kan skalas upp för storskalig drift. Med hjälp av Raman-spektroskopi, GC-FID, FTIR och SEM undersöktes och karakteriserades fem distinkta material och deras relativa membranförändringar, inklusive enzymet på det översta lagret. FTIR-resultaten indikerade att för några av dem var porerna effektivt fuktade med reaktanterna, och membranens hydrofobicitet ökades genom att applicera lämplig beläggning, som även innefattade enzymet. Även om varken syntes av butylbutyrat eller passage genom de olika skikten av membranen inträffade, visade Raman-spektroskopi och GC-FID att et av membranen var en möjlig kandidat för förestring. Dessutom kunde inte alla testade membran motstå reaktionsmediet eftersom SEM-bilder avslöjade att vissa områden av ytan och strukturen hade ändrats något. Mer studier behövs för att fastställa att det med lovande membranet och dess modifieringar, särskilt den hydrofoba naturen och det immobiliserade enzymet, är acceptabla för butylbutyratsyntes. Ytterligare forskning kan utforska processoptimering, enzymimmobiliseringstekniker och reningsmetoder nedströms för att förbättra den totala effektiviteten och ekonomiska bärkraften för denna enzymatiska förestringsprocess.

Einleitung: Monocarboxylattransporter (MCT) 1 & 4 sind in zahlreichen Geweben als Kotransporter für Monocarboxylate und Protonen beschrieben. Auch im Pansenepithel werden MCT benötigt, um kurzkettige Fettsäuren (SCFA) aus dem Pansenlumen in die Pansenepithelzelle aufzunehmen (MCT4) und um SCFA und deren Metabolite aus der Pansenepithelzelle in das Blut auszuschleusen (MCT1). Die transepitheliale Permeation von SCFA über die Pansenwand ist von enormer Bedeutung, da sie die wichtigste Energiequelle der Wiederkäuer darstellen. Die beteiligten Transportprozesse müssen dementsprechend einer Anpassung an variierende Mengen von SCFA unterliegen. Bisherige Studien bei anderen Spezies deuten auf eine Regulation des MCT1 auf mRNA Ebene über den Peroxisom-Proliferator-aktivierten Rezeptor α (PPARα) hin. Ziele der Untersuchung: Das Ziel dieser Arbeit war herauszufinden, ob MCT1 in ovinen Pansenepithelzellen über PPARα reguliert wird und ob auch MCT4 dieser Regulation unterliegt. Eine gleichzeitige Regulation beider Transporter läge nahe, da sie gemeinsam an der transepithelialen Permeation beteiligt sind. Die Auswirkungen solch einer Regulation auf die Proteinexpression und die Transportleistung der MCT sollte charakterisiert werden. Ebenfalls war das Potenzial der bei erhöhter Kraftfutterfütterung vermehrt anfallenden SCFA Butyrat auf die MCT1 Expression zu untersuchen. Material & Methoden: Aus dem Vorhof von Schafen wurden Pansenepithelzellen gewonnen und entsprechend einer bereits etablierten Methode kultiviert. Nach einer Subkultivierung wurden die Zellen immunzytochemisch mit Antikörpern gegen MCT1, MCT4 und Na+/K+-ATPase untersucht, um deren Lokalisation in den kultivierten Pansenepithelzellen zu bestimmen. Weiterhin erfolgte eine Behandlung mit WY 14.643, einem spezifischen, synthetischen PPARα Agonisten, sowie mit GW 6471, einem Antagonisten des PPARα. Mittels qPCR wurden die relativen mRNA Mengen von MCT1, MCT4, ACO, CPT1A und CACT bestimmt und auf die Referenzgene GAPDH und Na+/K+-ATPase normalisiert. Die Proteinexpression von MCT1 und MCT4 wurde mittels Western Blot bestimmt. Zur funktionellen Quantifizierung wurde der intrazelluläre pH-Wert der Zellen mittels Spektrofluorometrie gemessen und der laktatabhängige Protonentransport als Vergleichswert zwischen den Behandlungen genutzt. Um den MCT-abhängigen Teil des Transportes zu bestimmen, wurde ein spezifischer MCT1 & 4 Inhibitor, die p-Hydroxymercuribenzensulfonsäure (pHMB) eingesetzt. Die Zellen wurden mit Butyrat über einen Zeitraum von 6 und 48 h induziert. Die Erfassung der MCT1 Expression erfolgte mittels semiquantitativer PCR. Ergebnisse: MCT1 & 4 sind sowohl in der Zellmembran als auch intrazellulär in den Pansenepithelzellen lokalisiert. Die mRNA Expressionsdaten konnten zeigen, dass MCT1 und die PPARα Zielgene durch WY 14.643 hochreguliert werden konnten, wohingegen die MCT4 Expression keine eindeutige Antwort auf die Stimulation zeigt. Die Behandlung mit den Antagonisten zeigt eine Abhängigkeit der MCT1 Expression von PPARα, die MCT4 Expression konnte dagegen nicht beeinflusst werden. Mittels pHMB gelang es, den laktatabhängigen Protonenexport fast vollständig zu blocken. Sowohl laktatabhängiger Protonenexport als auch die Proteinexpression zeigten keine Änderung durch WY 14.643 Stimulation. Die Butyratexposition veränderte die Morphologie der Pansenepithelzellen und schien nicht geeignet für Untersuchungen der mRNA Expression zu sein. Schlussfolgerungen: Es konnte in dieser Arbeit erstmals gezeigt werden, dass MCT1 in Pansenepithelzellen über PPARα reguliert wird, nicht aber MCT4. PPARα scheint demnach einer der entscheidenden Angriffspunkte für die Regulation des SCFA Transportes zu sein, dessen natürliche Liganden im Pansen aber noch nicht bekannt sind. Damit legt diese Arbeit den Grundstein für regulative Studien am intakten Pansenepithel. / Introduction: Monocarboxylate transporters (MCT) 1 & 4 are cotransporters of monocarboxylates and protons in a variety of mammalian cell types. In the ruminal epithelium MCT are necessary to transport short-chain fatty acids (SCFA) from the lumen into the ruminal epithelial cell (MCT4) and to discharge SCFA and their metabolites from the cell into the blood (MCT1). Transepithelial permeation of SCFA is of great importance, because they are the main source of energy for ruminants. The regulation of appropriate transport proteins should thus be subject to the adaptation to varying SCFA amounts. Previous studies in other species suggested that gene expression of MCT1 is regulated by peroxisome proliferator-activated receptor α (PPARα), a ligand-activated nuclear receptor. Aims: The aim of the study was to examine if MCT1 in ruminal epithelial cells is regulated by PPARα and furthermore if MCT4 can be regulated by PPARα, as well. A simultaneous regulation seems likely, because both are acting jointly in the transepithelial transporting of SCFA. The implications of such a regulation on protein expression and transport capacity of MCT should be characterized. The effect of butyrate, a SCFA which increases under concentrate feeding, on MCT1 expression was determined. Materials & Methods: Ruminal epithelial cells of sheep were cultivated according to methods previously established. After subcultivation, immunocytochemistry with antibodies against MCT1, MCT4 and Na+/K+-ATPase was performed to determine their localization in ruminal epithelial cells. For studying the influence of PPARα, WY 14.643, a synthetic and selective ligand of PPARα, and GW 6471, a synthetic antagonist of PPARα, were applied to the culture medium of the cells. After processing the specimens, the relative amount of mRNA of MCT1, MCT4 and the target genes ACO, CPT1A and CACT were analyzed by qPCR and normalized on the reference genes GAPDH and Na+/K+-ATPase. Protein abundance of MCT1 & 4 was measured by using the Western Blot method. Functional quantification was measured by the intracellular pH (pHi) of cells using spectrofluorometry as well as comparing the effect of WY 14.643 treatment on lactate-dependent proton export. To determine the MCT-dependent part of the pHi recovery, p-hydroxymercuribenzoic acid (pHMB), a specific inhibitor of MCT1 & 4, was applied. Cells were also treated with butyrate for 6 h and 48 h and the mRNA abundance of MCT1 was analyzed by semiquantitative PCR. Results: Both MCT1 and MCT4 were localized in the cell membrane as well as in the cytoplasm of ruminal epithelial cells. By qPCR it could be demonstrated that the mRNA abundance of MCT1 and PPARα target genes in the ruminal epithelial cells was increased by WY 14.643 in comparison to untreated cells, whereas the response of MCT4 did not yield distinct results. Treatment with the PPARα antagonist pointed out, that MCT1 is influenced by PPARα, but not MCT4. Lactate-dependent proton export was blocked almost completely by pHMB. Both lactate-dependent proton export and protein expression were not altered by WY 14.643 treatment. Butyrate exposure changed the morphology of ruminal epithelial cells and seemed unsuitable for the analysis of mRNA expression. Conclusion: For the first time, it could be demonstrated, that MCT1 in ruminal epithelial cells is regulated by PPARα, but not MCT4. PPARα seems to be a vital target in the rumen for SCFA transport regulation, whose natural triggers have yet to be identified. Furthermore, this study provides the basis for regulative studies on intact ruminal epithelium.

Dois experimentos foram conduzidos com o objetivo de avaliar o efeito da inclusão de butirato de sódio, monensina sódica ou propionato de cálcio no concentrado inicial, sobre o desempenho, parâmetros sanguíneos e desenvolvimento ruminal de bezerros leiteiros. No primeiro experimento, 24 bezerras recém-nascidas da raça Holandesa foram alojadas em abrigos individuais até a 10a semana de vida, com livre acesso à água, sendo alimentadas com 4 litros de leite/dia e concentrado ad libitum, enquanto feno de capim-coast-cross foi fornecido após o desaleitamento. Os animais foram distribuídos em blocos de acordo com peso ao nascer e data de nascimento e alocados em um dos tratamentos, de acordo com o aditivo no concentrado: 1)Butirato de sódio (0,15%); 2)Monensina sódica (30 ppm); e 3)Propionato de cálcio (0,15%). Os animais foram pesados e avaliados quanto à altura na cernelha, largura do traseiro e perímetro torácico semanalmente. A partir da 4a semana foram realizadas colheitas semanais de amostras de sangue para determinação de glicose, ácidos graxos livres (AGL) e ?-hidroxibutirato (BHBA). Não foram observadas diferenças significativas entre os tratamentos para o consumo de concentrado ou de feno e para o peso e ganho de peso dos animais (P>0,05). As avaliações quanto à altura na cernelha e perímetro torácico também não apresentaram diferenças entre os tratamentos (P>0,05), entretanto as medidas de largura de traseiro foram menores para os animais do tratamento com adição de propionato de cálcio (P<0,05). As concentrações plasmáticas de glicose, AGL e BHBA não foram afetadas pelos tratamentos (P>0,05). Houve efeito significativo da idade (P<0,0001) para a concentração plasmática de glicose, sendo esta reduzida com a idade dos animais. No segundo experimento, 15 bezerros recémnascidos da raça Holandesa, recebendo o mesmo manejo nutricional, foram fistulados no rúmen e alojados em baias individuais até a 10ª semana de vida. A partir da 4ª semana, foram realizadas colheitas semanais de fluído ruminal para determinação de pH, ácidos graxos de cadeia curta (AGCC) e N-amoniacal; e de sangue para determinação de glicose. Ao completar dez semanas os animais foram abatidos para avaliação do desenvolvimento do trato digestório superior e de papilas ruminais. Não foram observadas diferenças significativas entre tratamentos para o consumo de concentrado e para o desempenho dos animais (P>0,05). Houve efeito significativo (P<0,05) de tratamento e horário de colheita para o pH ruminal. As concentrações de AGCC totais, bem como de cada ácido, não foram afetadas pelos tratamentos. As concentrações plasmáticas de glicose foram afetadas pelos tratamentos (P<0,05). O peso total do trato digestório superior, os pesos médios de cada compartimento e a capacidade máxima do retículo-rúmen não foram afetados pelos tratamentos, assim como os parâmetros de desenvolvimento do epitélio ruminal. Os aditivos incluídos no concentrado inicial se mostraram igualmente eficazes no que diz respeito aos seus efeitos no desempenho e desenvolvimento ruminal de bezerros em aleitamento. / Two trials were conducted in order to evaluate the effects of the addition of sodium butyrate, sodium monensin or calcium propionate in the starter feed on the performance, blood parameters and ruminal development of dairy calves. In the first experiment, 24 newborn Holstein calves were housed in individual hutches during ten weeks of life, with free access to water, being fed 4 liters of milk per day and starter ad libitum, with coast-cross hay offered only after weaning. The animals were blocked according to weight and date of birth and allocated in one of the treatments, according to the additive included in the starter feed: 1) sodium butyrate (0.15%); 2) sodium monensin (30 ppm); and 3) calcium propionate (0.15%). Animals were weighed and evaluated for whiter height, hearth girth and hip width weekly. From the fourth week of age blood samples were taken for glucose, free fatty acids and ?-hydroxybutyrate concentration determination. No significant differences were observed among treatments for starter or hay intake, and weight gain or live weight (P>0.05). Measurements of whiter height and hearth girth were also not affected by treatments (P> 0.05); however, measures of hip width of the animals were smaller for treatment with addition of calcium propionate (P<0.05). Plasma concentrations of glucose, free fatty acids and ?-hydroxybutyrate were not affected by treatment (P>0.05). There was significant effect of age (P<0.0001) for the plasma concentration of glucose, with reduction as animals aged. In the second experiment, 15 male newborns Holstein calves, receiving the same nutritional management, were ruminally fistulated and housed in individual pens during ten weeks of life. From the fourth week of age ruminal samples were taken weekly for the determination of pH, short-chain fatty acids (SCFA) and ammonia-N concentration. Blood samples were also taken weekly for glucose determination. By completing ten weeks of age, animals were slaughtered for forestomach growth and papillae development evaluation. No significant differences were observed among treatments for starter intake as well as for animal performance (P>0.05). The ruminal pH was significantly affected (P<0.05) by treatments and by sampling time. Concentrations of total SCFA and individual SCFA were not affected by treatments (P<0.05). Plasma concentrations of glucose were affected by treatments (P<0.05). The total forestomach weight, the average weight of each compartment and the maximum capacity of reticulum-rumen were not affected by treatments, as well as the parameters for ruminal epithelium development. The additives included in the starter feed were equally effective as regard to its effects on animal performance and rumen development of milk-fed dairy calves.

Obesity is pandemic. Pharmacological treatment development depends on modeling the regulation of feeding, particularly by free fatty acids (FFA). Most models have been employed in the rat in vivo, and show FFA-stimulated intestinal satiety signals are dependent on the fat’s acyl chain-length, involve cholecystokinin (CCK) secretion, and are mediated by vagal afferents. I hypothesized that an in vitro mouse model could be employed, with sensitivity to measure afferent responses to nutrient stimuli. Male C57BL/6N mice were killed, the intestine harvested en bloc, and a jejunal section dissected with neurovascular mesenteric arcade emanating centrally. The tissue was placed in a Krebs-superfused chamber, the lumen cannulated with the outlet open to drain, and Krebs or other mediators were continuously perfused intraluminally. The dissected afferent nerve was placed in a suction electrode for extracellular recording. Afferent responses to distension and the perfusion of mediators (e.g. CCK or FFA) were tested. Preparations from normal mice (no surgery), or from mice following chronic subdiaphragmatic vagotomy or sham operation, were used to assess vagal afferent contributions. Luminally-perfused CCK (100 nM) increased afferent firing. This response was abolished with the CCK-1 receptor antagonist lorglumide (10 µM). The short-chain fatty acid (SCFA) sodium butyrate (30 mM) potentiated firing. The long-chain fatty acid (LCFA) sodium oleate (1-300 mM) activated concentration-dependent firing (EC50=25.35 mM) that was significantly greater at 30 mM than that evoked by butyrate. Lorglumide (30 µM) abolished the oleate (30 mM) response. The L-type Ca2+ channel (LTCC) inhibitor nicardipine (3 µM), intraluminally, potentiated the oleate response, while bath application abolished it. Vagotomy attenuated the oleate response. Vagotomy abolished the intraluminal CCK (100 nM) response, and attenuated the response to bath-superfused CCK. These findings support FFA chain-length-dependent mesenteric afferent activation and CCK involvement in oleate-induced firing, and suggest LTCC mediation of excitatory and inhibitory oleate response transduction pathways. The murine oleate response was shown to be mostly vagally-mediated, with some spinal contribution, and both vagal and spinal contributions to CCK responses were suggested. These data provide a basis for further investigation in vitro of cellular and molecular mechanisms of afferent satiety signals, and ultimately of obesity pathogenesis. / Thesis (Master, Physiology) -- Queen's University, 2010-06-29 15:56:08.387

Dois experimentos foram conduzidos com o objetivo de avaliar o efeito da inclusão de butirato de sódio, monensina sódica ou propionato de cálcio no concentrado inicial, sobre o desempenho, parâmetros sanguíneos e desenvolvimento ruminal de bezerros leiteiros. No primeiro experimento, 24 bezerras recém-nascidas da raça Holandesa foram alojadas em abrigos individuais até a 10a semana de vida, com livre acesso à água, sendo alimentadas com 4 litros de leite/dia e concentrado ad libitum, enquanto feno de capim-coast-cross foi fornecido após o desaleitamento. Os animais foram distribuídos em blocos de acordo com peso ao nascer e data de nascimento e alocados em um dos tratamentos, de acordo com o aditivo no concentrado: 1)Butirato de sódio (0,15%); 2)Monensina sódica (30 ppm); e 3)Propionato de cálcio (0,15%). Os animais foram pesados e avaliados quanto à altura na cernelha, largura do traseiro e perímetro torácico semanalmente. A partir da 4a semana foram realizadas colheitas semanais de amostras de sangue para determinação de glicose, ácidos graxos livres (AGL) e ?-hidroxibutirato (BHBA). Não foram observadas diferenças significativas entre os tratamentos para o consumo de concentrado ou de feno e para o peso e ganho de peso dos animais (P>0,05). As avaliações quanto à altura na cernelha e perímetro torácico também não apresentaram diferenças entre os tratamentos (P>0,05), entretanto as medidas de largura de traseiro foram menores para os animais do tratamento com adição de propionato de cálcio (P<0,05). As concentrações plasmáticas de glicose, AGL e BHBA não foram afetadas pelos tratamentos (P>0,05). Houve efeito significativo da idade (P<0,0001) para a concentração plasmática de glicose, sendo esta reduzida com a idade dos animais. No segundo experimento, 15 bezerros recémnascidos da raça Holandesa, recebendo o mesmo manejo nutricional, foram fistulados no rúmen e alojados em baias individuais até a 10ª semana de vida. A partir da 4ª semana, foram realizadas colheitas semanais de fluído ruminal para determinação de pH, ácidos graxos de cadeia curta (AGCC) e N-amoniacal; e de sangue para determinação de glicose. Ao completar dez semanas os animais foram abatidos para avaliação do desenvolvimento do trato digestório superior e de papilas ruminais. Não foram observadas diferenças significativas entre tratamentos para o consumo de concentrado e para o desempenho dos animais (P>0,05). Houve efeito significativo (P<0,05) de tratamento e horário de colheita para o pH ruminal. As concentrações de AGCC totais, bem como de cada ácido, não foram afetadas pelos tratamentos. As concentrações plasmáticas de glicose foram afetadas pelos tratamentos (P<0,05). O peso total do trato digestório superior, os pesos médios de cada compartimento e a capacidade máxima do retículo-rúmen não foram afetados pelos tratamentos, assim como os parâmetros de desenvolvimento do epitélio ruminal. Os aditivos incluídos no concentrado inicial se mostraram igualmente eficazes no que diz respeito aos seus efeitos no desempenho e desenvolvimento ruminal de bezerros em aleitamento. / Two trials were conducted in order to evaluate the effects of the addition of sodium butyrate, sodium monensin or calcium propionate in the starter feed on the performance, blood parameters and ruminal development of dairy calves. In the first experiment, 24 newborn Holstein calves were housed in individual hutches during ten weeks of life, with free access to water, being fed 4 liters of milk per day and starter ad libitum, with coast-cross hay offered only after weaning. The animals were blocked according to weight and date of birth and allocated in one of the treatments, according to the additive included in the starter feed: 1) sodium butyrate (0.15%); 2) sodium monensin (30 ppm); and 3) calcium propionate (0.15%). Animals were weighed and evaluated for whiter height, hearth girth and hip width weekly. From the fourth week of age blood samples were taken for glucose, free fatty acids and ?-hydroxybutyrate concentration determination. No significant differences were observed among treatments for starter or hay intake, and weight gain or live weight (P>0.05). Measurements of whiter height and hearth girth were also not affected by treatments (P> 0.05); however, measures of hip width of the animals were smaller for treatment with addition of calcium propionate (P<0.05). Plasma concentrations of glucose, free fatty acids and ?-hydroxybutyrate were not affected by treatment (P>0.05). There was significant effect of age (P<0.0001) for the plasma concentration of glucose, with reduction as animals aged. In the second experiment, 15 male newborns Holstein calves, receiving the same nutritional management, were ruminally fistulated and housed in individual pens during ten weeks of life. From the fourth week of age ruminal samples were taken weekly for the determination of pH, short-chain fatty acids (SCFA) and ammonia-N concentration. Blood samples were also taken weekly for glucose determination. By completing ten weeks of age, animals were slaughtered for forestomach growth and papillae development evaluation. No significant differences were observed among treatments for starter intake as well as for animal performance (P>0.05). The ruminal pH was significantly affected (P<0.05) by treatments and by sampling time. Concentrations of total SCFA and individual SCFA were not affected by treatments (P<0.05). Plasma concentrations of glucose were affected by treatments (P<0.05). The total forestomach weight, the average weight of each compartment and the maximum capacity of reticulum-rumen were not affected by treatments, as well as the parameters for ruminal epithelium development. The additives included in the starter feed were equally effective as regard to its effects on animal performance and rumen development of milk-fed dairy calves.

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-10-26T17:28:34Z No. of bitstreams: 1 TeseJBCS.pdf: 2225419 bytes, checksum: d3ec7a73f461b658739175f8e171c762 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-11-08T18:27:30Z (GMT) No. of bitstreams: 1 TeseJBCS.pdf: 2225419 bytes, checksum: d3ec7a73f461b658739175f8e171c762 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-11-08T18:27:36Z (GMT) No. of bitstreams: 1 TeseJBCS.pdf: 2225419 bytes, checksum: d3ec7a73f461b658739175f8e171c762 (MD5) / Made available in DSpace on 2016-11-08T18:27:44Z (GMT). No. of bitstreams: 1 TeseJBCS.pdf: 2225419 bytes, checksum: d3ec7a73f461b658739175f8e171c762 (MD5) Previous issue date: 2016-08-25 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Cell-associated lipases have been considered biocatalysts economically advantageous because they are produced at low cost, avoiding further recovery or purification steps. Few studies regarding Streptomyces clavuligerus lipase have reported the production of extracellular enzyme, although at first this had been considered a cell-associated enzyme. In this context, the aim of this work was the production of the cell-associated lipase from Streptomyces clavuligerus (whole-cell lipase, Sc-WCL) by submerged fermentation, the biochemical characterization of the enzyme and its use in the synthesis of butyl butyrate, an aroma ester with industrial importance. The culture conditions on rotary shaker and the operational parameters for cultivation in bioreactor were evaluated in order to establish a protocol for Sc- WCL production. The conditions for S. clavuligerus cultivation in rotary shaker that resulted in maximal hydrolytic activity of Sc-WCL (3,000 U.L-1) were: baffled flask, free-glycerol production medium, pH 6.8 and 28 °C. The operational parameters in bench reactor that resulted in maximal volumetric productivity of Sc-WCL (54 U.L-1.h-1) were agitation of 400 rpm and aeration of 1 vvm. The maximal volumetric productivity in bioreactor operated under selected conditions (52.5 U.L-1.h-1) was reached after 24 h, while similar productivity in rotary shaker (54.8 U.L-1.h -1) was achieved only after 48 h fermentation. The catalytic potential of Sc-WCL in hydrolysis reactions was comparable to the commercial lipase preparations. Sc- WCL was more active at 60 °C and pH 10.7, and stable at 30-40 °C after 1 h incubation at pH 10. For butyl butyrate synthesis catalyzed by Sc-WCL, the reaction conditions that resulted in higher ester conversion (85%) were: 5 g of Sc-WCL/ L, molar ratio of fatty acid: alcohol 1:1 in heptane and 8 h reaction. The stability at alkaline pH and organic medium (leastwise in heptane and butanol), associated with the low cost, make Sc-WCL attractive in industrial applications, such as flavors synthesis, detergent formulations, hydrolysis of vegetable oils, among others. / Lipases associadas à célula têm se destacado como biocatalisadores economicamente vantajosos, pois são produzidas a baixo custo, dispensando etapas posteriores de recuperação ou purificação. Poucos estudos sobre lipase de Streptomyces clavuligerus relatam a produção da enzima extracelular, embora esta tenha sido, a princípio, considerada uma enzima associada à célula. Neste contexto, o objetivo deste trabalho foi a produção de lipase associada à célula de Streptomyces clavuligerus (lipase “whole-cell”, Sc-WCL) por fermentação submersa, a caracterização bioquímica da enzima e sua aplicação na síntese de butirato de butila, um éster de aroma com importância industrial. As condições de cultivo em shaker e os parâmetros operacionais para cultivo em biorreator foram avaliados com o intuito de estabelecer um protocolo para a produção de Sc-WCL. As condições de cultivo de S. clavuligerus em shaker que resultaram em maior atividade hidrolítica de Sc-WCL (3.000 U.L- 1) foram: frasco aletado, ausência de glicerol no meio de produção, pH 6,8 e 28 ºC. Os parâmetros operacionais em reator de bancada que resultaram em maior produtividade volumétrica de Sc-WCL (54 U.L-1.h-1) foram: agitação de 400 rpm e aeração de 1 vvm. A máxima produtividade volumétrica em biorreator operado nas condições selecionadas foi alcançada após 24 h de cultivo (52,5 U.L-1.h-1), enquanto que produtividade similar em shaker foi obtida somente após 48 h de cultivo (54,8 U.L-1.h-1). O potencial catalítico de Sc-WCL em reações de hidrólise foi comparável ao de preparações comerciais de lipase. Sc-WCL foi mais ativa a 60 ºC e pH 10,7, e mais estável na faixa de 30 a 40 ºC após 1 h de incubação a pH 10. Na síntese de butirato de butila catalisada pela Sc-WCL, as condições reacionais que resultaram em maior conversão (85%) foram: 5 g de Sc-WCL/ L, razão molar ácido graxo/álcool 1:1 em heptano e 8 h de reação. A estabilidade em pH alcalino e em meio orgânico (pelo menos em heptano e butanol), associada ao baixo custo, tornam a Sc-WCL atrativa em aplicações de interesse industrial, tais como, síntese de aromas, formulações de detergentes, hidrólise de óleos vegetais, dentre outras.

Orientadores: Maria Regina Wolf Maciel, Mario Eusebio Torres Alvarez / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-11T16:17:30Z (GMT). No. of bitstreams: 1 Araujo_WilsonAndaleciode_D.pdf: 2993508 bytes, checksum: 29abb03a65a80f6c9835367e0cb49d50 (MD5) Previous issue date: 2008 / Resumo: Os processos de separação com membranas (PSM) têm sido considerados como alternativa a processos clássicos de separação. Esta é uma área de estudo que apresenta uma forte interdisciplinaridade. Há um crescente interesse nestes processos para diversas aplicações como, por exemplo, tratamento de efluentes industriais, desalinização de águas, purificação e concentração de correntes da indústria alimentícia. A separação, em geral, não envolve mudança de fase, o que significa economia no consumo de energia e operações a temperaturas moderadas. Na tecnologia de separação com membranas, os componentes das misturas líquidas ou gasosas são separados ao permearem de forma seletiva através de uma membrana. As membranas podem ser poliméricas ou cerâmicas. A corrente de alimentação é dividida em duas correntes de saída: a que permeou através da membrana (permeado) e a corrente concentrada retida (¿retentate¿). Estes processos têm sido aplicados no processamento de bebidas, sucos, e aromas. Neste trabalho, dois PSM foram estudados, a Osmose Inversa (OI) e a Pervaporação (PV). Experimentos em escala piloto foram realizados empregando-se o processo de OI (membrana de poliamida) para a concentração de suco de laranja a 20°Brix. Avaliou-se a retenção de compostos de voláteis (acetaldeído, metanol e etanol) monitorando-se as correntes de alimentação e permeado. Os resultados de retenção de aromas obtidos não foram satisfatórios. A membrana apresentou baixas retenções para os voláteis monitorados na temperatura usada para realização dos experimentos. Na segunda etapa do trabalho, o processo de PV foi avaliado para recuperação de um importante éster do suco de laranja, o etil butirato. O software PERVAP, um simulador Fortran essencialmente preditivo, foi empregado no estudo de desempenho do processo para duas membranas, polidimetilsiloxano (PDMS) e polioctilmetilsiloxano (POMS). Realizou-se a predição de propriedades de membranas poliméricas para incremento da capacidade preditiva do simulador. Foram empregados métodos de contribuição de grupos para predição das propriedades dos polímeros. Os dados de viscosidade preditos para o POMS viabilizaram a realização de cálculos para obtenção de parâmetros requeridos para operação do simulador. A abordagem proposta proporcionou maior versatilidade ao simulador / Abstract: The membrane separation processes (MSP) have been considered as alternative for conventional separation processes. In this research area a strong interdisciplinarity is observed. There is an increasing of interest for these processes considering many aplications (e.g., industrial wastewater treatment, water desalination, purification and concentration of food industry streams). The separation usually does not requires phase change, which means energy savings and moderate temperatures. A membrane separation system separates an inlet stream into two effluent streams known as the permeate and the retentate. The permeate is the portion of the fluid that has passed through the membrane. Whereas the retentate stream contains the constituents that have been rejected by the membrane. The membrane can be polymeric or ceramic. These processes have been applied for processing beverages, juices and aromas. In this work, two of these processes were studied, Reverse Osmosis (RO) and Pervaporation (PV). Pilot scale experiments were accomplished using RO (poliamide membrane) for concentrating single strength orange juice at 20ºBrix. The retention of volatile compounds (acetaldehyde, methanol and ethanol) was evaluated by monitoring feed and permeate streams. The retention results obtained were unsatisfactory. The membrane presented low retention for monitored volatiles under studied temperature conditions. In the second stage of this work, the PV process was evaluated for recovering an important ester of orange juice, the ethyl butyrate. The PERVAP software, an essentially predictive Fortran simulator, was used for evaluating process performance considering two membranes, polydimethylsiloxane (PDMS) and polyoctylmethylsiloxane (POMS). It was accomplished the prediction of properties for polymeric membranes targeting the software predictivity improvement. Viscosity data predicted for POMS was crucial for calculating parameters required by simulator. The predictive approach proposed improved the software versatility / Doutorado / Desenvolvimento de Processos Químicos / Doutor em Engenharia Química

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-06-21T17:17:58Z No. of bitstreams: 2 Dissertação - Marília Ferreira Pires - 2016.pdf: 2038317 bytes, checksum: 6bf59849e1b382d63908376fa0df3f69 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Cláudia Bueno (claudiamoura18@gmail.com) on 2017-07-07T19:53:05Z (GMT) No. of bitstreams: 2 Dissertação - Marília Ferreira Pires - 2016.pdf: 2038317 bytes, checksum: 6bf59849e1b382d63908376fa0df3f69 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-07-07T19:53:05Z (GMT). No. of bitstreams: 2 Dissertação - Marília Ferreira Pires - 2016.pdf: 2038317 bytes, checksum: 6bf59849e1b382d63908376fa0df3f69 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-20 / The objective of the present study was to evaluate the effect of adding increasing levels of protected sodium butyrate in the diet of laying hens from 61 weeks of age on performance, egg quality and intestinal parameters. The treatments were 0, 105, 210 and 300 g / t of sodium butyrate protected in the feed. To evaluate the performance and quality of eggs, 320 laying hens were used as Dekalb Whyte lineage (61-76 weeks old) in cages and distributed design in blocks (with the weight of the blocky birds), with four treatments and eight replicates of 10 birds. The experiment was 16 weeks divided into four periods of 28 days each. The 76 weeks of age were separated 48 birds for metabolism trial with four treatments and six replicates of two birds. At the end of the assay was sacrificed a repeat per bird for intestinal development analysis. Was conducted analysis of variance and polynomial regression of the data with the help of the statistical program R. Regarding the performance for the period 73-76 weeks of age was negative linear effect for feed conversion (kg feed / kg eggs). For quality variables eggs were observed increasing linear effect for shell thickness in the periods 61-64 and 65-68 weeks and during the periods 69-72 and 73-76 weeks were observed quadratic effect with maximum point 199 and 174 g / t sodium butyrate protected, respectively. For specific gravity quadratic effect was found with a maximum point at 177 g / t sodium butyrate protected only in the period 73-76 weeks. To the percentage of peeling was found quadratic effect on periods 69-72 and 73 to 76 weeks, with a maximum at point 89 and 172 g / t sodium butyrate protected, respectively. To yolk index found a quadratic effect in the period 69-72 weeks of age with a minimum point at 189 g / t of protected sodium butyrate. When evaluating albume index, found a negative linear effect in the period 69-72 weeks of age. For Haugh unit regression was effective only in the period 65-68 weeks with a quadratic effect, with maximum point at 142 g / t of sodium butyrate. In total period was observed quadratic effect for shell thickness, percentage of peel and breakage resistance, in which maximum points were found in 193; 136 and 198 g / t sodium butyrate protected, respectively. When evaluating yolk index in the total period, found a quadratic effect with minimum point at 181 g / t. For intestinal development variables it was found negative linear effect for the length of the jejunum. For height of villi, it found a linear increase in the duodenum and jejunum. For crypt depth, quadratic effect was found in the jejunum with a minimum point at 151 g / t sodium butyrate protected. For the variable villous / crypt ratio was found linear increase in the duodenum and jejunum, and the ileum quadratic effect, with maximum point at 171 g / t. For BN was found quadratic effect with maximum point at 93 g / t, and for BEE was found increasing linear effect. For EMA and EMAn variables were found increasing linear effect. The addition of protected sodium butyrate in laying hens diet did not influence the performance of the birds, but improved the quality of skin and intestinal parameters (intestinal development and metabolization of nutrients). It is recommended to improve the quality of shell eggs in layers in the final production phase, the level of 150 g / t sodium butyrate protected. For intestinal development and diet metabolizable energy level of 300 g / t of sodium butyrate protected. / Objetivou-se com o presente estudo avaliar o efeito da adição de níveis crescentes de butirato de sódio protegido na ração de poedeiras comerciais, a partir de 61 semanas de idade, sobre o desempenho, qualidade de ovos e parâmetros intestinais. Os tratamentos foram: 0, 105, 210 e 300 g/t de butirato de sódio protegido na ração. Para avaliação do desempenho e qualidade de ovos, foram utilizadas 320 poedeiras comerciais da linhagem Dekalb Whyte (61 a 76 semanas de idade) alojadas em gaiolas e distribuídas em delineamento em blocos ao acaso (sendo o peso das aves blocado), com quatro tratamentos e oito repetições de 10 aves. O experimento teve duração de 16 semanas divididas em quatro períodos de 28 dias cada. As 76 semanas de idade foram separadas 48 aves para o ensaio metabólico, com quatro tratamentos e seis repetições de duas aves. Ao final do ensaio metabólico foi sacrificada uma ave por repetição para análise de desenvolvimento intestinal. Foi realizado análise de variância e regressão polinomial dos dados com auxílio do programa estatístico R. Com relação ao desempenho, para o período de 73 a 76 semanas de idade houve efeito linear negativo para conversão alimentar (kg ração/kg de ovos). Para as variáveis de qualidade de ovos foram observados efeito linear crescente para espessura da casca nos períodos de 61 a 64 e 65 a 68 semanas, e nos períodos de 69 a 72 e 73 a 76 semanas foram observados efeito quadrático com ponto de máxima em 199 e 174 g/t de butirato de sódio protegido, respectivamente. Para gravidade específica foi encontrado efeito quadrático com ponto de máxima em 177 g/t de butirato de sódio protegido apenas no período de 73 a 76 semanas. Para a porcentagem de casca foi encontrado efeito quadrático nos períodos de 69 a 72 e 73 a 76 semanas, com ponto de máxima em 89 e 172 g/t de butirato de sódio protegido, respectivamente. Para índice de gema encontrou-se efeito quadrático no período de 69 a 72 semanas de idade com ponto de mínima em 189 g/t de butirato de sódio protegido. Ao avaliar índice de albúmen, encontrou-se efeito linear negativo no período de 69 a 72 semanas de idade. Para unidade Haugh houve efeito de regressão apenas no período de 65 a 68 semanas com efeito quadrático, apresentando ponto de máxima em 142 g/t de butirato de sódio. No período total foi observado efeito quadrático para espessura de casca, porcentagem de casca e resistência à quebra, nos quais foram encontrados pontos de máxima em 193; 136 e 198 g/t de butirato de sódio protegido, respectivamente. Ao avaliar índice de gema no período total, encontrou-se efeito quadrático com ponto de mínima em 181 g/t. Para as variáveis de desenvolvimento intestinal, foi encontrado efeito linear negativo para comprimento do jejuno. Para altura de vilos, foi encontrado efeito linear crescente no duodeno e jejuno. Para profundidade de criptas, foi encontrado efeito quadrático no jejuno, com ponto de mínima em 151 g/t de butirato de sódio protegido. Para a variável relação vilo/cripta, foi encontrado efeito linear crescente no duodeno e jejuno, e efeito quadrático no íleo, com ponto de máxima em 171 g/t. Para BN foi encontrado efeito quadrático com ponto de máxima em 93 g/t, e para BEE foi encontrado efeito linear crescente. Para as variáveis EMA e EMAn foram encontrados efeito linear crescente. A adição de butirato de sódio protegido na dieta de poedeiras comerciais não influenciou no desempenho das aves, mas melhorou a qualidade de casca e os parâmetros intestinais (desenvolvimento intestinal e metabolizabilidade de nutrientes). Recomenda-se, para melhorar a qualidade de casca de ovos em poedeiras na fase final de produção, o nível de 150 g/t de butirato de sódio protegido. Para desenvolvimento intestinal e metabolizabilidade da energia da dieta o nível de 300 g/t do butirato de sódio protegido.

Résumé<p>L’objectif de ce travail a été d’évaluer la toxicité du butyrate de sodium (NaBu), un inhibiteur des<p>histones désacétylases (HDACs), et ses mécanismes d’action sur les cellules de cancer du sein<p>humain, les cellules MCF-7 déficientes pour la caspase-3, et lignées dérivées :les cellules MCF-<p>7/caspase-3, et les cellules VCREMS résistantes à la vincristine, et dans une moindre mesure à la<p>doxorubicine. La contribution de l’apoptose dans la létalité induite par le NaBu a été recherchée<p>dans les cellules MCF-7wt en estimant l’exposition de la phosphatidylsérine ainsi que le clivage de<p>la PARP. La présence de caspase-3, n’a ni amplifié ni accéléré l’apoptose qui a impliqué le<p>rhéostat Bax/Bcl-2 en faveur d’une induction de Bax. La cytostasie du NaBu dans les cellules<p>MCF-7 s’est manifestée par un blocage des cellules en phase G2/M. L’évaluation du niveau<p>d’expression des régulateurs du cycle cellulaire dans les cellules MCF-7wt et MCF-7/caspase-3 a<p>montré une surexpression de p21, de façon indépendante de p53. L’action cytostatique du NaBu<p>a été associée à une accumulation légère et modeste des formes non-phosphorylées de pRB, un<p>facteur dont la phosphorylation par les complexes cycline D/cdk4,6 et cycline E/cdk2 est<p>nécessaire à la transition G1/S. Dans ces conditions, les niveaux de cdk2 et de Cdc25A, une<p>oncoprotéine activatrice de cdk2, sont restés stables. Le NaBu est une molécule à effet<p>pléïotropique, l’utilisation de la trichostatine A, inhibiteur par excellence des HDACs, a permis<p>d’établir la relation de causalité entre l’inhibition des HDACs et la toxicité du NaBu. La plupart<p>des inhibiteurs des HDACs induisent l’apoptose en perturbant le métabolisme oxydatif de la<p>mitochondrie ce qui pourrait modifier le statut redox cellulaire. Nous avons cherché une<p>implication du métabolisme du glutathion (GSH), le thiol anti-oxydant non-protéique majoritaire<p>de la cellule, dans la toxicité induite par le NaBu. Les résultats montrent que le NaBu induit une<p>déplétion du GSH dans les cellules MCF-7wt et dérivées de façon dose-dépendante, corrélée avec<p>la mortalité cellulaire. Devant l’éventualité d’une consommation accrue de GSH par les enzymes<p>associées à son métabolisme, nous avons évalué le niveau des activités des enzymes glutathion<p>peroxydase, glutathion réductase et glutathion S-transférases. Dans les cellules MCF-7, le NaBu a<p>induit de façon significative ces enzymes anti-oxydantes, à l’exception des GSTs, de même que la<p>catalase, une enzyme indépendante de ce système. Les expériences visant à libérer le pool de<p>GSH lié aux protéines ont montré que la déplétion du GSH intracellulaire est parallèle à celle du<p>GSH lié aux protéines. Par conséquent, la consommation du GSH est réellement la cause de la<p>chute du niveau de GSH générant un stress oxydant. La doxorubicine, un inhibiteur des<p>topoisomérases, a une utilisation clinique limitée en raison de ses effets secondaires irréversibles<p>(cardiotoxicité entre autres). Dans le but d’améliorer son efficacité, nous avons expérimenté des<p>combinaisons NaBu/doxorubicine sur les cellules VCREMS et MCF-7, étant donné la capacité<p>du NaBu à induire l’expression des topoisomérases et favoriser la conformation déployée de la<p>chromatine. L’utilisation de la technique isobologramme nous a permis de déterminer les index<p>de combinaison pour une application simultanée ou séquentielle des drogues. Les résultats<p>indiquent que le NaBu sensibilise les cellules VCREMS et MCF-7 à l’action de la doxorubicine.<p>Dans les cellules VCREMS, cet effet s’est produit en dépit de la stimulation des enzymes de<p>détoxication, GSTs et GPX. L’ensemble de ces résultats indique que l’utilisation du NaBu en<p>combinaison avec certains anticancéreux constitue une stratégie très intéressante en<p>cancérothérapie. / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished