Made available in DSpace on 2014-06-11T19:29:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-26Bitstream added on 2014-06-13T19:38:29Z : No. of bitstreams: 1 tetzner_tad_me_jabo.pdf: 994984 bytes, checksum: dc3b9f9b3b09f1b6ee7040b88cd31ad8 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O presente trabalho objetivou avaliar os efeitos da substituição do SFB e do BSA pela OVA na PIV, por estudos nas etapas de maturação, fecundação e cultivo in vitro. Observamos que durante a etapa de maturação, a concentração 4mg/mL de OVA não afetou a maturação nuclear (82,66%) e a migração de grânulos corticais (GC) para a periferia dos oócitos (54,21%), sendo, portanto, utilizada nos experimentos subseqüentes. Foi observado que as fontes protéicas SFB, BSA, OVA, e BSA com OVA (BO) não afetaram (p>0,05) as taxas de maturação nuclear e migração de GC. Para a sigla dos tratamentos, as fontes protéicas foram identificadas pelas iniciais (SFB=S, BSA=B e OVA=O). Cada tratamento foi abreviado com a primeira letra referente à etapa de maturação, a segunda à fecundação, e a terceira ao cultivo. Quanto às taxas de formação de pronúcleo (PN) observamos que o grupo SO (76,67% de 2 PN) foi semelhante (p>0,05) ao grupo controle (82,95% de 2 PN). A etapa de CIV permitiu avaliar que os diferentes tratamentos foram semelhantes (p>0,05) quanto à taxa de clivagem. Entretanto, quanto à taxa de produção de blastocistos, o grupo OOO (26,0%) foi semelhante (p>0.05) aos grupos SOS (33,8%), BBB (35,8%), BOB (32%), e OBO (33%), mas foi inferior (p<0,05) aos grupos CONT (45%) e SBS (42,8%). Quanto à taxa de eclosão, o grupo OOO (20,4%), foi inferior (p<0,05) aos grupos CONT (46,2%), SBS (43,4%), SOS (38,4%), BBB (41,6%), e semelhante aos grupos BOB (28,2%) e OBO (25,4%). Concluímos que é possível produzir embriões bovinos na ausência de SFB e/ou BSA, com a fonte protéica OVA, embora tanto a quantidade como a qualidade dos blastocistos tenham se apresentado inferiores em relação àqueles produzidos na presença de SFB e/ou BSA. / The present work aimed to evaluate the effects of FCS and BSA substitution for OVA on in vitro production of bovine embryos, studying in vitro maturation, fertilization, and development. For maturation, we observed that the concentration 4mg/mL of OVA didn't affect nuclear maturation (82.66%), and cortical granule (CG) migration (54.21%), which indicated its use in the subsequent experiments. It was observed that the protein sources FBS, BSA, OVA and BO didn't significantly affect (p>0.05) the rates of nuclear maturation and CG migration. For evaluation of pronucleus (PN) formation rates, we observed that the SO group (the first letter means the protein source for the maturation stage, the second for the fertilization, and the third for the development), (76.67% of 2 PN) was similar (p>0.05) to the control group (82.95% of 2 PN). However, two pronucleus formation rates in BB (56.98%), BO (39.02%), OB (37.36%) and OO (39.24%) were different (p<0.05) from the control group (82.95% of 2 PN). Different treatments (CONT, SBS, SOS, BBB, BOB, OOO, OBO) were similar (p>0.05) in cleavage rates. However, regarding to blastocyst production rates, the OOO group (26.0%) was similar (p>0.05) to the SOS group (33.8%), BBB (35.8%), BOB (32%), and OBO (33%) groups, but it was inferior (p <0.05) compared to the CONT (45%) and SBS (42.8%) groups. In relation to blastocyst hatching rate, the OOO group (20.4%) was inferior (p<0.05) when compared to the CONT (46.2%), SBS (43.4%), SOS (38.4%), BBB (41.6%) groups, and it was to BOB (28.2%) and OBO (25.4%) groups. We concluded that it s possible to produce bovine embryos in the absence of FBS and/or BSA using ovalbumin (OVA) as the protein source, even though decreased quantity and quality rates of bovine blastocysts were observed.

Introdução: Há crescentes evidências que sinalizam o papel do estresse crônico como desencadeante e mesmo perpetuador de crises asmáticas, bem como a importância do parênquima pulmonar na piora funcional da asma. Objetivos: Deste modo, consideramos relevante avaliar em cobaias com inflamação alérgica crônica pulmonar como o estresse físico repetido, induzido pela natação forçada, modula a responsividade do parênquima pulmonar, ainfiltração eosinofílica, ativação da via de estresse oxidativo e o remodelamento, bem como o papel da ativação de enzima óxido nítrico sintase induzida nestas respostas. Métodos: Os animais receberam inalações duas vezes por semana durante quatro semanas com doses crescentes de ovoalbumina ou solução fisiológica Após 24 horas da quarta inalação os animais foram separadas em dois grupos onde metade destes animais foi submetido ao protocolo de natação forçada por dez dias com intervalo de dois dias, para a indução do estresse, enquanto a outra metade dos animais permaneceu sem a indução de estresse. Para avaliar a participação da ativação de iNOS nesta resposta as cobaias foram novamente divididas em grupos, onde parte foi tratada com 1400-W (inibidor específico de iNOS, 2mg/kg ip/dia/4 dias) ou veículo no mesmo período, iniciando 30 minutos antes da sétima inalação. Para avaliação das repercussões comportamentais da indução de estresse os animais foram submetidos ao teste de campo aberto, sendo obtidos os seguintes parâmetros: distância percorrida, velocidade média total, tempo de movimentação total. Foi também coletada uma amostra de sangue no dia da realização da mecânica pulmonar para a dosagem do cortisol. Após 72h da sétima inalação, os animais foram anestesiados, exsanguinados e fatias de tecido pulmonar periférico foram retiradas e suspensas em banho orgânico de Krebs, e a resistência (Rt) e elastância (Et) do tecido pulmonar periférico foram avaliadas em condição basal e após desafio com ovoalbumina (1%). Foi também obtida a medida de histerisividade. Após a realização da mecânica oscilatória, as mesmas fatias de tecido pulmonar periférico foram submetidas à avaliação histopatológica. Resultados: Nos animais submetidos a natação forçada foi observado um aumento no peso da adrenal, no cortisol sérico, na distância percorrida e no tempo de movimentação em campo aberto o que demonstrou que a natação forçada foi eficaz como indutor de estresse. Os animais expostos às inalações com ovalbumina apresentaram valores maiores de porcentagem de aumento da resistência e da elastância tecidual após desafio com ovoalbumina (p<0.05). Os animais expostos às inalações com ovalbumina e submetidos a estresse induzido por natação forçada apresentaram um aumento da resistência e elastância teciduais (p<0,05), enquanto que o tratamento com 1400W reverteu esse aumento tanto nos animais sensibilizados quantos nos sensibilizados submetidos a estresse. Houve aumento do número de eosinófilos (P<0.05), de células iNOS positivas (P<0,05), da deposição de fibras colágenas (P<0,05), no conteúdo de actina (P<0,05) e de 8-epi-PGF2a (P<0,05) no septo alveolar. Nos animais sensibilizados e submetidos ao estresse houve aumento todos os parâmetros morfológicos descritos anteriormente (P<0,05) exceto pelo depósito de fibras colágenas. A administração de 1400W reduziu todos estes parâmetros funcionais e morfológicos (P<0,05) com exceção do conteúdo de actina no septo alveolar, da infiltração eosinofílica e dos níveis de cortisol sérico. Conclusões: Neste modelo experimental, o bloqueio específico da iNOS atenuou a constrição, a inflamação eosinofílica, a expressão de iNOS, o remodelamento no parênquima pulmonar tanto nos animais apenas sensibilizados quanto nos animais sensibilizados e submetidos ao estresse. Estas alterações podem estar relacionadas aos efeitos da ativação da óxido nítrico sintase induzida na modulação da via do estresse oxidativo, sinalizada pelos efeitos na produção de isoprostano 8. O presente estudo sugere que a inibição específica da iNOS pode amplificar as estratégias terapêuticas utilizadas na abordagem de doenças inflamatórias crônicas pulmonares / Introduction: There is growing evidence to indicate the role of chronic stress even perpetuating as triggering asthma attacks and the importance of functional lung parenchyma in worsening of asthmatic responses. Objective: We considered relevant to evaluate in guinea pigs with chronic allergic pulmonary inflammation if repeated physical stress, induced by forced swimming, modulates the responsiveness of the distal lung parenchyma, eosinophilic infiltration, oxidative stress pathway activation and extracellular matrix remodeling as well as the role of the activation of induced nitric oxide synthase in these responses. Methods: The animals received inhalations twice a week for four weeks with increasing doses of ovalbumin or normal saline. After 24 hours of the fourth inhalation, the animals were separated into two groups where half of these animals were subjected to the protocol of forced swimming for ten days with an interval two days, while the other half of the animals remained without stress induction. In order to evaluate the involvement of iNOS activation in this response, guinea pigs were again divided in two groups, one part of them were treated with 1400W (specific inhibitor of iNOS, 2mg/kg ip/day/4 days) or vehicle in the same period, starting 30 minutes before the seventh inhalation. To evaluate the behavioral impact of stress induction, the animals were subjected to open field test, and we obtained the following parameters: distance traveled, average speed, total time of handling. It was also collected a blood sample on the day of pulmonary mechanics to measure cortisol. After 72h of the seventh inhalation, animals were anesthetized, and exsanguinated and slices of peripheral lung tissue were removed, suspended in a Krebs bath, and resistance (Rt) and elastance (Et) of peripheral lung tissue were evaluated either at baseline condition and after oavalbumin challenge (1%). It was also measured the histerisivity. After the end of the mechanical oscillatory evaluation, the same slices of peripheral lung tissue were submited to histopathological analysis. Results: We observed that animals submitted to forced swimming had an increase in adrenal weight, in the serum cortisol, in the distance and time of movement in the open field, showing that forced swimming was effective as stressor. The animals exposed to inhalations with ovalbumin and submited to stress induced by forced swimming had an increase in pulmonary tissue resistance and elastance (p <0.05), had an increased in the number of eosinophils (P <0.05), iNOS positive cells (P <0.05), actin (P <0.05) and 8-epi-PGF2a content (P <0.05) in alveolar septum compared to ovalbumin exposed animals (OVA group). Treatment with 1400W reversed this response in sensitized animals submitted or not to stress induction (P <0.05) There was no difference in the collagen deposition. Administration of 1400W for sensitized and stressed animals reduced all these functional and morphological parameters (P <0.05) except for the actin content of the alveolar septum, eosinophilic infiltration and cortisol levels. Conclusions: In this experimental model, the specific iNOS inhibition attenuated the constriction, the eosinophilic inflammation, the iNOS expression, the lung parenchyma remodeling in animals sensitized and submitted or not to stress induction. These changes may be related to the effects of inducible nitric oxide activation in the modulation of oxidative stress pathway. This study suggests that specific inhibition may amplify the therapeutic strategies to chronic inflammatory lung diseases

The interaction between ovalbumin (OVA) and four sulphonamides named sulfathiazole (S1), sulfaquinoxaline (S2), sulfadimethoxine (S3) and sulfamethazine (S4) was investigated employing spectroscopic techniques (molecular fluorescence, UV-Vis and 1H NMR) emulating in natura egg conditions. For the interaction study, the intrinsic fluorescence of the protein (λex = 280 nm / λem= 336 nm) was explored in the ligand’s presence and absence (SFs). The OVA spectrometric titrations (2 μM) with different ligands (0-200 μM) were performed in pH 7.4 (Tris-HCl buffer solution 50 mM, NaCl 100 mM). For the evaluation of the interaction process, it was noticed a decrease in the OVA fluorescent emission as SFs excess was added, due to an energy transfer process (quenching). By using these data, the Stern Volmer constant (Ksv), binding constant (Kb) and stoichiometric ratio (n) were calculated. The Kb values ranged from 3.98 to 27.4 x 105 L mol-1 (30 ºC) and by applying these information, it was observed that the interaction magnitude followed the order: S3 < S4 < S2 < S1. The number of binding sites in every condition applied was close to one. For all SFs evaluated the increase in temperature lead to a decrease in both Ksv and Kb values, indicating that the static quenching mechanism is preferable, with the formation of a non-fluorescent molecular complexes. This result was further confirmed by UV-Vis studies with formation of a fundamental state complex. According to thermodynamic parameters, ΔG < 0 was obtained for all SFs evaluated, indicating that the interaction is thermodynamically spontaneous. For S2 and S3, the main forces acting in the interaction are hydrogen bonds and Van der Waals forces, whereas S1 and S4 have electrostatic forces, predominantly. Through 3D fluorescence studies, it was observed a decrease in the fluorescence intensity of peak 2 and 3, which is related to the tyrosine and tryptophan residue emissions associated with the protein secondary structure in the presence of SFs, respectively. This result indicates that there were conformational changes in the protein native structure. By using synchronized fluorescence, it was possible to deduce that the interaction with OVA exposes the tyrosine and tryptophan residues, increasing the polarity of the micro–environment of those amino acids. Through studies based on FRET (Fluorescence resonance energy transfer), it was possible to calculate the intermolecular distance between the SFs (energy acceptors) and OVA (energy donor), which were smaller than 10 nm. The competition assay with ANS probe, which control the protein hydrophobic regions, indicates that only S2 was capable to shift this marker. Metallic ions were also evaluated and it was observed that Ca(II), Mg(II) and Fe(III) can facilitate the interaction between sulphonamides (S1 and S2) and OVA. The 1H NMR studies showed a signal shift for aromatic hydrogens present in S2 and S3. In nature ovalbumin (from egg white) interacts more effectively with SFs than commercial ovalbumin. Biological studies have shown that E. coli and B. megaterium are sensitive to the sulfonamides tested and the MIC values of these antibiotics in the absence and presence of OVA do not present significant difference. This way, the results indicate that there was interaction between SFs and macromolecule and, thus, conformational changes in the protein structure were identified. So, the presence of SFs in aliments shows a risk to food security, since the OVA has its polypeptide chain modified and can act/treat as a foreigner and cause allergic reactions. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A interação da ovalbumina (OVA) com quatro sulfonamidas, nomeadamente sulfatiazol (S1), sulfaquinoxalina (S2), sulfadimetoxina (S3) e sulfametazina (S4) foi investigada empregando técnicas espectroscópicas (fluorescência molecular, UV-vis e RMN 1H) simulando as condições in natura do ovo. Para o estudo de interação, explorou-se a fluorescência intrínseca da proteína (λex = 280 nm / λem = 336 nm) na ausência e presença dos ligantes (SFs). As titulações espectrofluorimétricas da OVA (2 μM) com os diferentes ligantes (0-200 μM) foram realizadas em pH 7,4 (tampão Tris 50 mM, NaCl 100 mM). Na avaliação do processo de interação notou-se diminuição na intensidade da emissão da fluorescência da OVA à medida que os excessos das SFs foram adicionados, devido ao processo de transferência de energia (quenching). Por meio destes resultados foi possível calcular a constante de Stern Volmer (Ksv), constante de ligação (Kb) e proporção estequiométrica (n). Os valores de Kb variaram de 3,98 a 27,4x105 L mol-1 (30 ºC) e a partir destes dados observou-se que a magnitude da interação seguiu a seguinte ordem: S1 > S2 > S4 > S3. O número de sítios de ligação em todas as condições foi próximo à unidade. Para todas as SFs avaliadas o aumento da temperatura levou a diminuição dos valores de Ksv e Kb, indicando que o mecanismo de quenching preferencial é estático, com formação de complexos supramoleculares não-fluorescentes. Este resultado foi confirmado pelos estudos de UV-vis com formação do complexo no estado fundamental. Quanto aos parâmetros termodinâmicos, obteve-se ΔG < 0 para todas as SFs avaliadas, indicando que a interação é termodinamicamente espontânea. Para S2 e S3, as forças predominantes na interação são ligações de hidrogênio e forças de Van der Waals, enquanto que para S1 e S4 as forças preferenciais foram eletrostáticas. Através dos estudos por fluorescência 3D observou-se a redução na intensidade de fluorescência dos picos 2 e 3, relativos à emissão dos resíduos de tirosina e triptofano e emissão associada a estrutura secundária da proteína na presença das SFs, respectivamente. Este resultado indica que houve mudanças na estrutura nativa da proteína. Por meio de fluorescência sincronizada foi possível inferir que a interação com a OVA expõe os resíduos de triptofano e tirosina aumentando a polaridade do microambiente destes aminoácidos. Através de estudos baseados em FRET (Fluorescence resonance energy transfer), foi possível calcular a distância intermolecular entre as SFs (receptoras de energia) e a OVA (doadora de energia), as quais foram inferiores a 10 nm. Os estudos de competição com a sonda ANS que monitora regiões hidrofóbicas da proteína indicaram que S2 foi a única sulfa capaz de deslocar este marcador. Já com os íons metálicos, observa-se que Ca(II), Mg(II) e Fe (III) favorecem a interação das sulfonamidas (S1 e S2) com a OVA. A partir dos estudos por ressonância magnética nuclear de hidrogênio (RMN 1H) verificou-se deslocamento dos sinais para os hidrogênios aromáticos presentes em S2 e S3. Além disto, foi verificado que a ovalbumina in natura (obtida da clara do ovo) interage com as SFs em maior magnitude quando comparado com a OVA comercial. Por meio de estudos biológicos constatou-se que a E. Coli e B. megaterium são sensíveis as sulfonamidas testadas e que os valores de MIC destes antibióticos na ausência e presença da OVA não diferem. Dessa forma, os resultados obtidos indicam que houve interação entre as SFs e a macromolécula e, consequentemente, mudanças conformacionais na estrutura da proteína foram identificadas. Logo, a presença das SFs em alimentos pode apresentar um risco a segurança alimentar, pois uma vez que há mudança conformacional na cadeia polipeptídica da OVA, esta pode comportar-se como um corpo estranho no organismo e causar/potencializar reações alérgicas.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Com o objetivo de verificar a influÃncia dos hormÃnios tireoideanos no desenvolvimento de hiperreatividade traqueal, ratos machos (200 - 250 g) eutireÃideos, hipotireÃideos (propiltiouracil [PTU] - v.o. 0,05% p/v, durante 4 semanas) ou hipertireÃideos (L- tiroxina [T4] - 0,5 mg/kg s.c., por 7 ou 9 dias) foram sensibilizados à ovalbumina (OVA) e, 14 dias depois, desafiados atravÃs da inalaÃÃo de OVA (1 mg/ml, seguida de 5 mg/ml, 15 min cada). O sacrifÃcio dos animais para a realizaÃÃo dos experimentos ocorreu 24 h apÃs o desafio antigÃnico por anestesia com hidrato de cloral (400 mg/Kg). A traquÃia isolada foi montada em cubas contendo 10 ml de Krebs-Henseleit modificado (37 oC, 5% de CO2 em O2). Foram obtidas curvas concentraÃÃo-efeito (CCE) para cloreto de potÃssio (KCl), carbacol (CCh) ou serotonina (5-HT). TambÃm foram realizadas CCE ao Ca2+ em preparaÃÃes estimuladas por KCl, CCh ou 5-HT e mantidas em soluÃÃo sem Ca2+. A reapresentaÃÃo do antÃgeno promoveu significativo aumento (p < 0,05, two-way ANOVA) da resposta mÃxima (RM) das CCE ao KCl de 0,96  0,10 gF para 1,53  0,11 gF (n = 7), ao CCh de 1,98  0,06 gF para 2,92  0,07 gF (n = 7) e à 5-HT de 1,64  0,14 gF para 2,41  0,15 (n = 6) nos tecidos obtidos de animais sensibilizados ou desafiados, respectivamente. As traquÃias tambÃm apresentaram aumento (p < 0,05, two-way ANOVA) da RM ao Ca2+ quando estimuladas com KCl de 0,54  0,06 gF para 0,86  0,07 gF (n = 6), com CCh de 1,20  0,14 gF para 1,77  0,14 gF (n = 6) ou com 5-HT de 0,59  0,10 gF para 1,15  0,05 gF (n = 6) nos tecidos obtidos de animais sensibilizados ou desafiados, respectivamente. O hipotireoidismo nÃo alterou significativamente a RM induzida por KCl e CCh, enquanto que aquela induzida pela 5-HT foi reduzida de 1,64  0,14 gF nos animais eutireÃideos para 0,34  0,07 gF nos animais hipotireÃideos (p < 0,001, two-way ANOVA). ApÃs desafio, a 5-HT produziu 0,56  0,11 gF (n = 7) no tecido hipotireÃideo (p < 0,001, two-way ANOVA). O hipotireoidismo aboliu o desenvolvimento de hiperreatividade para KCl e CCh. Ocorreu um aumento na CE50 nas CCE obtidas ao CCh de 0,49 x 10-6M para 4,65 x 10-6M (P < 0,05, two-way ANOVA ). O desafio reduziu a CE50 novamente para 1,53 x 10-6M (n = 6, p<0,05, two-way ANOVA). As traquÃias de animais hipotireÃideos desafiados apresentaram diminuiÃÃo da RM ao Ca2+ quando estimuladas com KCl, CCh e 5-HT. Ocorreu aumento na CE50 nas CCE ao Ca2+ em traquÃias desafiadas e estimuladas com CCh de 5,77 x 10-4 M para 22,50 x 10-4 M (n = 6, p<0,01 two-way ANOVA). O hipertireoidismo promoveu um significativo aumento na RM das CCE apenas ao KCl (0,96  0,10 gF no controle versus 1,58  0,15 no tecido hipertireÃideo). NÃo houve desenvolvimento de hiperreatividade apÃs o desafio antigÃnico (RM = 1,87  0,14 gF). Ocorreu diminuiÃÃo da CE50 ao CCh de 0,67 x 10-4 M no controle para 0,14 x 10-4 M apÃs tratamento com T4. Os resultados mostram que hà envolvimento dos hormÃnios tireoideanos no desenvolvimento de hiperreatividade em traquÃia de rato, induzida apÃs reapresentaÃÃo do antÃgeno a animais previamente sensibilizados. / In other to verify the influence of thyroid hormones on the tracheal hyperreactivity development, euthyroid, hypothyroid (propiltiouracil [PTU] - p.o. 0.05% w/v, 4 weeks) or hyperthyroid (L-tiroxine [T4] â 0.5 mg/kg s.c.,7 or 9 days) male rats (200 - 250 g) were sensitized to ovalbumine (OVA) and, 14 days later, challenged with OVA inhalation, (1 mg/ml, followed by 5 mg/ml, 15 min each). Animals sacrifice was carried out 24 later by means of anaesthesia with chloral hydrate (400 mg/Kg). Isolated trachea was mounted in 10 ml bath chamber filled with modified Krebs-Henseleit (37 oC, 5% de CO2 em O2). Concentration-effect curves (CEC) were carried out for potassium chloride (KCl), carbachol (CCh) or serotonin (5-HT). CEC to Ca2+ added in tissues maintained in Ca2+-free medium stimulated with KCl, CCh or 5-HT also were carried out. Antigenic challenge produced significant increase (p < 0.05, two-way ANOVA) of the maximal response (Emax) of the CCE for KCl from 0.96  0.10 gF to 1.53  0.11 gF (n = 7), for CCh from 1.98  0.06 gF to 2.92  0.07 gF (n = 7) and for 5-HT from 1.64  0.14 gF to 2.41  0.15 (n = 6) in tissues obtained from sensitized or challenged animals, respectively. Tracheae also showed increase on the Emax to Ca2+ (p < 0.05, two-way ANOVA) when stimulated with KCl from 0.54  0.06 gF to 0.86  0.07 gF (n = 6), with CCh from 1.20  0.14 gF to 1.77  0.14 gF (n = 6) or with 5-HT from 0.59  0.10 gF to 1.15  0.05 gF (n = 6) on sensitized or challenged tissues, respectively. The hypothyroidism did not modify significantly the KCl- or CCh-induced Emax, while the 5-HT-induced contractile effect was reduced from 1.64  0.14 gF in euthyroid tissues to 0.34  0.07 gF in hypothyroid tissues (p < 0.001, two-way ANOVA). After challenge, 5-HT produced in hypothyroid tissues a contraction corresponding to 0.56  0.11 gF (n = 7, p < 0.001, two-way ANOVA). Hypothyroidism prevented hyperreactivity development for KCl and CCh. It was observed an increased EC50 value in CCE for CCh from 0.49 x 10-6M to 4.65 x 10-6 M (p < 0,05, two-way ANOVA). After challenge, CE50 value was reduced to 1.53 x 10-6 M (n = 6, p < 0,05, two-way ANOVA). Tracheae from challenged hypothyroid animals showed decreased Emax to Ca2+ when they were stimulated with KCl, CCh and 5-HT. It was observed an increased EC50 value in CCE to Ca2+ in challenged tissues stimulated with CCh from 5.77 x 10-4 M to 22.50 x 10-4 M (n = 6, p < 0.01, two-way ANOVA). Hyperthyroidism significantly increased Emax of the KCl-induced CCE (0.96  0,10 gF in control versus 1.58  0.15 on hyperthyroid tissue). Hyperreactivity was not showed after antigenic challenge (Emax = 1.87  0.14 gF). It was observed a reduction of the EC50 value to CCh from 0.67 x 10-4 M in control to 0.14 x 10-4 M after T4 treatment. Our results show that there is a putative thyroid hormones involvement in hyperreactivity development on rat trachea, after an antigenic challenge.

Nesse estudo in vitro foi avaliado o potencial erosivo do suco de laranja modificado pela adição de caseína, ovalbumina e a combinação entre elas, sobre o esmalte e a dentina humanos. Duas proteínas da dieta, 0.2 g/l de caseína (CAS), 2.0 g/l de ovalbumina (OVA) e a combinação entre elas (CAS + OVA) foram adicionadas a um suco de laranja disponível comercialmente. O suco de laranja sem aditivos foi utilizado como controle negativo (C-) e o suco de laranja com adição de cálcio, também disponível comercialmente, como controle positivo (C+). O potencial erosivo dos sucos experimentais foi primeiramente comparado utilizando o método do pHStat, e em seguida, através de um modelo in vitro de erosão-remineralização. 55 espécimes de esmalte e 55 de dentina radicular (4 x 4 x 2mm) foram obtidos e incluídos em um bloco de resina acrílica. Esses blocos foram então planificados com discos de lixa abrasivos e polidos com disco de feltro e pasta diamantada. As superfícies polidas receberam a aplicação de fitas adesivas, expondo uma janela de 4 x 1mm. Os espécimes foram aleatoriamente distribuídos entre os 5 grupos experimentais (n = 11), e imersos nos respectivos sucos por 5 min, 6x ao dia, durante 5 dias. Entre as imersões e durante o período noturno, os espécimes permaneceram armazenados em saliva artificial. Após a ciclagem, os espécimes de esmalte foram analisados através de perfilometria óptica e microdureza (50 g, 15 s), enquanto que os espécimes de dentina foram analisados apenas por perfilometria óptica. Para o método do pH-Stat foi calculada a média do volume de HCl obtida em triplicata. Para a análise dos dados obtidos através de perfilometria e microdureza, foi utilizado o teste de Análise de Variância, um fator, seguido pelo teste complementar de Tukey, adotando um nível de significância de 5%. As médias do volume de HCl (em ml) obtidas no método do pH-Stat foram: C+ 0,46 (± 0,03); CAS 1,22 (± 0,06); OVA 1,10 (± 0,10); CAS+OVA 1,08 (± 0,01) e C- 1,07 (± 0,02). Na avaliação do esmalte, a perda de estrutura (m) observada foi de: C+ 0,09 (± 0,20); CAS -0,40 (± 0,32); OVA -0,44 (± 0,26); CAS+OVA -0,39 (± 0,25) e C- -1,04 (± 0,36). Quanto a microdureza superficial, os valores de dureza Knoop obtidos foram: C+ 312,68 (± 20,45); CAS 121,99 (± 10,70); OVA 108,87 (± 11,16); CAS+OVA 102,57 (± 11,89) e C- 101,94 (± 8,56). Para a dentina, a perda de estrutura observada foi de: C+ -0,82 (± 0,28); CAS -7,26 (± 0,65); OVA -6,74 (± 1,18); CAS+OVA -7,16 (± 0,75) e C- -7,51 (± 1,26). Conclui-se que para o esmalte, os sucos de laranja modificados pela adição de proteínas apresentaram um potencial erosivo reduzido. A caseína mostrou uma melhor proteção da desmineralização subsuperficial do esmalte; a sua combinação com a ovalbumina não apresentou nenhum benefício adicional. Para a dentina, nenhuma redução no potencial erosivo foi observado para os sucos de laranja modificados pela adição de proteínas. / The erosive potential of a modified orange juice by addition of casein, ovalbumin and its combination, on human enamel and root dentin was evaluated in this in vitro study. Two dietary proteins, 0.2 g/l casein (CAS), 2.0 g/l ovalbumin (OVA) and their combination (CAS + OVA) were added to a commercially available orange juice. The juice with no additives was used as negative control (C-) and a commercially available calcium-modified juice as positive control (C+). The erosive potential of the experimental juices was initially compared by the pH-Stat method, and then, by an in vitro erosion-remineralization cycling model. 55 enamel and 55 root dentin specimens (4 x 4 x 2mm) were obtained and embedded in acrylic resin blocks. These blocks were ground flat with abrasive discs and polished with felt paper and diamond paste. The polished surfaces were covered with an adhesive tape, leaving a central area of 4 x 1mm exposed. The specimens were randomly allocated within the 5 experimental groups (n=11), and immersed in the respective juices for 5 min, 6x/day, for 5 days. Between the immersions and overnight they were stored in artificial saliva. After the cycling, the enamel specimens were analyzed by surface Knoop microhardness (50g, 15s) and optical profilometry, while dentin specimens were analyzed only by profilometry. The mean volume of HCl obtained in triplicate were calculated for the pH-Stat method. The data obtained for profilometry and microhardness were statistically analyzed using ANOVA, one-way, followed by Tukeys test considering a significance level of 5%. The mean volume of HCl (ml) obtained for the pH-stat method were: C+ 0,46 (± 0,03); CAS 1,22 (± 0,06); OVA 1,10 (± 0,10); CAS+OVA 1,08 (± 0,01) e C- 1,07 (± 0,02). For enamel, the surface loss (m) was: C+ 0,09 (± 0,20); CAS -0,40 (± 0,32); OVA -0,44 (± 0,26); CAS+OVA -0,39 (± 0,25) e C- -1,04 (± 0,36). Regarding microhardness, the Knoop hardness values were: C+ 312,68 (± 20,45); CAS 121,99 (± 10,70); OVA 108,87 (± 11,16); CAS+OVA 102,57 (± 11,89) e C- 101,94 (± 8,56). For dentin, the surface loss (m) was: C+ - 0,82 (± 0,28); CAS -7,26 (± 0,65); OVA -6,74 (± 1,18); CAS+OVA -7,16 (± 0,75) and C- -7,51 (± 1,26). It was concluded that protein-modified orange juices presented reduced erosive potential on enamel. Casein showed a better subsurface demineralization protection, and its combination with ovalbumin did not lead to additional benefits. For dentin, any reduction on the erosive potential was observed for protein-modified orange juices.

O ferro é essencial para a vida do ser humano, desempenhando um papel fundamental no metabolismo. Contudo, quando não armazenado em compartimentos biológicos adequados, o metal apresenta um potencial tóxico ao organismo, uma vez que contribui para a formação de espécies reativas de oxigênio. A sobrecarga de ferro é uma condição desfavorável para portadores de algumas disfunções genéticas, como a hemocromatose, ou de anemias crônicas que requeiram transfusões de sangue periódicas, como é o caso da talassemia. Os fármacos atuais que controlam a patologia, como a desferrioxamina (DFO), requerem infusão subcutânea lenta, causando desconforto em pacientes e podendo trazer um série de complicações, como insuficiência hepática e renal. A modificação dessas moléculas com biopolímeros é uma proposta para minimizar efeitos colaterais e aumentar a biodisponibilidade do fármaco no organismo. Dentre esses biopolímeros, destacam-se as albuminas proveniente do soro bovino (BSA) e do ovo (OVA), que têm baixa toxicidade, baixo custo e abundância de sítios reativos, que quando modificados, favorecem reação com a desferrioxamina. Como resultado, houve a reação dos biopolímeros com a desferrioxamina, com mudanças em suas estruturas secundárias e possível dimerização, resultando na formação de conjugados possuem afinidade com íon ferro e capacidade antioxidante semelhante ao fármaco original, características que tornam os compostos bons candidatos a uma alternativa à terapia de quelação. Os conjugados BSA-DFO e OVA-DFO podem reagir, além do ferro, com gadolínio, fazendo com o que os complexos tenham uma potencial aplicação como agentes de contraste em ressonância magnética de imagem (MRI). Neste trabalho, vimos que o complexo entre Gd(III) e BSA-DFO apresentou uma relaxatividade de 52,92 s-1 mM-1 para T2 e 45,37 s-1 mM-1 para T1 , um valor bem superior aos fármacos disponíveis no mercado, que apre-sentam relaxatividade entre 4 e 5 s-1 mM-1, o que foi explicado por sua elevada massa molecular, indicando que poderia ter bons efeitos na qualidade de MRI, com menores doses. / Iron is essential for human life, playing a fundamental role in metabolism. However, when not stored in appropriate biological compartments, the metal presents a toxic potential to the body, contributing to the formation of reactive oxygen species (ROS). Iron overload is an unfavorable condition for people with certain genetic disorders, such as hemochromatosis, or chronic anemias that require periodic blood transfusions, as thalassemia. Current drugs that control the pathology, as desferrioxamine, require slow subcutaneous infusion, causing discomfort in patients and may lead to a number of complications, such as hepatic and renal failures. As a result, the biopolymers were reacted with desferrioxamine, with changes in their secondary structures and possible dimerization, resulting in the formation of conjugates with iron ion affinity and antioxidant capacity similar to the original drug, characteristics that make the compounds good candidates for an alternative chelation therapy As a result, the reaction of the biopolymers with desferrioxamine caused a change in the secondary structure, with possible formation of dimers and showing different mobility when exposed to an electric potential difference. Not all polymer chains have reacted with DFO, however BSA-DFO complex has antioxidant capacity similar to the original drug. The BSA-DFO and OVA-DFO conjugates can react, in addition to iron, with gadolinium, making the complexes potential contrast agents for magnetic resonance imaging (MRI). In this work, the complex between Gd(III) and BSA-DFO presented a relaxativity of 52,92 s-1 mM-1 for T2 and 45,37 s-1 mM-1 for T1, values higher than the available drugs in the market (4 - 5 s-1 mM-1) which was explained by the high molecular weight, indicating a good effects on the quality of MRI, with lower doses.

With the aim to develop and characterize a functionalized highly magnetic polymer carrier of micrometer size and of a narrow particle size distribution that will be suitable for biological application, hypercrosslinked microspheres were prepared. Simultaneously, the relation between structure and properties of product was observed. Condition of dispersion polymerization were optimized to obtain starting monodisperse poly(styrene-co-divinylbenzene) [P(St-DVB)] microspheres. The P(St-DVB) microspheres of different degree of crosslinking were prepared and effect of some polymerization parameters such as type of solvent, initiator, concentration and mode of DVB addition on morphology, size and particle size distribution were investigated. The starting microspheres were hypercrosslinked to obtain microporous inner structure. Hyperosslinked particles had very large specific surface area (> 1000 m2/g) and a high content of micropores (ca. 0.6 ml/g). First, P(St-DVB) microspheres were chloromethylated using three different chloromethylation agents to regulate their porous properties. Hypercrosslinking was achieved by the addition of stannic chloride as a catalyst and by increasing a temperature. The hypercrosslinked microspheres were then functionalized with sulfo- or aminogroups. The functional groups captured precipitated iron oxide inside the porous structure of the microspheres and also served as a reactive site for intended immobilization of the protein. A solution of ferrous and ferric chloride was imbibed under vacuum into the porous structure and the iron oxide was precipitated by an aqueous ammonia solution. Finally, the magnetic functionalized hypercrosslinked micropsheres were integrated into a biosensor for qualitative detection of ovalbumin.

L’objectif de cette thèse est la fabrication d’un microdispositif magnétique pour la détection et la manipulation d’éléments biologiques à base de nanoparticules magnétiques en conditions microfluidiques. Il a pour but d’intégrer des fonctions de base de contrôle et détection magnétique, pour atteindre des mesures spécifiques, stables, rapides et reproductibles. En effet, la technique d’immunodosage couplée à des nanoparticules magnétiques, bien connue dans la littérature, nécessite un contrôle du déplacement de ces dernières pour les fonctionnaliser efficacement et créer un complexe biologique encapsulant une molécule cible (biomarqueur). Dans notre cas une molécule modèle pour le domaine de la biodéfense a été utilisée : l’ovalbumine. Pour contrôler le champ magnétique nécessaire pour la capture des complexes magnétiques, nous avons opté pour l’utilisation de microbobines intégrées aux dispositifs fluidiques et comparé cette technique originale avec d’autres plus conventionnelles. Pour détecter un complexe biologique, la fluorescence est largement utilisée en biologie, mais cette technique ne permet pas une intégration complète pour un dispositif autonome. Dans cette optique, nous proposons la détection des complexes à base de nanoparticules magnétiques en relevant la variation de l’inductance d’un microcircuit magnétique refermant une chambre microfluidique contenant ces complexes immunologiques. Le dimensionnement des microbobines de contrôle par simulation a permis de déterminer les paramètres permettant d’obtenir le champ magnétique le plus adapté au contrôle des complexes biologiques. Dans le cas des microbobines utilisées pour la détection, des branches magnétiques micrométriques ont été insérées autour des microbobines pour créer un circuit de détection magnétique encore plus sensible. La réalisation de ces dispositifs a impliqué l’intégration de matériaux et de structures de nature fortement hétérogène, et leur assemblage a nécessité de résoudre de nombreux verrous technologiques. L’enjeu a été de déterminer l’ensemble des étapes successives et nécessaires pour un procédé de microfabrication fiable et reproductible. Pour montrer l’intérêt des dispositifs de capture des nanoparticules magnétiques, des tests immunologiques ont été réalisés tout d’abord en microtubes pour les comparer à ceux réalisés dans un circuit fluidique à l’aide d’aimant externe puis de microbobines intégrées. Dans ce dernier cas, une optimisation considérable a été validée en termes de réduction de temps d’incubation, de reproductibilité des mesures et de limites de détection équivalentes à l’état de l’art pour l’ovalbumine. Pour le dispositif de détection magnétique, des premières expériences de caractérisation électrique ainsi que des études en concentration de nanoparticules magnétiques ont été réalisées et comparées aux résultats obtenus par simulation. Pour la preuve de concept, un démonstrateur de détection de complexes magnétiques a été également finalisé validant la possibilité d’intégration du microcircuit magnétique dans un dispositif fluidique. Il a validé également l’obtention d’une gamme de sensibilité remarquable corrélée à la présence des complexes magnétiques. Ses caractéristiques ont été confrontées à celles obtenues par les simulations et discutées en tenant compte de toutes les étapes critiques du procédé de microfabrication. / RésuméThe objective of this thesis is the fabrication of a magnetic microdevice for the detection and manipulation of biological elements based on magnetic nanoparticles under microfluidic conditions. It aims to integrate basic functions of control and magnetic detection, to achieve specific, stable, fast and reproducible measurements. Indeed, the immunoassay technique coupled to magnetic nanoparticles, well known in the literature, requires a control of the displacement of magnetic nanoparticles to effectively functionalize them and create a biological complex to encapsulate a target molecule (biomarker). In our case, a model molecule for the field of biodefense was used: ovalbumin. To control the magnetic field which is necessary for the capture of magnetic complexes, we opted for the use of microcoils integrated in the fluidic devices and compared this original technique with other more conventional ones. To detect a biological complex, fluorescence is widely used in biology, but this technique does not allow a complete integration for an autonomous device. In this context, we propose the detection of complexes based on magnetic nanoparticles by observing the variation of the inductance of a magnetic microcircuit closing a microfluidic chamber containing these immunological complexes. The design of the control microcoils by simulation made it possible to determine the parameters allowing to obtain the most adapted magnetic field to the control of the biological complexes. In the case of microcoils used for detection, micrometric magnetic branches were inserted around the microcoils to create an even more sensitive magnetic sensing circuit. The realization of these devices involved the integration of materials and structures of highly heterogeneous nature, and their assembly has required to solve many technological locks. The challenge was to determine all the successive and necessary steps for a reliable and reproducible microfabrication process. To show the interest of magnetic nanoparticle capture devices, immunoassays were first performed in microtubes to compare with those made in a fluid circuit using external magnet and integrated microcoils. In the latter case, considerable optimization has been validated in terms of reduction of incubation time, reproducibility of measurements and detection limits equivalent to the state of the art for ovalbumin. For the magnetic detection device, first experiments of electrical characterization as well as concentration studies of magnetic nanoparticles were carried out and compared to the results obtained by simulation. For the proof of concept, a demonstrator of detection of magnetic complexes was also finalized validating the possibility of integration of the magnetic microcircuit in a fluidic device. It has also validated obtaining a remarkable range of sensitivity correlated with the presence of magnetic complexes. Its characteristics were compared to those obtained by the simulations and discussed taking into account all the critical steps of the microfabrication process.

No description available.

The work focused on addressing four main objectives. The first objective was to quantify protein and amino acid substitution reactions. Michael addition reactions were used to modify the amino acids and protein. Amino acids alanine, cysteine, and lysine, and protein ovalbumin (OA) were substituted with different concentrations of ethyl vinyl sulfone (EVS). The substituted products were analyzed using Raman spectroscopy and UV-spectroscopy based ninhydrin assay. In case of alanine, Raman and UV results correlated with each other. With cysteine at lower EVS substitutions amine on the main chain was the preferred site while the substitution shifted to thiols at higher substitutions. This could only be discerned using Raman spectroscopy. Lysine has amines on the main chain and side chain while main chain amine was the most reactive site at lower concentrations of EVS while at higher concentrations side chain amines were also substituted. This information could be discerned using Raman spectroscopy only and not UV spectroscopy. In case of protein as observed by Raman and UV spectroscopy the reaction continued at higher concentrations of EVS indicating the participation of glutamine and asparagines at higher substitutions. However, the reaction considerably slowed down at higher EVS substitutions. The second objective of the study was to decrease the glass transition temperature (Tg) of OA through internal plasticization and also study the effects of the substituents on the thermal stability of OA. The hypothesis was by covalently attaching substituents to OA, number of hydrogen bonds can be reduced while increasing the free volume and this would reduce Tg. EVS, acrylic acid (AA), butadiene sulfone (BS) and maleimide (MA) were the four groups used. EVS was the most efficient plasticizer of all the four substituents. The Tg decreased with the increasing concentration of EVS until all of the reactive of groups on OA were used up. Tg decreased slightly with AA and BS while no change was observed with MA. However, the substituents showed exact opposite trend in thermal stability as measured using thermogravimetric analysis (TGA). The thermal stability of MA substituted OA was the highest and that of EVS substituted OA was least. FT-IR spectroscopy results indicated that all four substituents caused structural changes in OA. This implied that there were intermolecular interactions between substituted protein chains in case of AA, BS, and MA. This caused an increase in the thermal stability. EVS on the other hand is a linear chain monomer with a hydrophobic end group and hence could not participate in the intermolecular interactions and hence caused a decrease in Tg. As mentioned above the limitation to this technique is the number of available reactive groups on the protein. However, we successfully demonstrated the feasibility of this method in decreasing Tg of protein. The third objective was to create hydrogels by crosslinking OA with divinyl sulfone (DVS). Protein hydrogels due to their biocompatible nature find applications in drug delivery and tissue engineering. For tissue engineering applications the hydrogels need to be mechanically stable. In this study the protein was substituted with EVS or AA and then crosslinked with DVS. The swelling ratio was measured as a function of pH. All the hydrogels showed the same trend and swelled the least at pH 4.5 which is the isoelectric point of the protein. At basic pH conditions EVS substituted hydrogels swelled the most while AA substituted hydrogels showed least swelling. The static and dynamic moduli of the hydrogels were determined using tensile tester and rheometer respectively. The static modulus values were three times the dynamic modulus. The modulus of the control which is crosslinked OA was least and that of AA substituted OA was highest. The stress relaxation test also showed similar results in which AA substituted OA relaxed the most and the control relaxed the least. FT-IR of the dry hydrogels showed that the amount of hydrogen bonding increased with AA substitution. The hydrophilic AA end groups interacted with each other forming hydrogen bonds. These hydrogen bonds served as additional crosslinks there by increasing the modulus of the hydrogels. EVS on the other hand was incapable of interactions due to the lack of hydrophilic end groups. We were successfully able to create protein hydrogels and control the swelling and mechanical properties by varying the amount of substituted group. The final objective of the study was to create and characterize microstructures from substituted alanine and lysine. Alanine and lysine were substituted with different concentrations of EVS. Bars and fibers were observed for alanine at moderate substitutions while at higher concentrations random structures were observed using scanning electron microscopy (SEM). Lysine formed tubes at moderate EVS substitutions and rosettes at high concentrations of EVS as evidenced by SEM. FT-IR results suggested that instead of carbonyl one of sulfonyl bonded to the available amine in modified amino acids. And only in this case fibers, tubes and rosettes were observed. X-ray diffraction (XRD) results supported this observation. Using these results we hypothesized that the self assembled structures very much depended on the amount of EVS present in the substituted product and sulfonyl forming β-sheet analogs with amine. / Ph. D.