Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-11T18:57:33Z No. of bitstreams: 2 Dissertação - Lorena Lopes Ferreira - 2013.pdf: 653277 bytes, checksum: 4c9aa7d38ca40c4e856a2bcb3c40a6f2 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-11T20:01:36Z (GMT) No. of bitstreams: 2 Dissertação - Lorena Lopes Ferreira - 2013.pdf: 653277 bytes, checksum: 4c9aa7d38ca40c4e856a2bcb3c40a6f2 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-11T20:01:36Z (GMT). No. of bitstreams: 2 Dissertação - Lorena Lopes Ferreira - 2013.pdf: 653277 bytes, checksum: 4c9aa7d38ca40c4e856a2bcb3c40a6f2 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-09-20 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Ticks are external parasites distributed in the world. Its parasitism causes direct and indirect damage to their hosts. Rhipicephalus microplus is the principal ectoparasite of the livestock causing economic and sanitary implications in the tropics regions. Control strategies are still a challenge and new alternatives are being sought to avoid the use of chemical methods that are reprehensible in relation to animal and human health, and also the environment. It is known that Bos indicus are more resistant to ticks than Bos taurus, while their crosses have a moderate resistance. The olfactory and gustatory senses allow these parasites to seek hosts, mating partners and distinguish between food and harmful substances. The receptors of these sensory organs are present throughout the body and appendages of arthropods, being pore sensilla in the chelicerae important when it comes to ticks taste receptors. Electrophysiology assists in the discovery of active principles that attract or repel these parasites and this will be proven after a behavioral test. This work was performed in order to characterize R. microplus electrophysiological responses to phagostimulants in different concentrations like salts (NaCl and KCl, 10-3 a 10-1M), glucose (10-4 a 10-1M), adenosine triphosphate (ATP, 10-6 a 10-2M), reduced glutathione (GSH, 10-6 a 10-2M), a mixture of substances (MIX, glucose1M+ATP10- 2M+GSH10-3M) and sera of susceptible and resistant cattle to ticks. A single cell electrophysiology technique was used in the taste sensila of R. microplus. Engorged nymphs of R. microplus were collected from naturally infested bovines. Females ticks were fed on rabbits for a period of three to five days. The females were removed and taken directly to the electrophysiology room in order to be prepared for the test. At least 10 females were tested per substance. The shape, frequency and amplitude of spikes were analyzed with the program Autospike. The statistical analysis was performed with the software R applying ANOVA followed by Tukey HSD post-hoc (5%). In relation to the phagostimulants it was observed that the taste receptors of the chelicerae were responsive for all substances tested, having a more intense responses at higher concentrations. The frequency of responses to salts ranged from 11.09 ± 0.98 to 28.50 ± 3.49 spikes/s; to glucose from 25.40 ± 2.33 to 37.15 ± 4.47 spikes/s, to ATP from 17.91 ± 1.28 to 37.30 ± 4.77 spikes/s; to GSH from 19.10 ± 2.84 to 56.6 ± 5.08 spikes/s and the MIX 50.60 ± 4.42 spikes/s. The responses were uni or multicellular depending on the concentration. Sera from resistant cattle xiii triggered a higher frequency of spikes/s (average of 53.48 ± 1.89 spikes/s, ranging from 48.45 ± 4.28 to 61.05 ± 5.14 spikes/s) when compared with the susceptible cattle (average of 40.33 ± 1.63 spikes/s, ranging from 33.30 ± 3.02 to 49.05 ± 5.20 spikes/s). The records analyzed showed that R. microplus was more stimulated by sera from resistant cattle than those from susceptible animals. It is known that in the serum has phagostimulants which probably triggered spikes in both groups. On the other hand, the greater number of spikes to resistant bovine serum might be due to the presence of deterrents compounds. Both sera showed a multicellular response. It was also observed that there were variations in the spikes frequency between animals of the same group according to the characteristic of spikes recorded. It was concluded that R. microplus respond in a dose dependent pattern to the phagostimulants tested. And also that, this tick was able differentiated the sera of susceptible to sera of resistant bovines. / Carrapatos são ectoparasitas obrigatoriamente hematófagos de diversas espécies e estão distribuídos mundialmente. Seu parasitismo acarreta danos diretos e indiretos a seus hospedeiros. Rhipicephalus microplus é o principal ectoparasita da pecuária causando problemas econômicos e sanitários nas regiões tropicais e subtropicais. O seu controle ainda é um desafio e novas alternativas estão sendo buscadas para evitar o uso de métodos químicos que são condenáveis em relação à saúde animal, humana e também ao meio ambiente. Sabe-se que os bovinos Bos indicus são mais resistentes que os Bos taurus, enquanto que seus cruzamentos possuem uma resistência moderada. A olfação e gustação permitem que esses parasitas busquem por hospedeiros, parceiros para cópula e distingam entre alimentos e substâncias nocivas a eles. Há receptores desses sentidos distribuídos por todo o corpo e apêndices dos artrópodes, sendo as porossensilas das quelíceras dos carrapatos importantes quando se refere a receptores gustativos. A eletrofisiologia auxilia na descoberta de compostos químicos que atraem ou repelem esses parasitos. Este trabalho foi feito com intuito de caracterizar as respostas eletrofisiológicas das porossensilas de R. microplus frente a alguns fagoestimulantes em diferentes concentrações como sais (NaCl e KCl, 10-3 a 10-1M), glicose (10-4 a 10-1M), adenosina trifosfato (ATP, 10-6 a 10-2M), glutationa reduzida (GSH, 10-6 a 10-2M) e uma mistura de substâncias (MIX, Glicose1M + ATP10-2M + GSH10-3M). Objetivou ainda avaliar as respostas eletrofisiológicas para soros de bovinos sensíveis (Girolando) e resistentes (Nelore) ao carrapato. Para isso foi utilizada a técnica de registro em sensila única nas porossensilas de fêmeas de R. microplus. Fêmeas alimentadas em coelhos por três a cinco dias foram usadas. Foram testadas no mínimo dez fêmeas por concentração e substância. Após o registro, analisou-se a forma, frequência e amplitude de spikes com o auxílio do programa Autospike. A análise estatística foi feita com o software R aplicando ANOVA seguido de Teste de Tukey com nível mínimo de significância de 5%. As porossensilas foram responsivas a todos os fagoestimulantes testados, mostrando resposta mais intensa nas maiores concentrações. A frequência de spikes/s para os sais variou de 11,09 ± 0,98 a 28,50 ± 3,49; para glicose 25,40 ± 2,33 a 37,15 ± 4,47; para o ATP de 17,91 ± 1,28 a 37,30 ± 4,77; para o GSH de 19,10 ± 2,84 a 56,6 ± 5,08 e o MIX de 50,60 ± 4,42. As respostas foram uni ou multicelulares. Os soros dos bovinos resistentes xi desencadearam uma maior frequência de spikes/s (média de 53,48 ± 1,89 spikes/s, variando 48,45 ± 4,28 a 61,05 ± 5,14 spikes/s) quando comparado com o grupo dos soros dos bovinos sensíveis (média de 40,33 ± 1,63 spikes /s, variando de 33,30 ± 3,02 a 49,05 ± 5,20 spikes/s). Os registros analisados mostraram que as porossensilas de R. microplus foram mais estimuladas pelo soro de animais resistentes do que aqueles de animais sensíveis. A maior frequência de spikes desencadeada pelos soros de bovinos resistentes pode ter ocorrido pela presença de compostos deterrentes além dos fagoestimulantes existentes no soro. Para ambos os soros foi observada uma resposta multicelular. Observou-se também que dentro de um mesmo grupo houve variações em relação à frequência dos spikes, que foi atribuído à resistência dos animais. Concluiu-se que as porossensilas de R. microplus responderam de forma dose dependente aos fagoestimulantes testados e também apresentaram uma maior atividade para soros de bovinos resistentes do que soros de bovinos sensíveis.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-06-06T12:34:21Z No. of bitstreams: 1 2010 - Alessandra Medeiros Ribeiro .pdf: 4846590 bytes, checksum: 525d2754e1be01d1117fe6e9f3362d1f (MD5) / Made available in DSpace on 2017-06-06T12:34:21Z (GMT). No. of bitstreams: 1 2010 - Alessandra Medeiros Ribeiro .pdf: 4846590 bytes, checksum: 525d2754e1be01d1117fe6e9f3362d1f (MD5) Previous issue date: 2010-03-19 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior, CAPES, Brasil. / Spectroscopic studies for several comercial flavonoids (flavone (FVA), alphanaphthoflavone (?-NAF), beta-naphthoflavone (?-NAF), thioflavone (TFA), S,Sdioxythioflavone (SDF), flavanone (FNA) and quercetin (QUE)), natural flavonoids (biflavonoids such as agatisflavone (ATF), 7?-O-methylagatisflavone (OMA), amentoflavone (AMF) and (DOF)) and thiochromanone (TCR) were performed in different solvents (acetonitrile (ACN), ethanol (ETOH), cyclohexane (CEX), dichloromethane (DCM) and milli-Q water (AD)). Irradiation of TFA, SDF and TCR in acetonitrile, employing the nanosecond laser flash photolysis, lead to the formation of their corresponding triplet excited state. Fluorescence emission spectroscopy studies showed that commercial and natural flavonoids and thiochromanone are not fluorescent. UV/visible spectroscopy studies for QUE, ATF, OMA, AMF and DOF, in the same previous solvents, revealed that for these flavonoids the ground-state absorption spectrum in polar solvents, such as water or PBS (pH=7.4), is completely different than the obtained in dichloromethane. This difference is more pronounced for ATF. For DOF the absorption spectrum in water shows remarkable variations when compared to that in PBS. The interaction between BSA and the flavonoids QUE, ATF, OMA, AMF and DOF in PBS solution, pH = 7.4, was studied by UV/visible spectroscopy, fluorescence emission spectroscopy, circular dicroism and molecular modelling. From these studies it was clearly demonstrated that the interaction observed was directly dependent on the flavonoid concentration and almost independent on temperature variation. The ground state absorption spectrum for BSA showed a hypsochromic effect on the absorption band around 208 nm, corresponding to the n?* transition of the BSA ?-helix structure, as a function of flavonoid concentration. Similar behavior was observed for the absorption at 280 nm, corresponding to the tryptophan absorption in BSA. The fluorescence emission spectrum for BSA in the presence of QUE, ATF, OMA, AMF and DOF, in PBS, at T = 22?C, 27?C, 32?C, 37?C and 42?C, shows a blue-shift on the protein emission as a function of flavonoid concentration. These results suggest that the BSA chromophore is in a more hydrophobic environment when compared with that sensed by the protein in the absence of the flavonoid. In this case, quenching of BSA fluorescence (tryptophan residues) was clearly observed with the high values obtained for the quenching rate constant kq (? 1013 to 1014 L/mol.s) indicating a static quenching process. The distance (r) observed for the tryptophan residues and the flavonoids was smaller than 7 nm, which indicates that there is a reasonable probability for a non-radiative energy transfer process between tryptophan and the flavonoids, based on the F?rster theory for energy transfer. Circular dicroism results at T = 25?C, 37?C and 42?C revealed a significant decrease on the ?-helix percentage for BSA at 208 nm and 222 nm, corresponding to the n?* transition for the secondary structure of BSA, as a function of flavonoid concentration. These effects can be attributed to the formation of a complex BSA/flavonoid which can induce conformational variations on the BSA structure. Molecular modelling indicates that the main regions for the interaction between flavonoids and ASB are located in hydrophobic cavities on the sub-domains IB and IIA, which contain tryptophan residues (Trp-158 and Trp-237). A large hydrophobic cavity containing the Trp-237 is present in the sub-domain IIA, which is responsible for the formation of the complex flavonoid-BSA through a strong interaction flavonoid-tryptophan. / Estudos espectrosc?picos para diversos flavon?ides comerciais (flavona (FVA), alfanaftoflavona (?-NAF), beta-naftoflavona (?-NAF), tioflavona (TFA), S,S-di?xidotioflavona (SDF), flavanona (FNA) e quercetina (QUE)), flavon?ides naturais (biflavon?ides como agatisflavona (ATF), 7?-O-metilagatisflavona (OMA), amentoflavona (AMF) e diidroochnaflavona (DOF)) e tiocromanona (TCR), foram realizados em diferentes solventes (acetonitrila (ACN), etanol (ETOH), cicloexano (CEX), diclorometano (DCM) e ?gua millliQ (AD)). A irradia??o de TFA, SDF e TCR, em acetonitrila, por fot?lise por pulso de laser de nanossegundo, levou ? forma??o de seus respectivos estados excitados triplete. Por espectroscopia de fluoresc?ncia, verificou-se que os flavon?ides comerciais e naturais, e a tiocromanona n?o apresentam emiss?o de fluoresc?ncia. Por espectroscopia de absor??o no ultravioleta/vis?vel (UV-Vis) para QUE, ATF, OMA, AMF e DOF, nestes solventes, percebeu-se que os espectros em presen?a de solventes polares, como AD, foram bem diferentes dos espectros em DCM, principalmente, para ATF, e os espectros em solu??o de tamp?o PBS (pH = 7,4) foram semelhantes aos em AD, exceto para DOF, apresentando mudan?as substanciais. A intera??o entre ASB e os flavon?ides (QUE, ATF, OMA, AMF e DOF) em solu??o tamponada (PBS, pH = 7,4) foi estudada por espectroscopia no ultravioleta/vis?vel, espectroscopia de emiss?o de fluoresc?ncia, dicro?smo circular e modelagem molecular sendo diretamente dependente da concentra??o adicionada de flavon?ides e muito pouco dependente com a varia??o da temperatura. No UV-Vis ocorreu deslocamento para o azul das bandas de absor??o pr?ximas a 208 nm (correspondente a ASB, referente ?s transi??es n?* da estrutura ?-h?lice da albumina) e 280 nm (correspondente ao triptofano da ASB), em fun??o do aumento de concentra??o dos flavon?ides. Na espectroscopia de fluoresc?ncia (T = 22?C, 27?C, 32?C, 37?C e 42?C) houve deslocamento para o azul na emiss?o da prote?na com o aumento da concentra??o dos flavon?ides, sugerindo que o crom?foro da ASB est? em um ambiente mais hidrof?bico em rela??o ?quele quando para ASB livre. Neste caso, observou-se supress?o da fluoresc?ncia de ASB (res?duos de triptofano), como consequ?ncia de um processo de supress?o est?tica como demonstrado pelos altos valores observados para kq (? 1013 a 1014 L/mol.s). A dist?ncia entre os res?duos de triptofano e os flavon?ides (r) foi menor que 7 nm, um indicativo da grande probabilidade de ocorrer transfer?ncia de energia entre ASB e flavon?ides, de acordo com a teoria de transfer?ncia de energia n?o-radiativa de F?rster (Teoria de F?rster). No dicro?smo circular (T = 25?C, 37?C e 42?C) foi verificada uma diminui??o do % de ?-h?lice da ASB em 208 nm e 222 nm (regi?es de transi??o n?* da estrutura secund?ria ?-h?lice da ASB no espectro de absor??o UV), devido ao aumento de concentra??o dos flavon?ides. Esses efeitos podem ser atribu?dos ? forma??o de um complexo flavon?ide-ASB que pode estar induzindo varia??es conformacionais na ASB. Por modelagem molecular, atrav?s do programa docking, percebeuse que as regi?es principais para a liga??o dos flavon?ides com os s?tios de liga??o da ASB est?o localizadas em cavidades hidrof?bicas nos subdom?nios IB e IIA (consistentes com os s?tios I e II) e os res?duos de triptofano (Trp-158 e Trp-237) de ASB est?o nesses subdom?nios, respectivamente. Existe uma grande cavidade hidrof?bica presente no subdom?nio IIA, onde os flavon?ides podem se ligar com o res?duo de triptofano Trp-237 (melhor s?tio de liga??o), formando o complexo flavon?ide-ASB.

The emergence of microneedle (MN) technologies offers a route for a pain free, straightforward and efficient way of transdermal drug delivery, but technological barriers still exist which pose significant challenges for manufacture of MN systems with high volume outputs at low cost. The main aim of this research was to develop new ways for MN manufacture primarily using micro-injection moulding processes with high performance engineering thermoplastics. During the moulding process these polymeric melts will be subjected to extreme stress and temperature gradients and detailed material characterisation combined with in-line monitoring is desirable to optimise the moulding parameters and will help in achieving sharp microneedles with acceptable quality. Hence high shear rheology of these selected materials was performed at wall shear rates carried out in excess of 107 s-1 over a range of temperatures to predict the flow behaviour of polymer melts at such high shear strain rates. This information was fed into injection moulding simulation software tools (Moldflow) to assist the MN production process design. The optimal design was then used to produce a full 3D solid model of the injection mould and mould insert. Furthermore various design of experiments were conducted considering input parameters such as injection pressure, injection speed, melt temperature, filling time and mould cavity temperature. Response variables including product quality and data acquired from the cavity pressure and temperature transducers were used to optimise the manufacturing process. The moulded MNs were geometrically assessed using a range of characterisation techniques such as atomic force microscopy, confocal microscopy and scanning electron microscopy. An attempt to make hollow MNs was performed and encountered many challenges like partial cavity filling and part ejection during processing. Studies were carried out to understand the problem and identified the major problem was in tool design and improvements to the moulding tool design were recommended. Plasma treatment and mechanical abrasion were employed to increase the surface energy of the moulded polymer surfaces with the aim of enhancing protein adsorption. Sample surface structures before and after treatment were studied using AFM and surface energies have been obtained using contact angle measurement and calculated using Owens-Wendt theory. Adsorption performance of bovine serum albumin and release kinetics for each sample set was assessed using a Franz diffusion cell. Results indicate that plasma treatment significantly increases the surface energy and roughness resulting in better adsorption and release of BSA. To assist design-optimisation and to assess performance, a greater understanding of MN penetration behaviour is required. Contact stiffness, failure strength and creep behaviour were measured during compression tests of MN against a steel surface, and in-vitro penetration of MNs into porcine skin. The MN penetration process into porcine skin was imaged using optical coherence tomography. Finally, a finite element model of skin was established to understand the effect of tip geometry on penetration. The output of findings from this research will provide proof of concept level development and understanding of mechanisms of MN penetration and failure, facilitating design improvements for micro-injection moulded polymeric MNs.

Submitted by Samira Ramos (samira.ramos@unifenas.br) on 2018-04-25T21:05:10Z No. of bitstreams: 1 TAIS APARECIDA PATROCINIO.pdf: 726977 bytes, checksum: 8cef7ddcd6970bf3d2ccd912eb038060 (MD5) / Made available in DSpace on 2018-04-25T21:05:10Z (GMT). No. of bitstreams: 1 TAIS APARECIDA PATROCINIO.pdf: 726977 bytes, checksum: 8cef7ddcd6970bf3d2ccd912eb038060 (MD5) Previous issue date: 2017-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG / This study evaluated the effect of AR33 (patent-pending formula), a resveratrol analogue, in the culture of in vitro fertilized embryos. Cumulus-oocyte complexes (COCs) recovered from bovine ovaries collected at the slaughterhouse were matured in vitro for 24 h and fertilized in vitro for 20 h, both at 38.8 °C under 5% CO2 in air and high humidity. Probably partially nude zygotes were randomly distributed in two experiments. Experiment 1: 0 (control, n = 347), 0.1 μM (n = 337), 0.5 μM (n = 277) and 2.5 μM AR33 (n = 343) with 2.5% fetal bovine serum (FBS) and experiment 2: 2.5 μM AR33 (n = 381), 0.5 μM resveratrol (n = 381), both with 2.5% SFB and 0 (control, n = 341) with 10% FBS. The base medium for all treatments was SOFaa and incubation conditions were 38.8 °C under 5% CO2 in air and high humidity. Half of the culture medium was fed on days 3 and 5 after fertilization. The cleavage rate was evaluated on day 3 and the blastocyst rate (B1) on days 7 and 8 post-fertilization. At day 8, the blastocysts were fixed and subsequently submitted to analysis of the number of cells and apoptotic index. Cleavage and blastocyst rates were analyzed by logistic regression models (Proc Logistic), and the number of cells and apoptotic index by mixed linear models (Proc Mixed) using the SAS statistical package. In experiment 1, the cleavage rate (P <0.05) was higher at 2.5 μM (69.0 ± 4.4%) than at 0, 0.1 and 0.5 μM AR33 (62.1 ± 2.0%, 60.7 ± 5.9% and 56.7 ± 5.8%, respectively). At day 7, the Bl rate was similar (P> 0.05) between 0.1, 0.5 and 2.5 μM (18.1 ± 5.4%, 17.5 ± 2.9% and 19.4 ± 3.3%, respectively) and all were higher (P <0.05) 05) at 0 μM AR33 (12.4 ± 2.5%). At day 8, only 0.1 and 2.5 μM (21.0 ± 5.0% and 24.6 ± 3.3%) were higher than 0 μM AR33 (15.2 ± 2.5%). There was no difference (P> 0.05) between treatments regarding total cell number (TC) and internal cell mass (MCI); However, the apoptotic index in CT and MCI was higher for 0 and 2.5 μM (11.36 and 9.89%, 20.52 and 15.85%) than in 0.1 and 0.5 μM AR33 (4.66 and 4.82%, 8.47 and 10.92%). In the experiment 2 the cleavage rate (P <0.05) was higher in the control (80.8 ± 3.4%) than in the treatment with 0.5 μM resveratrol (76.4 ± 3.6%), but similar to 2.5 μM AR33 (76.9 ± 1.2%). There were no differences (P> 0.05) for the Bl rate on days 7 and 8. The apoptotic index in the CT and MCI was higher in the control (8.9 and 14.9%, respectively) than in the 2.5 μM AR33 and 0.5 μM resveratrol (6.4 and 5.5%, 11.1 and 8.8%, respectively). In conclusion, resveratrol and its synthetic analogue tested in this study improve bovine embryonic development in culture medium supplemented with 2.5% FBS under 5% CO2 in air. / Este estudo avaliou o efeito de AR33 (fórmula com patente-pendente), um análogo de resveratrol, no cultivo de embriões fecundados in vitro. Complexos cumulus-oócitos (COCs) recuperados de ovários bovinos coletados no matadouro, foram maturados in vitro durante 24 h e fertilizados in vitro por 20 h, ambos em 38.8 °C sob 5% de CO2 em ar e alta umidade. Prováveis zigotos parcialmente desnudos foram distribuídos aleatoriamente em dois experimentos. Experimento 1: 0 (controle, n=347), 0.1 µM (n=337), 0.5 µM (n=277) e 2.5 µM de AR33 (n=343) com 2,5% de soro fetal bovino (SFB), e experimento 2: 2.5 µM de AR33 (n=381), 0.5 µM de resveratrol (n=381), ambos com 2,5% SFB e 0 (controle, n=341) com 10% SFB. O meio base para todos os tratamentos foi SOFaa e as condições de incubação foram de 38.8 °C sob 5% de CO2 em ar e alta umidade. Metade do meio de cultura foi renovado (feeding) nos dias 3 e 5 após a fertilização. A taxa de clivagem foi avaliada no dia 3 e a taxa de blastocisto (Bl) nos dias 7 e 8 pós-fecundação. No dia 8, os blastocistos foram fixados e posteriormente submetidos a análise do número de células e índice apoptótico. As taxas de clivagem e de blastocistos foram analisadas por modelos de regressão logística (Proc Logistic), e o número de células e índice apoptótico por modelos lineares mistos (Proc Mixed) usando o pacote estatístico SAS. No experimento 1, a taxa de clivagem (P<0.05) foi maior para 2.5 µM (69.0±4.4%) do que para 0, 0.1 e 0.5 µM de AR33 (62.1±2.0%, 60.7±5.9% e 56.7±5.8%, respectivamente). No dia 7, a taxa de Bl foi semelhante (P>0.05) entre 0.1, 0.5 e 2.5 µM (18.1±5.4%, 17.5±2.9% e 19.4±3.3%, respectivamente) e todos eles foram superiores (P<0,05) à 0 µM AR33 (12.4±2.5%). No dia 8, apenas 0.1 e 2.5 µM (21.0±5.0% e 24.6±3.3%) foram maiores do que 0 µM AR33 (15.2±2.5%). Não houve diferença (P>0,05) entre tratamentos quanto ao número de células totais (CT) e da massa celular interna (MCI); contudo, o índice apoptótico nas CT e na MCI foram maiores para 0 e 2.5 µM (11.36 e 9.89%; 20.52 e 15.85%) do que em 0.1 e 0.5 µM AR33 (4.66 e 4.82%; 8.47 e 10.92%). No experimento 2 a taxa de clivagem (P<0,05) foi maior no controle (80.8±3.4%) do que no tratamento com 0.5 µM resveratrol (76.4±3.6%), e este último semelhante à 2.5 µM AR33 (76.9±1.2%). Não houve diferenças (P>0,05) para a taxa de Bl nos dias 7 e 8. O índice apoptótico nas CT e MCI foi maior no controle (8.9 e 14.9%, respectivamente) do que para 2.5 µM AR33 e 0.5 µM resveratrol (6.4 e 5.5%; 11.1 e 8.8%, respectivamente). Em conclusão, o resveratrol e o seu análogo sintético testado neste estudo melhoram o desenvolvimento embrionário bovino em meio de cultura suplementado com 2,5% SFB sob 5% de CO2 em ar.

The Caco-2 cell assay is widely used for in vitro permeability measurements. However, a draw back with the assay that this study will focus on improving, is compound adsorption to the plastic material. Lipophilic compounds such as Cyclosporin A and Peptide J, that will be used in this study, tend to bind to the plastic material in the assay. This can result in poor recovery and misleading permeability predictions. Bovine serum albumin (BSA) is an alternative used today to prevent this but is not always successful.    The aim of this study is therefore to improve the Caco-2 permeability assay by adding phospholipid covered silica beads (PLB) to the basolateral chamber. The role of the PLB is to bind the compound of interest and decrease the amount of compound bound to the plastic material and thus better predict the permeability of the compound of interest.   The PLB was produced using phosphatidylcholine and silica beads. Caco-2 cells were seeded and maintained for 21-29 days ahead of the experiment. PLB concentration of 20, 60 and 100 mg/ml were prepared. Samples were analyzed with HPLC-MSMS. The results showed that with increasing PLB concentration we had a significantly decrease in non-specific plastic binding resulting in reliable permeability predictions, concluding that the hypothesis was correct.

Diploma thesis deals with analysis of coefficient of friction in total hip prosthesis for several materials bearing with presence of bovine serum as substitute of synovial fluid occurred in natural joint. Behavior of coefficient of friction is observed on Mini Traction Machine. Results are plotted at graphs representing dependence of coefficient of friction on time. There are described effects of several kinematic conditions, the influence of used material and the effect of the method of contact lubrication. The experiments analyze how the formation of lubricant film with a layer of adsorbed protein affects coefficient of friction.

No description available.

Background: Mesenchymal stromal cells (MSCs) are multipotent cells that can differentiate into various cell types and possess immunomodulatory and anti-inflammatory effects, making them interesting candidates for therapeutic applications. MSCs are present in small quantities in tissues like bone marrow and therefore need to be expanded while preserving their essential characteristics. They should adhere to plastic, differentiate into osteocytes, adipocytes and chondrocytes and express specific cell surface markers. Currently, the “golden standard” culture media supplement is fetal bovine serum (FBS). However, there is a potential contamination risk of MSCs by xenogeneic and zoonotic infectious agents, which can trigger an immune response. As an alternative, xeno-free serum supplements derived from human sources, e.g., human serum (HS) can be used.  Aim: This study aimed to identify and characterize human bone marrow derived MSCs and examine the effects different supplements have on the cells.  Methods: MSCs were cultured in 10% FBS, 2% FBS and 10% HS for 20 -21 days. Differentiation was induced and the potential was detected with immunocytochemistry. Cell surface markers CD73, CD90, CD105 and CD45 were identified with flow cytometry.  Results and Conclusion: There was no significant difference in morphology, differential potential or immunophenotype between the different serum conditions. However, HS-supplemented culture media resulted in a significantly higher number of cells with 1 x 107 cells after 20 days without affecting their differentiation potential and immunophenotype in comparison to 10% FBS with 2.2 x 106 cells (p=0.0004). MSCs cultured in 2% FBS resulted in the least number of cells (9.9 x 105) after 21 days of expansion.

I characterize the behavior of microcantilever arrays which utilize the in-plane photonic transduction that I've previously developed and evaluate the performance of the microcantilever arrays in simple sensing scenarios with integrated microfluidics. First the thermal responses of microcantilevers with a variety of patterns of deposited gold films are compared. Using a scanning electron microscope, I observe the deflection thermal sensitivities of 300 µm long microcantilevers to be -170.82 nm/K for a full gold coating and -1.93 nm/K for no gold coating. Using the photonic transduction method I measure a thermal sensitivity of -1.46 nm/K for a microcantilever array with no gold. A microcantilever array integrated with microfluidics is exposed to a solution of bovine serum albumin (BSA) followed by solutions of various pH's. In all cases I observe a previously unreported transient deflection response. We find that the transient response is due to temporary nonuniform concentration distributions. In response to nonspecific binding of BSA, I observe a transient surface stress of -0.23 mN/m that agrees well with the -0.225 mN/m predicted by simulations. We hypothesize that the deflection response to pH changes is due to stress generated by conformational changes of bound BSA.The deflection response of an integrated microcantilever array to different types of flow and different flow rates is observed. Simulations of the deflection response match well with experimental results but disagree at higher flow rates. For flow rates greater than 200 µL/min, the limitation of the differential signal's dynamic range becomes apparent. We then investigate flow driven by an on-chip reciprocating reservoir pump. We demonstrate that it is possible to use the reciprocating pump to achieve high flow rates while making deflection measurements in-between reservoir actuations. Investigations of the microcantilever array noise show that flicker noise dominates below 10 Hz, while above 10 Hz, readout noise dominates. A minimum deflection noise density of 15 pW/√Hz is achieved. To improve the signal-to-noise ratio I develop algorithms for a digital lock-in amplifier with a digital phase-lock loop. In simulation the lock-in amplifier is able to improve the SNR by up to a factor of 6000, and self-lock to a noisy carrier signal without an external reference signal.

Natural Killer (NK) cells can recognize and lyse a large variety of tumor cells and have been of interest as a potential cancer treatment option. Our group has developed a particle-based NK cell expansion method that utilizes plasma membrane particles (PM-particles) derived from K562 cells genetically engineered to express membrane bound IL21 and 41BBL(K562-mbIL21-41BBL), two proteins that stimulate growth and activity of NK cells. This method selectively expands highly cytotoxic NK cells > 400-fold in 14 days of culture. Currently NK cells are expanded in vitro using Fetal Bovine Serum (FBS) as a serum-supplement to promote cell growth. While effective, the use of animal products is not preferred in cell cultures grown for clinical purposes. This project tested Human Platelet Lysates (HPL) as a potential replacement for FBS in NK cell culture. NK cells were expanded using PM21-particle based expansion method with either FBS or HPL as supplements. Their growth characteristics, phenotype and functionality were assessed and compared. Results of this study determined that HPL is a viable option to replace FBS in NK cell culture for clinical applications, as there was no significant difference between the two serum supplements.