A exposição de camundongos neonatos ao poluente 1,2-naftoquinona (1,2-NQ) induziu maior suscetibilidade dos machos jovens (mas não fêmeas) à asma. O objetivo deste estudo foi investigar o impacto da 1,2-NQ no pulmão de camundongos jovens machos e fêmeas e averiguar as associações entre os receptores de potencial transitório TRPA1 e a 1,2-NQ. Camundongos machos e fêmeas C57Bl/6 (2-5 g) foram expostos a 1,2-NQ (100 nM). Maior atividade da catalase e expressão (RNAm) do Nrf2 foi observado no pulmão das fêmeas 24 h após a exposição da 1,2-NQ. O estímulo alérgico na puberdade aumentou a atividade da glutationa peroxidase, redutase e S-transferase nas fêmeas que, diferentemente dos machos, não exibiram exacerbação da asma, mas mostraram maior nitração e carbonilação proteica, expressão da eNOS e TRPA1. O antagonismo dos TRPA1 reduziu a eosinofilia pulmonar nos machos e inibiu a [Ca2+]i em cultura de neurônios frente a 1,2-NQ. A ausência susceptibilidade em fêmeas se deve a maior defesa antioxidante e a maturidade pulmonar destas. / The mice exposure to 1,2-naphthoquinone (1,2-NQ), during postnatal period induced increased susceptibility of males (but not females) to asthma. The aim of this study was to investigate the intensity of lung damage to impact of 1,2-NQ on young mice of both sexes, and to investigate the associations between TRPA1 and 1,2-NQ. Male and female C57Bl/6 mice (2-5g) were exposed to 1,2-NQ (100 nM). After 24 h postnatal exposure to 1,2-NQ, only female lungs showed increased catalase activity and Nrf2 mRNA expression. The allergic stimuli at puberty led to increased glutathione peroxidase, reductase and S-transferase activities only in female lung, which, unlike male, did not exhibit exacerbation of asma, but showed increased pulmonary nitration and protein carbonylation, and increased mRNA expression of eNOS and TRPA1. The TRPA1 antagonist reduced eosinophilia in male lung and inhibited the increased [Ca2+]i in dorsal root ganglion neurons culture to 1,2-NQ. The lack of susceptibility in female might be linked to increased antioxidant defenses and the pulmonary maturity.

Στόχος της παρούσας διπλωματικής εργασίας είναι η μελέτη μέτρησης της διασποράς μεγέθους λιποσωμάτων με την τεχνική της συντονιζόμενης ανίχνευσης παλμών ωμικού ρεύματος, σε σύγκριση με την μέθοδο της δυναμικής σκέδασης φωτός, που χρησιμοποιείται συνήθως. Για τον σκοπό αυτό, μελετήθηκε η διασπορά μεγέθους διαφόρων λιποσωμικών διασπορών, σε διάφορους χρόνους μετά την παρασκευή τους, πριν και μετά από λυοφιλοποίηση, καθώς και όταν επωάστηκαν (για διάφορες χρονικές περιόδους) παρουσία πρωτεϊνών. Όλοι οι τύποι λιποσωμάτων χαρακτηρίστηκαν ως προς το μέγεθος και τον δείκτη πολυδιασποράς και με τις δύο τεχνικές: Δυναμική σκέδαση φωτός (DLS) και την τεχνική συντονισμένης ανίχνευσης παλμών ωμικού ρεύματος (TRPS). Από την μελέτη σταθερότητας μεγέθους των λιποσωμάτων μετά από λυοφιλοποίηση (freeze-drying), φαίνεται ότι η σταθερότητα του μεγέθους, εξαρτάται από το υδατικό μέσο στο οποίο βρίσκονται και από την λιπιδική συγκέντρωση του δείγματος. Ακόμα, έγινε προσπάθεια να διερευνηθεί αν το ποσοστό του φορτίου που φέρουν τα λιποσώματα, επηρεάζει την σταθερότητα του μεγέθους, και παρασκευάστηκαν SUV λιποσώματα με διαφορετικό φορτίο. Σύμφωνα με τα αποτελέσματα, η σταθερότητα μεγέθους των λιποσωμάτων με την πάροδο του χρόνου, δεν εξαρτάται από τη συγκέντρωση της διασποράς (τουλάχιστον στην περιοχή συγκεντρώσεων που μελετήθηκαν), αλλά από το φορτίο που φέρουν. Πιο συγκεκριμένα, στα λιποσώματα με 20% PG, το μέγεθος αυξήθηκε κατά 5-22%, σε αντίθεση με τα λιποσώματα που φέρουν 10% PG, στα οποία το μέγεθος αυξήθηκε κατά 2,5-10%. Τέλος, η επώαση παρουσία πρωτεϊνών, φάνηκε να επηρεάζει σημαντικά το μέγεθος των λιποσωμάτων (με και χωρίς φορτίο) μετά από 48 ώρες. / The aim of this study is to investigate the results of a TRPS technique to measure the size distribution of various types of liposomes in comparison with those obtained by the classically used DLS methodology. For this, the size stability of liposomes was studied (using both techniques) before and after a freeze drying cycle, and also before and after their incubation (for various time periods) in presence of proteins. All types of liposomes were characterized for their size and polydispersity index, by using the two techniques: Dynamic light scattering (DLS) and Tunable Resistive Pulse Sensing (TRPS). The size stability study of liposomes, after freeze-drying, was found to depend on the aqueous medium in which they are dispersed (buffer or glucose) and (more) on the lipid concentration of the dispersions. The effect of adding a charged lipid in the liposome bilayer on their size stability was additionally investigated. For this, charged and non-charged SUV liposomes were prepared. According to the results the size stability of liposomes is affected by their charge. Specifically, liposomes with 20% PG, size increased by 5-22 % and liposomes with 10% PG, size increased by 2,5-10 %. Finally, the size stability of liposomes in the presence of proteins was studied, by using two different liposome types, with or without charge. The results showed that the size distribution remained stable during the first 48 hours of incubation but after that, liposome size was significantly increased.

A adaptação temporal é fundamental para a sobrevivência de espécies que precisam coordenar sua fisiologia e comportamentos ajustando-se a sinais externos. Ritmos biológicos não são simplesmente uma resposta às mudanças de 24 horas no ambiente físico impostas pela rotação da Terra sobre o seu próprio eixo, ao contrário, surgem a partir de um sistema de cronometragem endógeno. No teleósteo Danio rerio, ainda não foi identificada a presença de uma região que atue como relógio central; alguns estudos têm evidenciado a existência de células e tecidos que contêm relógios circadianos autônomos, fotossensíveis, comprovando um outro tipo de regulação dos ritmos circadianos onde a percepção do ambiente e o ajuste do período circadiano são efetivados diretamente em nível celular. As consequências deletérias do aumento da temperatura são impedidas, em certa medida, por uma resposta adaptativa que assegura a sobrevivência celular na presença de calor. Esta via de sobrevivência ativada por calor, conhecida como resposta ao choque térmico, é composta por uma cascata de eventos que conduzem à indução de proteínas de choque térmico (HSPs) que minimizam a lesão celular aguda. Acredita-se que os sistemas de percepção dos ciclos diários de temperatura e luminosidade sofreram as mesmas pressões seletivas em sua co-evolução, resultando em sua associação. As bases da sensação térmica estão em um grupo de canais altamente conservados, presente em todos os metazoários estudados até o momento e envolvidos em uma série de modalidades sensoriais, os canais de potencial receptor transiente (TRP); os que respondem a estímulos térmicos foram agrupados em uma subfamília e denominados termoTRPs. O objetivo deste trabalho foi investigar a influência do pulso de temperatura (33 ºC) na expressão de genes de relógio e de proteínas de choque térmico, bem como o papel do canal TRPV1, em células embrionárias de blástula de Danio rerio, denominadas ZEM-2S, submetidas a escuro constante (DD) ou ciclos claro-escuro (LD 12:12). Através de PCR em tempo real (quantitativo) demonstrou-se que as células ZEM-2S expressam os genes dos seguintes canais TRP: trpA1a, trpA1b, trpV1/2, trpV4, trpC6, trpM2, trpM4a, trpM4b/c e trpM5. Após um pulso de temperatura, observou-se um aumento no transcrito de hsp90 aa1 em células mantidas tanto em DD como em LD, sendo a expressão de hsp90 aa1 em LD, no ponto uma hora, duas vezes menor quando comparada a sua expressão no mesmo ponto temporal em DD. O pulso de temperatura não promoveu efeito em nenhum dos genes do relógio estudados (bmal1a, bmal1, bmal2, cry1a, cry1b, per1, per2) quando as células foram mantidas em DD. Porém, o transcrito de per2 aumentou em resposta ao pulso de temperatura quando as células foram sincronizadas pelos ciclos claro-escuro. A inibição do canal TRPV1 não alterou o efeito induzido pelo pulso de temperatura na expressão do gene hsp90 aa1 em células ZEM-2S mantidas em DD. Por outro lado, nossos dados permitem afirmar que o mesmo participa parcialmente na indução do aumento da expressão do gene per2 pelo estímulo térmico em células mantidas em LD, tendo em vista um decaimento significativo na resposta deste gene. Os dados obtidos neste trabalho abrem uma nova perspectiva sobre a investigação da relação temperatura e genes de relógio, colocando um novo “ator” na regulação deste fenômeno: o canal TRPV1 / Temporal adaptation is essential for the survival of species which need to coordinately adjust their physiology and behavior to external signals. Biological rhythms are not just a response to the 24 hour changes in the physical environment imposed by the rotation of the Earth around its own axis, but they arise from an endogenous timing system. In the teleost Danio rerio, there has not been identified so far a region in the nervous system that could act as a central clock; some studies have reported the existence of cells and tissues which contain photosensitive, autonomous circadian clocks, demonstrating the existence of another type of circadian rhythm regulation in which environment perception and entrainment of the circadian period are directly effected at cell level. The deleterious consequences of temperature increase are prevented by an adaptive response which assures cell survival in the presence of heat. This survival pathway activated by heat, known as response to temperature shock, is signaled by a cascade of events leading to the induction of thermal shock proteins (HSPs) which attenuate the acute cell lesion. It is believed that the systems perceiving temperature and light daily cycles were subject to the same selective pressures during their co-evolution, resulting in their association. The base of thermal sensation is a family of highly conserved channels, present in all metazoans studied to date, and involved in a variety of sensorial modalities, the transient receptor potential channels (TRP); those responding to thermal stimuli were grouped in a sub-family named thermo-TRPs. The aim of this work was to investigate the influence of a temperature pulse (33 ºC) on the expression of clock and heat shock protein genes, as well as the role of TRPV1 channel, in blastula embryonic cells of Danio rerio, named ZEM-2S, subject to constant dark (DD) or light-dark cycles (LD). Using quantitative PCR, we demonstrated that ZEM-2S cells express genes for the following TRP channels: trpA1a, trpA1b, trpV1/2, trpV4, trpC6, trpM2, trpM4a, trpM4b/c and trpM5. After the pulse of temperature, we observed an increase of hsp90 aa1 transcripts in DD as well as in LD; hsp90 aa1 expression 1 hour after the stimulus was two-fold lower in LD than in DD. Temperature pulse did not affect the expression of any of the studied clock genes (bmal1a, bmal1, bmal2, cry1a, cry1b, per1, per2), when the cells were kept in DD. However, per2 transcript increased in response to the temperature pulse when the cells were synchronized by light-dark cycles. Inhibition of TRPV1 channel did not change the effect induced by the temperature pulse on hsp90 aa1 in ZEM-2S cells kept in DD. On the other hand, our data suggest that this channel participates, at least partially, in the temperature-induced increase of per2 in cells maintained in LD, as indicated by the significant decay observed in the gene response in the presence of the inhibitor. Our results open new investigative perspective about the relationship between temperature and clock genes, placing a new “actor” in the regulation of the phenomenon: the TRPV1 channel

A adaptação temporal é fundamental para a sobrevivência de espécies que precisam coordenar sua fisiologia e comportamentos ajustando-se a sinais externos. Ritmos biológicos não são simplesmente uma resposta às mudanças de 24 horas no ambiente físico impostas pela rotação da Terra sobre o seu próprio eixo, ao contrário, surgem a partir de um sistema de cronometragem endógeno. No teleósteo Danio rerio, ainda não foi identificada a presença de uma região que atue como relógio central; alguns estudos têm evidenciado a existência de células e tecidos que contêm relógios circadianos autônomos, fotossensíveis, comprovando um outro tipo de regulação dos ritmos circadianos onde a percepção do ambiente e o ajuste do período circadiano são efetivados diretamente em nível celular. As consequências deletérias do aumento da temperatura são impedidas, em certa medida, por uma resposta adaptativa que assegura a sobrevivência celular na presença de calor. Esta via de sobrevivência ativada por calor, conhecida como resposta ao choque térmico, é composta por uma cascata de eventos que conduzem à indução de proteínas de choque térmico (HSPs) que minimizam a lesão celular aguda. Acredita-se que os sistemas de percepção dos ciclos diários de temperatura e luminosidade sofreram as mesmas pressões seletivas em sua co-evolução, resultando em sua associação. As bases da sensação térmica estão em um grupo de canais altamente conservados, presente em todos os metazoários estudados até o momento e envolvidos em uma série de modalidades sensoriais, os canais de potencial receptor transiente (TRP); os que respondem a estímulos térmicos foram agrupados em uma subfamília e denominados termoTRPs. O objetivo deste trabalho foi investigar a influência do pulso de temperatura (33 ºC) na expressão de genes de relógio e de proteínas de choque térmico, bem como o papel do canal TRPV1, em células embrionárias de blástula de Danio rerio, denominadas ZEM-2S, submetidas a escuro constante (DD) ou ciclos claro-escuro (LD 12:12). Através de PCR em tempo real (quantitativo) demonstrou-se que as células ZEM-2S expressam os genes dos seguintes canais TRP: trpA1a, trpA1b, trpV1/2, trpV4, trpC6, trpM2, trpM4a, trpM4b/c e trpM5. Após um pulso de temperatura, observou-se um aumento no transcrito de hsp90 aa1 em células mantidas tanto em DD como em LD, sendo a expressão de hsp90 aa1 em LD, no ponto uma hora, duas vezes menor quando comparada a sua expressão no mesmo ponto temporal em DD. O pulso de temperatura não promoveu efeito em nenhum dos genes do relógio estudados (bmal1a, bmal1, bmal2, cry1a, cry1b, per1, per2) quando as células foram mantidas em DD. Porém, o transcrito de per2 aumentou em resposta ao pulso de temperatura quando as células foram sincronizadas pelos ciclos claro-escuro. A inibição do canal TRPV1 não alterou o efeito induzido pelo pulso de temperatura na expressão do gene hsp90 aa1 em células ZEM-2S mantidas em DD. Por outro lado, nossos dados permitem afirmar que o mesmo participa parcialmente na indução do aumento da expressão do gene per2 pelo estímulo térmico em células mantidas em LD, tendo em vista um decaimento significativo na resposta deste gene. Os dados obtidos neste trabalho abrem uma nova perspectiva sobre a investigação da relação temperatura e genes de relógio, colocando um novo “ator” na regulação deste fenômeno: o canal TRPV1 / Temporal adaptation is essential for the survival of species which need to coordinately adjust their physiology and behavior to external signals. Biological rhythms are not just a response to the 24 hour changes in the physical environment imposed by the rotation of the Earth around its own axis, but they arise from an endogenous timing system. In the teleost Danio rerio, there has not been identified so far a region in the nervous system that could act as a central clock; some studies have reported the existence of cells and tissues which contain photosensitive, autonomous circadian clocks, demonstrating the existence of another type of circadian rhythm regulation in which environment perception and entrainment of the circadian period are directly effected at cell level. The deleterious consequences of temperature increase are prevented by an adaptive response which assures cell survival in the presence of heat. This survival pathway activated by heat, known as response to temperature shock, is signaled by a cascade of events leading to the induction of thermal shock proteins (HSPs) which attenuate the acute cell lesion. It is believed that the systems perceiving temperature and light daily cycles were subject to the same selective pressures during their co-evolution, resulting in their association. The base of thermal sensation is a family of highly conserved channels, present in all metazoans studied to date, and involved in a variety of sensorial modalities, the transient receptor potential channels (TRP); those responding to thermal stimuli were grouped in a sub-family named thermo-TRPs. The aim of this work was to investigate the influence of a temperature pulse (33 ºC) on the expression of clock and heat shock protein genes, as well as the role of TRPV1 channel, in blastula embryonic cells of Danio rerio, named ZEM-2S, subject to constant dark (DD) or light-dark cycles (LD). Using quantitative PCR, we demonstrated that ZEM-2S cells express genes for the following TRP channels: trpA1a, trpA1b, trpV1/2, trpV4, trpC6, trpM2, trpM4a, trpM4b/c and trpM5. After the pulse of temperature, we observed an increase of hsp90 aa1 transcripts in DD as well as in LD; hsp90 aa1 expression 1 hour after the stimulus was two-fold lower in LD than in DD. Temperature pulse did not affect the expression of any of the studied clock genes (bmal1a, bmal1, bmal2, cry1a, cry1b, per1, per2), when the cells were kept in DD. However, per2 transcript increased in response to the temperature pulse when the cells were synchronized by light-dark cycles. Inhibition of TRPV1 channel did not change the effect induced by the temperature pulse on hsp90 aa1 in ZEM-2S cells kept in DD. On the other hand, our data suggest that this channel participates, at least partially, in the temperature-induced increase of per2 in cells maintained in LD, as indicated by the significant decay observed in the gene response in the presence of the inhibitor. Our results open new investigative perspective about the relationship between temperature and clock genes, placing a new “actor” in the regulation of the phenomenon: the TRPV1 channel

Trichorhinophalangeal syndrome is a malformation syndrome characterized by craniofacial and skeletal abnormalities and is inherited in an autosomal dominant manner. We distinguish free subtypes on clinical and molecular level - TRPS I, TRPS II, TRPS III. All TRPS patients have sparse hair, a pear-shaped nose, a long flat philtrum, a thin upper lip and protruding ears. Skeletal abnormalities include cone-shaped epiphyses at the phalanges, hip malformations and short stature are present. The subgroups TRPS I and TRPS III are result of the mutated TRPS1 gene, which is maped into the 8q24 region. This gene is situated proximal of the EXT1 gene, both genes are affected in a subgroup of patients with TRPS II. These patients suffer more from multiple (cartilaginous) exostoses and mental retardation. In this work we performed molecular genetic analysis of a sample of 16 patients, 8 probands showed a TRPS phenotype and 8 probands had only isolated exostoses. The peripheral venous blood of patients was used to gain purified DNA, which was subsequently used to investigate the chromosome 8q24 region using MLPA ("multiplex ligation-dependent probe amplification"). This analysis revealed a deletion in 1 TRPS patient and 1 patient with exostoses. Sequencing of the TRPS1 gene coding exons in remaining 7 TRPS...