Rol de los factores de crecimiento Wnt sobre el desarrollo de polaridad neuronal y crecimiento axonal. Role of Wnt growth factors on neuronal polarity and axon growth.

El desarrollo y funcionamiento del sistema nervioso dependen de la formación de circuitos neuronales específicos y de programas intrínsecos y extrínsecos que actúan como moduladores del desarrollo neuronal. Inicialmente, los neuroblastos "sensan" a través de receptores específicos, la presencia en el medio de factores de crecimiento, como neurotrofinas clásicas (BDNF, NGF, etc), IGF-1, factores Wnts, que regulan la diferenciación neuronal, polarización, migración, etc. Hasta hace pocos años, las funciones específicas de los diferentes sistemas de factor de crecimiento-receptor en el establecimiento de polaridad y la regulación del crecimiento axonal eran mayormente desconocidas. Más recientemente, trabajos de nuestro y otros grupos de investigación han aportado significativamente al conocimiento de los mecanismos que involucran los sistemas IGF-1-receptor de IGF-1, BDNF-TrkB y NGF-TrkA sobre el desarrollo de polaridad neuronal. Sin embargo, si bien se conoce que los factores de crecimiento Wnt cumplen un rol crucial en eventos que ocurren durante la maduración neuronal (dendritogénesis, sinaptogénesis) poco se sabe sobre los mecanismos por los cuales estos factores regularían el establecimiento inicial de polaridad y el crecimiento axonal. Los factores Wnt como así también su primer efector intracelular Dishevelled (DVL) y sus cascadas de señalización participan de procesos como neurogénesis, guiado axonal, desarrollo dendrítico y formación y mantenimiento de sinápsis. Por estas razones, para el desarrollo del presente proyecto planeamos estudiar los efectos de los factores Wnts, su receptor Frizzled (Fz) y su efector DVL sobre el establecimiento de polaridad y la regulación del crecimiento axonal. También compararemos los efectos de los factores Wnt con los de IGF-1 (el único factor de crecimiento conocido esencial para el establecimiento de polaridad). Finalmente, intentaremos determinar cuál o cuáles de las cascadas intracelulares de señalización activadas por los Wnts están involucradas en sus efectos axogénicos. La metodología a utilizar se basará en el empleo de cultivos primarios de neuronas de hipocampo de embriones de rata de 18 días de gestación, los que serán expuestos a los factores Wnt y/o IGF-1. Se diseñarán experimentos tendientes a evaluar los efectos de dichos factores durante los diferentes estadíos de diferenciación neuronal que se analizarán por microscopía de fluorescencia confocal. Al mismo tiempo se realizarán ensayos de subfraccionamiento que permitan purificar conos de crecimiento aislados en los que se evaluará el rol local de Wnt y sus efectores sobre la fosforilación de quinasas que median la adición local de membrana y elongación axonal. Se examiná el rol de DVL sobre la especificación axonal a través de la expresión epistática en neuronas no diferenciadas como así también se bloqueará su expresión a tavés del uso de siRNA o cDNAs que actúen como dominantes negativas. Finalmente, se examinará una posible "transactivación" por IGF-1 o Wnts de sus receptores o primeros efectores intracelulares específicos, IRS-1- PI3K para IGF-1 y Dishevelled para Wnts. Para ello, se diseñarán experimentos en los que se utilizarán inhibidores farmacológicos específicos y se realizan ensayos de fosforilación en conos de crecimiento aislados y en cultivos neuronales. Los resultados serán cuantificados y sometidos softwares estadísticos adecuados.El desarrollo de estos experimentos nos permitirá examinar posibles paralelismos entre la activación del sistema Wnt-Frizzled-Dishevelled y del sistema IGF-1-Receptor de IGF-1-PI3K, el único sistema factor de crecimiento-receptor conocido esencial para el establecimiento de la polaridad neuronal y así poder lograr un acercamiento al/los posible mecanismo/s que regula/n la diferenciación neuronal y el crecimiento axonal.

PARTICIPACIÓN DEL RECONOCIMIENTO INMUNE INNATO EN LA RESPUESTA DE CARDIOMIOCITOS DURANTE LA INFECCIÓN EXPERIMENTAL CON TRYPANOSOMA CRUZI. Characterization of the cardiac innate immune response during Trypanosoma cruzi experimental infection.

La enfermedad de Chagas, causada por Trypanosoma cruzi, constituye la principal miocarditis infecciosa a nivel mundial. Crecientes evidencias revelan que la respuesta inmune innata tendría un rol determinante en la fisiopatología de las enfermedades cardiovasculares. La inmunidad innata es la primera línea de defensa, no específica, preprogramada para combatir agentes infecciosos. Este sistema censa la presencia de antígenos extraños a través de los receptores tipo toll (TLR) produciendo citoquinas y activando mecanismos microbicidas. Sin embargo, los TLRs también se hayan distribuidos en las células parenquimales no inmunes, jugando un importante rol tanto en la defensa como en la homeostasis de cada tejido. Durante la etapa aguda de la infección, el T. cruzi invade y se replica dentro de una amplia variedad de células y tejidos. Pero posteriormente, los parásitos son efectivamente eliminados de la mayoría de los tejidos persistiendo durante toda la vida en las células del músculo cardíaco y esquelético de los pacientes infectados. Debido a que el mantenimiento de la célula cardíaca infectada es crítica para la patogénesis de la enfermedad, los mecanismos que participan en la sobrevida de los cardiomiocitos están siendo foco de nuestro estudio. Hemos demostrado, que la infección ejerce efectos antiapoptóticos sobre células cardíacas aisladas. Nuestra hipótesis es que la inmunidad innata cardíaca estaría involucrada en el mantenimiento de la sobrevida de los miocitos así como en la defensa contra el parásito. Objetivo general: determinar la participación de la respuesta inmune innata cardíaca en el desarrollo de la enfermedad de Chagas experimental murina. Objetivos específicos: 1) Analizar el compromiso de TLRs en la respuesta anti-apoptótica y de autofagia de cardiomiocitos aislados de ratones salvajes y de ratones deficientes en TLR4, TLR2 y en MyD88, molécula adaptadora de la señalización por TLRs, sometidos a la infección con el parásito. 2) Determinar la importancia de la actividad cisteín proteasa parasitaria en el grado de infectividad y la sobrevida de cultivos primarios de ratones salvajes infectados con parásitos transgénicos que poseen disminuída o nula actividad cisteín proteasa. 3) Establecer la cinética de expresión de TLR2/TLR6, TLR4 y TLR9, factores antiapoptóticos (Bcl-2, Bcl-xL, etc.), daño cardíaco y la carga parasitaria en el tejido cardíaco de ratones infectados salvajes y/o deficientes antes mencionados. Materiales y Métodos: Los animales serán infectados i.p. con 5x103 parásitos y se determinará la cinética de expresión de los mediadores mencionados por western blot e inmunofluorescencia, la carga parasitaria será determinada por qRT-PCR. Como controles se procesarán animales inyectados con solución salina. En cultivos primarios de cardiomiocitos de ratones neonatos salvajes y deficientes infectados se estudiará la carga parasitaria, la activación de los mecanismos microbicidas (producción de óxido nítrico, metabolitos reactivos del oxígeno y del nitrógeno, ciclooxigenasa, etc.), producción de citoquinas y expresión de moléculas anti-apoptóticas (Bcl-2, Bcl-xL, Bax, etc.). Se explorará la tasa de apoptosis en cultivos deprivados de suero. La autofagia se analizará por microscopia electrónica. Cultivos controles serán mantenidos en medio o tratados con ligandos de los diferentes TLRs. Resultados preliminares sugieren que tanto TLR2 como Bcl-2 se incrementan en tejido cardíaco infectado. Esto nos lleva a profundizar en los mecanismos observados en cultivos y estudiarlos en un modelo in vivo, analizando la posible importancia que tiene la inmunidad innata cardíaca en el control del establecimiento de la infección. La comprensión de los mecanismos que mantienen la sobrevida de los cardiomiocitos y su respuesta a la infección es importante ya que el conocimiento de las bases moleculares es fundamental para el desarrollo de nuevos agentes quimioterapéuticos. Chagas disease is endemic in Central and South America and causes the most common myocarditis worldwide. We have previously reported that the cardiotrophic parasite Trypanosoma cruzi, its etiological agent, protects cardiomyocytes against apoptosis induced by growth factor deprivation activating the PI3K/Akt and MEK1/ERK signaling pathways. Recent studies have shown that local innate immunity plays a key role in initiating and coordinating homeostatic as well as defense responses in the heart. One of the mechanisms by which the innate immune system senses the presence of foreign antigens is through TLRs. The stimulation of these receptors leads to the activation and nuclear translocation of NF-kB transcription factor and the production of cytokines. Proinflammatory cytokines, in turn, appear to play a central role in the orchestration and timing of the intrinsic cardiac stress response providing, under different situations, instantaneous anti-apoptotic cytoprotective signals, which allow tissue repair and/or remodeling. The aim of the present project is to study the cardiomyocyte innate immune responses to T. cruzi infection and its role in target cell protection from apoptosis. Specific objectives: 1) Study the mechanism triggered by TLR in the anti-apoptotic response and parasite load of infected cardiomyocyte primary cultures from wild type and mice deficient in TLR2, TLR4 or MyD88. 2) Determine the effect of parasite cisteín protease activity on primary cultures from wild type mice. 3) Determine the TLR signaling-involvement in parasite load and survival indicators in deficient mice. Preliminary results showed us that cardiac-TLR2 may be involved in the anti-apoptotic effect elicited by the parasite and prompted us to establish the mechanisms triggered by the innate immunity that mediate parasite persistence within the host cell.

Validação da versão em português do Minnesota Living with Heart Failure Questionnaire

FUNDAMENTO: O Minnesota Living with Heart Failure Questionnaire (MLHFQ) é uma importante ferramenta de avaliação da qualidade de vida em pacientes com insuficiência cardíaca. Apesar de amplamente usado em nosso meio, não contávamos com a sua tradução e validação em língua portuguesa. OBJETIVO: Este estudo pretendeu traduzir e validar a versão em português do MLHFQ em pacientes com insuficiência cardíaca. MÉTODOS: Quarenta pacientes com insuficiência cardíaca (30 homens, FEVE 30±6%, 55% de etiologia isquêmica, NYHA I a III) com estabilidade clínica e terapia medicamentosa otimizada realizaram teste cardiopulmonar máximo para avaliação da capacidade física. Logo após, o MLHFQ, devidamente traduzido, foi aplicado por um mesmo pesquisador. A classe funcional NYHA foi encaminhada pela equipe medica. RESULTADOS: A versão em português do MLHFQ apresentou-se com a mesma estrutura e métrica da versão original. Não houve dificuldade na aplicação e compreensão do questionário por parte dos pacientes. A versão em português do MLHFQ mostrou-se concordante com o pico de VO2, o tempo de exercício do teste cardiopulmonar e com a classificação funcional da NYHA. Não houve diferença da média do escore do questionário entre os grupos de etiologia isquêmica e não-isquêmica. CONCLUSÃO: A versão em língua portuguesa da MLHFQ, proposta no presente estudo, demonstrou ser válida em pacientes com insuficiência cardíaca, constituindo uma nova e importante ferramenta para avaliar a qualidade de

Defining habitat characteristics influencing the distribution, density and growth of Plaice (Pleuronectes Platessa) and Dab (Limanda Limanda) on west of Ireland nursery grounds

Plaice (Pleuronectes platessa, L.) and dab (Limanda limanda, L.) are among the most abundant flatfishes in the north-eastern Atlantic region and the dominant species in shallow coastal nursery grounds. With increasing pressures on commercial flatfish stocks in combination with changing coastal environments, better knowledge of population dynamics during all life stages is needed to evaluate variability in year-class strength and recruitment to the fishery. The aim of this research was to investigate the complex interplay of biotic and abiotic habitat components influencing the distribution, density and growth of plaice and dab during the vulnerable juvenile life stage and to gain insight in spatial and temporal differences in nursery habitat quality along the west coast of Ireland. Intraspecific variability in plaice diet was observed at different spatial scales and showed a link with condition, recent growth and morphology. This highlights the effect of food availability on habitat quality and the need to consider small scale variation when attempting to link habitat quality to feeding, growth and condition of juvenile flatfish. There was evidence of trophic, spatial and temporal resource partitioning between juvenile plaice and dab allowing the co-existence of morphologically similar species in nursery grounds. In the limited survey years there was no evidence that the carrying capacity of the studied nursery grounds was reached but spatial and interannual variations in fish growth indicated fluctuating environments in terms of food availability, predator densities, sediment features and physico-chemical conditions. Predation was the most important factor affecting habitat quality for juvenile plaice and dab with crab densities negatively correlated to fish condition whereas shrimp densities were negatively associated with densities of small-sized juveniles in spring. A comparison of proxies for fish growth showed the advantage of Fulton’s K for routine use whereas RNA:DNA ratios proved less powerful when short-term environmental fluctuations are lacking. This study illustrated how distinct sets of habitat features can drive spatial variation in density and condition of juvenile flatfish highlighting the value of studying both variables when modeling habitat requirements. The habitat models generated in this study also provide a powerful tool to predict potential climate and anthropogenic impacts on the distribution and condition of juveniles in flatfish nurseries. The need for effective coastal zone management was emphasized to ensure a sustainable use of coastal resources and successful flatfish recruitment to the fishery.

Age, growth and reproductive biology of whiting Merlangius merlangus (Linnaeus 1758) in the Celtic Sea

Age, growth and reproductive biology were investigated for whiting (Merlangius merlangus) captured from the Celtic Sea (ICES division Vllg), for the period January 2001 to January 2002. Females dominated the sex ratio of 1: 2.25. The relative abundance of females exceeded the number of males in all length classes. The relationship between weight (g) and total length (cm) was the same for male and female whiting. A total of 973 fish were aged and the maximum age recorded was 11 years. Results from an intercalibration exercise showed 87% agreement in age readings between the author and an expert in ageing whiting at the Marine Institute. Females were dominated by 2 year olds, while males were dominated by 3 year olds. Lx was estimated as 38cm and a growth rate was calculated K = 0.3769 year'1. Females were fully recruited to the fishery at 3 years of age, while the age at full recruitment (tr) for males was 4 years. Female whiting spawned from late February to June 2001 and matured at a total length of 23 cm in their first year. Female whiting reached L5o at a total length of 28 cm and 2.7 years of age. Male whiting spawned from February to June 2001. They matured at a total length of 21 cm and in their first year. Male whiting reached L50 at a total length of 30.4 cm and 3.6 years of age. The following critical points should be taken into account in the management of the Celtic Sea whiting stock: An Fpa should be established in order to assess the current level of fishing mortality; The maturity ogives need further study; The extent of gutting of large fish before landing by fishers in the fleet should be investigated and the apparent decline in size of 4 - 7 year old fish in the Celtic Sea between 1996 and 2001 needs to be assessed.

Age, growth and population dynamics of lemon sole Microstomus kitt (Walbaum 1792) sampled off the West Coast of Ireland

The age, growth, maturity and population dynamics of lemon sole (Microstomus kitt), captured off the west coast of Ireland (ICES division Vllb), were determined for the period November 2000 to February 2002. The maximum age recorded was 14 years. Males of the population were dominated by 4 year olds, while females were dominated by 5 year olds. Females dominated the sex ratio in the overall sample, each month sampled, at each age and from 22cm in total length onwards (when N > 20). Possible reasons for the dominance of females in the sex ratio are discussed. Three models were used to obtain the parameters of the von Bertalanfly growth equation. These were the Ford-Walford plot (Beverton and Holt 1957), the Gulland and Holt plot (1959) and the Rafail (1973) method. Results of the fitted von Bertalanffy growth curves showed that female lemon sole o f f the west coast of Ireland grew faster than males and attained a greater size. Male and female lemon sole mature from 2 years of age onwards. There is evidence in the population o f a smaller asymptotic length (L«, = 34.47cm), faster growth rate (K = 0.1955) and younger age at first maturity, all of which are indicative o f a decrease in population size, when present results are compared to data collected in the same area 22 years earlier. Results of the yield per recruit curve indicate that lemon sole are currently being over-fished o f f the west coast of Ireland. Problems of selectivity within the sampling method, particularly at the discarding stage, may have influenced the outcome of results of the models used in the assessment of this stock. Therefore, additional/future work on this species should include catch data which incorporates discards and not landings data alone.

The production of vermicompost from dairy sludge and its value as a plant growth medium

Dairy sludge generated at Glanbia Ingredients Ltd., Kilkenny has up until now been landspread. This study investigated the feasibility of using earthworms to vermicompost the sludge as an alternative method of treatment. It was found that high levels of ammonia in the sludge led to earthworm fatality but that by manually aerating the sludge the ammonia could be volatilised or by adding zeolite the ammonia could be absorbed, thus solving the problem. In a medium scale trial, the earthworm species Dendrobaena veneta and Eisenia fetida dominated the polyculture. Earthworms grew and generated cocoons during vermicomposting. During vermicomposting no leachate was generated. Nutrient changes took place during vermicomposting. There were high levels of nitrate, increased calcium and sulphate in the vermicomposted dairy sludge. The amount of magnesium, potassium and chloride did not change, while phosphate was undetectable after vermicomposting. The levels of nitrate and phosphate were good indicators of the extent of vermicomposting. The vermicomposted dairy sludge provided improved growth and yields of radishes and barley compared to the dairy sludge and control. Compared to the vermicompost, the dairy sludge provided heavier ryegrass yields and more marigolds with larger flower diameters. Generally, it is the amount of phosphate in dairy sludge that dictates how much can be applied as a fertiliser on land. Vermicomposting reduced the amount of phosphate to an undetectable level but on the other hand created a problem of high nitrate levels. In a pot trial with grass grown in vermicompost the nitrate leached from the vermicompost. In field conditions the leaching of nitrate might occur and could cause an increased risk of contamination of groundwater and watercourses.

Data for the sustainable management of an emerging fishery: Age, growth and stock structure of Atlantic saury Scomberesox saurus saurus (Walbaum), in the Northeastern Atlantic Ocean

Almost half of Ireland’s commercial stocks face overexploitation. As traditional species decrease in abundance and become less profitable, the industry is increasingly turning to alternate species. Atlantic saury (Scomberesox saurus saurus (Walbaum)) has been identified as a potential species for exploitation. Very little information is available on its biology or population dynamics, especially for Irish waters. This thesis aims to obtain sound scientific data, which will help to ensure that a future Atlantic saury fishery can be sustainably managed. The research has produced valuable data, some of which contradicts previous studies. Growth of Atlantic saury measured using otolith microstructure is found to be more than twice that previously calculated from annual structures on scales and otoliths. This results in a significant reduction of the expected life span from five to about two years. Investigation of maturity stage at age indicates that Atlantic saury will reproduce for the first time at age one and will survive for one or at most two reproduction seasons. It is concluded that a future Irish fishery will target mostly fish prior to their first reproduction. Finally the thesis gives some insights into the population structure of Atlantic saury, by analysis of otolith morphometric. Significant differences are detected between Northeastern Atlantic and western Mediterranean Sea specimens of the 0+ age class (less than one year old). The implications of these results for the management of an emerging fishery are discussed.

Ireland’s understudied flatfish: reproduction, age and growth of the dab Limanda limanda (L.) in Irish coastal waters

In Ireland, although flatfish form a valuable fishery, little is known about the smallest, the dab Limanda limanda. In this study, a variety of parameters of reproductive development, including ovarian phase description, gonadosomatic index (GSI), hepatosomatic index (HSI), relative condition (Kn) and oocyte size were analysed to provide information on the dab’s reproductive cycle and spawning periods. Sampling were collected monthly over an 18-month period using bottom trawls of the Irish coastline. A six phase macroscopic guide was developed for both sexes of dab, and verified using histology. In comparisons of macroscopic and microscopic phases, there was high agreement in the proposed female guide (86%), with males demonstratively lower (62%). No significant bias was observed between the the two reproductive methods. When the male macroscopic guide was examined, misclassification was high in phase 5 and phase 5 (41%), with 96% of misclassification occurring in adjacent phases. The sampled population was primarily composed of females, with ratios of females to males 1:0.6, although the predominance of females was less noticeable during the reproductive season. Oocyte growth in dab follows asynchronous development, and spawn over a protracted period indicating a batch spawning strategy. Spawning occurred mainly in early spring, with total regeneration of gonads by May. The length at which 50% of the population was reproductively mature was identified as 14cm and 17cm, for male and female dab, respectively. Precision and bias in age determinations using whole otoliths to age dab was investigated using six age readers from various institutions. Low levels of precision were obtained (CV: 10-23%) inferring the need for an alternative methodology. Precision and bias was influence by the level of experience of the reader, with ageing error attributed to interpretative differences and difficulty in edge determination. Sectioned otolith age determinations were subsequently compared to whole otolith age determinations using two age readers experienced in dab ageing. Although increased precision was observed in whole otoliths from previous estimates (CV=0%, 0% APE), sectioned otoliths were used for growth models. This was based on multinominal logistic regression on age length keys developed using both ageing methods. Biological data (length and age) for both sexes was applied to four growth models, where the Akaike criterion and Multi model Inference indicated the logistic model as having the best fit to the collected data. In general, female dab attained a longer length then males, with growth rates significantly different between the two sexes. Length weight relationships between the two sexes were also significantly different.