Milk is provided to new borns as their first food source and it contains essential nutrients, vitamins and other beneficial components, such as enzymes and antibodies that are required for rapid growth and development of the new born and for sustained growth over time. Milk contains two main types of proteins; casein proteins and whey proteins. Although casein proteins account for up to 80% of the proteins found in bovine milk, it is the whey protein that has become of high interest because of its bioactive content. Whey, a very watery mixture of lactose, proteins, minerals and trace amounts of fat, is formed from milk when the milk is coagulated and/or the casein proteins are removed from the milk. Bovine whey protein, including both the acidic and basic fractions (low and high isoelectric point, respectively), has previously been studied in vitro (cell based) and in vivo (using rats) for its impact on bone to determine if it can help improve bone mineral density and help reduce the risk of developing bone diseases, such as osteoporosis. Bone is constantly undergoing a remodelling process of being dissolved and reformed and the two main cell types responsible for this bone remodelling process are mature osteoclasts, which dissolve (resorb) bone, and osteoblasts, which reform the bone. Prior work has shown that acidic protein fractions derived from different sources of whey protein concentrate (WPC) have both in vivo and in vitro activity on bone, particularly anti-resorptive properties. However, the component(s) which confer activity have not yet been identified. In this thesis, work was undertaken to better understand the analytical composition of three types of WPC (cheese, mineral acid and lactic acid) and their associated acidic protein fractions and relate this to bone activity in the hope of identifying where the activity lies. Bone activity was assessed using in vitro screening with osteoblast cells (MC3T3-E1) and osteoclast cells (RAW 264.7). Comparison of the cell-based bone activity of the parent WPCs and corresponding acidic fractions indicated that the acidic fractions derived from both mineral acid and lactic WPC were superior in their ability to inhibit osteoclast development. Although compositional data was complex and definitive correlations with both bone bioactivities could not be made, it appeared that elements common to both the acidic fractions were a higher proportion of GLYCAM-1 and bone sialoprotein-1 (osteopontin). Further studies to more closely investigate the bone bioactivity of the acidic fractions are warranted.

Milk is provided to new borns as their first food source and it contains essential nutrients, vitamins and other beneficial components, such as enzymes and antibodies that are required for rapid growth and development of the new born and for sustained growth over time. Milk contains two main types of proteins; casein proteins and whey proteins. Although casein proteins account for up to 80% of the proteins found in bovine milk, it is the whey protein that has become of high interest because of its bioactive content. Whey, a very watery mixture of lactose, proteins, minerals and trace amounts of fat, is formed from milk when the milk is coagulated and/or the casein proteins are removed from the milk. Bovine whey protein, including both the acidic and basic fractions (low and high isoelectric point, respectively), has previously been studied in vitro (cell based) and in vivo (using rats) for its impact on bone to determine if it can help improve bone mineral density and help reduce the risk of developing bone diseases, such as osteoporosis. Bone is constantly undergoing a remodelling process of being dissolved and reformed and the two main cell types responsible for this bone remodelling process are mature osteoclasts, which dissolve (resorb) bone, and osteoblasts, which reform the bone. Prior work has shown that acidic protein fractions derived from different sources of whey protein concentrate (WPC) have both in vivo and in vitro activity on bone, particularly anti-resorptive properties. However, the component(s) which confer activity have not yet been identified. In this thesis, work was undertaken to better understand the analytical composition of three types of WPC (cheese, mineral acid and lactic acid) and their associated acidic protein fractions and relate this to bone activity in the hope of identifying where the activity lies. Bone activity was assessed using in vitro screening with osteoblast cells (MC3T3-E1) and osteoclast cells (RAW 264.7). Comparison of the cell-based bone activity of the parent WPCs and corresponding acidic fractions indicated that the acidic fractions derived from both mineral acid and lactic WPC were superior in their ability to inhibit osteoclast development. Although compositional data was complex and definitive correlations with both bone bioactivities could not be made, it appeared that elements common to both the acidic fractions were a higher proportion of GLYCAM-1 and bone sialoprotein-1 (osteopontin). Further studies to more closely investigate the bone bioactivity of the acidic fractions are warranted.

Thesis (MSc)--Stellenbosch University, 2009. / ENGLISH ABSTRACT: Biotechnological applications are useful for adding value to the local medicinal plants and may provide an alternative source of pharmacologically-active compounds thus assisting with the conservation of wild populations. Salvia africana-lutea aromatic herb has long been used in folk medicine by traditional healers in the Western Cape Province (South Africa) for various ailments. As an in vitro conservation strategy, a continuous S. africana-lutea shoot culture was successfully established in solid MS medium containing BA (0.5 mg L-1) and NAA (0.2 mg L-1). The regeneration rate of the S. africana-lutea plants was high which produced approximately 720 plantlets in 20 culture bottles over a four week cycle. The microshoots were rooted in the MS medium without PGRs prior to acclimatisation. A survival rate of 92% was recorded for the greenhouse-acclimatised shoots. / AFRIKAANS OPSOMMING: Biotegnologiese toepassings is nuttig vir waarde toevoeging tot inheemse medisinale plante en kan ‘n alternatiewe bron van farmakologies aktiewe verbindings verskaf wat bydrae tot die bewaring van populasies in die natuur. Die aromatiese krui Salvia. Africana-lutea is reeds vir ‘n lang tydperk in volks medisyne deur tradisionele geneesheers in die Wes Kaap provinsie (Suid Afrika) vir ‘n verskeidenheid kwale gebruik. ‘n Kontinu S. africana-lutea lootkultuur in soliede Murashige en Skoog (1962) (MS) media wat BA (0.5 mg L-1) en NAA (0.2 mg L-1) bevat, is suksesvol as ‘n in vitro konservasie strategie ontwikkel. Die regenerasie tempo van die S. africana-lutea plante was hoog en het ongeveer 720 plante in 20 kultuur bottels tydens ‘n vier week siklus gelewer. Die mikrolote is op plant groei reguleerder vrye MS media gewortel voordat plante geaklimatiseer is. ’n Oorlewingstempo van 92% is vir die glashuis geaklimatiseerde lote waargeneem.

Chez la brebis, les BMPs inhibent la synthèse et la libération de FSH. La détection des ARNm de certaines BMPs dans l’hypophyse et de leurs récepteurs sur les cellules gonadotropes suggère une action paracrine/autocrine des BMPs sur la production de FSH. L’objectif de cette thèse a visé à déterminer l’importance des BMPs hypophysaires, en particulier BMP-4, dans la régulation de la sécrétion de FSH chez la brebis. Nous montrons que l’expression des ARNm du système BMP-4,analysée par RT-PCR en temps réel, ne varie pas au cours du cycle oestrien ou in vitro quelque soit le temps d’incubation (6h-48h) et le traitement (GnRH, oestradiol, activine) des cellules hypophysaires.Afin de déterminer si les cellules hypophysaires ovines sécrètent des BMPs, des milieux conditionnés(MC) hypophysaires ont été soumis à un test d’activité biologique reposant sur des cellules embryonnaires transfectées avec un élément de réponse BMPs couplé au gène rapporteur luciférase.Aucun effet des MC n’a été observé sur l’activité luciférase comparé au milieu non conditionnésuggérant l’absence ou la très faible activité BMP des MC, et cela quelque soit le temps d’incubation(6h-48h) et le traitement (GnRH, oestradiol, activine) des cellules hypophysaires. Toutefois, nous détectons une activité inhibitrice de BMPs qui est augmentée par la GnRH et l’oestradiol suggérant l’implication d’inhibiteurs de BMPs dans la régulation de la production de FSH. En conclusion,l’ensemble de nos résultats n’est pas en faveur d’un rôle de BMP-4 hypophysaire dans la synthèse de FSH chez l’adulte. Un rôle des BMPs au niveau hypophysaire via la voie endocrine peut s’envisager puisque nous avons mis en évidence une bioactivité de type BMP dans le sérum ovin. Les inhibiteurs BMPs produits par l’hypophyse, qui restent à identifier, pourraient moduler la biodisponibilité des BMPs atteignant l’hypophyse par la voie sanguine. / BMPs inhibit FSH synthesis and release in ewe. The detection of BMP mRNAs in the pituitary as well as the colocalisation of the two types of BMP receptors on gonadotrope cells suggest that these BMPs can exert paracrine/autocrine actions on FSH production. The aim of this thesis work was to determine the importance of pituitary BMPs in the regulation of FSH production in the ewe. We showed that the level of mRNAs for BMP-4 system, analyzed by real-time RT-PCR, did not vary across the oestrous cycle or in vitro whatever the incubation period (6h-48h) and the treatment (GnRH, oestradiol, activin) of pituitary cells. By using a bioactivity test based on embryonic mesenchymal cells transfected with an expression construct containing a BMP-responsive element fused to firefly luciferase reporter gene,we detected no BMP activity within conditioned media from pituitary cells whatever the incubation period (6h-48h) and the treatment (GnRH, oestradiol, activin) of the pituitary cells. However, we detected a BMP inhibitory activity which is increased by GnRH and oestradiol suggesting the implication of BMP inhibitor(s) in FSH regulation. In conclusion, the results are not in favor of a role for pituitary BMP-4 in the regulation of FSH synthesis in adult ewe. An endocrine action of BMPs at pituitary level can be evoked since BMP bioactivity was detected within ovine serum. BMP inhibitors that remain to be identified can modulate the bioavailability of BMPs reaching the pituitary by the blood way.

Le Titane (Ti) est un matériau biocompatible largement utilisé dans le domaine biomédical, notamment pour les implants orthopédiques (prothèse de hanche ou du genou par exemple). Il se distingue plus particulièrement par son excellente résistance à la corrosion et sa capacité d’ostéo-intégration. Cependant, une surface plane de Ti n’est pas assez bioactive pour être implantée ; il est nécessaire d’avoir recours à un traitement de surface pour améliorer ses propriétés. La modification de la surface du titane par anodisation permet la synthèse de nanotubes (NTs) de TiO2 ordonnés et verticalement alignés. Cette technique, peu couteuse, rapide et facile à mettre en œuvre permet également de contrôler finement la morphologie des NTs (diamètre, longueur, aspect des parois…). De plus, les NTs étant amorphes après anodisation, la structure cristalline peut être ajustée par un traitement thermique, conduisant à une structure anatase (450°C) ou à une structure mixte d’anatase et de rutile (550°C).Ainsi, des surfaces de différentes morphologies et différentes structures cristallines ont été synthétisées dans ce travail de thèse, afin d’évaluer l’influence des caractéristiques morpho-structurelles sur la résistance à la corrosion en milieu physiologique et sur la bioactivité (formation d’hydroxyapatite et réponse cellulaire).Nous avons démontré dans cette étude que les NTs cristallisés présentent une plus grande résistance à la corrosion et une meilleure bioactivité que les NTs amorphes (ou que les surfaces planes de Ti). Plus précisément, en tenant compte des aspects électrochimiques, thermiques, mécaniques, chimiques et de bioactivité, il semblerait que des NTs mixtes d’environ 720 nm de long et 90 nm de diamètre constituent une surface optimale pour les applications visées.D’autre part, dans le cadre de l’étude des propriétés électrochimiques de l’interface, une attention toute particulière a été accordée dans ce travail de thèse à la modélisation des résultats de mesures par Spectroscopie d’Impédance Electrochimique (SIE). Il s’avère que la réponse en impédance des NTs de TiO2 en milieu physiologique correspond au modèle d’électrode poreuse de De Levie. Toutefois, pour des électrodes poreuses non-idéales, cette théorie peut être généralisée et modélisée par des lignes de transmission. Ainsi, le modèle de ligne de transmission proposé dans cette étude s’ajuste de façon très satisfaisante aux mesures expérimentales. Il permet notamment de mettre en évidence la nature très peu réactive des NTs de TiO2, justifiant ainsi leurs applications dans le domaine biomédical. / Titanium (Ti) is a biocompatible material widely used in the biomedical field, especially for orthopedic implants (for instance hip or knee replacement). It is particularly corrosion resistant and shows remarkable osseointegration properties. However, plane Ti surfaces are not bioactive enough to be implanted; they need to be improved by surface treatments. Surface modification of Ti by anodization enables to synthesize self-organized and vertically aligned TiO2 nanotubes (NTs). This cheap, fast and easily implementable technique also permits a fine tuning of NTs morphology (diameter, length, wall look, etc.). Moreover, since as-anodized NTs are amorphous, crystalline structure may be adjusted by heat treatment, producing anatase structure (450°C) or a mixed structure of anatase and rutile (550°C).Therefore, surfaces of different morphologies and crystalline structures have been synthesized in order to evaluate the influence of these characteristics on corrosion resistance in physiological medium and on bioactivity (hydroxyapatite formation and cell response).We demonstrated that crystallized NTs are more corrosion resistant and more bioactive than amorphous ones or even than flat Ti surfaces. More precisely, considering electrochemical, thermal, mechanical, chemical and bioactive aspects, it seems that mixed NTs of about 720 nm in length and 90 nm in diameter constitute an optimal surface for the present applications.Additionally, in the frame of electrochemical investigations, we focused on modeling experimental results from Electrochemical Impedance Spectroscopy (EIS) measurements. The impedance response of TiO2 NTs in physiological conditions can correspond to porous electrode model as developed by De Levie. However, for non-ideal electrodes, this theory can be generalized and modeled by transmission lines. Thus, the transmission line model developed in this study fits very well with experimental measurements. It suggests that TiO2 NTs are almost non-reactive, justifying their applications in biomedical fields.

Made available in DSpace on 2016-06-02T20:18:11Z (GMT). No. of bitstreams: 1 2456.pdf: 6548607 bytes, checksum: 486173aee7443e35a2910af043a12256 (MD5) Previous issue date: 2009-04-03 / Universidade Federal de Sao Carlos / Bioactive materials have the ability to bond and to integrate with bone tissue by forming a biologically active bonelike apatite layer, which has chemical and structural properties equivalent to the mineral phase of living bone. This process is determined by chemical reactions, whose products also influence the attachment, the proliferation, the differentiation and the mineralizing capacity of bone cells. Cellular responses contribute to the bioactive behavior, which is known for being higher in glass materials. However, as low mechanical properties are also inherent characteristics of glasses, researchers from Federal University of Sao Carlos were stimulated to develop nucleation and growth thermal treatments for the obtainment of the Biosilicate®, a fully-crystallized bioactive glassceramic of the quaternary system P2O5-Na2O-CaO-SiO2. Although a high in vitro osteogenic potential of this novel glass-ceramic has been previously demonstrated, its in vivo effects have not been investigated yet. To contribute to this knowledge, two studies were developed. The first one aimed to investigate the in vivo biological performance of Biosilicate® in bone defects of rat tibias, by means of hystomorphometric and biomechanical analyses 20 days after the surgical procedure. This study revealed that the fully-crystallized Biosilicate® has good bone-forming and bone-bonding properties. Hence, the second study aimed to compare the kinetics of the bone reactions to two different granulometric distributions of this novel glass-ceramic. Although they were both efficient for bone formation, smaller-sized particles of Biosilicate® showed partial reabsortion, which was accompanied by a more pronounced osteogenic activity within the period of time studied. Since positive results were obtained, the search for scaffolds that could serve as supports for the guided bone regeneration had started. A third study preliminarily evaluated cell culture and cocultures in porous structures made of Biosilicate® and of other chemical compositions that were specifically developed for this purpose. The findings suggest that, when in adjusted conditions, the scaffolds can create favorable cellular responses for bone tissue engineering purposes. Taken togheter, these studies point to a promising potential and provide directives for the use of Biosilicate® in bone regenerative processes. / Materiais bioativos possuem a capacidade de se ligar ao tecido ósseo por meio da formação de uma interface apatítica que apresenta similaridade química e estrutural com a fase mineral dos ossos. Esse processo ocorre devido a uma série de reações químicas, cujos produtos também influenciam a adesão, a proliferação, a diferenciação e a capacidade de mineralização da matriz pelas células ósseas. As respostas celulares contribuem para o comportamento bioativo, que é conhecido por ter maiores índices em materiais vítreos. No entanto, como baixas propriedades mecânicas também são características inerentes aos vidros, pesquisadores da Universidade Federal de São Carlos foram estimulados a empregarem nucleação e tratamentos térmicos especiais para o desenvolvimento do Biosilicato®, uma vitrocerâmica biotiva, totalmente cristalina, pertencente ao sistema quaternário P2O5-Na2O-CaO-SiO2. Embora um elevado potencial osteogênico in vitro tenha sido demonstrado para esta vitrocerâmica, seus efeitos in vivo ainda não são conhecidos. Para auxiliar este entendimento, foram desenvolvidos dois estudos. O primeiro teve como objetivo investigar o desempenho biológico in vivo do Biosilicato® particulado em defeitos ósseos em tíbias de ratos, por meio de análises histomorfométricas e biomecânicas 20 dias após o procedimento cirúrgico. Este estudo evidenciou que o Biosilicato® parece favorecer a formação óssea in vivo e o estabelecimento de fortes ligações com o tecido neoformado. Com isso, o objetivo do segundo estudo foi comparar a cinética das reações ósseas frente a duas diferentes distribuições granulométricas desta nova vitrocerâmica. Embora ambas tenham sido eficientes para a formação óssea, as partículas de Biosilicato® com menores diâmetros demonstraram reabsorção parcial no período estudado, que foi acompanhada de uma maior atividade osteogênica. Com os resultados positivos obtidos nestas investigações, iniciou-se uma busca para o desenvolvimento de matrizes porosas que pudessem servir de suporte para a regeneração guiada do tecido ósseo. Um terceiro estudo preliminarmente avaliou monoculturas e coculturas celulares em matrizes porosas de Biosilicato® e de outras novas composições químicas desenvolvidas especificamente para este propósito. Os achados sugerem que, quando em condições adequadas, as matrizes avaliadas podem produzir respostas celulares favoráveis ao seu emprego na engenharia do tecido ósseo. Estes estudos, de maneira conjunta, apontam para um potencial promissor e fornecem diretrizes para o emprego do Biosilicato® no favorecimento de processos regenerativos ósseos.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nectandra megapotamica (Lauraceae) is abundant in the Deciduos Forest in Santa Maria-RS. Its essential oil (EO) has been studied for biological activities due to its anti-inflammatory, antifungal, antimicrobial, and cytotoxic effects against tumor cells, among others. The presence of phenylpropanoids as well as sedative and anesthetic activities in R. quelen were observed in the prospect of EO of Nectandra megapotamica. As a hypothesis, we considered the seasonal variability and the occurrence of chemical phenotypes for the individuals in the same population of the species with different biological activities for the EO. The objectives of this study were to define the potential of N. megapotamica in a population in Santa Maria-RS for the production of EO; to determine the yield (Y), chemical composition and the seasonal variation of EO; to evaluate the possible occurrence of chemical phenotypes (differentiated chemical composition); to isolate compounds of interest; and to perform tests of biological activities for the EO and for the isolated compound. The EO samples were obtained from fresh leaves, which were initially separated in young (YL) and old leaves (OL) by hydrodistillation in triplicate for three hours. The collections occurred in function of the seasons in two locations in Santa Maria-RS (Morro do Elefante, and in the proximity of the 318 km of BR 158). The respective Y values were determined, and EO samples were analyzed by GC/MS. Some samples were also analyzed by GC-FID, in triplicate. The chemical composition of the EO was analyzed and grouped by multivariate techniques, main components and hierarchical grouping. The EO was also fractionated by CC, for the isolation of a compound, whose identification happened by NMR. The discrepant groups (CG2 and CG5) were tested for allelopathic, insecticide and anesthetic activities in fish. The compound obtained from CG5 was identified as isoelemicin (phenylpropanoid) and also tested for the anesthetic activity. The largest Y for the EO was observed in spring (YL 0.24% and OL 0.14%) during flowering, fruiting and foliation, and the lowest in autumn (YL 0.13% and OL 0.11%) in the rustification and at rest. The major constituents were α-pinene with greater Y in spring and summer (YL 30.81% and OL 35.86%), and bicyclogermacrene with greater Y in autumn and winter (YL 24.19% and OL 21.73%), which formed two large CG. In the CG of bicyclogermacrene, the presence of phenylpropanoids was observed and therefore it constituted a specific group (CG5). However, CG2 presented higher concentration of monoterpenoids. In the biological tests, the allelopathic activity (lower seed germination and seedling development) for L. sativa var. regina, B. subalternans, G. max, A. strigosa, L. multiflorum and O. sativa was observed when the seeds were submitted to the EO. Moreover, the insecticidal activity was observed in N. viridula, and also sedation in R. quelen. The CG5 was superior than CG2, concerning allelopathy in O. sativa; however, both groups were active at the concentration of 15% EO for the other species, which inhibits 100% germination of B. subalternans (QG5) and A. strigosa (both groups). Regarding the insecticidal action of EO at 5%, the CG5 promoted 46.67% mortality of N. viridula, and CG2 only 14.44%. Concering R. quelen, we observed toxicity associated with sedation, both by the EO similar to CG5 and by isoelimicin. The stage of anesthesia was observed at the concentracion of 500 mg L-1 EO; however, the effect was lower than that observed for eugenol (positive control). In conclusion, the species in the population assessed has the potential to produce EO as a source of new bioactive molecules and biological action mechanisms that should consider the toxicity component, which requires further studies. / Nectandra megapotamica (Lauraceae) é abundante na Floresta Estacional Decidual em Santa Maria-RS. O seu óleo essencial (OE) vêm sendo estudado quanto às atividades biológicas, devido a sua ação anti-inflamatória, antifúngica, antimicrobiana, citotóxica contra células tumorais, dentre outras. Na prospecção do OE foram encontrados fenilpropanoides, além de atividades sedativas e anestesicas em Rhamdia quelen. Como hipótese, considerou-se que haja variabilidade sazonal e ocorrência de fenótipos químicos para os indivíduos na mesma população da espécie, com atividade biológica diferenciada para o OE. Os objetivos deste trabalho foram pesquisar o potencial de N. megapotamica, em uma população em Santa Maria, para a produção de OE; determinar o rendimento (R), a composição química e sua variação sazonal; avaliar a possível ocorrência de fenótipos químicos (composição química diferenciada); isolar constituintes de interesse; e por fim, realizar testes de atividades biológicas, para o OE e para o isolado. Os OE foram obtidos de folhas frescas, inicialmente separadas em jovens (FJ) e velhas (FV), por hidrodestilação em triplicata por três horas. As coletas ocorreramem função das estações do ano, em dois locais em Santa Maria-RS (Morro do Elefante e, na proximidade do km 318 da BR 158). Foram determinados os respectivos R, e os OE foram analisados por CG-EM. Algumas amostras também foram analisadas por CG-DIC, em triplicata. A composição química do OE dos indivíduos coletados foi analisada e agrupada por técnicas multivariadas, componentes principais e agrupamento hierárquico. Foi também realizado fracionamento do OE por CC, para o isolamento de uma substância, cuja identificação se deu por RMN. Determinados os grupos químicos (GQ) e o constituinte isolado, os grupos mais discrepantes (GQ2 e GQ5) foram testados quanto a atividade alelopática, inseticida e anestésica em peixes. A isoelemicina foi avaliada para esta última. O maior R do OE foi observado na primavera (FJ 0,24% e FV 0,14%), durante a floração, frutificação e foliação, e o menor no outono (FJ 0,13% e FV 0,11%), na rustificação e repouso. Os constituintes majoritários foram α-pineno, com maior R na primavera e verão (FJ 30,81% e FV 35,86%), e biciclogermacreno, com maior R no outono e inverno (FJ 24,19 e FV 21,73%). Estes formaram dois grandes GQ. No GQ do biciclogermacreno foi observada a presença de fenilpropanoides, que constituíram um grupo específico (GQ5). Já o GQ2 apresentou maiores teores de monoterpenoides. Do OE do GQ5 foi isolada a isoelemicina (fenilpropanoide). Nos testes biológicos realizados foram observadas atividade alelopática (menor germinação de sementes e desenvolvimento de plântulas) para Lactuca sativa cv. regina, Bidens subalternans, Glicine max, Avena strigosa, Lolium multiflorum e Oryza sativa; atividade inseticida em Nezara viridula e sedação em R. quelen. O GQ5 foi superior ao GQ2, nos testes de alelopatia em O. sativa, mas ambos os grupos foram ativos na concentração de 15% de OE para as demais espécies, inibindo100% da germinação de B. subalternans (GQ5) e de A. strigosa (ambos os grupos). Na ação inseticida com 5% do OE, o GQ5 promoveu 46,67% de mortalidade de N. viridula, e o GQ2 apenas 14,44%. Em R. quelen foi observada sedação associada à toxicidade, tanto pelo OE similar ao GQ5 (obtido do segundo local de coleta) como pela isoelemicina. O estágio de anestesia foi observado na concentração de 500 mg L-1 de OE, mas o efeito foi inferior ao observado para o eugenol com 50 mg L-1 (controle positivo). Conclui-se que a espécie, na população avaliada, possui potencial para produzir OE, como fonte de novas moléculas bioativas e mecanismos de ação biológica, que devem considerar a componente de toxicidade, o que exigirá novas pesquisas.

Pour combiner les bonnes propriétés mécaniques des matériaux métalliques et obtenir des surfaces bioactives, des revêtements de phosphate de calcium (Ca-P) ont été développés. Le but est d’améliorer la biocompatibilité et la bioactivité du système et créer une barrière contre l’éventuelle libération d’ions toxiques du substrat métallique. Notre démarche a été de développer une nouvelle voie de synthèse. Après un polissage mécanique et décapage chimique du substrat de Titane ou Ti–6Al–4V une couche intermédiaire en titanate de sodium a été obtenue par un prétraitement alcalin sur les substrats. Elle a été ensuite soumise à un traitement thermique afin de créer une couche alvéolaire nanométrique. Celle-ci facilite la croissance une couche de phosphate de calcium par voie autocatalytique d'une manière similaire au procédé de formation de l'os naturel. La caractérisation et l’étude des revêtements des Ca-P obtenus par les trois bains (acides, alcalins et oxydant) sur les substrats métalliques ont été réalisés. Les dépôts ont été étudiés d’un point de vue structural et morphologique. La stabilité des couches de Ca-P a été mesurée dans le fluide corporel (SBF) pour des périodes différentes utilisant des analyses biochimiques. En conclusion, cette méthode est une solution peu coûteuse, fiable, et utilisable à l'échelle industrielle. La couche de Ca-P aussi bien que le titanate de sodium obtenus peuvent permettre d’être imprégnés par des agents actifs comme un ou plusieurs agents antibactériens. / To combine the good mechanical properties of metallic substrate with the bioactivity of some ceramics, to obtain bioactive surface, a calcium phosphate (Ca-P) coating has been developed. The goal is to improve the biocompatibility and bioactivity of metallic implant and create a barrier against the possible release of toxic ions from the metallic substrate. Our approach has been to develop a new synthetic route. After mechanical polishing and chemical etching of the titanium or Ti-6Al-4V substrates, an interlayer of sodium titanate was obtained by an alkaline pretreatment of the substrates. Then, it was subjected to a heat treatment to create a nanometeric layer. The latter facilitates the growth of a layer of Ca-P by means of an electroless manner similar to the forming process of the natural bone. Characterization and study of Ca-P coatings obtained by the three baths (acids, alkalis and oxidizing) on metallic substrates have been made. The deposits were studied from a structural and morphological point of view. The stability of Ca-P layers was measured in simulated body fluid (SBF) for different periods using biochemical analyzes. In conclusion, this method is a cheap and reliable solution that can be used on an industrial scale. The Ca-P as well as the sodium titanate layers can afford to be impregnated with active agents such as one or more antibacterial agents.

Submitted by (lucia.rodrigues@ufrpe.br) on 2016-06-06T14:55:01Z No. of bitstreams: 1 Vilma Sobral Bezerra.pdf: 3241045 bytes, checksum: 1dbe7e33df34dacca3124c036d6370bd (MD5) / Made available in DSpace on 2016-06-06T14:55:01Z (GMT). No. of bitstreams: 1 Vilma Sobral Bezerra.pdf: 3241045 bytes, checksum: 1dbe7e33df34dacca3124c036d6370bd (MD5) Previous issue date: 2011-12-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Dairy proteins have bioactive peptides production great potential, however, their bioactivity is achieved only after enzymatic hydrolysis, which produces substances beneficial to health when incorporated into food or pharmaceuticals. Among them, casein has been used for this purpose. This study aims to determine peptide profile and amino acid sequence of bioactive peptides derived from Moxotó goat milk casein hydrolysis, using proteolytic enzymes such as trypsin, pepsin, papain and a protease extracted from Penicillium aurantiogriseum URM 4622. Enzymatic hydrolysis was performed using a statistical experimental design, which independent variables were pH, enzyme substrate (E: S), temperature and reaction time in Moxotó goat milk casein hydrolysis. Hydrolysis products were visualized in SDS-PAGE electrophoresis. Hydrolysates subjected to ultrafiltration (cut off 3000Da) were used to determine biological properties. Antioxidant activity was evaluated by ABTS + [2,2 '-Azin-bis (3-ethylbenzothiazoline) 6-sulfonic acid] method, using the pool of peptides (permeate <3000Da; retentate >3000Da). Antimicrobial activity was determined by the Clinical and Laboratory Standards Institute. Binding through the zinc solubility of zinc in the sample by Mass Spectrometry Inductively Coupled Plasma. Peptide profile and amino acid sequences were determined by mass spectrometry MALDI-TOF-MS/MS. The best hydrolysis degree (38.27%) was obtained with pepsin enzyme pH 3.0, 1:100 E: S, temperature of 40°C and 5 hours time reaction. However, a high hydrolysis degree precluded the use of these hydrolysates for bioactive peptides obtention. Casein tryptic hydrolysates demonstrated antioxidant activity up to 3242.3 μmol.L-1TROLOX/mg peptide in retentate (> 3000Da). By papain use, we obtained an activity up to 2329.6 μmol.L-1TROLOX /mg of peptide in permeate (<3000Da). The peptides produced by P. auratiogriseum protease action showed activity from 843.17 to 2587.30 μmol.L-1of Trolox / mg of 10 and 29% peptides hydrolysates, respectively, which were compatible with natural antioxidants, such as C vitamin and α-tocopherol. Goat casein tryptic hydrolysates demonstrated antimicrobial activity against Enterococcus faecalis ATCC 6057, Escherichia coli ATCC 2508, Klebisiela pneumoniae ATCC 29665, Bacillus subtilis ATCC 6633. Casein hydrolysates showed IC50 of 4.46 mg/g for zinc binding. Mass spectrometry MALDI TOF MS\MS allowed the visualization of caprine casein peptides in permeate (<3000Da), ranging from 568 to 2923 Da. It also showed LLYQEPVLGPV and HPINHQGLSPEVPNENLLR amino acids sequences for αs1 and β-casein, respectively, from casein tryptic hydrolysates; LLYQEPVLGPV sequences of β-casein, the NPWDQVK αs2 NENLL-casein and-casein in the αS1 casein hydrolysates by papain use; LLYQEPVLGPVRGPFPI β-casein sequence from casein hydrolysates obtained by the use of P. aurantiogriseum URM 4622 protease. The casein hydrolysates bioactive properties are referent to the prevalence of hydrophobic amino acids, which possibility of their use as bioactive peptides. / Proteínas lácteas apresentam grande potencial na produção de peptídeos bioativos. A caseína se destaca na produção de bioativos. Entretanto, a sua bioatividade só é conseguida após hidrólise enzimática, ao qual se produz substâncias benéficas à saúde quando incorporados em alimentos ou produtos farmacêuticos. O objetivo deste trabalho foi o de caracterizar e a avaliar atividades biológicas e determinando o perfil peptídico e a sequência de aminoácidos dos peptídeos bioativos obtidos a partir de hidrólise da caseína do leite de cabra Moxotó, usando enzimas proteolíticas tripsina, pepsina, papaína e protease extraída de Penicillium aurantiogriseum URM 4622. A hidrólise enzimática foi realizada através de planejamento experimental estatístico, os quais as variáveis independentes foram pH, relação enzima substrato (E:S), temperatura e tempo de reação na hidrólise da caseína do leite de cabra Moxotó. Os produtos de hidrólise foram visualizados em eletroforese SDS‑PAGE. Os hidrolisados submetidos à ultrafiltração (Cut off 3000Da) foram utilizados para determição das propriedades biológicas. A atividade antioxidante foi avaliada no pool de peptídeos, permeado (<3000Da) e retentado (>3000Da) usando-se o método ABTS+ [2,2’-azino-bis (3-etilbenzotiazolin) 6-ácido sulfônico]. A atividade antimicrobiana foi determinada pelo método do Clinical and Laboratory Standards Institute. O carreamento de zinco através da solubilidade do zinco na amostra através da Espectrometria de Massa em Plasma Indutivamente Acoplado. O perfil peptídico e as sequências de aminoácidos foram determinados por espectrometria de massa MALDI-TOF-MS/MS. O melhor grau de hidrólise (38,27%) foi obtido com a enzima pepsina usando pH 3,0, E:S de 1:100, 40ºC e 5 horas de reação. Entretanto, um alto grau de hidrólise impossibilitou o uso desses hidrolisados para obtenção de peptídeos bioativos. Os hidrolisados trípicos da caseína mostraram atividade antioxidante de até 3.242,3μmol.L-1TROLOX/mg de peptídeo no retentado (>3000Da). Com o uso da papaína, obteve-se uma atividade de até 2.329,6μmol.L-1TROLOX/mg de peptídeo no permeado (<3000Da). Os peptídeos produzidos pela ação da protease produzida por P. auratiogriseum apresentaram atividade de 843,17 a 2.587,30μmol.L-1de TROLOX/mg de peptídeos para os hidrolisados com 10 e 29% respectivamente, os quais foram compatíveis a antioxidantes naturais, como a vitamina C e α-tocoferol. Os hidrolisados trípticos da caseína caprina apresentaram atividade antimicrobiana frente a Enterococcus faecalis ATCC 6057, Escherichia coli ATCC 2508, Klebisiela pneumoniae ATCC 29665, Bacillus subtilis ATCC 6633. Os hidrolisados de caseína mostraram IC50 de 4,46mg/g para carreamento de zinco. A espectrometria de massa MALDI TOF MS permitiu visualizar os peptídeos no permeado (<3000Da) da caseína caprina, na faixa de 568 a 2.923 Da. Mostraram as sequências LLYQEPVLGPV e HPINHQGLSPEVPNENLLR e da β- e αs1-caseina para hidrolisados trípticos da caseína; as sequências LLYQEPVLGPV da β-caseína, NPWDQVK da αs2-caseina e NENLL da αS1-caseína nos hidrolisados da caseína com o uso da papaína e a sequência LLYQEPVLGPVRGPFPI da β-caseina no hidrolisado da caseína com o uso da protease produzida por P. aurantiogriseum URM 4622. As propriedades bioativas dos hidrolisados da caseína referem-se à prevalência de aminoácidos hidrofóbicos, o que possibilita o uso destes como peptídeos bioativos.

Submitted by (edna.saturno@ufrpe.br) on 2016-12-02T11:16:10Z No. of bitstreams: 1 Marcileyne Pessoa Leite de Lima.pdf: 517075 bytes, checksum: b7ddab703563527f0c2b8e77f710122c (MD5) / Made available in DSpace on 2016-12-02T11:16:11Z (GMT). No. of bitstreams: 1 Marcileyne Pessoa Leite de Lima.pdf: 517075 bytes, checksum: b7ddab703563527f0c2b8e77f710122c (MD5) Previous issue date: 2008-02-04 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The synthetic insecticide is the main method used to control Spodoptera frugiperda (J.E. Smith). However, the inadequate application of the insecticide has contributed to increase costs, reduction of the natural enemies, and selection of resistant populations, among other problems. Attempting to obtain alternative control of S. frugiperda more ecologically acceptable, the present work investigated the effects of neem (Azadirachta indica A. Juss) and Bacillus thuringiensis subsp. aizawai (Bta) on S. frugiperda under laboratory, greenhouse and field conditions. In the field, the commercial formulations of Natuneem® (10 mL/L in 2005 and 15 mL/L in 2006) and Neemseto® (5 and 10 mL/L in 2005, and 10 and 15 mL/L in 2006) and the biological insecticide Xentari® (10 g/L in 2005 and 2006) were applied at 15, 30 and 45 and at 15, 25 e 35 days after plant emergency in 2005 and 2006, respectively. Thee evaluations consisted of using a scale of foliar injury from 0 to 5 prior each spray. In 2005, only Neemseto® 10 mL/L was efficient at 30 days. In 2006, Neemseto® 15 mL/L and Xentari® with average score of 0.93 and 0.98, respectively, were different from the control, which produced a score of 2.38 at 35 days after plant emergency. The greenhouse assay consisted of treatment of plants 40-days old with the insecticides Natuneem®, Neemseto® and Xentari® followed by artificial infestation with newly-hatched larvae of S. frugiperda. After 10 days, it was verified that the treatment were equally efficient, differing from the control, regarding to the injury score, as well as, the number of surviving larvae. In the last bioassays, in the laboratory, 10-days old S. frugiperda larvae were fed with corn leaves treated with the insecticides Natuneem®, Neemseto® and Xentari® at 2.5; 5.0; 7.5; 10 mL or g/mL and control (water). The effect of the insecticides increased as function of increasing concentration, causing higher larval mortality, longer period and lower larval weight. In some cases, reduction of pupa weight and viability and longevity of adults were also verified. Larvae 0-24 h old were more susceptible to the insecticides, as compared with 10-days old larvae. / A utilização de inseticidas sintéticos tem sido o principal método de controle de Spodoptera frugiperda (J.E. Smith), mas o uso inadequado tem contribuindo para o aumento do custo de produção, redução de inimigos naturais, seleção de populações resistentes, entre outros problemas. Desta forma, buscando alternativas de controle mais ecológicas, estudou-se no presente trabalho os efeitos de nim (Azadirachta indica A. Juss) e Bacillus thuringiensis subsp. aizawai (Bta) sobre S. frugiperda em experimentos de laboratório, casa de vegetação e campo. Foram testadas em campo formulações comerciais de inseticidas botânicos Natuneem® (10 mL/L em 2005 e 15 mL/L em 2006) e Neemseto® (5 e 10 mL/L em 2005, e 10 e 15 mL/L em 2006) e do inseticida biológico Bta (Xentari® 10 g/L em 2005 e 2006), aplicados aos 15, 30 e 45 e aos 15, 25 e 35 dias após a emergência das plantas em 2005 e 2006, respectivamente. Antes de cada pulverização foram atribuídas notas de 0 a 5 para as injúrias foliares. Em 2005, somente Neemseto® 10 mL/L foi eficiente aos 30 dias. Em 2006, Neemseto® 15 mL/L e Bta 10 g/L, com notas médias de 0,93 e 0,98, diferiram da testemunha, com nota 2,38, na avaliação realizada aos 35 dias. Plantas de milho, com cerca de 40 dias de idade, foram pulverizadas em casa de vegetação com os inseticidas Natuneem®, Neemseto e Bta, e posteriormente infestadas,artificialmente, com lagartas de S. frugiperda recém-eclodidas. Após 10 dias verificou-se que os tratamentos foram igualmente eficientes, diferindo da testemunha, tanto em relação às notas, quando ao número de lagartas sobreviventes. Nos últimos bioensaios lagartas de S. frugiperda com 10 dias de idade foram alimentadas com folhas de milho submersas na calda inseticida nas concentrações 2,5; 5,0; 7,5; 10 mL ou g/L e testemunha (água). Os efeitos dos inseticidas dependeram da concentração utilizada, pois provocaram mortalidade crescente das lagartas, alongaram o período e reduziram o peso larval. Em alguns casos, também, reduziram o peso e a viabilidade pupal e a longevidade de adultos. Lagartas com 0-24 h de idade foram mais susceptíveis aos inseticidas, em relação às com 10 dias de idade.