Chloride channel family has been shown to play a significant role in physiological homeostasis processes. The function mechanism of these proteins has not yet been clearly understood. Their deficiency or mutation causes serious human illnesses. Our understanding of the chloride channels' transporting mechanisms can lead to better treatment of these illnesses. As mammalian chloride channels are difficult to prepare in laboratory, the experiments are usually done on homologous chloride channels from prokaryotic organisms. The structures of prokaryotic chloride channels have been solved and moreover they are produced with high yields. Most experiments currently use protein crystallography and provide a static picture of the system. This thesis is focused on the study of structural changes of an E. coli chloride channel using hydrogen/deuterium exchange. This method enables us to monitor dynamic conformation changes dependent on pH and exchanged ions. The measurements were done for the protonated (pH 4.5) and deprotonated state (pH 7.5) and/or in the presence of various anions: Cl− , SCN− , I− , F− , TAR. (tartaric anion). The obtained results justified the theories explaining the function of chloride channel as Cl− /H+ antiporter and provided new findings. Subject words biochemistry, protein...

Orientador: Alexandre Secorun Borges / Resumo: A principal causa de miotonia não distrófica hereditária ocorre devido à mutações no gene CLCN1, codificante para a proteína CLC1 que forma o canal iônico seletivo para o íon cloreto predominante no tecido muscular esquelético. Mutações no gene CLCN1 foram descritas como causadoras de miotomia hereditária em humanos e em várias espécies animais. Não existe descrição de miotonia hereditária na espécie suína. O objetivo deste estudo foi realizar a caracterização clínica e molecular de uma forma de miotonia hereditária em suínos. A hipótese desse estudo foi que animais com sinais clínicos compatíveis apresentavam a miotonia hereditária. Esses animais foram avaliados sob aspectos clínicos, eletromiográficos, histopatológicos e moleculares. Os sinais clínicos verificados foram hipertrofia e rigidez musculares, miotonia com startle response formação de dimples e fenômeno warm-up evidentes. Não foi constatada distrofia muscular ao exame histopatológico. Ao exame eletromiográfico foram demonstradas descargas miotônicas clássicas com formação de som característico diver bomb. A nível molecular foi verificada a ausência dos nucleotídeos referentes aos éxons 15 e 16 utilizando amostras de cDNA dos animais afetados. No DNA genômico foi encontrada uma grande deleção de 4165pb (g. NC_010460.4 del 6912538_6916702) na região do gene CLCN1. Análises de expressão relativa demonstraram níveis de expressão em tecido muscular de animais wild type para um transcrito associado a miotonia hereditári... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The major cause of hereditary non-dystrophic myotonia occurs due to mutations in the CLCN1 gene, coding for the CLC1 protein that forms the ionic channel selective for the predominant chloride ion in skeletal muscle tissue. The resulting hereditary disease is called congenital myotonia in human medicine. Mutations in the CLCN1 gene have been described as causing hereditary myotomy in several animal species, but in the swine species, no mutation in this gene has been described. The objective of this study was to perform the clinical and molecular characterization of hereditary myotonia in swine. The hypothesis of this study was that animals with compatible clinical signs had hereditary myotonia. These animals were evaluated under clinical, electromyographic, histopathological and molecular aspects. The clinical signs verifed were muscular hypertrophy and stifness, myotonia with startle response and formation of dimples. The phenomenon warm-up was evident. No muscular dystrophy was observed at the histopathological examination. Electromyographic examination showed classic myotonic discharges with characteristic sound. At the molecular level, the absence of nucleotides from exons 15 and 16 was verifed using cDNA samples of afected animals. In genomic DNA a large deletion of 4165bp (g NC_010460.4 del 6912538_6916702) was found in the region of the CLCN1 gene. Relative expression analyzes demonstrated expression levels of wild type animals for a transcript associated with heredita... (Complete abstract click electronic access below) / Doutor

Caligid copepods, also called sea lice, are ectoparasites of marine fish, with Lepeophtheirus salmonis (Krøyer, 1837) emerging as a problem for mariculture of Atlantic salmon (Salmo salar Linnaeus, 1758) in the northern hemisphere. Annual costs of sea lice to global salmon farming was estimated to be in excess of €300 million in 2006, with the majority of this accounted for through expenses accrued from chemical treatments. Only a limited range of anti-sea louse drugs are available and licensed for the treatment of fish, and the continued use of only a few compounds creates a situation potentially favouring the development of drug resistance. Emamectin benzoate (EMB) is currently used as a salmon delousing agent, being employed as a 0.2 % in-feed pre-mix (SLICE®). Atlantic salmon farmers have reported increased incidence of reduced L. salmonis sensitivity to SLICE®, which has highlighted the requirement for further research into the molecular mechanisms controlling salmon louse resistance to EMB. Genomic and transcriptomic research concerning L. salmonis drug resistance mechanisms has not often been reported, with previous transcriptomic studies using candidate gene approaches and genetic studies focussing on population genetics. Drug resistance in ecdysozoan invertebrates is associated with a variety of molecular mechanisms including target site mutations and changes in the expression of components in drug detoxification pathways. The research reported in this thesis was aimed at the exploration of mechanisms employed by L. salmonis to reduce the toxicity of EMB exposure, following a transcriptomic approach that utilised custom oligonucleotide (oligo) microarrays and a genetic approach that utilised Restriction-site associated DNA sequencing (RAD-seq) to identify Single Nucleotide Polymorphism (SNP) markers. An EMB-resistant (PT) and drug-susceptible (S) L. salmonis laboratory-maintained strain were to be used as a model for this research, as these two strains differ in EMB susceptibility (~ 7-fold) and show stable susceptibility profiles through multiple generations, suggesting that this drug resistance phenotype may be a heritable trait. Sequence resources available for salmon lice are limited as an annotated L. salmonis genome is currently under construction. Therefore, a significant amount of this study involved creating new resources to facilitate the analysis of EMB susceptibility. Suppression subtractive hybridisation (SSH) was used to enrich for transcripts that were differentially expressed between strains PT and S, which provided sufficient target sequence for the development of 15K oligo microarrays when combined with sequences assembled from existing L. salmonis ESTs. Additionally, transcripts were generated through sequencing a pooled sample representing key developmental stages of the L. salmonis life cycle, which were later used in the construction of a 44K oligo microarray. The toxicity of EMB and other avermectins (AVMs) against ecdysozoan invertebrates is reported to be based mainly on their interaction with ligand-gated ion channels (LGIC), specifically glutamate-gated chloride channels (GluCl). However, -aminobutyric acid (GABA)-gated chloride channels (GABA-Cls) are also believed to be targeted by AVMs and neuronal acetylcholine receptors (nAChRs) can be allosterically modulated by the AVM compound ivermectin. Transcriptional responses in PT and S salmon lice were investigated using custom 15K L. salmonis oligo microarrays. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains. GABA-Cl and nAChR subunits showed significantly lower transcript levels in PT compared to S lice, which was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR using RT-qPCR, suggesting their involvement in AVM toxicity in caligids. Although, salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 µg L-1 of EMB, a drug concentration tolerated by PT lice, but toxic for S lice. RAD-seq analysis of both genders from L. salmonis strains S and PT identified 15 RAD-markers that show complete association with salmon louse strain, although these preliminary results will need further analysis to confirm marker association with reduced EMB susceptibility. Additionally, RAD marker Lsa101901 showed complete association with sex for all individuals analysed, being heterozygous in females and homozygous in males. Using an allele-specific PCR assay, this SNP association pattern was further confirmed for three unrelated salmon louse strains. Marker Lsa101901 was located in the coding region of the prohibitin-2 gene, which showed a sex-dependent differential expression, with mRNA levels determined by RT-qPCR about 1.8-fold higher in adult female than adult male salmon lice. In conclusion, the identification of decreased transcript abundances for LGIC subunits in EMB-resistant salmon lice, and polymorphic SNP markers showing complete association with L. salmonis strains S or PT, provides suitable candidates for further investigation into their association with reduced EMB susceptibility. Further analysis will also be required to confirm whether EMB-induced mechanisms are not associated with reduced EMB susceptibility in L. salmonis. Additionally, the identification of sex-linked SNP Lsa101901 suggests that sex determination in the salmon louse is genetic and follows a female heterozygous system, with marker Lsa101901 providing a tool to determine the genetic sex of salmon lice. Improved knowledge of L. salmonis biology and the mechanisms potentially involved in EMB resistance, obtained during this study, may provide molecular markers that contribute to successful monitoring and management of this commercially important parasite of Atlantic salmon.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease with an annual mortality rate of 15% despite current therapies. Uncontrolled proliferation of pulmonary artery smooth muscle cells (PASMCs) results in adverse vascular remodeling contributing to PAH. Understanding the mechanisms of PASMC proliferation may identify new targets for treatment. Chloride currents/channels (ICl) are expressed in PASMCs and their roles in proliferation have been suggested based on their importance in resting membrane potential and cell volume regulation. The present study explored the role of ICl in proliferation in rat and human PASMCs. We found that either nonspecific ICl inhibitors (DIDS or NPPB) or a putative specific blocker of swelling-activated ICl (ICl,swell) reduced proliferation of PASMCs cultured in serum-containing media. Patch-clamp studies showed that proliferating PASMCs had increased baseline ICl and ICl,swell in association with depolarized membrane potentials. Quantitative real-time RT-PCR studies identified expressions of CLC-3, a candidate gene of ICl,swell, and several other CLC genes in proliferating PASMCs. While selective knockdown of CLC-3 with lentiviral shRNA reduced PASMC proliferation, it had no effect on ICl,swell. These findings are consistent with the conclusion that ICl regulate proliferation of PASMCs and suggest that selective ICl inhibition may be useful in treating pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease with an annual mortality rate of 15% despite current therapies. Uncontrolled proliferation of pulmonary artery smooth muscle cells (PASMCs) results in adverse vascular remodeling contributing to PAH. Understanding the mechanisms of PASMC proliferation may identify new targets for treatment. Chloride currents/channels (ICl) are expressed in PASMCs and their roles in proliferation have been suggested based on their importance in resting membrane potential and cell volume regulation. The present study explored the role of ICl in proliferation in rat and human PASMCs. We found that either nonspecific ICl inhibitors (DIDS or NPPB) or a putative specific blocker of swelling-activated ICl (ICl,swell) reduced proliferation of PASMCs cultured in serum-containing media. Patch-clamp studies showed that proliferating PASMCs had increased baseline ICl and ICl,swell in association with depolarized membrane potentials. Quantitative real-time RT-PCR studies identified expressions of CLC-3, a candidate gene of ICl,swell, and several other CLC genes in proliferating PASMCs. While selective knockdown of CLC-3 with lentiviral shRNA reduced PASMC proliferation, it had no effect on ICl,swell. These findings are consistent with the conclusion that ICl regulate proliferation of PASMCs and suggest that selective ICl inhibition may be useful in treating pulmonary arterial hypertension.

In the skeletal muscle cell membrane, the voltage gated chloride channel, CIC-1, maintains as unusually high resting membrane conductance and thereby prevents myotonic skeletal muscle disease. Protein crystallization experiments with bacterial CIC proteins, provide the information for the three dimensional (3D) structure of CIC chloride channels. / PhD Doctorate

Inorganic ions are one of the most important group chemicals. Their importance is crucial for all living organisms on the planet and monitoring of their content is essential for this purpose in clinical or environmental analysis but also in the food industry, agriculture or many other industries. Many analytical methods for ion detection have been designed and used, depending on the area of their use. Recently, numerous methods have been adapted to areas where not primarily intended. One such methods may be, for example, laser ablation with inductively coupled plasma mass spectrometry, which has recently begun to affect the analysis of biological samples. As a complementary method, able to detect also non-metallic ions is capillary electrophoresis. In this master thesis, the possibility of applying capillary electrophoresis with indirect photometric detection for the separation of inorganic ions in cell lysates of HeLa cells by capillary electrophoresis was verified. Main attention was paid to chloride ions. The ability to separate inorganic compounds in cellular material using salicylic acid as an absorbing anion in the base electrolyte was tested.

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