The effect of temperature on the gill morphology and ion transporter distribution in the branchial epithelium of a freshwater teleost, the Koi carp (Cyprinus carpio, L.) was examined. Three different water temperatures were used to detect changes in expression level of transporter proteins in the gill epithelial cells. With increasing temperature, the expression level of all three ion transporters studied declined, and the gill lamellae protruded out of the cell mass, thus increasing the surface area of the branchial epithelium. A hypothetical organization of the transporter proteins within the ionocytes is proposed.

Fungos patogênicos, como Cryptococcus neoformans e Candida albicans, são uma das mais frequentes causas de infecções oportunistas em todo o mundo, sendo capazes de sobreviver, proliferar e escapar de macrófagos, células de primeira linha da resposta imune de hospedeiros mamíferos. Macrófagos geralmente expõem o patógeno intracelular a um ambiente tóxico, caracterizado por pH ácido, presença de espécies reativas, como também de peptídeos antimicrobianos. Neste contexto, há uma competição entre o patógeno intracelular e o hospedeiro mamífero por nutrientes essenciais, como por exemplo, metais de transição. Cobre é um metal de transição essencial para a vida aeróbica, porém pode ser tóxico em altas concentrações e, devido a isto, diversos organismos desenvolveram mecanismos regulatórios para controlar suas concentrações. O objetivo deste trabalho foi avaliar a modulação da homeostase de cobre durante a interação entre macrófagos e células de C. neoformans ou C. albicans. Neste trabalho, foi possível demonstrar que a presença de cobre contribui para uma menor atividade fungicida de macrófagos infectados. Também, o pré-carregamento das células fúngicas com cobre alterou a sensibilidade de ambas leveduras patogênicas à atividade anti-fúngica. Além disso, foi demonstrado que a expressão de transportadores de cobre (CTR1 e ATP7A) e proteínas ligadoras de cobre (ceruloplasmina e metalotioneína 1) de macrófagos foram moduladas em resposta à infecção por estes fungos patogênicos. Entretanto, essa regulação pode envolver mecanismos mais complexos, como estratégias do patógeno para subverter a ação antimicrobiana de macrófagos. / Pathogenic yeasts, such as Cryptococcus neoformans and Candida albicans, are one of the most frequent causes of the opportunistic infections worldwide, being able to survive, proliferate and escape from macrophages, the first line cells engaged in the immune defense of the mammalian host. Macrophages generally expose the intracellular pathogen to a toxic environment, which is characterized by acid pH, presence of reactive species, as well the presence of antimicrobial peptides. In this context, there is a competition between intracellular pathogen and mammalian host for essential nutrients, such as transition metals. Copper is an essential transition metal for aerobic life, but can be toxic at high concentrations and, therefore, many organisms have evolved highly regulated mechanisms for controlling its concentration. In this work, we aim is evaluate a modulation of copper homeostasis during interaction between macrophages and C. neoformans or C. albicans cells. This work demonstrated that the presence of copper resulted in a lower fungicidal activity of infected macrophages. Also, the pre-loading of fungal cells with copper can alter the sensitivity of both pathogenic yeasts to an antifungal activity. In addition, we showed that the expression of macrophage copper transporters (CTR1 and ATP7A) and copper-binding proteins (ceruloplasmin and metallothionein 1) are modulated in response infection by pathogenic fungi. However, this regulation may involve more complex mechanisms, such as strategies of pathogen to subvert the antimicrobial action of macrophages.

Fungos patogênicos, como Cryptococcus neoformans e Candida albicans, são uma das mais frequentes causas de infecções oportunistas em todo o mundo, sendo capazes de sobreviver, proliferar e escapar de macrófagos, células de primeira linha da resposta imune de hospedeiros mamíferos. Macrófagos geralmente expõem o patógeno intracelular a um ambiente tóxico, caracterizado por pH ácido, presença de espécies reativas, como também de peptídeos antimicrobianos. Neste contexto, há uma competição entre o patógeno intracelular e o hospedeiro mamífero por nutrientes essenciais, como por exemplo, metais de transição. Cobre é um metal de transição essencial para a vida aeróbica, porém pode ser tóxico em altas concentrações e, devido a isto, diversos organismos desenvolveram mecanismos regulatórios para controlar suas concentrações. O objetivo deste trabalho foi avaliar a modulação da homeostase de cobre durante a interação entre macrófagos e células de C. neoformans ou C. albicans. Neste trabalho, foi possível demonstrar que a presença de cobre contribui para uma menor atividade fungicida de macrófagos infectados. Também, o pré-carregamento das células fúngicas com cobre alterou a sensibilidade de ambas leveduras patogênicas à atividade anti-fúngica. Além disso, foi demonstrado que a expressão de transportadores de cobre (CTR1 e ATP7A) e proteínas ligadoras de cobre (ceruloplasmina e metalotioneína 1) de macrófagos foram moduladas em resposta à infecção por estes fungos patogênicos. Entretanto, essa regulação pode envolver mecanismos mais complexos, como estratégias do patógeno para subverter a ação antimicrobiana de macrófagos. / Pathogenic yeasts, such as Cryptococcus neoformans and Candida albicans, are one of the most frequent causes of the opportunistic infections worldwide, being able to survive, proliferate and escape from macrophages, the first line cells engaged in the immune defense of the mammalian host. Macrophages generally expose the intracellular pathogen to a toxic environment, which is characterized by acid pH, presence of reactive species, as well the presence of antimicrobial peptides. In this context, there is a competition between intracellular pathogen and mammalian host for essential nutrients, such as transition metals. Copper is an essential transition metal for aerobic life, but can be toxic at high concentrations and, therefore, many organisms have evolved highly regulated mechanisms for controlling its concentration. In this work, we aim is evaluate a modulation of copper homeostasis during interaction between macrophages and C. neoformans or C. albicans cells. This work demonstrated that the presence of copper resulted in a lower fungicidal activity of infected macrophages. Also, the pre-loading of fungal cells with copper can alter the sensitivity of both pathogenic yeasts to an antifungal activity. In addition, we showed that the expression of macrophage copper transporters (CTR1 and ATP7A) and copper-binding proteins (ceruloplasmin and metallothionein 1) are modulated in response infection by pathogenic fungi. However, this regulation may involve more complex mechanisms, such as strategies of pathogen to subvert the antimicrobial action of macrophages.

Previous epidemiological studies have shown that the diabetic population has a poor oral status compared to healthy subjects. While changes of the local microcirculation observed during diabetes can somehow explain periodontal pathologies this hypothesis does not stand for the carious decay. We propose the hypothesis that the increase of carious incidence might be linked to changes in the salivary composition, such as an increase of salivary glucose concentrations.The amount of glucose in saliva has been widely discussed in various scientific articles. Subsequently, we performed an initial study that allowed us to confirm that the concentration and excretion of glucose in the saliva were higher in diabetic patients compared to normal patients. In diabetic patients, the relative increase in salivary glucose concentration is even comparable to the rise of blood glucose.We conducted experimental studies in animals in order to investigate the presence and the level of expression of glucose transporters (GLUT1, GLUT2, GLUT4, SGLT1) in rat parotid glands. Through immunohistochemical labeling techniques, gene expression studies and protein expression studies, we demonstrated that GLUT1, GLUT4 and SGLT1 were found in rat parotid glands of normal and diabetic rats. Functional studies targeting the activity and the flux through those glucose transporters strengthened our hypothesis on the potential role of glucose transporter in salivary glucose.This original work introduce for the first time the hypothesis of a secretion mechanism of glucose by the salivary glands through GLUT1 & SGLT1 transporters, the first one being located on the apical and baso-lateral membranes of acinar cells, while the second one is located on the baso-lateral membrane. Secretion of glucose by the parotid gland is no longer at any doubt even though further studies must now specify the specific mechanisms. / Doctorat en Sciences dentaires / info:eu-repo/semantics/nonPublished

Research in drug development meets many challenges including lengthy, complex and costly procedures to identify novel pharmacotherapies. In our lab, we developed a method for fast screening of small molecules that interact with monoamine transports – dopamine and serotonin (DAT, SERT). These membrane proteins play important roles in brain neurotransmission responsible for cognition, motion and pleasure. Dysfunction in dopaminergic and serotonergic systems result in neurological disorders such as depression, Attention Deficit Hyperactivity Disorder (ADHD), schizophrenia and addiction. DAT and SERT are responsible for uptake of dopamine (DA) or serotonin (5HT) into the synapse and they limit neurotransmitter signaling. Drugs that mimic or antagonize actions of endogenous neurotransmitters (DA and 5HT) increase the concentrations of DA and/or 5HT either by blocking the transporter (blockers) or by competing uptake with neurotransmitter (substrate). The uptake of substrates is associated to an inward current that depolarizes the cell membrane. Voltage-gated calcium channels (CaV) can respond to small changes in membrane potential. In our method, we combined permanent cell line expressing the human dopamine transporter (hDAT) or the human serotonin transporter (hSERT) (FlpIn TREx expression system) with transient transfection of CaV. This system works as a tightly electrically coupled system. Cells challenged with substrate of the transports produce detectable Ca2+ signal while monoamine transporter blockers can inhibit these Ca2+ signals. The novelty of this method relies on the ability to discriminate between substrate and blockers of monoamine transporters. Preliminary experiments measuring our optimized cell system in a Flex Station 3 plate reader suggest that the co-expression of a voltage-gated Ca2+ channel, a monoamine transporter and a genetically encoded Ca2+ sensor constitute a rapid screening biosensor to identify active drugs at monoamine transporters. Our novel methodology can rapidly assess drug-effect profile on monoamine transporters and benefit development of new psychotherapeutics for treatment of mental illnesses. It can also be used to characterize mechanism of action of emerging drug of abuse, as well as to discover small molecules with novel drug-effect profile useful in basic neuroscience research.

No description available.

Arbuscular mycorrhizal (AM) fungi are obligate biotrophs that formsymbiosis with plants by colonizing their roots. The fungus supplies thehost plant with various nutrients, including phosphorus. Phosphorus iscrucial for the development of plants and is hard to acquire in soilsince it can be scarce and has a slow motility. The fungus utilizes itslong hyphal threads to contact more soil to obtain phosphorus andtransport it back to the plant. It does so with its use of differentphosphorus transporters (PTs) located in its membranes. Here we havedone a phylogenetic analysis of different PTs from a variety of fungifrom different phyla together with plants and new sequence data from AMfungi. In total, 955 genomes were screened, 26 of which belong to AMfungi. This work resulted in a database of 1351 PT sequences, 907 fromfungi (243 from AM) and 444 from plants, and two phylogenetic trees tovisualize the data. One phylogeny was made of the branch of the PT Pho87which was selected for building a Hidden Markov model, which canfacilitate future searches of PTs.

Orientador: Eleni Gomes / Resumo: O conhecimento das propriedades inerentes aos microrganismos assimiladores de pentoses deve ser obtido para que se desenvolva novas possibilidades de aplicação da biomassa lignocelulósica. A hemicelulose (polissacarídeo rico em pentoses) corresponde em média 25 % da massa da biomassa vegetal e sua hidrólise gera uma mistura de xilose e arabinose. Muitas leveduras, que utilizam pentoses como fonte de carbono, poderiam ser utilizadas direta ou indiretamente (através da expressão heteróloga de seus genes) na bioconversão dessas pentoses em combustível ou em outros produtos de elevado valor mercadológico. Portanto, o objetivo desse trabalho foi avaliar o potencial das cepas Sporidiobolus pararoseus U3, Pichia terricola G20 e Metschnikowia koreensis G18 de crescerem em xilose e arabinose nos pHs 4,5, 6,5 e 8,5, nas temperaturas 29 e 32 °C, na presença de compostos potencialmente tóxicos (8 compostos em três concentrações cada) e em hidrolisados de bagaço de cana. Dados sobre o efeito do pH, nutrientes e oxigenação e fontes de nitrogênio (nitrato de amônio, sulfato de amônio e ureia) em diferentes concentrações no crescimento e produção de ácidos orgânicos foram obtidos para da cepa M. koreensis G18. Todas as leveduras foram capazes de crescer em todos os pHs testados, com destaque as cepas M. koreensis G18 e P. terricola G20, uma vez que a primeira cresceu consideravelmente tanto em 29 °C como em 32 °C e a segunda por crescer mais rápido com arabinose, embora somente a 32 °C. A me... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The knowledge of the properties inherent to the pentoses assimilating microorganisms must be obtained in order to develop new possibilities for the application of lignocellulosic biomass. The hemicellulose (polysaccharide rich in pentoses) corresponds in average 25 % of the mass of the vegetal biomass and its hydrolysis generates a mixture of xylose and arabinose. Many yeasts, which use pentoses as a carbon source, could be used directly or indirectly (through the heterologous expression of their genes) in the bioconversion of these pentoses to fuel or other products of high market value. Therefore, the aim of this work was to evaluate the growth potential of strains Sporidiobolus pararoseus U3, Pichia terricola G20 and Metschnikowia koreensis G18 to grow in xylose and arabinose at pHs 4.5, 6.5 and 8.5 at temperatures of 29 and 32 °C in the presence of potentially toxic compounds (8 compounds in three concentrations each) and sugar cane bagasse hydrolysates. Data on the effect of pH, nutrients and oxygenation and sources of inorganic nitrogen (ammonium nitrate, ammonium sulfate and urea) at different concentrations in the growth and production of organic acids were obtained for the strain M. koreensis G18. All yeasts were able to grow in all tested pHs, with emphasis on the M. koreensis G18 and P. terricola G20 strains, since the former grow considerably both at 29 °C and at 32 °C and the second grow at a faster rate with arabinose, although only at 32 °C. The best pentose an... (Complete abstract click electronic access below) / Doutor

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Mgr. Lenka Ťupová Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of doctoral thesis: Interactions of selected antiretroviral drugs and methylmercury with placental membrane transporters. Pregnant women are especially in developed countries exposed to high amount of various xenobiotic including environmental pollutants and drugs. Antiretroviral therapy (ART) is administered to HIV positive pregnant women for the purpose of prevention of HIV mother- to-child-transmission. Pharmacokinetics of many antiretrovirals is limited or enhanced by activity of ATP-binding cassette (ABC) or Solute carrier's transporters, of which many are expressed also in placental tissue. ART therapy usually consists of combination of 3 - 4 antiretroviral drugs, thereby leading to higher risk for development of drug-drug interactions on ABC and SLC transporters. In this study we described influence of non-nucleoside reverse transcriptase inhibitors etravirin and rilpivirin on BCRP- and MDR1-mediated transport of tenofovir disoproxil fumarate (TDF) and/or abacavir. Etravirin showed potent inhibition of BCRP transporter significantly changing transport of both, TDF and abacavir, across monolayers of MDCKII-BCRP...

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Mgr. Ondřej Martinec Supervisor: Assoc. Prof. PharmDr. Lukáš Červený, Ph.D. Title of doctoral thesis: Interactions of antiretroviral drugs with membrane transporters Oral delivery is the most common, convenient, and economical form of drug administration. Absorption of orally administered drugs occurs mainly in the intestine. Intestinal absorption can be reduced by the activity of efflux drug ABC transporters, mainly p-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2), located in the apical membrane of the intestinal epithelium. HIV-infected patients are dependent on lifelong pharmacotherapy, which includes a combination of three or more antiretroviral drugs. Hepatitis C (HCV) is a common co-infection of HIV. In addition, the HIV-positive population is aging, which is associated with burden of other comorbidities. This results in an indication of polypharmacy and thus an increased risk of drug-drug interactions. Many antiretroviral drugs used are substrates, inhibitors and /or inducers of ABCB1, so they might quantitatively affect the intestinal absorption of co-administered drugs (ABCB1 substrates), thereby affecting the efficacy/safety of treatment. As part of this...