Dissertations / Theses on the topic 'PIG-1'

The Human Immunodeficiency Virus (HIV) epidemic still poses a problem with approximately 2 million new infections reported worldwide in 2014. New strategies are required to alleviate this burden. Our laboratory has previously shown that crude saliva and purified mucins from cervical plug mucin, saliva and breast milk inhibit HIV-1 infection in vitro. This project investigates purified mucins sourced from pig and horse mucus, as an alternative and abundant source of material for anti-HIV-1 research. Pig gastric and cervico-vaginal mucus was collected and stirred overnight in 6M guanidine hydrochloride with 10mM Na₂HPO4, 10mM EDTA, 1mM PMSF and 5mM NEM. Gastric and cervicovaginal mucus was purified by density gradient ultracentrifugations in CsCl at 105 000g for 48 hours, twice, and mucin rich fractions were separated by size exclusion column chromatography. Mucin-rich materials eluting in the void volume (V₀) were reduced with 10mM dithithreitol (DTT) or subjected to proteolysis with trypsin. Pig saliva was collected in 0.2M NaCl:0.02% sodium azide and horse saliva (due to its viscous nature) was collected and stirred overnight in 6M guanidine hydrochloride with 10mM Na₂HPO4, 10mM EDTA, 1mM PMSF and 5mM NEM. Pig and horse saliva samples underwent size exclusion column chromatography, where the V₀ fractions of both were purified with one density gradient ultracentrifugation and then dialysed and freeze dried, after which aliquots were treated with either DTT or trypsin. At every stage of purification, lyophilized aliquots of all mucin sources were tested on a luciferase based replication defective HIV neutralization assay on a CD4 expressing HeLa cell line. Luciferase expression quantified as relative light units by a luminometer was used to calculate percentage neutralization. Log dose response curves were constructed to extrapolate the half maximal inhibitory concentrations (IC₅₀) on GraphPad Prism. Samples were tested on an MTT cell toxicity assay. Pig gastric and cervicovaginal mucins were added to a simulated vaginal fluid to make gels (at a concentration of 30mg of mucin per ml of buffer). These gels were tested on the neutralization, MTT assays and the pig gastric mucin gel then underwent particle tracking and nanoparticle diffusion assays at varying pH. Pig gastric and cervicovaginal mucin showed good inhibition and low toxicity, with pig gastric mucin V₀ having the best IC₅₀ (1.668μg/ml). Pig and horse saliva showed inhibition but low cell viability. Pig gastric and cervicovaginal mucin gels exhibited good IC₅₀'s but pig gastric mucin had the best neutralization and lowest toxicity (PGM in Gel Solution 4 IC₅₀: 20.23μg/ml). HIV particle tracking and nanoparticle diffusion assays showed that the pig gastric mucin gel inhibited HIV-1 at low pH and existed as a soft gel. This project shows the efficacy of pig gastric mucin to possibly being a component of an anti-HIV-1 vaginal microbicide.

Diabetes can be considered to be one of the main health epidemics of the 21st century. Studies conducted by the World Health Organisation (WHO) indicate that the number of people with diabetes in the year 2000 was 171 million and this is projected to increase to 366 million by 2030 (Wild et al. 2004). The increasing incidence of both Type 1 and Type 2 diabetes is due to population growth, aging, urbanisation, obesity and physical inactivity. The current treatment by insulin injections for individuals with Type 1 diabetes fails to overcome the long term microvascular and macrovascular complications associated with the disease. A major challenge in the treatment of diabetes is to provide patients with an insulin source that is capable of regulating blood glucose levels (BGL) on a minute to minute basis. Advances in medical research have enabled the investigation of a variety of potential alternative therapies that may provide Type 1 diabetic patients with a more superior control of BGL and consequently minimise complications. The utilisation of pancreases obtained from fetal pigs offers potential therapeutic value in the treatment of Type 1 diabetes. Islet-like cell clusters (ICCs) are obtained from such tissue following partial mechanical and enzymatic digestive procedures. ICCs are primarily composed of immature duct cells which, when transplanted, will mainly differentiate into insulin producing ?? cells. Such cells are able to normalise BGL in immunodeficient diabetic recipients and in immunocompetent recipients when anti-rejection drugs are administered. This study investigates microencapsulation as an immunoprotective strategy that has the potential to remove the need for immunosuppression when such cells are transplanted. A review of the literature related to current medical research in the field of diabetes is presented in Chapter 1. In order to achieve the aims of the study, an understanding of how fetal pig ICCs behave when placed within a barium alginate microcapsule both in vitro and in vivo is essential and this data is presented in Chapter 3. This chapter demonstrates that ICCs will survive and differentiate in their typical manner when enclosed within microcapsules and transplanted. Such encapsulated cells will function to normalise BGL when transplanted into diabetic immunodeficent mice for at least 25 weeks and the animals exhibit increased bodyweight. Microcapsules retrieved at this time point were observed to be intact with no breakages or evidence of cellular overgrowth. Transplantation of encapsulated insulin-producing cells into immunocompetent mice are described in Chapter 4. Allotransplantation of a microencapsulated mouse insulin-producing cell line into these diabetic mice also exhibited graft function, resulting in normal BGL in recipients. Large animal experiments are described in Chapter 5. Allotransplantation of microencapsulated fetal pig ICCs into diabetic pig recipients displayed evidence of transient graft function in terms of lower BGL and reduced exogenous insulin requirements. The xenotransplantion of encapsulated fetal pIg ICCs into diabetic immunocompetent mice described in Chapter 4 proved to be more challenging. The transplantation of such cells in this environment did not yield particularly positive results. BGL remained elevated in these recipients and the animals lost bodyweight post transplantation. This area of research warrants further investigation as it is likely that further measures such as transient immunosuppression in combination with microencapsulation will allow fetal pig ICCs to function in a xenograft setting.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Mapeamento de loci de caracaterística quantitativas (QTL) geralmente resultam na detecção de regiões genômicas que explicam parte da variação quantitativa da característica. Entretanto essas regiões são muito amplas e não permitem uma acurada identificação dos genes. Dessa forma, torna-se necessário o estreitamento dos intervalos onde os QTL estão localizados. Com a seleção genômica ampla (GWS), foram desenvolvidas ferramentas estatísticas de forma a se estimar os efeitos de cada marcador. A partir dos valores desses efeitos, pode-se analisar quais são os marcadores de maiores efeitos. Assim, objetivou-se realizar o mapeamento fino dos cromossomos suínos 1, 4, 7, 8, 17, e X, usando marcadores microsatélites e polimorfismo de base única (SNP), em uma população F2 produzida pelo cruzamento de varrões da raça naturalizada brasileira Piau com fêmeas comerciais, associados com características de desempenho, carcaça, orgãos internos, cortes e qualidade de carne. Também objetivou-se estimar os efeitos dos marcadores SNP nas características que tiveram QTL detectados, analisar quais são os mais expressivos e verificar se eles estão localizados dentro do intervalo de confiança do QTL. Os QTL foram identificados por meio do método regressão por intervalo de mapeamento e as análises foram realizadas pelo software GridQTL. O efeito de cada marcador foi estimado pela regressão de LASSO Bayesiano, usando o software R. No total, 32 QTL foram encontrados ao nível cromossômico de significância de 5%, destes, 12 eram significativos ao nível cromossômico de 1% e 7 destes eram significativos ao nível genômico de 5%. Seis de sete QTL apresentaram marcadores de efeito expressivo dentro do intervalo de confiança do QTL. Resultados deste estudo confirmaram QTL de outros trabalhos e identificaram vários outros novos. Os resultados encontrados utilizando marcadores microsatélites junto com SNPs aumentaram a saturação do genoma levando a um menor intervalo de confiança dos QTL encontrados. Os métodos usados foram importantes para estimar os efeitos dos marcadores, e também para localizar aqueles com efeitos mais expressivos dentro do intervalo de confiança do QTL, validando os QTL encontrados pelo método da regressão.
Quantitative Trait Loci (QTL) mapping efforts often result in the detection of genomic regions that explain part of the quantitative trait variation. However, these regions are very large and do not allow accurate gene identification, hence the interval must be narrowed where the QTL was located. With the genome wide selection (GWS), many statistical tools have been developed in order to estimate the effects for each marker. With the marker effects values it is possible to analyze which markers have large effects. Hence, the objective of this investigation was to fine map pig chromosomes 1, 4, 7, 8, 17 and X, using microsatellites and SNP markers, in a F2 population produced by crossing naturalized Brazilian Piau boars with commercial females, associated with performance, carcass, internal organs, cut yields and meat quality traits. A further aim was to estimate the effects of single nucleotide polymorphism (SNP) markers on traits with detected QTL, analyze the most expressive ones and verify whether the markers with larger effects were indeed within the QTL confidence interval. QTL were identified by regression interval mapping using the GridQTL software. Individual marker effects were estimated by Bayesian LASSO regression using the R software. In total, 32 QTL for the studied traits were significant at the 5% chromosome-wide level, including 12 significant QTL at the 1% chromosome-wide level and 7 significant at the 5% genome-wide level. Six out of seven QTL with genome-wide significance had markers of large effect within their confidence interval. These results confirmed some previous QTL and identified numerous novel QTL for the investigated traits. Our results have shown that the use of microsatellites and SNP markers that increase the genome saturation lead to QTL of smaller confidence intervals. The methods used were also valuable to estimate the marker effects and to locate the most expressive markers within the QTL confidence interval, validating those QTL found by the regression method.

Immunological stressors, in the form of clinical and sub-clinical disease are currently controlled using both prophylactic antibiotics in-feed, and therapeutic antibiotic treatment. Respiratory disease, primarily Actinobacillus pleuropneumoniae (App) infection, is recognised as a major factor causing reduced productivity in pigs. This thesis reports investigations into the use of novel immunomodulators in particular Interleukin 4 (IL-4) and Interleukin 1 receptor antagonist (IL-1ra) as alternatives to antibiotics to treat App infection. Immunological and molecular biological assays were used to investigate and accumulate data. An in vitro study undertaken to find potential anti-inflammatory substances, revealed that Interleukin 8 (IL-8) mRNA production stimulated by PMA or LPS in whole pigs' blood was suppressed by IL-4. IL-1ra also suppressed stimulated IL-8 mRNA production by heat killed App bacteria (KB) in vitro. An acute LPS challenge in pigs in vivo however, showed no variation in illness or weight loss between pigs treated prophylactically with anti-inflammatory substance (IL-4 and IL-1ra) and saline treated pigs. The use of plasmids as a delivery system for anti-inflammatory substance did not show promise since it did not enhance growth or prolong the expression of the substances in the pigs. However, in the chronic App challenge model IL-4 and IL-1ra administered prophylactically in vivo showed an ability to improve growth. The therapeutic administration of IL-4 and IL-1ra to App challenged pigs showed no difference in pigs' growth, regardless of the treatment or control administered. To conclude, IL-4 and IL-1ra showed promise when administered prophylactically and improved growth and abrogated disease under conditions of App challenge. However when IL-4 and IL-1ra where administered therapeutically they did not perform as well. Moreover these compounds have potential as a commercial application to reduce the growth reduction caused by disease such as App.

Los objetivos de la tesis fueron el estudio y revisión de los actuales conocimientos sobre el metabolismo y las acciones de IGF-I en la producción porcina y más específicamente la investigación de los efectos que un suplemento alimenticio proveniente de la fermentación de la proteína de patata puede tener sobre los niveles de IGF-I en diferentes estadios productivos, y si estas modificaciones tienen alguna repercusión práctica sobre la producción. En el estudio realizado con las cerdas lactantes, el objetivo fue estudiar si la adición en la dieta de las cerdas lactantes durante 5 días antes del destete y 5 días post destete de la proteína fermentada de patata con o sin glucosa tenía algún efecto sobre los días no productivos entre el destete y su cubrición, sobre la fertilidad y sobre el número de lechones nacidos totales en el subsiguiente parto y si estos parámetros productivos estuvieron relacionados con niveles plasmáticos de IGF-I. Para ello se eligió una granja altamente productiva con un alto status sanitario porque se consideró que era la mejor opción para conocer los efectos de la proteína fermentada de patata sobre los parámetros estudiados. Se usaron un total de 183 cerdas de 3 grupos de cerdas destetadas de forma consecutiva y se distribuyeron en 4 grupos de tratamiento teniendo en cuenta el ciclo de parto, el número de lechones destetados en el ciclo anterior y el número de lechones paridos en el ciclo actual. El tratamiento consistió en la adición de la proteína de patata fermentada, con y sin glucosa, que se consideró como el control positivo y un grupo de cerdas que actuó como control negativo al que no se le suministró ningún producto. Los tratamientos se iniciaron 5 días antes del destete y concluyeron 5 días post destete. Se registraron todos los parámetros productivos para alcanzar los objetivos y se obtuvieron muestras de sangre de un subgrupo de cerdas para determinar el nivel de IGF-I. No se encontraron diferencias significativas en cuanto a los niveles de IGF-I al finalizar los tratamientos ni tampoco se encontraron diferencias significativas en los parámetros productivos estudiados. Un hallazgo no contemplado en el estudio pero encontrado en el análisis fue que las cerdas que fueron nodrizas durante la lactación en la que se inició el tratamiento tenían mayor nivel de IGF-I que las que no lo fueron. Este hallazgo confirma que el nivel de IGF-I depende el status nutricional y metabólico de los animales ya que las cerdas que actuaron como nodrizas tuvieron un gasto metabólico menor que las que no lo fueron y pudieron recuperar mejor su status metabólico. A pesar de ello no existieron diferencias de productividad tras el subsiguiente parto. En el estudio con los lechones lactantes el objetivo fue estudiar el efecto de la administración oral de la proteína fermentada de patata a lechones durante las primeras 12 horas tras el nacimiento sobre los niveles plasmáticos de IGF-I, sobre la mortalidad y sobre la ganancia de peso vivo desde el nacimiento al hasta el destete. Este estudio se realizó en una granja con productividad media, con una sanidad considerada como habitual en la producción porcina y que podía ser catalogada como una granja estándar. Se usaron 542 lechones nacidos en 3 grupos consecutivos de parto. Los lechones fueron clasificados según su peso al nacimiento entre lechones grandes, con 1,2 kg de peso o más al nacimiento y lechones pequeños con menos de 1,2 kg de peso al nacimiento porque se consideró que el peso al nacimiento podría ser una variable que influyera en el estudio. Se distribuyeron en 4 grupos de tratamiento dentro de cada grupo de peso. Los tratamientos consistieron en el suministro oral de la proteína de patata fermentada bien en una sola dosificación (primer grupo) o bien partida en media dosis separadas de un intervalo de 12 h (segundo grupo) suministrando a un tercer grupo de animales glicerol en una sola toma, grupo que se consideró como el control positivo. El cuarto grupo fue el control negativo y los lechones de este grupo no recibieron ningún tratamiento. No se encontraron diferencias significativas de forma global ni dentro de cada grupo de peso en ninguna de las variables estudiadas. Los niveles de IGF-I fueron mayores en los animales del grupo de peso grande cuando se compararon con los lechones pequeños a los 7 días del estudio, pero no al finalizar el mismo. En el estudio con los lechones destetados, el objetivo fue estudiar si la adición en la dieta de lechones de proteína fermentada de patata tras su destete en diferentes proporciones podía reemplazar el uso de plasma porcino y si las modificaciones estaban relacionadas con los niveles plasmáticos de IGF-I. Este estudio se realizó en una granja de destete de lechones. Los lechones provenían de la granja utilizada en el estudio de lechones lactantes. Se realizaron dos estudios consecutivos, el primero para conocer los efectos de la proteína de patata fermentada sobre los lechones y el segundo para conocer el nivel necesario de incorporación a las dietas para substituir al plasma porcino. Se usaron 200 lechones en el primer estudio distribuyéndose homogéneamente según su peso y edad al destete y su sexo. Se distribuyeron en 5 grupos de tratamiento donde un grupo fue el control negativo sin plasma ni proteína de patata fermentada, un grupo como control positivo con plasma en la dieta y 3 dosificaciones crecientes de proteína de patata. En el segundo estudio se usaron 1036 lechones distribuidos homogéneamente según su peso y edad al destete y su sexo. Se distribuyeron en 6 grupos de tratamiento donde un grupo fue el control negativo sin plasma ni proteína de patata fermentada, un grupo como control positivo con plasma en la dieta y 4 grupos con cantidades crecientes de proteína de patata fermentada. Los lechones alimentados con plasma en el primer estudio tuvieron el día 4 del mismo un nivel mayor de IGF-I que el resto siendo los lechones que fueron alimentados con la dosis inferior de proteína de patata fermentada los que tuvieron el nivel de IGF-I inferior teniendo el resto de grupos un nivel intermedio de IGF-I. Los lechones que más pienso consumieron fueron los lechones que consumieron el menor nivel de proteína de patata y los que menos los del grupo control negativo. Estas diferencias de consumo no se vieron reflejadas en un mayor crecimiento diario ni en una mejor conversión alimenticia al final del estudio. Los lechones que consumieron proteína de patata tuvieron mayor peso y ganancia media diaria que los animales del control negativo al finalizar el segundo estudio. En cuanto al consumo, los animales con las 3 dosis más bajas de proteína de patata tuvieron mayor consumo que el resto teniendo una mejor conversión alimenticia los lechones que consumieron bien proteína de patata o plasma que los lechones del control negativo. Los resultados de la presente tesis demuestran que el suministro de un suplemento alimenticio derivado de la fermentación de la proteína de patata i) no mejora la productividad de las cerdas en el subsiguiente parto, ii) no mejora la mortalidad de los lechones durante la lactación ni tampoco su peso a destete y iii) puede reemplazar al plasma animal en las dietas de lechones destetados. El nivel de IGF-I no se ve modificado en ningún estudio al suministrar la proteína de patata fermentada por lo que el modo de acción de este producto debe ser investigado con estudios futuros.
Els objectius de la tesi van ser l'estudi i revisió dels actuals coneixements sobre el metabolisme i les accions d'IGF-I en la producció porcina i més específicament la investigació dels efectes que un suplement alimentari provinent de la fermentació de la proteïna de patata pot tenir sobre els nivells d'IGF-I en diferents estadis productius, i si aquestes modificacions tenen alguna repercussió pràctica sobre la producció. En l'estudi realitzat amb les truges lactants, l'objectiu va ser estudiar si l'addició a la dieta de les truges lactants durant 5 dies abans del deslletament i 5 dies post deslletament de la proteïna fermentada de patata amb o sense glucosa tenia algun efecte sobre els dies no productius entre el deslletament i la cobrició, sobre la fertilitat i sobre el nombre de garrins nascuts totals en el subsegüent part i si aquests paràmetres productius van estar relacionats amb nivells plasmàtics d'IGF-I. Per a això es va triar una granja altament productiva amb un alt estatus sanitari perquè es va considerar que era la millor opció per conèixer els efectes de la proteïna fermentada de patata sobre els paràmetres estudiats. Es van usar un total de 183 truges de 3 grups de truges deslletades de forma consecutiva i es van distribuir en 4 grups de tractament tenint en compte el cicle de part, el nombre de garrins deslletats en el cicle anterior i el nombre de garrins parits en el cicle actual. El tractament va consistir en l'addició de la proteïna de patata fermentada, amb i sense glucosa, que es va considerar com el control positiu i un grup de truges que va actuar com a control negatiu al qual no se li va subministrar cap producte. Els tractaments es van iniciar 5 dies abans del deslletament i van concloure 5 dies post deslletament. Es van registrar tots els paràmetres productius per assolir els objectius i es van obtenir mostres de sang d'un subgrup de truges per determinar el nivell d'IGF-I. No es van trobar diferències significatives pel que fa als nivells d'IGF-I en acabar els tractaments ni tampoc es van trobar diferències significatives en els paràmetres productius estudiats. Un resultat no contemplat en l'estudi però trobat en l'anàlisi va ser que les truges que van ser alletants durant la lactació en què es va iniciar el tractament tenien major nivell d'IGF-I que les que no ho van ser. Aquesta troballa confirma que el nivell d'IGF-I depèn l'estatus nutricional i metabòlic dels animals ja que les truges que van actuar com dides van tenir una despesa metabòlica menor que les que no ho van ser i van poder recuperar millor el seu status metabòlic. Malgrat això no van existir diferències de productivitat després del subsegüent part. En l'estudi amb els garrins lactants l'objectiu va ser estudiar l'efecte de l'administració oral de la proteïna fermentada de patata a garrins durant les primeres 12 hores després del naixement sobre els nivells plasmàtics d'IGF-I, sobre la mortalitat i sobre el guany de pes viu des del naixement al fins al deslletament. Aquest estudi es va realitzar en una granja amb productivitat mitjana, amb una sanitat considerada com habitual en la producció porcina i que podia ser catalogada com una granja estàndard. Es van usar 542 garrins nascuts en 3 grups consecutius de part. Els garrins van ser classificats segons el seu pes al naixement entre garrins grans, amb 1,2 kg de pes o més al naixement i garrins petits amb menys de 1,2 kg de pes al naixement perquè es va considerar que el pes al naixement podria ser una variable que influís en l'estudi. Es van distribuir en 4 grups de tractament dins de cada grup de pes. Els tractaments van consistir en el subministrament oral de la proteïna de patata fermentada bé en una sola dosificació (primer grup) o bé partida en mitja dosi separades d'un interval de 12 h (segon grup) subministrant a un tercer grup d'animals glicerol en una sola presa, grup que es va considerar com el control positiu. El quart grup va ser el control negatiu i els garrins d'aquest grup no van rebre cap tractament. No es van trobar diferències significatives de forma global ni dins de cada grup de pes en cap de les variables estudiades. Els nivells d'IGF-I van ser majors en els animals del grup de pes gran quan es van comparar amb els garrins petits als 7 dies de l'estudi, però no al finalitzar el mateix. En l'estudi amb els garrins deslletats, l'objectiu va ser estudiar si l'addició a la dieta de garrins de proteïna fermentada de patata després de la seva deslletament en diferents proporcions podia reemplaçar l'ús de plasma porcí i si les modificacions estaven relacionades amb els nivells plasmàtics d'IGF-I. Aquest estudi es va realitzar en una granja de transició. Els garrins provenien de la granja utilitzada en l'estudi de garrins lactants. Es van realitzar dos estudis consecutius, el primer per conèixer els efectes de la proteïna de patata fermentada sobre els garrins i el segon per conèixer el nivell necessari d'incorporació a les dietes per substituir al plasma porcí. Es van usar 200 garrins en el primer estudi distribuint homogèniament segons el seu pes i edat al deslletament i el seu sexe. Es van distribuir en 5 grups de tractament on un grup va ser el control negatiu sense plasma ni proteïna de patata fermentada, un grup com a control positiu amb plasma en la dieta i 3 dosificacions creixents de proteïna de patata. En el segon estudi es van usar 1036 garrins distribuïts homogèniament segons el seu pes i edat al deslletament i el seu sexe. Es van distribuir en 6 grups de tractament on un grup va ser el control negatiu sense plasma ni proteïna de patata fermentada, un grup com a control positiu amb plasma en la dieta i 4 grups amb quantitats creixents de proteïna de patata fermentada. Els garrins alimentats amb plasma en el primer estudi van tenir el dia 4 del mateix un nivell major d'IGF-I que la resta sent els garrins que van ser alimentats amb la dosi inferior de proteïna de patata fermentada els que van tenir el nivell d'IGF-I inferior tenint la resta de grups un nivell intermedi d'IGF-I. Els garrins que més pinso consumir van ser els garrins que van consumir el menor nivell de proteïna de patata i els que menys els del grup control negatiu. Aquestes diferències de consum no es van veure reflectides en un major creixement diari ni en una millor conversió alimentària al final de l'estudi. Els garrins que van consumir proteïna de patata van tenir més pes i guany mitjà diari que els animals del control negatiu al final del segon estudi. Pel que fa al consum, els animals amb les 3 dosis més baixes de proteïna de patata van tenir major consum que la resta tenint una millor conversió alimentària dels garrins que van consumir bé proteïna de patata o plasma que els garrins del control negatiu. Els resultats de la present tesi demostren que el subministrament d'un suplement alimentari derivat de la fermentació de la proteïna de patata i) no millora la productivitat de les truges en el subsegüent part, ii) no millora la mortalitat dels garrins durant la lactació ni tampoc el seu pes a deslletament i iii) pot reemplaçar al plasma animal en les dietes de garrins deslletats. El nivell d'IGF-I no es veu modificat en cap estudi al subministrar la proteïna de patata fermentada per la qual cosa la manera d'acció d'aquest producte ha de ser investigat amb estudis futurs.
The objectives of this thesis were the study and review of the current knowledge about the metabolism and actions of IGF-I in pig production. The thesis also studied the effects that an additive coming from the fermentation of the potato protein can have on the IGF-I levels in the different pig production phases and if these modifications have some practical consequence in the pig production. In the lactating sows study, the objective was to assess if the introduction of the fermented protein potato in the diet of the sows 5 days before and 5 days after weaning, with or without glucose, had some effect on the non-productive days between weaning and mating, the fertility and the number of piglets total born in the subsequent litter, and if these productive parameters were in relation with plasma IGF-I levels. It was chosen a high productive sow farm with a high health status because it was considered the best option to assess the effects of the fermented protein potato over the studied parameters. 183 sows coming from 3 consecutive weaned batches sows were distributed in 4 treatment groups taking into account the parity, the number of piglets weaned in the former litter and the number of piglets born in the current litter. The dietary treatments were arranged as a 2x2 complete factorial design with the factors being the addition or not of fermented protein potato and with or without glucose. The treatment started 5 days before weaning and ends 5 days after weaning. There were recorded all the production parameters to get the targets and blood samples were obtained from a subgroup of sows to analyze IGF-I level. No statistical differences were found in IGF-I levels at the end of the treatment nor in the productive parameters recorded. An outcome not considered in the design of the study was that nursing sows had higher IGF-I levels. This finding confirms that IGF-I level depends on the nutritional and metabolic status. Sows that were nursing an extra litter (small piglets) in the current lactation had lower metabolic expense that normal sows during the nursing time and there could recover their metabolic status. In spite of, there were not found productive differences in the next parity. In the study with milking piglets the objectives were to assess if the oral administration of the fermented potato protein in piglets during the first 12 h of live had some effects on the plasma levels of IGF-I, the pre weaning mortality and the average daily gain from birth to wean. This study was carried out in a sow farm with the country average production, with a standard health status. 542 piglets from 3 consecutive batches were used. Piglets were classified into two groups depending on their weight at birth because it was considered that weight at birth could influence the results. The cut-off weight was 1.2 kg. The treatments were: a single oral dose of fermented protein potato, a split half dose in 12 h interval of fermented protein potato, and a group of piglets that received glycerol as positive control group. One group of piglets with no treatment was considered negative control group. No differences were found in any group neither of treatment nor within the weight groups. IGF-I levels were higher in heavier piglets at 7 day of study but not at the end. In the study with weaned piglets the objectives were to assess if the introduction in the diet of the fermented potato protein in weaned piglets at different ratios could substitute the use of animal plasma and if this modification was linked with the plasma level of IGF-I. This study was carried out in a nursery using piglets from the sow farm used in the milking piglets study. There were conducted 2 consecutive experiments. The first of them was carried out to assess the effects of the fermented protein potato on the productive parameters and the second to assess the level to substitute animal plasma. In the first experiment, 200 piglets were distributed taking into account their age, sex and weight at weaning in 5 experimental groups. One group was considered as negative control group with neither fermented protein potato nor animal plasma, one group was the positive control group with animal plasma and 3 other groups with different levels of fermented protein potato. In the second experiment, 1036 piglets were distributed taking into account their age, sex and weight at weaning in 6 experimental groups. One group was the negative control group without animal plasma or fermented protein potato, one group as positive control group with animal plasma and 4 groups with increasing ratios of fermented protein potato. IGF-I levels at day 4 of the first study were higher in piglets fed with animal plasma being the lowest IGF-I level for the piglets fed the lower ratio of fermented protein potato. The rest of the groups had intermediate IGF-I levels. The highest daily feed intake was achieved by the piglets fed with the lowest ratio of fermented protein potato. This high daily feed intake was not achieving a high daily gain or a better feed conversion ratio at the end of the study. Piglets fed with fermented protein potato were heavier and growth faster than piglets in the negative control group at the end of the second study. The highest daily feed intake was achieved by the piglets fed the 3 lower doses of fermented protein potato. Feed conversion ratio was improved in piglets fed either fermented protein potato or animal plasma than piglets in the negative control group. The results of the studies of this thesis show that feeding a fermented protein potato i) do not improve the productivity of the sows in the subsequent parity, ii) do not improve pre weaning piglet mortality nor their weaning weight, iii) can substitute animal plasma in the post weaning diets. The IGF-I levels were not modified in any study when fermented potato was administered orally either to weaned sows, pre weaning piglets or weaned piglets. The mode of action of fermented protein potato should be researched in future experiments.

Les mycotoxines sont des métabolites secondaires toxiques produits par certains champignons filamenteux. Selon leur toxicité et leur occurrence, certaines d'entre elles dont le déoxynivalénol (DON), l'une des toxines les plus répandues dans l'alimentation humaine et animale, sont été réglementées au sein de l'Union Européenne. D'autres métabolites secondaires découverts récemment ou encore peu étudiés sont appelées mycotoxines émergentes et ne sont ni détectés en routine ni réglementés. Les denrées alimentaires destinées à l'homme et à l'animal peuvent être naturellement contaminées par plusieurs mycotoxines et le risque lié à une exposition à des mélanges de mycotoxines est préoccupant. Parmi les animaux d'élevage, le porc est une espèce très sensible aux mycotoxines. De par son alimentation riche en céréales, il peut être exposé à de fortes concentrations de ces contaminants. 524 échantillons d'aliments complets pour porcs prélevés dans le monde entier ont été analysés par une technique de chromatographie en phase liquide couplée à la spectrométrie de masse en tandem (LC-MS/MS) pour plus de 800 métabolites. 88 % des échantillons étaient co-contaminés avec du DON et d'autres mycotoxines réglementées et émergentes. La toxicité du DON et des 10 mycotoxines émergentes les plus répandues a été évaluée en mesurant la viabilité de cellules épithéliales intestinales porcines (IPEC-1) après 48 h d'exposition. Trois mycotoxines émergentes (brevianamide F, cyclo-(L-Pro-L-Tyr) et tryptophol) n'ont pas eu d’impact sur la viabilité cellulaire. Les autres toxines ont été classées dans l'ordre de toxicité suivant: apicidine> enniatine A1> DON> beauvéricine> enniatine B> enniatine B1> émodine> aurofusarine. La toxicité combinée du DON et des 10 mycotoxines émergentes a été évaluée en fonction de leurs concentrations réelles dans les aliments analysés. Nous avons observé que malgré la très forte fréquence des co-contaminations, la corrélation entre les concentrations de DON et des mycotoxines émergentes étudiées était faible. Nous avons donc évalué les effets toxiques de trois mélanges correspondant à des situations auxquelles les animaux peuvent être exposés. Le ratio n°1 a été calculé en utilisant la concentration P25 (1er quartile) de la mycotoxine émergente et la concentration P75 (3ème quartile) du DON. Le ratio n°3 correspondait au scénario inverse du ratio n°1. Le ratio n°2 a été calculé en utilisant la concentration médiane (2ème quartile) du DON et de chaque mycotoxine émergente. Pour la plupart des mélanges, la cytotoxicité combinée était similaire à celle du DON seul. Pour ce qui concerne la santé des animaux, ces résultats ont montré que lorsque ces mycotoxines émergentes sont présentes avec le DON, elles n'exacerbent pas la toxicité du DON. Outre l'intestin, le foie est le principal site de détoxification des xénobiotiques, y compris des mycotoxines, et représente un organe cible des contaminants alimentaires. Nous avons donc mis au point un nouvel outil, les Precision Cut Liver Slices (PCLS), des explants de foie d’épaisseur définie. Elles ont été utilisées pour évaluer la toxicité du DON (3 et 10 µM) à différents temps d'incubation (0 à 20 h), en étudiant l'expression génique, le contenu en ATP et en protéines totales. Le milieu d'incubation a permis d'évaluer la qualité des PCLS en mesurant les marqueurs de dommage hépatique (phosphatase alcaline, lactate déshydrogénase, alanine aminotransférase, aspartate aminotransférase et protéines totales). Nous avons montré que ces marqueurs n’étaient impactés ni par le temps d'incubation, ni par le traitement. Les PCLS traitées avec 10 µM de DON pendant 4 h ou plus, montrent une altération de l’expression de certains gènes. Ces expériences préliminaires ont montré que les PCLS représentent un modèle prometteur pour évaluer la toxicité hépatique des mycotoxines ou d'autres contaminants alimentaires
Mycotoxins are toxic secondary metabolites, produced by several filamentous fungi. Depending on their toxicity and occurrence, some of them, including deoxynivalenol (DON), one of the most common toxin in food and feed, have been regulated in the European Union. Other secondary metabolites, which neither routinely determined nor regulated, are called emerging mycotoxins because they have been recently discovered or poorly investigated. Food and feed can be naturally contaminated by several mycotoxins and concern about the hazard of exposure to mycotoxin mixtures is increasing. Among farm animals, pig is one of the most sensitive farm animal to mycotoxins and it can be exposed, through its rich cereal diet, to high concentrations of mycotoxins. In total, 524 finished pig feeds samples from worldwide were analyzed for more than 800 metabolites using, LC- MS/MS (liquid chromatography tandem-mass spectrometry) method. Eighty-eight percent of the samples were co-contaminated with DON and other regulated and emerging mycotoxins. The toxicity of DON and the 10 most common emerging mycotoxins was analyzed on the viability of porcine intestinal epithelial cells (IPEC-1) over a 48 h period. Among the emerging mycotoxins, 3 of them (brevianamide F, cyclo-(L-Pro-L-Tyr), and tryptophol) did not alter cells viability. The other mycotoxins were ranked in the following order of toxicity: apicidin> enniatin A1> DON> beauvericin> enniatin B> enniatin B1> emodin> aurofusarin. The combined toxicity of DON and the 10 emerging mycotoxins was assessed based on their actual ratios found in pig feed. We observed that, despite the very high frequency of co- contamination, there was a poor correlation between the concentrations of DON and emerging mycotoxins. Thus, we assessed the toxic effects of three mixtures corresponding to situations to which animals may be exposed. Ratio #1 was calculated using the P25 (1st quartile) concentration of the emerging mycotoxin and P75 (3rd quartile) concentration of DON. Ratio #2 was calculated using the median (2nd quartile) concentration of DON and each emerging mycotoxin. Ratio #3 was the reverse scenario of ratio #1. Cytotoxicity analyses showed that, in most of the mixtures, the combined toxicity was similar to the one of DON alone. These results demonstrated that, when these emerging mycotoxins are present with DON, in terms of pig health, it does not exacerbate the problem of the toxicity of DON. In addition to intestine, liver is the main site of detoxification for xenobiotics, including mycotoxins and represents a target organ for food contaminants. Hence, we developed a new tool the Precision Cut Liver Slices (PCLs) an ex vivo explants of liver with a well-defined thickness. This tool was used to assess the toxicity of DON (3 and 10 µM) at different incubation times (0 to 20 h), by studying gene expression, ATP and total protein contents. The incubation medium was used to assess the quality of PCLS by measuring liver damage markers (alkaline phosphatase, lactate dehydrogenase, alanine transaminase, aspartate transaminase and total proteins). We showed that these markers were not affected by either incubation time or treatment. PCLS treated with 10 µM DON for 4 h or more, showed an alteration in the expression of certain genes. These preliminary experiments demonstrated that PCLS represent a promising model for assessing the hepatic toxicity of mycotoxins or other food contaminants

Le diabète de type 1 est une maladie chronique causée par la destruction auto-immune des cellules bêta productrices d'insuline dans les îlots pancréatiques. L'allotransplantation d'îlots pancréatiques est déjà une réalité clinique pour le traitement de cette maladie. Cependant, cette stratégie est limitée par la faible disponibilité de pancréas humains et la nécessité d'une immunosuppression à vie. Le pancréas bioartificiel (BAP), basé sur l'encapsulation d'îlots allo- ou xénogéniques dans un matériau immunoprotecteur, est une alternative prometteuse permettant de lever ces barrières. Cependant, l'apport d’02 après transplantation et avant la vascularisation autour du greffon reste un défi majeur pour maintenir l'efficacité thérapeutique du BAP. En effet, l'hypoxie induit une perte de fonction et à terme la mort des cellules des îlots. Dans ce contexte, nos travaux ont pour but de développer et d'évaluer des stratégies pour remédier à l'hypoxie dans le BAP. Dans un premier temps, les îlots de porcs nouveau-nés ont été évalués dans le BAP en tant que source d'îlots résistants à l'hypoxie. Par la suite, une stratégie d'oxygénation efficace pour le BAP a été développée en combinant le transporteur d'02, Hemoxcell (hémoglobine marine, Hemarina) et un générateur chimique d'02 composé de peroxyde de calcium encapsulé en silicone. Enfin, un BAP à géométrie plane régulé en 02 a été conçu et optimisé afin de permettre le maintien d'îlots encapsulés à forte densité en situation d'hypoxie. L'ensemble des résultats obtenus devrait permettre d'améliorer l'efficacité thérapeutique du BAP et d'extrapoler de dispositifs plus réalistes pour la clinique humaine en maintenant l'équilibre en 02
Type 1 diabetes is a chronic disease caused by the autoimmune destruction of beta cells within the pancreatic islets. The transplantation of pancreatic alloislets has already entered the clinics for this disorder. However, the shortage of human donor organs and the need for lifelong immunosuppressive treatments to prevent graft rejection strongly limit the extension of this approach to a larger patient population. The development of a bioartificial pancreas (BAP), based on the encapsulation of allo-or xenogeneic islets in an immunoprotective membrane, is gathering interest to overcome the main hurdles of islet allotransplantation. However, maintaining the 02 supply after transplantation and before graft vascularisation remains the major challenge to ensure the BAP therapeutic efficacy over extended periods of lime. lndeed, hypoxia induces islet cell dysfunction and finally cell death. In this context, this work aims to develop and evaluate strategies to overcome the hypoxia-induced damages in the BAP. First, neonate pig islets were evaluated in the BAP as a promising islet source exhibiting resistance to hypoxic damages. Then, an efficient oxygenation strategy for the BAP was developed by combining the 02 carrier HEMOXCell (marine haemoglobin, Hemarina) with an 02 generator composed of silicone-encapsulated calcium peroxide. Finally, a planar device based on this oxygenation strategy was designed and optimised to maintain the 02 balance in the BAP supporting a high islet density in a hypoxic environment. This work should allow to improve the BAP therapeutic efficacy and to scale a more realistic device for human clinical applications by maintaining the 02 balance

COORDENAÇÃO DE APERFEIÇOAMENTO DO PESSOAL DE ENSINO SUPERIOR
Tubulações industriais são a maneira mais segura, rápida e econômica de se transportar diferentes produtos, principalmente aqueles relacionados com a industria do petróleo. Essas tubulações sempre envolvem altos investimentos financeiros sendo, portanto, fundamental a garantia de um funcionamento contínuo, com o mínimo de interrupções. Para isso são usados Pigs. Pigs são dispositivos utilizados com muita frequência e com várias finalidades, tais como limpeza e desobstrução de tubulações, retirada de água, vedação de linhas, separação de diferentes produtos, remoção de condensado, inspeção e em testes hidrostáticos. O modelo aqui desenvolvido utiliza as equações unidimensionais da conservação de massa e da quantidade de movimento linear, para simular o movimento transiente do fluido. A equação de conservação de quantidade de movimento para o Pig é resolvida simultaneamente. Uma equação auxiliar para prever a vazão de fluido através do by-pass é proposta e acoplada ao sistema. Vários casos típicos são estudados, obtendo-se excelentes resultados.
Industrial pipelines are the safest, fastes and the most economical way to transport a large range of products, mostly those related to the oil industry. Pipeline systems are usually associated with huge financial investiments, being of fundamental importance to sustain their continuous operation, and maintainning them, reducing the number of interruptions or accidents at a minimum level. To accomplish that, pigs are used. Pigs are devices frequently used for many different purposes, a few of them are clemaming and dewatering a pipeline, sealing of a papeline, transport of different products, inspection and hydraulic tests all of them being accomplished without the interruption of the production lines. The developed model uses the unidimensional conservation equation of mass and linear momentum to simulate the transient movement of the fluid. The conservation of linear momentum of the Pig is also solved, Auxiliary equation to predict the by-pass flow is also incorporated into the system. several special cases are studied with very good results.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2007.
Title from first page of PDF file (viewed September 4, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 90-103).

PETRÓLEO BRASILEIRO S. A.
Na indústria do petróleo, a passagem de pigs em dutos tem sido largamente aplicada com diferentes propósitos: limpeza do tubo, inspeção, remoção de líquido e separação de produtos, entre outros. A eficiência e segurança de uma operação com pig demandam que diversos parâmetros operacionais, tais como pressões máximas e mínimas no duto e velocidade de movimentação do pig, sejam bem avaliados durante a etapa de planejamento e mantidos dentro de determinados limites durante o acompanhamento da operação. Tendo como objetivo a obtenção de uma ferramenta eficiente para ajudar no controle e projeto das operações de passagem de pigs, desenvolveu-se um código numérico baseado no método de diferenças finitas para a simulação de escoamentos transientes de dois fluidos, podendo estes ser líquido-líquido, gás-gás ou líquido-gás. Módulos para controle automático das variáveis do processo foram incluídos, visando à previsão do escoamento mediante diferentes estratégias para alcançar uma operação eficiente. Problemas teste foram realizados, validando a metodologia. Por fim, os resultados obtidos com o simulador são comparados com um caso real de esvaziamento de um trecho do oleoduto OSPAR, pertencente à Petrobras, com 30`` de diâmetro e extensão de 60 km.
In the oil industry, pigging operations in pipelines have been largely applied for different purposes: pipe cleaning, inspection, liquid removal and product separation, among others. Pigging operations to be efficient and safe require a number of operational parameters, such as maximum and minimum pressures in the pipeline and pig velocity, to be well evaluated during planning stage and maintained within stipulated limits while the operation accomplishment. With the objective of providing an efficient tool to assist in the controlling and designing of pig operations through pipelines, a numerical code based on a finite difference scheme for a two-fluid transient flow simulation was developed. The model accounts for liquid-liquid, gas-gas and liquid-gas products in the pipeline. Automatic control modules for some process variables were included to permit the flow prediction by means of different strategies to reach an efficient operation. Test problems were performed to validate the methodology. At last, simulation results were compared with an actual liquid displacement operation at a 60 km segment of the 30`` diameter OSPAR pipeline, owned by Petrobras.

PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO
Este trabalho apresenta a análise do sistema de medição utilizado pelo pig instrumentado tipo palito usado para detecção e dimensionamento de perda de espessura em dutos provocada por corrosão interna. A aplicação de testes experimentais de vibração, perfilagem geométrica e análise por elementos finitos têm objetivo de avaliar o sistema de medição dos sensores palito do Pig Palito, assim como mostrar os resultados obtidos com esta nova tecnologia de dimensionamento de micro geometria. Os dutos submarinos (offshore) empregam tradicionalmente as mesmas tecnologias de inspeção usadas em dutos terrestres (onshore) e uma dessas tecnologias é o pig instrumentado. No entanto, é encontrada uma vasta quantidade de dutos offshore com diferentes tipos de obstáculos que inviabilizam o uso dos pigs instrumentados convencionais, como os Pigs Magnéticos M.F.L. (Magnetic Flux Leakage) e Ultrassônicos. Os fatores relevantes que dificultam a inspeção, especialmente em dutos offshore, são os multi-diâmetros, raios de curvatura acentuados, equipamentos instalados ao longo do duto, alta espessura de parede do duto, escoamento multifásico, etc. Atualmente, o uso de Pigs Ultrassônicos e Magnéticos são as técnica disponíveis no mercado para inspeção da corrosão. Contudo, esses pigs possuem suas próprias limitações. Dentro deste contexto, foi desenvolvido um novo pig instrumentado, chamado de Pig Palito, para detecção e dimensionamento de perda de espessura em dutos com corrosão interna. Esta ferramenta foi desenvolvida para superar diversas limitações que outros pigs instrumentados convencionais têm durante a inspeção. Os resultados obtidos pela instrumentação do sensor palito na análise experimental indicam que a vibração dos sensores pode interferir na medição de micro geometria gerando erros de interpretação. A possibilidade de detecção, minimização e/ou eliminação desta possível deficiência do sensor palito são factíveis e abordadas na presente dissertação. Os bons resultados das inspeções de campo com os Pigs Palito comprovam o sucesso e viabilidade técnica no emprego desta tecnologia.
This work presents the analysis and study of the technology used by the instrumented pig called Feeler Pig, used for detection and measurement of loss of wall thickness in pipelines due to internal corrosion. Those study´s objective are the evaluation of dynamic measurement of Feeler Pig´s sensors, through the application of experimental vibration tests, geometric evaluation`s tests and finite elements analysis, so that are showed the results rewarded by this new micro geometric measurement technique. Submarine pipelines (offshore pipelines) inspection traditionally employs the same technologies used for onshore pipelines and one of such technologies is the instrumented pig. However, it is very common to find offshore pipelines with many kinds of obstacles that may prevent the use of conventional instrumented pigs, like MFL (Magnetic Flux Leakage) pigs and ultrasonic ones. The relevant factors that make the inspection difficult, particularly in offshore pipelines, are the different diameters along the pipeline, small radius bends, equipments installed in the pipeline (such as manifolds and valves), increased wall thickness, multi-phase fluids, etc. Currently available techniques in the market to inspect these pipelines are ultrasonic and magnetic pigs, which, nevertheless, have their own limitations. Focusing on this context, a new tool was developed to detect and measure the loss of wall thickness in pipelines due to internal corrosion. This tool, called Feeler Pig, was designed to be able to overcome some of the limitations of conventional inspection pigs. The results achieved by instrumentation of the feeler type sensor`s body, experimental analysis tests, proved that vibration modes of the sensor interferes in the measurement of micro geometric. The possibility of detection, mitigation and / or elimination of the deficiencies of sensor`s issues are viable and addressed in this work. These, coupled with the excellent results of Feeler Pigs field inspections prove the technical feasibility and success in using this technology.

Phosphodiesterases (PDEs) play a key role in the control of cardiac contraction. PDE inhibitors are associated with cardiotoxicity, and increased knowledge of cardiomyocyte PDEs is required for improved pharmaceutical development. PDE activity and function were compared in animal and human cardiomyocytes in order to determine a suitable model for the human response, and to generate new insights into the role of cardiomyocyte PDEs. Ventricular myocytes were isolated from rat, guinea pig and diseased human heart. PDE gene expression was measured using RTPCR; PDE activity was measured using a scintillation proximity assay and isoform-specific PDE inhibitors. Effects of PDE inhibition on cardiomyocyte contractility were measured using electrically paced isolated cells. The human cardiomyocyte PDE expression and activity profile was closer to the guinea pig than the rat. High levels of PDE1 activity against cAMP and lower levels of possible PDE5 activity were found in guinea pig and human cardiomyocytes, but not rat. Non-specific PDE inhibition increased basal cardiomyocyte percentage shortening in cells from all three species; PDE3 inhibition only increased percentage shortening in guinea pig and human cardiomyocytes; PDE4 inhibition did not affect basal percentage shortening in cardiomyocytes from any of the three species. Further studies using rat cardiomyocytes found that PDE3 and PDE4 inhibition both significantly increased percentage shortening when cAMP levels were raised; rat β1- and β2- adrenergic receptors were also differentially regulated by PDE3 and PDE4. The functional effects of PDE1 inhibition were investigated in guinea pig cardiomyocytes but no significant effects were measured. Dual PDE1/PDE5 inhibition was shown to have a small negative effect on isoprenaline-stimulated contractility in rat, guinea pig and human cardiomyocytes. This study has revealed new insights into cardiomyocyte PDEs and demonstrated that the guinea pig may be a more suitable model for diseased human cardiomyocytes than the rat.

Les mycotoxines sont des métabolites secondaires de moisissures contaminant de façon naturelle de nombreuses denrées alimentaires, notamment les céréales. Le déoxynivalénol (DON), produit par Fusarium sp., est la mycotoxine la plus répandue dans le monde. Du fait de sa grande stabilité chimique, le DON est difficile à éliminer, et se retrouve dans les céréales et les produits finis ou il induit des effets toxiques pour l'homme et l'animal. De nouvelles stratégies de lutte sont mises en places, telle la transformation biologique utilisant des bactéries ou des plantes. En effet certaines bactéries possèdent des enzymes capables de transformer le DON en de nouveaux composés, le déepoxy-déoxynivalénol (DOM-1) et le 3-épi-déoxynivalénol (3-epi-DON). De plus, certaines plantes sont naturellement capables de transformer le DON dans le but de l'éliminer et de le détoxifier, formant ainsi le deoxynivalénol-3-ß-D-glucoside (D3G). L'objectif de cette thèse était d'évaluer la toxicité de ces dérivés du DON au niveau de l'intestin et du système immunitaire par le biais d'analyses in silico, in vitro, ex vivo et in vivo. Les tests de toxicité in vitro sur la lignée humaine intestinale cellulaire Caco-2 montrent que le DOM-1, le 3-epi-DON et le D3G n'étaient pas cytotoxiques, ils ne modifiaient ni la viabilité, ni la fonction de barrière des cellules, mesurée par la résistance électrique transépithéliale. Les tests de toxicité ex vivo sur des explants jéjunum porcin ont montré que le DOM-1, le 3-epi-DON ou le D3G n'induisaient pas de modifications histomorphologiques. En revanche, les explants exposés au DON montraient des lésions morphologiques et une régulation positive de l'expression des cytokines pro-inflammatoires. L'impact de ces trois dérivés a été également analysé sur l'expression de l'ensemble des gènes du tissu, avec une analyse microarray. Ceci a montré que ces dérivés du DON n'induisaient aucun changement dans l'expression des gènes par rapport au groupe contrôle. Le DON quand a lui exprimait différentiellement 747 sondes, correspondantes à 333 gènes impliqués dans l'immunité, la réponse inflammatoire, le stress oxydatif, la mort cellulaire, le transport moléculaire et la fonction mitochondriale. L'analyse in silico a montré que le D3G, contrairement au DON était incapable de se lier au site-A du ribosome, principale cible de la toxicité pour le DON. Les deux dérivés microbiens eux, étaient capables de se fixer au site-A au sein du ribosome, mais contrairement au DON ils ne formaient que deux liaisons hydrogènes au lieu de trois. De plus, ces trois dérivés n'induisaient pas de stress ribotoxique, d'activation des MAPKs (mitogen-activated protein kinases), et de réponse pro-inflammatoire. Une étude complémentaire a été menée in vivo pour évaluer la toxicité du DOM-1 chez le porc (gavage pendant 21 jours avec .0.14mg / kg de poids vif). Les résultats ont montré que le DOM-1, contrairement au DON n'induisait pas les effets toxiques du DON au niveau des paramètres zootechniques (pas de vomissements, aucune diminution de la consommation alimentaire ou de perte de poids), sur l'intestin et le foie (pas de dommages tissulaires), ou sur la réponse immunitaire (pas de réponse inflammatoire induite). En conclusion, nos résultats montrent l'efficacité de ces transformations enzymatiques. La déepoxydation et l'épimérisation bactérienne, ainsi que la glycosylation par les plantes permettent de sensiblement diminuer la toxicité du DON, passant par une absence de toxicité sur le ribosome avec une absence d'activation des MAPKs et de réponses inflammatoires. Dans ce contexte de contamination par les mycotoxines, ces méthodes de luttes alternatives semblent être des approches prometteuses
The Fusarium sp. mycotoxin deoxynivalenol (DON) is one of the most frequently widespread mycotoxin worldwide. Due to its high structural stability, the elimination of DON, once present in cereals or feed materials, becomes difficult. Thereby, it is present in many cereals and final feed products, inducing several toxic effects on human and animals, and causing big economic losses. New strategies of to fight against mycotoxins were developed, as biological transformation, either by the use of bacteria or plants. Indeed, some microorganisms are able to transform DON in new products, by enzymatic reaction, forming the deepoxy-deoxynivalenol (DOM-1) and the 3-epi-deoxynivalenol (3-epi-DON). Moreover, some plants naturally own the capacity to glycosylate DON in the aim to detoxify it, forming the deoxynivalenol-3-ß-D-glucoside (D3G). The aim of this thesis was to assess the toxicity of these DON derivatives, on the intestine and immune response, using several approaches such as in silico, in vitro, ex vivo and in vivo models. On the human intestinal Caco-2 cell line, DOM-1, 3-epi-DON and D3G were not cytotoxic; they did not alter its viability and barrier function, as measured by the trans epithelial electrical resistance. The expression profile of DOM-1, 3-epi-DON and D3G-treated jejunal explants was similar to that of controls and these explants did not show any histomorphology alteration. On the other hand, the treatment of intestinal explants with DON, induced morphological lesions and upregulated the expression of proinflammatory cytokines. The impact of these three derivatives was also studied on intestinal explants with a pan-genomic transcriptomic analysis. Results show that the derivatives of DON did not induce any change on the gene expression in comparison to the control-treated explants. In contrary, DON-treated explants differentially expressed 747 probes, representing 323 genes involved in immune and inflammatory responses, oxidative stress, cell death, molecular transport and mitochondrial function. In silico analysis revealed that D3G, opposing to DON, was unable to bind to the A site of the ribosome, which is the main target for DON toxicity. Both DOM-1 and 3-epi-DON were able to fit into the pockets of the A site of the ribosome but only by forming two hydrogen bonds, while in this position, DON forms three hydrogen bonds. Moreover, the three derivatives do not elicit a ribotoxic stress, MAPKinase activation, and inflammatory response. Then, an in vivo study was carried out to assess the toxicity of DOM-1 on pig (feed forced during 21 days at 0.14 mg/Kg BW). The results showed that DOM-1 does not have as much toxic effects as DON on zootechnical parameters (no emesis induced, no decrease of food consumption or weight loss observed), on intestine and liver (no tissues damages), or on the immune response (no inflammatory response induced). Our data demonstrate that bacterial de-epoxidation or epimerization of deepoxy-DON modified its interaction with the ribosome, leading to an absence of MAPKinase activation and toxicity; and that the glycosylation of DON suppresses its ability to bind to the ribosome and decreases its intestinal toxicity. The mycotoxin deoxynivalenol (DON) remains an important challenge in many regions in the world. Thus, these biological detoxifications of DON seem to represent a new promising approach helping manage the problem of its contamination

The increasing demand for organs has stimulated the necessity in xenotransplantion, which promises an unlimited supply of organs for clinical use. However, coagulopathy of xenografts remains a major hurdle to successful pig-to-primate xenotransplantation. The ability to generate pigs expressing a human complement-regulatory protein (hCRP) and/or pigs in which the α1,3-Galactosyltransferase gene has been knocked-out (GT-KO) has largely overcome the barrier of hyperacute rejection (HAR) of a pig organ transplanted into a primate. However, acute humoral xenograft rejection (AHXR), presenting as microvascular thrombosis with/without consumptive coagulopathy (CC), ensures and results in graft failure. The causes of coagulopathy were believed to be humoral response-dependent. Xenoreactive antibodies (Abs) and activation of complement provoke porcine endothelial cells (ECs) from an anticoagulant to a procoagulant phenotype. In this study, I demonstrated that recipient platelets and monocytes were activated to express tissue factor (TF), an initiator of the coagulation cascade, after incubation with porcine ECs through humoral immune response-independent process. These observations were mirrored in the animal studies. Kidneys or livers from GT-KO pigs that express a hCRP transplanted into nonhuman primates were not susceptible to HAR. Nevertheless, most recipients developed CC, even when the grafts were still functioning. Activation of graft ECs and the measurable immune response were minimal. TF expression on recipient platelets plays a pivotal role in initiating CC. Therefore, understanding the interactions between porcine ECs and primate platelets may be crucial to prevent coagulopathy. On the other hand, the generation of GT-KO pigs has directed interest to the role of anti-nonGal Abs in intravascular thrombosis. My study revealed that anti-nonGal Abs activated porcine ECs to express TF, independent of complement activation. I also demonstrated that anti-P-selectin and vWF Abs and some anti-platelet agents, such as clopidogrel and eptifibatide, prevented TF expression on platelets after incubation with porcine ECs. Porcine ECs from pigs that expressed tissue factor pathway inhibitor (TFPI) were resistant to the activation induced by primate serum even with high titre anti-nonGal Abs. Atorvastatin not only inhibited this activation of platelets but also prevented the activation of porcine ECs induced by primate serum. Coagulopathy is increasingly recognized as barriers to successful xenotransplantation, many mechanisms of which are not associated with humoral immune response. Further manipulation of the immune response alone, with the risk of inducing infection and other complications, does not appear likely to resolve the challenge of xenograft coagulopathy. My results provide evidence for further genetic manipulation or systemic pharmaceutical treatment to prevent coagupolpathy seen after pig-primate xenotransplantation.

The bacterium Actinobacillus pleuropneumoniae is an important respiratory tract pathogen of pigs leading to major swine health problems and huge economic losses to farmers worldwide. It is divided into at least 16 distinct capsular serovars and has highly diverse pathogenicity. A safe vaccine that offers complete protection against all serovars has yet not reached the market. In this study, a multi-strain genomic approach was used to screen universal vaccine candidates against A. pleuropneumoniae. Roary was used to identify core genes between 221 A. pleuropneumoniae strains from serovars 1-5, 5b, 6-16, and K2:O7 and nontypeables strains. By cross-referencing with previous literature, it was identified that 34 of the A. pleuropneumoniae conserved genes are predicted to code for virulence factors. These included genes involved in cell wall biogenesis, anaerobic respiration, metabolism, secretion and ATP synthesis. Of the 34 genes, nine genes (ompW, prc, rbsB, tatB, atpG, dmsA, nrfAB, napB and ccmH) were identified to encode for surface proteins or secretory proteins. These surface or secretory proteins are potential candidates for subunit vaccine. Another 19 genes (murD, glpR, rfaC, malP, secB, dnaK, aspA, lpdA, purF, guaB, atpGH, moaA, moaE, lldD, frdA, hemA, hemB, ureABC and ureEG) were identified to encode for cytoplasmic proteins. These cytoplasmic proteins are potential candidates for live-attenuated or differentiating infected from vaccinated animals (DIVA) vaccines. Diversity between different A. pleuropneumoniae serovars was also examined by reconstructing phylogenetic trees based on core genes and accessory genes separately. The core gene tree clearly divided the isolates into two main groups. The genomic relationships between A. pleuropneumoniae strains belonging to the same serovar were also studied. Mostly, the isolates of the same serovar were found to be closely related to each other. The exceptions were serovars 6, 12, K2:O7 and nontypeables, which showed greater genetic variation. Due to the presence of genetic variation between and within serovars, the identification of universal vaccine candidates is the best way forward to develop subunit or DIVA based vaccines against A. pleuropneumoniae. The isolates of A. pleuropneumoniae were also investigated by formulat- ing a multilocus sequence typing (MLST) scheme based on partial sequences of the genes gdhA, infB, recA, mdh, frdB, atpG and gph and 32 sequence types (STs) were observed. The analysis of these STs using eBURST revealed six clonal complexes and suggested that A. pleuropneumoniae is an intermediately clonal bacterium. Both the MLST and the core-gene phylogeny results also revealed a possibility of capsule switching in A. pleuropneumoniae which has implications for whole-cell bacterin vaccines. Additionally, an evolutionary genomics approach was used to identify core genes that show evidence of recombination and positive selection in A. pleuropneumoniae. Approximately, 61% of the core genes showed strong signals for homologous recombination (q-value < 0.05). Furthermore, the selection analysis indicated that 25 genes are under significant selection pressure. Extensive functional analysis of the positively selected genes demonstrated that genes coding for products relevant to bacterial cell membrane, metabolism, transcription, secretion and transportation are prone to positive selection pressure. This information will be useful for researchers for novel drug development against A. pleuropneumoniae.

PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO
COORDENAÇÃO DE APERFEIÇOAMENTO DO PESSOAL DE ENSINO SUPERIOR
PROGRAMA DE EXCELENCIA ACADEMICA
A utilização de PIG em oleodutos e gasodutos tem uma grande importância para a manutenção da integridade estrutural de tubulações, e pode ser utilizado para inspeção, limpeza e separação de interface. Neste contexto é importante que a passagem de PIG ocorra de modo controlado causando o menor impacto possível à operação, respeitando os limites operacionais da tubulação e dentro dos limites de velocidade de deslocamento do PIG. Caso o PIG possua uma velocidade baixa, ele pode ficar preso à tubulação, podendo ser difícil soltá-lo. Por outro lado, caso a velocidade seja elevada o PIG pode danificar a tubulação em função dos impactos gerados. Com o objetivo de atender os requisitos referente a velocidade recomendável de deslocamento do PIG referente aos limites operacionais existentes e não danificá-lo, foi desenvolvido um simulador termo hidráulico de deslocamento de PIGs. O simulador prevê escoamentos transientes isotérmico ou térmico acoplado ao deslocamento do PIG. Para controlar a velocidade do PIG, uma válvula de controle foi instalada no corpo do próprio PIG. Sua velocidade de deslocamento é função da abertura do furo de bypass, ou seja, quanto maior a velocidade de escoamento na tubulação, maior a abertura e maior o fluxo através do PIG para manter a velocidade controlada. As equações de conservação de massa, quantidade de movimento e energia, acopladas com um balanço de forças no PIG, foram discretizadas pelo método das diferenças finitas e resolvidas de forma acoplada. Para o controle da velocidade optou-se pelo método de controle PID. Investiga-se o deslocamento do PIG tanto para escoamento de líquido quanto de gás, considerando ou não a perda de calor para o ambiente. Diversos casos são apresentados demonstrando a eficácia do método de controle modelado.
The use of PIG in oil and gas pipelines has great important to maintain the structural integrity of pipelines, and it can be used for inspection, cleaning and interface separation. In this context, it is important that the PIG displacement happen in a controlled way causing the lowest possible impact to the operation, respecting the operating limits of the pipe and within the limits of the PIG s speed of displacement. If the PIG has a low speed, it can be trapped in the tubing and it can be difficult to release it. On the other hand, if the speed is high the PIG can damage the tubing in function of the generated impacts. In order to respect the requirements for the recommended PIG velocity displacement, and operational pipelines limits requirements, a thermo hydraulic PIG simulator was developed. The simulator predicts isothermal or thermal transient flow coupled with the PIG displacement. To control the PIG velocity, a control valve is installed in the body of the PIG. The PIG speed is as a function of the bypass aperture inside the PIG s body, ie, the greater the flow velocity in the pipe, the greater is the opening and the greater the flow through the PIG. The conservation equations of mass, momentum and energy coupled with force balance at the PIG were discretized by the finite difference method, and solved in coupled manner. A PID control method was employed to control the PIG velocity. It is investigated the displacement of PIG in both liquid and gas flow, considering or not the loss of heat for the environment. Several cases are presented demonstrating the effectiveness of the control method modeled.

Prolactinomas account for 40% of all tumors in the pituitary, an important gland of the endocrine system, and secrete excess amount of prolactin hormone. We recently identified a member of the TGF-beta superfamily of growth factors, activin, as an important regulator of prolactin expression and cell growth. Activin inhibits prolactin expression through inhibition of expression of a pituitary-specific transcription factor, named Pit-1. Pit-1 plays major roles in the development and maintenance of the anterior pituitary gland integrity. In this study, I investigated the intracellular signaling pathways and transcriptional mechanisms by which activin controls Pit-1 activity. I demonstrate that in addition to the down-regulation of mRNA and proteins levels of Pit-1, a functional p38 MAPK signaling pathway is required for activin to down-regulate human Pit-1 gene promoter activity in a Smad-independent manner. This study allows a better understanding of the regulatory effects of activin in the pituitary gland.

Vertical transmission of Aujeszky's Disease virus (ADV) was studied in pigs with two British strains. The strains used were both isolated from an outbreak of disease in Yorkshire. In vivo experiments involving intranasal infection of 10-week old pigs revealed that one strain was highly virulent (Leeds-1) while the other was of low virulence (Leeds-2). Both experimental and natural infections were evaluated by the clinical response and by the detection of specific immune responses. The techniques used included class-specific ELISA tests, lymphocyte transformation and antibody-dependent cellular cytotoxicity. Twelve pregnant sows were infected intranasally at 85+/-1 days of gestation. Evidence of transplacental infection was found in only one of the ten surviving sows. The litter consisted of 8 mummified fetuses. ADV antigens were detected in liver sections with fluorescent antibodies. In contrast the incidence of perinatal infection was high. Colostrum-deprived piglets born to infected sows developed antibodies to the virus, providing evidence that they were infected at birth. All naturally-reared piglets, were sero-positive after ingestion of colostrum from their dams. Litters born to sows infected with the low virulence strain were poorly protected and succumbed to the infection, whereas no postnatal mortality was recorded among the litters born to sows infected with the high virulence strain, which induced high levels of colostral immunity. This emphasised the importance of maternal protection in the natural disease and brings into consideration the possibility that such low-virulence strains, which do not evoke clinical disease in mature animals, may be important as the cause of outbreaks of disease in breeding herds. In vitro studies of strain characteristics revealed that sensitivity to trypsin or to heat did not correlate with the virulence of the strains. Studies of the replication of the viruses in the pig alveolar macrophages, however, showed between-strain differences which correlated with their virulence for pigs. The Leeds-1 and Leeds-2 strains were also clearly differentiated by restriction enzyme analysis of their DNA.

The paper describes an attempt to gain insight into the relationship between cash and futures markets for US lean hogs and EU live pigs, and the opportunity of arbitrage hedging. In doing so, the authors use newer methods of threshold cointegration analysis for time series from 1999 until 2008. Besides the existence of a long-run equilibrium, asymmetric price adjustments can be demonstrated. This is especially the case for the EU live pigs, where price variations of the basis are higher and exhibit lower standard deviation. The results also perfectly show that cash prices follow the futures market more than the other way round. Furthermore, a grid search has revealed that the residual-based threshold in either market is near zero and therefore coherent with economic interpretation. Thus, at least theoretically, arbitrageurs in those markets are able to exploit the price differences between the two markets and reap no-risk monetary benefit. Hence, the results are in line with the statement that "speculating the basis" generates a better return. (authors' abstract)

The Pim family of serine/threonine protein kinases (Pim 1, 2, and 3) contribute to cellular transformation by regulating glucose metabolism, protein synthesis, and mitochondrial oxidative phosphorylation. Drugs targeting the Pim protein kinases are being tested in phase I/II clinical trials for the treatment of hematopoietic malignancies. The goal of these studies was to identify Pim substrate(s) that could help define the pathway regulated by these enzymes and potentially serve as a biomarker of Pim activity. To identify novel substrates, bioinformatics analysis was carried out to identify proteins containing a consensus Pim phosphorylation site. This analysis identified the insulin receptor substrate 1 and 2 (IRS1/2) as potential Pim substrates. Experiments were carried out in tissue culture, animals, and human samples from phase I trials to validate this observation and define the biologic readout of this phosphorylation. Our study demonstrates in both malignant and normal cells using either genetic or pharmacological inhibition of the Pim kinases or overexpression of this family of enzymes that human IRS1S1101 and IRS2S1149 are Pim substrates. In xenograft tumor experiments and in a human phase I clinical trial, a pan-Pim inhibitor administered in vivo to animals or humans decreased IRS1S1101 phosphorylation in tumor tissues. This phosphorylation was shown to have effects on the half-life of the IRS family of proteins, suggesting a role in insulin or IGF signaling. These results demonstrate that IRS1S1101 is a novel substrate for the Pim kinases and provide a novel marker for evaluation of Pim inhibitor therapy.

In many organisms, the most fundamental event during embryogenesis is differentiating between germline cells and specialized somatic cells. In C. elegans, PIE-1 functions to protect the germline from somatic differentiation and appears to do so by blocking transcription and by preventing chromatin remodeling in the germline during early embryogenesis. Yet the molecular mechanisms by which PIE-1 specifies germline remain poorly understood. Our work shows that SUMOylation facilitates PIE-1-dependent germline maintenance and specification. In vivo SUMO purification in various CRISPR strains revealed that PIE-1 is SUMOylated at lysine 68 in the germline and that this SUMOylation is essential for forming NuRD complex and preserving HDA-1 activity. Moreover, HDA-1 SUMOylation is dependent on PIE-1 and enhanced by PIE-1 SUMOylation, which is required for protecting germline integrity. Our results suggest the importance of SUMOylation in the germline maintenance and exemplify simultaneous SUMOylation of proteins in the same functional pathway.

Background: Stem cells are a promising therapy for regeneration following myocardial infarction (MI). Another therapy currently under investigation for MI is glucagon-like peptide-1 (GLP-1), a natural incretin hormone that has cardio-protective properties, although a short half-life in vivo. GLP-1 CellBeads are a novel therapy, combining stem cells and GLP-1. Human mesenchymal stem cells (MSCs) were immortalised, engineered to secrete a fusion protein of GLP-1 and encapsulated in alginate. We have previously demonstrated that GLP-1 CellBeads significantly reduce infarct size and improve ejection fraction post-MI, but the underlying mechanisms are unclear. The therapy was assessed in an in vivo pig MI model and an in vitro cardiomyocyte ischaemia model. Methods: GLP-1 CellBeads were delivered to coronary artery branches in pigs, creating micro-infarcts, as determined by echocardiography. Cell-free beads (Beads) and CellBeads containing hMSCs without GLP-1 (Beads-MSC) were delivered as controls (n=3-5/group). Pigs were sacrificed one and four weeks post-MI. Tissue was analysed for: apoptosis, collagen, cardiomyocyte cross sectional area and myofibroblasts. The localised response around the beads was also measured using immunohistochemistry. Atomic force microscopy (AFM) was used to examine the ultra-structure of the collagen scar. The expression profiles of genes involved in collagen remodelling were measured using qRT-PCR. Viability of MSCs was measured using GFP-tagging and confirmed using qRT-PCR. To examine effects on apoptosis in vitro, human adult cardiomyocytes underwent ischaemia for 1 hour before incubation with: media conditioned with MSCs or MSC+GLP-1, GLP-1, Exendin-4 or media. Apoptosis and viability were measured at 24 and 48 hours respectively. Results: In the in vivo pig model, significant increases in apoptosis were observed in the infarct of all groups one week post-MI, with no differences between treatments. Despite decreased numbers of myofibroblasts, significantly more collagen was observed in MSC treated groups, with increased collagen fibril periodicity and a more organised collagen scar. The altered scar structure was reflected in differences in gene expression between groups, with an accelerated healing response in the MSC groups. However, significantly fewer myofibroblasts were observed in the MSC treated groups. Viability of MSCs was confirmed up to four weeks post-infusion, with GLP-1 secretion confirmed up to one week. In the in vitro ischaemia model, MSC+GLP-1 conditioned media significantly reduced cardiomyocyte apoptosis 24 hours post-ischaemia, compared to media alone. All agonists (GLP-1, MSC media and MSC+GLP-1 media) significantly improved viability compared to media alone 48 hours post-ischaemia. Conclusions GLP-1 CellBeads have a beneficial effect on healing following MI by significantly decreasing infarct size and improving ejection fraction post-MI. these benefits are associated with decreased cardiomyocyte apoptosis and altered collagen scar formation. The CellBeads act as local hubs for regeneration and are viable up to one month post-infusion. The effects observed are due to a combination of the GLP-1 and paracrine factors released from the hMSCs.

Ce travail de thèse est consacré à la conception et à la synthèse d’une nouvelle famille d’inhibiteurs de la sérine/thréonine kinase Pim-1 qui a été identifiée comme une cible prometteuse pour le traitement de certains cancers. Ces molécules, dont le pharmacophore principal est l’acide 8-hydroxy-7-quinoléinecarboxylique, inhibent sélectivement la kinase Pim-1. Nous appuyant sur des études de modélisation moléculaire réalisées avec le logiciel GOLD V, nous avons pu proposer un mode de fixation plausible de ces molécules au sein du site catalytique de la kinase Pim-1 et suggérer de nouveaux inhibiteurs potentiels. Parmi ceux-ci, les plus importants portent un groupement aryle ou hétéroaryle en C-5 sur le noyau quinoléine. Leur synthèse a été réalisée selon une séquence dont l'étape clé est un couplage de Suzuki utilisant les organotrifluoroborates de potassium comme nucléophiles. Une réaction d’hydrogénolyse du groupe protecteur benzylique en C-8, sous activation micro-onde a complété la synthèse. Les nouveaux inhibiteurs ont été évalués sur la kinase Pim-1 et les relations structure-activité discutées en s'appuyant sur la modélisation
This thesis describes the design and the synthesis of a new family of inhibitors of the serine/threonine kinase Pim-1 which as been recognized as a promising target for cancer drug therapy. These molecules, whose main pharmacophore is the 8-hydroxy-7-quinoléinecarboxylic acid moiety, selectively inhibit the kinase Pim-1. Relying on molecular modeling studies performed using the software GOLD V, we have proposed a binding mode of these molecules within the catalytic site of the kinase Pim-1 and we have suggested new potential inhibitors. The most important ones display an aryl or a heteroaryl ring at the C-5 carbon of the quinoline nucleus. Their synthesis was achieved using a sequence involving a Suzuki cross-coupling reaction using organotrifluoroborate as key step. O-debenzylation at C-8 by transfer hydrogenation under microwave heating has completed the synthesis. The new inhibitors have been tested on the kinase Pim-1 and the structure-activity relationships are discussed based on docking studies

The transcription factor, Pit-1, is involved in the transcriptional regulation of the mammalian prolactin (Prl), growth hormone (GH) and thyroid stimulating hormone b-subunit genes (TSHb) as well as its own gene. The role of Pit-1 in avian species is unknown. Three turkey (t) Pit-1 isoforms have been identified that arise from alternative transcription initiation and alternative splicing. Splicing of exon 1 to an alternative acceptor splice site in exon 2 results in a 28 amino acid insertion in tPit-1β* relative to tPit-1*. Both isoforms initiate transcription at exon 1. A tPit-1 transcript unique to the turkey has been identified and arises following transcription initiation upstream of the alternative acceptor splice site in exon 2. Western blot analysis of pituitary extracts has revealed two isoforms of 37 and 40 kDa. The ability of Pit-1 to transactivate the Prl, GH, and Pit-1 promoters was determined with cotransfection assays. The tPrl, tGH, tPit-1 and rat (r) Prl promoters were cloned upstream of the luciferase gene in a reporter construct. Turkey Pit-1 isoforms and rPit-1 were expressed under the control of the Avian Sarcoma Virus Long Terminal Repeat (ASVLTR) promoter. Cotransfection analyses in mouse L cells indicate that tPit-1* activates the tPrl, tGH, tPit-1 and rPrl promoters 4.6-, 3.8-, 1.7-, and 29.0-fold, respectively. Similar results were observed when cotransfection assays were performed in a turkey pituitary-derived cell line and in primary turkey pituitary cells. These results indicate that tPit-1 is not a strong activator of the tPrl, tGH, or tPit-1 genes, whereas Pit-1 does activate these genes in mammals. A point mutation at amino acid position 176 (ser ⟹ leu) in the POU-homeodomain results in a mutant tPit-1 that shows decreased activity on all promoters tested. Turkey Pit-1* (ser-176) activates the rPrl promoter 14-fold lower than the wild type tPit-1* (leu-176).
Master of Science

Membrane-based gas separation processes are an area of interest owing to their high industrial demand for a wide range of applications, such as natural gas purification from CO2 or H2, and N2 or O2 separation from air. This thesis is focused on developing and investigating polymeric-based membranes. Firstly, novel mixed matrix membranes (MMMs) were prepared, incorporating few-layer graphene in the polymer of intrinsic microporosity PIM-1. Secondly, novel polyphenylene-based polymers of intrinsic microporosity (PP-PIMs) were synthesised. An optimum preparation method of graphene/PIM-1 MMMs (GPMMMs) was established from numbers of experiments. In this study, graphene exfoliation was a step towards GPMMM preparation. Starting from graphene exfoliation in chloroform, as a good solvent for PIM-1, enhancement in graphene dispersibility was obtained with addition of PIM-1. This result helped in GPMMM preparation with high graphene content (up to 4 wt.%). Characterizations techniques such as Raman spectroscopy and scanning electron microscopy (SEM) of GPMMMs, confirmed the few layer graphene content, with morphology changes in the polymeric matrix compared to pure PIM-1.Gas permeability results of GPMMMs showed an enhancement in permeability with low loading graphene (0.1 wt.%) using a relatively low permeability PIM-1 batch, due to high water content. However, less influence of graphene incorporation on permeability was observed with a highly permeable PIM-1 batch. Reduction in permeability over time, termed an ageing effect, is known for a polymer of high-free volume like PIM-1. However, the enhancement of GPMMMs permeability after eight months storage was shown to be retained. Novel PP-PIMs were prepared from novel precursors using a series known organic reactions. PP-PIMs were divided into two groups of polymers based on their polymerization reactions. A group of polymers were prepared from condensation polymerization between bis-catecol monomers and tetrafluoroterephthalonitrile (TFTPN). Another group of polymers were prepared from Diels Alder polymerization between monomers of terminal bisphenylacetylene groups and bis tetraphenylcyclopentadienones (TPCPDs). All of which yielded polymers with apparent BET surface area in the range 290-443 m2 g-1.

In response to the ever-increasing global energy demand and the need to move away from non-renewable and CO2-emitting fossil fuels as the primary energy production method, renewable energy sources have become more and more viable as energy production methods. However, given the unreliable and instantaneous nature of these energy sources, reliable, renewable energy storage methods are required. Hydrogen is an excellent candidate as a chemical energy store, as it is highly abundant, relatively easily produced as diatomic hydrogen (including from water electrolysis), and only produces water upon its complete combustion. Hydrogen also has the highest gravimetric energy density of any known chemical fuel, meaning that not very much of it is required relative to other chemical fuels. However, hydrogen gas is incredibly sparse, and therefore hydrogen has a very low volumetric energy density, making storage of the material a key challenge in the development of the so-called “hydrogen economy”. Most commonly, hydrogen is stored by compressing it to 70 MPa. However, this technique has a number of flaws, including the high expense of strong tanks (and in the case of light duty vehicles, lightweight materials are also required), and the inherent safety risks that high pressure, highly flammable gas poses. One of the alternatives to compression is to store hydrogen by adsorption, which uses high surface area materials to densify hydrogen via the formation of weak physical bonds. This research line is well developed, and a number of different materials has been created that show good potential as hydrogen storage materials, such as activated carbons and metal organic frameworks. However, the vast majority of materials developed for this purpose are tailored only with the hydrogen uptake in mind, which can cause issues as the focus of development shifts from small scale tests to full tank scale. One adsorptive that shows a number of highly useful engineering properties on the large scale, such as good thermal resistance and solution processability, is the polymer of intrinsic microporosity PIM-1. This material can be processed into a number of morphologies without losing porosity, and shows good thermal and mechanical resistance. However, its adsorption capacity is rather limited, with the BET surface area generally reported in the 700 – 800 m2 g-1 range, and hydrogen uptake of 1.45 wt% at 77 K and 1 MPa. This thesis presents two separate studies on attempting to improve the hydrogen uptake of PIM-1 without adversely affecting the material properties that make it attractive. The first of these was the creation of mixed-matrix-membrane style composite films solution cast from PIM-1 and the metal organic framework MIL-101. PIM-1 proved slightly difficult to synthesise consistently with high molecular weight, but MIL-101 is an easy hydrothermal synthesis. Film casting was successfully performed, producing flat, homogeneous films that maintained the MOF crystallinity. These materials were tested for their thermal properties – thermal decompositions proceeded according to the rule of mixtures of the two starting materials, whilst an increasing concentration of MOF was shown to decrease the specific heat capacity. Both PIM-1 and MIL-101 were shown to adsorb nitrogen as previously reported. The composites showed increasing uptake with MIL-101 content, but at a lower rate than the rule of mixtures. This was a common theme for the N2 (77 K), CO2 (293 K) and low pressure H2 isotherms performed. High pressure isotherms up to 17 MPa were performed on PIM-1 for the first time, showing a maximum excess uptake of 1.8 wt% on the powder and 1.6 wt% on the film, both at 77 K. The composites showed improved uptake with increasing MIL-101, but the maximum uptakes did not meet the rule of mixtures. The uptakes at the highest pressure did, however. Multiple temperature isotherm sets were performed on the PIM-1 film and powder, as well as the 30 wt% composite. These data sets were hampered largely by machine faults, but contained sufficient valuable data to be able to proceed with parameter fitting. The sensitivity of the isotherms produced in this study to the value of skeletal density is also examined closely. The second theme of improved H2 uptake in PIM-1 was to carbonise the material. TGA studies on PIM-1 showed good thermal stability in anoxic conditions, and TGA twinned with mass spectroscopy was able to confirm a previously proposed mechanism of thermal decomposition. Carbonised and activated PIM-1 film samples, and a carbonised powder, were produced using physical activation methods. The adsorption performance of the carbons was disappointing, as the uptakes of N2 and H2 (< 0.1 MPa) were reduced post-carbonisation, with little recovery in the activated film. CO2 uptakes were improved, however. High pressure H2 isotherms on both the carbonised and activated films showed unusual ‘stepping’ behaviour in the adsorption curve, but maximum uptakes for both (1.0 – 1.3 wt%) were less than that seen for PIM-1 alone. Parameter fitting was performed on all of the high pressure H2 isotherms performed in this study, using a method previously proposed by the Mays group. The parameter fits all showed effective hydrogen densification in the adsorbate layer, although the repeatability of parameter values, and the smoothness of the parameters as a function of temperature were undermined by the low quality of some of the isotherms. Using the parameters acquired, it was possible to calculate the isosteric enthalpy of adsorption for PIM-1 powder (-9.5 kJ mol-1), film (- 8.0 kJ mol-1) and the 30 wt% composite (-9.3 kJ mol-1). The stored and deliverable hydrogen contained within tanks featuring the tested materials were estimated, although only the MIL-101 powder on its own competes with other hydrogen storage adsorbents currently reported.

This thesis describes the synthesis of soluble Polymer of Intrinsic Microporosity (PIM-1), fluoro-endcapped PIM-1 (F-PIM-1) and copolymers of F-PIM-1 with poly(ethylene glycol) monomethyl ether (MeOPEG). The main aim of the project was to alter the porosity of microporous PIM-1 in three ways: (a) synthesis of copolymers of F-PIM-1 with MeOPEG (b) blending of PIM-1 with MeOPEG in various proportions; and (c) adsorption of MeOPEG from aqueous solution byPIM-1. PIM-1 and F-PIM-1 were synthesized by step growth polymerization of tetrafluoroterephthalonitrile (TFTPN) with 5,5',6,6'-tetrahydroxy-3,3,3',3'-tetramethyl-1,1'-spirobisindane (THSB), using the conventional method and a newly reported high shear mixing method. F-PIM-1 oligomers were then coupled to poly(ethylene glycol) monomethyl ether (MeOPEG). The products were analyzed by NMR, IR, MALDI ToF MSS, TGA and polystyrene based GPC as well as multidetector GPC techniques. The high shear technique generally produced high molar mass products and yields. This method was also more successful for copolymerization.Blending of PIM-1 and MeOPEG in different proportions resulted in macrophase separation. Copolymer products were used to facilitate mixing of blends (as compatibilizers), however only 5% of MeOPEG could be solubilised into a PIM-1 phase. The effect of compatibilizer was found to be affected by interaction between PIM-1 and copolymer. However, N2 adsorption studies showed that after thermal removal of MeOPEG, PIM-1 regained stable porosity with significant BET surface area.Fluorescence studies were aimed at applications of PIM-1 and copolymers in sensors. PIM-1 and copolymers, spin-coated on the polyester-based substrate Melinex, were studied with and without methanol treatment in an environment of different solvent vapours. The effect of time and volume on wavelength shift and change in intensity was studied. Polar solvents tended to cause a red shift with decrease in intensity while less polar solvents behaved otherwise. Based on fluorescence experiments, solvent profiles for PIM-1 and copolymers were established.