Avaliação da inervação entérica e análise proteômica do intestino delgado de ratos expostos à dose aguda ou crônica de fluoreto

O trato gastrointestinal (TGI) é a principal rota de exposição ao fluoreto (F) e o seu mais importante sítio de absorção. Acredita-se que a toxicidade do F comprometa a fisiologia do intestino, devido à relevante sintomatologia gastrointestinal relatada em consequência da exposição excessiva ao F. A função intestinal é controlada por uma complexa rede neuronal interligada e incorporada à parede deste órgão, denominada Sistema Nervoso Entérico (SNE). Embora os efeitos tóxicos do F sobre o Sistema Nervoso Central sejam descritos na literatura, não há estudos relacionados à sua toxicidade sobre o SNE. Neste estudo realizado em ratos, foi avaliado o efeito da exposição aguda ou crônica ao F, sobre a população geral de neurônios entéricos e sobre as subpopulações que expressam os principais neurotransmissores entéricos: Acetilcolina (ACh), Óxido Nítrico (NO), Peptídeo Vasoativo Intestinal (VIP), Peptídeo Relacionado ao Gene da Calcitonina (CGRP) e Substância P (SP). Os animais foram divididos em 5 grupos: 3 destinados à exposição crônica (0 ppm, 10 ppm ou 50 ppm de F na água de beber) e 2 à exposição aguda (0 ou 25 mgF/Kg por gavagem gástrica). Foram coletados os 3 segmentos do intestino delgado (duodeno, jejuno e íleo) e processados para a detecção da HuC/D, ChAT, nNOS, VIP, CGRP e SP, através de técnicas de imunofluorescência, no plexo mioentérico. Foram obtidas imagens para a realização da análise quantitativa dos neurônios da população geral (HuC/D) e nitrérgicos (imunorreativos à nNOS); e morfométrica dos neurônios imunorreativos à HuC/D ou nNOS; e das varicosidades imunorreativas à ChAT, VIP, CGRP ou SP. Amostras dos 3 segmentos intestinais foram preparadas e coradas em Hematoxilina e Eosina para análise histológica da morfologia básica. O segmento intestinal considerado mais afetado na análise morfométrica da população geral de neurônios, o duodeno, foi selecionado para a realização da análise proteômica, com o objetivo de oferecer o seu perfil proteico e determinar diferenças na expressão proteica em decorrência da exposição crônica ou aguda ao F. A análise da concentração de F no plasma sanguíneo foi realizada para a confirmação da exposição. Na análise quantitativa, o grupo de 50 ppm F, apresentou uma diminuição significativa na densidade da população geral de neurônios do jejuno e do íleo e na densidade dos neurônios imunorreativos à nNOS no duodeno e no jejuno. Quanto à análise morfométrica, a população geral e as subpopulações neuronais entéricas avaliadas apresentaram alterações morfológicas significativas, tanto após a exposição crônica quanto a aguda. Para a análise proteômica do duodeno, verificou-se que da associação de seus genes a um termo, e assim classificadas de acordo com diferentes processos biológicos. No caso do grupo da dose aguda, o processo biológico com a maior porcentagem de genes associados foi a geração de metabólitos precursores e energia (27% das proteínas); enquanto para os grupos de 10 e 50 ppm F foram o processo metabólico da piridina (41%) e a polimerização proteica (33%), respectivamente.

Efeitos dos agrotóxicos Kraft®36EC e Score®250EC (e seus princípios ativos) em ecossistemas aquáticos: análises comparativas e ecossistêmicas

Com o advento da agricultura ampliou-se a produção alimentar e os bens de consumo, no entanto, os riscos ambientais também foram maximizados em função da adoção de técnicas produtivas baseadas no uso intensivo de insumos agrícolas. Esta problemática é mundial, embora mais evidenciada nos países em desenvolvimento e que tem, na produção agrícola, a base de sua economia. O Brasil enquadra-se nesta situação e desde 2009 é considerado o maior consumidor de agrotóxicos do mundo, criando um cenário de risco ambiental e de saúde humana. Os efeitos ambientais, base deste estudo, estão relacionados não somente à perda de espécies não-alvo, uma vez que os agrotóxicos não são seletivos, mas também as alterações em nível ecossistêmico, a qual se relaciona com as perdas das funções e dos serviços gerados pelos sistemas naturais. Adiciona-se a esta complexidade, a forma de ação de cada agrotóxico, a distribuição dos mesmos nos diferentes compartimentos (ar, solo e água), o período de permanência de cada um, as relações sinérgicas decorrentes das interações entre diferentes produtos, a formação de subprodutos no processo de degradação, entre outros fatores, como as diferenças existentes entre o ingrediente ativo e a formulação comercial, na qual existem os chamados ingredientes inertes em sua composição, os quais podem ser muito mais tóxicos para espécies e ecossistemas. Considerando esta abordagem, a presente pesquisa foi desenvolvida com base na realidade de um local de referência, o município de Bom Repouso (MG/BR), no qual a intensificação da produção de morango e batata tem trazido uma série de riscos sociais e ambientais. Semelhante a outras regiões produtivas do país, o uso de agrotóxicos é recorrente, amplo e irrestrito, com destaque para as formulações comerciais Kraft®36EC e Score®250EC, as quais, juntamente com seus respectivos ingredientes ativos (abamectina e difenoconazol), foram avaliadas por meio de testes de toxicidade com espécies de diferentes níveis tróficos representativas de um ecossistema aquático, gerando informações que foram avaliadas em nível de espécie e de ecossistema, simulando o cenário de aplicação dos produtos no local de referência. Os resultados obtidos permitiram concluir sobre as diferenças de sensibilidade das espécies e quais seriam as mais indicadas para se avaliar os efeitos tóxicos de ambos os agrotóxicos; os efeitos diferenciados entre a formulação comercial e os ingredientes ativos; bem como as respostas em termos de espécies e de ecossistemas, demonstrando a necessidade de que ambas as análises sejam consideradas na avaliação de risco ecológico.

Comparação de métodos ecotoxicológicos na avaliação de sedimentos marinhos e estuarinos

A amônia não ionizada pode ser interferente nos testes de toxicidade com a água intersticial de sedimentos marinhos e estuarinos. Com o objetivo de se encontrar em uma alternativa que minimizasse o efeito da amônia avaliou-se, em paralelo à água intersticial, o método que utiliza a interface sedimento/água. Foram analisadas 25 amostras de sedimento através do teste de toxicidade crônica de curta duração com Lytechinus variegatus. Foi observada correlação significativa entre os efeitos tóxicos para os diversos estágios embrio-larvais e a concentração de amônia não ionizada para os dois métodos avaliados. Quando comparadas com a interface sedimento/água, foram observadas concentrações superiores de amônia não ionizada em todas as amostras de água intersticial, que isoladamente, poderiam causar os efeitos tóxicos observados. Conclui-se que o teste de toxicidade na interface sedimento/água é o método mais adequado para avaliação de sedimentos marinhos e estuarinos, pois possibilitou a utilização de um maior número de resultados de testes de toxicidade do que aquele com água intersticial, uma vez que minimizou a influência da amônia. Foi evidenciada correlação significativa entre os resultados das concentrações de HPAs determinadas nas amostras de sedimento bruto e a toxidade observada nos diversos estágios embrio-larvais, pelo método da água intersticial. Para os metais, obteve-se correlação significativa com os efeitos tóxicos nos referidos estágios determinados na interface sedimento/água. Não foi evidenciada correlação entre a granulometria e o efeito tóxico nas amostras. O registro dos efeitos nos diferentes estágios embrio-larvais mostrou-se bastante útil, pois além de oferecer informações a respeito da toxicidade da amostra (ausência ou presença) possibilitou a hierarquização, em termos da intensidade de efeito adverso, daquelas amostras tóxicas.

Aplicação de testes de toxicidade com organismos marinhos para a análise de efluentes industriais lançados em áreas estuarinas

Com o objetivo de aplicar e avaliar a viabilidade de uso dos métodos disponíveis com organismos marinhos, no controle da toxicidade de efluentes líquidos que são lançados em ambientes estuarinos, foram realizados testes de toxicidade aguda com os crustáceos Mysidopsis juniae, Artemia sp, Temora stylifera e Acartia IiIljeborgi e testes de toxicidade crônica de curta duração com o equinodermo Lytechinus variegatus, utilizando-se os efluentes industriais de uma indústria siderúrgica, COSIPA e uma fábrica de fertilizantes, ULTRAFÉRTIL/JARDIM SÃO MARCOS, ambos lançados no estuário do Rio Cubatão. Dentre os organismos-testes utilizados, para avaliação do efeito tóxico agudo, o misidáceo M. juniae foi o mais sensível para ambos os efluentes, sendo que Artemia sp foi o menos sensível. Testes de toxicidade crônica com L. variegatus também se mostraram bastante úteis para avaliação de efeitos subletais. Os efluentes analisados apresentaram grande variabilidade durante o período de estudo, o que foi evidenciado através do cálculo do coeficiente de variação para testes com M. juniae. Foi avaliado, também, o efeito da salinidade sobre a sensibilidade dos crustáceos M. juniae e Artemia sp a agentes químicos (zinco e DSS) e aos efluentes industriais. A salinidade não interferiu significativamente nos resultados observados, com exceção de um experimento realizado a 15x10-3 com Artemia sp, com o efluente da COSIPA. Verificou-se, ainda, o possível efeito da utilização de salmoura obtida através dos processos de congelamento e evaporação da água do mar, sendo que o primeiro processo foi indicado para salinização de efluentes.

Electrohydrodynamic Atomization for Improved Macromolecular Drug Delivery

With advances in drug research, the use of biological therapeutics is becoming a reality. Unfortunately, methods for processing and delivering these fragile macromolecules often limit their therapeutic potential. For this dissertation, we explore the aerosolization of macromolecules by way of electrohydrodynamic atomization (EHDA) and how this method can be used to process and deliver therapeutics. EHDA employs a high voltage to break a column of liquid into drops. It was unknown if or how the residual charge left of the resulting droplets would affect lung cells. An in vitro experiment was conducted to spray aerosolized DNA, by way of EHDA, onto human derived lungs cells to test for immunogenic and toxic effects. The lung cells displayed no immunogenic or toxic response to the DNA or high voltage. Previous researchers have used EHDA to aerosolize proteins with mixed results. This work sets forth a simplified thermodynamic theory and provides recommendations to pharmaceutical companies on how to design more stable protein formulations for aerosol processing or delivery. Finally, a new method of producing liposomes was created. It constructs the liposome one layer at a time. The inside of the liposome is sprayed by EHDA, with the lipid and drug in solution together. As the sprayed monolayer passes through a pool containing a solution of lipid in water, the second part of the bilayer attaches to the inner layer creating a complete bilayer liposome.

Non-Viral Gene Therapy to Cells of the bovine Intervertebral Disc

Background. Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and degeneration. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM).1 Further studies showed that growth factors from transforming growth factor β (TGFβ) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC).2 Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods. Bovine nucleus pulposus (bNPC) and annulus fibrosus cells (bAFC) were harvested from bovine coccygeal IVD. Primary cells were then electroporized with plasmid GDF6 (Origene, vector RG211366) by optimizing parameters using the Neon Transfection system (Life Technologies, Basel). After transfection, cells were cultured in 2D monolayer or 3D alginate beads for 7, 14 or 21 days. Transfection efficiency of pGDF6 was analyzed by immunohistochemistry and fluorescent microscopy. Cell phenotype was quantified by real-time RT-PCR. To test a non-viral gene therapy applied directly to 3D whole organ culture, coccygeal bovine IVDs were harvested as previously described. Bovine IVDs were transfected by injection of plasmid GDF6 into the center. Electroporation was performed with ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) using 2-needle array electrode or tweezertrodes. 72 h after tranfection discs were fixed and cryosectioned and analyzed by immunofluorescence against GDF6. Results. RT-PCR and immunohistochemistry confirmed up-regulation of GFP and GDF6 in the primary bNPC/bAFC culture. The GFP-tagged GDF6 protein, however, was not visible, possibly due to failure of dimer formation as a result of fusion structure. Organ IVD culture transfection revealed GDF6 positive staining in the center of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GDF6 positive cells. Conclusion. Non-viral transfection is an appealing approach for gene therapy as it fulfills the translational safety aspects of transiency and lacks the toxic effects of viral transduction. We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgements. This project was funded by the Lindenhof Foundation (Funds “Research & Teaching”) Project no. 13-02-F. The imaging part of this study was performed with the facility of the Microscopy Imaging Center (MIC), University of Bern. References. Roughly PJ (2004): Spine (Phila), 29:2691-2699 Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014), Arthritis Research & Therapy, 16:R67

Surgery alone versus chemoradiotherapy followed by surgery for resectable cancer of the oesophagus: a randomised controlled phase III trial

Background Resection remains the best treatment for carcinoma of the oesophagus in terms of local control, but local recurrence and distant metastasis remain an issue after surgery. We aimed to assess whether a short preoperative chemoradiotherapy regimen improves outcomes for patients with resectable oesophageal cancer. Methods 128 patients were randomly assigned to surgery alone and 128 patients to surgery after 80 mg/m(2) cisplatin on day 1, 800 mg/m(2) fluorouracil on days 1-4, with concurrent radiotherapy of 35 Gy given in 15 fractions. The primary endpoint was progression-free survival. Secondary endpoints were overall survival, tumour response, toxic effects, patterns of failure, and quality of life. Analysis was done by intention to treat. Findings Neither progression-free survival nor overall survival differed between groups (hazard ratio [HR] 0.82 [95% CI 0.61-1.101 and 0.89 [0.67-1.19], respectively). The chemoradiotherapy-and-surgery group had more complete resections with clear margins than did the surgery-alone group (103 of 128 [80%] vs 76 of 128 [59%], p=0.0002), and had fewer positive lymph nodes (44 of 103 [43%] vs 69 of 103 [67%], p=0.003). Subgroup analysis showed that patients with squamous-cell tumours had better progression-free survival with chemoradiotherapy than did those with non-squamous tumours (HR 0.47 [0.25-0.86] vs 1.02 [0.72-1.44]). However, the trial was underpowered to determine the real magnitude of benefit in this subgroup. Interpretation Preoperative chemoradiotherapy with cisplatin and fluorouracil does not significantly improve progression-free or overall survival for patients with resectable oesophageal cancer compared with surgery alone. However, further assessment is warranted of the role of chemoradiotherapy in patients with squamouscell tumours.

Comparison of active venom components between Eastern brown snakes collected from South Australia and Queensland

The abundance and activity of the prothrombin activator (pseutarin C) within the venom of the Eastern brown snake (Pseudonaja textilis textilis) is the primary determinant of its coagulation potency. Textilinin-1, also in this venom, is a plasmin inhibitor which is thought to exert its toxic effects through the slowing of fibrinolysis. The aim of this report is to determine if there are differences in the potency of the venom from Eastern brown snakes collected from South Australia (SA) compared to those from Queensland (QLD). A concentration of 0.4 mu g/ml venom protein from six QLD specimens clotted citrated plasma in an average time of 21.4 +/- 3.3 s compared to 68.7 +/- 2.4 s for the same amount of SA venom (averaged for six individuals). The more potent procoagulant activity of the QLD venom was measured between 0.4 and 94 mu g/ml venom protein in plasma. The anti-plasmin activity of textilinin was also greater in the venom of the snakes collected from QLD, causing full inhibition of plasmin at approximately 1.88 mu g/ml of venom protein compared to approximately 7.5 mu g/ml for the SA venoms. It is concluded that geographic differentiation of the Eastern brown snakes results in significant differences venom potency.

Determinants of biological activity of nanomaterials in innate immunity cells: the role of aspect ratio, environmental contaminants and protein corona

As defined by the European Union, “ ’Nanomaterial’ (NM) means a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or agglomerate, where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm-100 nm ” (2011/696/UE). Given their peculiar physico-chemical features, nanostructured materials are largely used in many industrial fields (e.g. cosmetics, electronics, agriculture, biomedical) and their applications have astonishingly increased in the last fifteen years. Nanostructured materials are endowed with very large specific surface area that, besides making them very useful in many industrial processes, renders them very reactive towards the biological systems and, hence, potentially endowed with significant hazard for human health. For these reasons, in recent years, many studies have been focused on the identification of toxic properties of nanostructured materials, investigating, in particular, the mechanisms behind their toxic effects as well as their determinants of toxicity. This thesis investigates two types of nanostructured TiO2 materials, TiO2 nanoparticles (NP), which are yearly produced in tonnage quantities, and TiO2 nanofibres (NF), a relatively novel nanomaterial. Moreover, several preparations of MultiWalled Carbon Nanotubes (MWCNT), another nanomaterial widely present in many products, are also investigated.- Although many in vitro and in vivo studies have characterized the toxic properties of these materials, the identification of their determinants of toxicity is still incomplete. The aim of this thesis is to identify the structural determinants of toxicity, using several in vitro models. Specific fields of investigation have been a) the role of shape and the aspect ratio in the determination of biological effects of TiO2 nanofibres of different length; b) the synergistic effect of LPS and TiO2 NP on the expression of inflammatory markers and the role played therein by TLR-4; c) the role of functionalization and agglomeration in the biological effects of MWCNT. As far as biological effects elicited by TiO2 NF are concerned, the first part of the thesis demonstrates that long TiO2 nanofibres caused frustrated phagocytosis, cytotoxicity, hemolysis, oxidative stress and epithelial barrier perturbation. All these effects were mitigated by fibre shortening through ball-milling. However, short TiO2 NF exhibited enhanced ability to activate acute pro-inflammatory effects in macrophages, an effect dependent on phagocytosis. Therefore, aspect ratio reduction mitigated toxic effects, while enhanced macrophage activation, likely rendering the NF more prone to phagocytosis. These results suggest that, under in vivo conditions, short NF will be associated with acute inflammatory reaction, but will undergo a relatively rapid clearance, while long NF, although associated with a relatively smaller acute activation of innate immunity cells, are not expected to be removed efficiently and, therefore, may be associated to chronic inflammatory responses. As far as the relationship between the effects of TiO2 NP and LPS, investigated in the second part of the thesis, are concerned, TiO2 NP markedly enhanced macrophage activation by LPS through a TLR-4-dependent intracellular pathway. The adsorption of LPS onto the surface of TiO2 NP led to the formation of a specific bio-corona, suggesting that, when bound to TiO2 NP, LPS exerts a much more powerful pro-inflammatory effect. These data suggest that the inflammatory changes observed upon exposure to TiO2 NP may be due, at least in part, to their capability to bind LPS and, possibly, other TLR agonists, thus enhancing their biological activities. Finally, the last part of the thesis demonstrates that surface functionalization of MWCNT with amino or carboxylic groups mitigates the toxic effects of MWCNT in terms of macrophage activation and capability to perturb epithelial barriers. Interestingly, surface chemistry (in particular surface charge) influenced the protein adsorption onto the MWCNT surface, allowing to the formation of different protein coronae and the tendency to form agglomerates of different size. In particular functionalization a) changed the amount and the type of proteins adsorbed to MWCNT and b) enhanced the tendency of MWCNT to form large agglomerates. These data suggest that the different biological behavior of functionalized and pristine MWCNT may be due, at least in part, to the different tendency to form large agglomerates, which is significantly influenced by their different capability to interact with proteins contained in biological fluids. All together, these data demonstrate that the interaction between physico-chemical properties of nanostructured materials and the environment (cells + biological fluids) in which these materials are present is of pivotal importance for the understanding of the biological effects of NM. In particular, bio-persistence and the capability to elicit an effective inflammatory response are attributable to the interaction between NM and macrophages. However, the interaction NM-cells is heavily influenced by the formation at the nano-bio interface of specific bio-coronae that confer a novel biological identity to the nanostructured materials, setting the basis for their specific biological activities.

Characterization of tumour necrosis factor alpha release by human granulocytes in response to procainamide challenge

The role of human granulocytes in the promotion of procainamide (PA) toxicity in vitro has been studied and one of the agents responsible for DNA strand scission and cell death in human target cells has been characterized. Crude peripheral blood mononuclear cells (cPBMNs) isolated by density centrifugation, and the lymphocyte cell lines--CCRF-HSB2 and WIL-2NS--were exposed to PA, and DNA strand breaks were quantified by fluorescent analysis of DNA unwinding. Therapeutic plasma concentrations of PA (0-50 microM) caused dose-dependent cytotoxicity, determined by dye exclusion, and strand breaks in cPBMNs incubated for 3 and 1.5 hr at 37 degrees, respectively. Using 50 microM PA a five-fold increase in DNA strand breaks was observed after 1.5 hr, with significant induction of strand breaks also being observed for 10 and 25 microM concentrations. Toxicity was much reduced in lymphocyte cell lines (maximal killing = 3.0% at 50 microM PA compared with 13.2% in cPBMNs). A similar decrease in toxicity was observed where N-acetyl procainamide (NAPA) was substituted for PA (less than 50% of strand breaks at all concentrations). Further investigations showed that the presence of a contaminating granulocyte population in the cPBMN fraction was responsible for the induction of PA toxicity. Incubation of a highly enriched granulocyte population with PA for 1 hr prior to exposure to purified peripheral blood mononuclear cells (pPBMNs) led to the complete restoration of the toxic effects. The resulting cyto- and genotoxicity were not significantly different to levels observed in cPBMNs. Significantly, incubation of granulocytes with NAPA did not induce toxicity in target pPBMNs. Ultrafiltration of granulocyte supernatants led to the identification of two toxic fractions of < 3000 and > 30,000 Da. Temporal studies showed that the toxicity associated with the < 3000 Da fraction appeared during the first 10-15 min incubation with PA whereas the > 30,000 Da fraction did not display significant toxicity until the 40-60 min period. Further assessment of the nature of these agents indicated that the 30,000 Da fraction was a protein. SDS-PAGE analysis showed an inducible 17,800 Da species appearing in granulocyte supernatants after 40 min incubation with PA. Dot blot analysis indicated that tumour necrosis factor alpha (TNF alpha) was present in the > 30,000 Da fraction. Evidence that TNF alpha was the high-molecular weight species responsible for PA-induced toxicity was obtained from neutralization assays employing an anti-TNF alpha antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

An age-related increase in resistance to DNA damage-induced apoptotic cell death is associated with development of DNA repair mechanisms

Neurons in the developing brain die via apoptosis after DNA damage, while neurons in the adult brain are generally resistant to these insults. The basis for this resistance is a matter of conjecture. We report here that cerebellar granule neurons (CGNs) in culture lose their competence to die in response to DNA damage as a function of time in culture. CGNs at either 1 day in vitro (DIV) or 7 DIV were treated with the DNA damaging agents camptothecin, UV or gamma-irradiation and neuronal survival measured. The younger neurons were effectively killed by these agents, while the older neurons displayed a significant resistance to killing. Neuronal survival did not change with time in culture when cells were treated with C2-ceramide or staurosporine, agents which do not target DNA. The resistance to UV irradiation developed over time in culture and was not due to changes in mitotic rate. Increases in DNA strand breakage, up-regulation of the levels of both p53 and its phosphorylated form and nuclear translocation of p53 were equivalent in both older and younger neurons, indicating a comparable p53 stress response. In addition, we show that treatment of older neurons with pharmacological inhibitors of distinct components of the DNA repair machinery promotes the accumulation of DNA damage and sensitizes these cells to the toxic effects of UV exposure. These data demonstrate that older neurons appear to be more proficient in DNA repair in comparison to their younger counterparts, and that this leads to increased survival after DNA damage.

Aspects of the feeding behaviour of intensively reared trout

Feeding behaviour of trained rainbow trout was investigated by the use of demand feeders, under different light conditions. The effects of the energy content of diet, and the size, colour and texture of feed pellets, on the feeding behaviour, were studied. An attempt was made to locate the assumed centres for feeding and satiety in the hypothalamus of brain by the intraperitoneal injections of goldthioglucose. Feeding under nine different constant photoperiods at 160 lux, at a temperature of 13.5°C, showed that trout exhibit a rhythmic pattern of feeding behaviour in all photoperiods except in continuous darkness.Feeding rhythms of trout attributable to the degree of gut distension were formed every eight to ten hours. Further studies by varying levels of light intensity revealed the interaction of light intensity and photoperiod. At shorter photoperiods lower levels of light intensity decreased the feeding activity in terms of food intake but by increasing the photoperiod the same feeding activity was accomplished as by the fish subject to a short photoperiod but under higher light intensity.Simulated effect of increasing and decreasing daylengths did not affect the overall food intake and growth performance. Trout are quite efficient in adjusting their food intake in terms of energy content. Colour, size and texture of feed pellets affect the feeding responses and elicit preferential food selection behaviour in trout. Goldthioglucose induced some reversable toxic effects upon general physiology of trout and did not produce any lesions in the assumed areas of feeding and satiety centres in the brain.  It was concluded that the feeding behaviour of trout exhibited selective preferences according to the physical nature of food items and those preferences could be further influenced by the biotic and abiotic factors, light being one of the most important abiotic factors.

Investigation of potential intervention targets to improve insulin action: a therapeutic approach

Impaired insulin action (insulin resistance) is a key factor in the pathogenesis of diabetes mellitus. To investigate therapeutic targets against insulin resistance, this thesis explores the mechanism of action of pharmacological agents and exogenous peptides known or suspected to modify insulin action. These included leptin, a hormone primarily involved in the regulation of body weight; sibutramine, an antiobesity agent; plant-derived compounds (pinitol and chamaemeloside) and agents known to affect insulin sensitivity, e.g. metformin, tolbutamide, thiazolidinediones, vanadyl sulphate and thioctic acid. Models used for investigation included the L6 skeletal muscle cell line and isolated skeletal muscles. In vivo studies were undertaken to investigate glycaemia, insulinaemia, satiety and body weight in streptozotocin-induced diabetic mice and obese (ob/ob) mice. Leptin acutely altered insulin action in skeletal muscle cells via the short form of the leptin receptor. This direct action of leptin was mediated via a pathway involving PI 3-kinase but not Jak2. The active metabolites of sibutramine had antidiabetic properties in vivo and directly improved insulin sensitivity in vitro. This effect appeared to be conducted via a non-PI 3-kinase-mediated increase in protein synthesis with facilitated glucose transport, and was independent of the serotonin and noradrenaline reuptake inhibition produced by sibutramine. Pinitol (a methyl inositol) had an insulin mimetic effect and was an effective glucose-lowering agent in insulinopenic states, acting directly on skeletal muscle. Conversely chamaemeloside appeared to improve glucose tolerance without directly altering glucose transport. Metformin directly increased basal glucose uptake independently of PI 3-kinase, possibly via an increase in the intrinsic activity of glucose transporters. Neither tolbutamide nor thiazolidinediones directly altered insulin sensitivity in L6 skeletal muscle cells: however vanadyl sulphate and thioctic acid increased glucose transport but appeared to exert toxic effects at therapeutic concentrations. Examination of glucose transport in skeletal muscle in this thesis has identified various components of post-receptor insulin signalling pathways which may be targeted to ameliorate insulin resistance. Type 2 Diabetes Mellitus Obesity L6 Skeletal Muscle Cells Glucose Transport Insulin Signalling 2

The importance of Na(plus)-K(plus)-Cl- cotransport in nitrogen mustard induced cell death

The incubation of murine leukaemic L1210 cells in vitro for 4 hours (hr) with 10uM nitrogen mustard (HN2), a bifunctional alkylating agent, inhibited the influx of the potassium congener, 88rubidium+ ( 86Rb+) by the selective inhibition of the Na+-K+-CI- cotransporter. The aim of this project was to investigate the importance of this lesion in HN2-induced cytotoxicity. 86Rb+ uptake in human erythrocytes was inhibited by high concentrations of HN2 (2mM) and occurred in two phases.In the first hour both the Na+/K+ ATPase pump and the Na+-K+-CI- cotransporter were equally inhibited but after 2 hrs exposure to 2mM HN2, the Na+ -K+ -CI- cotransporter was significantly more inhibited than the Na+/K+ ATPase pump. In contrast, both potassium transport systems were equally inhibited in L1210 cells incubated for 10 minutes with 1mM HN2. The selective inhibition of the Na+-K+-CI- cotransporter, after a 3 hrs exposure to 10uM HN2, was not absolved by coincubation with 5ug/ml cycloheximide (CHX), an inhibitor of protein synthesis. Incubation of L1210 cells with concentrations of diuretics which completely inhibited Na+-K+-CI- cotransport did not enhance the cytotoxicity of either HN2 or its monofunctional analogue 2-chloroethyldimethylamine (Me-HN1). The incubation of L1210 cells with a twice strength Rosewell Park Memorial Institute 1640 media did not enhance the toxicity of HN2. An L1210 cell line (L1210FR) was prepared which was able to grow in toxic concentrations of furosemide and exhibited a similiar sensitivity to HN2 as parental L1210 cells. Treatment of L1210 cells with 10uM HN2 resulted in a decrease in cell volume which was concurrent with the inhibition of the Na+-K+-CI- cotransporter. This was not observed in L1210 cells treated with either 1 or O.SuM HN2. Thus, possible differences in the cell death, in terms of necrosis and apoptosis, induced by the different concentrations of HN2 was investigated. The cell cycle of L1210 cells appeared to be blocked non-specifically by 10uM HN2 and in S and G2/M by either 1 or 0.5uM HN2. There were no significant changes in the cytosolic calcium concentrations of L1210 cells for up to 48 hrs after exposure to the three concentrations of HN2. No protection against th_ toxic effects of HN2 was observed in L1210 cells incubated with 5ug/ml CHX for up to 6 hrs. Incubation for 12 or 18 hrs with a non-toxic concentration (5mM) of L-Azetidine-2- carboxylic acid (ACA) enhanced the toxicity of low concentrations (<0.5uM) of HN2.

Reduced metal transferrin binding in neurological diseases

By employing G75 gel-filtration chromotography, it has been demonstrated that human plasma gallium speciation (and by implication, Al speciation) is bimodal. Normally, gallium was predominantly bound to a high molecular weight fraction which was presumably transferrin. Literature reviews and experimental work throughout this thesis provided evidence to support this idea. An aluminium-transferrin species was assumed to be relatively non-toxic and a protective function for this complex has been suggested. A second, low molecular weight species of gallium was observed and its identity has been suggested to be citrate. The results of this thesis support the concept citrate was a gallium binding ligand present in the plasma, but there was another species (tentatively identified as phosphate) which bound gallium to a much greater degree than did citrate in the majority of samples studied. The consequence of a low molecular weight species of aluminium is the possibility that this leads to a more rapid, uncontrolled deposition of the metal in the brain compared to a transferrin mediated mechanism. Plasma speciation studies in Alzheimer's disease, Parkinson's disease, Down's syndrome, and neonates has revealed an altered ratio of the two gallium species found in control subjects. In all groups there was an increase in the potentially more neurotoxic low molecular weight species. These observations have led to a suggested mechanism of accumulation of metals in the brain, which is known to occur in the first three groups. Possible pathogenic mechanisms are described. The results can also offer an explanation to the reported increased sensitivity to the toxic effects of aluminium in the neonate. Speciation studies on normal plasma has shown the balance between high and low molecular weight species of gallium to be influenced by many physiological factors. There appears to be a fine equilibrium between both species which can be altered without any great difficulty. Therefore, in the diseased groups studied, it is possible that there are subtle biochemical changes within the circulatory system to affect the equilibrium which results in an increased low molecular weight species of aluminium. Furthermore, it has been demonstrated that there is a group of normal controls with no clinical signs of Alzheimer's or Parkinson's disease which have reduced transferrin binding. This indicates there is a population of healthy people who are at risk to the development of either disease.