Dissertations / Theses on the topic 'Peptones'

Orientador: Maria Helena Andrade Santana<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química<br>Made available in DSpace on 2018-08-20T01:59:45Z (GMT). No. of bitstreams: 1 Benedini_LeandroJunqueira_M.pdf: 2379873 bytes, checksum: 6e878845e2b38c395cbc006441849b4c (MD5) Previous issue date: 2012<br>Resumo: O ácido hialurônico (AH) é um polissacarídeo linear com vasta gama de aplicações médicas e farmacêuticas. Por esta razão, é essencial a obtenção do produto com elevado grau de pureza. Atualmente, há grande foco de pesquisa direcionado ao AH produzido por rota microbiana, utilizando extrato de leveduras (EL) como fonte de nitrogênio. Disso resulta uma quantidade relevante de proteínas e peptídeos totais (TPP) remanescentes no meio de cultivo. Este trabalho visa estudar o efeito da substituição do EL e de fontes de origem animal, por peptonas vegetais no cultivo do Streptococcus zooepidemicus para a produção do AH, com finalidades de reduzir a quantidade de TPP nos meios e eliminar possíveis contaminantes. Na primeira etapa deste trabalho, foram realizadas fermentações com peptonas de trigo (PT), de batata (PB), de soja (PS) e uma mistura de peptonas vegetais (MPV). O extrato de leveduras (EL) foi usado como controle. Os melhores resultados foram obtidos em meio suplementado com PS e com PT. Meio composto com a primeira peptona (PS) produziu 0,28 g.'L POT. -1'de AH com 1,4 g de TPP por g de AH, enquanto meio suplementado com EL apresentou 0,21 g.'L POT. -1' de AH com 4,1 g de TPP por g de AH. Meio composto com a segunda peptona (PT) produziu menor quantidade de AH (0,05 g.'L POT. -1'), porém com maior massa molar (5,06.106 Da) que a observada em AH proveniente de outras fontes de nitrogênio (3,2 - 3,7 x '10 POT. 6' Da). Numa segunda etapa, foram realizadas novas fermentações com finalidade de se realizar um estudo cinético do metabolismo microbiano com as peptonas mais promissoras da etapa anterior (PS e PT). Novamente, meio suplementado com PS apresentou o melhor desempenho para a produção. Enquanto isso, PT apresentou menor desempenho para produção de AH, mas AH com maior massa molar média (4,76 x '10 POT. 6' Da). Além disso, análises cinéticas demonstraram que quanto maior quantidade de AH produzido, menor a sua massa molar. Estes resultados reforçam os benefícios de utilização PS para produção de grandes quantidades de AH, e os benefícios da utilização de PT para produção de AH com elevado peso molecular médio. Numa terceira etapa, foi estudado o efeito da substituição dos meios BHI (Brain and Heart Infusion) e sangue de carneiro por PS no preparo de placas utilizadas para adaptação e crescimento celular em etapa inicial da inoculação. Quando somente BHI foi substituído por PS, e a presença de sangue de carneiro foi mantida, quantidade semelhante de AH foi produzida em relação ao controle. A remoção de sangue de carneiro das placas com a melhor concentração de de quantidade semelhante de AH com maior massa molar média (3,50 - 3,60 x '10 POT. 6' Da). Portanto, foi concluído que PS e PT substituem EL em meio de fermentação, produzindo maior quantidade de AH, e AH com maior massa molar, respectivamente. Além disso, PS se demonstrou efetiva para crescimento e desenvolvimento de células em placas, já que substitui BHI e sangue de carneiro<br>Abstract: Hyaluronic acid (HA) is a linear polysaccharide with a wide range of medical and pharmaceutical applications. For this reason, it is essential to obtain a product with high purity. Currently, HA is produced by microbial route, using yeast extract (YE) as nitrogen source. This results in a significant amount of total proteins and peptides (TPP) remaining in the culture medium. It was studied effects of replacing YE by vegetable peptones in the cultivation of Streptococcus zooepidemicus for HA production, with purposes of reducing the amount of TPP in the media and to remove potential contaminants. In the first step of this work, fermentations were performed during 24 hours in 100 ml of medium with wheat (WP), potato (PP) and soy (SP) peptones and a mixture of vegetable peptones (MPV). The yeast extract (YE) was used as control. The best results were obtained in medium supplemented with SP and WP. The first peptone (SP) produced 0.30 g.'L POT. -1' of HA with 1.4 g of TPP per g of HA, while medium supplemented with YE produced 0.21 g.'L POT. -1' of HA with 4.1 g of TPP per g of HA. The second peptone (WP) produced less amount of HA (0.05 g.'L POT. -1'), but with higher molecular weight (5.06 x '10 POT. 6' Da) than observed in HA from other sources of nitrogen (3.2 - 3.7 x '10 POT. 6' Da). In a second step, fermentation was carried out with 340 ml of medium, during 33 hours, with the purpose of conducting a microbial kinetics study with the most promising peptones of the previous step (SP and WP). Again, SP showed the best performance for production. Meanwhile, WP showed lower performance for HA production, but higher HA average molar mass. These results underscore the benefits of using SP for the production of large amounts of HA and WP utilization for production of HA with high average molecular weight. In a third step, it was studied the effect of replacing the BHI media ("Brain and Heart Infusion") and sheep blood by SP in the preparation of Petri plates used for adaptation and cell growth in the initial stage of inoculation. When only BHI was replaced by 67 g.'L POT. -1' SP, and the presence of sheep blood was maintained, 0.27 g.'L POT. -1' of HA was produced in the fermentation (24 hours, 100 ml medium), against 0.29 g.'L POT. -1' using 37 g. 'L POT. -1' of BHI and sheep blood. Removal of sheep blood from Petri plates with the best concentration of SP (67 g.'L POT. -1') led to the production of 0.30 g.'L POT. -1' HA. Furthermore, HA in fermentation from SP plates performed a higher average molar mass (from 3.50 to 3.60 x '10 POT. 6' Da) compared with HA obtained from plates with sheep blood and BHI (3.09 x '10 POT. 6' Da ). These results also demonstrated effectiveness of SP on the growth of Streptococcus zooepidemicus on plates. It was concluded that SP and WP replace YE in fermentation medium, producing higher amount of HA and HA with higher molecular weight, respectively. Furthermore, SP has been shown effective for cell growth and development<br>Mestrado<br>Desenvolvimento de Processos Biotecnologicos<br>Mestre em Engenharia Química

Les fractions protéose-peptones (non hydrophobes et hydrophobes) ont été isolées à partir de lait bovin, ovin et caprin. Ces fractions sont ensuite caractérisées en analysant leurs comportements électrophorétiques (page-native, page-sds, fie, électrophorèse bidimensionnelle), chromatographiques (interactions hydrophobes, échange d'ions, perméation sur sephacryl s 200 et superose 12) ainsi que leurs compositions chimiques (acides amines, glucides, phosphore). Les fractions homologues des espèces considérées présentent une analogie. Seule la faible mobilité en page-native du pp3 des laits ovin et caprin parait caractéristique. Cette différence est mise à profit pour l'élaboration d'une méthode de détection du lait de vache dans celui de la chèvre. Une activité mitogénique des fractions hydrophobes des protéose-peptones des trois types de lait a été mise en évidence en culture de cellules eucaryotes sur l'hybridome mark 3. L'origine et l'évolution de la fraction hydrophobe contenant le composant 3 sont étudiées en relation avec des traitements physico-chimiques (agitation mécanique énergique et homogénéisation à forte pression) et enzymatiques (hydrolyse par la plasmine et la trypsine) qui affectent les membranes des globules gras. Cette étude apporte des éléments supplémentaires qui confortent l'origine membranaire du composant-3

La fraction composant 3 des protéose peptonés est une fraction protéique mineure du lait de vache. Une première étude immunologique a montré une relation antigénique entre la fraction hydrophobe des protéose-peptones contenant le composant 3 et la membrane des globules gras du lait. Un protocole à 3 étapes a été établi pour isoler une fraction contenant le composant 3, sans traitement thermique du lait. Il comprend une étape de précipitation au sulfate d'ammonium et 2 étapes successives de chromatographie sur colonne d'hydroxyapatite et de chromatographie en phase liquide sur colonne échangeuse d'anions. La fraction contenant le composant 3 ainsi isolée contient 2 glycoprotéines majeures associées à des protéines mineures de hauts poids moléculaires. Une comparaison des caractéristiques physico-chimiques et des compositions protéiques a été réalisée sur la fraction composant 3, obtenue avec ou sans chauffage du lait ; elle a montré l'hétérogènéité de cette fraction. Une seconde étude immunologique a montré une réaction antigénique entre la fraction contenant le composant 3 et le sérum sanguin bovin. L'électroimmunodiffusion a permis l'évaluation de la proportion de cette fraction dans différents milieux

Les protéose-peptones préparées par thermocoagulation du lait sont séparées par FPLC d'interactions hydrophobes. Les fractions obtenues sont alors caractérisées par électrophorèse bidimensionnelle. Le composant-3, concentré dans la fraction hydrophobe, est constitué par agrégation de trois sous-unités glycoprotéiques de 11, 19, et 29 kiloDaltons environ. L'étude de la partie glucidique révèle que le composant-3 est N-glycosylé. La structure du N-glycanne est de type lactosaminique complexe. Le fucosyl-lacto-N-tétraose a été également identifié dans la fraction hydrophobe. Il semble présenter une forte affinité pour le composant-3. L'origine de ce dernier est liée aux protéines des membranes de globules gras du lait. Les sous-unités du composant-3 pourraient être des fragments de N-glycosylprotéines membranaires obtenus par action de la plasmine à la surface des globules gras. Le mécanisme de l'inhibition de la lipolyse par le composant-3 est étudié dans un système modèle émulsifié. Il est montré que l'inhibition n'est pas due à une interaction directe entre la lipase et le composant-3, mais résulte d'un changement de la qualité de l'interface huile/eau de l'émulsion après adsorption du composant-3.

Orientador: Maria Helena Andrade Santana<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química<br>Made available in DSpace on 2018-08-23T09:43:37Z (GMT). No. of bitstreams: 1 Martinez_RenataMiliani_M.pdf: 2560469 bytes, checksum: b2891306de29653ef82ffcbbab2a7cb3 (MD5) Previous issue date: 2013<br>Resumo: O uso de matérias-primas sustentáveis, biodegradáveis e biocompatíveis é de grande interesse em aplicações farmacêuticas e cosméticas. O mercado cosmético situa-se em pleno processo de desenvolvimento, principalmente na área de produtos capilares, apresentando vendas expressivas no Brasil. Esse crescimento acarreta na necessidade de produtos inovadores, polivalentes e seguros para oferecimento aos consumidores. Neste âmbito, as partículas poliméricas coloidais têm ocupado posição de destaque no cenário mundial, marcando a presença da nanotecnologia em produtos cosméticos. Uma grande variedade de matérias-primas naturais ou sintéticas são polieletrólitos, cuja reticulação eletrostática em condições controladas produz partículas coloidais. A pectina cítrica e as peptonas vegetais fazem parte dos polieletrólitos, cuja origem vegetal tem sido preferida para as aplicações em cosméticos, em substituição a produtos de origem animal ou inorgânica. Neste trabalho, foi estudada a produção de partículas coloidais de pectina cítrica e das peptonas de soja e trigo, reticuladas com cloreto de cálcio e goma guar quaternizada. Em todos os casos, a produção das partículas foi realizada em processo descontínuo, simples, escalonável e na ausência de solventes orgânicos, envolvendo o gotejamento de solução do agente reticulante sobre a solução do polieletrólito sob agitação mecânica. Os resultados mostraram que para a pectina cítrica, o tamanho e a polidispersidade das partículas foram controlados pelo grau de esterificação, agente reticulante e concentração inicial de eletrólito em solução. Para obtenção de partículas em escala nanométrica, a concentração da pectina foi de 10,0 g/l e a concentração do agente reticulante foi 1,0% (m/v). As partículas obtidas conferiram viscoelasticidade à mistura de solução de pectina e copolímero catiônico (poliquaternium-7). A viscoelasticidade foi dependente da proporção e do diâmetro das partículas de pectina, com contribuição semelhante das partículas micro e nanométricas para formações viscoelásticas. No caso das peptonas vegetais, as de trigo tiveram o seu melhor desempenho na formação de nanopartículas com menor polidispersidade. As partículas obtidas atenderam às características físico-químicas de composição, tamanho e polidispersidade requeridas para aplicações em cosméticos. As partículas de pectina, em particular, apresentaram-se promissoras para aplicações em produtos cosméticos capilares devido tamanho reduzido e potencial viscoelástico. Os fios de cabelo submetidos aos processos de transformação capilar foram o foco para a aplicação dessas partículas na tentativa de minimizar os danos pré-existentes<br>Abstract: The use of sustainable, biodegradable and biocompatible raw materials show great interest for pharmaceutical and cosmetic applications. The cosmetic market is situated in development process, particularly in the area of hair products, with significant sales in Brazil. This growth brings the need for innovative, versatile and safe products to consumers. In this context, polymeric colloidal particles have occupied a prominent position on the world stage, marking the presence of nanotechnology in cosmetics. A variety of materials are natural or synthetic polyelectrolytes, whose electrostatic crosslinking in controlled conditions produces colloidal particles. The citrus pectin and vegetables peptones are part of polyelectrolytes, whose vegetable source has been preferred for applications in cosmetics, replacing animal products or inorganic materials. In this work, we studied the production of colloidal particles of citrus pectin and soybean and wheat peptones, all crosslinked with calcium chloride and quaternized guar gum. In all cases, production of particles was performed in a batch process, simple, scalable and in the absence of organic solvents, involving the drip of crosslinking agent on the polyelectrolyte solution under mechanical stirring. The results showed that for citrus pectin, size and polydispersity of the particles was controlled by the degree of esterification, crosslinking agent and initial concentration of electrolyte in solution. To obtain nanometer scale particles the concentration of the pectin was 10.0 g/l and the concentration of crosslinking agent was 1.0% (m / v). The particles obtained impart viscoelasticity to the mixture of pectin and cationic copolymer (polyquaternium-7). The viscoelasticity depended on the proportion and the particle diameter of pectin, as the micro and nanometric particles were similar to viscoelastic formations. In the case of peptones, wheat had the better performance in the formation of nanoparticles with low polydispersity. The obtained particles met the physico-chemical composition, size and polydispersity required for applications in cosmetics. The pectin particles, in particular, were very promising for applications in cosmetic hair due small size and potential viscoelastic. Capillary transformation processes were the focus for the application of these particles in an attempt to minimize the pre-existing damaged hair<br>Mestrado<br>Engenharia de Processos<br>Mestra em Engenharia Química

La microfiltration tangentielle 0,1 µm de lait écrémé est une opération employée en industrie pour la séparation des micelles de caséine et des protéines sériques. Pourtant la conception et les conditions de fonctionnement de l’opération ne sont pas optimisées. L’objectif de cette thèse est d’apporter des connaissances pour améliorer les performances de la microfiltration 0,1 µm de lait écrémé. L’approche a été double en considérant les caractéristiques du lait à traiter et les paramètres de conduite de l’opération. Il a été montré que la durée de stockage à froid et les traitements de stabilisation bactériologique appliqués au lait avant la microfiltration ont un impact sur les performances de l’opération. La diminution des performances (pression, récupération des protéines sériques)est attribuée aux effets cumulatifs des bactéries et fragments bactériens et les protéines sériques dénaturées/ agrégées. La diafiltration à l’eau améliore les performances des membranes organiques (augmentation des flux de perméation et de la transmission des protéines sériques), lorsqu’elle est réalisée sur des rétentats concentrés et avec des pressions transmembranaires faibles. Les résultats s’expliquent par des changements de propriétés du concentré et du dépôt de micelles de caséines à la membrane. Ces résultats associés à l’analyse de composition et de fonctionalité des fractions obtenues avec membranes céramiques et organiques soulignent la nécessité d’une optimisaiton multi-objective de la microfiltration de lait<br>Crossflow microfiltration of skimmed milk using 0.1 µm pore size is commonly used in dairy industry to separate casein micelles from serum proteins. However, the design and the conduct of this operation are not optimized. The objective of this thesis is to provide knowledge to improve the performance of 0.1µm-microfiltration skimmed milk. We considered both the characteristics of skimmed milk to be microfiltered and the operating conditions of the operation. This work shows that the duration of cold-storage and the method for microbiological stabilization performed prior microfiltration impact the performance of the operation. The decrease of performance (transmembrane pressure, serum protein recovery)is attributed to the cumulative effect of bacteria/bacterial fragments and modifications of serum proteins (denaturation and aggregation). Performance of microfiltration using polymeric membrane are considerably improved (increase of permeation flux and transmission of serum proteins) using diafiltration mode with reverse osmosis water realized on concentrated retentate and with low transmembrane pressure. The modifications observed can be explained by changes on the properties of the fluid (viscosity) and of the deposit of casein micelles accumulated at the membrane (swelling). These results and the analyses of composition and functionalities of fractions obtained with ceramic and polymeric membranes highlight the real necessity, in the futur, of multi-objective optimization of skimmed milk microfiltration

O etanol é o combustível que mais se destaca como alternativa à gasolina no setor do transporte. No entanto, a melhoria da eficiência da fermentação, aprimorando o processo de conversão de biomassa em etanol combustível e reduzindo o custo de produção são desafios ainda pertinentes. Neste cenário, as fermentações com alta concentração de açúcar ou VHG (very high gravity) estão sendo cada vez mais estudadas. A adição de compostos como fonte de nitrogênio pode gerar benefícios para a produção de etanol, entretanto, a maioria dos estudos com suplementação de fontes nitrogenadas é realizada em sistema descontínuo simples e em escala laboratorial, embora os processos fermentativos industriais sejam conduzidos em sistema descontínuo alimentado. Neste contexto, este trabalho visou avaliar o perfil fermentativo da linhagem CAT-1 em mosto contendo alta concentração de açúcar fermentescível e suplementado com as fontes nitrogenadas peptona e sulfato de amônio, nas concentrações 5 e 10 g/L, em sistema de descontínuo simples e descontínuo alimentado com reciclo de células, utilizando diferentes escalas produtivas. Nos experimentos em sistema descontínuo simples, os tratamentos controle e peptona (5 g/L) apresentaram uma melhor produção de etanol, maior rendimento e produtividade em comparação com os demais tratamentos. Nos experimentos em sistema descontínuo alimentado não houve diferença significativa entre os tratamentos controle e peptona 5 e 10g/L, porém, o tratamento com sulfato de amônio (10 g/L) apresentou parâmetros fermentativos de produção de etanol e produtividade superiores ao tratamento controle, o oposto do descontínuo simples. Os resultados mostraram que quando as células de levedura foram submetidas a concentrações crescentes de açúcar, como é o caso do sistema descontínuo alimentado, ocorre uma adaptação das mesmas, proporcionando melhores resultados, com uma redução no tempo de fermentação para 16h. Além disso, a alta concentração de etanol no meio fermentativo causa um efeito prejudicial às células, já que nos tratamentos onde houve teores mais elevados de etanol ocorreu menor viabilidade e maior quantidade de açúcar residual.<br>Ethanol is the fuel that stands out as an alternative to gasoline in the transportation sector. However, improving the efficiency of fermentation, improving the process of converting biomass to fuel ethanol and reducing the cost of production are still relevant challenges. In this scenario, fermentations with high sugar concentration or very high gravity (VHG) are being studied more and more. The addition of compounds as a source of nitrogen can generate benefits for ethanol production, however, most of the studies with nitrogen supplementation are carried out in a simple discontinuous system and in laboratory scale, although the industrial fermentation processes are conducted in a fed batch system . In this context, the objective of this work was to evaluate the fermentation profile of the CAT-1 strain in must containing high fermentable sugar concentration and supplemented with nitrogen peptone and ammonium sulfate at 5 and 10 g / L in a simple discontinuous system and fed batch system, fed with recycle of cells, using different productive scales. In the simple discontinuous system experiments, the control and peptone treatments (5 g / L) presented better ethanol production, higher yield and productivity in comparison to the other treatments. However, the treatment with ammonium sulfate (10 g / L) showed fermentative parameters of ethanol production and productivity higher than the control treatment, the opposite of simple discontinuity. The results showed that when the yeast cells were submitted to increasing concentrations of sugar, as is the case of the fed batch system, an adaptation occurs, providing better results, with a reduction in the fermentation time to 16h. In addition, the high concentration of ethanol in the fermentation medium causes a detrimental effect on the cells, since in the treatments with higher levels of ethanol; less viability and more residual sugar were observed.

Les peptoïdes (oligomères de glycine N-substituées) sont une classe importante de foldamères capables d'adopter une gamme de structures secondaires uniques basées sur les géométries cis/trans de leurs liaisons amides tertiaires. Le mode de repliement des chaînes peptoïdes en structure ordonnée et leur stabilité conformationnelle sont étroitement liés à la séquence. Dans ce contexte, mon travail s'est principalement focalisé sur la conception de nouvelles séquences d’oligomères peptoïdes pouvant servir de plateformes présentatrices de macrocycles photosensibilisateurs (phtalocyanines/porphyrines). Une première partie importante de la thèse a porté sur la conformation des oligopeptoïdes. Nous avons élaboré des peptoïdes structurés en hélice de type polyproline I (PPI) et présentant une très grande stabilité conformationnelle à partir d’unités aliphatiques chirales et achirales. Dans une seconde grande partie, des hélices de type PPI possédant des chaînes latérales fonctionnelles ont été construites et conjuguées à un ou deux macrocycles phtalocyanines/porphyrines. Nous avons également synthétisé des peptoïdes cycliques en tant que plateformes présentatrices de photosensibilisateurs. Différents squelettes (α, β et α,β-peptoïde) ont été envisagés pour obtenir après conjugaison des édifices présentant des tailles de cycles et des topologies différentes<br>Peptoids (N-substituted glycine oligomers) are an important class of foldamers capable of adopting a range of unique secondary structures based on the cis/trans conformation of their constituting main chain tertiary amide bonds. The folding mode of peptoid oligomers in orderly secondary structures and their conformational stability are closely related to their sequence. In this context, my work has focused on the development of new peptoid oligomer sequences as chemical platforms for the topological presentation of photosensitizer agents (phthalocyanin/porphyrin). A first important part of the thesis concerned an in-depth study of the conformation of oligopeptoids. We have developed Polyproline I (PPI) helical structured peptoids with high conformational stability from chiral and achiral aliphatic units. Helical peptoids have been conjugated to one or two macrocyclic photosensitizers. In a second part, cyclic peptoids with functionalpendant side chains have been conceived for the presentation of photosensitizers. Various backbones (α, β and α,β-peptoid) were considered to obtain conjugated molecules displaying diverse ring sizes and topologies

Foram avaliados 25 reservatórios de água dos equipos odontológicos da Clínica de Dentística/Endodontia da FOB/USP com relação à presença de micro-organismos e a ação do ultrassom (US) na remoção do biofilme. Amostras de 10ml de água foram obtidas e alíquotas de 25l in natura e diluída até 10-4 foram semeadas pela técnica da gota nos meios: R2A Agar (R2A), Plate Count Agar (PCA), Peptona Diluída (PD) e Sabouraud Dextrose Agar com cloranfenicol a 1% (SDA), incubadas a 24º C por 72 horas. A água dos reservatórios foi descartada e 500 ml de água destilada esterilizada foi adicionada, sendo submetidos à ação do ultrassom (US) por 15 minutos, seguidos do mesmo procedimento descrito anteriormente. As colônias de bactérias foram quantificadas e os fungos foram identificados por micro-cultivo. A média da detecção de UFC/ml antes e após o US foi de 173.787 e 15.841 para o R2A, 104.873 e 3.034 para o PCA e de 245.824 e 8.231 para o PD. A média de fungos foi de 52,4 antes e 19,2 UFC/ml após ação do US. Fungos foram detectados em 20 reservatórios antes e em 12 deles após uso do US. O Penicillium sp apresentou prevalência de 36% nos reservatórios de água avaliados. Os resultados obtidos permitem concluir que o US foi eficiente em desestruturar o biofilme, embora não o elimine por completo, apresentando maior efetividade na desestruturação de bactérias.<br>A total of 25 waterline unit reservoirs of the odontological sets from the Dentistry/Endodontic Clinic of FOB/USP were assessed, in relation to the presence of microorganisms and the ultrasound action (US) on the biofilm removal. Waterline samples of 10ml were obtained from aliquots of 25l in natura and diluted until 10-4, then, they were spread using the dripping technique on the means: R2A Agar (R2A), Plate Count Agar (PCA), diluted Peptone (DP) and Sabouraud Dextrose Agar with cloranfenicol at 1% (SDA), being incubated at 24º C for 72 hours. The waterline units of the reservoirs were discarded and 500 ml of sterilized distilled water was added, submitted to ultrasound action (US) for 15 minutes, following the same procedure described afore. The bacteria colonies were quantified and the fungi were identified through micro-culture. The average of detection of UFC/ml before and after US was 173.787 and 15.841 for R2A, 104.873 and 3.034 for PCA and of 245.824 and 8.231 for PD. The fungi average was 52,4 before and 19,2 UFC/ml after the action of US. Fungi were detected in 20 reservoirs before and 12 after using US. Penicillium sp showed a prevalence of 36% in the waterline reservoirs assessed. The results obtained, led to the conclusion that US was efficient to break the structure of the biofilm, although it did not eliminate it completely, showing more effectiveness to break the bacteria structure.

The food-borne pathogen Listeria monocytogenes is more salt tolerant in the complex medium brain heart infusion (BHI, 2.0M NaCI upper limit for growth) than in a chemically-defined medium (DM, 1.0M NaCI upper limit for growth). The components in BHI responsible for the characteristic salt tolerance of L. monocytogenes are peptone and glycine betaine. At high osmolarity, the growth stimulation by peptone was higher than expected from nutritional supplementation, indicating that an osmoprotective mechanism was also at play. Peptone provided a higher level of osmotic protection than the compatible solute glycine betaine which was a moderate osmoprotectant. Our growth data demonstrated that of the free amino acids and peptides contained in peptone it is the peptides which are the osmoprotectants for L monocytogenes. Furthermore, specific peptides, such as PGG (prolyl-glycyl- glycine) and PHP (prolyl-hydroxyproline), behaved in growth experiments as the compatible solute glycine betaine, i.e. stimulation of growth at high osmolarity and no effect at low osmolarity. Our analysis of the changes in the intracellular pools of amino acids, under conditions of sosmotic stress, when peptone or specific peptide are supplied to the growth medium, has shown the following features in the mechanism of adaptation of L. monocytogenes to osmotic stress: i) Peptides are taken up, by at least two specific transport systems. ii) Subsequently, peptides are hydrolysed intracellularly by peptidases. iii) As a consequence of ii), a significant increase in the pool of free amino acids occurs. Osmoadaptation in L. monocytogenes iv) We have also demonstrated that depending on the nature of the constituent amino acids, some peptides are not fully hydrolysed which leads to the accumulation of an intracellular peptide pool in L. monocytogenes. v) Proline, glycine and hydroxyproline are the amino acids preferentially accumulated as free amino acids or as part of peptides. vi) The intracellular accumulation of free amino acids and peptides is positively correlated to an increase in the external osmolarity and has an important role in the osmoadaptation of L. monocytogenes.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-21T13:39:52Z No. of bitstreams: 1 mariziatrevizani.pdf: 1220027 bytes, checksum: 70abc77309c910bd7507cd8ebeed0526 (MD5)<br>Rejected by Adriana Oliveira (adriana.oliveira@ufjf.edu.br), reason: on 2017-08-24T11:31:11Z (GMT)<br>Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-24T15:20:42Z No. of bitstreams: 0<br>Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-30T14:33:33Z (GMT) No. of bitstreams: 0<br>Made available in DSpace on 2017-08-30T14:33:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-02-22<br>CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior<br>A evolução das técnicas de cultura de células levaram à necessidade de eliminar componentes de origem animal, de modo a evitar a contaminação bacteriana, viral ou priônica. As células-tronco da polpa dentária são células-tronco multipotentes que apresentam alta plasticidade, sendo capazes de se diferenciar em linhagens osteogênica, adipogênica e condrogênica. O presente trabalho tem como objetivo testar a utilização de peptonas vegetais (ervilha, trigo e soja) nas concentrações de 0,5%; 1% e 5% como possíveis substitutos para 10% de soro bovino fetal (SFB) em cultura de CTs. A proliferação celular foi avaliada pelo método de MTT analisado em espectrofotómetro (VarioSkan). Realizou-se a diferenciação osteogênica e empregou-se a coloração Vermelho de Alizarina para testar o suporte de diferenciação. Medimos a concentração de deposição de cálcio ressuspendendo o conteúdo de Vermelho de Alizarina em solução alcoólica e analisando espectrofotometricamente. O ensaio de MTT e ensaios de diferenciação osteogênica permitiram concluir que a peptona de trigo a 1% foi a mais eficiente nas condições empregadas. A concentração de 5% de todas as peptonas utilizadas foi tóxica. Ao analisar os aminogramas das três peptonas, foi possível inferir que a maior eficiência da peptona de trigo pode estar relacionada à maior proporção de prolina e ácido glutâmico.<br>The evolution of cell culture techniques led to the necessity of eliminating components with animal origin, in order to prevent bacterial, viral or prionic contamination. Deciduous teeth dental pulp stem cells are multi potent stem cells which present high plasticity, being able to differentiate into osteogenic, adipogenic and chondrogenic lineages. The present work aims at testing the utilization of vegetal peptones (pea, wheat and soy) at the concentrations 0.5%; 1%, and 5% as possible substitutes for 10% Fetal Bovine Serum (FBS) in SCs culture. Cell proliferation was assayed by the MTT method analyzed on spectrophotometer (VarioSkan). We performed osteogenic differentiation and employed Alizarin Red staining in order to test the differentiation support. We measured the concentration of calcium deposition by ressuspending the alizarin red content in alcoholic solution and by analyzing spectrophotometrically. MTT and osteogenic differentiation essays allowed to conclude that wheat peptone at 1% was the most efficient in the conditions employed. The concentration of 5% of all the peptones utilized was toxic. Upon analyzing the aminograms of the three peptones, it was possible to infer that the higher efficiency of wheat peptone may relate to the greater proportion of proline and glutamic acid.

Orientador: Maria Helena Andrade Santana<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química<br>Made available in DSpace on 2018-08-25T22:51:52Z (GMT). No. of bitstreams: 1 Oliveira_RhelvisdeCampos_M.pdf: 1444700 bytes, checksum: 22be4f55322956156176e0226207310e (MD5) Previous issue date: 2014<br>Resumo: O ácido hialurônico (AH) é um polissacarídeo linear com diversas aplicações nas áreas médica e farmacêutica. A produção de ácido hialurônico por via fermentativa vem sendo muito estudada atualmente e modificações no meio de cultura tornaram - se uma das principais alternativas para se atingir um produto com altos níveis de rendimento e massa molar (MM). A primeira etapa deste trabalho teve como objetivo estudar os efeitos de duas peptonas de soja, cuja principal diferença se encontra nas concentrações dos aminoácidos aspartato glutamina e glutamato (AGG), além das proporções de aminoácidos livres e totais. O microrganismo Streptococcus zooepidemicus ATCC 39920 foi cultivado por 24h em frascos agitados contendo uma concentração inicial de 25g L-1 de glicose e razões Carbono:Nitrogênio (C:N) de 3,4:1, 4,1:1 e 5,5:1 para a peptona rica em AGG e 4,1:1, 4,9:1 e 6,3:1 para a peptona pobre em AGG. Os resultados mostraram que a maior produção de AH foi de 1,37g L-1 à razão C:N de 4,1:1 para a peptona pobre em AGG, com MM média de 8,9x10 3 Da e cerca de 50 % das frações de MM na casa de 104 Da, enquanto que para a peptona rica em AGG esses valores foram de 0,70g L-1 e 4,1:1 em termos de produção de AH e razão C:N respectivamente, com MM média de 1,19x106 Da e cerca de 67 % das frações de MM na casa de 105 Da. Na segunda parte deste trabalho foi feita uma comparação do comportamento cinético de fermentações realizadas com as duas peptonas de soja em ensaios com aeração forçada. Os modelos logísticos de Verhulst para o crescimento celular, um análogo ao modelo de Luedeking-Piret incorporando o modelo logístico de Verhulst para o consumo de glicose, Verhulst modificado e Luedeking¿Piret para a produção de AH foram utilizados para estimar os parâmetros cinéticos. Os ajustes dos modelos apresentaram coeficientes de correlação maiores que 0,93. Os resultados mostraram que crescimento celular, consumo de glicose e produção de AH ocorreram durante as 24h de cultivo em meio de cultura contendo a peptona rica em AGG, enquanto que para a peptona pobre em AGG esse tempo reduziu para 10h. A quantidade produzida de AH foi semelhante para ambas as peptonas, entretanto, utilizando a peptona pobre em AGG, 60% da concentração final de AH veio da fase anterior à etapa de fermentação propriamente dita. Os parâmetros cinéticos apresentaram um coeficiente de manutenção maior para o cultivo com a peptona pobre em AGG e maior consumo de glicose foi verificado no cultivo com a peptona rica em AGG, refletindo-se no no rendimento YX/S, enquanto que o rendimento YP/X foi semelhante nos cultivos com ambas as peptonas. Esses resultados mostram que as peptonas não são somente fontes de nitrogênio, mas desempenham papéis específicos no cultivo celular<br>Abstract: Hyaluronic acid (HA) is a linear polysaccharide with many applications in the medical and pharmaceutical fields. The production of hyaluronic acid by fermentation pathway has been widely studied nowadays, and modifications made in the culture medium is one of the main alternatives to achieve a product with high levels of yield and molecular weight (MW). The first step of this work was to study the effects of two soy peptones, whose main difference is the amino acids concentrations of aspartate, glutamine and glutamate (AGG), and the proportions of free and total amino acids. The microorganism Streptococcus zooepidemicus ATCC 39920 was grown over 24 hours in shake flasks containing initial glucose concentration of 25 g L-1 and the same carbon: nitrogen (C: N) ratios 3.1:1, 1.7:1 and 1.0:1 for both peptones. The results showed that the higher production of HA was 1.37g L-1 with C:N ratio of 1.0:1 for poor AGG peptone with average MW of 8.9 x 103 Da and about 50 % of the MW fraction in the range of 104 Da, while rich AGG peptone showed values of 0.70 g L-1 and 1.7:1 in terms of HA production and C:N ratio respectively, with average MW of 1.19 x 106 Da and about 67% of the fractions of MW in the range of 105 Da. In the second part of this work was made one comparison of the kinetic behavior of cultivations using both peptones in assays with forced aeration. The logistic models of Verhulst for cell growth, analogous to Luedeking-Piret model incorporating the logistic model of Verhulst for glucose consumption, Verhulst modified and Luedeking-Piret for the production of HA were used to estimate the kinetic parameters. The adjustments of the models had correlation coefficients greater than 0.93. The results showed that cell growth, glucose consumption and HA production occurred over the 24 hours of cultivation in culture medium with rich AGG peptone, while with poor AGG peptone this time reduced for 10h. The amount of HA produced was similar for both peptones, however, using poor AGG peptone 60% of the final HA concentration came from the previous stage of the fermentation. The kinetic parameters showed higher coefficient of maintenance for cultivation with poor AGG peptone and increased glucose consumption was observed in cultivation with rich AGG peptone, reflected in terms of YX/S yield, while the yield YP/X was similar in cultivations with both peptones. These results show that peptones are not only sources of nitrogen but play specific roles in cell culture<br>Mestrado<br>Desenvolvimento de Processos Biotecnologicos<br>Mestre em Engenharia Química

A água residual do processamento do Minced de tilápia (Oreochromis niloticus) é descartada sem qualquer aproveitamento, constituindo um efluente de potencial impacto ao ambiente. O objetivo desse estudo foi avaliar este efluente, caracterizar sua fração proteica, verificar seu potencial antioxidante e sua eficiência como meio de cultura para o crescimento microbiano. A carne mecanicamente separada (CMS) de tilápia foi submetida à operação de clarificação em água destilada a 10ºC, na proporção 1:3, processo que se constituiu em homogeneização por 3 min, repouso por 3 min, sendo o Minced acondicionado e prensado em tecido de algodão esterilizado para drenagem da água residual. O efluente gerado pela etapa de lavagem do Minced apresentou 8854,5 mgO2/L de DQO e 4656,88 mgO2/L de DBO, valores considerados elevados dentre os padrões de qualidade, devido ao alto teor de matéria orgânica. O efluente foi submetido à centrifugação e a fração aquosa foi liofilizada, sendo designada de peptona e submetida à análise eletroforética que apresentou maior concentração de proteínas na faixa de 47-60 kDa. A atividade antioxidante foi avaliada em diferentes concentrações utilizando ensaios in vitro como o método do radical 2,2-azinobis-(3-etilbenzotiazoline-6-ácido sulfônico) (ABTS) e 1,1-difenil-2-picrilhidrazil (DPPH), sendo que, em ambos, o aumento da atividade antioxidante acompanhou a concentração. Com destaque para o extrato 15 mg/ml que propiciou 46,23% de inibição do radical ABTS, e 83,22% em DPPH. Em relação ao teste de efetividade, foram comparadas peptonas comerciais e a peptona da água residual liofilizada, sendo avaliada a curva de crescimento pela densidade óptica, produção de biomassa e detecção por plaqueamento da Escherichia coli e Staphylococcus aureus. A performance obtida nas peptonas da água residual foram superiores às comerciais, evidenciando o vantajoso reuso desse efluente como coproduto destinado ao cultivo de microrganismos. O desempenho do peptídeo residual permite que se recomende o seu uso por parte das empresas que buscam a ecoeficiência<br>The wastewater of minced tilapia (Oreochromis niloticus) processing is discarded without any use, constituting a effluent of potential impact on the environment. The aim of this study was to evaluate this effluent, characterize its protein fraction, verify its antioxidant potential and its efficiency as a culture medium for microbial growth. Mechanically separated meat (CMS) tilapia was submitted to clarification operation in distilled water at 10°C in a 1:3 ratio, a process that constitutes homogenizing for 3 minutes, rest for 3 minutes, and the minced packed and compressed in a sterilized cotton fabric for drainage of wastewater. The effluent generated by the minced washing step showed 8854.5 mgO2 / L of COD and 4656.88 mgO2 / L BOD, values considered among high quality standards due to the high content of organic matter. The effluent was subjected to centrifugation and the aqueous fraction was lyophilized, and designated peptone and subjected to electrophoresis analysis showed a higher concentration of proteins in the 47-60 kDa range. The antioxidant activity was evaluated at different concentrations using in vitro assays such as radical method 2,2-azinobis- (3-sulphonic acid 6-etilbenzotiazoline) (ABTS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH), wherein, in both the increased antioxidant activity followed the concentration. Especially the extract 15 mg / ml which provided 46.23% inhibition of ABTS radical, and 83.22% in DPPH. Regarding the effectiveness test, they were compared to commercial peptones and peptone lyophilized wastewater, assessing growth curves by optical density biomass production by plating and detection of Escherichia coli and Staphylococcus aureus. The performance achieved in peptones wastewater were higher than commercial, showing the beneficial reuse of the effluent as a co-product intended for microorganisms cultivation. The performance of the residual peptide allows recommending its use by businesses seeking eco-efficiency

The emergence of antimicrobial resistance is a severe threat to global health and new classes of antibiotics are desperately needed. Peptoids, or oligo-N-substituted glycines, are a group of peptidomimetics with increased structural stability and resistance to protease degradation compared to peptide analogues. In Chapter 1, peptoids are introduced and their antimicrobial properties reported to date are summarised. The synthesis and characterisation of one of the largest library of antimicrobial peptoids in existence is outlined in Chapter 2, comprising linear sequences and cyclic compounds. The development of synthetic methodology that allows the on-resin synthesis of novel peptoids containing both lysine- and arginine-type monomers is also described. In Chapter 3, the antiparasitic activity of the peptoid library is assessed against a variety of clinically relevant protozoan targets; including Leishmania mexicana, the causative agent of the neglected tropical disease cutaneous leishmaniasis. Active peptoids were identified against the insect and mammalian life stages of this parasite, including several with low micromolar potency against L. mexicana infected macrophages, an in vivo model of the disease. Additionally, peptoids that have selective activity at sub-micromolar concentrations against Plasmodium falciparum have been identified. Chapter 4 discusses the potent antibacterial and antifungal properties of the peptoid library against planktonic bacteria and also against mixed species, cross kingdom biofilms using a new quantitative polymerase chain reaction approach. Evaluation of peptoid toxicity to mammalian cells is also considered and conjugation of active sequences to the lantibiotic nisin is evaluated as a method to increase peptoid selectivity. To rationalise the activity of the peptoid library, Chapter 5 investigates the relationship between peptoid hydrophobicity, secondary structure and biological activity using circular dichroism spectroscopy and partitioning experiments. Finally, the antimicrobial mode of action is also examined using confocal fluorescence microscopy.

Les peptoïdes sont une classe de peptidomimétiques pour lesquels les chaînes latérales de chaque résidu sont déplacées du carbone α sur l'azote d'amide adjacent. Le travail présenté dans ce document s'intéresse à l'étude et à l'utilisation de plateformes moléculaires de géométrie contrôlée de type β- et α,β-peptoïdes alternés. Une nouvelle méthode de synthèse "submonomer" en solution des différentes familles de peptoïdes a tout d'abord été mise au point. Elle permet de s'affranchir des purifications intermédiaires par chromatographie, grâce à l'utilisation d'amines primaires volatiles et de solvants permettant des précipitations sélectives, et ainsi d'accéder rapidement à des tétrapeptoïdes simples. Sur ce type de composés, la possibilité d'introduire de la diversité chimique par des post-modifications sur les chaînes latérales a été étudiée. Une plateforme β-peptoïde modèle pouvant présenter trois pharmacophores différents a été obtenue grâce à des ligations orthogonales sur les chaînes latérales par Cycloaddition 1,3-dipolaire catalysée au cuivre entre un alcyne et un azoture (CuAAC), couplage thiol-ène photochimique (TEC) et alkylation d'amine tertiaire. Une méthode efficace d'accès à des glycoclusters a également été développée : elle fait intervenir la ligation multivalente de 1-thiosucres non protégés sur des plateformes peptoïdes portant des chaînes latérales allyles grâce à la réaction de TEC photochimique. Dans un deuxième temps, une étude sur le contrôle l'isomérie cis / trans, corollaire du lien amide tertiaire présent dans les peptoïdes, est présentée. Elle a permis d'observer qu'un groupement triazolium sur la chaîne latérale permet de sélectionner de façon très efficace la géométrie cis grâce à des interactions électroniques. Le groupement tert-butyle a une influence similaire grâce à des effets stériques. La dernière partie décrit la synthèse d'analogues de la Somatostatine (hormone humaine impliquée dans la régulation de nombreuses fonctions physiologiques) possédant un squelette β-peptoïde en vue d'étude pharmacologique<br>Peptoids are a class of peptidomimetics in which the side chains of each residue are moved from the α-carbon to the adjacent amide nitrogen. The work presented in this document focuses on the study and use of β-peptoid and α,β-alternating peptoid molecular scaffolds with controlled geometry. Firstly, a new method for solution-phase synthesis of peptoids was optimised. This method derives from the submonomer synthesis of peptoids and allows suppression of intermediate chromatography purifications thanks to the use of volatile primary amines and solvents allowing selective precipitations. It gives rapid access to simple tetrapetoids on which the potential of side chains post-modifications was investigated. A functionalised β-peptoid scaffold was decorated through orthogonal ligations on side chains using the Copper-catalysed Alkyne-Azide Cycloaddition (CuAAC), thiol-ene coupling (TEC) and tertiary amine alkylation reactions. An efficient method allowing access to glycoclusters was also developed : it consists in multivalent ligation on allyl fonctionalised peptoid scaffolds with unprotected 1-thiosugars using the photochemical TEC reaction. Secondly, a study was conduced on the control of the subsequent cis / trans isomerisation of tertiary amide linkage present in peptoids. It allowed us to observe that a triazolium side chain induces efficient selection of the cis geometry due to electronic interactions. The tert-butyl group has a similar effect due to steric effects. The last part of the document describes the synthesis of somatostatin (human hormone involved in the regulation of numerous physiological functions) analogs possessing α,β-peptoid scaffold with the aim of pharmacological studies

Made available in DSpace on 2016-06-02T19:56:48Z (GMT). No. of bitstreams: 1 4595.pdf: 1437433 bytes, checksum: f83e0ea8c49064050b3f382c7d942d28 (MD5) Previous issue date: 2012-08-27<br>Financiadora de Estudos e Projetos<br>Diseases caused by Streptococcus pneumoniae are one of the main problems of public health in the world. The pneumococcal surface protein A(PspA) is a potential canditate as carrier in a conjugate vaccine against this bacteria. Considering the inherent high losses of the purification and conjugation steps, it is fundamental to adopt a strategy of cultivation and expression that allows the obtainance of large quantities of protein. Thus, the use of Escherichia coli as expression system as well as its cultivation in complex medium constitutes promising alternatives for reducing the cost and increasing the productivity of the process. The goal of this work was to study the influence of the temperature and cultivation medium composition over the production of a PspA belonging to clade 4 protein fragment (PspA4Pro) during rE coli cultivations, aiming at to evaluate the possibility of employing vegetable-based nitrogen sources (soybean protein hydrolisates) instead of the Triptona, an animal-derived nitrogen source. The experiments were carried out in both shakers and benchscale bioreactor, using a complex medium which contained glucose and glycerol as carbon sources, lactose as inducer and Soytone, Phytone or Triptone as nitrogen sources, besides yeast extract. Samples were collected during the experiments to follow the cell growth (measurements of absorbance, dry cell weight and permittivity signal from biomass sensors), the carbon sources consumption and the production of organic acids by HPLC analysis. The stability of the plasmid (agar plates with or without kanamycin) and the production of recombinant protein (Bradford and SDS-PAGE electrophoresis followed by densitometry) were also evaluated. Preliminary experiments were performed in shake flasks, incubated at 300rpm and 37ºC, employing both complex and defined media. The highest productivity was achieved in complex medium, with a 42% superior protein production. Subsequently, nine complementary experiments were conducted in shake flasks with complex medium, under the agitation of 300rpm and temperatures of 37ºC (growth phase) and 25, 31 or 37ºC (induction phase). The largest specific production of soluble PspA4Pro was verified at 25ºC, reaching, respectively, 209±6, 192±5mg/g dry cell mass for Phytone and Triptone, with final absorbance values (after 12h of induction) of 9.0±0.4 and 8.5±0.4. The best protein production for Soytone (124±4mg/g dry cell weight) was observed at 31ºC, yielding a final absorbance 8.0±0.4. From the results obtained in the preliminary tests, the nitrogen source Phytone was selected for experiments in bioreactor. Four batch cultures were conducted in bench-scale bioreactor (5L), containing a modified auto-induction complex medium (10g/L glucose, 60g/L glycerol and 20g/L lactose), being three of them with Phytone and one with Triptone, for comparison. The best results in terms of protein production (245±7mg of PspA4Pro soluble/g dry mass) were obtained in the presence of Phytone, corresponding to an increase of 16% towards the maximum value achieved in the cultivation with Triptone. These results demonstrate the potential of vegetable-based nutrients as alternatives to animal-derived nitrogen sources in complex media, contributing to adequate these media formulations to the current guidelines of good manufacturing practices.<br>Doenças causadas por Streptococcus pneumoniae constituem um dos principais problemas de saúde pública mundial. A proteína A de superfície de pneumococo (PspA) é candidata em potencial a ser carreadora em vacina conjugada contra essa bactéria. Considerando as altas perdas inerentes às etapas de purificação e conjugação da proteína, é fundamental adotar uma estratégia de cultivo e expressão que permita obter grandes quantidades de proteína. Nesse sentido, o emprego da bactéria Escherichia coli como sistema de expressão e o cultivo da mesma em meio complexo se apresentam como alternativas promissoras para redução do custo e aumento da produtividade do processo. O objetivo do presente trabalho foi estudar a influência da temperatura e da composição do meio de cultivo sobre a produção do fragmento da proteína PspA do clado 4 (PspA4Pro) em cultivos de rE. coli, visando avaliar a viabilidade de utilização de fontes de nitrogênio de origem vegetal (hidrolisados protéicos de soja) em substituição à Triptona, de origem animal. Os experimentos foram realizados em câmara incubadora e em biorreatores de bancada, utilizando meio complexo contendo glicose e glicerol e lactose como fontes de carbono, lactose como indutor e Soytone, Phytone ou Triptona como fontes de nitrogênio, além de extrato de levedura. Amostras foram coletadas ao longo dos experimentos para acompanhamento do crescimento celular (medida de absorbância, massa seca e permissividade por sensor de biomassa), do consumo das fontes de carbono e da produção de ácidos orgânicos por análises em cromatografia líquida de alto desempenho. A estabilidade do plasmídeo (plaqueamento em meio contendo ou não canamicina) e a produção de proteína recombinante (Bradford e eletroforese SDS-PAGE seguida por densitometria) também foram avaliadas. Experimentos preliminares foram realizados em frascos agitados e incubados a 300rpm e 37oC, empregando tanto o meio complexo como o definido. A maior produtividade foi obtida em meio complexo, a qual foi 42% superior a alcançada com meio definido. Em seguida, nove experimentos complementares foram conduzidos em frascos agitados em meio complexo sob agitação de 300rpm e à temperatura de 37ºC (fase de crescimento) e de 25, 31 ou 37ºC (fase de indução). Verificou-se que a temperatura de 25ºC proporcionou a maior produção específica de PspA4Pro solúvel, alcançando-se, respectivamente, 209±6, 192±5mg/g massa seca para o Phytone e para a Triptona, com absorbâncias finais (após 12h de indução) de 9,0±0,4 e 8,5±0,4. Já para o Soytone, a melhor produção de proteína (124±4mg/g massa seca) foi observada à temperatura de 31ºC, obtendo-se uma absorbância de final de 8,0±0,4. A partir dos resultados obtidos nos ensaios preliminares, a fonte de nitrogênio de origem vegetal Phytone foi selecionada para experimentos em biorreator. Quatro cultivos em batelada foram conduzidos em biorreator de bancada (5L), contendo meio complexo de autoindução modificado (10g/L glicose, 60g/L glicerol e 20g/L lactose), sendo 3 com Phytone e um com Triptona, para comparação. Os melhores resultados em termos de produção de proteína (245±7mg de PspA4Pro solúvel/g massa seca) foram obtidos na presença de Phytone, correspondendo a um aumento de 16% em relação ao valor máximo alcançado no cultivo com Triptona. Esses resultados comprovam o potencial dos nutrientes de origem vegetal como alternativa às fontes de nitrogênio de origem animal em meios complexos, contribuindo para adequar as formulações desses meios às atuais diretrizes de boas práticas de fabricação.

2010 - 2011<br>Le strutture e le attività metaboliche di tutte le cellule si basano su una vasta gamma di molecole comprendenti: amminoacidi, carboidrati, lipidi e nucleotidi, insieme alle forme polimeriche di questi composti. Ognuna di queste molecole presenta una caratteristica struttura chimica, una determinata capacità di interagire con altre molecole e una specifica funzione fisiologica. È stato visto che la struttura chimica e le attività fisiologiche sono strettamente correlate tra loro, a tal punto che senza una giusta conformazione le suddette attività possono venir meno. Pertanto, negli ultimi anni si sta investigando su composti che possano mimare le molecole biologiche in tutte le loro funzioni e allo stesso tempo avere le proprietà di un biopolimero non naturale. Una di queste classi di molecole biologiche è quella dei nucleotidi, i quali prendono parte attiva in molti aspetti della vita cellulare, infatti partecipano a reazioni di biosintesi e di ossidoriduzione, al trasferimento di energia, svolgono una serie di funzioni strutturali e catalitiche. Un ruolo ancora più importante rivestono i loro polimeri, gli acidi nucleici, ovvero DNA ed RNA. In accordo con il dogma centrale della biologia molecolare, infatti, la natura ha scelto questi due composti per l’immagazzinamento (DNA) e il trasferimento (RNA) dell’informazione genetica nelle cellule viventi... [abstract a cura dell'autore]<br>X n.s.

Les organismes vivants produisent des peptides antimicrobiens (PAMs) pour se protéger contre les microbes. La résistance croissante aux antibiotiques nécessite le développement de nouvelles stratégies thérapeutiques et les PAMs sont des candidats prometteurs pour résoudre ce problème. Ils possèdent une activité à large spectre et leur principal mécanisme d'action par perméation de la membrane engendre peu de phénomènes de résistance. Néanmoins, leur faible biodisponibilité empêche leur utilisation. Certaines limitations peuvent être surmontées en développant des mîmes de PAMs qui conservent leur activité mais avec un potentiel thérapeutique accru. Les peptoïdes (oligomères de N-alkylglycine) structurés en hélice cationique amphiphile sont de bons mimes de PAMs. Les peptoïdes sont plus flexibles que les peptides en raison de l'isomérie cis/trans des amides N,N-disubstitués ; cependant la conformation des amides peut être contrôlée par un choix judicieux des chaînes latérales. Le but de cette thèse est d'étudier l'influence de chaînes latérales(hydrophobes ou cationiques) bloquant la conformation des amides en cis et induisant une structure hélicoïdale de type PolyProline I (PPI) robuste, sur l’activité antibactérienne et la sélectivité de peptoïdes. La conception, la synthèse et l’étude conformationnelle de nouveaux oligomères peptoïdes cationiques portant des chaînes latérales de type tert-butyle et/ou triazolium ont été réalisées. Dans un premier temps, la synthèse en solution d'oligomères à base de tert-butyle a été développée puis une stratégie de synthèse en phase solide a été mise en place pour accéder aux oligomères à base de 1,2,3-triazolium. Ensuite, ces nouveaux oligomères ont été évalués pour leur activité vis à vis d’un panel de bactéries Gram-positive et Gram-négative, leur l'activité antibiofilm et leur sélectivité cellulaire. Enfin, pour visualiser les effets des peptoïdes amphiphiles sur les bactéries, une étude de microscopie a été réalisée<br>Living organisms produce antimicrobial peptides (AMPs) to protect themselves against microbes.The growing problem of antimicrobial resistance calls for new therapeutic strategies and the natural AMPs have shown ground-breaking potential to address that issue. They show broad-spectrum activity and their main mechanism of action by bacterial cell membrane disruption implies low emergence of resistance which makes them potent candidates for replacing conventional antibiotics. Nevertheless, few hurdles are impeding their use, notably poor bioavailability profile. Some of these limitations can be overcome by developing peptidomimetics of AMPs which exhibit antibacterial activities together with enhanced therapeutic potential. Peptoids (i.e. N-alkyl glycine oligomers) adopting cationic amphipathic helical structures are mostly competent AMP mimetics. From a conformational point of view, peptoids are fundamentally more flexible than peptides primarily due to the cis/trans isomerism of N,N-disubstituted amides but studies in this area have shown that cis amide conformation can be controlled by careful choice of side-chain to set a PolyProline I-type helical structure of peptoids. In this thesis, the genesis of novel amphipathic cationic peptoids carrying cis-directing tert-butyl and/or triazolium-type side-chains and their untapped potential to act against bacteria will be discussed comprehensively. First, the solutionphase synthesis of tert-butyl-based oligomers was developed. Second, novel method of solid-phase submonomer synthesis was optimised to access 1,2,3-triazolium-based oligomers. Then, the synthesised cationic oligomers were evaluated for their antibacterial potential, followed by antibiofilm activity and cell selectivity assays. In the end, to have insights on the mode of action of amphipathic peptoids, microscopy was carried out

Os resíduos de pescado são descartados, em sua maioria, gerando um alto impacto ao ambiente; ou subutilizados em co-produtos de baixo valor agregado. O presente estudo objetivou a obtenção de hidrolisados proteicos a partir de resíduos sólidos oriundos do processamento industrial de tilápia vermelha (Oreochromis niloticus var.), avaliando sua capacidade antioxidante e eficiência como fonte suplementar de nitrogênio para o crescimento de bactérias e leveduras. Um total de 30 carcaças inteiras não evisceradas, sem filé e pele, com peso médio de 741,53g, foram mecanicamente homogeneizadas. A composição centesimal apresentou 54,34±0,31g/100 g de umidade, 8,46±0,72 g/100 g de cinza, 27,03±2,94 g/100 g de proteína e 10,17±0,65 g/100 g de lipídeos, assim como a carga microbiana do homogeneizado estava dentro dos limites de tolerância preconizados pela legislação vigente para consumo humano. Foram otimizadas as condições de hidrólise proteica quanto à concentração de substrato, enzima e tempo de hidrólise, dos resíduos sólidos de tilápia vermelha, resultando na obtenção de modelos preditivos para o grau de hidrólise (GH) produzido tanto pelas enzimas endógenas como pelas enzimas comerciais neutrase e papaína. OGH diminuiu significativamente (p < 0,05) com o incremento da concentração de substrato, exceto na hidrólise produzida pelas enzimas endógenas sob condições ótimas para papaína. Não obstante, o GH produzido pelas enzimas endógenas aumentou significativamente (p < 0,05) com o incremento do tempo de hidrólise, enquanto que o incremento na concentração de papaína também aumentou significativamente (p <0,05) o GH nos resíduos. Rendimento de 80,95 ± 3,12%, e GH de 45,33 % foram alcançados sob condições ótimas para neutrase (22 g substrato/100 mL e 0,277 g enzima/100 g proteína durante 3 min), enquanto que a papaína teve um rendimento de 82 ± 2,21% e GH de 42,37% sob condições ótimas (18,5 g substrato/100 mL e 0,570 g enzima/100 g proteína durante 3 min). As peptonas produzidas apresentaram atividade antioxidante quanto à capacidade de sequestro dos radicais livres ABTS e DPPH, sendo esta incrementada significativamente (p < 0,05) com o aumento da concentração de peptona. As peptonas também foram altamente eficientes para o crescimento das bactérias Escherichia coli, Listeria monocytogenes e Staphylococcus aureus, e para a levedura Saccharomyces cerevisiae, sendo similares ou mesmo superiores quando comparadas às três peptonas comerciais. Os resultados obtidos no presente trabalho demonstraram, portanto, que os hidrolisados proteicos obtidos a partir de resíduos sólidos de tilápia vermelha podem ser uma fonte alternativa de antioxidantes e de nitrogênio para crescimento de microrganismos.<br>Fish wastes are mostly discarded in Brazil, causing a high environmental impact, or underused in low-valuable coproducts. The present study aimed to obtain protein hydrolysates from solid wastes of red tilapia (Oreochromis niloticus var.), evaluating their antioxidant activity as well as their efficiency as supplemental source of nitrogen for the growth of bacteria and yeasts. Thirty carcasses with viscera of red tilapia (i.e. tilapia without fillet and skin), with an average weight of 741.53 g, were mechanically homogenized. Proximate composition and microbial quality were determined, containing 54.34±0,31g/100 g of moisture, of ash, 27.03±2,94 g/100 g of protein and 10.17±0,65 g/100 g of lipids, as well as microbial levels in concordance with the legal limits in force. Conditions for the proteolysis of solid wastes of red tilapia (concentration of substrate, concentration of enzyme and time of hydrolysis) were optimized. Predictive models for the degree of hydrolysis (DH) generated by both endogen enzymes and thecommercial enzymes neutrase and papain were obtained. DH decreased significantly (p < 0.05) with the increase of the concentration of substrate, except in the hydrolysis caused by the endogen enzymes under optimal conditions for papain. However, DH produced by the endogen enzymes increased significantly (p < 0.05) with the increment of the time of hydrolysis. Meanwhile, DH increase significantly (p < 0.05) with the increase of the concentration of papain. A yield of 80.95 ± 3.12% and a DH of 45.33 % were achieved under optimized conditions for neutrase (i.e. 22 g substrate/100 mL and 0.277 g enzyme/100 g protein for 3 min). Moreover, proteolysis with papain reached a yield of 82 ± 2.21% and a DH of 42.37% under optimized conditions (i.e. 18.5 g substrate/100 mL and 0.570 g enzyme/100 g protein for 3 min). Obtained peptones showed antioxidant activity in terms of inhibition of free radicals of ABTS and DPPH, being increased significantly (p < 0.05) with the increment of the concentration of peptone. Peptones also had a high efficiency for the growth of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Saccharomyces cerevisiae, being comparatively similar or higher than commercial peptones. Therefore, results obtained in the present study demonstrated that protein hydrolysates from solid wastes of red tilapia could be an alternative source of antioxidants and nitrogen for the use in bioprocess.

2014 - 2015<br>My PhD research activity aimed to enrich the realm of peptoids1 with novel compounds and new insights into their behavior and properties, investigating their potential applications. A new class of cyclic “arylopeptoids” was synthesized by the insertion of an aromatic ring and a methylene unit into the peptoidic backbone (Chapter 2). Tested as ion transporters of alkali metal cations, protons, and a series of anions through a phospholipid membrane, they highlighted the strong influence of the size of the macrocycle on the ion transport activity. Exploiting the ion complexation properties of cyclic peptoids, two cyclic hexamers were synthesized with three carboxyethyl side chains each, to promote the formation of Gd3+-complex as MRI-probes (Chapter 3). When carboxyethyl side chains were alternated with methoxyethyl side chains, the cyclic peptoid coordinated Gd3+ and displayed good relaxometric properties, as revealed by 1H-relaxometric investigations.2 The versatility of peptoids enabled us to explore the field of glycoscience as well. A library of cyclic peptoids, ranging from 4-mer to 16-mer, with appended propargyl groups was synthesized and underwent click chemistry with DNJ azido-derivatives (Chapter 4). This is the first example of cyclopeptoid-based iminosugar click-clusters that showed activity towards α-mannosidases inhibition and the highest multivalent effect in the correspondence of the 36-valent ligand.3 This impressive outcome is the result of the modular synthetic approach of peptoids that enables for the insertion of an unlimited numbers and types of side chains.4 Taking advantage of such feature, we synthesized a library of cyclic hexapeptoids with methoxyethyl and propargyl side chains, varying in the relative content and positions (Chapter 5). Studying their role in the solid-state assembly of cyclic hexapeptoids, they showed to promote a columnar arrangement in which the propargyl groups are the pillars and the methoxyethyl chains provide intercolumnar interactions and side-by-side contacts.5 [edited by author]<br>XIV n.s.

Este trabalho buscou avaliar a influência da relação C/N e da fonte de carbono no processo de nitrificação e desnitrificação simultâneas em reator de fluxo contínuo e leito fixo estruturado. Foi utilizado um reator vertical de acrílico, com volume total de 11,0 L e volume útil de 5,5 L, com hastes cilíndricas verticais de espuma de poliuretano como suporte para a biomassa. O sistema foi operado com TDH de 11,2±0,6 horas, aeração intermitente (2 horas com aeração e 1 hora sem aeração) e razão de recirculação de efluente igual a 5. A carga carbonácea afluente foi mantida constante (1,07 kg DQO.m-3.dia-1), ao longo de todo o período experimental, sendo as relações C/N testadas (9,7±1; 7,6±1; 2,9±1 e 2,9±0,4) obtidas a partir da variação na carga nitrogenada aplicada. Duas fontes orgânicas foram avaliadas: sacarose e peptona de carne. A eficiência média de remoção de DQO manteve-se acima de 90%. A eficiência máxima de remoção de N-total (84,6±10,1%) foi obtida para relação C/N de 2,9±1, com concentrações efluentes médias de NTK e N-NH4 de 5,9 e 4,3 mg.L-1, respectivamente. A análise estatística da eficiência de remoção de N-total confirmou que a fonte de carbono não exerceu influência sobre os processos de remoção. A obtenção de eficiências de desnitrificação superiores às calculadas estequiometricamente, em função da fonte de carbono, indicou a ocorrência de possíveis vias complementares para remoção de nitrogênio, como o processo anammox. As velocidades de nitrificação e desnitrificação obtidas nos ensaios cinéticos foram similares para as duas fontes de carbono e para as relações C/N estudadas e da mesma ordem de grandeza das apresentadas na literatura, reforçando a ideia de que a configuração de reator utilizada, aliada às adequadas condições operacionais, permitiu a remoção concomitante de matéria carbonácea e nitrogenada.<br>This study aimed to evaluate the influence of COD/N ratio and organic source on the simultaneous nitrification and denitrification (SND) in an up-flow structured-bed reactor. The reactor was made of acrylic with a total volume of 11 L and a working volume of 5.5 L and filled with cylinders of polyurethane foam as support for biomass growth. The system was continuously operated with an HRT of 11.2±0.6 h, intermittent aeration (2h with aeration and 1h without aerationd) and a liquid recycle ratio equal to 5. The organic load rate was constant (1.07 kg COD.m-3d-1) during the entire experiment. The COD/N ratios (9.7±1; 7.6±1; 2.9±1 and 2.9±0,4) were obtained from the variation of nitrogen load rate. Two organic sources were evaluated: sucrose and meat peptone. The average COD removal efficiency was above 90%. The maximum total nitrogen removal efficiency was 84.6±10.1%, with average TKN and NH4+-N effluent concentrations of 5.9 and 4.3 mg.L-1, respectively. Statistical analysis of total nitrogen removal efficiency confirmed that the carbon source did not influence over the removal processes. The denitrification efficiencies higher than the stoichiometrically calculated in function of the carbon source, indicated the occurrence of possible paths for nitrogen removal of nitrogen as anammox process. The nitrification and denitrification rates obtained in kinetic experiments were similar for the two sources of carbon and all C/N ratio studied at the same order of magnitude in relation to those reported in the literature, enhancing that the reactor configuration tested combined with the proper operational conditions allowed the organic matter and nitrogen removal simultaneously.

L'interès principal d'aquesta tesi es centra en la utilització de les tècniques combinatòries per a dissenyar i sintetitzar una quimioteca de peptoides. Els peptoides són un tipus de peptidomimètics amb una estructura general basada en un esquelet de glicinies N-substituídes. Tenen una estructura molt apropiada per a ser sintetitzats en fase sòlida seguint l'estratègia combinatòria del rastreig posicional, basada en la utilització de mescles de reactius en les etapes d'introducció de diversitat. Seguint aquesta estratègia s'ha sintetitzat una quimioteca de mescles controlades de 5120 peptoides (trímers d'N-alquilglicina). <br/><br/>Els peptoides tenen un gran interès en l'àrea biomèdica. Així doncs, el cribratge de la quimioteca de peptoides davant diverses dianes biològiques d'interès terapèutic ha permès la identificació de dos potents neutralitzadors del fenotip d'MDR en l'àrea del càncer, així com de dos potents inhibidors d'LPS en l'àrea del shock sèptic. Aquests compostos han representat un punt de partida per a l'optimització de les seves estructures per a dissenyar nous compostos conformacionalment més restringits que augmentin l'activitat i millorin la selectivitat cap a la diana d'interès.<br/><br/>Per altra banda, s'ha portat a terme l'optimització d'un procés per a obtenir peptoides amb un extrem carboxílic terminal (peptoides àcids), seguint dues estratègies: per hidròlisi de peptoides en dissolució i per síntesi en fase sòlida. L'optimització del procés d'hidròlisi en dissolució s'ha dut a terme mitjançant un disseny d'experiments factorial, obtenint excel·lents resultats de pureses i rendiments, mentre que la síntesi de peptoides àcids en fase sòlida ha implicat treballar amb diversos tipus de resines, sent la clorur de 2-clorotritil la que va donar els millors rendiments i pureses. <br/><br/>L'interès d'obtenir aquest tipus de derivats àcids radica en el fet de poder acoplar els peptoides a suports sòlids, com columnes d'agarosa o sefarosa, així com poder biotinilitzar-los per acoplar-los a columnes d'agarosa amb avidina. Amb aquest tipus de columnes amb el peptoide immobilitzat s'ha utilitzat la tècnica de la cromatografia d'afinitat per a identificar les dianes potencials d'un peptoide d'un extracte proteic que contenia proteïnes de diferent naturalesa.<br/><br/>A partir de l'estudi realitzat per a obtenir peptoides àcids, s'ha dut a terme la realització d'un estudi per obtenir peptoides dímers i de peptoides dímers-cíclics, en fase sòlida i en dissolució. Aquests estudis han constituït un punt d'inici per al plantejament del disseny i síntesi de noves quimioteques combinatòries de mescles, que es realitzarà en un futur pròxim en el nostre grup d'investigació. Aquest tipus de molècules poden resultar ser molt interessants en estudis d'interacció proteïna-proteïna.<br/><br/>Dins del context del cribratge de la quimioteca davant diferents dianes biològiques, s'ha intentat desenvolupar un assaig de fluorescència per a cribrar la quimioteca de peptoides davant la seva capacitat d'inhibir la fosforilació d'un substrat peptídic fluorescent per la proteïna cinasa A. Malauradament, s'ha comprovat que el grup fluoròfor escollit, la 7-amino-4-metilcumarina, adjacent al lloc de fosforilació del pèptid, no ha induït pràcticament cap diferència d'intensitat de fluorescència després de la reacció de fosforilació per la proteïna.<br/><br/>Globalment, aquest treball reflecteix la validesa de la utilització i aplicació de la química combinatòria, en concret de quimioteques de mescles controlades, per a la recerca de compostos amb activitats biològiques. No obstant, en funció de les dades que es coneguin del receptor i/o dels llocs d'interacció, és convenient dissenyar la quimioteca de forma més focalitzada o de forma més diversa. En aquest disseny, tant si és inicial com si és d'optimització d'un cap de sèrie, és cada vegada més evident la importància i necessitat de l'ús d'eines computacionals i d'estudis in silico per cobrir el màxim espai de diversitat i estalviar temps i costos d'experimentació al laboratori.<br>Peptoids are oligomers of N-substituted glycines residues that have been developed as peptidomimetics but with improved stability and better pharmacological properties. For these reasons, peptoids have also been subjected of application of combinatorial approaches to identify bioactive molecules. <br/><br/>In this context, we have synthesised a combinatorial library of peptoids containing 5120 compounds in 52 controlled mixtures, constructed on solid-phase by using the positional scannig format. <br/><br/>The screening of the peptoid library against different biological targets have identified potent hits. To sum up, two molecules have been rescued from the library with a potent chemosensitiser activity against the MDR phenotype, and two other molecules have been identified with an in vivo neutralizing activity against the Gram-negative lipopolysaccharide (LPS). Optmisation of these hits is in progress to find lead compunds that may become potential drugs.<br/><br/> On the othe hand, we have been interested in expanding the combinatorial chemistry of peptoids to those analogues bearing free carboxylic acid groups, prepared in solution and on solid phase. These compounds are attractive to link to solid-support columns in order to use the affinity cromatography to identify potential targets. As well as this, we have studied the conditions for the efficient linear condensation and cyclocondensation of these acid peptoids.<br/><br/>Finally, we have tried to develop a fluorescent assay for the screening of the peptoid library against the phosphorylation of a fluorescent peptide by the protein Kinase A. However, there has no been detected any difference in the fluorescent intensity with the group 7-amino-4-methylcoumarin, located next to the phosphorilation site.

Chemistry<br>Ph.D.<br>Binding to protein surfaces or shallow grooves with synthetic molecules poses a unique challenge, since this inherently requires large areas to facilitate interactions. Peptoids have been shown to interact with proteins, and combinatorial libraries of peptoids have been proven to be effective in discovering new ligands for protein binding. Unfortunately, most peptoids are flexible and lack the surface area required to compete with larger protein interactions. To combat these problems, we have created spiroligomers that have a rigid backbone, exhibit functionality comparable to proteins, and are resistant to proteases. To facilitate the rapid installment of spiroligomers into peptoid subunits, we required a more streamlined approach for functionalization of spiroligomers. To this end we applied a single-pot alkylation method, with which we installed over 25 unique functional groups onto different spiroligomer hydantoins. These spiroligomer hydantoins are spirocycles that possesses two stereocenters, of which we have complete control, as well as a protected proline amino acid. These new proline amino acids (enhanced prolines) have been incorporated into peptides via Fmoc-SPPS. Finally, we have functionalized these enhanced proline residues with another functional group and a protected primary amine, which allow for their use in peptoid synthesis. We developed methods to tether multiple spiroligomers together utilizing a peptoid backbone, as well as being able to incorporate spiroligomers into peptoid macrocycles. These spiroligomer-peptoid hybrids are large, diverse, and preorganized structures that have a large potential interacting surface area for binding to protein surfaces or shallow grooves.<br>Temple University--Theses

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico. Programa de Pós-Graduação em Engenharia de Alimentos.<br>Made available in DSpace on 2012-10-23T11:30:49Z (GMT). No. of bitstreams: 1 255934.pdf: 897286 bytes, checksum: a9c37d8b67e86e7e36ff1b0b486aaa09 (MD5)<br>A hidrólise enzimática da lactose presente no soro lácteo é uma das possibilidades mais atrativas para utilização deste composto que é produzido em milhões de toneladas anualmente em todo o mundo. Atualmente, parte deste soro é utilizada para nutrição humana e animal e o restante é descartado no meio ambiente. Além da lactose, o soro apresenta em sua composição proteínas de alto valor biológico que podem ser concentradas por evaporação, precipitação ou ultrafiltração. Uma das possibilidades para valorização do soro lácteo com relação à lactose é sua hidrólise em reatores a membrana que apresenta a vantagem de permear continuamente o hidrolisado, mantendo-se a enzima ativa no retentado. Desta forma, maior quantidade de lactose pode ser hidrolisada por unidade de enzima. Sabe-se que enzimas podem interagir com superfícies hidrofóbicas, que são comuns em muitas membranas poliméricas. Portanto, é importante pesquisar se esta interação pode levar a uma perda de atividade catalítica da enzima adsorvida. Isto pode ocorrer devido à possibilidade da enzima utilizar o seu sítio ativo na adsorção, perdendo parte de sua atividade. Este trabalho teve como objetivo estudar a hidrólise da lactose a partir do permeado resultante da concentração de leite por ultrafiltração, verificando-se a influência das peptonas sobre a ação da enzima ß-galactosidase. Verificou-se, também, a atividade da enzima adsorvida na superfície da membrana e a capacidade de retenção da enzima pelas membranas. A hidrólise da lactose foi estudada em reator a membrana de fluxo perpendicular. Foram utilizadas membranas planas de polifluoreto de vinilideno (PVDF) e de poliétersulfona (PES), ambas com ponto de corte de 10 kDa. Foram utilizadas duas marcas comerciais de ß-galactosidase (Kluyveromyces lactis), Lactozym® 3000 L HP-G e Maxilact® L-5000. A taxa de hidrólise foi determinada através da pesquisa de glicose, um dos produtos da hidrólise, através de kit colorimétrico-enzimático. A maior atividade da enzima Lactozym foi na concentração de 0,4 g.L-1, a 40°C e pH 6,8 e a melhor atividade da enzima Maxilact, na concentração de 0,2 g.L-1, a 35°C e pH 6,8. Os resultados mostraram que a membrana foi capaz de reter totalmente a enzima lactase no reator, conseguindo-se até 95% de hidrólise da lactose após 90 minutos de processo, à temperatura de 35oC e pH 6,8. As peptonas do permeado foram removidas através de aquecimento e centrifugação e seu efeito sobre o fluxo permeado e a taxa de hidrólise foi investigado. A remoção das peptonas do soro fez aumentar o fluxo permeado em até 150% e a taxa de hidrólise em até 20%. Ficou evidenciado que a enzima adsorvida na superfície da membrana apresentou pouca atividade hidrolítica.

Background: Acute lung injury (ALI) and nosocomial pneumonia are major causes of morbidity and mortality. There are 200,000 cases per year of ALI in the US with a mortality of 40%. On the intensive care unit (ICU), ALI accounts for over 40% of all ventilated patients at any one time. Despite this huge burden on healthcare and the relatively high prevalence, no therapies currently exist in clinical practice that attenuate the condition. The pathophysiology and aetiology of ALI is multifactorial but neutrophilic influx and consequent damage to the endothelial-epithelial interface are regarded as central features. Alongside neutrophils, peripheral blood monocytes (PBMs) are recruited to the acutely inflamed lung. The role played by PBMs in perpetuating the pathogenic neutrophilic influx remains poorly characterised. Nosocomial pneumonia is also a major problem with drug resistant organisms. With the increasing prevalence of antibiotic resistance and the paucity of novel antimicrobials being generated by pharmaceutical companies, there is real concern that the end of the ‘antibiotic era’ may be approaching. AIMS 1) To develop murine models of lung inflammation and infection 2) To establish the role of the PBM in perpetuating the neutrophilic response in ALI 3) To develop non-invasive methodologies to study the trafficking of cells and molecular events within the inflamed lung 4) To apply a novel antimicrobial to prevent and treat nosocomial pneumonia Methods: A murine model of ALI was utilised using direct intratracheal instillation of lipopolysaccharide. To this model 3 different PBM depletion strategies were applied to study the effect on neutrophil recruitment and consequent lung injury. Non invasive optical imaging was utilised to study the effect of PBM depletion on proteolytic events within the murine lung. To understand cellular trafficking, cell labeling strategies were compared for primary murine macrophages with whole body optical imaging in mice. Murine models of Staphylococcus aureus, Pseudomonas aeruginosa and Burkholderia cepacia were established and a novel antimicrobial agent called the nonalysine like peptoid (NLLP) tested in vitro and in vivo for efficacy. Results: PBM depletion significantly attenuated neutrophil recruitment in an established model of ALI. Near infrared (NIR) optical imaging permitted the non invasive tracking of primary murine cells. A non toxic peptidomimetic agent (NLLP) possessed antimicrobial activity against gram positive and gram negative pathogens with therapeutic and prophylactic efficacy in vivo. Conclusions: PBM depletion is a potential therapeutic strategy for treating ALI. Further studies are required to determine the exact mechanism by which PBMs orchestrate neutrophil recruitment. Optical imaging is a versatile platform for molecular imaging. A novel antimicrobial agent termed NLLP has been discovered with therapeutic and prophylactic efficacy against multi-drug resistant pathogens.

Il campo dei foldameri è in evoluzione. I ricercatori stanno sviluppando sistemi con ogni possibile combinazione di elementi e strutture chimiche, allo scopo di progettare molecole artificiali altamente ordinate, capaci di mimare il comportamento conformazionale dei biopolimeri. β-Peptidi, Υ-peptidi, peptoidi, noti come "peptidomimetici", sono tra i più interessanti foldameri poichè possiedono sequenze specifiche in grado di interagire con i sistemi biologici e formare strutture secondarie ben definite. Nella mia tesi sono stati approfonditi β-peptidi ciclici e peptoidi. Nel primo caso, utilizzando una sintesi peptidica in fase solida ed alternando monomeri con diversa chiralità, sono stati sintetizzati e analizzati strutturalmente due modelli di β-peptidi con catene laterali diversamente costrette: gli oligomeri 1 e 2 (H-[(1R,2R)-BACHC-(1S,2S )-BACHC]n-NH2) e gli oligomeri 3 e 4 (H-[(1R,2S)-ACHEC-(1S,2S)-BACHC]p-NH2). 2D NMR (TOCSY, ROESY, COSY), scambi NH/ND e spettri ECD hanno mostrato che l’oligomero con elica H10/12 più stabile è l’esamero 4. La seconda parte è stata dedicata ai peptoidi. È stato preparato un nuovo monomero β-peptoide, conformazionalmente bloccato da un anello ϒ-lattamico. Tale unità è stata utilizzata per le successive sintesi peptidiche in fase omogenea: un oligomero peptoide e un oligomero ibrido peptide/peptoide. La sintesi del peptoide è stata complicata dalla bassa resa nelle reazioni di coupling e da una difficoltosa analisi strutturale, a causa della costante presenza di rotameri. Il dimero peptoide è stato caratterizzato tramite esperimenti 2D NMR, che hanno messo in evidenza una conformazione completamente estesa. L’ibrido peptide/peptoide, invece, è stato preparato con successo. Esso è formato da due unità di peptoide che si alternano in una sequenza di β-amino acidi ciclici. Lo spettro 1H indica la presenza una struttura conformazionalmente stabile, ma le analisi al CD chiariranno definitivamente la sua struttura secondaria.<br>Foldamers is a field in progress; researchers are developing systems with any possible combination of elements and chemical structures, with the aim to design artificial highly-ordered molecules that exhibit biopolymer-like conformational behavior. Systems as β-peptides, ϒ-peptides, peptoids, so-called “peptidomimetics”, are among the most attractive foldamers because they possess specific sequences to interact with biological systems and they are able to form well-defined secondary structures. In my thesis β-peptides have been analyzed, in particular conformationally rigidified β-peptides and peptoids. In the first case, utilizing a peptide synthesis in solid phase and an alternating heterochiral approach, two patterns of β-peptides with different constrained side-chains have been synthesized: the (H-[(1R,2R)-BACHC-(1S,2S)-BACHC]n-NH2) oligomers 1, 2 and the (H-[(1R,2S)-ACHEC-(1S,2S)-BACHC]p-NH2) oligomers 3, 4. 2D NMR analyses (TOCSY, ROESY, COSY), NH/ND exchanges and ECD spectra, performed to study their conformation, have revealed that the hexamer 4 is the best in 10/12-Helix folding. The second part has been focused in peptoids. In good yield a new β-peptoid monomer, with conformational constraint due to ϒ-lactamic ring, has been prepared. This unit has been utilized for next peptide syntheses in homogeneous phase: a peptoid oligomer and a hybrid peptide/peptoid oligomer. The peptoid oligomeric synthesis has been complicated from low yield in coupling step and difficult structural analysis, due to the consistent presence of rotamers, but the dimer, characterized trough 2D NMR, has displayed a fully extended conformation. The hybrid peptide/peptoid, instead, has been prepared successfully. The mixed backbone has exhibited two alternated peptoids units in a sequence of conformationally rigidified β-amino acids; the 1H spectrum has shown a conformationally stable structure; finally ECD analysis will clarify definitively its secondary structure.

A collection of twenty three selectively mono-protected di- and triamines, masked with the Boc, Fmoc or Ddiv protecting groups, were synthesised via continuous flow synthesis in a self-assembled meso-scale PTFE flow reactor. The continuous flow strategy offered direct access to the mono-protected compounds in good yields, especially in the case of the Fmoc carbamates which circumvented the use of another sacrificial protecting group. Two of the mono-Boc-protected carbamates were used as starting materials to generate N-alkylglycine monomers; synthesised via tandem mono-alkylation and Fmoc carbamation, linked by an in-line scavenging protocol using a silica-based trisamine scavenger resin. The final step of the monomer synthesis employed catalytic transfer hydrogenolysis using 20% Pd(OH)2/C and 1,4- cyclohexadiene. The three-step flow procedure gave access to two monomers, with one of them being a novel N-alkylglycine unit bearing a triethylene glycol bridge. The monomers were used as building blocks to assemble new oligo-N-alkylglycines (peptoids) via microwave-assisted solid phase synthesis. Three different types of peptoids were synthesised: (i) oligo-N-(6-aminohexyl)glycines (“standard” peptoids), (ii) oligo-N-{2-[2-(2-aminoethoxy)ethoxy]ethyl}glycines (“triethylene glycol” [TEG] peptoids) and (iii) hetero-oligomers of alternating “standard” and “TEG” monomers (“hybrid” peptoids). The peptoids were evaluated for their cellular permeability and cytotoxicity with HeLa, HEK-293 and CHO cells. All the peptoids were shown to be non-cytotoxic at 10 μM based on cell proliferation assays. In general, it was found that the cellular uptake of the hybrid peptoids outperformed their standard and TEG analogues. Flow cytometry and confocal microscopy results revealed that the hybrid nonamer had the highest cellular uptake efficiency of all the peptoids synthesised. At a concentration of 1 μM, it outperformed the second best molecular transporter (standard nonamer) by a factor of seven.

Recent approaches to constraining peptide sequences into more structurally-defined α- helical secondary structures, so-called peptide stapling, are discussed. Stapled peptides are a class of therapeutics that have been shown to more effectively target protein-protein interactions, which are harder to target using a classical small-molecule therapeutic approach. Stapling a peptide constrains it into a well-defined secondary structure. This more accurately mimics the protein-protein interaction making the peptide a more viable therapeutic. Starting from the p53-MDM2 interaction, a protein-protein interaction with important implications in cell health, a known peptidyl inhibitor of this interaction was stapled and analysed for increased α-helicity. This was achieved by using monomers that utilise the copper (I) alkyne azide cycloaddition as a cross-linking methodology, which has been less well researched in the context of peptide stapling. The viability of a novel stapled peptomer inhibitor approach, accomplished using a new, optimised monomer synthesis, is investigated. Additionally, the synthesis of a ligand series designed for use in the copper(I) alkyne azide cycloaddition is also discussed.

Forward osmosis (FO) is an emerging technology for water treatment due to their ability to draw freshwater using an osmotic pressure gradient across a semi-permeable membrane. However, the lack of draw agents that could both produce reasonable flux and be separated from the draw solution at a low cost stand in the way of widespread implementation. This study had two objectives: evaluate the performance of three materials — peptone, carboxymethyl cellulose (CMC), and magnetite nanoparticles (Fe3O4 NPs) — as potential draw agents, and to use multi-criteria decision matrices to systematically prioritize known draw agents from literature for research investigation. Peptone showed water flux and reverse solute flux values comparable to other organic draw agents. CMC’s high viscosity made it impractical to use and is not recommended as a draw agent. Fe3O4 NPs showed average low fluxes (e.g., 2.14 LMH) but discrete occurrences of high flux values (e.g., 14 LMH) were observed during FO tests. This result indicates that these nanoparticles have potential as draw agents but further work is needed to optimize the characteristics of the nanoparticle suspension. Separation of the nanoparticles from the product water using coagulation was shown to be theoretically possible if only electrostatic and van der Waals forces are taken into account, not steric repulsion. If coagulation is to be considered for separation, research efforts on development of nanoparticle suspensions as FO draw agents should focus on development of electrostatically stabilized nanoparticles. A combination of Fe3O4 NP and peptone showed a higher flux than Fe3O4 NPs alone, but did not produce additive or synergistic flux. This warrants further research to investigate more combinations of draw agents to achieve higher flux than that obtained by individual draw agents. Potential draw agents were prioritized by conducting a literature review of draw agents, extracting data on evaluation criteria for draw agents developed over the past five years, using these data to rank the draw agents using the Analytical Hierarchy Process (AHP) and Technique for Order of Preference by Similarity to Ideal Solutions (TOPSIS). The evaluation criteria used in the ranking matrices were water flux, reverse solute flux, replenishment cost, regeneration cost, and regeneration efficacy. The results showed that the top five ranked draw agents were P-2SO3-2Na, TPHMP-Na, PEI-600P-Na, NaCl, and NH4-CO2. The impact of the assumption made during the multi-criteria decision analysis process was evaluated through sensitivity analyses altering criterion weighting and including more criteria. This ranking system provided recommendations for future research and development on draw agents by highlighting research gaps.

Made available in DSpace on 2016-06-02T19:56:39Z (GMT). No. of bitstreams: 1 3049.pdf: 2440029 bytes, checksum: f8b9ee5d47547364d040e6797729060f (MD5) Previous issue date: 2010-03-19<br>Universidade Federal de Sao Carlos<br>Pork is one of the most widely eaten meats in the world and pig farming is an economic activity booming in Brazil and the world. Several efforts have been made to develop more effective vaccines for major diseases that are affecting livestock such as swine erysipelas, caused by the bacterium Erysipelothrix rhusiopathiae. The currently available vaccines for the prevention of erysipelas are produced with culture broth of this microorganism inactivated or attenuated. The main antigenic agent identified is an protein fraction of 64-69 kDa, present in cell wall of bacteria and the supernatant of the culture. Given the accumulated knowledge of the studies conducted by Silva (2007), this study aimed to: i) study the conditions employed in the preparation of cell suspension for freezing and formation of culture stocks in crytubes and the stage of their activation; ii) studying the growth of E. rhusiopathiae, the formation of lactic acid and expression of antigen in culture media containing carbon sources alternative to glucose and in culture media containing nitrogen sources of plant origin; iii) to study the behavior of the microorganism in the new culture medium, animal-free, in a bioreactor. The studies for the improvement of the medium formulation were carried out in flasks incubated at static condition or under agitation of 200 rpm. The temperature was set at 37°C and the initial pH at 8,0 in all experiments. The studies in bioreactor were made using a 4.0 L stirred-tank bioreactor, with an agitation frequency kept between 100 and 700 rpm and air flow rate of 1.0 L/min. Samples of cell extracts made with choline chloride were analyzed by electrophoresis under denaturating conditions (SDS-PAGE) to evaluate the antigen expression. Studies of activation of the criotubes containing frozen cell suspensions led to the standardization of this step, with high reproducibility, and reduced activation time by 50%. The studies were grew with different carbon sources, showing that E. rhusiopathiae is able to assimilate galactose, lactose, and glucose. However, there was no assimilation of glycerol. The replacement of proteose peptone, a nitrogen source animal widely used in the cultivation of E. rhusiopathiae to produce bacterins, by Soytone, a soy peptone, animal-free, was a promising alternative for the production of the inactivated vaccine, helping to increase the specific growth rate and substrate conversion of cells in relation to values obtained in conventional medium. In batch cultivation performed in a bioreactor with medium containing glucose and Soytone, it was reached a biomass concentration of 10 g / L at 5 hours of cultivation. For the conventional medium, containing proteose peptone, the maximum cell concentration reported for the test batch in a bioreactor was approximately 2 g / L, which was reached after 7 hours of culture. Note also that a higher level of expression of antigenic protein in relation to those observed with peptone of animal origin was achieved in cultures performed with medium containing soytone.This result shows that could be incorporate the best practices of manufacturing practices for pharmaceutical and veterinary products, subject to the productivity of the process and with significant cost reduction.<br>A carne suína é a mais consumida no mundo e a suinocultura é uma atividade econômica em franca expansão no Brasil e no mundo. Diversos esforços vêm sendo realizados para o desenvolvimento de vacinas mais eficientes para as principais doenças que afetam os rebanhos, como a erisipela suína, causada pela bactéria Erysipelothrix rhusiopathiae. As vacinas disponíveis atualmente para a prevenção da erisipela são produzidas com o caldo de cultivo deste microrganismo inativado ou atenuado. O principal agente antigênico identificado é uma fração protéica de 64-69 kDa, presente tanto na parede celular da bactéria quanto no sobrenadante do cultivo. Diante do conhecimento acumulado ao longo dos estudos conduzidos por Silva (2007), o presente trabalho teve como objetivos: i) estudar as condições empregadas na preparação das suspensões celulares para armazenamento na forma de cultura estoque em criotubos assim como a etapa de ativação dos mesmos; ii) estudar o crescimento de E. rhusiopathiae, a formação de ácido lático e a expressão do antígeno SpaA em meios de cultivo contendo fontes de carbono alternativas à glicose e fontes de nitrogênio de origem vegetal; iii) estudar o comportamento do microrganismo no meio de cultura novo, livre de substratos de origem animal, em biorreator. Os experimentos foram conduzidos em câmara incubadora, em cultivos estáticos ou com agitação de 50 ou de 200 rpm. A temperatura utilizada foi de 37°C e o pH inicial foi de 8,0 em todos os ensaios realizados. Os estudos em biorreator foram realizados em biorreator de 5,0 L, com agitação entre 100 a 700 rpm e vazão de ar de 1,0 a 2 L/min. Amostras retiradas durante os cultivos foram empregadas para análise da densidade ótica (a 420 nm) e das concentrações de glicose, biomassa e metabólitos. A expressão do antígeno foi avaliada por eletroforese em condições desnaturantes (SDS-PAGE) a partir de extratos das células preparados com solução de cloreto de colina. Os estudos envolvendo a ativação dos criotubos contendo as suspensões celulares congeladas levaram à padronização desta etapa, com alta reprodutibilidade, e à diminuição do tempo de ativação em 50 %. Os cultivos realizados com diferentes fontes de carbono, mostraram que E. rhusiopathiae é capaz de assimilar galactose e lactose, além de glicose. No entanto, não foi verificada assimilação de glicerol. A substituição da proteose peptona, fonte de nitrogênio de origem animal amplamente utilizada nos cultivos de E. rhusiopathiae para produção de bacterinas, pela peptona de soja hidrolisada Soytone, de origem vegetal, mostrou-se um alternativa promissora para a produção da vacina de células inativadas, contribuindo para aumento da velocidade específica de crescimento e da conversão de substrato em células em relação aos valores obtidos no meio convencional. Em cultivo descontínuo realizado em biorreator de bancada com o meio contendo glicose e Soytone, foi alcançada uma concentração de biomassa de 10 g/L em 5 horas de cultivo. Para o meio convencional, contendo proteose peptona, a máxima concentração celular relatada para ensaio em batelada em biorreator de bancada foi aproximadamente 2 g/L, a qual foi atingida após 7 horas de cultivo. Destaca-se ainda que um nível superior de expressão da proteína antigênica em relação aos observados com a peptona de origem animal foi alcançado nos cultivos realizados com o meio contendo soytone. Esse resultado mostra ser possível incorporar as boas práticas de manufatura recomendadas para produtos farmacêuticos e veterinários, sem prejuízo à produtividade do processo e com significativa redução do custo do meio.

[EN] High Hydrostatic Pressure technology emerged several years ago in response to consumer interest for products having greater organoleptic and nutritional quality than those traditionally processed by heat. The food industry initially improved preservation processes developing continuous heat treatment HTST, UHT and aseptic packaging. However, despite these improvements foodstuffs preserved by heat still have quality losses. Although initially the application of High Hydrostatic Pressure in the field of food was focused towards preservation, with the development of technology and research around new applications raised that make it very interesting for the sector. In this context, the aim of this thesis has been the application of High Hydrostatic Pressure on the preservation of fresh cheese and the revalorization of by-products from cheese factories. For this, the effect of treatments by high hydrostatic pressure (HHP) on pathogenic microorganisms (S. aureus, L. monocytogenes) and spoilage microorganisms (Rhodotorula glutinis, aurantiogriseum Penicillium, Pseudomonas fluorescens) that are present in lactic products is evaluated as well as the effect of various factors (sodium chloride (NaCl), initial treatment temperature) on microbial inactivation. To study this effect peptone water at concentrations of 0; 0.1; 0.5; 0.8% NaCl was used as reference medium and fresh cheese as complex matrix containing 0.5% NaCl. Pressure ranges between 200 and 550 MPa (Mega Pascals) were used with short treatment times (in the order of seconds to several minutes), combined with a moderate initial temperature of 20 to 50 °C.The results of microbial inactivation were fitted to the mathematical model that best described the behavior of inactivation curves, allowing predicting the behavior of the microorganisms being treated by HHP under different environmental factors. Treatments by HHP of R. glutinis and P. aurantiogriseum around 350 - 400 MPa generated more than 5 log10 reductions on the initial contamination. The NaCl concentration present in the reference medium for these two microorganisms was an additional control factor. Regarding the effect of the substrate significant differences between the mean values obtained for the reference medium and the complex matrix (cheese) were observed, fresh cheese showed a lower reduction in the initial inoculated load. Due to the production processes of cheesemaking large amount of residual whey generated is a problem under the environmental point of view. In this thesis the use of residual liquid whey was also studied in conjunction with whey protein isolates (WPI) for the purpose of producing gels with a high biological value through various HHP processes. The various mixtures of whey and different percentages of WPI (5, 7, 11, 15% w/v) processed by HHP resulted in gels with different characteristics. The pH values, color (L * a * b *) vary slightly directly depending on the WPI concentration and the storage time. The hardness values and gumminess were modified depending on the liquid medium and the concentration of WPI. The use of cheese whey to gelation favored water retention, significantly reducing the syneresis under refrigeration. This study shows the potential of HHP as control measure, demonstrating the effectiveness of these treatments to prolong the shelf life of fresh cheeses and as unconventional technique to generate new textures that can have great industrial interest at the same time that revalorize some wastes.<br>[ES] La tecnología de las Altas Presiones Hidrostáticas (HHP) emergió hace varios años como respuesta al interés de los consumidores por disponer de productos de una mayor calidad organoléptica y nutricional que los tradicionalmente procesados por calor. La industria agroalimentaria inicialmente mejoró sus procesos de conservación por calor desarrollando los tratamientos continuos HTST, UHT y el envasado aséptico. Sin embargo, a pesar de estas mejoras los productos alimenticios conservados por calor siguen presentando pérdidas en su calidad final. Aunque al principio la aplicación de las Altas Presiones Hidrostáticas en el campo de la alimentación se enfocó hacia la conservación, con el desarrollo de la tecnología y la investigación alrededor de ella han surgido nuevas aplicaciones que la hacen muy interesante para el sector. En este contexto, el objetivo de la presente tesis doctoral ha sido la aplicación de las Altas Presiones Hidrostáticas en la conservación de queso fresco y en la revalorización de subproductos de queserías. Para ello, se evalúa el efecto de los tratamientos por Altas Presiones Hidrostáticas (HHP) sobre microorganismos patógenos (Staphylococcus aureus, Listeria monocytogenes) y alteradores (Rhodotorula glutinis, Penicillium aurantiogriseum, Pseudomonas fluorescens) de productos lácteos y el efecto de diversos factores presentes en los alimentos (cloruro sódico (NaCl), temperatura inicial de tratamiento) sobre la inactivación microbiana. Para estudiar este efecto se utilizó como medio de referencia agua peptona a concentraciones de 0; 0,1; 0,5; 0,8% de NaCl y como matriz compleja queso fresco con un contenido de 0,5% de NaCl. Se utilizaron intervalos de presión entre 200 y 550 MPa (Mega Pascales) con tiempos de tratamiento cortos (en el orden de segundos a varios minutos), combinados con una temperatura inicial moderada de 20 a 50 °C. Los resultados de inactivación microbiana se ajustaron al modelo matemático que mejor describía el comportamiento de las curvas de inactivación, lo que permite predecir el comportamiento de los microorganismos frente a las HHP y bajo los diferentes factores. En cuanto a las HHP para R. glutinis y P. aurantiogriseum tratamientos en torno a los 350 - 400 MPa generaron más de 5 reducciones log10 de la carga inicial. La concentración de NaCl presente en el medio de referencia para estos dos microorganismos fue un factor adicional de control. Con respecto al efecto del medio de trabajo se observaron diferencias significativas (p<0,05) entre los valores medios obtenidos para el medio de referencia y la matriz compleja (queso fresco): el queso fresco presentó una menor reducción en la carga inicial inoculada.Debido a los procesos productivos de la fabricación del queso se genera gran cantidad de suero lácteo residual que es un problema bajo el punto de vista medioambiental. En esta tesis también se estudió la utilización del suero lácteo líquido residual en conjunto con aislados proteicos del suero (WPI) con la finalidad de producir geles con un elevado valor biológico mediante diversos procesos de HHP. Las diversas mezclas de suero de queso junto con los porcentajes de 5, 7, 11, 15% p/v de WPI procesadas por HHP dieron lugar a geles con diversas características. Los valores de pH, color (L*a*b*) variaron ligeramente dependiendo directamente tanto de la concentración de WPI como del tiempo de almacenamiento. Los valores de dureza y gomosidad sufrieron modificaciones dependiendo del medio líquido y la concentración de WPI. La utilización de suero de queso para la formación del gel favoreció la retención de agua, reduciendo significativamente la sinéresis en refrigeración.Este estudio muestra el potencial de las HHP como una medida de control, demostrándose la efectividad de estos tratamientos para prolongar la vida útil de los quesos frescos y como técnica no convencional para generar nuevas texturas que pueden tener gran inte<br>[CAT] La tecnologia de les altes pressions hidrostàtiques va emergir fa diversos anys com a resposta a l'interés dels consumidors per disposar de productes d'una qualitat organolèptica i nutricional major que la dels processats per calor tradicionalment. La indústria agroalimentària inicialment va millorar els seus processos de conservació per calor desenvolupant els tractaments continus HTST, UHT i l'envasat asèptic. No obstant això, malgrat aquestes millores els productes alimentaris conservats per calor continuen presentant pèrdues en la seua qualitat final.Tot i que al principi l'aplicació de les altes pressions hidrostàtiques en el camp de l'alimentació es va enfocar cap a la conservació, amb el desenvolupament de la tecnologia i la investigació al voltant d'ella han sorgit noves aplicacions que la fan molt interessant per al sector. En aquest context, l'objectiu de la present tesi doctoral ha sigut l'aplicació de les altes pressions hidrostàtiques en la conservació del formatge fresc i en la revalorització de subproductes de formatgeries. Per a això, s'avalua l'efecte dels tractaments per altes pressions hidrostàtiques (HHP) sobre microorganismes patògens (Staphylococcus aureus, Listeria monocytogenes) i alteradors (Rhodotorula glutinis, Penicillium aurantiogriseum, Pseudomonas fluorescens) de productes lactis i l'efecte de diversos factors presents als aliments (clorur sòdic (NaCl), temperatura inicial de tractament) sobre la inactivació microbiana. Per a estudiar aquest efecte es va utilitzar com a medi de referència aigua peptona a concentracions de 0; 0,1; 0,5; 0,8% de NaCl i com a matriu complexa formatge fresc amb un contingut de 0,5% de NaCl. Es van utilitzar intervals de pressió entre 200 y 550 MPa (Mega Pascals) amb temps de tractament curts (en l'orde de segons a diversos minuts), combinats amb una temperatura inicial moderada de 20 a 50 °C. Els resultats d'inactivació microbiana es van ajustar al model matemàtic que millor descrivia el comportament de les corbes d'inactivació, el que permet predir el comportament dels microorganismes front a les HHP i baix els diferents factors. En referència a les HHP per a R. glutinis i P. aurantiogriseum tractaments al voltant dels 350 - 400 MPa van generar més de 5 reduccions log10 de la càrrega inicial. La concentració de NaCl present en el medi de referència per a aquestos dos microorganismes va ser un factor addicional de control. Respecte a l'efecte del medi de treball es van observar diferències significatives (p<0,05) entre els valors mitjans obtinguts per al medi de referència i la matriu complexa (formatge fresc): el formatge fresc va presentar una menor reducció en la càrrega inicial inoculada. Degut als processos productius de la fabricació del formatge es genera gran quantitat de sèrum lacti residual que és un problema baix el punt de vista mediambiental. En aquesta tesi també es va estudiar la utilització del sèrum lacti líquid residual en conjunt amb aïllats proteics del sèrum (WPI) amb la finalitat de generar gels amb un elevat valor biològic mitjançant diversos processos de HHP. Les diverses mescles de sèrum de formatge juntament amb els percentatges de 5, 7, 11, 15% p/v de WPI processades per HHP van generar gels amb diverses característiques. Els valors de pH, color (L*a*b*) van variar lleugerament depenent directament tant de la concentració de WPI com del temps d'emmagatzematge. Els valors de duresa i gomositat van sofrir modificacions depenent del medi líquid i la concentració de WPI. La utilització del sèrum de formatge per a la formació del gel va afavorir la retenció d'aigua, reduint significativament la sinèresi en refrigeració.Aquest estudi mostra el potencial de les HHP com a una mesura de control, demostrant-s'hi l'efectivitat d'aquestos tractaments per a prolongar la vida útil dels formatges frescos i com a tècnica no convencional per a generar noves textures que poden tindre gran interés<br>Torres Bello, EF. (2016). Efecto del tratamiento por altas presiones hidrostáticas (HHP) en la calidad de queso fresco y en las proteínas de suero de queserías [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/72866<br>TESIS

Die SPOT-Synthese an Zellulosemembranen wurde 1992 als eine hocheffiziente Methode zur parallelen Synthese von Peptiden beschrieben. Die wichtigste Anwendung der so synthetisierten Verbindungen ist das direkte Festphasen-Screening. Im Rahmen dieser Arbeit ist es gelungen, das Anwendungsgebiet der SPOT-Methode von Peptiden auf verschiedene Peptidmimetika auszudehnen und durch Screening entsprechender Bibliotheken bioaktive Substanzen zu identifizieren. (1) Peptoid-Synthese an Zellulosemembranen Die Ähnlichkeit von Oligo-N-alkylglycinen (Peptoiden) zu Peptiden sowie die Vereinbarkeit ihrer Synthese mit den Bedingungen der SPOT-Technik ließen sie als besonders geeignete Kandidaten für eine Erweiterung der SPOT-Synthese von Peptiden auf Peptidmimetika erscheinen. Die Peptoide wurden nach der 1992 für die Synthese am Harz beschriebenen Sub-Monomer-Methode synthetisiert, bei der die N-Alkylglycin-Monomere zweistufig durch Bromacetylierung und nachfolgende Bromsubstitution durch ein primäres Amin aufgebaut werden. Die Kernaufgabe bei der Anpassung der Synthesebedingungen an Zellulosemembranen war dabei die Entwicklung einer N/O-selektiven Bromacetylierungsmethode, da die Anwesenheit freier Membran-Hydroxyfunktionalitäten ein Reagenz erfordert, welches eine N-Acylierung in Anwesenheit von O-Nukleophilen zuläßt. Durch Untersuchung mehrerer Aktivester der Bromessigsäure konnte gezeigt werden, daß der kristalline Bromessigsäure-2,4-dinitrophenylester im Hinblick auf Ausbeute und N/O-Selektivität optimale Eigenschaften besitzt. Im Anschluß an die Bromacetylierungsmittel wurden 46 primäre Amine auf ihre Anwendbarkeit bei der Synthese von Modell-Tripeptoiden untersucht. Aus den Ergebnissen konnten Gesetzmäßigkeiten abgeleitet werden, die eine Abschätzung der Verwendbarkeit von Aminen für die Peptoidsynthese im Hinblick auf Flüchtigkeit, sterischen Anspruch, Nukleophilie des Stickstoffatoms sowie vorhandene funktionelle Gruppen in Seitenketten ermöglichen. (2) Synthese und Screening von Peptoid-Bibliotheken Unter den optimierten Synthesebedingungen wurden zwei Bibliotheken mit jeweils 8000 Tri- bzw. Hexapeptoiden synthetisiert. Die Trimeren-Bibliothek beinhaltete dabei den gesamten Sequenzraum basierend auf 20 Bausteinen, während die Verbindungen der Hexameren-Bibliothek aus einem wesentlich größeren, auf 40 Bausteinen basierenden Sequenzraum statistisch ausgewählt wurden. Um zu überprüfen, ob sich die Bibliotheken zur "de novo" Auffindung von Protein-Liganden eignen, wurden sie auf Bindung zum monoklonalen Antikörper Tab-2 untersucht. Es konnten in beiden Fällen bioaktive Oligomere identifiziert werden (Trimere: KD >= 87 µM, Hexamere: KD >= 2.7 µM), die sich vom Peptid-Epitop des Antikörpers [VVSHFND] deutlich unterschieden. (3) Rückgratmodifizierte Peptoide Mit dem Ziel, Rückgratmodifikationen in Peptoide einzufügen, wurden neun Biselektrophile im Rahmen eines "chemischen Screenings" zur Synthese eines Modell-Trimers verwendet. Vier der Bausteine waren geeignet und ermöglichten damit die Einführung von beta-Peptoid-, m- und p-Aminomethylbenzoesäure- sowie Carbamat-Einheiten in Peptoide. Beim Versuch, in analoger Weise auch Harnstoffe zugänglich zu machen, wurde unter den Linker-Spaltungsbedingungen eine Cyclisierung zu Hydantoinen beobachtet. Diese interessante Reaktion wurde näher untersucht, um die SPOT-Methode auf die Synthese von Hydantoinen als heterocyclische Struktur zu erweitern. (4) Synthese von Hydantoinen an Zellulosemembranen Die Bildung von Hydantoinen in einer Cyclisierungsreaktion, bei der Ammoniak aus einem Amid freigesetzt wird, wurde an fester Phase noch nicht genutzt, während dieser Reaktionstyp in Lösung bereits intensiv untersucht wurde. Durch eine Optimierung der Cyclisierungsbedingungen ließ sich die zunächst unvollständige Reaktion zur Vollständigkeit bringen. Auch C-substituierte Hydantoine konnten durch Verwendung von alpha-Aminosäureamiden bzw. -tert.-butylestern enantiomerenrein zugänglich gemacht werden.<br>SPOT-synthesis on cellulose membranes was introduced as a highly efficient method for the parallel synthesis of peptides in 1992. The most important applications of libraries synthesized by SPOT-synthesis are solid phase binding assays. Within this work the extension of the SPOT-method to the synthesis of various peptidomimetics and the identification of bioactive substances by screening of corresponding libraries is described. (1) peptoid synthesis on cellulose membranes The similarity of oligo-N-alkylglycines (peptoids) and peptides as well as the compatibility of their synthesis with the conditions of the SPOT-technique made them ideally suited for the extension of the SPOT-synthesis from peptides to peptidomimetics. The peptoids were synthesized by the sub-monomer approach originally developed for the synthesis on standard resins in 1992. N-alkylglycine monomers are hereby synthesized in a stepwise manner by bromoacetylation and subsequent substitution of the bromine atom by a primary amine. The most critical point in the adaptation of the synthesis conditions was the development of an N/O-selective reagent for bromoacetylation due to the presence of free hydroxyl functionalities of the membrane support requiring a reagent suitable for N-acylation in the presence of O-nucleophiles. Several active esters of bromoacetic acid were synthesized and tested whereby crystalline 2,4-dinitrophenylbromoacetate gave the best results with respect to yield and N/O-selectivity. After optimization of bromoacetylation 46 primary amines were applied to the synthesis of model tripeptoids. Rules for the applicability of amines in peptoid synthesis with respect to volatility, sterical demand, nucleophilicity of the nitrogen atom and compatibility with sidechain functional groups were derived from the results. (2) synthesis and screening of peptoid libraries Two libraries consisting of 8000 tri- and hexapeptoids respectively were synthesized under optimized conditions. The library of trimers displayed the entire sequence space based on 20 building blocks, whereas the sequences of the hexamers were selected statistically from the sequence space based on 40 building blocks. In order to examine the suitability of the libraries for the "de novo" identification of protein ligands they were screened for binding to the monoclonal antibody Tab-2. Bioactive peptoids could be identified in both cases (trimers: KD >= 87 µM, hexamers: KD >= 2.7 µM) both differing significantly from the peptide epitope [VVSHFND]. (3) backbone modified peptoids In order to introduce backbone modifications into peptoids nine biselectrophiles were applied in the synthesis of model trimers in a chemical screening. Four of the building blocks were well suited allowing the incorporation of beta-peptoid, m- and p-aminomethylbenzoic acid and carbamate units into peptoids. When the introduction of urea-units in a similar approach was attempted hydantoins were formed during cleavage from the solid support. This interesting reaction was examined in detail in order to extend SPOT-synthesis to the synthesis of heterocycles. (4) synthesis of hydantoins on cellulose membranes The formation of hydantoins from terminal amides was not yet described in a solid phase synthesis, whereas it was examined intensively in solution. By optimizing the conditions of cyclization the reaction could be driven to completion. C-substituted hydantoins were obtained as single enantiomers, when alpha-amino acid-amides or -tert. butylesters were used in the synthesis.

碩士<br>國立成功大學<br>環境工程學系碩博士班<br>92<br>The multiple substrates, starch and peptone, were investigated for anaerobic hydrogen production in this study. This study included the compare of different enrich cultures, the effects of substrate composition on hydrogen fermentation and the performance evaluation of a mesophilic biohydrogen reactor. 　　The seeding sludge was enriched from five different sources of anaerobic sludge. After 3 times of successfully transferring enrichment cultures to new medium and growing up, the recovery of hydrogen gas from multiple substrates of starch and peptone would reach similar and stable levels. DGGE fingerprint of the bacteria domain 16S rDNA fragments revealed that the diversity of different inoculia were almost similar. 　　Among of all the organic substances in Nature, carbohydrates have been proved to be a suitable substrate for anaerobic hydrogen production. However, protein is a good substrate for cell growth and can promote hydrogen production. Besides, the presence of protein in the substrate would enlarge the buffer capacity of the broth. Therefore, the multiple substrates of carbohydrate and protein would be the better choice for hydrogen fermentation. The ratios of starch and peptone in the multiple substrates would also affect the rate of fermentation noticeably. When the content of starch increased to 60 % in the multiple substrates, the hydrogen production would increase noticeably. But without peptone in the multiple substrates, the bacteria would degenerate and lose the ability to produce hydrogen. 　　A hydrogen fermentor which fed with 12,000 mg/L corn starch and 8,000 mg/L peptone was operated at different sludge retention time (SRT) and hydraulic retention time (HRT). A micro-filtration membrane was employed for the separation and recovery of the biomass. The result revealed that starch gelatinizing was an important process for hydrogen production in continuous flow operation. After the influent changed to raw starch (without gelatinizing) in the same organic loading rate, 20 kg/m3-day, hydrogen production rate dropped obviously. At the same time, the starch conversion ratio would drop to 40%. The compositions of the volatile fatty acids in the effluent shifted to acetic acid and propoinic acid. SRT and HRT were important operational parameters to recovery the hydrogen production in the hydrogen fermentor. The ammonia in the effluent decreased as the SRT decreased. This result implied that longer SRT could enhance the degradation of peptone. But the metabolism of peptone would lead to hydrogen consumption because of no hydrogen in biogas. However, hydrogen production and consumption did occur in the batch test. As the HRT decreased to 6 hours, the hydrogen gas could be detected in the biogas and lactic acid could be detected in the effluent. Furthermore, lactic acid was an important intermediate which could be detected in the shock loading phase and in the batch test. Lactate produced along with the hydrogen production, and consumed consequently. This indicated that the consortium in the fermentor was very complex. And shorten HRT could be a strategy for hydrogen recovery.

碩士<br>國立成功大學<br>環境工程學系碩博士班<br>93<br>This study is to describe the different hydrogen fermentation mechanism between monosaccharide (glucose) mixed with peptone and polysaccharide (starch) mixed with peptone, as artificial multiple substrates, in the batch test. This study also covered the performance evaluation of continuous hydrogen fermentation system feed with starch and peptone. Finally extended to the feasibility study of apply substrate-food waste. The first part studied glucose and peptone fermentation mechanism by batch test. After fermentation, butyrate is the major by-product in the bulk solution, and the yield is 156 mg Hbu/ g CODa. The hydrogen producing rate is 122.41mL H2 /gVSS-hr. Use peptone as sole substrate in batch test, the hydrogen consuming phenomena is observed. The major by-product is acetate. The molecular method, terminal restriction fragment length polymorpholysium (T-RFLP), was used to investigate the dynamic of microbial ecology of the biohydrogen fermentation process. The TRFLP results indicated that Clostridium clusters I and II presented in the fermentor at all HRT conditions. Desulfotomaculum – like can also be found in some of these operation periods. The multiple substrate of glucose and peptone is also be used to study the effect of NaCl in hydrogen fermentation. The result shows it comes the maximum hydrogen yield and producing rate when the additional NaCl concentration is 10 g/L. In this study of starch and peptone fermentation, the hydrogen consuming phenomena is observed in batch test. After fermentation, propionate is the major by-product in the bulk solution, and the yield is 99.37 mg HPr/ g CODa. The hydrogen producing rate is 25mL H2 /gVSS-hr. To detect the amylase activity, it is found that α-amylase characteristics of HPBs is more cell bound type and growth associated. By the data of reducing sugar analysis, the hydrolysis mechanism of HPBs can be generally described. The performance of the anaerobic hydrogen fermentation process was evaluated at different hydraulic retention times (HRTs) using a continuous stirred tank reactor (CSTR) type fermentor fed with starch and peptone. The results of anaerobic fermentor operations indicated that hydrogen production performance were strongly dependent on HRTs. At HRTs longer than 9 hours, no net hydrogen production was observed in the fermentor although enormous amount of starch and peptone was consumed. At HRT of 3 hours, the CSTR had a hydrogen production rate of 435 mmol/L/d which was significantly higher than those observed at other HRT conditions. The TRFLP results indicated that Clostridium clusters I and II presented in the fermentor at all HRT conditions and were, presumably, responsible for hydrogen production from starch fermentation. The SEM observation, discovered the microbial morphology is rod-like shape and supposed to be Clostridium-like microorganism. This study include the feasibility of food waste thermophilic hydrogen fermentation. The sludge from the food waste methanogenesis tank is taken as the inoculation, and the content of the vegetarian lunch box is mashed as the substrate. it gets great hydrogen yield and great hydrogen producing rate. By T-RFLP method, it can find Clostridium cluster I, cluster II, and cluster III, which is similarity to hydrogen producing bacterium.

Pedobacter lusitanus NL19 is a Gram -negative bacterium from the famil y Sphingobacteriaceae, which was isolated from a deactivated uranium mine in Portugal. This strain produces non ribosomal peptides (NRPs), calle d pedopeptins. The production of these peptides is repressed by high concentrations of peptone from casein (PC). In addition to biosynthetic gene clusters (BGCs) encoding NRPs, the NL19 strain genome also has BGCs of other secondary metabolites (SMs), incl uding lanthipeptides (4 BGCs: ped8, ped 14, ped15 and ped17). Lanthipeptides are ribosomally synthesized and post -translationally modified peptides (RiPPs), which exhibit a wide variety of biological activities, including antimicrobial and antiallodyni c. Lanthipeptides are characterized by the presence of lanthionine (Lan) and meth yllanthionine (MeLan) residues and are divided into four classes, defined by the enzymes that catalyze the reactions needed for the installation of these residues. T his s tudy focused on the NL19 strain and the main objectives were to determine the effect of high concentrations of PC: i) on the transcription of lanthipeptides and ii) on the proteome of the strain, with special interest in proteins involved in the biosy nthesis of SMs. Due to the COVID -19 pandemics and confinement, a third objective was defined , w hich aimed to identify and analy z e, in silico, lanthipeptide BGCs from the genomes of other genera of th e family Sphingobacteriaceae. Objective i) involved the transcriptional analysis of BGC ped 1 5 that encodes two precursor peptides and other biosynthetic proteins. It was necessary to s e quence the upstream region of this cluster, through primer walking, whi ch allowed the identification of six other structural peptide genes. The transcriptional analysis was performed by RT -qPCR and revealed that high concen trations of PC do not affect the expression of the BGC ped15, contrary to what was found fo r the pedope ptin s BGC . In general, the RT -qPCR results validated the available RNA -seq results and showed that the transcriptional repression caused by high concentrations of P C is not transversal to t he production of other SMs. Objective ii) involved t he analysis of the proteome of the NL19 strain grow n in high concentrations of PC (and its control) by nano LC -ESI -MS/MS and allowed the detection of the differential expression of some proteins relat ed to the biosynthesis of SMs, including the pedopeptins nonribo somal peptide synth etases and a lanthipeptide precursor encoded in the ped8 BGC. Objective iii) involved the analysis of 446 BGCs of the family Sphingobacteriaceae with the antiSMASH and Bi G -SCAPE tools. Class I and class III lanthipeptide BGCs were ide ntified in the gene ra Mucilaginibacter and Sphingobacterium. Class III BGCs encode La nKC enzymes with slightly different lyase domains, which may indicate that these enzymes use a different me chanism for the installation of Lan /MeLan. This stu dy contributes to the bo dy of knowledge of the bacterial response to the manipulation of culture media, in particular in the production of SMs with biotechnological potential as lanthipeptide s. In addition, this study identified the potential of bacterial genera already known, but still unde rex plored, for the production of new lanthipeptides, whose biosynthetic, structural and functional characterization is unknown.<br>Pedobacter lusitanus NL19 é uma bactéria de Gram-negativo, da família Sphingobacteriaceae, que foi isolada de uma mina de urânio desativada em Portugal. Esta estirpe produz péptidos não ribossomais (NRPs), designados por pedopeptinas. A produção destes péptidos é reprimida em meio com elevadas concentrações de peptona de caseína (PC). Para além de clusters biosintéticos (BGCs) que codificam NRPs, o genoma da estirpe NL19 também possui BGCs de outros metabolitos secundários (SMs), incluindo lantipéptidos (4 BGCs: ped8, ped14, ped15 e ped17). Lantipéptidos são péptidos de síntese ribossomal com modificações pós-traducionais (RiPPs), que exibem uma ampla variedade de atividades biológicas, incluindo antimicrobiana e antialodínica. Estes péptidos são caracterizados pela presença de resíduos de lantionina (Lan) e metillantionina (MeLan) e dividem-se em quatro classes, definidas pelas enzimas que catalisam as reações que originam esses resíduos. Este estudo focou-se na estirpe NL19 e os principais objetivos foram determinar o efeito de elevadas concentrações de PC: i) na transcrição de lantipéptidos e ii) no proteoma da estirpe, em particular proteínas envolvidas na síntese de SMs. Devido à pandemia COVID-19 e consequente confinamento, foi definido um terceiro objetivo, que consistiu na identificação e análise in silico de BGCs de lantipéptidos presentes nos genomas de outros géneros da família Sphingobacteriaceae. O objetivo i) envolveu a análise transcricional do BGC ped15, que codifica dois péptidos percursores e outras proteínas biosintéticas. Para tal, procedeu-se à sequenciação da região a montante deste cluster, por primer walking, o que permitiu identificar outros seis genes de péptidos percursores. A análise transcricional foi realizada por RT-qPCR e revelou que elevadas concentrações de PC não alteram a expressão do BGC ped15, ao contrário do que acontece com o BGC das pedopeptinas. De uma forma geral, os resultados de RT-qPCR validaram os resultados de RNA-seq disponíveis, e mostram que o efeito repressor da PC não é transversal à produção de todos os SMs. O objetivo ii) incluiu a análise do proteoma da estirpe NL19 cultivada com elevadas concentrações de PC (e respetivo controlo) por nano LC-ESI-MS/MS e permitiu detetar a expressão diferencial de várias proteínas relacionadas com a biosíntese de SMs, incluindo as péptido sintetases não ribossomais das pedopeptinas e de um precursor de lantipéptido codificado no BGC ped8. O objetivo iii) envolveu a análise de 446 BGCs da família Sphingobacteriaceae com as ferramentas bioinformáticas antiSMASH e BiG-SCAPE. Foram identificados BGCs de lantipéptidos de classe I e classe III nos géneros Mucilaginibacter e Sphingobacterium. Os BGCs de classe III codificam enzimas LanKC com domínios liase um pouco distintos, o que pode indicar que estas enzimas utilizam um mecanismo de formação de Lan/MeLan relativamente diferente daquele já conhecido. Este estudo contribui para o conhecimento da resposta bacteriana à manipulação de meios de cultura, em particular na produção de SMs com potencial biotecnológico como os lantipéptidos. Para além disso, permitiu identificar o potencial de géneros bacterianos já conhecidos, mas até agora inexplorados, para produzir novos lantipétidos, cuja caracterização biosintética, estrutural e funcional é ainda desconhecida.<br>Mestrado em Biotecnologia

Over the years, the Burgess group has been focusing on the preparation and testing of small molecules that mimic protein secondary structures for protein-protein interactions. The most successful compounds made are C10 peptide macrocycles that effectively mimic β-turns and have given promising results from biological testing. These peptide macrocycles have also been dimerized to give even more effective ligands for protein-protein interaction. The successes of the peptide macrocycles have enabled us to look into increasing the chemical diversity of our libraries. This we believe will not only improve our ability to obtain high affinity ligands for the receptors of interest, but will also allow us to investigate other receptors. To achieve this, peptoids were incorporated into the C10 system to replace the peptides in the i+1 and i+2 positions. With the help of Microwave irradiation, semi-peptoid macrocycles were synthesized with a total reaction time of less than 2 h. These compounds were characterized and found to mimic β-turn, and show promising biological activity towards the Insulin-like growth factor 1 receptor (IGF-IR).

This study reports a series of promising and structurally diverse potential HIV-1 protease inhibitors. Human Immunodeficiency Virus (HIV) is the causative agent of Acquired Immune Deficiency Syndrome (AIDS). HIV infection disrupts the immune system and makes the body susceptible to opportunistic infections. If untreated, AIDS is generally fatal. Today, AIDS has become a long lasting pandemic. According to the World Health Organization (WHO) and Joint United Nations Program (UNAIDS-2009) report, it is estimated that 33.3 million men, women and children worldwide are infected with HIV. This situation is steadily deteriorating in some parts of the world compared to the previous years. One of the major drawbacks associated with the currently FDA-approved anti-HIV drugs are severe side effects, toxicities, high dosage and high treatment cost. Thus, an urgent need for new drugs to combat HIV is apparent. In the first part of the study, research efforts were focused to synthesize potent pentacycloundecane (PCU) derived peptide and peptoids as protease inhibitors. It is proposed that these inhibitors bind to wild type C-South African HIV protease (C-SA) catalytic site via a non-cleavable or non-hydrolysable cyclic ether bond for the first polycyclic cage compound and via a dihydroxylethelene type functional group for the second cage compound. The desired compounds were synthesized by coupling of the peptides and peptoids to the PCU derived cage. Second part of the study involves, biological evaluation against wild type C-SA enzyme and characterization of the synthesized compounds by Nuclear Magnetic Resonances (NMR). All the synthesized novel compounds were evaluated against wild type C-SA enzyme for their ability to inhibit 50% of the enzyme’s activity (IC50). Some of the compounds reported herein showed promising activity by inhibiting the enzyme activity at concentrations of less than 0.6 nM. 2D NMR investigations employing a new Efficient Adiabatic Symmetrized Rotating Overhauser Effect Spectroscopy (ROESY / NOESY) technique enabled the attainment of vital information about the 3D structure of these small linear peptides and peptoids in solution. The activity could be related to conformations induced by the PCU moiety on the coupled peptide side chain. Further quantum mechanics/molecular mechanics/molecular dynamics (QM/MM/MD) simulations were carried out to confirm the observed NMR experimental results. Docking studies were performed for the synthesized compounds. Binding energies obtained from the docking calculations were then used to further validate the experimental IC50 results. These experimental and theoretical methods provided valuable insight into the interaction mode of these cage peptide and peptoids inhibitors with the enzyme.<br>Thesis (Ph.D.)-Unversity of KwaZulu-Natal, Westville, 2012.