Σκοπός της συγκεκριμένης ερευνητικής εργασίας ήταν η αποσαφήνιση του ρόλου της Κυκλοσπορίνης στην πρόκληση νεφρικής ίνωσης, διαμέσου του συστήματος ΕΤ-1/ΝΟ. Για το λόγο αυτό πραγματοποιήθηκαν in vitro πειράματα, στα οποία χρησιμοποιήθηκαν ΗΚ-2 κύτταρα και διερευνήθηκε η επίδραση της κυκλοσπορίνης στην βιωσιμότητα των κυττάρων, στην παραγωγή ΝΟ, στην συσσώρευση των mRNA των et-1, etr-a, etr-b, enos και inos καθώς και τη συσσώρευση των αντίστοιχων πρωτεϊνών τους. Προέκυψε ότι η κυκλοσπορίνη τροποποιεί την έκφραση των γονιδίων et-1 και των υποδοχέων της etr-a και etr-b, των συνθετασών του ΝΟ, enos και inos, τη συσσώρευση των αντίστοιχων πρωτεϊνών, καθώς και του παραγόμενου NO. Επίσης, η αλβουμίνη δεν επηρεάζει τη συσσώρευση της ακτίνης, παρουσιάζει ωστόσο κυτταροτοξική δράση. / The aim of this research was the elucidation of the role of Cyclosporine in the induction of renal fibrosis, through the activation of the ET-1/NO system. We performed in vitro experiments using HK-2 cells (human proximal tubular epithelial cells) and we studied the impact of Cyclosporine on cell viability production of NO, expression of et-1, etr-a, etr-b, enos and inos and the accumulation of the subsequent proteins, as well as actin. Cyclosporine was found to alter the expression of et-1, etr-a, etr-b and enos and the accumulation of the subsequent proteins. Moreover, it affects the amount of the synthesized and secreted NO, while having no effect on the accumulation of actin.

O balanço do fluoreto (F) dentro do corpo é modulado por sua ingestão, absorção e remoção, e os rins, responsáveis pela sua excreção do organismo, são particularmente vulneráveis à toxicidade do F. Os efeitos nefrotóxicos do F envolvem mudanças estruturais marcantes nos rins, além disso, estudos proteomicos têm mostrado grandes alterações no perfil de proteínas envolvidas em pontos chave na transdução de sinal. Estes efeitos podem influenciar negativamente no transporte iônico nos rins. A vista deste fato, o transporte iônico no canal de sódio epitelial (ENaC) é limitante para a taxa de reabsorção de sódio nos rins, sendo essencial para a manutenção do equilíbrio eletrolítico e homeostase do corpo. Assim, este trabalho objetivou investigar os efeitos do F, utilizando concentrações semelhantes às que podem ser encontradas no néfron durante a fluorose dentária, na viabilidade celular e expressão de transportadores de membrana (ENaC) em linhagem celular renal M-1. Para os ensaios de viabilidade das células da linhagem M-1 foi empregado os testes colorimétricos Cristal Violeta e MTT, utilizando as concentrações de tratamento com fluoreto de sódio (NaF) a 10, 40, 100, 200 e 400 M, durante períodos experimentais de 24, 48, 72 e 96 h. As mesmas concentrações e os mesmos tempos foram utilizados no tratamento com cloreto de sódio (NaCl), utilizado como controle na possível interferência de Na+ na modulação das células. Para a investigação da influência do F: 1) nos canais ENaC foi utilizada a técnica de imunofluorescência e 2) para a análise de expressão gênica das subunidades que formam o ENaC, foi utilizada a técnica RT-PCR. Em nossos resultados pudemos observar que as maiores concentrações tanto de NaF quanto de NaCl provocaram a diminuição da viabilidade celular para ambos os ensaios de viabilidade, no entanto, foi possível observar algumas diferenças na resposta do tratamento com NaF em comparação com NaCl, por meio do ensaio Cristal Violeta. Não foi observado diferenças nas imagens de imunofluorescêcia, mas outros aspectos morfológicos foram vistos nessas imagens, como o aparecimento de domes celular, sugerindo que até mesmo a maior concentração de F não foi capaz de inibir a proliferação celular. Nosso resultado mais significativo foi em relação à expressão das subunidades dos canais de ENaC, onde a concentração de 400 M foi capaz de diminuir bruscamente a expressão das três subunidades do ENaC, enquanto as concentrações de 100 e 200 M mostraram apresentar expressão igual e em alguns casos até maior que o grupo controle. Pudemos concluir que doses de F na ordem de micromolares podem modular a expressão das subunidades formadoras do ENaC, positivamente quando em baixas concentrações e negativamente quando em concentrações elevadas. / The balance of fluoride (F) within the body is modulated by its ingestion, absorption and removal, and the kidneys, responsible for their excretion of the organism, are particularly vulnerable to the toxicity of F. The nephrotoxic effects of F involve marked structural changes in the kidneys , in addition, proteomic studies have shown large changes in the profile of proteins involved in key points in signal transduction. These effects may negatively influence ion transport in the kidneys. In view of this fact, the ionic transport in the epithelial sodium channel (ENaC) is limiting to the rate of sodium reabsorption in the kidneys, being essential for the maintenance of the electrolyte balance and homeostasis of the body. Thus, the objective of this work was to investigate the effects of F, using concentrations similar to those found in the nephron during dental fluorosis, cell viability and expression of membrane transporters (ENaC) in renal cell line M-1. For the M-1 cell line viability assays, the Crystal Violet and MTT colorimetric assays were used, using the 10, 40, 100, 200 and 400 M sodium fluoride (NaF) treatment concentrations during experimental periods of 24, 48, 72 and 96 h. The same concentrations and the same times were used in the treatment with sodium chloride (NaCl), used as control in the possible interference of Na + in the modulation of the cells. For the investigation of the influence of F: 1) in the ENaC channels, the immunofluorescence technique was used and 2) for the analysis of gene expression of the subunits that form the ENaC, the RT-PCR technique was used. In our results it was observed that the higher concentrations of NaF and NaCl caused a decrease in cell viability for both viability assays, however, it was possible to observe some differences in NaF treatment response in comparison to NaCl, through test Crystal Violet. No differences were observed in immunofluorescence images, but other morphological aspects were seen in these images, such as the appearance of cellular \"domes\", suggesting that even the highest F concentration was not able to inhibit cell proliferation. Our most significant result was the expression of the subunits of the ENaC channels, where the concentration of 400 M was able to decrease expression of the three subunits of the ENaC, whereas the concentrations of 100 and 200 M showed equal expression and in some cases even higher than the control group. We can conclude that doses in the order of micromolar the F can modulate the expression of the ENaC forming subunits, positively when in low concentrations and negatively when in high concentrations.

Assessement of effects of cadium chloride exposure on the anaphylaxis reaction to food was done on six week old Swiss and BALB/c female mice. The animals were exposed to cadium as cadium chloride for either three days or six weeks. Intra-peritonal dose of cadium chloride was injected once a day, five days per week for three successive weeks. The animals were then sensitised to cow's milk by force-feeding with cow's milk for three consecutive days. Oral exposure of mice to a high dose of cadium resulted in cytotoxicity of liver and kidney cells. Retardation in growth rate and haematology change were detected. Proliferative response to the T-cell epitope from the circumsporozoite protein of plasmodium falsiparum was decreased in cultures of lymph node cells from cadium chronically treated mice and sensitised with the same peptide. In contrast, an increase of cell proliferation was observed when cow's milk was used instead. Significant increase in Immunoglobulin E level and Anaphylactic reaction dependent on the quantity of cadium exposed were recorded. No protective effect of ascorbic acid or zinc acetate on cadium alteration of immune response was observed / Master of Science (Hons) (Food Science)

O balanço do fluoreto (F) dentro do corpo é modulado por sua ingestão, absorção e remoção, e os rins, responsáveis pela sua excreção do organismo, são particularmente vulneráveis à toxicidade do F. Os efeitos nefrotóxicos do F envolvem mudanças estruturais marcantes nos rins, além disso, estudos proteomicos têm mostrado grandes alterações no perfil de proteínas envolvidas em pontos chave na transdução de sinal. Estes efeitos podem influenciar negativamente no transporte iônico nos rins. A vista deste fato, o transporte iônico no canal de sódio epitelial (ENaC) é limitante para a taxa de reabsorção de sódio nos rins, sendo essencial para a manutenção do equilíbrio eletrolítico e homeostase do corpo. Assim, este trabalho objetivou investigar os efeitos do F, utilizando concentrações semelhantes às que podem ser encontradas no néfron durante a fluorose dentária, na viabilidade celular e expressão de transportadores de membrana (ENaC) em linhagem celular renal M-1. Para os ensaios de viabilidade das células da linhagem M-1 foi empregado os testes colorimétricos Cristal Violeta e MTT, utilizando as concentrações de tratamento com fluoreto de sódio (NaF) a 10, 40, 100, 200 e 400 M, durante períodos experimentais de 24, 48, 72 e 96 h. As mesmas concentrações e os mesmos tempos foram utilizados no tratamento com cloreto de sódio (NaCl), utilizado como controle na possível interferência de Na+ na modulação das células. Para a investigação da influência do F: 1) nos canais ENaC foi utilizada a técnica de imunofluorescência e 2) para a análise de expressão gênica das subunidades que formam o ENaC, foi utilizada a técnica RT-PCR. Em nossos resultados pudemos observar que as maiores concentrações tanto de NaF quanto de NaCl provocaram a diminuição da viabilidade celular para ambos os ensaios de viabilidade, no entanto, foi possível observar algumas diferenças na resposta do tratamento com NaF em comparação com NaCl, por meio do ensaio Cristal Violeta. Não foi observado diferenças nas imagens de imunofluorescêcia, mas outros aspectos morfológicos foram vistos nessas imagens, como o aparecimento de domes celular, sugerindo que até mesmo a maior concentração de F não foi capaz de inibir a proliferação celular. Nosso resultado mais significativo foi em relação à expressão das subunidades dos canais de ENaC, onde a concentração de 400 M foi capaz de diminuir bruscamente a expressão das três subunidades do ENaC, enquanto as concentrações de 100 e 200 M mostraram apresentar expressão igual e em alguns casos até maior que o grupo controle. Pudemos concluir que doses de F na ordem de micromolares podem modular a expressão das subunidades formadoras do ENaC, positivamente quando em baixas concentrações e negativamente quando em concentrações elevadas. / The balance of fluoride (F) within the body is modulated by its ingestion, absorption and removal, and the kidneys, responsible for their excretion of the organism, are particularly vulnerable to the toxicity of F. The nephrotoxic effects of F involve marked structural changes in the kidneys , in addition, proteomic studies have shown large changes in the profile of proteins involved in key points in signal transduction. These effects may negatively influence ion transport in the kidneys. In view of this fact, the ionic transport in the epithelial sodium channel (ENaC) is limiting to the rate of sodium reabsorption in the kidneys, being essential for the maintenance of the electrolyte balance and homeostasis of the body. Thus, the objective of this work was to investigate the effects of F, using concentrations similar to those found in the nephron during dental fluorosis, cell viability and expression of membrane transporters (ENaC) in renal cell line M-1. For the M-1 cell line viability assays, the Crystal Violet and MTT colorimetric assays were used, using the 10, 40, 100, 200 and 400 M sodium fluoride (NaF) treatment concentrations during experimental periods of 24, 48, 72 and 96 h. The same concentrations and the same times were used in the treatment with sodium chloride (NaCl), used as control in the possible interference of Na + in the modulation of the cells. For the investigation of the influence of F: 1) in the ENaC channels, the immunofluorescence technique was used and 2) for the analysis of gene expression of the subunits that form the ENaC, the RT-PCR technique was used. In our results it was observed that the higher concentrations of NaF and NaCl caused a decrease in cell viability for both viability assays, however, it was possible to observe some differences in NaF treatment response in comparison to NaCl, through test Crystal Violet. No differences were observed in immunofluorescence images, but other morphological aspects were seen in these images, such as the appearance of cellular \"domes\", suggesting that even the highest F concentration was not able to inhibit cell proliferation. Our most significant result was the expression of the subunits of the ENaC channels, where the concentration of 400 M was able to decrease expression of the three subunits of the ENaC, whereas the concentrations of 100 and 200 M showed equal expression and in some cases even higher than the control group. We can conclude that doses in the order of micromolar the F can modulate the expression of the ENaC forming subunits, positively when in low concentrations and negatively when in high concentrations.

Fluoxetine (FLX), the active ingredient of Prozac™, is a member of the selective serotonin reuptake inhibitor (SSRI) class of anti-depressant drugs and is present in aquatic environments worldwide. Previous studies reported that FLX is an endocrine disruptor in fish, bioconcentrating in tissues including the brain. Evidence implicates that serotonin influences the activity of the hypothalamo-pituitary-interrenal (HPI) stress axis, thus exposure to FLX may disrupt the teleost stress response. This study examined in vitro cortisol production in rainbow trout (Oncorhynchus mykiss) head kidney/interrenal cells exposed to FLX and 14C-pregnenolone metabolism in head kidney microsome preparations of FLX-exposed trout. Results indicated that cells exposed in vitro to increasing concentrations of FLX had lower cortisol production and cell viability (versus control) and microsomes isolated from trout exposed to 54 μg/L FLX had higher pregnenolone metabolism versus those of control and low FLX-exposed (0.54 μg/L) trout.

Fluoxetine (FLX), the active ingredient of Prozac™, is a member of the selective serotonin reuptake inhibitor (SSRI) class of anti-depressant drugs and is present in aquatic environments worldwide. Previous studies reported that FLX is an endocrine disruptor in fish, bioconcentrating in tissues including the brain. Evidence implicates that serotonin influences the activity of the hypothalamo-pituitary-interrenal (HPI) stress axis, thus exposure to FLX may disrupt the teleost stress response. This study examined in vitro cortisol production in rainbow trout (Oncorhynchus mykiss) head kidney/interrenal cells exposed to FLX and 14C-pregnenolone metabolism in head kidney microsome preparations of FLX-exposed trout. Results indicated that cells exposed in vitro to increasing concentrations of FLX had lower cortisol production and cell viability (versus control) and microsomes isolated from trout exposed to 54 μg/L FLX had higher pregnenolone metabolism versus those of control and low FLX-exposed (0.54 μg/L) trout.

Fluoxetine (FLX), the active ingredient of Prozac™, is a member of the selective serotonin reuptake inhibitor (SSRI) class of anti-depressant drugs and is present in aquatic environments worldwide. Previous studies reported that FLX is an endocrine disruptor in fish, bioconcentrating in tissues including the brain. Evidence implicates that serotonin influences the activity of the hypothalamo-pituitary-interrenal (HPI) stress axis, thus exposure to FLX may disrupt the teleost stress response. This study examined in vitro cortisol production in rainbow trout (Oncorhynchus mykiss) head kidney/interrenal cells exposed to FLX and 14C-pregnenolone metabolism in head kidney microsome preparations of FLX-exposed trout. Results indicated that cells exposed in vitro to increasing concentrations of FLX had lower cortisol production and cell viability (versus control) and microsomes isolated from trout exposed to 54 μg/L FLX had higher pregnenolone metabolism versus those of control and low FLX-exposed (0.54 μg/L) trout.

Fluoxetine (FLX), the active ingredient of Prozac™, is a member of the selective serotonin reuptake inhibitor (SSRI) class of anti-depressant drugs and is present in aquatic environments worldwide. Previous studies reported that FLX is an endocrine disruptor in fish, bioconcentrating in tissues including the brain. Evidence implicates that serotonin influences the activity of the hypothalamo-pituitary-interrenal (HPI) stress axis, thus exposure to FLX may disrupt the teleost stress response. This study examined in vitro cortisol production in rainbow trout (Oncorhynchus mykiss) head kidney/interrenal cells exposed to FLX and 14C-pregnenolone metabolism in head kidney microsome preparations of FLX-exposed trout. Results indicated that cells exposed in vitro to increasing concentrations of FLX had lower cortisol production and cell viability (versus control) and microsomes isolated from trout exposed to 54 μg/L FLX had higher pregnenolone metabolism versus those of control and low FLX-exposed (0.54 μg/L) trout.

The causative role of the huntingtin (HTT) gene in Huntington's disease (HD) has been identified more than 25 years ago. The extension of CAG repeat stretch over 39 repeats in exon 1 of one HTT allele results in full penetrance of this neurodegenerative disorder. While the identification of the causative mutation raised hopes that development of the therapeutic compound will be easily achievable, the patients and their families are still waiting for treatment until now. The main reason for that might be the complex cellular function HTT that makes the determination of the pathologic mechanism difficult and the development of treatments even more challenging. Although a lot of different animal models have been generated until now, establishing a suitable model has still not been achieved yet. Due to its anatomy, physiology, and genetics, the minipig seems to be a suitable candidate for neurodegenerative disease models. Indeed, the existing Transgenic (Tg) Libechov minipig model manifests signs typical for HD in patients, but on the other hand significant inconsistencies have also been observed. The finding of malformation that partially shows the situation in human patients is true for both, the male reproductive tract as well as for the brain. The reason for this might be the fact the genetic...

The causative role of the huntingtin (HTT) gene in Huntington's disease (HD) has been identified more than 25 years ago. The extension of CAG repeat stretch over 39 repeats in exon 1 of one HTT allele results in full penetrance of this neurodegenerative disorder. While the identification of the causative mutation raised hopes that development of the therapeutic compound will be easily achievable, the patients and their families are still waiting for treatment until now. The main reason for that might be the complex cellular function HTT that makes the determination of the pathologic mechanism difficult and the development of treatments even more challenging. Although a lot of different animal models have been generated until now, establishing a suitable model has still not been achieved yet. Due to its anatomy, physiology, and genetics, the minipig seems to be a suitable candidate for neurodegenerative disease models. Indeed, the existing Transgenic (Tg) Libechov minipig model manifests signs typical for HD in patients, but on the other hand significant inconsistencies have also been observed. The finding of malformation that partially shows the situation in human patients is true for both, the male reproductive tract as well as for the brain. The reason for this might be the fact the genetic...