Compounds containing a quinone moiety represent an important class of biologically active molecules that are widespread in nature, displaying anticancer, antibacterial, antimalarial, and fungicidal activities. In the course of designing 2,3-disubstituted-1,4-naphthoquinones derivatives as potential cysteine protease inhibitors, two naphtho[2,3-d]isoxazole-4,9-dione-3-carboxylates, 1a and 1b, were obtained. The antiapoptotic potential of 1a and 1b was then evaluated and compared to that of naphthoquinone 4. Primary rat hepatocytes were incubated with synthesized naphthoquinone derivatives and then exposed to the apoptotic stimulus camptothecin. Our results indicate that naphtho[2,3-d]isoxazole-4,9-dione-3-carboxylates 1a and 1b exerted a potent protective role in camptothecin-induced apoptosis in primary rat hepatocytes. Both 1a and 1b significantly increased cell viability, while reducing nuclear fragmentation, caspase-3, -8 and -9 activation, and cytochrome c release induced by camptothecin. In addition, 1a and 1b were shown to up-regulate Bcl-XL, a pro-survival member of the Bcl-2 family of proteins, which modulates the mitochondrial pathway of apoptosis. Similar protective effects of quinone derivatives were seen in HuH-7 and PC12 cells incubated with distinct apoptotic stimuli, such as camptothecin, TGF-β1, or rotenone. Our results suggest that naphtho[2,3-d]isoxazole-4,9-dione-3-carboxylates 1a and 1b may act as potent, cytoprotective agents, through modulation of apoptotic pathways.

CONTEXT: Ketamine is an anaesthetic and analgesic drug used in research and clinical practice. Little is known about the effects of different doses of this drug on memory and brain cellular death. OBJECTIVE: To study the effects of different doses of ketamine on working and reference memory, and neurodegeneration in adult mice. DESIGN: A randomised study. SETTINGS: The study was carried out in a basic science laboratory, between March 2011 and August 2012. ANIMALS: Forty-eight 7-month-old, male C57BL/6 mice were used. INTERVENTION: Animals received a single intraperitoneal injection of physiological saline solution or one of three doses of ketamine (25, 75 or 150 mg kg). Each group consisted of 12 animals (seven animals for behavioural tests and five animals for histopathological and immunohistochemical studies). The animals used for histopathology studies were sacrificed 3 h after anaesthesia. MAIN OUTCOME MEASURES: Working and reference memories were assessed using the radial-maze test over 12 consecutive days. The equilibrium was tested using the vertical pole (4 and 24 h after injection), whereas locomotion was assessed using the open field (24, 48 and 72 h after injection). Histopathological (haematoxylin-eosin staining) and immunohistochemical analyses (procaspase-3 and activated caspase-3 detections) were performed 3 h after injection to assess neurodegeneration in the retrosplenial and visual cortices, pyramidal cell layer of the cornu Ammonis 1 and cornu Ammonis 3 areas of the hippocampus, in the granular layer of the dentate gyrus, in the laterodorsal thalamic nucleus, striatum and accumbens nucleus. RESULTS: No significant differences were observed between the groups regarding the number of dead cells and cells showing positive immune-reactivity in the different regions of the brain studied. The performance in the vertical pole test and the number of reference and working memory errors in the radial-maze were similar in all groups. Nevertheless, the animals treated with ketamine 75 mg kg were transiently more active, walking a greater total distance at a greater speed in the open field than other groups (power of 0.96). CONCLUSION: These data indicate that a single intraperitoneal injection of ketamine at subanaesthetic and anaesthetic doses does not impair working memory, reference memory or neurodegeneration in adult mice, but an intermediate dose of ketamine produces transitory hyperlocomotion.

Ganoderma lucidum is one of the most extensively studied mushrooms as a functional food and as a chemopreventive agent due to its recognized medicinal properties. Some G. lucidum extracts have shown promising antitumor potential. In this study, the bioactive properties of various extracts of G. lucidum, from both the fruiting body and the spores, were investigated. The most potent extract identified was the methanolic fruiting body extract, which inhibited the growth of a gastric cancer cell line (AGS) by interfering with cellular autophagy and cell cycle.

A variety of the classic green tea plant, Camellia sinensis, was developed and is exclusive to Kenya. Due to high content of anthocyanin polyphenols in its leaves, the beverage obtained from this variety is purple in color and is the origin of the name purple tea. This work had two main purposes. The first one was to identify and quantify the major anthocyanin polyphenols in a hot water aqueous extract of the purple tea leaves. The second one was to test the hypothesis if this extract is capable of inhibiting triglyceride absorption considering that anthocyanin polyphenolics have been frequently associated to antilipidemic effects. Parallel experiments were always done with a similar green tea extract for comparison purposes. The antioxidant, anti-inflammatory, and cytotoxic activities of both tea varieties are similar. The purple tea extract, however, was strongly inhibitory toward the pancreatic lipase (minimal IC50 = 67.4 mu g mL(-1)), whereas the green tea preparation was a weak inhibitor. Triglyceride digestion in mice was inhibited by the purple tea extract starting at 100 mg kg(-1) dose and with a well-defined dose dependence. Green tea had no effect on triglyceride digestion at doses up to 500 mg kg(-1). The latter effect is probably caused by several components in the purple tea extract including non-anthocyanin and anthocyanin polyphenols, the first ones acting solely via the inhibition of the pancreatic lipase and the latter by inhibiting both the lipase and the transport of free fatty acids from the intestinal lumen into the circulating blood. The results suggest that the regular consumption of Kenyan purple tea can be useful in the control of obesity.

Pleurotus sajor-caju (PSC) is an edible mushroom used in food supplements, presenting antitumor properties through induction of cell death pathways. The PSC potential against colorectal cancer was analyzed by exposing HCT116wt cells to different PSC extracts. The PSC n-hexane extract (PSC-hex) showed the highest cytotoxicity effect (IC50 value 0.05 mg/mL). The observed cytotoxicity was then associated to apoptosis-promoting and cell cycle-arrest pathways. PSC-hex was able to induce apoptosis related to breakdown of mitochondrial membrane potential and ROS generation. The absence of cytotoxicity in HTC116-p53 and HTC116-Bax cells, alongside with an increase in p53, Bax and Caspase-3 expression, and decrease in Bcl-2 expression, supports that the proapoptotic effect is probably induced through a p53 associated pathway. PSC-hex induced cell cycle arrest at G2/ M in HCT116wt without cytotoxicity in HTC116-p21 cells. These findings suggest that a p21/p53 cell cycle regulation pathway is probably disrupted by compounds present on PSC-hex. Identification of the major components was then performed with ergosta-5,7,22-trien-3β-ol representing 30.6% of total weight. In silico docking studies of ergosta-5,7,22-trien-3β against Bcl-2 were performed and results show a credible interaction with the Bcl-2 hydrophobic cleft. The results show that PSC-hex can be used as supplementary food for adjuvant therapy in colorectal carcinoma.

Several novel methyl 7-[(hetero)arylamino]thieno[2,3-b]pyrazine-6-carboxylates were synthe-sized by Pd-catalyzed C–N Buchwald–Hartwig cross-coupling of either methyl 7-aminothieno[3,2-b] pyrazine-6-carboxylate with (hetero)arylhalides or 7-bromothieno[2,3-b]pyrazine-6-carboxylate with (hetero)arylamines in good-to-excellent yields (50% quantitative yield), using different reaction conditions, namely ligands and solvents, due to the different electronic character of the substrates. The antitumoral potential of these compounds was evaluated in four human tumor cell lines: gastric adenocarcinoma (AGS), colorectal adenocarcinoma (CaCo-2), breast carcinoma (MCF7), and non-small-cell lung carcinoma (NCI-H460) using the SRB assay, and it was possible to establish some structure–activity relationships. Furthermore, they did not show relevant toxicity against a non-tumor cell line culture from the African green monkey kidney (Vero). The most promising compounds (GI50 ≤ 11 µM), showed some selectivity either against AGS or CaCo-2 cell lines without toxicity at their GI50 values. The effects of the methoxylated compounds 2b (2-OMeC6 H4 ), 2f and 2g (3,4-or 3,5-diOMeC6 H3, respectively) on the cell cycle profile and induction of apoptosis were further studied in the AGS cell line. Nevertheless, even for the most active (GI50 = 7.8 µM) and selective compound (2g) against this cell line, it was observed that a huge number of dead cells gave rise to an atypical distribution on the cell cycle profile and that these cells were not apoptotic, which points to a different mechanism of action for the AGS cell growth inhibition.

Mushrooms are known as a powerful source of bioactive compounds including antioxidants, inhibitors of human tumour cell lines growth, inducers of apoptosis and enhancers of immunity. Indeed, many pre-clinical studies have been conducted in human tumour cell lines and in some cases a number of compounds isolated from mushrooms have followed to clinical trials. The Northeast of Portugal is one of the European regions with higher wild mushrooms diversity. However, to our knowledge, no studies had been conducted so far to verify their bioactivities. The main aim of this work was the evaluation of the bioactive properties (antioxidant properties and growth inhibitory potential on human tumour cell lines) of wild edible mushrooms collected in the Northeast of Portugal. Once properly identified, methanolic, ethanolic and boiling water extracts were prepared from thirty eight wild mushroom species collected in that region. Chemical characterization was obtained by high performance liquid chromatography (HPLC) coupled to a photodiode array detector (DAD) or to a refraction index detector (RI). Antioxidant activity assays were carried out in those extracts, including evaluation of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging capacity, reducing power and inhibition of β-carotene bleaching. Extract-induced cell growth inhibition was assessed with the sulforhodamine B assay in four human tumour cell lines (NCI-H460 - lung cancer, MCF-7 -breast cancer, HCT-15 -colon cancer and AGS - gastric cancer). The effects on cell cycle profile and apoptosis were evaluated by flow cytometry and the effect on the expression levels of proteins related to cell cycle and apoptosis was further investigated by Western blotting. Three wild edible mushroom species revealed growth inhibitory activity in the studied human tumour cell lines: Clitocybe alexandri ethanolic extract, Lepista inversa methanolic extract and Suillus collinitus methanolic extract. C. alexandri ethanolic extract induced an S-phase cell cycle arrest and increased the percentage of apoptotic cells, in the NCI-H460 cell line. The analysed mushroom species also provided interesting antioxidant potential, mainly the boiling water extract of L. inversa which showed the highest DPPH radical scavenging activity, reducing power and β-carotene bleaching inhibition. S. collinitus methanolic extract induced a slight increase in the number of cells in G1, with a concomitant decrease in the percentage of cells in the S phase of the cell cycle and an increase in the percentage of apoptotic cells, in the MCF-7 cell line. The combined use of the S. collinitus methanolic extract and etoposide caused a greater decrease in the percentage of cell growth, when compared to either of them used individually, indicating the potential benefit of this combination. The tested extracts were chemically characterized and protocatechuic, p-hydroxybenzoic, p-coumaric and cinnamic acids were the main compounds identified on the phenolic (methanolic and ethanolic) extracts, while mannitol, trehalose and arabinose were the main sugars found in the polysaccharidic (boiling water) extracts after hydrolysis. The individual compounds identified in the extracts were submitted to a screening of tumour cells growth inhibitory activity, but only the phenolic acids and a related compound, cinnamic acid, presented activity. This compound was found to be the most potent one regarding cell growth inhibition in the NCI-H460 cell line. The effect of the individual and combined treatment with the identified compounds was also evaluated. Cinnamic and protochatequic acids caused a statistically significantly reduction in the number of viable cells. In addition, p-hydroxybenzoic acid did not show any significantly reduction in the viable cell number. Nevertheless, it was verified that the concomitant use of the three compounds provided the strongest decrease in the viable cell number, suggesting a possible concomitant effect of those compounds. Overall, the present work has contributed to further understand the bioactive potential of wild edible mushrooms from the Northeast of Portugal. This study allowed to identify some species with antioxidant or tumour cell growth inhibitory potential.

Vemurafenib (VB), a BRAF inhibitor and a first-line treatment for unresectable or metastatic melanoma, is strongly phototoxic towards normal skin cells. Herein, we show that in cultured HS 68 human diploid dermal fibroblasts, low concentrations of VB suffice to promote photosensitization to low doses of UVA (∼ 5 J/cm2), as evidenced by a significant decrease in cell viability. In contrast to data obtained in chemico our results support a role for ROS (reactive oxygen species). Indeed, peroxidation of cellular lipids was observed which could be alleviated by the lipophilic antioxidant BHT (2,6-di-tert-butyl-4-methylphenol). Using in vivo confocal laser scanning microscopy and vital fluorescent probes it was shown at the single cell level that the plasma membrane and lipid-rich organelles, namely mitochondria, endoplasmic reticulum, and lysosomes, as well as actin filaments, were severely damaged by the UVA-induced VB-photosensitization. Finally, we showed that mitochondrial impairment was concurrent with caspase 3/7 activation and cell death by apoptosis.

Constitutively active phosphoinositide 3-kinase (PI3K) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it upregulates cell proliferation, survival and drug resistance. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the pan-PI3K inhibitor NVP-BKM120 (BKM120), an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in G2/M phase cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T lymphoblasts, and promoting a dose- and time-dependent dephosphorylation of Akt and S6RP. BKM120 maintained its pro-apoptotic activity against Jurkat cells even when cocultured with MS-5 stromal cells, which mimic the bone marrow microenvironment. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. Moreover, in vivo administration of BKM120 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth, thus prolonging survival time. Taken together, our findings indicate that BKM120, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment for T-ALLs that have aberrant upregulation of the PI3K signaling pathway.

A doença de Parkinson (DP), a segunda doença neurodegenerativa mais comum, é impulsionada pela perda progressiva de neurónios dopaminérgicos na substantia nigra e no estriado. A etiologia da DP é desconhecida, mas tanto fatores genéticos como ambientais parecem desempenhar um papel preponderante na patogénese da doença. Além disso, os microRNAs (miRNAs ou miRs) encontram-se frequentemente desregulados em doenças neurodegenerativas, incluindo na DP. Mais especificamente, resultados do nosso laboratório demonstraram que o miR-335 está significativamente diminuído em diferentes modelos experimentais que mimetizam a DP, bem como em doentes. Em termos de mecanismo, o miR-335 tem como alvo direto Leucin-rich repeat kinase 2 (LRRK2), cuja sobre-expressão tem sido associada a uma maior suscetibilidade para desenvolver a doença. Também demonstrámos que, em linhas celulares de microglia e neuronais. o miR-335 inibe a neuroinflamação induzida por estímulos inflamatórios clássicos, como lipopolisacáridos (LPS) e alfa-sinucleína, e até mesmo pela sobre-expressão de LRRK2. Com este trabalho, pretendemos investigar o potencial do miR-335 para aliviar a morte neuronal induzida por 1-methyl-4-phenylpyridinium (MPP+), uma neurotoxina amplamente usada para mimetizar a DP in vitro. Como esperado, o tratamento de células SH-SY5Y de neuroblastoma humano com 2.5 mM MPP+ durante 48 h, induziu níveis significativos de morte celular determinados pela libertação de adenilato cinase (AK) e ensaios do metabolismo de MTS. Paralelamente, testámos no nosso modelo o potencial neuroprotetor do composto X. Curiosamente, os nossos resultados indicaram que o composto X, estruturalmente semelhante ao fármaco citoprotetor UDCA, mas de uma rota sintética diferente, foi capaz de proteger parcialmente as células da morte celular induzida por MPP+ e o seu efeito protetor foi mais acentuado do que no caso do UDCA e TUDCA. Além disso, estudos realizados fora do âmbito desta tese, também demonstraram a atividade antiapoptótica do composto X em outros tipos de células não neuronais e estímulos tóxicos, o que reforça ainda mais os nossos resultados. No futuro, estudos de otimização química devem ser realizados de forma a melhorar a atividade do composto X e torná-lo mais adequado para ensaios in vivo. Curiosamente, o inibidor geral de caspases, zVAD, reverteu os efeitos tóxicos da neurotoxina MPP+. Para melhor caracterizar o tipo de morte celular associada à toxicidade do MPP+, medimos a atividade e ativação das caspase-3 e -7, moléculas-chave para a execução da apoptose, que se verificaram estar aumentadas. A ativação da apoptose foi ainda confirmada pela avaliação da morfologia nuclear após a coloração com o marcador fluorescente Hoechst. Curiosamente, também descobrimos que a incubação concomitante de células com necrostatina-1 (Nec-1), um inibidor potente da necroptose, abolia a toxicidade mediada pelo MPP+ sugerindo assim um papel adicional para este tipo de morte celular regulada neste modelo. No entanto, nem a ferrostatina-1 nem a liproxstatina-1, fortes inibidores da ferroptose, demonstraram qualquer efeito protetor no nosso modelo, o que nos indica que possivelmente este tipo de morte celular não está a ocorrer, pelo menos nestas condições experimentais. É importante referir que quando transfectámos transitoriamente as células com um precursor específico do miR-335-3p (pre-miR-335-3p), 24 h antes da exposição ao MPP+, os níveis de morte celular diminuíram em comparação com as células transfectadas com um controlo negativo. No que diz respeito à necroptose, a exposição de células transfectadas com controlo negativo ao MPP+ resultou num aumento de 50% na razão entre a proteína MLKL fosforilada (pMLKL) e a sua forma total, e um aumento de 10 vezes na expressão de RIP3, no proteoma insolúvel das células, sugerindo assim a ativação de necroptose. Em contrapartida, o tratamento com MPP+ não alterou os níveis de RIP1, o que não invalida a existência de necroptose, porque estudos indicam que este tipo de morte celular pode ocorrer de forma independente da RIP1. Contudo, é curioso que a Nec-1, um inibidor da atividade cinase da RIP1, proteja as células da toxicidade provocada por MPP+. Podemos especular que estes efeitos resultam de atividades off-target da molécula. Em geral, os nossos resultados indicam um papel importante do miR-335 na proteção contra a morte celular induzida por MPP+. Enquanto que a morte celular por apoptose parece ser a via de sinalização induzida preferencialmente pela neurotoxina e o alvo do miR-335, os resultados da ativação da necroptose requerem confirmação adicional. Para caracterizar ainda mais o mecanismo de ação do miR-335 e porque a necroptose é um tipo de morte celular inflamatória, colocámos a hipótese de que a inibição da necroptose pelo miR-335 também poderia resultar na diminuição da inflamação. Uma característica marcante da DP é a neuroinflamação crónica caracterizada pela ativação microglial e níveis mais elevados de mediadores pró-inflamatórios. Por outro lado, o MPP+ parece promover a expressão dos marcadores pró-inflamatórios TNFα, COX-2 e NLRP3, em células SH-SY5Y. Em conjunto, os nossos resultados parecem sugerir um papel para o miR-335 na atenuação da inflamação associada ao MPP+ no nosso modelo; no entanto, são necessárias mais experiências para confirmar estas descobertas. A ativação da morte celular por piroptose também merece ser explorada no nosso modelo. Finalmente, seria interessante avaliar a ativação de vias de sinalização inflamatória clássicas, incluindo JNK, p38 e ERK1/2, bem como a ativação NF-κB, que é um mediador-chave na resposta inflamatória. Compreender a interação entre a diferentes vias de sinalização é essencial para a criação de novas e mais eficazes estratégias terapêuticas. Com este trabalho foi implementado e otimizado um modelo robusto para o estudo dos mecanismos de morte celular associados à DP, usando a linha celular de neuroblastoma humano, SH-SY5Y, exposta à neurotoxina MPP+, e contribuímos com novo conhecimento sobre o potencial neuroprotetor do miR-335 na patogénese desta doença. Em particular, avançámos com resultados in vitro que indicam que a sobre-expressão do miR-335 pode vir a traduzir-se numa abordagem terapêutica promissora e inovadora para travar a neurodegenerescência associada à DP, através da inibição da morte celular por apoptose e possivelmente necroptose, bem como da neuroinflamação.

The unstrained S-allyl cysteine amino acid was site-specifically installed on apoptosis protein biomarkers and was further used as a chemical handle and ligation partner for 1,2,4,5-tetrazines by means of an inverse-electron-demand Diels-Alder reaction. We demonstrate the utility of this minimal handle for the efficient labeling of apoptotic cells using a fluorogenic tetrazine dye in a pre-targeting approach. The small size, easy chemical installation, and selective reactivity of the S-allyl handle towards tetrazines should be readily extendable to other proteins and biomolecules, which could facilitate their labeling within live cells.

Colon cancer (CC) remains highly ranked in incidence and mortality worldwide. 5-Fluorouracil (5-FU) has been the mainstay for the treatment of CC for several decades. Nevertheless, chemoresistance remains a significant drawback to its clinical success. Therefore, a better understanding of the molecular mechanisms by which tumor cells circumvent 5-FU-induced cytotoxicity may lead to the identification of new therapeutic targets to improve CC treatment. Aberrant MEK5/ERK5 signaling has been reported in several types of human cancer, being associated with increased cell proliferation, survival, and chemoresistance. In CC, MEK5 overactivation was correlated with disease stage progression. Moreover, recent data from our group demonstrated that MEK5 and ERK5 expression is increased in human colon adenomas and adenocarcinomas, suggesting that MEK5/ERK5 signaling overactivation may contribute to CC initiation and progression. In the present study, using HCT116 and SW620 cells, we produced two stable CC models with differential activation of the MEK5/ERK5 pathway to investigate its role in cell proliferation and response to 5-FU. Our results demonstrate that MEK5 constitutive activation increased CC cell proliferation and KRAS expression, in both HCT116 and SW620 models. In turn, in the HCT116 model, MEK5/ERK5 signaling inhibition, using a dominant-negative MEK5, increased p53 and p21 expression, as well as cell death following 5-FU exposure, which was further associated with increased caspase-3/7 activation and apoptosis. Conversely, constitutive activation of MEK5/ERK5 signaling increased 5-FU chemoresistance, reducing 5-FU-induced cytotoxicity and apoptosis. Interestingly, 5-FU exposure markedly decreased the levels of endogenous MEK5 and ERK5 expression and activation. Finally, our results show that MEK5/ERK5 activation may modulate cell proliferation and sensitivity to 5-FU by downregulating the expression of the tumor-suppressors miR-143/-145/-34a. Collectively, these data unravel an important interplay between the MEK5/ERK5 pathway and the mechanisms of 5-FU-induced cytotoxicity, suggesting that ERK5-targeted inhibition may provide a promising therapeutic approach for CC treatment, warranting further investigation.

Rheumatoid arthritis is the most common inflammatory joint disease. The etiopathogenesis of this condition has been classically explained by a T cell-driven process. However, recent studies have highlighted the possible contribution of neutrophils for the early phases of RA physiopathology. These cells are phagocytic leukocytes that play crucial roles in the acute defense against pathogens while modulating the function of other immune cells and contributing to the perpetuation of an initial inflammatory response. The herein article reviews recent progresses in the understanding of the immunopathology of RA with a special emphasis on the role of neutrophils.

Organisms living in volcanic environments are chronically exposed to metals, either as particles or associated with gases, from volcanic emissions, being therefore potential sentinels of the effects derived from such exposure. Concentrations of Ca, Cd, Cu, Mg, Mn, Pb, Rb, and Zn were measured in soil, grass (Lolium perenne), and larvae of Pseudaletia uninpuncta captured in sites exposed and non-exposed to volcanic activity. The midgut epithelial cell morphometry and apoptosis of P. unipuncta larvae were also analyzed. Larvae from the site with volcanic activity showed higher levels of Cu, Mn, Rb and Zn. Metals such as Pb, Cd and Mg levels of P. unipuncta larvae were similar between sites. Apoptosis was higher in cells from digestive epithelium of larvae exposed to volcanic activity. Soils and grass not exposed to volcanic activity showed higher levels for most of the analyzed elements with the exception of Rb. Such result when compared with metal levels of larvae may reveal that bioavailability of elements differs between sites. The higher levels of Cd, Zn and Mg in soils and grass from the site with no volcanic activity are probably related to the severe artificial fertilization in the studied pastures. Such result, when compared with metal levels of larvae, suggest that the bioavailability of metals differs between sites.

The chloragogenous tissue and the intestinal epithelium of adult earthworms, Lumbricus terrestris, sampled from sites with and without volcanic activity in the Azores were submitted to hematoxylin/eosin staining, autometallography and TUNEL-test in order to quantify the radial thickness of both tissues, their relative abundance of metals and apoptosis levels. Metals were visualized, through light microscopy, as black silver deposits (BSD) mostly in the chloragogenous tissue. The lowest radial thickness values of both tissues were found in the active volcanic sites, as well as the highest BSD and apoptosis levels. The BSD extent in the chloragogenous tissue, semi-quantified by stereology, exhibited a positive correlation with the apoptosis levels and a negative one with the radial thickness of both tissues. Thus, the variation of the radial thickness of both tissues, but especially of the chloragogenous tissue, which could reflect different cellular turnover rates caused by exposure to metals, is suggested as a biomarker of effect for metal exposure in terrestrial worms inhabiting volcanic environments.

The pressure exerted by shallow water hydrothermal vents on edible gastropods and their cellular responses triggered by these stresses are almost unknown. The aims of this study were to evaluate the bioavailability of metals in the Macaronesian endemic limpet Patella candei gomesii living close to shallow water hydrothermal vents, and the structural differences in their digestive gland as well as the levels of apoptosis in that organ. Limpets were sampled in four sites, two with the presence of hydrothermalism and the other two without it. Whole body concentrations of several metals (Ca, Cd, Cs, Co, Cu, Fe, Hg, Mg, Mn, Pb, Rb, Se, Sr, and Zn) were obtained, morphometry analysis of the digestive gland and TUNEL test for apoptosis were also performed. Results revealed that the presence of shallow water hydrothermal vents is a source of chronic metal stress to limpets, imposing modifications in the morphometry and cell composition of the digestive gland of those limpets that may constitute cell and tissue adaptations to the environment they live in. This study sets up new baseline data for further research on the influence of shallow water hydrothermal vents over communities living in these habitats.

The influence of extreme environments of volcanic origin over vertebrates and the cellular responses that these may give are almost unknown. The main objectives were to evaluate the exposure of mice to metals in the interior of houses of a small village settled inside a volcanic crater (Furnas, Azores), and the levels of apoptosis and metallothionein in the organs (lung, liver, and kidney) of those animals. Adult mice (Mus musculus) were captured in two areas, one with volcanic activity and the other without it over the last three centuries. In the excised organs, analysis of metals (Al, Cd, Pb, Zn), TUNEL assay for apoptosis, and immunohistochemistry for metallothionein were undertook. Mice from the area with volcanic activity presented higher levels of apoptosis and metallothionein than those from the area without volcanic activity. Such results were in agreement with the differences in metal burdens of the three organs, and interestingly these concentrations were similar to or higher than others found in heavily polluted areas outside the Azores. Thus, there may be a high risk of harmful effects for organisms, including humans, inhabiting areas with volcanism, where hazardous gases and metals in the air are very common during the entire day or even all year round.

O principal objetivo deste estudo consistiu em avaliar os efeitos da exposição crónica a diferentes práticas agrícolas (agricultura convencional e orgânica) sobre o sistema reprodutor masculino, particularmente em termos de dano testicular. Três grupos de 12 machos da espécie Mus musculus, pertencentes a populações naturais dos locais de estudo, foram capturados vivos para posterior avaliação do dano testicular e determinação das doses hepáticas de metais essenciais e traço, entre os quais Se, Pb, Hg e Cd. O primeiro grupo foi capturado numa exploração agrícola convencional na qual os solos são frequentemente tratados com agroquímicos. O segundo grupo foi capturado numa exploração orgânica certificada, em que o tratamento dos solos está limitado a fertilizantes orgânicos. O terceiro grupo (controlo) foi capturado numa reserva natural cujos solos nunca foram utilizados para fins agrícolas. Após eutanização, os testículos de cada indivíduo foram extraídos, fixados e processados para análise histológica, enquanto o resíduo seco dos fígados foi sujeito à quantificação de metais traço. A avaliação do dano testicular foi levada a cabo através da análise da densidade volumétrica relativa de diferentes estágios celulares espermatogénicos, de espaço intersticial e espaço luminal, juntamente com uma avaliação qualitativa do dano nos túbulos seminíferos e com a quantificação de células espermatogénicas em apoptose (TUNNEL assay). A proporção de espaço intersticial foi significativamente superior nos ratos de ambas as explorações em relação ao grupo controlo, sendo superior no grupo da exploração convencional, enquanto a proporção de espermátides tardias e espermatozoides foi significativamente inferior neste mesmo grupo, o qual não só apresentou um número significativamente superior de túbulos seminíferos desprovidos de espermatozoides e com evidências de dano estrutural, mas também uma quantidade significativamente superior de células apoptóticas, em comparação com os outros dois grupos. Estes resultados sugerem que ambas as práticas agrícolas, especialmente a agricultura convencional, originam dano testicular em ratos que se encontram naturalmente e cronicamente expostos a ambientes agrícolas enriquecidos em metais traço, confirmando a adequação da espécie Mus musculus como bioindicadora dos potenciais efeitos e consequentes riscos para a fertilidade masculina de agricultores continuamente expostos a esses ambientes.

O cancro colorretal (CRC, do inglês colorectal cancer) é o terceiro tipo de cancro com maior incidência sendo responsável por 10% dos diagnósticos e por 9% das mortes associadas. Apesar da existência de uma abordagem terapêutica multidisciplinar intensiva, somente 60% dos pacientes ocidentais consegue atingir a meta dos 5 anos de sobrevivência. A resistência aos regimes terapêuticos é uma ocorrência frequente em quimioterapia podendo ser hereditária ou desenvolvida pelas células cancerígenas por estas após exposição aos fármacos. Estas células também podem desenvolver uma múltipla resistência, a nível estrutural e funcional, a diversos fármacos em simultâneo. Esta problemática é bastante complexa de solucionar devido à heterogeneidade populacional e sub-populacional dos tumores, os quais apresentam diferentes características genéticas e epigenéticas promovendo a agressividade do mesmo. Outras estratégias de resistência consistem na transição epitelial para mesenquimal, desregulação de importantes vias de sinalização, resistência metabólica, mutações adquiridas, evasão da apoptose, ativação de resposta a danos de ADN, assim como readaptações nas células estaminais cancerígenas (CSC, do inglês cancer stem cells). As CSCs são caracterizadas por uma distinta capacidade de autorrenovação, associada à sua tumorigénese. Esta população específica de células faz parte do microambiente tumoral, participando na intercomunicação com as células vizinhas. Estas interações têm como objetivo recrutar e regular os componentes celulares do microambiente tumoral e reestruturar os constituintes da matriz extracelular. Assim, são reunidos os fatores necessários ao desenvolvimento do tumor através do aprimoramento das propriedades intrínsecas das CSCs, como estaminalidade, sobrevivência, capacidade angiogénica, transição epitelial-mesenquimal e metastática. Como tal, o desenvolvimento de novas abordagens terapêuticas, incluindo fármacos com mecanismos de ação distintos é da maior importância. O ambiente marinho, com uma elevada diversidade biológica e química, tem demonstrado ser uma fonte singular de novos metabolitos com características químicas incomuns, distintas e únicas, exibindo novos mecanismos de ação. Os referidos compostos são chamados de metabolitos secundários e estão distribuídos por diferentes classes químicas, tais como alcalóides, terpenóides, esteróides, policetídeos, açúcares, peptídeos, derivados do ácido chiquímico e uma infinidade de outros metabolitos. Estes produtos naturais são comumente produzidos pelos organismos marinhos como resposta a diferentes pressões relacionadas com fatores bióticos e abióticos, como variações de salinidade, pressão e temperatura, exposição à luz ultravioleta, mas também como resposta face à competição pelo espaço, predação, reprodução entre outros. Por este motivo, de entre os organismos marinhos, os vi invertebrados sésseis e os seus microorganismos simbiontes são apontados como importantes alvos de pesquisa farmacológica devido à sua necessidade de readaptação constante a estes fatores. Estudos anteriores sugerem a relação simbiótica entre fungos marinhos e outras espécies de organismos como responsáveis pela produção destes compostos bioativos, pois muitas vezes estes microrganismos são encontrados, como endófitos, em relações simbióticas com, principalmente, invertebrados sésseis. Compostos isolados a partir destes organismos têm sido associados a atividades antibacterianas, antivirais, antifúngicas e anticancerígenas, entre outras. Com a presente dissertação pretendeu-se avaliar o potencial citotóxico de extratos de fungos, isolados do ambiente marinho da costa portuguesa, em diferentes modelos celulares do CRC, mas também tentar compreender os seus mecanismos de ação. Mais especificamente, quatro espécies de fungos isolados do ambiente marinho (Aspergillus pseudoglaucus, Aspergillus chevalieri, Aspergillus fructus e Lindra obtusa) foram cultivadas. De seguida a citotoxicidade dos seus extratos foi avaliada em modelos 2D e num modelo de esferóides de CRC enriquecido em CSCs e, por fim, os efeitos em biomarcadores relacionados com o stress oxidativo, disfunção mitocondrial e apoptose foram avaliados. A biomassa dos fungos foi sujeita a uma extração líquido/ líquido com acetato de etilo obtendose rendimentos entre os 10 e os 82% sendo o mais alto proveniente da espécie Aspergillus chevalieri. Após esta extração, foi realizado um screening em duas linhas celulares de CRC (HCT-15 e Caco-12) e uma linha celular não-tumoral de fibroblastos do cólon (Ccd-18CO) através da avaliação do metabolismo de MTT. O extrato Aspergillus pseudoglaucus foi o mais citotóxico (IC50: 27.93 µg/mL) nas células cancerígenas, enquanto, na linha celular nãotumoral Ccd-18CO, não houve uma sensibilidade significativa à exposição ao extrato nas dosagens testadas. A partir deste screening, a linha celular que se mostrou mais sensível à atividade dos extratos, HCT-15, e o extrato que demonstrou ter uma atividade citotóxica mais marcada, Aspergillus pseudoglaucus, foram selecionados para os ensaios subsequentes. O extracto Aspergillus pseudoglaucus, induziu um aumento da morte celular, na linha HCT15, nos tempos de incubação mais longos (72h, 48h e 24h) sendo que, tais observações, foram corroborados através da diminuição da hidrólise de calceína-AM e do aumento da libertação de LDH. Para além disso, foram avaliados vários marcadores relacionados com a apoptose como o potencial da membrana mitocondrial, produção de espécies reativas de oxigénio e a atividade da caspase-3. Verificou-se um aumento no potencial da membrana mitocondrial de forma dependente da dose do extrato a partir das 3 horas após a incubação sendo que tal efeito foi- vii se perdendo nos tempos de incubação mais longos, possivelmente refletindo uma resposta adaptativa. A tendência para um aumento na produção de espécies reativas de oxigénio foi evidente após as 6 e as 3 horas de incubação. A tendência para um aumento na atividade de caspase-3 foi clara tanto à concentração de IC50 como à metade do IC50, ao fim de 12 horas de exposição ao extrato. A capacidade citotóxica do extrato Aspergillus pseudoglaucus foi também avaliada na linha celular de CRC HCT116 numa cultura celular 2D de monocamada, através do metabolismo de MTS, e num modelo 3D de tumoresferas enriquecidas em CSCs utilizando o metabolismo de ATP. Comparando os dois modelos celulares, foi observável uma melhor atividade do extrato nos esferoides enriquecidos em CSCs, já que o valor IC50 obtido foi 2.84 µg/mL (95% IC = 2.14 – 3.74), enquanto no modelo tradicional 2D, o valor obtido foi de 43.04 µg/mL (95% IC = 26.91 – 55.89). Os resultados mostraram ainda uma redução na área dos esferoides através da ação do extrato Aspergillus pseudoglaucus. De seguida, de modo a confirmar que o mecanismo de ação do extrato Aspergillus pseudoglaucus tinha por base a via da apoptose, as células HCT116 foram co-incubadas com um inibidor de apoptose, Zvad. Com este inibidor foram realizados ensaios de citotoxicidade e viabilidade celular, mas também de fragmentação nuclear e de ADN, após exposição à concentração de IC50 e a metade do IC50 do extrato. Na presença do inibidor Zvad, foi observada uma tendência para a redução na fragmentação de DNA e na morte celular sendo possível deduzir que a apoptose pode ter um papel importante na morte celular causada pelo extrato. Os objetivos principais desta dissertação de mestrado foram compridos já que o potencial citotóxico das espécies Aspergillus pseudoglaucus, Aspergillus chevalieri, Aspergillus fructus e Lindra obtusa, derivadas do ambiente marinho, foram avaliadas em diferentes linhas celulares de CRC, mas também numa linha celular não tumoral do colon. Para além disso, os dados obtidos sugerem o possível envolvimento da produção de ROS, alterações na MMP e da ativação da Casp3 na citotoxicidade mediada pelo extrato de Aspergillus pseudoglaucus. Foi observada uma possível maior especificidade do extrato no modelo de esferoides de CRC enriquecido em CSCs em relação ao modelo 2D de células HCT116. O envolvimento da apoptose observada também pode ser observado através da co-incubação com inibidor de apoptose Zvad. Estes resultados sugerem que o extrato Aspergillus pseudoglaucus ativa a via de sinalização da apoptose com uma possível maior especificidade para as CSCs. Estes resultados enaltecem o potencial do extrato Aspergillus pseudoglaucus como fonte de compostos citotóxicos para o desenvolvimento de novos agentes terapêuticos para o tratamento do CRC.