Gene gun assisted micro-particle delivery system is an excellent method for the delivery of DNA into target tissue so as to carry out gene transfection in the target cells. The gene gun is primarily a particle accelerator which accelerates DNA-coated micro-particles to sufficient velocities to breach the target layer enabling the micro-particles to penetrate to a desired depth and target the cells of interest to achieve gene transfer. However, an inevitable problem in this process is the tissue/cell damage due to the impaction of the pressurized gas and micro-particles on the target. The purpose of this research is developing a new conceptual system which improves the penetration depth of micro-particles at less imposed pressure and particle injection velocity. This is achieved by applying a microneedle array and ground slide in the gene gun system, thus a study involving microneedle assisted micro-particle delivery is conducted in this work. Microneedle array is used to create holes in the target which allows a number of micro-particles to penetrate through the skin which enhances the penetration depth inside target. The ground slide is used to load a pellet of the micro-particles and prevent the pressurized gas to avoid the impaction on the target. The operation principle is that the pellet is attached to ground slide which is accelerated to a sufficient velocity by the pressurized gas. The pellet is released from the ground slide which separates into individual micro-particles by a mesh and penetrates to a desired depth inside the target. An experimental rig to study various aspects of microneedle assisted micro-particle delivery is designed in this PhD research. The passage percentage of the micro-particles and size of the separated micro-particles are analysed in relation to the operating pressure, mesh pore size and Polyvinylpyrrolidone (PVP) concentration to verify the applicability of this system for the micro-particle delivery. The results have shown that the passage percentage increases from an increase in the mesh pore size and operating pressure and a decrease in PVP concentration. A mesh pore size of 178 μm and pellet PVP concentration of 40 mg/ml were used for the bulk of the experiments in this study as these seem to provide higher passage percentage and the narrow size distribution of the separated micro-particles. In addition, the velocity of the ground slide is detected by the photoelectric sensor and shown that it increases from an increase in operating pressure and reaches 148 m/s at 6 bar pressure, A further analysis in the penetration depths of the micro-particles to determine whether they achieve enhanced penetration depths inside the target after using microneedles is carried out. A skin mimicked agarose gel is obtained from comparing the viscoelastic properties of various concentration of agarose gel in comparison with the porcine skin, which is assumed to mimic the human skin. These experiments are used to relate the micro-particle penetration depth with the operating pressure, microneedle length and particle size. In addition, a theoretical model is developed based on the experimental data to simulate the microneedle assisted micro-particle delivery which provide further understanding of the microneedle assisted micro-particle delivery. The developed model was used to analyse the penetration depth of micro-particles in relation to the operation pressure, target properties, microneedle length and particle size and density. The modelling results were compared with the experimental results to verify the feasibility of the microneedle assisted micro-particle delivery for micro-particles delivery. As expected, both experimental and theoretical results show that the micro-particles achieve an enhanced penetration depth inside target. The maximum penetration depth of micro-particles is increased from an increase in operating pressure, microneedle length, particle size and density.

Das porzine Parvovirus (PPV), 2013 vom International Committee on taxonomy of Viruses (ICTV) in ungulate Protoparvovirus 1 umbenannt, ist ein unbehülltes, einzelsträngiges DNA Virus und gehört innerhalb der Familie Parvoviridae zur Subfamilie Parvovirinae. Es ist weltweit in allen Bereichen der Schweinehaltung endemisch und verursacht große wirtschaftliche Verluste in den Betrieben (TRUYEN und STRECK 2012). Anders als die verwandten caninen und felinen Parvoviren (seit 2013 arnivore Protoparvovirus 1) ist es nicht durch zum Teil tödlich verlaufende Durchfallerkrankungen, sondern durch Fruchtbarkeitsstörungen wie Abort, Mumifikation und Unfruchtbarkeit, auch bekannt als SMEDI – Syndrom (Stillbirth = Totgeburt, Mummification =Mumifikation, Embryonic Death = embryonaler Tod und Infertility = Unfruchtbarkeit), gekennzeichnet. Die Schwere des Verlaufs hängt dabei wesentlich vom Zeitpunkt sowie von dem, für die Infektion verantwortlichen Isolats ab. Als besonders gefährdet gelten ungeimpfte Jungsauen, die innerhalb der ersten 70 Tage der Trächtigkeit in Kontakt mit dem Virus treten. Das Virus verfügt über eine ausgesprochen hohe Tenazität gegenüber äußeren Einflüssen. Es ist hitzestabil, unempfindlich gegenüber pH-Werten zwischen 3-9 sowie äther- und chloroformresistent (CARTWRIGHT und HUCK 1967, MAYR et al. 1968, JOHNSON und COLLINGS 1969, BACHMANN 1970, MORIMOTO 1972). Einmal im Bestand bleibt es somit über Monate infektiös. Es stehen für die Bekämpfung nur wenige Mittel zur Verfügung. Eine entscheidende Möglichkeit ist die Einhaltung eines strikten Impfregimes, wobei Impfstoffe zum Einsatz kommen, die seit etwa 3 Jahrzehnten auf den gleichen inaktivierten Virus-Isolaten beruhen. In den letzten zehn Jahren wurden zunehmend neue Isolate entdeckt, die sich, wie das hochvirulente Isolat Kresse und das wenig virulente Isolat NADL2, nur in wenigen Aminosäuren unterscheiden. Zum Teil weisen sie aber gravierende Unterschiede in ihrer Pathogenität auf. Daraus ergeben sich neben dem dringenden Rat zur Beobachtung der aktuellen Entwicklung mehrere Fragen hinsichtlich der zukünftigen Handhabung des Virus (SOARES et al. 2003, ZIMMERMANN et al. 2006). So sollte geklärt werden: • wie verbreitet sind diese neuen Isolate • was könnte ihre Entwicklung begünstigt haben • wie effizient ist der Schutz, den herkömmliche Impfstoffe gegen die neuen Isolate bieten • kann eines der Isolate eine Grundlage für einen neuen, effizienteren Impfstoff liefernDiese Dissertation umfasst insgesamt drei Veröffentlichungen, welche versuchen, die gestellten Fragen zu beantworten. Im ersten Artikel wird die Wirksamkeit eines neuen Impfstoffes auf Grundlage des hochvirulenten, vorherrschenden Isolat 27a untersucht. Im zweiten Manuskript wird mit Hilfe von in vitro- und in silico- Modellen die Populationsdynamik demonstriert. Die dritte Veröffentlichung widmet sich der Beschreibung der neuen Parvotypen (PPV2, PPV3 und PPV4), welche aus Herzen und Tonsillen von deutschen, klinisch gesunden Schlachtschweinen isoliert werden konnten.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Master of Science / Department of Grain Science and Industry / Cassandra K. Jones / Animal food manufacturing facilities need to evaluate biological hazards within their facility due to their severity and probability to cause illness or injury in humans or animals. Control of biological hazards, including Salmonella and Porcine Epidemic Diarrhea Virus (PEDV), in animal food manufacturing facilities may require a preventative control to mitigate the risk of the hazard. Thermal processing is an effective point-in-time control, but does not prevent cross-contamination during drying, cooling, and packaging/load-out of animal food. Therefore, it may be appropriate to sanitize surfaces to prevent cross-contamination of animal food during manufacturing. The objective of the first experiment was to evaluate surface decontamination strategies for Porcine Epidemic Diarrhea Virus (PEDV) using chemical disinfectants to reduce viral RNA on various manufacturing surfaces. Concentrated liquid formaldehyde and sodium hypochlorite reduced the quantity of viral PEDV RNA on all tested surfaces. Rubber belting from a bucket elevator retained the most PEDV RNA, while the polyethylene tote bag retained the least. In the second experiment, surface decontamination was evaluated for Salmonella Typhimurium using liquid and dry chemical sanitizers on various manufacturing surfaces. Surfaces treated with concentrated commercial formaldehyde had no detectable Salmonella after treatment, and surfaces treated with medium chain fatty acids (MCFA) had at least a 4-log reduction compared to the control. The dry commercial acidulant, sodium bisulfate, was the most effective dry sanitizer tested, but had limited efficacy depending on surface type. Experiment 3 further tested the application of two chemical sanitizers against Salmonella Enteritidis on residual surface and feed contamination in pilot-scale mixers. Manufacturing sequence, but not treatment impacted feed and surface contamination of Salmonella Enteritidis. Specifically, there was Salmonella-positive residue in the batch of feed manufactured immediately after the positive control batch. However, no Salmonella residue was detected in batches of feed treated with either concentrated commercial essential oil blend or rice hulls treated with 10% MCFA. Low levels of Salmonella residues were observed from feed and surfaces manufactured after Sequence 1, but no residues were observed by Sequence 2. This data suggests that sequencing of feed during manufacturing can reduce Salmonella-positive contamination within animal food and on manufacturing surfaces, particularly after the second batch or with the use of chemical treatments. In summary, liquid sanitizers have been shown to be effective at reducing Salmonella spp. and PEDV contamination on a variety of animal food manufacturing surfaces, but application and practicality may be limited.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233 amino acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs, and compare these responses to pigs diagnosed with porcine circovirus-associated disease (PCVAD). The approach was to react porcine sera with different CP polypeptide fragments that each contained one or more immunoreactive regions. Expression of polypeptides was performed using E.coli. Initial results showed that sera from vaccinated pigs preferentially recognized only the largest CP(43-233) polypeptide fragment and showed low levels of binding to other CP polypeptide fragments. The results of sera from pigs diagnosed with PMWS showed only minimal reactivity with CP polypeptide fragments, including the largest CP(43-233). PCV2 infected or PDNS diagnosed pigs reacted to all CP polypeptides: however, the strongest reactivity was primarily directed towards CP polypeptides containing residues in the 160-180 region. For this purpose, finer mapping studies were performed. These experiments involved reacting sera from experimentally infected PCV2 pigs and PDNS pigs with overlapping oligopeptides that covered amino acids 141-200. Overall, the results showed a subset of experimentally infected pigs and pigs with PDNS preferentially recognized the CP oligopeptide, 169-STIDYFQPNNKR-180. Alanine scanning identified Y-173, F-174, Q-175 and K-179 as important for antibody recognition. The results from this study support the notion of PCV2 modulation of immunity, including antibody responses that may represent a precursor for disease. The results from this study support the notion of PCV2 modulation of immunity. Furthermore, the methods incorporated in this study provide a means for characterizing the immune response upon vaccination, natural infection and disease.

Atualmente, a avaliação das propriedades biomecânicas da córnea vem sendo considerada um parâmetro importante a ser determinado, uma vez que está relacionado a diversos procedimentos (diagnósticos e cirúrgicos) e oftalmopatias. Devido à complexa disposição de suas lamelas, o estroma corneal é considerado a camada que exerce maior influência sobre as propriedades elásticas da córnea. A busca por modelos experimentais no estudo das propriedades biomecânicas da córnea têm aumentado ultimamente, devido à dificuldade em se obter amostras de córnea humana para fins científicos. Logo, estudos comparativos entre a córnea humana e a suína vêm sendo desenvolvidos, e algumas similaridades foram identificadas entre estas duas espécies. O presente estudo tem como objetivo avaliar as propriedades biomecânicas de diferentes regiões da córnea suína por meio da microscopia da força atômica. Dezesseis bulbos oculares não escaldados, de oito animais da espécie suína, foram adquiridos em frigorífico local. Animais de diferentes raças, faixas de peso e idade foram utilizados neste estudo. Bulbos oculares frescos foram submetidos ao debridamento da camada epitelial da córnea, sendo posteriormente imersos em solução de dextran a 25%. Mensurações da paquimetria corneal em regiões central, superior, inferior, nasal e temporal foram realizadas em cada etapa do preparo das amostras. Após 24 horas submersas em solução de dextran, as córneas foram excisadas em fragmentos de aproximadamente 3 x 3 mm, conforme as regiões acima descritas. Tais fragmentos foram submetidos à avaliação pelo microscópio de força atômica, imersos em solução de dextran a 25%. Os valores do módulo de Young para cada fragmento foram obtidos com base no modelo de elasticidade de Hertz. O armazenamento de amostras de córnea em solução de dextran preveniu a hidratação excessiva destas, mantendo a paquimetria dentro dos valores considerados normais. Tanto a paquimetria quanto o módulo de elasticidade corneais não variaram dentre as regiões central, superior, inferior, nasal e temporal da córnea. A espessura e a elasticidade da córnea não diferiram frente à comparação de olhos contralaterais. Devido à facilidade de aquisição e aos resultados obtidos, a córnea suína pode ser empregada como modelo experimental na avaliação das propriedades biomecânicas corneais. / Currently, the assessment of corneal biomechanical properties has been considered an important parameter to be determined, since it is related to several procedures (diagnostic and surgical) and ocular diseases. Due to the complex arrangement of its lamellae, the corneal stroma is considered the layer that exert more influence on the elastic properties of the cornea. The demand for experimental models to study the biomechanical properties of the cornea has recently increased due to the difficulty in obtaining samples of human cornea for scientific purposes. Therefore, comparative studies between human and porcine cornea have been developed, and some similarities were identified between these two species. This study aims to evaluate the biomechanical properties of different regions of the porcine cornea using atomic force microscopy. Sixteen eyes, enucleated from eight animals, were purchased at a local slaughterhouse. Animals of different breeds, age and weight ranges were used in this study. Fresh eyeballs underwent debridement of the corneal epithelial layer, and subsequently immersed in 25% dextran solution. Measurements of corneal pachymetry in the central, superior, inferior, nasal, and temporal regions were performed at each stage of sample preparation. After 24 hours submerged in dextran solution, the corneas were excised into fragments of approximately 3 x 3 mm, according to the regions described above. These fragments were analysed by atomic force microscope immersed in 25% dextran solution. The values of Young modulus for each fragment were obtained from the elasticity model of Hertz. The storage of samples in dextran solution prevented their excessive hydration, keeping the pachymetry values within normal limits. Both corneal thickness and elastic modulus did not vary among the central, superior, inferior, nasal and temporal regions of the cornea. The thickness and elasticity of the cornea did not differ between right and left eyes. Due to the facility of acquisition and the results obtained, porcine cornea can be used as experimental model for assessment of corneal biomechanical properties.

A dexmedetomidina, fármaco da classe dos α2-agonistas, é amplamente empregada no paciente gravemente enfermo por seus efeitos de analgesia e sedação, porém seu comportamento nas situações de sepse e choque séptico é pouco estudado. O objetivo deste estudo foi avaliar os efeitos hemodinâmicos, metabólicos e inflamatórios da dexmedetomidina em modelo suíno de choque séptico. Foram utilizadas 18 fêmeas suínas (peso médio 23,5 kg), divididos em três grupos. O grupo SHAM foi submetido apenas à anestesia. O grupo CH recebeu infusão intravenosa de E. coli O55 (3x109 ufc/mL) de 0,75 ml/kg durante 1 hora. Os animais do grupo CHDEX receberam a mesma infusão de bactérias vivas, associada à infusão de dexmedetomidina 0,7 μg/kg em 10 minutos seguida de 0,5 μg/kg/h até o fim do experimento. Foi considerado T0 o momento do fim da infusão da bactéria, e os animais foram observados por 240 minutos. Os animais receberam tratamento com fluidoterapia e/ou norepinefrina para manutenção da PVC 8 mmHg e PAM 65 mmHg. O modelo proposto mimetizou as alterações presentes na sepse grave e choque séptico, com colapso circulatório, lesão pulmonar aguda e acidose metabólica. Houve tendência de maior depressão cardiovascular em CHDEX (índice cardíaco em T240: 2,8±0,5 L.min-1.m-2 para CHDEX; 3,6±1,7 L.min-1.m-2 para CH e 4,7±1,1 L.min-1.m-2 para SHAM). Não houve diferença estatística entre os grupos CH e CHDEX, à exceção da SvO2 (62,5±9,0% para CHDEX, 74,2±9,1% para CH em T180), do consumo de oxigênio (149,90±25,62 mL.min-1.m-2 para CHDEX, 111,49±21,59 mL.min-1.m-2 para CH em T0), da taxa de extração de oxigênio (43±20% para CHDEX, 25±11% para CH em T240) e do Pr-Pa (53±14mmHg para CHDEX, 35±11mmHg para CH em T0). A diminuição no IC justifica a diminuição dos parâmetros de oxigenação. Os níveis plasmáticos de TNF-α, IL-1β, IL-6 e IL-10 aumentaram de forma mais pronunciada em CH, mas não houve diferença estatisticamente significativa com relação a CHDEX. Ambos os grupos não diferiram em relação ao débito urinário e tratamento de ressuscitação empregado. Frente aos resultados obtidos pode-se pressupor que a dexmedetomidina pode desencadear desequilíbrio entre oferta e consumo de oxigênio por afetar diretamente a microcirculação. Sugere-se que o emprego da dexmedetomidina no choque séptico seja alvo de mais estudos e discussões. / The use of dexmedetomidine to achieve sedation, anxiolysis, analgesia and allowance to mechanical ventilation has increased in critical ill patients, but only a few data are available regarding the effects of dexmedetomidine in situations of sepsis and septic shock. The aim of this study was to assess hemodynamic, metabolic and inflammatory effects of dexmedetomidine in a porcine model of septic shock. Eighteen sows with mean weight of 23,5 kg were included in the study, and allocated in three groups. Animals in the SHAM group underwent a standard anesthetic protocol. The CH group, in addition to anesthesia, received an intravenous infusion of live E. coli O55 (3x109 cfu/mL), total volume of 0,75 ml/kg in 1 hour. The CHDEX group underwent the same treatment of the CH group plus 0,7 μg/kg of dexmedetomidine over 10 minutes and a constant rate of infusion of 0,5 μg/kg/h until the end of the experiment. T0 was considered the end of bacteria infusion and animals were monitored during 240 minutes. Fluid therapy and/or norepinephrine infusion were given as needed, aiming to maintain central venous pressure 8 mmHg and mean arterial pressure 65 mmHg. Typical septic shock symptoms were shown in animals receiving bacteria infusion, such as cardiovascular collapse, acute lung injury and metabolic acidosis. It was observed a greater cardiovascular depression in the CHDEX group (T240 cardiac index: 2,8±0,5 L.min-1.m-2 within CHDEX; 3,6±1,7 L.min-1.m-2 within CH e 4,7±1,1 L.min-1.m-2 within SHAM). There was not statistical difference between CH and CHDEX, but concerning SvO2 (62,5±9,0% within CHDEX, 74,2±9,1% within CH: moment T180) oxygen consumption (149,90±25,62 mL.min-1.m-2 within CHDEX, 111,49±21,59 mL.min-1.m-2 within CH: moment T0), oxygen extraction rate (43±20% within CHDEX, 25±11% within CH: moment T240) and Pr-Pa (53±14mmHg within CHDEX, 35±11mmHg within CH: moment T0). Impaired cardiac index justified changes in oxygenation parameters. TNF-α, IL-1β, IL-6 and IL-10 plasma levels were increased in a more pronounced way in the CH group, although no statistical differences were found when compared with the CHDEX group. Both groups presented similar results concerning urine output, fluid therapy and norepinephrine additional treatments. Dexmedetomidine is likely to cause a mismatch between oxygen delivery and consumption by affecting microcirculation in critical ill patients. Further investigations on the use of dexmedetomidina in septic shock are required.

O Circovírus suíno tipo 2 (PCV-2) pertence ao gênero Circovirus da família Circoviridae. É considerado “vírus emergente", tendo sido associado, principalmente, à Síndrome de Refugagem Multissistêmica dos suínos (SRM). O genoma circular é composto por: (i) ORF-1 que codifica a proteína replicativa; (ii) ORF-2 que codifica a proteína estrutural formadora do capsídeo, (iii) região não codificadora que intercala as ORF-1 e 2 e contem a origem da replicação, denominada IGS-1 e (iv) região que intercala as ORF-1 e 2, denominada IGS-2. Oito amostras, denominadas amostras completas, tiveram mais de 1705 nucleotídeos seqüenciados (945 da ORF-1, 699 da ORF-2, 20 da IGS-1 e 39 da IGS-2); duas amostras tiveram em média 1692 nucleotídeos seqüenciados (945 da ORF-1 e 699 da ORF-2, os restantes correspondem às regiões IGS-1 e 2); uma amostra teve 1392 nucleotídeos seqüenciados (945 da ORF-1, 414 da ORF-2, 9 da IGS-1 e 24 da IGS-2) e nove amostras tiveram em média 970 nucleotídeos seqüenciados (196 da ORF-1 e 699 da ORF-2, os restantes correspondem às regiões IGS-1 e 2). Portanto, a partir dessas 20 amostras em estudo, foram obtidas: (i) oito amostras completas; (ii) 11 seqüências completas de ORF-1 e (iii) 19 seqüências completas de ORF-2, as quais foram analisadas. A identidade de nucleotídeo variou de: (i) 99,7% a 100% entre as oito amostras completas; (ii) 99,3% a 100% entre as 11 seqüências completas de ORF-1 e (iii) 91,9% a 100% entre as 19 seqüências completas de ORF-2. A topologia das árvores genealógicas utilizando as oito seqüências completas e as 11 seqüências completas de ORF-1 foi similar, agrupando todas as amostras em estudo em um só grupo denominado subtipo PCV-2a. Pela análise da genealogia da ORF-1 observou-se que todas as amostras agruparam-se com uma amostra de PCV-2 associada à Síndrome de Dermatite e Nefropatia suína (PDNS), formando um grupo separado das amostras de PCV-2 associadas à Síndrome de Refugagem Multissistêmicas dos suínos (SRM) e abortamento. A genealogia proposta para as 19 amostras que tiveram a ORF-2 seqüenciada, dividiu as amostras em dois grupos, sendo que 14 amostras agruparam-se num grupo denominado subtipo PCV-2a e 5 no subtipo PCV-2b. Os resultados mostraram a circulação de, pelo menos, dois subtipos de PCV-2 no Brasil / Porcine circovirus 2 belongs to Circovirus genus and Circoviridae family. It is considered an “emerging virus" being associated to Postweaning Multisystemic Wasting Syndrome (PMWS). The circular viral genome is formed by: (i) ORF-1 coding for the replicative associated proteins; (ii) ORF-2 encoding the structural proteins of the viral capsid; (iii) a non-coding intergenic sequence between ORF-1 and ORF-2 containing the replicative origin of the viral genome (IGS-1) and (iv) a second non-coding intergenic sequence between ORF-1 and ORF-2 (IGS-2). Eight samples, named complete sequences, had about 1705 nucleotides determined (945 from ORF-1, 699 from ORF-2, 20 from IGS-1 and 39 from IGS-2); two samples had about 1692 nucleotides sequenced (945 from ORF-1, 699 from ORF-2, and the rest from IGS-1 and 2); one sample had 1392 nucleotides sequenced (945 from ORF-1, 414 from ORF-2, 9 from IGS-1 and 24 from IGS-2) and nine samples had about 970 nucleotides sequenced (196 from ORF-1, 699 from ORF-2, and the rest from IGS-1 and 2). Therefore, from the 20 samples it was obtained: (i) eight complete sequences; (ii) 11 complete sequences of ORF-1 and (iii) 19 complete sequences of ORF-2. The identity at nucleotide level was from: (i) 99,7% to 100% among the eight complete sequences; (ii) 99,3% to 100% among the 11 sequences of ORF-1 and (iii) 91,9 to 100% among the 19 sequences of ORF-2. The topology of the tree generated by the ORF-1 analysis revealed a cluster formed by the Brazilian samples of PCV-2 and the samples associated to PDNS and another cluster formed by the PCV-2 associated to other syndromes. The genealogy presented for the 19 samples using the data from ORF-2 revealed the presence of two clusters, one of them formed by 14 samples named subtype PCV-2a and the other formed by 5 samples, named PCV-2b. The results demonstrated that, at least, two subtypes of PCV-2 circulate in Brazil

A aprotinina, um inibidor de serinoproteinase ácido resistente de massa molar de 7 kDa, é utilizada como insumo ou medicamento. O objetivo principal deste trabalho foi purificar a aprotinina a partir de pulmão suíno. Três procedimentos foram utilizados. O primeiro procedimento utilizou a coluna de tripsina-agarose, o segundo procedimento utilizou a filtração tangencial e coluna de tripsina-Sepharose. O terceiro procedimento utilizou três cromatografias: filtração em gel, troca-iônica e afinidade (tripsina-agarose). A aprotinina suína foi purificada de pulmão utilizando o terceiro procedimento. A seqüência parcial do gene da aprotinina suína apresentou 74% de identidade com a seqüência do gene da aprotinina bovina. Outros dois inibidores de serinoproteinases ácido resistentes foram purificados, são eles: o fragmento ativo do segundo domínio do inibidor de leucoprotease secretada (SLPI), e um segundo inibidor de alta massa molecular, provavelmente bikunina. O protocolo de purificação utilizado neste trabalho recuperou 85mg de aprotinina suína por kg de pulmão. / Aprotinin, an acid stable serine proteinase inhibitor with a molecular mass of 7 kDa, is used as a reagent or drug. The purification of the aprotinin from porcine lungs was the main objective of this work. Three procedures were used. The first one utilized the trypsin-agarose column. The tangential ultra filtration and trypsin-Sepharose column were used in the second procedure. And finally, the gel filtration, ion-exchange and affinity chromatography were employed in the third procedure. The porcine lung aprotinin was purified using the third procedure. The partial sequence of the aprotinin gene was obtained and showed 74% of the identity with the aprotinin bovine gene sequence. Another two acid stable serine proteinase inhibitors were purified: the active fragment of the secretory leukoprotease inhibitor second domain, and one high molecular mass inhibitor, probably bikunina. The purification protocol used in this work recovered 85mg of the porcine aprotinin from kg of lung.

A potencialidade do uso de uma matriz de colágeno e de um creme preparado pela mistura de um creme hidratante comercial com um gel de colágeno (90:10 m/m) foi avaliada no processo de cicatrização de feridas planas produzidas em pele de ratos. A matriz acelular de colágeno e o gel de colágeno foram obtidos a partir de serosa porcina por meio de um tratamento alcalino que não altera a estrutura do colágeno nativo e remove componentes celulares. Este estudo fez um comparativo macroscópico e histológico do processo cicatricial das feridas planas tratadas com soro fisiológico ou creme comercial (controles) e as tratadas com a sutura de uma membrana de colágeno (matriz) ou creme com colágeno. As feridas foram produzidas pela retirada de um flap de pele de 20 'MM POT.2' e receberam curativos diários. O material para histologia foi coletado nos 3º, 5º, 7º e 9º dias pós-cirurgia. Apesar de não ter havido uma acentuada diferença na cicatrização das feridas planas dos dois grupos de controle e no grupo que recebeu tratamento do creme contendo colágeno, a presença deste no creme indicou uma pequena diferença no grau de colageinização, o que demonstra serem válidas mais investigações nesta direção, buscando uma melhor proporção creme:gel e/ou diferentes concentrações para o gel de colágeno. A membrana demonstrou ser uma ótima opção para o reparo de lesões por ser de fácil obtenção e armazenamento, ter baixo custo e ser excelente para manuseio (maleável e resistente), além de atender às principais exigências mencionadas na literatura para qualquer curativo biológico oclusivo / The potentiality of the use of a collagen based matrix and of a cream prepared by the mixture of a commercial cream plus collagen (90:10 w/w) were evaluated in the healing process of rats’ skin. The acellular collagen matrix and the collagen gel were obtained by an alkaline treatment of porcine serosa which does not damage the native collagen structure and removes cellular components. This study compared by macroscopy analysis and histology the skin healing repair of wounds treated with physiological solution or commercial cream (control groups) and those treated with collagen based matrix suture or commercial cream plus collagen mix. The wounds were made by removing a skin flap with 20 'MM POT.2' and have received treatment every day. The material for histology was retired on the 3rd, 5th, 7th and 9th days after surgery. Even without an accentuated difference in the healing process of both control groups and the wounds treated with the commercial cream plus collagen, its presence in the cream showed a small difference of the collagen level in the new skin what validate more investigations on this way, searching better cream:gel proportion and/or different gel concentration. The matrix demonstrated to be a very good option to help wound healing because it is easily shelf able and obtainable, it has cheap cost and it is extremely nice to handle (resistant and manipulation able), besides to follow the main requirements present in the literature citation for any biologic occlusive dressing