A infecção crônica das vias aéreas por Pseudomonas aeruginosa (PA) é a principal causa de morbidade e mortalidade em pacientes com fibrose cística (FC), devido à contínua degradação do tecido pulmonar, que leva ao declínio da função pulmonar, gerada pela infecção e pelo processo inflamatório. O objetivo do presente estudo foi analisar características genéticas de PA que levam à sua adaptação às vias aéreas destes pacientes com infecção pulmonar crônica, atendidos no Centro de Referência em FC do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto - USP e relacionar com resultados de tipagem molecular, resistência a antibióticos, cronicidade e dados clínicos dos pacientes em acompanhamento clínico no período de julho/2011 a abril/2014. As características genéticas dos isolados investigados englobam pesquisa de 18 genes de virulência e genes do sistema quorum sensing (genes lasR e rhlR), associação entre mutações e conversão para fenótipo mucoide (operon algTmucABD) e caracterização de linhagens hipermutantes (genes mutS e mutL). A identificação de P. aeruginosa foi realizada por PCR e MALDI-TOF, que mostraram alta concordância. Foram considerados os dados clínicos dos pacientes: índice de massa corpórea, escore de Shwachman, medidas de capacidade vital forçada e volume expiratório forçado no primeiro segundo. A porcentagem de pacientes com infecção pulmonar crônica por PA observada foi similar aos dados disponíveis na literatura, entretanto, a alta incidência em pacientes jovens foi preocupante. O perfil de macrorrestrição do DNA genômico por PFGE se mostrou útil para definição de colonização crônica/intermitente em associação com critérios clínicos e, juntamente com a detecção de mutações nos genes mucA e mucD confirmaram transmissão interpacientes. Foi observada alta ocorrência dos genes de fatores de virulência pesquisados para grande maioria dos isolados de pacientes crônicos. A resistência aos antibióticos pesquisados dos isolados de P. aeruginosa foi baixa e está de acordo com a literatura nacional e internacional e com a antibioticoterapia adotada no hospital. Não foi observada resistência aos carbapenêmicos e às fluoroquinolonas devido à presença de genes de resistência plasmideais. As mutações no gene mucA foram o principal mecanismo de conversão para o fenótipo mucoide e o fenótipo revertente não-mucoide ocorreu principalmente por mutações no gene algT. Foram detectadas novas mutações nos genes mutS e mutL que também suportam a ideia que hipermutação em PA está associada com mutações do sistema mismatch de reparo do DNA. O sistema quorum sensing dos isolados estudados está parcialmente prejudicado devido às várias mutações no gene lasR, mas todos conservam o gene rhlR intacto, que sustenta alguma atividade quorum sensing envolvida na produção de fatores de virulência importantes. Pacientes com infecção pulmonar crônica por PA com isolamento de outros bacilos gram-negativos não-fermentadores apresentaram maior alteração da função pulmonar quando comparados com pacientes com infecção pulmonar crônica por PA com ou sem isolamento de Staphylococcus aureus. As alterações presentes no operon algTmucABD, quorum sensing e hipermutabilidade contribuem para a cronicidade dos pacientes com FC em relação à infecção por P. aeruginosa. / The chronic airway infection by P. aeruginosa (PA) is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients, due to continuous degradation of the pulmonary tissue. This leads to decline in lung function, which is generated by the related infection and inflammation. The aim of this study was to analyze genetic characteristics associated with the adaptation of PA to the airways of patients with chronic pulmonary infection attended at the CF Reference Center from the Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto - USP; and to correlate these findings with the results of molecular typing, antibiotic resistance, chronicity and clinical data of patients in clinical follow-up from July/2011 to April/2014. The genetic characteristics of isolates investigated includes the research of 18 virulence genes and the quorum sensing system genes (lasR and rhlR genes), association between mutations and conversion to the mucoid phenotype (algTmucABD operon), and characterization of hypermutable strains (mutS and mutL genes). Identification of PA was performed by PCR and MALDI-TOF, which showed a high correlation. The patients\' clinical data considered were: body mass index, Shwachman score, forced vital capacity measures and forced expiratory volume in one second. The percentage of patients with chronic PA infection observed was similar to the data available in the literature; however, a worrying high incidence in young patients was noticed. The macrorestriction profile of genomic DNA by PFGE proved to be useful to define chronic/intermittent colonization in association with clinical criteria and it confirmed interpatient transmission, in combination with the detection of mutations in the mucA and mucD genes. High occurrence of virulence genes was detected for the vast majority of isolates from chronic CF patients. Antibiotic resistance for PA isolates was low and is in accordance with national and international literature and antibiotic therapy adopted in the hospital. There was no resistance to carbapenems and fluoroquinolones by the presence of plasmid mediated resistance genes. Mutations in the mucA gene were the main mechanism to conversion to mucoidy, and the non-mucoid revertants occurred mainly by mutations in the algT gene. New mutations in mutS and mutL genes were detected, which support the idea that hypermutation in PA is associated with mutations in the DNA mismatch repair system. The quorum sensing system of the isolates is partially damaged due to several mutations in the lasR gene, but all isolates maintain an intact rhlR gene, which holds some quorum sensing activity with production of important virulence factors. Patients with chronic PA infection with isolation of other non-fermenting gram-negative rods had greater change in lung function compared with patients with chronic PA infection with or without isolation of Staphylococcus aureus. The changes presented in the algTmucABD operon, quorum sensing and hypermutability contribute to the chronicity of CF patients in relation to infection by P. aeruginosa.

As infecções pneumocócicas são uma importante causa de morbi-mortalidade entre as crianças. Até 2000, era disponível apenas a vacina pneumocócica polissacarídica 23valente, de uso a partir dos 2 anos de idade. Essa vacina era recomendada para crianças com fibrose cística (FC) e para as asmáticas em uso de corticóide oral, dentre outras recomendações. A partir de 2000, licenciou-se a vacina pneumocócica conjugada 7valente, com grande impacto contra infecções causadas pelos sorotipos vacinais. Nos países onde as crianças não são universalmente vacinadas com essa vacina, as recomendações permanecem as mesmas. Atualmente, os adultos asmáticos estão incluídos nas recomendações para vacinação pneumocócica nos EUA. Há poucos estudos sobre o risco de doença pneumocócica em crianças asmáticas per si e naquelas com fibrose cística e sobre a resposta à vacina pneumocócica conjugada. Salienta-se que ainda não há um critério estabelecido para avaliar a resposta sorológica a essa vacina. Recentemente, foi sugerido o critério de 0,35mcg/ml por Elisa para se correlacionar com proteção para doença invasiva pneumocócica. Objetivou-se determinar a concentração dos anticorpos contra os sorotipos vacinais contidos na vacina pneumocócica conjugada 7valente em crianças com asma moderada em uso de corticóide inalatório e em crianças com fibrose cística; avaliando-as pelos critérios de 0,35mcg/ml, 1,3mcg/ml e aumento de 4 vezes o título pós em relação ao pré-vacinal, para cada sorotipo e para a vacina, considerando-se a positividade para 5 sorotipos. Foram avaliadas 18 crianças em cada grupo. A mediana da idade foi de 82,5m nas asmáticas e 69,5m naquelas com FC. Foi colhida amostra para sorologia pré-vacinação e outra após 2 doses da vacina conjugada. As concentrações de anticorpos para os sorotipos vacinais foram quantificadas pelo Elisa. Para 0,35mcg/ml de corte, a grande maioria nos dois grupos já era positiva à inclusão para os sorotipos vacinais e à vacina. Considerando-se o valor de 1,3mcg/ml, entre os que eram negativos, as crianças asmáticas responderam entre 66,7% (9V) e 100% (14), e as com FC, entre 50% (19F) e 100% (6B e 14); e, em relação à resposta vacinal para esse nível, as asmáticas apresentaram 81,8% de resposta, enquanto as com FC, 91,7%. Avaliando-se pelo aumento de 4 vezes o título pós em relação ao pré-vacinal, a melhor resposta aos sorotipos, nos asmáticos, foi de 33,3% (4, 6B, 14 e 18C), e a nos com FC, 61,1% para o 6B; em termos de resposta vacinal, obteve-se 16,7% e 44,4%, para as asmáticas e aquelas com FC, respectivamente. Não houve interferência da vacinação prévia com a vacina pneumocócica polissacarídica. As medianas dos títulos pós em relação aos pré-vacinais, para os sorotipos, nos dois grupos, apresentaram um aumento significante. Apesar de boa parte das crianças apresentarem uma positividade elevada à inclusão, aquelas que eram negativas tenderam a apresentar uma boa resposta à vacina. / Pneumococcal infections are an important morbi-mortality cause among children. Until 2000, it was only available the 23-valent polysaccharide pneumococcal vaccine for children over two years old. This vaccine was recommended for cystic fibrosis (CF) children and to asthmatics children in use of oral corticosteroids, among other recommendations. From 2000, it was licensed the 7-valent conjugated pneumococcal vaccine, with a great impact against the infections caused by the vaccine serotypes. In the countries that dont make a universally use of this vaccine for children, the recommendations remain the same. At the present time, asthmatic adults are included for the pneumococcal vaccine recommendations in the United States. There are few studies about pneumococcal disease risk with cystic fibrosis children and asthmatics, per si, and about the conjugated pneumococcal vaccine response. It points out that there are no a definitive criteria or evaluation established for the serology response for this vaccine. It was suggested, recently, that the level of 0,35mcg/ml, measured by ELISA, is adequate to correlate with the invasive pneumococcal disease protection. The goal of this study was to determine the antibodies concentration of the seven vaccine serotypes from 7-valent conjugated pneumococcal vaccine among children with moderate asthma in use of inhaled corticosteroids and with cystic fibrosis. It was considered the dosage 0,35mcg/ml and 1,3mcg/ml levels and the four-fold increase between pre- and post-immunization concentrations levels, to each serotype and to the vaccine (positivity for five serotypes or more) for positivity. Eighteen children were included in each study group. The age median was 82,5 months for the asthmatics and 69,5 months for the CF children. A blood sample was taken for pre-immunization serology and a second one after the second vaccine dose was given. The antibodies concentrations for the vaccine serotypes were measured by ELISA. Considering the 0,35mcg/ml levels, the majority of children, in both groups, was positive for vaccine serotypes and for the vaccine as well in the beginning. At the 1,3mcg/ml level, among the children with negative serology, asthmatic children responded between 66,7% (9V) and 100% (14), and those with CF, between 50% (19F) and 100% (6B e 14). Related to the vaccine response for this level, the asthmatics had a 81,8% response, while the CF childrens response was 91,7%. Evaluating for the four-fold increase between pre- and post-immunization concentrations, the best response observed for the vaccine serotypes was 33,3% (4, 6B, 14 e 18C) for the asthmatics. In the CF group the best result was 61,1% (6B). In terms of the vaccine response, it was observed that 16,7% and 44,4% were the results for both the asthmatics and CF group, respectively. The polysaccharide vaccine didnt interfere in the results. The medians of the pre- and post-immunization antibodies concentrations for the vaccine serotypes, in both groups, were significantly increased. Despite those children that were already positive for the criteria evaluated, at the first moment of the study, for those children that were negative, the majority had a positive serology towards the vaccination response.

Protein stability is critical for the proper function of all proteins in the cell. Ubiquitin is a key post-translational modification that serves as a universal regulator of protein turnover and has emerged as a highly sought-after signal for biological inquiry and drug development. Yet the pervasive role of ubiquitin signaling has given rise to the fundamental challenge of selectively manipulating a widespread signal: current pharmacological and genetic tools that target the ubiquitin-proteasome system (UPS) broadly alter cellular proteostasis with confounding side effects. Ion channels are essential proteins that regulate fundamental cellular properties including; electrical activity, fluid homeostasis, muscle contraction, neuronal firing, gastric acidification, and gene expression. Enhanced or reduced ion channel expression represents a pathological signature for a myriad of disease states, from chronic pain to cardiac arrhythmias, epilepsy, and cystic fibrosis. Although ubiquitin represents a critical mediator of ion channel expression, the inability to precisely manipulate ubiquitin modifications in situ has limited mechanistic insight and opportunities for therapeutic intervention. To address this barrier, I developed a novel nanobody-based toolset to selectively – and bidirectionally – manipulate the ubiquitin status and functional expression of target ion channels for basic study and therapeutic rescue.

Background: Body mass index (BMI) is the primary accepted method to determine nutrition status in children with cystic fibrosis (CF); however, lean body mass (LBM) is more strongly associated with pulmonary function. Handgrip strength (HGS) measures muscle function and is reflective of LBM. The aims of this study were to assess if there was a relationship among HGS, nutrition status, and pulmonary function, to assess if HGS changed after hospitalization, and to assess if there was a relationship between HGS and nutrient intake. Methods: Twenty-three children with CF ages 6-18 years participated. BMI z-scores, nutrition risk scores, and pulmonary function were assessed about five months before, day 5-7 of, and about six weeks after hospitalization. HGS z-scores and arm anthropometrics were measured during and after hospitalization. Nutrient intakes were assessed during hospitalization. Results: Mean dominant HGS z-score was -1.95 ± 0.92 at hospitalization and -1.59 ± 1.06 at follow-up (p=0.007). Mean BMI z-score was -0.09 ± 0.64 at hospitalization and 0.06 ± 0.54 at follow-up (p=0.178). No significant relationship was found between HGS z-scores and BMI z-scores (p=0.892) or HGS z-scores and pulmonary function (p=0.340). Conclusions: HGS z-scores were lower than the standard even though mean BMI z-scores classified participants as normal nutrition status. Further research should be done utilizing a larger sample size in order to better examine HGS's potential as a nutrition assessment tool in this population.

Les niveaux de chlorure sont rigoureusement régulés par des canaux de chlorure tels que le régulateur transmembranaire de mucoviscidose (CFTR), la famille de canaux CLC ou les canaux de chlorure activés par le calcium. Un dérèglement des concentrations de chlorure ou du transport de ce dernier a été rapportée pour être lié à plusieurs maladies telles que la mucoviscidose, la myotonie, l'épilepsie, l'hyperekplexie ou la surdité. Toutes ces maladies appartiennent à la famille des pathologies qui présentent un déficit de la fonction lysosomale et qui sont caractérisées par un défaut du processus autophagique.Les cellules épithéliales pulmonaires des malades atteints de mucoviscidose montrent également un défaut d’autophagie. L’association de deux produits : la cysteamine et l’epigallocathecin gallate (EGCG) permet de restaurer la fonction autophagique de ces cellules et améliore les symptômes de la maladie. Nous avons tenté d'améliorer cette combinaison en criblant des inducteurs de l'autophagie pour leur capacité d’interaction avec la cystéamine et pour une meilleure efficacité de traitement. Nous avons ainsi trouvé que l'amiodarone, de manière similaire à l'EGCG, était capable d'engager une interaction coopérative avec la cystéamine pour stimuler l'autophagie dans les lignées cellulaires. De plus, l'amiodarone s’est révélé relativement efficace pour restaurer l'expression d’une protéine Del 508 CFTR mature et fonctionnelle dans les cellules épithéliales.Dans la deuxième partie de cette thèse, nous avons exploré les relations entre la modulation des concentrations intracellulaires des ions chlorure et l'autophagie. Trois approches complémentaires ont été utilisées (i) l’appauvrissement / réduction du chlorure dans le milieu de culture des cellulaires (ii) la réduction de l'expression du gène CLCN7 codant pour le seul transporteur de chlorure connu pour être présent dans les lysosomes et (iii) l’utilisation de transporteurs synthétique des ions chlorure. Nous avons montré que la modulation de la concentration des ions Cl- régulait l'autophagie. Cela a été observé lorsque nous avons déplété le chlorure du milieu de culture cellulaire ou lorsque nous avons aboli le gradient de chlorure en utilisant des transporteurs de chlorure synthétique (SCTs). Nous avons également montré que les STCs modifiaient la structure et la fonction des mitochondries. De plus, nous avons montré que les antioxydants restauraient la fonction mitochondriale et inhibaient la stimulation de l'autophagie, ce qui permet de voir sous un jour nouveau le rôle que le chlorure pourrait jouer dans la signalisation de différentes voies cellulaire de réponse au stress. / Chloride levels are stringently regulated by chloride channels such as cystic fibrosis transmembrane regulator (CFTR), the CLC family of channels or calcium activated chloride channels. A dysregulation of chloride concentrations or transport has been reported to be linked to several diseases including cystic fibrosis (CF), myotonia, epilepsy, hyperekplexia or deafness. All these diseases belong to the family of lysosomal storage pathologies which are characterized by a defect in the autophagic process.The pulmonary epithelial cells of CF patients show a defect of autophagy. The combination of two products: cysteamine and epigallocathecin gallate (EGCG) has been shown to restore the autophagic function of these cells and to improve the symptoms of the disease. We have tried to enhance this combination by screening inducers of autophagy for their ability to interact with cysteamine and for better treatment efficacy. We found that amiodarone, similarly to EGCG, was able to engage in a cooperative interaction with cysteamine to stimulate autophagy in cell lines. In addition, amiodarone has been found to be relatively effective in restoring expression of a mature and functional Del F508 CFTR protein in epithelial cells.In the second part of this thesis, we explored the relationships between the modulation of intracellular chloride concentration and autophagy. Three complementary approaches were used (i) the depletion/reduction of chloride in the cell culture medium (ii) the reduction of the expression of the CLCN7 gene encoding the only chloride transporter known to be present in lysosomes and (iii) the use of synthetic carriers of chloride ions. We have shown that the modulation of the concentration of Cl- modulates autophagy. This was observed when we depleted chloride from the cell culture medium or when we abolished the chloride gradient using Synthetic Chloride Transporters (SCTs). We have also shown that SCTs modify the structure and function of mitochondria. In addition, we have shown that anti-oxidants restore mitochondrial function and inhibit the stimulation of autophagy, allowing us to see in a new light the role that chloride could play in different stress response pathways.

La mucoviscidose (ou cystic fibrosis, CF) est une maladie caractérisée par une inflammation pulmonaire chronique qui contribue à la dégradation progressive de l’épithélium des voies aériennes des patients. Les glucocorticoïdes (GC) représentent un outil essentiel pour le traitement du patient mais leur efficacité et leur rapport bénéfice/risque restent cependant controversés. Les effets secondaires provoqués par l’administration de ces molécules pourraient être diminués par l’utilisation de leur protéine d’adressage, la corticosteroid-binding globulin (CBG). L’objectif de ce travail était d’étudier l’expression de la CBG chez les patients CF, afin d’optimiser leur traitement anti-inflammatoire par GC. Nous avons montré, dans un premier temps, que la synthèse hépatique de CBG était augmentée, tandis que son taux plasmatique était conservé chez les patients CF, comparé aux patients non-CF. Dans un second temps, nous nous sommes intéressés à l’expression de la CBG au niveau pulmonaire. L’inflammation chez les patients CF étant principalement pulmonaire, l’expression locale de CBG à ce niveau pourrait moduler l’efficacité du GC, en le recaptant. Nos données montrent que l’expression de cette CBG pulmonaire est diminuée chez les patients CF. Les études sur des modèles in vitro hépatiques et pulmonaires n’ont pas permis d’expliquer les résultats obtenus et ont souligné la limite des modèles et des outils à disposition. Le maintien de la concentration plasmatique de CBG et la diminution de la CBG pulmonaire chez les patients CF suggèrent ainsi que la CBG pourrait être utilisée comme outil thérapeutique dans le contexte de la mucoviscidose, afin d’optimiser le traitement par GC. / Cystic fibrosis (CF) is characterized by a chronic pulmonary inflammation, responsible of the progressive degradation of the airways epithelium. In CF, glucocorticoids (GC) are widely used but their efficiency and benefit/risk ratio are still discussed. The side effects, caused by the administration of these molecules, might be decreased by the use of their delivery protein, corticosteroid-binding globulin (CBG). The aim of the work was to study the expression of CBG in CF patients, in order to optimise their anti-inflammatory treatment by GC. First, we showed that the hepatic synthesis of CBG was increased while its plasmatic level was preserved in CF patients, compared to non-CF. Second, we were interested by the expression of CBG at the pulmonary level. The inflammation in CF patients being primarely pulmonary, the local expression of CBG in the lung could modulate the efficiency of GC through recapture. Our data showed that this expression of this pulmonary CBG was decreased in CF patients. The studies conducted in vitro on hepatic and pulmonary models did not explained our results and highlighted the limit of the models and tools at our disposal. The maintained plasmatic concentration of CBG and the decrease of pulmonary CBG in CF patients suggest that CBG might be useful as a therapeutic tool in the CF context, in order to optimise the GC treatment.

Vitamin K belongs to the family of fat-soluble vitamins, which is not determinated in clinical laboratories. It is a cofactor necessary for posttranslational γ-carboxylation of glutamyl residues in selected proteins such as the osteocalcin, and procoagulation factors II, VII, IX, X. Vitamin K deficient individuals appear to have more undercarboxylated proteins, which are functionally defective. Lack of this vitamin has been associated with risk of developing osteoporosis and cardiovascular diseases. The aim of this work was to develop and validate the HPLC method and the LC-MS/MS method for determination of three vitamin K's forms - vitamin K1, MK-4 and MK-7 in serum. After successful validation of both methods, patient samples and healthy population samples were measured. There were measured 350 patient samples by HPLC method. These samples were divided into two groups - patients with diagnostic of osteoporosis and patients without osteoporosis. We measured 946 samples by LC-MS/MS method. Samples were divided into groups: patients with osteoporosis, patients without osteoporosis, healthy population, patients with osteopenia and patients with cystic fibrosis. The reference range of vitamin K in healthy population was obtained by LC-MS/MS method. The next aim was to compare the effectiveness of...

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The aim of this study was to evaluate the relationship of oxidative stress (OS) marker s in exhaled breath condensate (EBC) in adult patients with cystic fibrosis (CF) to the severity of lung disease, nutritional status and systemic antioxidants and inflammatory markers, as well as to short - and medium - term development of pulmonary function and nutritional status, and finally to assess the response to treatment with inhaled corticosteroids (ICS). Methods: CF patients were examined in a stable phase of the disease during routine outpatient controls. EBC was collected using E C oScreen device (J aeger) in CF patients and in control group members. Nitrites and nitrates and 8 - isoprostane were examined using liquid chromatography and competitive enzyme immunoanalysis, respectively, in EBC as OS markers. Demographic data including the dominant pathogen of airway colonization and ICS treatment were recorded in CF patients. Lung function tests, chest X-ray s, nutritional statuses and systemic antioxidants and inflammatory markers were also examined using standard methods. The values of OS markers in EBC in patients with CF were compared with the control group and correlated to clinical parameters. Lung function tests and nutritional status es in CF patients were examined in one, three and five years intervals...

The primary clinical characteristics of cystic fibrosis (CF) are malnutrition caused by malabsorption secondary to pancreatic insufficiency, chronic pulmonary infections, and male infertility. The major cause of morbidity and mortality are bronchiectasis and obstructive pulmonary disease. Lung disease in CF is manifested by this chronic lung disease progression, with intermittent episodes of acute worsening of symptoms called pulmonary exacerbations. Once the patient has stabilized, and if suitable care can be arranged, these interventions are often transitioned to the home. This review summarizes important points pertinent to the use of intravenous and inhaled antimicrobials that may be encountered by prescribers, nurses, technicians, and case managers in the home health setting. Appropriate dosing, indications, adverse drug reactions, monitoring parameters, and practicality of both intravenous and inhaled antimicrobials are discussed.