<p>The engineering of bacterial surfaces has in recent yearsattracted a lot of attention with applications in manydifferent areas of bioscience. Here we describe the use of twodifferent surface display systems for the gram-positivebacteria Staphylococcus carnosus and Staphylococcus xylosus invarious biotechnological applications.</p><p>Environmental microbiology currently attracts a lot ofattention since genetically engineered plants and bacteriamight be used as bioadsorbents for sequestration of toxicmetals. Bacterial surface display of metal-binding peptidesmight enable recycling of the biomass by desorption ofaccumulated heavymetals. In an attempt to recruitstaphylococcal display systems for bioremediation purposes,polyhistidyl peptides were successfullly displayed on thesurface of recombinant S. carnosus and S. xylosus cells.Whole-cell Ni2+-binding assays demonstrated that therecombinant cells had gained metal-binding capacity compared towild-type cells.</p><p>Tailor-made, metal-binding staphylococci was created using apreviously constructed phage-display combinatorial proteinlibrary based on a fungal cellulose-binding domain (CBD)derived from the cellobiohydrolase Cel7A of Trichoderma reseii.Novel metal-binding CBDs were generated through a phagemediated selection procedure. Selected CBD variants, now devoidof cellulose binding, were randomly selected and sequenceanalysis of selected variants revealed a marked preference forhistidine residues at the randomized positions. Surface displayof these novel CBD variants resulted in recombinantstaphylococci with increased metal-binding capacity compared tocontrol strains, indicating that this could become a generalstrategy to engineer bacteria for improved binding to specificmetal ions.</p><p>Directed immobilization of cells with surface displayedheterologous proteins have widespread use in modernbiotechnology. Among other things they could provide aconvenient way of generating biofilters, biocatalysts orwhole-cell diagnostic devices. It was therefore investigatedwhether directed immobilization of recombinant staphylococci oncotton fibers could be achieved by functional display of afungal cellulose-binding domain (CBD). Recombinant S. carnosuscells with surface anchored CBDs from Trichoderma reseii Cel6Awere found to efficiently bind to cotton fibers creating almosta monolayer on the fibrous support. The co-expression of thisCBD together with previously described metal-binding proteinson the surface of our staphylococci would create means fordeveloping effective bioadsorbents for remediationpurposes.</p><p>The original plasmid vector, designed for heterologoussurface display on recombinant S. carnosus cells has exhibitedproblems related to structural instability, possibly due to thepresence of a phage f1 origin of replication in the vectorsequence. This would be a problem if using the vector systemfor library display applications. Therefore, novel surfacedisplay vectors, lacking the phage ori were constructed andevaluated by enzymatic and flow cytometric whole-cell assays.One such novel vector, pSCXm, exhibited dramatically increasedplasmid stability with the retained high surface density ofexpressed heterologous proteins characteristic for the originalS. carnosus display vector, thus making it potentially moresuitable for library display applications.</p><p>The successful engineering of our staphylococcal displaysystem encouraged us to further evaluate the potential to usethe staphylococcal system for display of combinatorial proteinlibraries and subsequent affinity based selections using flowcytometric cell sorting. A model system of recombinant S.carnosus cells with surface displayed engineered protein Adomains was constructed. It was demonstrated that target cellscould be sorted essentially quantitatively from a moderateexcess of background cells in a single sorting-step.Furthermore, the possibility of using staphylococcal surfacedisplay and flow cytometric cell sorting also for specificenrichment of very rare target cells by multiple rounds ofcell-sorting and in between amplification was demonstrated.</p><p><b>Key words:</b>affibody, albumin binding protein, bacterialsurface display, cell immobilization, bioremediation,combinatorial protein engineering, flow cytometry,Gram-positive, metal binding, staphylococcal protein A,Staphylococcus carnosus, Staphylococcus xylosus, whole-celldevices</p>

A new Laser Induced Fluorescence Capillary Zone Electrophoresis (LIF-CZE) bioassay to detect and study the catalytic activity of the sulfur assimilating enzyme commonly found in E. coli species; alkanesulfonate monooxygenase (EC 1.14.14.5) is described for the first time. This technique enables the possibility for direct injection onto a capillary for detection without the need for pre-concentration of sample and with minimal sample preparative steps prior to analysis. In this bioassay, a group of Fischer based cyanine dyes and two Oxazine (Nile red) derivatives were designed for further optimization as key Vis-NIR fluorescent substrate. In developing this technique, the test dyes were first assessed for their photophysical properties, based on four criteria; (1) photostable (2) solvatochromism (3) binding affinity towards both the monooxygenase active site and serum albumin and (4) chemical stability in strong electric field strength. Applying key dye characterization procedures including; molar absorptivity determination, quantum yield determination, photostability, solvatochromism and protein interaction studies it was determined that the Fischer indolium cyanine dyes were most suitable for the method development. The data revealed that under the test conditions, reduced flavin, the oxidative monooxygenase catalytically specifically converts the alkylsulfonate substituted cyanine dyes to the corresponding aldehyde. This new bioassay has proven to be quick, portable, sensitive, reliable and the exhibit the possibility of ‘on-the-spot’ detection; advantages not readily realized with other commonly applied techniques such as PCR, SPR, ELISA and GC used to study bacterial sulfur assimilation processes. In addition, recent literature results proposed by other research groups developing similar techniques showed strong reliance on GC analyses. Those assays involve the use of low molecular weight straight chain non-emissive alkanesulfonate substrates. Once enzyme catalysis occurs the aldehyde is formed becomes rather volatile and requires complex and tedious headspace sampling for GC analyses. This feature limits the in vitro applicability and eliminated the possibility in vivo development. Our goal is to further develop, optimize and present this CZE based bioassay as a suitable alternative to the current trends in the field while creating a more robust and sensitive in vitro monooxygenase detection method with the possibilities of in vivo application.

Drug transport across the intestinal epithelium is roughly predicted from permeability values obtained from Caco-2 cell monolayers. This thesis examines the important role of pH and extracellular additives for increasing the reliability and predictivity of the in vitro screening system, Caco-2. It was shown that the passive transport of ionizable compounds may be biased by a false efflux or uptake component, when applying a physiological pH-gradient across the membrane. pH also affected the amount of compound available at the transporter-binding site. Therefore, pH dependence should be considered in studies of such compounds and of drug-drug interactions involving efflux transporters. It was also shown that proton-dependent apical uptake or basolateral efflux should be studied both with and without a pH gradient over the whole monolayers. The two extracellular additives, bovine serum albumin (BSA) and the solubilizing agent, Cremophor® EL, also influenced Caco-2 permeabilities. BSA applied to the receiver side increases, and to the donor side decreases drug permeation according to the drug’s protein binding capacity. Thus, the absorptive transport for both passive and active compounds is favoured, giving a physiologically sound improvement of the Caco-2 cell model. Inclusion of BSA increased both the predictivity and quality of permeability studies, particularly of highly lipophilic, BCS class II compounds. Passive and active transport processes could also be distinguished after accounting for unbound concentrations. The overall effect of Cremophor® EL on the permeability to a drug was compound-specific and probably dependent on micellar incorporation. Cremophor® EL can therefore not be recommended. Neither pH nor BSA affect the functionality of transporters such as P-glycoprotein. However, efflux ratios of ionizable or protein bound drugs are altered in the presence of a pH-gradient or BSA, indicating that an experimental system without protein or pH gradient can over- or underestimate active and passive efflux in drug transport.

Particles and macromolecular components, including biopolymers (protein and carbohydrate), are viewed as the main foulants in the complex feed submerged membrane filtration systems such as membrane bioreactor (MBR). This work focused on two aspects of fouling in complex fluids: 1- Assessing fouling propensity and mechanisms for various model solutions. 2- Using of two specific solutions modelling biomass found in MBR for a better understanding of the fouling mechanisms in submerged MBR processes. Filtrations were carried out with 0.22 ??m PVDF hollow fibre membrane. Alginate was used as a model for polysaccharide, bovine serum albumin (BSA) as a model for protein, (un)washed yeast and bentonite were representing suspended solid contents. According to the data obtained during this study the fouling propensity of each model solution was classified as follow in a decreasing order: Alginate &gt unwashed yeast &gt washed yeast &gt BSA &gt bentonite for one-component solutions; and Alginate-washed yeast &gt Alginate-BSA &gt Alginate-bentonite &gt Alginate-unwashed yeast for two-component solutions. Introducing the alginate increased the reversible fouling (except BSA). Passive adsorption had a significant effect on fouling of alginate even before the beginning of the filtration. Washed yeast and a mixture of washed yeast + BSA were then used as model solutions to simulate the activated sludge found in MBR. The concentration of washed yeast and BSA used in this study were calculated in order for the characterisations of the two model solution to match (in terms of biopolymer contents) those of MBR biomasses reported in the literature. By rinsing, backwashing and chemical cleaning of the membrane, three fouling layers of upper, intermediate and lower were defined respectively. Results obtained from the analysis of the biopolymers found in the cleaning solutions allow a better understanding of the fouling mechanisms occurring for the two model solutions used in this study: for washed yeast, the lower layer and for washed yeast + BSA , the upper and intermediate layers were found to have relatively high biopolymeric composition. This was explained by higher concentration of solids on the membrane surface and by higher biopolymer interactions when washed yeast was mixed with BSA.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

Bovine serum albumin (BSA) is the most abundant protein in plasma and it is responsible for the transport of most of the exogenous and endogenous compounds present in blood. Roxarsone (RX) and its metabolite acetarsone (AC) are organic compounds of arsenic(V) used in poultry as a food additive. In this sense, the interaction process of RX and AC compounds with BSA was evaluated under physiological conditions using spectroscopic techniques. It was possible to determine the formation of a supramolecular complex between RX and AC ligands using molecular fluorescence. The interaction degree was moderate, with a binding constant (Kb) of 4.27x105 L mol-1 and 0,27x105 L mol-1 for RX and AC, respectively. The quenching mechanism involved in the interaction process for both ligands was static and, preferably, by electrostatic interactions to the BSA-RX complex and hydrogen bonds and Van der Waals forces to BSA-AC. Both the formation of the complex and the quenching mechanism were confirmed by UV-vis spectroscopy. The 3D fluorescence analysis evidenced structural changes in the BSA polypeptide chain, being more pronounced in the presence of the RX ligand in comparison to AC. In addition, the BSA-RX interaction process modified the BSA surface, leading to reduction of hydrophobic regions more effectively compared to BSA-AC. About the binding site, both ligands are preferably in the site II of BSA. From the results obtained by 1H NMR, greater chemical shift was observed in the presence of the AC ligand, being the region close to amide group interacting with BSA, while the RX ligand possibly interacts more superficially in the protein. Thus, the RX and AC ligands do not favor the kinetics of protein fibrillation, giving indications that the possible deleterious effects in the evaluated in vitro system are not related to this process. Alkaline phosphatase inhibition kinetics demonstrated inhibition of enzymatic activity from 5 min and signal stabilization in 55 min of analysis for both RX and arsenate (positive control). Finally, the evaluation of in vitro alkaline phosphatase activity showed inhibition of enzymatic activity in 42% in the presence of the RX ligand and 5% in the AC ligand, giving indications of more detrimental effects for RX when compared to AC. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A albumina do soro bovino (BSA) é a proteína mais abundante do plasma e responsável pelo transporte da maioria dos compostos exógenos e endógenos presentes no sangue. Roxarsone (RX) e seu metabólito acetarsone (AC) são compostos orgânicos de arsênio(V) empregados na avicultura como aditivo alimentar. Neste sentido, o processo de interação dos compostos RX e AC com a BSA foi avaliado sob condições fisiológicas utilizando técnicas espectroscópicas. Empregando fluorescência molecular foi possível determinar a formação do complexo supramolecular para os ligantes RX e AC. O grau de interação foi moderado, com constante de ligação (Kb) de 4,27x105 L mol-1 e 0,27x105 L mol-1 para RX e AC, respectivamente. O tipo de quenching envolvido no processo de interação para ambos os ligantes foi estático e, preferencialmente, por interações de natureza eletrostática para o complexo BSA-RX e ligações de hidrogênio e forças de Van der Waals para BSA-AC.Tanto a formação do complexo quanto o tipo de quenching foram confirmados por espectroscopia de UV-vis. A análise por fluorescência 3D evidenciou mudanças estruturais na cadeia polipeptídica da BSA, sendo mais pronunciadas na presença do ligante RX em comparação ao AC. Ademais, o processo de interação BSA-RX modificou a superfície da BSA, levando à redução de regiões hidrofóbicas de forma mais efetiva em comparação à BSA-AC. Com relação ao sítio de ligação, ambos os ligantes encontram-se preferencialmente no sítio II da BSA. A partir dos resultados obtidos por RMN 1H observou-se maior deslocamento químico na presença do ligante AC, estando a porção próxima ao grupamento amida interagindo com a BSA. Enquanto que o ligante RX possivelmente interage mais superficialmente na proteína. Além disso, os ligantes RX e AC não favorecem a cinética de fibrilação protéica, dando indícios que os possíveis efeitos deletérios no sistema in vitro avaliado não estão relacionados a este processo. A cinética de inibição da fostatase alcalina demonstrou inibição da atividade enzimática a partir de 5 min e estabilização do sinal em 55 min de análise tanto para o RX quanto para o arseniato (controle positivo). Por fim, a avaliação da inibição da atividade da fosfatase alcalina in vitro evidenciou inibição da atividade enzimática em 42% na presença do ligante RX e 5% para o ligante AC, dando indícios de efeitos mais prejudiciais para o RX quando comparado ao AC.

SOUZA, Pedro Filho Noronha de.Purificação, caracterização bioquímica e atividade antimicrobiana de uma nova albumina 2s da torta da mamoneira (Ricinus communis L.), com atividade inibitória contra tripsina. 2012. 114 f. Dissertação (Mestrado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2012. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-06-27T14:14:02Z No. of bitstreams: 1 2012_dis_pfnsouza.pdf: 1847174 bytes, checksum: 6675a05ab248cfb741080071e366161c (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:11:34Z (GMT) No. of bitstreams: 1 2012_dis_pfnsouza.pdf: 1847174 bytes, checksum: 6675a05ab248cfb741080071e366161c (MD5) / Made available in DSpace on 2016-08-02T20:11:34Z (GMT). No. of bitstreams: 1 2012_dis_pfnsouza.pdf: 1847174 bytes, checksum: 6675a05ab248cfb741080071e366161c (MD5) Previous issue date: 2012 / Castor bean (Ricinus communis L.) is an important crop for the Northeast of Brazil, which recently has been used to produce biodiesel. Around 90% of the castor bean production in Brazil is concentrated at Northeast region and the state of Ceará is the second largest producer. During the oil extraction process from the castor bean seeds, a resulting residue, called castor cake, is underutilized. However, this byproduct is rich in protein and micronutrients such as nitrogen, phosphorus and potassium, an attribute that qualifies it as a stuff that could be used as organic fertilizer or as animal feed, for example, bringing added value to it. Unfortunately, its use as food has not been possible because of the presence of toxic elements and allergens (ricin, ricinine, complex allergens) in its composition, unless it were previously submitted to a detoxification process. To date, the existing technologies to this end are not economically viable on an industrial scale. Therefore, studies to add value to this abundant byproduct generated in the castor bean biodiesel productive chain is of paramount importance. Thus, in this context, this study was performed to identify, isolate, purify and characterize new bioactive molecules of de-oiled castor cake with biotechnological potential. Through extraction of soluble proteins with 50 mM Tris-HCl pH 7.5, fractionation with ammonium sulfate (50-75%), following by hydrophobic interaction chromatography in Phenyl-Sepharose column and ion exchange chromatography (DEAE-Sepharose), a protein, named Rc-2S-Alb, able to inhibit trypsin was purified. Rc-2S-Alb has a molecular mass of approximately 75.8 kDa, as determined by SDS-PAGE, and under reducing conditions showed a large and a small protein band of 15.8 kDa and 10.5 kDa, respectively. Its NH2-terminal sequence showed similarity with the following proteins: putative 2S albumin precursor (89%), chain A of RicC3 (89%), and chain A of mabilin-1 (89%), all from R. communis seeds; and with the short chain of a "napin-like" protein from Brassica napus (89%). In addition, these similar proteins have two highly conserved domains: QEVQRKDLS and YLRQS. Comparison of the partial primary structure of Rc-2S-Alb generated by the ESI-Q-TOF MS/MS analysis of 17 tryptic peptides, showed 43% similarity to Mabinlin-1 (pI/Mr 6.7 and 29.3 kDa) from R. communis. There were also high similarities among the three-dimensional structures of Rc-2S-Alb, RicC3 and Mabinlin-1. Rc-2S-Alb did not inhibit the spore germination of the phytopathogenic fungi Fusarium oxysporum and Rizoctonia solani, but promoted aggregation of their respective spores. Moreover, Rc-2S-Alb did not inhibit the mycelial growth of Fusarium oxysporum, Fusarium solani, Rizoctonia solani and Collethotricum gloeosporioides. Contrary, Rc-2S-Alb was effective in inhibiting the growth of the human pathogenic bacteria Pseudomonas aeruginosae, Klebsiella pneumoniae and Bacillus subtilis, at low concentrations. In conclusion, it was established a protocol to purify a new 2S albumin from castor bean cake, which inhibits trypsin and has important antibacterial activity. Thus, the Rc-2S-Alb should be further studied in order to verify its effectiveness as new alternative therapeutic agent against resistant bacteria to commercial antibiotics, which will contribute to improve the human health. / A mamoneira (Ricinus communis L.) é uma cultura importante para a região Nordeste do Brasil, onde, recentemente, tem sido utilizada para produção de biodiesel. O Nordeste detém 90% da produção brasileira de mamona, sendo o Ceará, o segundo maior produtor. No processo de extração de óleo das sementes de mamoneira para produção de biodiesel, o resíduo resultante, denominado de torta da mamona, representa um subproduto pouco utilizado. Apesar disso, essa torta é rica em proteínas e micronutrientes, como nitrogênio, fósforo e potássio, atributo que a qualifica como um insumo que poderia ser utilizado como adubo orgânico, ou mesmo como ração animal, o que lhe agregaria valor comercial. Todavia, seu uso como alimento não tem sido possível por causa da presença de elementos tóxicos e alergênicos (Ricina, Ricinina, Complexos Alergênicos) na sua composição, a não ser que passasse por processamento para sua destoxificação. Infelizmente, tecnologia economicamente viável para esse fim, em escala industrial, ainda é inexistente. Portanto, há necessidade de pesquisas que venham a agregar valor a esse subproduto abundante da cadeia produtiva do biodiesel. Assim, nesse contexto, o presente trabalho está inserido num projeto cujo objetivo maior é identificar, isolar, purificar e caracterizar novas moléculas bioativas da torta delipidada de sementes de mamoeira com potencial biotecnológico. Através de extração de proteínas solúveis com tampão Tris-HCl, 50 mM, pH 7,5, e fracionamento do extrato obtido com sulfato de amônio (50-75%), cromatografia de interação hidrofóbica em coluna Phenyl-Sepharose e cromatografia de troca iônica (DEAE-Sepharose) foi possível purificar uma albumina 2S, denominada Rc-2S-Alb, capaz de inibir tripsina. A Rc-2S-Alb apresentou massa molecular de, aproximadamente, 75,8 kDa, determinada por SDS-PAGE, mas, em condições redutoras, apareceu como uma banda maior de 15,8 kDa e outra menor com 10,5 kDa. Sua sequência NH2-terminal revelou haver similaridade (89%) com o precursor putativo da albumina 2S de R. communis já descrita, com a cadeia A da estrutura da RicC3 (89%), com a cadeia A da mabilin-1, ambas, também, de R. communis (89%), com a cadeia pequena de uma proteina “napin-like”de Brassica napus (89%). Em todas essas proteínas similares, dois domínios, QEVQRKDLS e YLRQS, são altamente conservados. Comparação da estrutura primária gerada por ESI-Q-TOF MS/MS, a apartir de 17 peptídeos trípticos da Rc-2S-Alb, mostrou similaridade de 43% com a Mabinlin-1 (pI/Mr de 6,7 e 29.3 kDa) de R. communis. Em relação à estrutura tridimensional da Rc-2S-Alb, ela apresentou similaridade com a de Ric C3 e Mabinlin-1. A Rc-2S-Alb foi incapaz de inibir a germinação de esporos dos fungos fitopatogênicos Fusarium oxysporum e Rizoctonia solani, mas foi capaz de promover a aglomeração dos mesmos. A Rc-2S-Alb também não foi capaz de inibir o crescimento micelial dos fungos Fusarium oxysporum, Fusarium solani, Rizoctonia solani e Collethotricum gloeosporioides. Por outro lado, a Rc-2S-Alb foi eficiente em inibir o crescimento de Pseudomonas aeruginosae, Klebsiella pneumoniae e Bacillus subtilis, todas as bactérias patogênicas a seres humanos, quando em baixas concentrações. Como conclusão, foi possível a purificação de uma nova albumina 2S da torta da mamona, capaz de inibir tripsina e com atividade antibacteriana importante. Assim, a Rc-2S-Alb deve ser explorada no sentido de verificar sua eficácia como um novo agente terapêutico alternativo no combate a bactérias resistentes aos produtos disponíveis, hoje, no mercado, o que, se confirmado, poderá contribuir para a melhoria da saúde humana.

Made available in DSpace on 2016-12-23T13:53:36Z (GMT). No. of bitstreams: 1 Dione Henrique Breda Binoti.pdf: 341138 bytes, checksum: 6c66646819669adecffe7330b65615a7 (MD5) Previous issue date: 2011-08-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The dairy farming is practiced throughout the national territory and approximately 70% of milk production in Brazil comes from crossbred animals derived from European dairy cattle breeds with zebu breeds of excellent adaptation to tropical conditions. Due the metabolic profile studies have focused on specialized pure breeds in this research was aimed to assess the metabolic profile of protein in two genetic groups of Holstein x Gir cows, from the second lactation order, in two periods of lactation, during the dry season. This work was carried out at the Fazenda Santa Luzia, belonging to the Grupo Cabo Verde, Passos - MG. Data were collected between May and August 2009, from ½ HG cows (37) and ¾ HG cows (35). Two periods of lactation were studied: from 28 to 60 and from 110 to 130 days. The feeding of the herd was conducted in accordance with milk production. From each animal, fasting, 10 mL of blood without anticoagulant, and 4.5 mL, with EDTA anticoagulant were collected. Milk yield and body condition score were evaluated and serum levels of urea, albumin, total protein and hemoglobin determined using a manual spectrophotometer, based on specific procedures for each component. Results showed that milk production was higher in animals ¾ HG in both periods of lactation and body condition score higher in the animals of the second period of lactation, but similar when comparing genetic groups. Although had occurred differences among lactation periods to the genetic groups for urea, albumin and total protein, results for these variables were similar as well as for hemoglobin into genetic groups. However genetic groups presented different results from those obtained for purebreds, demonstrating the need particularly to reconsider the nutritional management of these groups, when related to the purebreds. So, it s suggested that others research could be done in these conditions in order to elucidate this uncertain and to enable to construct tables with specific reference values for these genetic groups / A atividade leiteira é praticada em todo o território nacional e aproximadamente 70% da produção de leite do Brasil provem de animais derivados de cruzamentos de raças européias especializadas para produção de leite com raças zebuínas de excelente adaptação às condições tropicais. Estudos de Perfil Metabólico têm se concentrado em raças especializadas puras, assim objetivou-se aferir o perfil metabólico protéico de dois grupos genéticos de vacas Holandês x Gir, de segunda ordem de lactação, em dois períodos da lactação, na estação seca do ano. Também se procurou relacionar os resultados com os de estudos com raças puras. Foi conduzido na Fazenda Santa Luzia, pertencente ao Grupo Cabo Verde, Passos - MG. Os dados foram coletados entre maio e agosto de 2009, em vacas ½ HG (37) e vacas ¾ HG (35). Foram estudados dois períodos da lactação: de 28 a 60 e de 110 a 130 dias. O manejo alimentar do rebanho foi conduzido de acordo com a produção de leite. Foram coletados, de cada animal, em jejum, 10 mL de sangue, sem anticoagulante, e 4,5 mL, com anticoagulante EDTA. Foram avaliados a produção de leite e o escore de condição corporal e determinados os teores séricos de uréia, albumina, proteínas totais e hemoglobina em espectrofotômetro manual, baseado em procedimentos específicos para cada componente. Os resultados mostraram que a produção de leite foi mais elevada em animais ¾ HG, em ambos os períodos da lactação e o escore de condição corporal mais elevado nos animais do segundo período da lactação, mas semelhantes quando se comparam grupos genéticos. Embora entre períodos da lactação tenham ocorrido diferenças dentro de cada grupo genético para uréia, albumina e proteínas totais, entre os dois grupos genéticos os resultados para essas variáveis, assim como para hemoglobina, foram semelhantes. Contudo, os dois grupos genéticos apresentaram resultados distintos dos obtidos com raças puras, demonstrando a necessidade de se reconsiderar o manejo, principalmente nutricional, desses grupos genéticos em relação às raças puras. Assim, sugere-se que outras pesquisas possam ser realizadas, visando a elucidar essa incerteza e possibilitar a construção de tabelas de valores de referência específicas para esses grupos genéticos

Submitted by JOÃO PAULO RAMOS COSTA null (jpramoscosta@hotmail.com) on 2016-02-25T13:30:07Z No. of bitstreams: 1 Tese_João_Paulo_Ramos_Costa.pdf: 3560400 bytes, checksum: 7b30b4c8363f3e9135f4b6977b1e5ac2 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-02-26T16:28:14Z (GMT) No. of bitstreams: 1 costa_jpr_dr_jabo.pdf: 3560400 bytes, checksum: 7b30b4c8363f3e9135f4b6977b1e5ac2 (MD5) / Made available in DSpace on 2016-02-26T16:28:14Z (GMT). No. of bitstreams: 1 costa_jpr_dr_jabo.pdf: 3560400 bytes, checksum: 7b30b4c8363f3e9135f4b6977b1e5ac2 (MD5) Previous issue date: 2016-02-02 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo foi avaliar o efeito da virginiamicina (VM) via suplemento mineral e altura pastos (AP) sobre o desempenho, parâmetros sanguíneos, fermentação ruminal, microorganismos ruminais e produção de N microbiano. O estudo foi dividido em dois experimentos. Capítulo 1: Oitenta tourinhos Nelore (inicial BW = 258 ± 15 kg) foram blocados por peso e distribuídos aleatoriamente em 16 piquetes (5 animais / piquete) arranjados em um fatorial 2 × 2: suplemento mineral sem VM (SVM) ou com VM e AP [15 e 35 cm (15AP e 35AP, respectivamente)]. Os animais que receberam VM tiveram maior GMD (P <0,01). Como esperado, o 35AP apresentou maior GMD (P <0,01). A VM não afetou a ingestão de suplemento mineral por PV (ISPV) nem por animal (ISA) (P = 0,49 e P = 0,55, respectivamente) e 15AP mostrou um maior ISPV e ISA (P <0,01 e P <0,01 , respectivamente). Albumina sanguínea foi maior em VM (P <0,01) e tendeu ser maior (P = 0,07) em 35AP. A concentração de ureia no sangue tendeu a diminuir em VM (P = 0,06) e foi maior (P <0,01) em 15AP. A concentrações de NEFA e de cálcio foram maiores (P = 0,007 e P = 0,038, respectivamente) em VM. Capítulo 2: Doze Touros Nelore (PV inicial = 334 ± 47 kg) canulados no rúmem foram designados no mesmo tratamento citados acima em três quadrados latino balanceado 4 × 4. O CMS foi maior no 15AP (P <0,01) do que em 35AP. A amônia-N tendeu a ser maior em VM (P = 0,07). A proporção de nenhuma das bactérias ruminais foram afetados (P > 0,10), excepto R. flavefaciens que tendeu a aumentar em 35AP (P = 0,08), enquanto que a contagem de protozoários totais aumentou (P = 0,03) e o género Entodinium tendeu a aumentar (P = 0,05) em VM. A VM aumento do ácido úrico (P <0,01) e tendeu a aumentar a excreção derivados de purinas (PD; P = 0,074), as purinas absorvidas (AP; P = 0,07), fluxo de N microbiano (NMIC; P = 0,07) e o índice de PDC (P = 0,07)]. Portanto, o efeito da VM parece estar mais relacionada ao aumento do fluxo de N microbiano para o intestino provocado pelo aumento dos protozoários ruminais do que a fermentação ruminal. / The objective was evaluate the effect of virginiamycin (VM) via mineral supplement and two swards height (SH) on performance, blood metabolites, ruminal fermentation, ruminal microorganisms and microbial N production. The trial was divided in two experiments. Chapter 1: Eighty Nellore young Bulls (initial BW = 258 ± 15 kg) were blocked by weight and randomly assigned into 16 paddocks (5 animals/paddock) arranged in a 2 × 2 factorial design: mineral supplement without VM (WVM) or with VM and two SH [15 and 35 cm (15SH and 35SH, respectively)]. The animals receiving VM had higher ADG (P < 0.01). As expected, the 35SH had higher ADG (P < 0.01). VM had no effect on mineral supplement intake by BW (MSIBW) or mineral supplement intake by animal (MSIA) (P = 0.49 and P = 0.55, respectively) and 15SH showed a greater MSIBW and MSIA (P < 0.01 and P < 0.01, respectively). Blood albumin was greater in VM (P < 0.01) and tended to be greater (P = 0.07) in 35SH. The blood urea concentration tended to decrease in VM (P = 0.06) and was higher (P < 0.01) in 15SH. Plasma NEFA and calcium concentrations were greater (P = 0.007 and P = 0.038, respectively) in VM. Chapter 2: Twelve Nellore steer (initial BW = 334 ± 47) ruminally cannulated were used in three 4 × 4 balanced Latin square were assigned in the same treatment arrangement cited above. The DMI was greater (P < 0.01) in 15SH than in 35SH. Ammonia-N tended to be higher in VM (P = 0.07). None ruminal bacteria proportion were affected (P > 0.10), except R. flavefaciens that tended to increase in IV 35SH (P = 0.08), while total protozoa counts increased (P = 0.03) and the Entodinium genus tended to increase (P = 0.05) in VM. The VM increased uric acid (P < 0.01) and tended to increase the purines derivatives excretion (PD; P = 0.074), absorbed purines (AP; P = 0.07), microbial N flow (NMIC; P = 0.07) and the PD: creatinine ratio index [PDC index (P = 0.07)]. Therefore, the VM effect seems to be more related to increasing the N microbial flow to the intestine caused by incresing of ruminal protozoa than the ruminal fermentation. / FAPESP: 2012/02166-1

Made available in DSpace on 2014-12-17T14:25:23Z (GMT). No. of bitstreams: 1 AbdoFN_TESE.pdf: 1739510 bytes, checksum: 7f520b9c4a32fd74e279d017c0bdd143 (MD5) Previous issue date: 2014-03-31 / Foam was developed as a novel vehicle for streptokinase with the purpose of increasing the contact time and area between the fibrinolytic and the target thrombus, which would lead to a greater therapeutic efficacy at lower doses, decreasing the drug s potential to cause bleeding. Fibrinolytic foams were prepared using CO2 and human albumin (at different v:v ratios), as the gas and liquid phases, respectively, and streptokinase at a low total dose (100,000 IU) was used as fibrinolytic agent conveyed in 1 mL of foam and in isotonic saline solution. The foams were characterized as foam stability and apparent viscosity. The thrombolytic effect of the streptokinase foam was determined in vitro as thrombus lysis and the results were compared to those of a fibrinolytic solution (prepared using the same dose of streptokinase) and foam without the fibrinolytic. In vitro tests were conducted using fresh clots were weighed and placed in test tubes kept at 37 ? C. All the samples were injected intrathrombus using a multiperforated catheter. The results showed that both foam stability and apparent viscosity increased with the increase in the CO2:albumin solution ratio and therefore, the ratio of 3:1 was used for the incorporation of streptokinase. The results of thrombus lysis showed that the streptokinase foam presented the highest thrombolytic activity (44.78 ? 9.97%) when compared to those of the streptokinase solution (32.07 ? 3.41%) and the foam without the drug (19.2 ? 7.19%). We conclude that fibrinolytic foam showed statistically significant results regarding the enhancement of the lytic activity of streptokinase compared to the effect of the prepared saline solution, thus it can be a promising alternative in the treatment of thrombosis. However, in vivo studies are needed in order to corroborate the results obtained in vitro / Uma espuma foi desenvolvida como novo ve?culo para a estreptoquinase com vistas a aumentar a ?rea de contato e o tempo de perman?ncia junto ao trombo, de modo a se obter maior efici?ncia terap?utica em doses menores, diminuindo suas potenciais complica??es hemorr?gicas. A espuma fibrinol?tica foi preparada com CO2, albumina humana e estreptoquinase, em dispositivo desenvolvido para tal fim, com diferentes raz?es de fases g?s/l?quido. Ensaios de estabilidade e viscosidade aparente foram realizados para caracteriza??o das espumas e a escolha da mais est?vel. A estreptoquinase em dose total reduzida (100.000 UI) foi utilizada como fibrinol?tico veiculado em 1 mL de espuma e em solu??o salina isot?nica (0,9%). A espuma sem fibrinol?tico tamb?m foi utilizada como comparativo. Testes in vitro foram realizados utilizando-se co?gulos frescos, que foram pesados e colocados em tubos de ensaio mantidos a 37?C. As espumas com e sem fibrinol?tico e a solu??o fibrinol?tica foram testadas por aplica??o intraco?gulo em doses id?nticas atrav?s de cateter multiperfurado e pistola de inje??o. Os resultados in vitro evidenciaram atrav?s da diminui??o dos pesos dos co?gulos, que a espuma trombol?tica apresentou atividade l?tica de 44,78 ? 9,97%, enquanto as mesmas doses da estreptoquinase em solu??o salina isot?nica promoveram 32,07 ?3,41% de lise dos co?gulos. Na espuma sem fibrinol?tico a redu??o do trombo foi de 19,2 ? 7,19%. Conclui-se que a espuma fibrinol?tica apresentou resultados estatisticamente significativos no tocante ? potencializa??o da atividade l?tica da estreptoquinase, quando comparado ao efeito da solu??o preparada com solu??o salina, podendo ser uma alternativa promissora nos tratamentos das tromboses. Os dados obtidos sinalizam para necessidade de estudos in vivo para comprova??o dos obtidos nos in vitro