L'hydrolyse pepsique de l’hémoglobine bovine purifiée ou à partir d'un coproduit des abattoirs : le cruor, peut être considérée comme une voie importante d'obtention de peptides antibactériens. Une étude cinétique nous a permis de maîtriser cette hydrolyse et de déterminer un modèle mathématique capable de prédire la concentration de chaque peptides antibactériens des deux familles de peptides α 1-32 et α 107-141 et ceux pour un intervalle de température de 15-37°C, de pH de 3,5-5,5 et de rapport enzyme/substrat de 1/5-1/20. Le calcul des énergies d'activation pour les différentes réactions impliquées dans le mécanisme était effectué grâce à l’équation d’Arrhenius qui a permis d’étudier l’effet de la température sur les différents coefficients cinétiques. L’effet du pH et du rapport E/S était également étudié et le modèle trouvé a démontré une augmentation linéaire de la vitesse d’hydrolyse en diminuant le pH (entre 3,5 et 5,5) et une vitesse invariable avec le rapport E/S (1/5-1/20). L'étude de la relation structure-fonction des peptides antibactériens α 1-32 et α 137-141 a été effectuée grâce à un suivie de la cinétique de K+ extracellulaire en présence de Lisrea innocua et le déterminant antibactérien minimal a été déterminé pour le peptide α 137-141. La possibilité de valoriser les peptides antibactérien α 1-32 et α 137-141 dans un emballage bioactif pour la conservation des aliments contre le développement de bactéries pathogènes a été étudiée par l'adsorption de ces peptides en surface d'un film de polyéthylène basse densité, traité avec le plasma froid. / The pepsin hydrolysis of purified bovine hemoglobin or from a co-product of slaughterhouses: cruor, can be considered as an important route for obtaining antibacterial peptides. A kinetic study has allowed us to control the hydrolysis and to determine a mathematical model able to predict the concentration of each antibacterial peptides of two families of peptides α 1-32 and α 107-141 and those of an interval of temperature 15-37°C, of pH 3,5-5,5 and ratio enzyme/substrate of 1/5-1/20 . The calculation of activation energies for the different reactions involved in the mechanism was made by the Arrhenius equation, which was used to study the effect of temperature on the various kinetic coefficients. The effect of pH and ratio E / S was also studied and the model found showed a linear increase in the rate of hydrolysis decreasing the pH ( between 3,5 and 5,5 ) and an invariable speed with the ratio E / S ( 1/5-1/20 ). The study of the structure-function relationship of antibacterial peptides α 1-32 and α 137-141 was carried out thanks to a followed the kinetics of extracellular K + in the presence of Lisrea innocua and the minimal determinant antibacterial was determined for the peptide α 137-141. The possibility to recovery the antibacterial peptides α 1-32 and α 137-141 in to a bioactive food packaging against the growth of pathogenic bacteria has been studied by the adsorption of these peptides on the surface of a low density polyethylene film treated with cold plasma.

Orientador: Sergio de Toledo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-04T01:47:57Z (GMT). No. of bitstreams: 1 Cruz_GabrielaAlessandrada_M.pdf: 15668447 bytes, checksum: 48a5ee54985687b46e68567ad7b84fbb (MD5) Previous issue date: 2004 / Resumo: Os substitutos ósseos são materiais utilizados em procedimentos periodontais regenerativos como uma alternativa para tratamento de defeitos ósseos. Neste estudo foram avaliados a morfologia, o tamanho e a composição química das seguintes partículas: osso bovino orgânico cortical e esponjoso com partículas microgranular medindo entre 0,25 e 1,0 mm e macrogranular medindo entre 1,0 e 2,0 mm, osso bovino cortical inorgânico com partículas medindo entre 0,25 e 1,0 mm, hidroxiapatita com partículas medindo entre 0,75 e 1,0 mm e osso humano descalcificado, congelado e seco medindo entre 0,25 a 0,5 mm. Para a analise da morfologia e do tamanho das partículas, as amostras foram preparadas em portaespécime, metalizadas em ouro e analisadas a vácuo em microscópio eletrônico de varredura (SEM). Para a análise da composição química, as partículas não foram metalizadas e foram analisadas por microanálise de raio X, por espectroscopia por dispersão de energia (EDS). A análise visual realizada por SEM, demonstrou que as partículas de osso bovino orgânico e inorgânico, osso humano e hidroxiapatita apresentaram formato irregular e tamanho variável, maior do que o mencionado no rótulo pelo fabricante. A análise da composição química realizada por microanálise (EDS) detectou a presença de elementos como: sódio, cálcio e fósforo, que são comuns à composição do tecido ósseo, porém revelaram a presença de elementos químicos nas partículas de osso bovino orgânico. Esses resultados sugerem que o osso bovino orgânico não é composto puramente por colágeno e proteína / Abstract: This article evaluates the morphologic and chemical composition of the following bone substitutes: cancellous and cortical organic bovine bone with micro and macro particles size ranging from 0,25 to 1,0 mm and 1,0 to 2,0 mm respectively, inorganic bovine bone with particles size ranging from 0,25 to 1,0 mm, hydroxyapatite with particles size ranging from 0,75 to 1,0 mm and demineralized freeze-dried bone allograft with particles size raging from 0,25 to 0,5 mm. The samples were coated with gold in an ion coater, the morphology was observed and the measurements of particle size were performed on vacuum condition by scanning electron microscopy (SEM). The chemical composition was evaluated by microanalysis EDS using samples without covering. The SEM analyses provided visual evidence that all materials analyzed have irregular shape and the particle size values were larger than the values mentioned by the manufacturer. The microanalysis EDS detected the presence of sodium, calcium and phosphorus that are common elements of the bone tissue. However, mineral elements were detected in all analyzed particles of organic bovine bone excepting for macro cancellous organic bovine bone. These results suggest that the evaluated organic bovine bone could not be considered a pure organic material / Mestrado / Periodontia / Mestre em Clínica Odontológica

Nanoparticles are currently used in a wide range of applications including industrially processes, consumer products, and as drug delivery vehicles. The potential toxicity of these nanoparticles in living organisms is concerning due to their ever-expanding applications and accumulation in the environment. The effects of properties of the human body on the potential harmful nature of these nanoparticles must be understood in order to ensure safety in workplaces and at-home products. In this thesis, the interactions between nanoparticles and the most abundant blood protein, serum albumin, were investigated. The effects of changing the aqueous environment was investigated over a range of different pH values and with different ionic salts dissolved in water. The effects of changing the nanoparticle substrate were investigated to determine if different nanoparticles affect proteins differently. Finally, the effects of changing the concentration of nanoparticles and the presence of protein were investigated in a model lung cell line in vitro. The studies over different pH values revealed that serum albumin was able to adsorb to the silica nanoparticle surface, and retained its secondary structure both as a function of pH and adsorption in a 2-hour time frame. However, adsorption was greater on the titanium dioxide nanoparticle surface and the protein lost secondary structure at acidic pH (pH 2.0). Studies with different ionic salts revealed a possible correlation between BSA adsorption and nanoparticle aggregation in that the attractive interactions between nanoparticles were least when the least amount of protein was adsorbed. To the nanoparticle surface. In vitro studies with A549 human adenocarcinoma lung cells were inconclusive in determining the potential toxicity of these nanoparticles, but preliminary results suggested that the addition of protein to the system decreased toxicity compared with nanoparticles alone. This research aims to inform the field of nanotechnology to investigate the safety and efficacy of nanoparticles before they reach the consumer.

La reprogrammation, qui est la réversion d’un noyau d’un état somatique vers un état moins différencié, constitue un enjeu majeur pour la thérapie cellulaire. Cependant, les mécanismes initiaux qui président à la reprogrammation restent mal connus. Le transfert nucléaire (clonage) met à profit les propriétés de reprogrammation uniques du cytoplasme ovocytaire, et constitue une approche expérimentale intéressante pour analyser ces processus. Le but de cette thèse est d’étudier la différence de capacité de cellules fibroblastiques à être reprogrammées efficacement, en tirant partie d’une situation-modèle d'efficacité différentielle de reprogrammation après clonage chez le bovin. Ce modèle est constitué de deux lots de fibroblastes donneurs de noyaux, qui forment des embryons clonés dont la différence d’efficacité de développement à terme varie d’un facteur 8. L’analyse des cellules donneuses a montré une augmentation des anomalies de ploïdie dans les cellules à faible potentiel, et la similitude transcriptomique entre les cellules donneuses, alors que la comparaison des transcriptomes des embryonsclonés a montré des différences de reprogrammation de l’expression génique dès le stade suivant l’activation du génome embryonnaire. Des différences de méthylation de l’ADN entre cellules donneuses ont été observées dans les promoteurs de gènes candidats différentiellement reprogrammés, ainsi que dans une analyse plus globale par RRBS. Nous avons enfin étudié la distribution des cellules filles des deux premiers blastomères au stade blastocyste, la distribution « orthogonale » et l’aptitude au développement à terme des embryons de souris clonés étant liées (Liu et al., 2012). Nous avons montré l’existence de trois distributions dans les embryons fécondés mais n’avons pas observé de différence de proportions de celles-ci entre embryons bovins clonés. En conclusion, dans notre modèle, la distribution des cellules filles des deux premiers blastomères au stade blastocyste ne semble pas associée à l’efficacité de reprogrammation dans les embryons bovins clonés, contrairement aux différences épigénétiques entre cellules donneuses. / Reprogramming, which is the return of a nucleus from a somatic state to a less differentiated state, is a major issue for cell therapy. However, the initial mechanisms governing the reprogramming remain poorly understood. Nuclear transfer (cloning) takes advantage of the unique reprogramming properties of the oocyte cytoplasm, and therefore is an interesting experimental approach to analyze these processes. The aim of this thesis is to study the difference in fibroblasts’ ability to be reprogrammed by taking advantage of a model-situation of differential reprogramming efficiency after cloning in cattle. This model consists of two batches of donor fibroblasts, which form cloned embryos having an 8 fold difference in development to term efficiency. Analysis of donor cells has shown increase ploidy abnormalities in cells of low potential, and transcriptomic similarity between the donor cells, whereas comparison ofcloned embryos transcriptomes showed gene expression reprogramming differences just after embryonic genome activation. Differences in DNA methylation between donor cells were observed in the promoters of candidate genes differentially reprogrammed and in a more comprehensive analysis by RRBS. Finally we studied the distribution of the first two blastomeres’ daughter cells at the blastocyst stage, as an "orthogonal" distribution and development to term of mice cloned embryos are linked (Liu et al., 2012). We have shown the existence of three distributions in the fertilized embryos but haven’t seen any difference of proportions between bovine cloned embryos. In conclusion, in our model, the distribution of the first two blastomeres’ daughter cells at the blastocyst stage does not seem related to the reprogramming efficiency in bovine cloned embryos, unlike epigenetic differences between donor cells.

Orientador: Rosangela Zacarias Machado / Resumo: Anaplasma marginale é uma bactéria, Gram negativa, intracelular obrigatória parasita de eritrócitos e o principal agente da Anaplasmose bovina. Esta doença causa anemia severa, provocando a redução do ganho de peso e da produção de leite e gerando grandes perdas econômicas na pecuária mundial. A diversidade genética desta bactéria vem sendo caracterizada com base na sequência das proteínas de superfície (MSPs), principalmente, na proteína MSP1α, sendo possível identificar as diferentes estirpes geográficas de acordo com as diferenças nas sequências de aminoácidos. O presente estudo teve como objetivo investigar a diversidade genética de A. marginale em bovinos de corte da raça Angus naturalmente infectados durante um surto da enfermidade. Vinte bovinos, com idades entre 6 e 9 meses, oriundos de uma fazenda no município de Itú, estado de São Paulo foram acompanhados por quatro meses e quatro colheitas de sangue foram realizadas. Amostras de soro foram submetidas à Ensaio Imunoenzimático Indireto (iELISA) para detecção de anticorpos IgG anti-A. marginale. As oitenta amostras de sangue total obtidas, foram submetidas à extração de DNA, PCR em tempo real quantitativa (qPCR) para o gene msp1β, semi-nested PCR (snPCR) para o gene msp1α, clonagem do fragmento alvo e sequenciamento pelo método de Sanger. As sequências obtidas foram submetidas à analises de diversidade genética pelo software RepeatAnalyzer. O ensaio Imunoenzimático Indireto (iELISA), utilizando Ag total, revelou baixa... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Anaplasma marginale is a Gram-negative, obligatory intracellular erythrocyte parasite and the main agent of bovine Anaplasmosis. This disease causes severe anemia, causing a reduction in weight gain and milk production and generating major economic losses in livestock worldwide. The genetic diversity of this bacterium has been characterized based on the sequence of surface proteins (MSPs), mainly in the protein MSP1α, making it possible to identify the different geographical strains according to the differences in the amino acid sequences. The present study aimed to investigate the genetic diversity of A. marginale in Angus beef cattle naturally infected during an outbreak of the disease. Twenty cattle, aged 6 to 9 months, from a farm in the city of Itú, state of São Paulo, were followed for four months and four blood samples were taken. Serum samples were subjected to the Indirect Immunoenzymatic Assay (iELISA) to detect antibodies IgG anti-A. marginale. The eighty whole blood samples obtained were subjected to DNA extraction, quantitative real-time PCR (qPCR) for the msp1β gene, semi-nested PCR (snPCR) for the msp1α gene, cloning of the target fragment and sequencing by the Sanger method. The obtained sequences were subjected to genetic diversity analysis using the RepeatAnalyzer software. The Indirect Immunoenzymatic assay (iELISA) revealed low seroprevalence in the sampled animals despite 100% positivity in the qPCR, with quantification between 103 and 107 number of DNA c... (Complete abstract click electronic access below) / Mestre

Orientador: Simone Biagio Chiacchio / Resumo: A pecuária no Brasil evolui a cada ano buscando avanços genéticos como, animais mais rústicos e precoces. A raça Nelore é à base do rebanho comercial de corte brasileiro, correspondendo a cerca de 80% do rebanho nacional, e ainda com poucos estudos sobre o período pré o pós-natal de bezerros desta raça que é extremamente adaptada às condições climáticas, resistência a ecto e endo parasitas, além de boa fertilidade mesmo em condições de escassez de alimento de alta qualidade. Buscando técnicas que proporcionam menor estresse no monitoramento gestacional, o acompanhamento via avaliação da frequência cardíaca materna fetal e a variabilidade da frequência cardíaca estão em desenvolvimento. A importância de procedimentos adequados durante as fases que antecedem o parto, período pré-natal e o período neonatal diminuem a mortalidade dos neonatos, melhorando a assistência veterinária neste momento crítico. Assim este estudo teve como objetivo avaliar parâmetros clínicos, índices eletrocardiográficos (ECG) e de variabilidade cardíaca (VFC) em vacas (n=10) (período pré e pós parto) e nos fetos (n=10), 35 dias pré parto, e na fase neonatal até 35 dias de idade. Os momentos de análise foram 35°, 28°, 21°, 14°, 7 ° 1° dia antes do parto e ao 1°, 7°, 14°, 21°, 28° e 35° dias pós-parto. Os neonatos apresentaram boa adaptação com o avançar da idade, foram verificadas poucas alterações no ECG desta categoria, com isso verificou-se que bezerros saudáveis se adaptam bem as alterações da fase fe... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Livestock in Brazil evolves every year looking for genetic advances such as more rustic and precocious animals. The Nellore breed is based on the commercial Brazilian beef bank, corresponding to about 80% of the national bank, and still with few studies on the pre or post-natal period of calves of this breed which is extremely adapted as climatic conditions, ecto resistance. and endo parasites in addition to good fertility even in conditions of scarcity of high quality food. Seeking techniques that provide less stress on pregnancy monitoring, or monitoring via maternal heart rate and heart rate variability are under development. The importance of the procedures used during the phases that precede birth, the prenatal period and the neonatal period decrease the mortality of neonates, improving veterinary assistance at this critical moment. Thus, this study aimed to evaluate clinical criteria, electrocardiographic indexes (ECG) and cardiac variability (HRV) in cows (n = 10) (pre and postpartum period) and in cases (n = 10), 35 days before delivery. pregnancy, and in the neonatal phase until they reach 35 days of life The moments of analysis were 35 °, 28 °, 21 °, 14 °, 7 ° 1 ° day before participating and on the 1 °, 7 °, 14 °, 21, 28 and 35 days postpartum. The newborns showed good adaptation with advancing age, less changes in the ECG were verified in this category, with this it was verified whether calves adapt as well as changes in the fetal to neonatal phase, resulting in a... (Complete abstract click electronic access below) / Mestre

A 30-mer oligonucleotide probe encoding the "A box" and anticodon loop regions of a human glycine tRNA gene was used to isolate a 581bp DNA fragment from a bovine genomic DNA library. Although the cross-hybridizing segment of DNA was found not to encode any tRNA gene or pseudogene, a region with homology to the "C-element" of the "BOV-tA" type Alulike artiodactyl retroposons was identified. This cross-hybridization was determined to be the result of conserved RNA polymerase III promoter elements in the probe portion of the tRNA gene and these repetitive elements. A microsatellite repeat (TC) was also found associated with this element. Future screening for bovine tRNA genes will require the use of a) longer probes and higher stringency hybridization conditions or b) the simultaneous screening with probes from the 5' and 3' ends of the gene which avoid the conserved Pol III promoter boxes.

Select epithelial regions of the bovine inner ear were established and maintained in cell culture. Marginal cells from the stria vascularis and dark cells from the posterior wall of the utricle were isolated, dissociated and placed in culture medium. Within 24 h, cellular islands of hexagonal-shaped, epithelial-like cells from both the stria vascularis and posterior utricular wall were readily identifiable by inverted light microscopy. Ultrastructural examination of both the cultured stria marginal cells and utricular dark cells revealed that both cell types had numerous microvilli on their apical surfaces and interdigitating infoldings of their basolateral surfaces. Apical tight junctional complexes were present between apposing cells. These findings demonstrate that inner ear bovine epithelial cells can be successfully isolated and maintained in culture, and that such cells retain certain of their in vivo morphological characteristics.

This study was conducted to confirm the presence of bovine tuberculosis (BTB) and determine its prevalence, based on skin test reactivity, in cattle reared under extensive farming conditions in the Govuro district, Inhambane province, Mozambique. The study was comprised of a primary screening test using the single intradermal test (SIT) in randomly selected animals from Colonato and Sede dip tanks in Govuro. Positive reactors to the SIT were tested again with bovine and avian tuberculin using the single intradermal comparative test (SICTT) 7 weeks after the SIT. The sample size was calculated using Win Episcope 2.0 based on 95% confidence to detect a 2% expected prevalence using the SIT, with a 1% accepted error and accounting for a total population size of 7208. The calculated sample size was 682 animals. To compensate for the probability of 20% default in reading, the sample size was increased to 853. During the testing process (SIT), it was evident from the first 3 reading days that the apparent prevalence (61, 94%) was higher than expected (2%), hence we decided to stop when the total number of cattle was 530. During the testing process (SIT), it was evident from the first 3 reading days that the apparent prevalence (61.94%) was far higher than expected (2%), hence we decided to stop when the total number of cattle was 530. This was due to the fact that, at such a high prevalence, it would not be necessary to achieve as high a precision as 1% accepted error. A sample size of 530 would be sufficient to achieve a precision of 4% accepted error, which was regarded as more than adequate. The 530 cattle, 3 or more years of age, were selected using systematic random sampling from the two dip tanks (Colonato 371 and Sede 159 animals). All animals were identified by numbers painted, dorsally on the sacral region. Out of 530 tested cattle by SIT, 268 were read, and 166/268 (61.94% with 95% confidence interval [CI]: 55.8 – 67.8%) were found positive, with visible swallow at the injection site. Apparent prevalence (AP) was found to be 61.94% while the true prevalence (TP) was 75.92%. The predictive value of a positive result (PV+) was found to be 87.9%. No significant difference in apparent prevalence between the two areas was detected by Fisher’s exact test (P = 0.11). By SICTT, out of 28 animals positive reactors to SIT, 21 were possible to read, and 13/21 (61.9%; 95% CI: 55.1 – 89.3%) were found positive. A three year old bull, positive reactor to the SIT, was slaughtered, and a detailed post mortem was carried out and organs with visible lesions were collected for further laboratory testing (histopathology, culture and isolation of M. Bovis and PCR). Later on, 30 more positive reactors to the SIT test were slaughtered: 25/30 (83.3%) showed visible lesions compatible with BTB, and total condemnation of carcass was made in 3/25 (12%) due to generalized lesions. The high prevalence rate of skin test positive animals as well as gross lesions and histopathology were confirmed to be BTB by the isolation and identification of M. Bovis by culture and PCR. Our results suggest that bovine tuberculosis is highly prevalent in Govuro district and may thus represent a potential health problem of zoonotic tuberculosis in humans. Our results suggest that BTB has reached the plateau phase of endemicity in cattle in Govuro district. In this context, the positive predictive value of the SIT is very high and thus the use of the SICTT as a confirmatory test has a limited value and should not be advocated. Our results further indicate that no other prevalence study of BTB should be conducted in the next few years in Govuro district, unless comprehensive control measures are implemented. The focus of further studies should be on the isolation and the molecular characterization of M. Bovis from cattle and humans in order to assess transmission routes and the role played by BTB in human TB cases in Govuro district. Copyright / Dissertation (MSc (Veterinary Tropical Diseases))--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

Embryonic stem cells are pluripotent cells isolated from morula stage embryos or the inner cell mass of blastocyst stage embryos. They are capable of differentiating into tissues of all three primary germ layers. In recent years pluripotent cell lines have been created from somatic cell types using various methods, the primary method being viral transduction of exogenous Oct4, Sox2, Klf4, and c-Myc or Oct4, Sox2, Nanog, and Lin28 transgene constructs. The resulting cell lines are termed induced pluripotency stem cells, and are similar to embryonic stem cells in many ways. However, these cell lines are not acceptable for clinical applications due to the use of both modified viral vectors and insertion of exogenous transgenes in their production. Recently the small molecule RepSox, a TGF-ß pathway inhibitor, was used to replace Sox2 during cellular reprogramming of murine embryonic fibroblasts. We evaluated the effects of RepSox on expression of pathways related to pluripotency in murine embryonic fibroblast, murine embryonic stem, and bovine embryonic fibroblast cells. Each cell type was treated with RepSox for 72 hours and subjected to standard qPCR for gene expression analysis. PCR arrays specific to stem cell pathways were used to initially evaluate the effects of RepSox on candidate genes. A subset of genes was then selected for further analysis based on these initial results. We report that RepSox inhibition of the TGF-ß pathway in murine embryonic fibroblasts results in significant upregulation of components of the Wnt, Notch, and Hedgehog signaling pathways, all of which have been linked to stem cell maintenance. In addition, we observed significant upregulation of genes associated with embryonic, mesenchymal, stem cell, and neural cell lineages, indicating that RepSox may be useful in direct reprogramming of murine cells to other somatic cell types. RepSox treatment of murine embryonic stem cells did not result in consistent upregulation of Wnt, Notch, or Hedgehog pathway components, but did result in upregulation of Sox2 and Klf4 expression. Lastly, RepSox treatment of bovine embryonic fibroblasts did not result in the same effects as seen in murine fibroblasts, indicating a need for further analysis to determine the effects of RepSox on bovine cells.