Objetivos: Avaliar a estatura final, mineralização e marcadores de mineralização óssea de adultos com síndrome nefrótica (SN) idiopática corticossensível na infância e adolescência e analisar a influência da doença, suas comorbidades e do alvo de estatura no crescimento e mineralização destes pacientes. Casuística: Avaliamos a estatura final de 60 pacientes (41 masculinos e 19 femininos) com idade mínima de dezenove anos ou desenvolvimento genital P4G4 nos masculinos e menarca nos femininos portadores de SN corticossensível na infância e adolescência. Realizamos a densitometria óssea (DMO=g/cm2) em 26 destes pacientes e em 35 controles, com análise concomitante dos níveis séricos de 25 OH vitamina D3 (25(OH)D), Paratormônio (PTH), telopéptido carboxiterminal do colágeno tipo 1( (CTx), Propeptídeo Aminoterminal do Colágeno Tipo I (P1NP) e Osteocalcina (OC) Resultados: A idade média inicial dos 60 pacientes foi de 5a3m e final de 20a5m, com acompanhamento médio de 15a2m. A dose média de prednisona utilizada foi de 1264 mg/kg. O Zscore médio da estatura inicial (-0,60; SD: 1,0) e final (0,64; SD: 0,92), não diferiu significativamente (Teste T: p=0,72) entre si. O Zscore estatura na idade adulta se correlacionou significativamente apenas com o Zscore estatura inicial e com o Zscore alvo de estatura. Seis pacientes atingiram Zscore estatura < -2 na idade adulta e este achado demonstrou forte correlação com o Zscore estatura inicial e com o Zscore alvo de estatura. A DMO e Zscore DMO de L1L4, Cabeça do fêmur e do Fêmur total dos pacientes e controles não diferiram significativamente. 6 pacientes e 2 controles apresentaram Zscore DMO < -2 (massa óssea reduzida) enquanto 2 pacientes e 1 controle demonstraram , Zscore DMO < -2,5 (osteoporose). Pacientes com massa óssea reduzida receberam 2189 mg/kg de prednisona durante 13 anos e aqueles com osteoporose, 2510 mg/kg durante 14 anos. Estes valores, comparados com aqueles de pacientes com massa óssea normal, mostraram significância estatística (p=0,01). Não houve correlação significativa entre as demais variáveis analisadas e a DMO. Os marcadores 25(OH)D, PTH, CTx, P1NP e OC dos pacientes e controles não diferiram significativamente. Quando analisados em relação à doença e suas comorbidades, DMO e estatura final não apresentaram significação estatística. Conclusões: 1. Os valores de Zscore estatura inicial e final se correlacionaram fortemente com o alvo de estatura. 2. Não houve associação entre as características clinicas da doença e a aquisição do alvo de estatura, neste grupo de pacientes. 3. A massa óssea e os marcadores de mineralização dos pacientes não diferiram quando comparados aos controles. 4. Os 6 pacientes com massa óssea reduzida (2 com osteoporose) utilizaram dose total e tempo de uso da prednisona significativamente maior que aqueles com massa óssea adequada 5. Não houve correlação entre os níveis séricos dos marcadores de mineralização óssea e a doença e suas comorbidades, a estatura final e a DMO dos pacientes adultos com SN na infância e adolescência / Objectives: The aim of the present study was to evaluate the final height, bone mineral density (BMD) and bone mineralization markers of adults with steroid responsive Idiopathic Nephrotic Syndrome (NS) in childhood and adolescence and to examine the influence of the disease, its co-morbidities and the patients\' target height in the final height and mineralization results. Patients and Methods: We have analyzed initial and final anthropometric data of 60 patients (41 male and 19 females) and / or their records, with a minimum age of nineteen years or fully developed pubertal status (P4G4 in males and menarche in females). BMD (g/cm2) was evaluated in 26 patients and in 35 controls, with a concomitant analysis, of serum levels of 25-OH Vitamin D (25(OH)D), Parathyroid Hormone (PTH); C-terminal telopeptide of type I collagen (CTx) and aminoterminal propeptide of type 1 procollagen (P1NP) and Osteocalcin (OC) Results: Mean age at first consultation was 5.3 years (SD: 2.4 yrs) and at last consultation was 20.4 yrs (SD: 3.0 yrs). The mean cumulative dose of prednisone was 1254 mg/kg (SD: 831.39 mg/kg). The mean initial height SDS was -0.60; (SD: 1.0) the final height SDS was -0.64; (SD: 0.92), (t-test: p=0.72). The final height SDS showed correlated significantly only with the initial height SDS and the target height SDS. Six patients achieved a final height SDS <-2 and this finding showed a strong correlation to the initial height SDS and to the target height SDS in the male patients. The patients\' and control subjects L1L4 head of the femur and the total femur BMD and BMD SDS did not differ significantly. 6 patients and 2 control subjects showed a BMD SDS <-2 (low bone mass) while 2 patients and 1 control subjects showed a BMD SDS <-2.5 (osteoporosis). Patients with BMD SDS <-2 received 2189 mg / kg of prednisone over 13 years while those with a BMD SDS <-2.5 received 2510 mg / kg prednisone for 14 years (p = 0.01 vs BMD SDS -2 ). No other studied variable correlated significantly with BMD. The studied bone biomarkers showed similar results in patients and control subjects without a significant correlation with disease activity, co-morbidities, and BMD or height parameters. Conclusion: 1. the initial and final height SDS were strongly correlated to the height target. 2. INS and its co-morbidities did not prevent the patients to reach their target height 3. The patients\' BMD and bone mineralization markers did not differ when compared to controls. 4. The 6 patients with low bone mass (2 with osteoporosis) used a total dose of prednisone for a longer period of time in relation to those with an adequate BMD 5. There was no correlation between bone mineralization markers, disease activity and its co-morbidities, final height and BMD of adult patients with INS in childhood and adolescence

O treinamento aeróbico (TA) traz benefícios para os asmáticos, porém os mecanismos antiinflamatórios não são conhecidos. Estudos experimentais de asma têm mostrado que o TA reduz a inflamação pulmonar alérgica crônica (IPAC) e a reposta Th2, no entanto, nenhum estudo explicou quando os efeitos protetores são iniciados e qual é a principal via anti-inflamatória desencadeada. Objetivo: Avaliar o efeito do TA ao longo do tempo em um modelo murino de asma visando identificar quando são iniciados os efeitos anti-inflamatórios e a reversão do remodelamento brônquico (RB). Métodos: BALB/c (160 animais) foram divididos em 4 grupos: Controle (CT): não induzidos à IPAC e não treinados; Treinamento Aeróbico (TA): não induzidos à IPAC e treinados; OVA: induzidos à IPAC e não treinados; OVA+TA: induzidos à IPAC e treinados. Em seguida foram criados outros subgrupos 1, 3, 7, 15 e 30 dias de TA, ou seja, cada grupo foi repetidos 5 vezes para investigação do efeito do TA ao longo do tempo. Os grupos OVA foram sensibilizados com i.p. (OVA+HidroxAlum), após foram induzidos à IPAC com aerosol de OVA (1-3%) iniciado no dia 21 (3 x semana; 30 min./sessão). A adaptação ao TA foi realizada entre os dias 21 a 23, no dia 25 foi realizado o teste físico, no dia 28 o TA foi iniciado (50% intensidade, frequência 5 x, por 4 semanas). Vinte quatro horas da ultima sessão de TA (1, 3, 7, 15 e 30 dias) os animas foram anestesiados, eutanizados e coletados o lavado broncoalveolar (LBA) (contagem celular total e diferencial), sangue para quantificação das imunoglobulinas (IgE e IgG1) por técnica de reação de anafilaxia cutânea passiva (PCA), o tecido pulmonar para avaliação dos mediadores: IL-4, IL-5, eotaxina, RANTES, ICAM-1, VCAM-1, TGF-b, VEGF, Osteopontina (OPN), NF-kB, FOXP3, receptor de glicocorticóide (RG) e anti-inflamatórias IL-10 e IL-1ra (imunohistoquímica e quantificação por morfometria) e foi coletado também o músculo quadríceps para avaliação da produção das miocinas (IL-10, IL-1ra e IL-6) (imunohistoquímica e quantificado por análise de imagem). O RB (músculo liso, epitélio, deposições das fibras de colágeno e elástica e produção de muco) também foi avaliado por análise de imagem. Resultados: Não foi observada produção das miocinas (p>0,05). Os níveis de IgE, IgG1, migração celular, produção dos mediadores inflamatórios e o RB foram aumentados nos grupos OVA (p<0,05), que ainda mostraram redução da produção do RG (p<0,05). O TA aumentou o RG no músculo liso das vias aéreas, as produções de IL-10 e IL- 1ra aumentaram a partir do 7º dia por células peribrônquicas, ao mesmo tempo que foram reduzidos o NF-kB, IL-4, IL-5, eotaxina, RANTES, ICAM-1, VCAM-1, VEGF, eosinófilos no LBA e foram revertidos o espessamento do músculo liso, do epitélio e as deposições de fibras de colágeno (p<0,05). Curiosamente, a diminuição de TFG-b ocorreu após o 3º dia, enquanto OPN, elástica e muco ocorreram após 15 dias de TA, enquanto IgE, IgG1 e neutrófilos apenas foram reduzidas ao final de 30 dias (p<0,05). Conclusão: A partir do 3º dia do TA foi iniciado o mecanismo anti-inflamatório pelo aumento do RG no músculo liso das vias aéreas, seguido pelo aumento de IL-10 e IL-1ra e pela redução de NF-kB a partir do 7º dia do TA, efeitos que reverteram a inflamação alérgica crônica e o RB / The aerobic training (AT) promotes benefits for asthmatics, but the anti-inflammatory mechanisms are not known. Experimental studies of asthma have shown that AT reduces the pulmonary allergic chronic inflammation (PACI) and response Th2, however no study has ever explained when the protective effects are initiated and which is the main anti-inflammatory pathway triggered. Aim: To evaluate the effect of AT over time in a murine model of asthma to identify when the anti-inflammatory effects is started and reverse bronchial remodeling (BR). Methods: BALB/c (160 mice) were divided into 4 groups: Control (CT): not induced to PACI and untrained; Aerobic Training (TA): not induced to PACI and trained; OVA: induced to PACI and untrained; OVA + TA: induced to PACI and trained. After that were created others subgroups 1, 3, 7, 15 and 30 days AT, that is, each group was repeated 5 times to investigate the effect of AT over time. The OVA groups were sensitized with i.p. OVA (OVA+AlumHidrox), and then the mice were induced after the PACI with aerosol of OVA (1-3%) started on the 21st day (3 x week, 30 min./Session). Adaptation to TA was held between 21-23, on the 25th day the physical test was performed, and on day 28 AT was begun (50% intensity, frequency x 5 for 4 weeks). Twenty four hours of the after last session of AT (1, 3, 7, 15 and 30 days) the mice were anesthetized, euthanized and the bronchoalveolar lavage fluid was collected (BALF) (Total and differential cell count) and blood was used to quantify immunoglobulins (IgE and IgG1) by passive cutaneous anaphylaxis reaction (PCA) technique, the pulmonary tissue was removed and used to evaluate the mediators IL-4, IL-5, eotaxin, RANTES, ICAM-1, VCAM-1, TGF-b, VEGF, osteopontin (OPN), NF-kB, FOXP3, glucocorticoid receptor (GR), and antiinflammatory IL-10 and IL-1ra (immunohistochemistry and quantified by morphometry), was also the quadriceps muscle to assess the expression of myokines (IL-10, IL-1ra and IL-6) (by immunohistochemistry and image analyses). The BR (smooth muscle, epithelium, collagen and elastic fibers deposition, and mucus production) was also evaluated by image analysis. Results: It was not observed any production of myokines (p>0.05). The levels of IgE and IgG1, cell migration, production of inflammatory mediators, and the BR were increased in the OVA groups (p<0.05); that still showed a decreased production of the GR (p<0.05). The AT promoted an increase of GR in the airway smooth muscle from the 3rd day, the production of IL-10 and IL- 1ra were increased from day 7 for cells peribronchial, while NF-kB, IL-4, IL-5, eotaxin, RANTES, ICAM-1, VCAM-1, VEGF, eosinophil counting in BALF were reduced, and reversed the smooth muscle thickening, epithelium and deposition of collagen fibers too (p<0.05). Interestingly, the decreasing of TGF-b occurred in the 3rd day, and OPN, elastic fibers, mucus occurred after 15 days of AT, while IgE and IgG1, and neutrophils were reduced only after 30 days (p<0.05). Conclusion: The anti-inflammatory mechanism by increasing the GR on the smooth muscle of the airways was initiated from the 3rd day of the AT, followed by an increase of IL-10 and IL-1ra and a reduction of NF-kB from the 7th day of the AT, reversed the effects of chronic allergic inflammation and bronchial remodeling

Los trastornos emocionales de tipo depresivo y la ansiedad son las formas más prevalentes de enfermedad mental y suponen un serio problema de salud en la sociedad occidental. Recientemente, se ha postulado que el sistema endocannabinoide pueda ser un importante sustrato en el desarrollo de estos trastornos dada su participación en el control de las emociones. Nuestros resultados demuestran que los animales carentes del receptor cannabinoide CB1 manifiestan un fenotipo de tipo depresivo asociado a una deficiencia del factor neurotrófico BDNF en el hipocampo, que podría estar causada por los elevados niveles de glucocorticoides liberados en respuesta al estrés en estos mutantes. Por otra parte, el sistema endocannabinoide participa en los efectos inducidos por la nicotina sobre la ansiedad y en la expresión del síndrome de abstinencia de esta droga. Así, la actividad del receptor CB1 alivia los efectos ansiogénicos de dosis elevadas de nicotina y facilita los efectos ansiolíticos de dosis bajas. Además, la administración del agonista cannabinoide &#61508;9-THC atenúa las manifestaciones somáticas y emocionales negativas de la abstinencia de nicotina. En general, considerando los resultados presentados en esta Tesis Doctoral, podemos afirmar que el receptor CB1 participa de forma determinante en la recuperación del balance homeostático del organismo tras la exposición a un estímulo emocional negativo, bien sea una situación estresante aguda o sostenida, o bien una droga que incrementa los niveles de ansiedad o cuya retirada produce abstinencia. / Mood disorders such as depression and anxiety are the most common mental diseases and they suppose a serious health problem in our society. Recently, endocannabinoid system has been postulated to be an important substrate in the development of such disorders taking into account the role exerted by this neuromodulatory system in mood and emotions. Our results demonstrate that CB1 knockout mice exhibit a depressive-like phenotype associated to a deficiency in the neurotrophic factor BDNF in the hippocampus, which could be a consequence of the increased glucocorticoid release in response to stress exposure. On the other hand, the endocannabinoid system participates in nicotine induced effects on anxiety and in the expression of nicotine withdrawal. Thus, CB1 receptor activity attenuates anxiogenic-like effects and facilitates anxiolytic-like responses induced by high or low doses of nicotine, respectively. Moreover, &#61508;9-THC administration ameliorates somatic and negative motivational signs of nicotine withdrawal. In summary, the results presented in this Doctoral Thesis indicate that CB1 receptor participates in the recovery of the homeostatic balance after the exposure to negative emotional stimuli, either acute or sustained stress or a drug which induced anxiety-like effects or withdrawal signs after the end of the exposure.

No description available.

Στην παρούσα διδακτορική διατριβή προτείνονται μεθοδολογίες υπολογιστικής νοημοσύνης για την επεξεργασία και ανάλυση δεδομένων γονιδιακής έκφρασης μικροσυστοιχιών cDNA. Πιο συγκεκριμένα, στο πρώτο σκέλος αναπτύσσονται δύο νέες προσεγγίσεις για την εύρωστη εκτίμηση και διόρθωση του θορύβου υποβάθρου: η διόρθωση υποβάθρου βάσει εκατοστημορίων και η διόρθωση υποβάθρου βάσει παλινδρόμησης loess. Οι προσεγγίσεις αυτές καινοτομούν κυρίως στο ότι χρησιμοποιούν μία εύρωστη εκτίμηση του θορύβου υποβάθρου, γεγονός που τις καθιστά ιδανικές σε περιπτώσεις, όπου τα δεδομένα είναι θορυβώδη. Επιπροσθέτως, αναπτύσσεται ένα νέο, γενικής χρήσεως, πλαίσιο για τη συστηματική αξιολόγηση του βαθμού επίδρασης των μεθόδων διόρθωσης υποβάθρου. Μέσω του πλαισίου αυτού, οι δύο προτεινόμενες προσεγγίσεις, καθώς και άλλες ευρέως χρησιμοποιούμενες μέθοδοι, αξιολογούνται βάσει εφαρμογής τους σε διαφορετικά σύνολα δεδομένων αυτο-υβριδοποίησης, με τις πρώτες να εμφανίζουν ιδιαιτέρως καλή απόδοση. Το πλαίσιο αυτό καινοτομεί στο ότι ενσωματώνει νέα κριτήρια και τρόπους γραφικής απεικόνισης. Τόσο οι προτεινόμενες μέθοδοι εκτίμησης και διόρθωσης θορύβου υποβάθρου, όσο και το πλαίσιο συστηματικής αξιολόγησής τους, συνιστούν μία νέα, ενδελεχή μελέτη που προσανατολίζει στην εφαρμογή ή απόρριψη μίας συγκεκριμένης προσέγγισης, συνεισφέροντας εν τέλει στην κατάκτηση καλλίτερης ποιότητας δεδομένων μικροσυστοιχιών. Επίσης, στο δεύτερο σκέλος της διατριβής αναπτύσσεται ένα νέο, ολοκληρωμένο και γενικής χρήσεως πλαίσιο ανάλυσης δεδομένων μικροσυστοιχιών ούτως, ώστε να διερευνηθεί το ζήτημα εάν στην T-λευχαιμική κυτταρική σειρά CCRF-CEM επικρατούν εγγενείς ή επίκτητοι μηχανισμοί αντοχής στην πρεδνιζολόνη. Συγκεκριμένα, καταλλήλως επιλεχθέντα δεδομένα μικροσυστοιχιών cDNA – που διευκολύνουν την εξέταση τόσο της εξαρτώμενης από τη συγκέντρωση δράσης, όσο και της δυναμικής της ανταπόκρισης στην πρεδνιζολόνη (πρώιμη και όψιμη δράση) – γίνονται αντικείμενο επεξεργασίας και ενδελεχούς ανάλυσης, και βάσει συγκεκριμένων, προ-διατυπωμένων συλλογισμών, προσεγγίζεται το εν λόγω ερώτημα. Το πλαίσιο αυτό είναι καινοτόμο, εφόσον, πέραν του ότι ενσωματώνει μία πρωτότυπη ακολουθία μεθόδων, προσεγγίζει συστηματικά το πρόβλημα της εγγενούς ή επίκτητης αντοχής, συνεισφέροντας, έτσι, στην ευρύτερη προσπάθεια διερεύνησης των επακριβών μηχανισμών αντοχής των λευχαιμικών κυττάρων στα γλυκοκορτικοειδή. Τα αποτελέσματα από την εφαρμογή του στα δεδομένα της εν λόγω κυτταρικής σειράς συνηγορούν υπέρ της ύπαρξης μίας σύνθετης ανταπόκρισης του υπό μελέτη συστήματος στα γλυκοκορτικοειδή, η οποία όμως τείνει περισσότερο προς έναν εγγενή μηχανισμό αντοχής. / In the present Ph.D. thesis, computational intelligence methods for processing and analyzing cDNA microarray gene expression data are designed and developed. More specifically, in the first part of this thesis, the problem of background estimation and correction of two-channel microarray data is addressed and two novel algorithms are proposed, namely the percentiles-based and the loess-based background correction methods. Both approaches are based on the multiplicative model of background, while utilizing robust background noise estimators, thus making them ideal for noisy datasets. Furthermore, a new, generic framework for the systematic evaluation of the impact of the background estimating methodologies is suggested, whereupon the aforementioned methods as well as other approaches are evaluated by application to various publicly available self-self hybridization datasets. As suggested by this thorough, comparative evaluation our algorithms perform very well regarding noise reduction. The evaluation framework, which is based mainly on different and widely used statistical measures, incorporates new criteria and visualization methods. Moreover, it represents a novel, detailed contribution to the examination of the impact of background correction methods to the final interpretation of microarray experiments, conferring explicit guidance on the pros and cons of them and when they should be applied. Additionally, in the second part of this thesis, a new, generic, computational microarray data analysis framework is described, in order to examine the hypothesis of whether the resistant T-cell leukemia cell line CCRF-CEM posses an intrinsic or exert an acquired mechanism of resistance and to investigate the molecular imprint of this, upon prednisolone treatment. More analytically, using the above explained computational analysis workflow, microarray data that enable the examination of both the dose effect of prednisolone exposure and the dynamics (early and late) of the molecular response of the cells at the transcriptomic layer, are systematically analyzed based on specific, predefined formulations. The analysis of the results supports a complex mechanism of action for the cells which seems to favor though more the intrinsic mechanism of resistance.

O treinamento aeróbico (TA) traz benefícios para os asmáticos, porém os mecanismos antiinflamatórios não são conhecidos. Estudos experimentais de asma têm mostrado que o TA reduz a inflamação pulmonar alérgica crônica (IPAC) e a reposta Th2, no entanto, nenhum estudo explicou quando os efeitos protetores são iniciados e qual é a principal via anti-inflamatória desencadeada. Objetivo: Avaliar o efeito do TA ao longo do tempo em um modelo murino de asma visando identificar quando são iniciados os efeitos anti-inflamatórios e a reversão do remodelamento brônquico (RB). Métodos: BALB/c (160 animais) foram divididos em 4 grupos: Controle (CT): não induzidos à IPAC e não treinados; Treinamento Aeróbico (TA): não induzidos à IPAC e treinados; OVA: induzidos à IPAC e não treinados; OVA+TA: induzidos à IPAC e treinados. Em seguida foram criados outros subgrupos 1, 3, 7, 15 e 30 dias de TA, ou seja, cada grupo foi repetidos 5 vezes para investigação do efeito do TA ao longo do tempo. Os grupos OVA foram sensibilizados com i.p. (OVA+HidroxAlum), após foram induzidos à IPAC com aerosol de OVA (1-3%) iniciado no dia 21 (3 x semana; 30 min./sessão). A adaptação ao TA foi realizada entre os dias 21 a 23, no dia 25 foi realizado o teste físico, no dia 28 o TA foi iniciado (50% intensidade, frequência 5 x, por 4 semanas). Vinte quatro horas da ultima sessão de TA (1, 3, 7, 15 e 30 dias) os animas foram anestesiados, eutanizados e coletados o lavado broncoalveolar (LBA) (contagem celular total e diferencial), sangue para quantificação das imunoglobulinas (IgE e IgG1) por técnica de reação de anafilaxia cutânea passiva (PCA), o tecido pulmonar para avaliação dos mediadores: IL-4, IL-5, eotaxina, RANTES, ICAM-1, VCAM-1, TGF-b, VEGF, Osteopontina (OPN), NF-kB, FOXP3, receptor de glicocorticóide (RG) e anti-inflamatórias IL-10 e IL-1ra (imunohistoquímica e quantificação por morfometria) e foi coletado também o músculo quadríceps para avaliação da produção das miocinas (IL-10, IL-1ra e IL-6) (imunohistoquímica e quantificado por análise de imagem). O RB (músculo liso, epitélio, deposições das fibras de colágeno e elástica e produção de muco) também foi avaliado por análise de imagem. Resultados: Não foi observada produção das miocinas (p>0,05). Os níveis de IgE, IgG1, migração celular, produção dos mediadores inflamatórios e o RB foram aumentados nos grupos OVA (p<0,05), que ainda mostraram redução da produção do RG (p<0,05). O TA aumentou o RG no músculo liso das vias aéreas, as produções de IL-10 e IL- 1ra aumentaram a partir do 7º dia por células peribrônquicas, ao mesmo tempo que foram reduzidos o NF-kB, IL-4, IL-5, eotaxina, RANTES, ICAM-1, VCAM-1, VEGF, eosinófilos no LBA e foram revertidos o espessamento do músculo liso, do epitélio e as deposições de fibras de colágeno (p<0,05). Curiosamente, a diminuição de TFG-b ocorreu após o 3º dia, enquanto OPN, elástica e muco ocorreram após 15 dias de TA, enquanto IgE, IgG1 e neutrófilos apenas foram reduzidas ao final de 30 dias (p<0,05). Conclusão: A partir do 3º dia do TA foi iniciado o mecanismo anti-inflamatório pelo aumento do RG no músculo liso das vias aéreas, seguido pelo aumento de IL-10 e IL-1ra e pela redução de NF-kB a partir do 7º dia do TA, efeitos que reverteram a inflamação alérgica crônica e o RB / The aerobic training (AT) promotes benefits for asthmatics, but the anti-inflammatory mechanisms are not known. Experimental studies of asthma have shown that AT reduces the pulmonary allergic chronic inflammation (PACI) and response Th2, however no study has ever explained when the protective effects are initiated and which is the main anti-inflammatory pathway triggered. Aim: To evaluate the effect of AT over time in a murine model of asthma to identify when the anti-inflammatory effects is started and reverse bronchial remodeling (BR). Methods: BALB/c (160 mice) were divided into 4 groups: Control (CT): not induced to PACI and untrained; Aerobic Training (TA): not induced to PACI and trained; OVA: induced to PACI and untrained; OVA + TA: induced to PACI and trained. After that were created others subgroups 1, 3, 7, 15 and 30 days AT, that is, each group was repeated 5 times to investigate the effect of AT over time. The OVA groups were sensitized with i.p. OVA (OVA+AlumHidrox), and then the mice were induced after the PACI with aerosol of OVA (1-3%) started on the 21st day (3 x week, 30 min./Session). Adaptation to TA was held between 21-23, on the 25th day the physical test was performed, and on day 28 AT was begun (50% intensity, frequency x 5 for 4 weeks). Twenty four hours of the after last session of AT (1, 3, 7, 15 and 30 days) the mice were anesthetized, euthanized and the bronchoalveolar lavage fluid was collected (BALF) (Total and differential cell count) and blood was used to quantify immunoglobulins (IgE and IgG1) by passive cutaneous anaphylaxis reaction (PCA) technique, the pulmonary tissue was removed and used to evaluate the mediators IL-4, IL-5, eotaxin, RANTES, ICAM-1, VCAM-1, TGF-b, VEGF, osteopontin (OPN), NF-kB, FOXP3, glucocorticoid receptor (GR), and antiinflammatory IL-10 and IL-1ra (immunohistochemistry and quantified by morphometry), was also the quadriceps muscle to assess the expression of myokines (IL-10, IL-1ra and IL-6) (by immunohistochemistry and image analyses). The BR (smooth muscle, epithelium, collagen and elastic fibers deposition, and mucus production) was also evaluated by image analysis. Results: It was not observed any production of myokines (p>0.05). The levels of IgE and IgG1, cell migration, production of inflammatory mediators, and the BR were increased in the OVA groups (p<0.05); that still showed a decreased production of the GR (p<0.05). The AT promoted an increase of GR in the airway smooth muscle from the 3rd day, the production of IL-10 and IL- 1ra were increased from day 7 for cells peribronchial, while NF-kB, IL-4, IL-5, eotaxin, RANTES, ICAM-1, VCAM-1, VEGF, eosinophil counting in BALF were reduced, and reversed the smooth muscle thickening, epithelium and deposition of collagen fibers too (p<0.05). Interestingly, the decreasing of TGF-b occurred in the 3rd day, and OPN, elastic fibers, mucus occurred after 15 days of AT, while IgE and IgG1, and neutrophils were reduced only after 30 days (p<0.05). Conclusion: The anti-inflammatory mechanism by increasing the GR on the smooth muscle of the airways was initiated from the 3rd day of the AT, followed by an increase of IL-10 and IL-1ra and a reduction of NF-kB from the 7th day of the AT, reversed the effects of chronic allergic inflammation and bronchial remodeling

Värmechockprotein 90 kDa (HSP90) bildar ett anmärkningsvärt komplicerat nätverk med en mängd olika cochaperones. Komplexet av FK506-bindande protein 51 kDa (FKBP51) och HSP90 förmedlar proteinveckning och funktion, främjar tau aggregation vid Alzheimers sjukdom och påverkar stressrelaterade störningar, fetma, typ två-diabetes, etc. I samarbete med den molekylära chaperonen HSP90, FKBP51 har nyligen föreslagits som ett lovande terapeutiskt mål för Alzheimers sjukdom (AD). Således skapades knock-in-musen med punktmutationer i tetratricopeptide repeat (TPR) domänen av FKBP51, vilket gör den oförmögen att interagera med HSP90, för att undersöka de potentiella terapeutiska målen för behandling av dessa sjukdomar. Glukokortikoidreceptorn (GR) fungerade traditionellt som utgångspunkten för de initiala studierna av FKBP51-funktion och mekanism som kan stimuleras av den syntetiska glukokortikoiden dexametason (Dexa). Det primära målet med projektet är att förstå den biologiska betydelsen av FKBP51-HSP90 interaktioner. Det är oklart hur FKBP51-mutation påverkar protein-protein-interaktionen och glukokortikoidsignalering. Här analyserades embryonala fibroblaster (MEF) isolerade från vildtyp och FKBP51 mutant mus med avseende på proteinlokalisering, proteinuttryck och genuttryck. Även om ingen säker skillnad mellan vildtyp och mutantmöss sågs i Dexa-medierad glukokortikoidsignalering, förekommer de posttranslationella modifieringarna (PTM) vid exponering för Dexa-behandling av FKBP51 i vildtypmöss i en signifikant högre utsträckning än i Fkbp51mute-möss.Fosforyleringsmodifieringen av FKBP51 antogs initialt och bekräftades av fosforyleringsanrikningsstrategier. Bekräftelse har dock ännu inte erhållits. / Heat shock protein 90 kDa (HSP90) forms a remarkably complicated network with a variety of cochaperones. The complex of FK506-binding protein 51 kDa (FKBP51) and HSP90 mediates protein folding and function, promoting tau aggregation in Alzheimer's disease and influencing stress-related disorders, obesity, type two diabetes, etc. In collaboration with the molecular chaperone HSP90, FKBP51 has recently been proposed as a promising therapeutic target for Alzheimer's disease (AD). Thus, the knock-in mouse harboring point mutations in the tetratricopeptide repeat (TPR) domain of FKBP51 rendering it unable to interact with HSP90 were created to investigate the potential therapeutic targets for the treatment of these diseases. Glucocorticoid receptor (GR) traditionally served as the starting point for the initial studies of FKBP51 function and mechanism which can be stimulated by the synthetic glucocorticoid, dexamethasone (Dexa). The primary goal of the project is to comprehend the biological significance of FKBP51-HSP90 interactions. It is unclear how FKBP51 mutation affects the protein-protein interaction and glucocorticoid signaling. Here, embryonic fibroblasts (MEFs) isolated from wildtype and FKBP51 mutant mouse were analyzed with respect to protein localization, protein expression, and gene expression. Although no certain difference between wildtype and mutant mice was seen in Dexa-mediated glucocorticoid signaling, the post-translational modifications (PTMs) in exposure to Dexa treatment of FKBP51 occur in wildtype mice to a significantly higher extent than in Fkbp51mute mice. The phosphorylation modification of FKBP51 was initially hypothesized and confirmed by phosphorylation enrichment strategies. However, confirmation has not yet been obtained.

Das Lipopolysaccharid Bindende Protein (LBP) ist ein überwiegend in der Leber synthetisiertes Akutphaseprotein. Es bindet den Zellwandbestandteil Lipopolysaccharid (LPS) Gram-negativer Bakterien und transportiert es zu zellulären Rezeptoren, wodurch das angeborene Immunsystem aktiviert wird. In dieser Arbeit wird die Regulation der LBP-Expression in Interleukin (IL)-1, IL-6 und Dexamethason (Dex) stimulierten humanen Hepatomzelllinien HuH-7 und HepG2 untersucht. Der wichtigste Stimulator ist dabei IL-6, dessen Wirkung über die Transkriptionsfaktoren (TF) Stat-3, C/EBP-beta und AP-1 vermittelt wird. Für alle 3 TF konnten aktive Bindungsstellen auf dem LBP-Promotor nachgewiesen werden. Für IL-1-Effekte die u. a. über den TF NF-kappaB vermittelt werden, konnten ebenfalls aktive Bindungsstellen nachgewiesen werden. Die Wirkung von Dex wird über Glucocorticoid Responsive Elements (GREs) vermittelt. Auf dem LBP-Promotor befinden, sich wie gezeigt werden konnte, mehrere aktive GREs, wobei einige verstärkend und einige hemmend wirken. Eine zu beobachtende Synergiewirkung von Dex und IL-6 wird durch die Aufregulation des IL-6-Rezeptors durch Dex verursacht. Die LBP-Expression kann durch TGF (Transforming Growth Factor)-beta gehemmt werden. Der TGF-beta-Signalweg über Smads ist in den Hepatomzellen aktiv, vermittelt aber nicht den TGF-beta-Hemmeffekt, sondern eine geringe stimulierende Wirkung, die bei alleiniger TGF-beta-Inkubation auftritt. Die inhibierende Wirkung von TGF-beta wird durch Gfi-1- und AP-1-Bindungsstellen vermittelt. Die Gfi-1-Bindungsstelle nimmt dabei, wie hier erstmals gezeigt werden konnte, eine herausragende Stellung ein. Die Aufklärung der LBP-Regulation und dabei besonders die Hemmung der LBP-Expression kann mittelfristig dazu beitragen, den klinischen Verlauf von inflammatorischen und infektiösen Erkrankungen zu beeinflussen und bietet daher Potenzial für neue Therapieansätze. / Lipopolysaccharide (LPS) binding protein (LBP) is an acute phase protein with the ability to bind and transfer LPS of Gram-negative bacteria. This soluble pattern recognition molecule represents an important defense principle of the host. Regulation of the hepatic acute phase response and its termination are important mechanisms for limiting systemic inflammatory activity of the host. Here were analyze the cooperation of Interleukin (IL)-1, IL-6, and Dexamethasone (Dex) at LBP expression in the hepatoma cell lines HuH-7 and Hep G2. The major inducer of LBP expression is IL-6. Within the LBP promoter numerously highly consensus binding sites such as AP-1, C/EBP-beta? and STAT3 are present, that confer transcriptional activity as shown by truncation and mutation experiments. Additionally, activate NF-kappaB sites activated by IL-1 were detected at the LBP promoter. By mutation experiments of the promoter furthermore were found differentially active glucocorticoid response elements (GREs). The promoter contains GREs enhancing the activity as well as inhibitory ones. The enhancing effect towards LBP expression by Dex was mediated by IL-6. Dex stimulated the expression of the IL-6 receptor and therefore upregulated the IL-6 pathway. Transforming Growth Factor (TGF)-beta is able to inhibit LBP expression in stimulated cells. An AP-1 binding site was identified mediating inhibitory TGF-beta effects towards LBP promoter activity. Furthermore it was shown that a growth factor independence (Gfi)-1 binding site localized near the AP-1 site is essential for mediating the TGF-beta inhibitory effect. The relevancy of the Gfi-1 site fore mediating TGF-beta effects indicates a novel mechanism for understanding inhibitory TGF-beta effects at the transcriptional level. In summary the complex regulation of LBP were elucidate which may help to eventually develop novel intervention strategies for acute phase, sepsis, and septic shock.