ABSTRACT PURIFICATION AND CHARACTERIZATION OF CYTOPLASMIC AND PROTEASOME ASSOCIATED CHYMOTRYPSIN-LIKE PROTEASES FROM THERMOPLASMA VOLCANIUM &Ouml / zdemir, F.inci Ph.D., Department of Biology Supervisor: Prof. Dr. Semra Kocabiyik September, 147 pages In this study, two novel cytoplasmic serine proteases were isolated and characterized from thermophilic archaea Thermoplasma volcanium. The first protease was purified by ion exchange and affinity chromatographies and identified as a chymotrypsin-like serine protease mainly based on its substrate profile and inhibition pattern. The presence of protease activity was analyzed by gelatin zymography which was detected as a single band (35 kDa). Optimum temperature was found to be 60oC for azocasein hydrolysis and 50oC for N-Suc-Phe-pNA hydrolysis. Optimum activity was observed in the pH range of 6.0-8.0 with a maximum value at pH 7.0. The Km and Vmax values for the purified protease were calculated to be 2.2 mM and 40 &micro / moles of p-nitroanilide released min-1.ml-1, respectively, for N-Suc-Phe-PNA as substrate. Ca2+ and Mg2+ at 4 mM concentrations were the most effective divalent cations in activating the enzyme. In the second stage of this study, 20S proteasome of Tp. volcanium with substantial chymotrypsin-like activity was purified and characterized. This enzyme complex was purified with 19.1 U/mg specific activities from cell free extract by a four-step procedure. SDS-PAGE analysis revealed two strong bands with relative molecular masses of 26 kDa (&amp / #945 / -subunit) and 21.9 kDa (&amp / #946 / -subunit). Tp. volcanium 20S proteasome predominantly catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residue Phe (chymotrypsin-like activity) in short chromogenic peptides. Low-level hydrolyzing activity was also detected carboxyl to basic residue Arg (trypsin-like activity). Chymotrypsin-like activity of Tp. volcanium 20S proteasome was significantly inhibited by chymotrypsin specific serine protease inhibitor chymostatin. When N-CBZ-Arg was used which is a substrate for trypsin, 20S proteasome was strongly inhibited by TLCK. The optimum temperature for Ala-Ala-Phe-pNA hydrolysis by the Tp. volcanium 20S proteasome was 55oC and the optimum pH was 7.5. The chymotryptic activity was significantly enhanced by divalent cations such as Ca+2 and Mg2+ at high concentrations, i.e. 125-250 mM. Keywords:Serine protease, 20S proteasome, archaea, thermophilic protease, Thermoplasma volcanium, chymotrypsin-like serine protease.

Under år 2008 användes globalt en energimängd motsvarande nästan 144 000 TWh ochav dessa stod fossila bränslen för 81 %. I Sverige uppgick energitillförseln under år2010 till totalt 616 TWh och av detta stod råolja/oljeprodukter för 30,4 %. Vidförbränning av fossila bränslen frigörs koldioxid, en gas som bidrar till att förstärkaväxthuseffekten. År 2000 uppmättes halten av koldioxid i atmosfären till 370 ppmv ochför att den globala temperaturen inte ska öka med mer än 2°C bör halten stanna på 450ppmv innan år 2100. Ett sätt att minska andelen av fossila bränslen är att öka andelen avförnybara energikällor, som t.ex. biogas, som i Sverige uppskattas kunna ge enenergimängd motsvarande 10-15 TWh/år i framtiden.Vid biogasanläggningen Kungsängens gård, Uppsala, samrötas slakteriavfall samtorganiskt avfall från hushåll och livsmedelsindustri i en termofil rötningsprocess. Underår 2011 producerades ca 3 400 000 Nm3 biogas och den största andelen uppgraderadestill fordonsgas. Behovet fordonsgas i Uppsala ökar och i detta examensarbete utreddestvå sätt att effektivisera processen och öka gasproduktionen. Dels undersöktes om enökad belastning skulle ge en ökad biogasproduktion utan att riskera processensstabilitet. Detta gjordes i två labskalereaktorer där belastningen ökades gradvis i denena. Dels studerades möjligheten att minska energiförbrukningen på anläggningengenom att byta hygieniseringsmetod. Innan substratet matas in i rötkamrarna måste dethygieniseras, vilket i dagsläget görs genom pastörisering (upphettning till 70oC under entimme). Då detta är väldigt energikrävande finns det planer på att byta metod ochistället låta substratet hygieniseras i rötkamrarna (52°C) i minst 10 timmar. Det är dockviktigt att beakta huruvida metanpotentialen för pastöriserat och opastöriserat materialskiljer sig åt, varför detta utreddes i sk satsvisa utrötningsförsök.Genom hela belastningsökningen (från 3 till 6 kg VS/m3,d) ökade biogasproduktionenoch vid den högsta belastningen var ökningen 100 % jämfört med dagens nivå. Andraviktiga processparametrar, så som specifik gasproduktion, kvoten CO2/CH4, pH,halterna av fettsyror och utrötningsgraden, låg på en jämn nivå under försöket, vilkettyder på att processen var stabil trots den ökade belastningen. Utrötningsförsöket visadeatt pastöriseringen inte hade någon effekt på metanproduktionen, troligtvis eftersomsubstratet redan var lättnedbrytbart. Beräkningen av energiförbrukning visade attenergianvändningen skulle minska med ca 33 % vid byte av hygieniseringsmetod. / During 2008 an amount of energy equivalent to almost 144,000 TWh was used globally,of which fossil fuels accounted for 81 %. In Sweden, during 2010, an amount of energyequivalent to 616 TWh was used, of which crude oil/oil products accounted for 30.4 %.Carbon dioxide, a gas that contributes to the global warming, is produced during thecombustion of fossil fuels. In 2000 the levels of carbon dioxide in the atmosphere wasmeasured to be 370 ppmv and if the global temperature is not to increase with morethan 2°C, the levels should stay at 450 ppmv by 2100. One way of decreasing the use offossil fuels is to increase the use of renewable energy, such as biogas. In the futurebiogas can, approx., provide with energy equivalent to 10-15 TWh/year in Sweden.At the biogas plant Kungsängens gård, in Uppsala, slaughterhouse byproducts are codigestedwith source separated household waste and waste from the food processingindustry in a thermophilic process. During the year 2011 approximately 3,400,000 Nm3of biogas was produced at the plant, of which most was upgraded to vehicle fuel. Theconsumption of vehicle fuel is increasing in Uppsala and thus there is a need forincreased biogas production. The aim of this master thesis was to investigate two waysto increase the efficiency and consequently the gas production at the biogas plant atKungsängens gård. Firstly, it was studied if an increased organic loading rate (OLR)would give an increased biogas production, without disturbing the process. This wasdone in two lab scale reactors, where the load was increased gradually in one. Secondly,the possibility to decrease the energy consumption by means of a change of sanitizationmethod was studied. The substrate has to be sanitized before it is fed to the digesters,currently this is done by pasteurization. This process is, however, energy-demandingand there are plans to change the method of sanitization. It is, however, important toconsider whether the methane potential differs for the pasteurized and the nonpasteurizedsubstrate. This was studied in small scale biogas batch reactors.Through all stages of increased OLR (from 3 to 6 kg VS/m3, d) the biogas productionincreased, and at the largest load the increase was 100 % compared to the present level.Other important process parameters, such as specific methane production, CO2/CH4-ratio, pH, levels of fatty acids and degree of digestion, were at regular levels and thisindicates that the process was maintained stable in spite of the increased load. The testin the small scale biogas batch reactors showed that pasteurization of the substrate hadno effect on the methane potential, probably due to that the substrate already is readilybiodegradable. The estimation of the energy consumption showed that the use of energyshould decrease with approx. 33 % if the sanitization was replaced.

In this study, the effects of bioprocess parameters on glucose isomerase roduction by Bacillus thermoantarcticus (DSMZ 9572) were investigated. For his purpose, firstly, in laboratory scale shake-bioreactors, a defined medium was designed in terms of its carbon and nitrogen sources, to achieve the highest glucose isomerase activity. Optimum concentrations of medium components were determined by a statistical approach, namely Response Surface Methodology (RSM). RSM defined the relationship between the response, glucose isomerase activity and the independent variables, medium components. The highest glucose isomerase volumetric activity was obtained as 1630 U L-1 in the optimized medium containing 10.64 kg m-3 xylan, 5.66 kg m-3 yeast extract, 5.92 kg m-3 (NH4)2SO4, 0.25 kg m-3 MgSO4.7H2O, 0.001 kg m-3 FeSO4.7H2O, 0.001 kg m-3 ZnSO4.7H2O , 0.000075 kg m-3 MnSO4. H2O, and 0.00001 kg m-3 CuSO4.5H2O at conditions: pH0= 6.0, T=55&deg / C, N=200 min-1. Then by using the designed medium, pH and oxygen transfer conditions of the bioprocess were investigated in 3.0 dm3 pilot scale bioreactor. The effect of pH was analyzed at pH=6 uncontrolled and controlled operations with the following conditions: air inlet rate of Q0/VR=0.5 vvm and agitation rate of N=500 min-1. The effects of oxygen transfer parameters were examined, at pH=6 controlled condition, at an air inlet rate of Q0/VR=0.5 vvm and the agitation rates of N=300,500,750 min-1. The variations in glucose isomerase activity, cell, amino acid and organic acid concentrations with the cell cultivation time, specific cell growth rate, the oxygen uptake rate, the liquid phase coefficient by using the dynamic method, maintenance coefficient for oxygen and yield coefficients were determined. The highest glucose isomerase volumetric activity and cell concentration, 1838 U L-1 and 2.26 kg m-3 at the 24th of the cell cultivation were attained at 0.5 vvm, 500 min 1 and pH=6 uncontrolled operation.

Made available in DSpace on 2014-06-11T19:23:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2002-09-23Bitstream added on 2014-06-13T20:10:52Z : No. of bitstreams: 1 rabalho_aa_me_sjrp.pdf: 3150566 bytes, checksum: 6d823b8dd0082afcc44630ccf0ecf00a (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Sabe-se que uma variedade de microrganismos produzem um ou mais tipos de amilases para degradar os amiláceos presentes no ambiente e que as amilases produzidas por microrganismos termofílicos apresentam características mais termoestáveis do que aquelas produzidas por mesófilos. Essas amilases termoestáveis são de grande interesse na indústria de processamento de amido, uma vez a temperatura do gelatinização do mesmo, etapa importante do processo, fica em torno de 70ºC. Além disso, os processos que ocorrem em altas temperaturas, têm menor risco de contaminação por mesófilos e a diminuição da viscosidade do meio permite trabalhar com elevadas concentrações de substrato. No presente trabalho foram isoladas, a partir de amostras de solos, compostagens e resíduos agro-industriais, 339 linhagens microbianas termofílicas (326 bacterianas e 13 fúngicas) capazes de crescer a 55ºC em meio contendo amido como única fonte de carbono. As 163 linhagens (162 bactérias e 1 fungo) que se destacaram quanto à produção de amilases e à velocidade de crescimento em meio sólido, foram selecionadas e submetidas às fermentações submersa (meio nutriente) e semi-sólida (farelo de trigo). As atividades enzimáticas foram determinadas pelo método dextrinizante (a- amilase), pela quantificação da glicose (glucoamilase) e de açúcares redutores liberados a partir da hidrólise do amido. Das linhagens testadas, nove mostraram-se boas produtoras de a-amilase em fermentação submersa e/ou semi-sólida, fato pelo qual foram selecionadas para produção de amilases em meios formulados a partir de resíduos líquidos da extração de mandioca e milho (fermentação submersa) e sólidos, constituídos de farelos de mandioca, milho e trigo (fermentação semi-sólida). Quatro linhagens se destacaram, sendo suas enzimas brutas caracterizadas quanto às propriedades físico-químicas... / It is known that some microorganisms produce one or more amylase types that degrade organic compound present in the environment. The amylases produced by thermophilic microorganisms present characteristics more thermostable than those produced by mesophilics. Since starch gelatinization temperature is around 70ºC, the thermostable amylases are very important to starch processing industry. Besides, the processes that happen in high temperatures have less risk of contamination for the mesophilic microorganisms, the decrease of the viscosity of the media and allows to work with high substrate concentrations. In the present work thermophilic microorganisms (326 bacteria and 13 fungi) were isolated from soil samples, decaying agricultural waste and agriculture-industrial residues. They are able to grow at 55ºC in media containing starch as only carbon source. 163 strains (162 bacteria and 1 fungus) that showed high amylase production and fast growth in solid media were selected and submitted to the submerged (nutrient media) and semi-solid (wheat bran) fermentations. The enzymatic activities were determined by dextrinogenic method (a-amylase), by the quantification of the glucose (glucoamylase) and of the reducing sugars released from the starch hydrolysis. Among the tested strains, nine have shown high a-amylase production in submerged and/or semi-solid fermentations and because of this they were selected for amylase production in medium formulated with liquid wastes of extraction starch from the cassava and corn (submerged fermentation), and solid wastes constituted of cassava, corn, and wheat brans (semi-solid fermentation). Among these strains, four were selected and their crude enzymes were characterized regarding to their physicochemical properties. The strains Bacillus sp A13-22 and Mucor sp A13-36 produced amylases with optimum temperature of 70 and 80 ºC...(Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-18Bitstream added on 2014-06-13T20:04:38Z : No. of bitstreams: 1 ferrarezi_al_dr_rcla.pdf: 2316153 bytes, checksum: 70628c1ba7de515160f7ca0246de5404 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As enzimas são catalisadores muito eficientes e de grande interesse na aplicação industrial. As lipases (E.C. 3.1.1.3) são serina-hidrolases que agem na hidrólise, esterificação e transesterificação de acilgliceróis de cadeia longa. Lipases microbianas têm sido amplamente usadas devido à sua especificidade. Na transesterificação moléculas de triacilglicerol reagem com um álcool na presença de um catalisador formando uma mistura de glicerol e ésteres de ácidos graxos. O biodiesel, definido como ésteres metílicos ou etílicos, tem atraído crescente interesse como uma fonte de energia renovável, substituindo o diesel produzido a partir de combustíveis fósseis. O presente trabalho tem como objetivo principal efetuar a prospecção de fungos termofílicos que apresentem produção significativa de lipase e, concomitantemente, atividade transesterificante. As linhagens foram selecionadas por detecção de atividade lipolítica em placas de ágar contendo Rodamina B, por fermentação submersa (FSm) e fermentação em estado sólido (FES). Foram testadas linhagens de fungos termofílicos da coleção do laboratório de Bioquímica e Microbiologia Aplicada, sendo Thermomucor indicae-seudaticae N31, Rhizomucor pusillus Myceliophtora sp F2.1.4, Myceliophtora sp M7.7, F2.1.1, F2.1.3 e Thermomyces lanuginosus TO-05 os que mostraram um maior potencial hidrolítico. Estudos adicionais avaliaram a produção de lipase através da modificação da fonte de componentes nutricionais e algumas propriedades físicas na produção de lipase por T. indicae-seudaricae N31 em FSm, e por R. pusillus e T. indicae-seudaticae N31 em FES. Os estudos dos processos fermentativos foram bem sucedidos, havendo um aumento de 16 vezes na produção de lipase de R. pusillus e de 36 vezes na lipase de T indicae-seudaticae... / Enzymes are efficient catalysts and interesting for industrial applications. Lipases (EC 3.1.1.3) constitute a group of serine hydrolases that catalyze the hydrolysis, esterification and transesterification reactions of long chain acylglycerols. Microbial lipases have been widely used for biotechnological applications due to their specificity. In transesterification, molecule of a tryacylglicerol react with an alcohol in the presence of catalyst, producing a mixture of fatty esters and glycerol, Biodiesel, defined as methyl or ethyl fatty esters and has attracted considerable attention as a renewable source of energy, in substitution of fossil fuels. The main goal of the present work is the screening of thermophilic fungi that present outstanding lipase production and in parallel able to perform transesterification reaction. Strains were screened for lipase activity on agar plates containing Rhodamine B, for submerged fermentation (SmF) and solid state fermentation (SSF). The tested thermophilic strains were from the collections of the Biochemistry and Applied Microbiology Laboratory, where Thermomucor indicae-seudancae N31, Rhizomucor pusillus Myceliophtora sp F2.1.4, Myceliophtora sp M7.7, F2.1.1, F2.1.3 and Thermomyces lanuginosus TO-O5 had the highest lipase production. Additional studies attempted to improve lipase production by nutrient source modifications and physicals conditions in FmS by T. indicae-seudaticae N31 and in FSS by T. indicae-seudaticae N31 and R. pusillus. The fermentations studies were successful, with a 16 fold enhancement in lipase yield compared to the initial medim from lipase R. pusillus and 36 fold for the lipase T. indicae-seudaticae N31, both in FES. The lipase from T. indicae-seudaticae N31 cultured in SSF and SmF, exhibited maximum lipolytic activity at 40°C and stability for the pH range from 4 to 8. The enzyme produced by FmS presented maximum activity... (Complete abstract click electronic access below)

abstract: Microbial Electrochemical Cell (MXC) technology harnesses the power stored in wastewater by using anode respiring bacteria (ARB) as a biofilm catalyst to convert the energy stored in waste into hydrogen or electricity. ARB, or exoelectrogens, are able to convert the chemical energy stored in wastes into electrical energy by transporting electrons extracellularly and then transferring them to an electrode. If MXC technology is to be feasible for ‘real world’ applications, it is essential that diverse ARB are discovered and their unique physiologies elucidated- ones which are capable of consuming a broad spectrum of wastes from different contaminated water sources. This dissertation examines the use of Gram-positive thermophilic (60 ◦C) ARB in MXCs since very little is known regarding the behavior of these microorganisms in this setting. Here, we begin with the draft sequence of the Thermincola ferriacetica genome and reveal the presence of 35 multiheme c-type cytochromes. In addition, we employ electrochemical techniques including cyclic voltammetry (CV) and chronoamperometry (CA) to gain insight into the presence of multiple pathways for extracellular electron transport (EET) and current production (j) limitations in T. ferriacetica biofilms. Next, Thermoanaerobacter pseudethanolicus, a fermentative ARB, is investigated for its ability to ferment pentose and hexose sugars prior to using its fermentation products, including acetate and lactate, for current production in an MXC. Using CA, current production is tracked over time with the generation and consumption of fermentation products. Using CV, the midpoint potential (EKA) of the T. pseudethanolicus EET pathway is revealed. Lastly, a cellulolytic microbial consortium was employed for the purpose ofassessing the feasibility of using thermophilic MXCs for the conversion of solid waste into current production. Here, a highly enriched consortium of bacteria, predominately from the Firmicutes phylum, is capable of generating current from solid cellulosic materials. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2015

Made available in DSpace on 2014-06-11T19:22:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-23Bitstream added on 2014-06-13T18:49:55Z : No. of bitstreams: 1 zanphorlin_lm_me_sjrp.pdf: 945446 bytes, checksum: 9959e6f0472c3ac5cdce9a3374c74387 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fungos termofílicos têm despertado grande interesse acadêmico e industrial por produzirem uma variedade de enzimas termoestáveis com potenciais aplicações em processos biotecnológicos como biocatálise nas indústrias de couro, farmacêutica, têxtil e alimentícia, e na preparação de produtos de limpeza e cosméticos. Particularmente, as proteases, além de participarem de inúmeros processos fisiológicos vitais como vias metabólicas, hemostasia e sinalização celular, também representam hoje cerca de 60% do mercado mundial de enzimas. Neste trabalho, descrevemos a produção, purificação e caracterização bioquímica de uma serino-protease produzida por um fungo termofílico do gênero Myceliophthora. As taxas de atividade proteolítica foram avaliadas através de fermentação em meio sólido (FES) e submerso (FSM) e observou-se um rendimento na atividade proteolítica 4,5 vezes maior para o meio sólido. A enzima bruta obtida por ambos os procedimentos (FES e FSM) exibiu a mesma temperatura ótima de 50 ºC, porém em relação ao pH ótimo houve um deslocamento de 7 (FSM) para 9 (FES) sugerindo que o perfil enzimático do fungo difere de acordo com suas condições de fermentação. Baseado nesses resultados prosseguiu-se os estudos com o extrato bruto obtido por FES. A imobilização da enzima bruta em esferas de alginato de cálcio resultou no aumento da temperatura ótima e na estabilidade térmica quando comparado com a enzima livre. O extrato bruto obtido por FES foi, então, fracionado por métodos cromatográficos como exclusão molecular e troca-iônica que resultaram na protease pura com peso molecular de 28,2 kDa determinado por espectrometria de massa. A protease pura demonstrou pH ótimo de 9,0 e temperatura ótima de 45 °C que corroboram... / Thermophilic fungi have attracted great academic and industrial interest because they produce a variety of thermostable enzymes with potential applications in biotechnological processes such as biocatalysis in the industries of leather, pharmaceutical, textile and food, and the preparation of detergents and cosmetics. In particular, proteases not only participate in many vital physiological processes such as metabolic pathways, cell signaling and homeostasis, but also currently represent about 60% of the world market of enzymes. In this work, we describe the production, purification and biochemical characterization of a serine protease produced by a thermophilic fungus of the genus Myceliophthora. The levels of proteolytic activity were evaluated either by solid fermentation (SSF) and submerged (SmF). The crude enzyme obtained by both procedures (SSF and SmF) exhibited similar optimum temperature of around 50 ºC, but in relation to the optimum pH was shifted of 7 (SmF) to 9 (SSF), suggesting that the enzymatic profile of the fungus differs from according to its fermentation conditions. Based on these results, the studies were followed with crude extract obtained by SSF. The immobilized enzyme on beads of calcium alginate resulted in increased optimum temperature and thermal stability when compared to the free enzyme. The crude extract obtained by SSF was then fractionated by chromatographic methods including molecular exclusion and ion-exchange that resulted in the pure protease with molecular weight of 28.2 kDa as determined by mass spectrometry. The pure protease showed optimum pH of 9.0 and optimum temperature of 45 °C that corroborate to the preliminary characterization of the crude extract. Inhibition tests resulted in complete inhibition by PMSF, a canonical... (Complete abstract click electronic access below)

O estudo das temperaturas preferenciais e comportamento termofílico é essencial na compreensão da termobiologia das serpentes, todavia, a maioria dos trabalhos tem focado somente os animais diurnos e de clima temperado e pouco se sabe a respeito das serpentes noturnas tropicais e sub-tropicais. O presente estudo utilizou uma arena com gradiente térmico monitorada por vídeo para mensurar o intervalo térmico preferencial procurado durante os períodos de atividade e inatividade (Tset) e a resposta termofílica pós-prandial frente a diferentes quantidades de ingesta em 29 serpentes Bothrops moojeni . Os resultados obtidos mostraram que o intervalo térmico preferencial de B.moojeni diferiu conforme o fotoperíodo (20,93°C a 22,20°C durante o dia e 22,81°C a 24,42°C durante a noite) e foi similar a outros intervalos encontrados para outras serpentes crotalinae. Os dados mostraram que há uma correlação inversa entre intensidade da resposta termofílica pós-prandial e quantidade de alimento ingerido, uma vez que os animais alimentados com uma porção correspondente a 10% de seu peso corporal apresentaram maior resposta termofílica pós-prandial quando comparados àqueles que foram alimentados com porções maiores (20 e 40% do peso corporal). Esta diferença parece estar relacionada com a diminuição de mobilidade e a maior SDA (Specific Dynamic Action) nos animais que ingeriram a maior quantidade de alimento. As serpentes crotalinae são tidas como animais preponderantemente noturnos sendo que a maioria dos dados a respeito dessas atividades foram obtidos em pesquisas de campo. A detecção do período de atividade, assim como o conhecimento de como o estado nutricional influencia a taxa de trânsito, são extremamente importantes na compreensão da ecofisiologia destes animais. No estudo foram utilizadas 29 exemplares de Bothrops moojeni, monitoradas quanto ao horário e o tempo de deslocamento durante o jejum e nos 5 dias subseqüentes a alimentação. A alimentação das serpentes variou entre 10, 20 e 40% do peso da própria serpente. Os resultados confirmaram Bothrops moojeni como espécie essencialmente noturna, sendo que a atividade foi aumentada em mais de 45 vezes durante o período. O pico de atividade ocorreu entre as 19:00 e 20:00 horas, não havendo diferença estatística entre machos e fêmeas. Com relação à atividade pós-prandial foi notada diferença estatística nas taxas de deslocamento dos animais que comeram as maiores proporções (20-40%). Os resultados obtidos corroboram com os dados prévios obtidos em estudos de campo que pontuam Bothrops moojeni como um animal noturno e de baixo grau de mobilidade. A interação entre a quantidade de alimento ingerido pela serpente, o balanço energético e o grau de mobilidade dos animais é bastante complexa e discutível. As serpentes foram consideradas por muito tempo como incapazes de gerar calor através de seu metabolismo aeróbico, à exceção das pythons no choco. Entretanto, estudos recentes mostraram que algumas espécies são capazes de aumentar a temperatura corporal sem recorrer a fontes externas de calor ou contrações musculares. Esta termogênese ocorre devido aos enormes níveis metabólicos atingidos durante a digestão (Specific Dynamic Action - SDA). Em geral as serpentes têm um baixo custo energético de manutenção e a maioria das espécies é capaz de permanecer longo período em jejum e depois se alimentar de uma grande ingesta. A energia produzida durante a digestão destas grandes refeições pode gerar um significativo aumento na temperatura corpórea dos animais. O presente estudo investigou a termogênese pós-prandial em Bothrops moojeni, através de imagens térmicas tomadas em um ambiente termoestável a 30°C. As 12 serpentes foram divididas em 2 grupos e acompanhadas pelas 72 horas após ingerirem refeições equivalentes a 10-60 20% e 30-40% de seu próprio peso, respectivamente. Os resultados mostraram que a resposta termogênica pós-prandial levou a um aumento de 1,6°C da temperatura da pele das serpentes. O pico térmico ocorreu entre 33 e 36 horas após a alimentação em ambos os grupos e a duração da termogênese variou conforme o tamanho da ingesta. Os resultados mostraram uma alta correlação entre a termogênese pós-prandial e a SDA das serpentes. O aumento significativo da temperatura corporal e a sua manutenção por certo tempo sugerem que estes animais são capazes de manter altas taxas metabólica durante a digestão independente da temperatura do ambiente. / The study of the preferred temperatures and thermophilic behavior is essential to understand the snakes thermal biology. Although some studies have been reported, most of them are focused in temperate and diurnal species. Thus, little is known about the tropical and sub-tropical nocturnal species. In the present study , a video-monitored arena with a thermal gradient was used to investigate the preferred thermal range during activity and inactivity (Tset) and the post-prandial thermophilic response after different levels of food intake in 29 Brazilian lanceheads (Bothrops moojeni). The results showed that the preferred thermal range of B.moojeni changes depending on the photoperiod (20.93°C to 22.20°C in daytime and 22.81°C to 24.42°C in night time), being similar to other crotalinae snakes. Our data suggests that there is an inverse correlation between the post-prandial thermophilic response intensity and food intake, as animals fed with a portion corresponding to 10% of its bodyweight presented a higher thermophilic response in comparison to those who were fed with larger portions (20 and 40% of bodyweight). This difference may be related to the decreased mobility and increased SDA (Specific Dynamic Action) in animals that consumed the largest amount of food. Crotalinae are considered to be mainly nocturnal and most of the gathered data on these snakes came from field work. The knowledge of activity pattern and how nutritional status affect wandering rate are determinant to understand the ecophysiology of the snakes. In this study the daily activity pattern was followed in 29 subjects of the lancehead Bothrops moojeni in order to record displacement rate in fasting and feed snakes for five days after feeding meal of 10, 20 and 40% of their own body weight. The results show that B.moojeni is prevalently nocturnal, and activity increased 45 times at night, peaking between 19 to 20 h, in both males and females. Activity level changed significantly after feeding in snakes which ate a larger meal (20-40%). The results confirm previous field data for B.moojeni as a nocturnal species with low mobility. The complex interactions between the amount of food taken by the snake, energetic balance and mobility are discussed. Snakes were for long considered incapable to generate heat fueled by aerobic metabolism, except for brooding pythons. However, recent studies proved that some species are able to elevate their body temperature without muscle contraction or external heat source. Such thermogenic capacity is supported by high metabolic rate that follows digestion and assimilation of food (Specific Dynamic Action - SDA). Snakes have a low maintenance cost and most species can stand for long fasting time, particularly after ingesting a great meal. The energy produced during digestion of such meal can generate significant increase in body temperature. The present study investigated thermogenic response after feeding in the Brazilian lancehead, Bothrops moojeni, using thermal images taken in temperature controlled environment at 30°C. The 12 snakes were divided into two groups and followed for 72 hours after fed a meal representing 10-20% and 30-40% of their body weight, respectively. The results showed thermogenic digestive response with an increase up to 1.6 °C of skin temperature. Thermal heat production peak occurred between 33 to 36 hours after feeding in both groups, and the duration of thermogeny varied with the meal size. The result showed high correlation between thermogenic data and SDA in snakes. The significant increase of body temperature after feeding and its maintenance for extended time suggest a physiological advantage to keep high metabolic rate despite of environmental temperature.

This diploma thesis focuses on the molecular characterization of selected PHA producers. Within this work, the PHA producing thermophilic isolates originating from the samples of activated sludge and compost were identified and characterized using molecular biological methods. By sequencing the 16S rRNA gene, the thermophilic isolates were identified and taxonomically classified into the Firmicutes bacterial phylum. In these bacterial isolates, the ability to produce PHA at the genotype level was determined by conventional PCR detection of the phaC gene encoding PHA synthase, which is a key enzyme in PHA biosynthesis. Class I, II and IV PHA synthases were detected in most of the isolated bacteria, wherein class I and II PHA synthases are not characteristic for these bacterial genera. The largest proportion of isolates was identified for the species of thermophilic bacterium Aneurinibacillus thermoaerophilus, in which class IV PHA synthase was detected. In the second part of the diploma thesis, the RT-qPCR method was implemented to study the expression of selected genes of the bacterium Cupriavidus necator H16 involved in PHA metabolism. As part of the implementation of this method, PCR-based detection of selected genes was optimized and quantification of genes using real-time PCR was performed. The tested method included steps of RNA isolation, cDNA synthesis and quantification of gene segments for which the critical points of the method were determined based on the obtained data.

The thermophilic Campylobacters, Campylobacter jejuni and Campylobacter coli are found as commensals in the intestinal tract of healthy mammals and birds. Campylobacter jejuni is one of the leading causes of sporadic food-borne bacterial disease in humans which is predominantly contracted from poultry products. Although the vast majority of these infections are mild, life-threatening complications should be treated with antimicrobials. Patients are usually treated with either macrolides of fluoroquinolones. However, globally there is an increased trend in the development of resistance to these antibiotics. This trend has also been observed in infection of poultry and pigs. The aim of this investigation was to determine antimicrobial sensitivity of thermophilic Campylobacters isolated from pigs and poultry by broth microdilution minimum inhibitory concentration testing. A total of 482 samples of the small intestinal content from poultry and pigs from the Western Cape and Gauteng Provinces were collected and analysed. Thirty-eight Campylobacter isolates were obtained. Statistical analyses included percentage resistance, minimum inhibitory concentrations (MIC50 and MIC90) as well as the distribution percentages of the MICs. The non-parametric Mann-Whitney U test was used to establish any significant differences at an interspecies, interhost and interprovincial level. Analyses of the data obtained revealed indications of decreasing susceptibility to several antibiotic groups including the tetracyclines, macrolides, erythromycin and tylosin, as well as the lincoasamides, and fluoroquinolones. It was found that isolates from the Western Cape were more likely to be resistant to the fluoroquinolones (p = 0.0392), macrolides (p = 0.0262), and lincoasmides (p = 0.0001) and, as well as to a certain extent the pleuromutulins (p = 0.0985), whereas isolates from Gauteng were more resistant to the tetracycyclines (p = <.0001). Poultry Campylobacter spp. were more prone to be resistant to enrofloxacin (p = 0.0021). Campylobacter jejuni, mainly isolated from poultry, was more liable to be resistant to the tetracyclines (chlrotetracycline p= 0.0307), whereas C. coli, predominatly isolated from pigs was more likely to be resistant to the macrolides (tylosin p= 0.063). Four of the bacteria isolated from the Western Cape were resistant to three or more antibiotic classes, namely; tetracyclines, macrolides, lincosamides, pleuromutulins and fluoroquinolones. No multi-resistant Campylobacter spp. were isolated from the flocks in Gauteng. With the exception of tiamulin, the bacterial populations could clearly be divided into resistant and susceptible populations. As consequence of the increased resistance to the antimicrobial classes used for human therapy and the geographical differences in antimicrobial susceptibility, it is recommended that an antimicrobial resistance monitoring system for the thermophilic Campylobacter spp. be initiated in the South Africa National Veterinary Surveillance and Monitoring Programme for Resistance to Antimicorbial Drugs (SANVAD) Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted