Signal transducer and activator of transcription (STAT) is a family of intracellular proteins that are responsible for carrying the signal from the cell surface to the nucleus in response to specific ligands. Once in the nucleus, STATs activate the transcription of specific genes. To date, seven human STATs have been identified. Among these STATs, STAT3 is considered as oncogenic. It activates genes that block apoptosis and inhibits antitumor immune responses (1). STAT3 is also essential in early embryogenesis and plays a role in cell growth and survival, differentiation and apoptosis depending on the target tissue. Analysing STAT3 signalling provides insights into pathology and can be used as a tool for diagnosis, prognosis and therapy development. Traditionally, western blot has been used to analyse cell signalling but it is impractical in analysing rare cell populations or providing information at the single cell level. Moreover, it is a demanding and time consuming technique that offers qualitative and less sensitive analysis. The rapid evolution in the multi-parametric flow cytometry and the availability of both epitope specific antibodies and sophisticated software facilitate the wide application of this technology in cell signalling studies. Flow cytometry has the ability to resolve different subcellular sets in a heterogeneous population, collects data at a single cell level and correlates multiple markers simultaneously. However, it requires highly standardized protocols for maximal sensitivity. The aim of this study was to assess the dose and the time response of both total STAT3 and pSTAT3 to in vitro stimulation with either IL-6 or IL-10 in peripheral blood mononuclear cells (PBMC). This assessment was done using both the flow cytometry and the western blot techniques. The results of this study showed that lower doses of IL-6 (1 & 10 ng/ml) were not sufficient to induce phosphorylation of STAT3. However, following stimulation with 100 ng/ml of IL-6, no significant change in the level of total STAT3 could be detected in either lymphocytes or monocytes from 3 different donors using either the FC500 or the Accuri cytometer. Using the FC500 cytometer, a small but insignificant increase in the pSTAT3 was seen in the lymphocytes and monocytes. A significant increase in STAT3 phosphorylation was only observed for monocytes after 15 minutes stimulation with 100 ng/ml of IL-6 using the Accuri flow cytometer. xii When the fluorescent labelled antibodies used in the flow cytometric assays were used for western blot probing, western blot analysis of stimulated cell lysates with 100 ng/ml IL-6 detects proteins of a low molecular weight than STAT3 or pSTAT3 which may explain the flow cytometric results of IL-6 stimulation. In IL-10 stimulation experiments, lower doses (1 and 10 ng/ml) tested by flow cytometric and western blot techniques demonstrated insignificant STAT3 phosphorylation induction. Following stimulation with either 50 or 100 ng/ml IL-10, no significant change in the total STAT3 was seen in either lymphocytes or monocytes when using the Accuri flow cytometer. However, stimulation with 100 ng/ml IL-10 induces STAT3 phosphorylation from 10 minutes through 30 minutes in both lymphocytes and monocytes. Longer times were required and high inter-individual variability was noticed for the activation of STAT3 after stimulation with 50 ng/ml IL-10. By using different antibodies from those used in the flow cytometric assay; the western blot results were comparable with the flow cytometric findings following stimulation with 100 ng/ml IL-10. The addition of phosphatase inhibitors during the flow cytometric protocol didn’t show any increase in the STAT3 phosphorylation. However, using paraformaldehyde for fixation and methanol for permeabilisation significantly decreased the mean fluorescence intensity of the PE conjugated antibodies comparing to the BD commercial fixation and permeabilisation buffers. The onset and the signal intensity of “in house” chemiluminescence mixture for western blot detection of STAT3 were comparable to the commercial ECL reagent used. However, the background of the “in house” mixture increased with time and was higher than with the commercial product. Upon longer exposure, the background increased enough to cause signal loss. In spite of the number of advantages of the flow cytometric assay compared to the western blot assay, these results are highly dependent on the specificity and the selectivity of the used antibodies. Furthermore, flow cytometry requires a highly standardized protocol to be able to assess the normal level of signalling proteins which could be later applied to detect abnormalities. It is suggested that the antibodies used in the flow cytometric assay be tested by western blot to confirm their selective detection of the target protein before their use in the flow cytometric analysis. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Pharmacology / unrestricted

Introdução: O Acidente Vascular Cerebral (AVC) é a maior causa de morte no Brasil e um dos maiores responsáveis por incapacitação e invalidez. Existem informações insuficientes quanto aos principais fatores associados à ocorrência de óbito nos pacientes vítimas desta enfermidade. Alguns escores preditores foram desenvolvidos porém não foram validados em população brasileira até o momento. Uma das ações mais importantes na redução do ônus do AVC é o atendimento sistematizado destes pacientes de forma mulltidisciplinar em Unidades de AVC (UAVC) com potencial aumento do uso da terapia trombolítica, além da estratificação dos pacientes, possibilitando decisões terapêuticas mais precoces. Este estudo traz informações sobre o perfil epidemiológico dos pacientes admitidos na UAVC do Hospital Geral de Fortaleza (HGF), bem como identifica fatores preditores de mortalidade e incapacidade até a alta hospitalar e busca validar o Escore de Risco do Registro da Rede Canadense de AVC (IScore), possibilitando a utilização desta ferramenta na estratificação de risco de morte e incapacidade em uma população distinta daquela originalmente realizada. Objetivos: avaliar perfil clínico-epidemiológico dos pacientes e identificar fatores preditores independentes de mortalidade e incapacidade (primários); validar o iScore para morte ou incapacidade e desenvolver um escore na amostra para morte e incapacidade (secundários). Métodos: Foram selecionados pacientes consecutivos admitidos na Unidade de AVC do HGF entre novembro de 2009 até maio de 2012 com diagnóstico clínico de AVC isquêmico. Os dados foram coletados por equipe treinada e através de um formulário específico. Foi realizada análise univariada (método do quiquadrado) e análise multivariada (com regressão logística, stepwise forwardbackward) para descrição das características e identificação dos fatores associados ao desfecho. Teste de correlação de Pearson e curva ROC foram utilizados para medidas de correlação e desempenho dos escores prognósticos. Resultados: no período entre novembro de 2009 e maio de 2012 foram elegíveis 1433 pacientes, sendo 780 analisados. Houve predomíno do sexo masculino e a média de idade (± desvio padrão) foi de 66,1 anos (± 15,44). A forma de apresentação mais comum foi a fraqueza muscular (653 pacientes, 83,6%). O desfecho combinado ocorreu em 423 pacientes (45,8%) e 40 pacientes (5,1%) morreram. Foram identificados 8 fatores preditores independentes para o desfecho. O iScore apresentou bom desempenho, com AUC de 0,797 e Correlação de Pearson de 0,989. Conclusão: Pacientes com AVCi tem altas taxas de incapacidade ou morte até a alta de uma unidade de AVC. Medidas populacionais de informação tem potencial para reduzir a ocorrência dos desfechos. Foram identificados oito fatores preditores de mortalidade ou incapacidade. O iScore apresentou bom desempenho na amostra e pode ser utilizado com acurácia na população brasileira como ferramenta prognóstica. / Intoduction: Stroke is the leading cause of death and one of the most important disease associated with disability in Brazil. There is insufficient information about factors associated with death in stroke patients. Some death risk score has been developed, but none of them were applied in the Brazilian population yet. One of the most important actions to be done to reduce the burden of the stroke is the multidisciplinary assessment of the patients in stroke units (UAVC), with the potential to improve the thrombolytic therapy utilization and the early stratification of patients, allowing earlier treatment decisions. The present study, provides information on the epidemiological profile of patients admitted to the stroke unit in the Hospital Geral de Fortaleza (HGF), identifies predictors of in-hospital mortality and disability and seeks to validate the IScore, allowing the use of this tool to stratify the risk of death and disability in a population different from that which was originally derived. Objectives: to evaluate patient epidemiologic and clinical patterns and factors independently associated with death and disability at hospital discharge (primary objectives); to validate the iScore fitness to predict mortality and/or disability and to develop a new risk score to predict mortality and disability at discharge (secondary objectives). Methods: all consecutive patients admitted to the Hospital Geral de Fortaleza Stroke Unit since November 2009 until May 2012 were elegible. Data were collected by a trained team and by using a specific clinical research form. Univariable analysis (by chi-square test) followed by multivariable analysis (with logistic regression) were performed to identify and establish the variables associated with the outcome (death or disability at hospital discharge). Additionally, Pearson correlation test and ROC curve to measure the iScore correlation and discrimination ability were conducted. Results: a total of 1433 patients were selected and 781 considered eligible were included for the analysis. Male gender were more frequent; mean age was 66,1 (± 15,44). The most common clinical pattern at hospital arrival was \"weakness\" (653 pacientes, 83,6%). Outcome occurred in 423 patients (58,6%) and 40 patients (5,1%) had died. Eight factors were independently associated with outcome. The iScore had good performance, with AUC of 0,797 and Pearson Correlation Test of 0,985. Conclusion: Stroke patients have substantial rate of death or disability at hospital discharge. Populationbased strategies to inform about the signs and symptoms of stroke have potential to decrease this rate. Eight factors were identified as predictors of death or disability and might be used to support patient risk stratification. The iScore had a good performance in the sample and can be used with accuracy as a prognostic tool in Brazil.

Introdução: O Acidente Vascular Cerebral (AVC) é a maior causa de morte no Brasil e um dos maiores responsáveis por incapacitação e invalidez. Existem informações insuficientes quanto aos principais fatores associados à ocorrência de óbito nos pacientes vítimas desta enfermidade. Alguns escores preditores foram desenvolvidos porém não foram validados em população brasileira até o momento. Uma das ações mais importantes na redução do ônus do AVC é o atendimento sistematizado destes pacientes de forma mulltidisciplinar em Unidades de AVC (UAVC) com potencial aumento do uso da terapia trombolítica, além da estratificação dos pacientes, possibilitando decisões terapêuticas mais precoces. Este estudo traz informações sobre o perfil epidemiológico dos pacientes admitidos na UAVC do Hospital Geral de Fortaleza (HGF), bem como identifica fatores preditores de mortalidade e incapacidade até a alta hospitalar e busca validar o Escore de Risco do Registro da Rede Canadense de AVC (IScore), possibilitando a utilização desta ferramenta na estratificação de risco de morte e incapacidade em uma população distinta daquela originalmente realizada. Objetivos: avaliar perfil clínico-epidemiológico dos pacientes e identificar fatores preditores independentes de mortalidade e incapacidade (primários); validar o iScore para morte ou incapacidade e desenvolver um escore na amostra para morte e incapacidade (secundários). Métodos: Foram selecionados pacientes consecutivos admitidos na Unidade de AVC do HGF entre novembro de 2009 até maio de 2012 com diagnóstico clínico de AVC isquêmico. Os dados foram coletados por equipe treinada e através de um formulário específico. Foi realizada análise univariada (método do quiquadrado) e análise multivariada (com regressão logística, stepwise forwardbackward) para descrição das características e identificação dos fatores associados ao desfecho. Teste de correlação de Pearson e curva ROC foram utilizados para medidas de correlação e desempenho dos escores prognósticos. Resultados: no período entre novembro de 2009 e maio de 2012 foram elegíveis 1433 pacientes, sendo 780 analisados. Houve predomíno do sexo masculino e a média de idade (± desvio padrão) foi de 66,1 anos (± 15,44). A forma de apresentação mais comum foi a fraqueza muscular (653 pacientes, 83,6%). O desfecho combinado ocorreu em 423 pacientes (45,8%) e 40 pacientes (5,1%) morreram. Foram identificados 8 fatores preditores independentes para o desfecho. O iScore apresentou bom desempenho, com AUC de 0,797 e Correlação de Pearson de 0,989. Conclusão: Pacientes com AVCi tem altas taxas de incapacidade ou morte até a alta de uma unidade de AVC. Medidas populacionais de informação tem potencial para reduzir a ocorrência dos desfechos. Foram identificados oito fatores preditores de mortalidade ou incapacidade. O iScore apresentou bom desempenho na amostra e pode ser utilizado com acurácia na população brasileira como ferramenta prognóstica. / Intoduction: Stroke is the leading cause of death and one of the most important disease associated with disability in Brazil. There is insufficient information about factors associated with death in stroke patients. Some death risk score has been developed, but none of them were applied in the Brazilian population yet. One of the most important actions to be done to reduce the burden of the stroke is the multidisciplinary assessment of the patients in stroke units (UAVC), with the potential to improve the thrombolytic therapy utilization and the early stratification of patients, allowing earlier treatment decisions. The present study, provides information on the epidemiological profile of patients admitted to the stroke unit in the Hospital Geral de Fortaleza (HGF), identifies predictors of in-hospital mortality and disability and seeks to validate the IScore, allowing the use of this tool to stratify the risk of death and disability in a population different from that which was originally derived. Objectives: to evaluate patient epidemiologic and clinical patterns and factors independently associated with death and disability at hospital discharge (primary objectives); to validate the iScore fitness to predict mortality and/or disability and to develop a new risk score to predict mortality and disability at discharge (secondary objectives). Methods: all consecutive patients admitted to the Hospital Geral de Fortaleza Stroke Unit since November 2009 until May 2012 were elegible. Data were collected by a trained team and by using a specific clinical research form. Univariable analysis (by chi-square test) followed by multivariable analysis (with logistic regression) were performed to identify and establish the variables associated with the outcome (death or disability at hospital discharge). Additionally, Pearson correlation test and ROC curve to measure the iScore correlation and discrimination ability were conducted. Results: a total of 1433 patients were selected and 781 considered eligible were included for the analysis. Male gender were more frequent; mean age was 66,1 (± 15,44). The most common clinical pattern at hospital arrival was \"weakness\" (653 pacientes, 83,6%). Outcome occurred in 423 patients (58,6%) and 40 patients (5,1%) had died. Eight factors were independently associated with outcome. The iScore had good performance, with AUC of 0,797 and Pearson Correlation Test of 0,985. Conclusion: Stroke patients have substantial rate of death or disability at hospital discharge. Populationbased strategies to inform about the signs and symptoms of stroke have potential to decrease this rate. Eight factors were identified as predictors of death or disability and might be used to support patient risk stratification. The iScore had a good performance in the sample and can be used with accuracy as a prognostic tool in Brazil.

Employees at Statoil Canada participated in an action research, cooperative inquiry-based program that was designed to facilitate the reduction of their office environmental footprint. The program was designed using a combination of techniques that have been shown in previous studies to produce pro-environmental change. Almost 90% of participants that were offered the program opted to take part. During a 4-month period, participants set up individualized programs to reduce their environmental footprint in a manner of their own choosing. Results were collected both formally, through self-report data from a web-based Action Tracker, through 1 on 1 interviews and through a facilitated discussion and informally, through impromptu conversations, emails and observation. The data suggests that the program was effective in reducing environmental footprint, although it remains to be seen if the changes will be long lasting.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

En chimie quantique, le principe variationnel est largement utilisé pour calculer la limite supérieure de l'énergie exacte d'un système atomique ou moléculaire. Des méthodes pour calculer la valeur limite inférieure de l'énergie existent mais sont bien moins connues. Une méthode précise pour calculer une telle limite inférieure permettrait de fournir une barre d'erreur théorique pour toute méthode de chimie quantique. Nous avons appliqué des méthodes de type variance pour calculer différentes énergies limites inférieures de l'atome d'hydrogène en utilisant des fonctions de base gaussiennes. L'énergie limite supérieure se trouve être toujours plus précise que ces différentes limites inférieures, i.e. plus proche de l'énergie exacte. L'importance de points singuliers sur l'évaluation de valeurs moyennes d'opérateurs quantiques a également été soulignée.Nous avons étudié les réactions d'adsorption d'un atome de fluor et d'un ion fluorure sur de petits agrégats de lithium Li$_n$ (n=2-15), à l'aide de méthodes de chimie quantique précises. Pour le plus petit système, nous avons montré que la formation de complexes stables Li$_2$F et Li$_2$F$^-$ se produit par un transfert d'électrons sans barrière et à longue portée, de Li$_2$ vers F pour le système neutre et l'inverse pour le système anionique. De telles réactions pourraient être rapides à très basse température. De plus, les complexes formés présentent des caractéristiques uniques de "longue liaison". Pour les systèmes plus gros Li$_n$F/Li$_n$F$^-$ ($n\geqslant4$), nous avons montré que les énergies d'adsorption peuvent être aussi grandes que 6~eV selon le site d'adsorption et que plus d'un état électronique est impliqué dans le processus d'adsorption. Les complexes formés présentent des propriétés intéressantes de "super alcalins" et pourraient servir d'unités de base dans la synthèse de composés à transfert de charge avec des propriétés ajustables. / In quantum chemistry, the variational principle is widely used to calculate an upper bound to the true energy of an atomic or molecular system. Methods for calculating the lower bound value to the energy exist but are much less known. An accurate method to calculate such a lower bound would allow to provide a theoretical error bar for any quantum chemistry method. We have applied variance-like methods to calculate different lower bound energies of a hydrogen atom using Gaussian basis functions. The upper bound energy is found to be always more accurate than the lower bound energies, i.e. closer to the exact energy. The importance of singular points on mean value evaluation of quantum operators has also been brought to attention.The adsorption reactions of atomic fluorine (F) and fluoride (F$^-$) on small lithium clusters Li$_n$ (n=2-15) have been investigated using accurate quantum chemistry ab initio methods. For the smallest system, we have shown that the formation of the stable Li$_2$F and Li$_2$F$^-$ complexes proceeds via a barrierless long-range electron transfer, from the Li$_2$ to F for the neutral and conversely from F$^-$ to Li$_2$ for the anionic system. Such reactions could be fast at very low temperature. Furthermore, the formed complexes show unique long bond characteristics. For the bigger Li$_n$F/Li$_n$F$^-$ systems ($n\geqslant 4$), we have shown that the adsorption energies can be as large as 6~eV depending on the adsorption site and that more than one electronic state is implied in the adsorption process. The formed complexes show interesting "superalkali" properties and could serve as building blocks in the synthesis of charge-transfer compounds with tunable properties.

Inflammatory bowel disease (IBD), characterized by chronic gastrointestinal inflammation, represents a significant childhood chronic disease. In this thesis, a case ascertainment definition of paediatric-onset IBD was validated using administrative data and developed the Ontario Crohn’s and Colitis Cohort (OCCC). The epidemiology of paediatric IBD in Ontario was described, demonstrating that Ontario has one of the highest worldwide incidence rates. Statistically significant increases in incidence were noted in 0-4 year olds (5.0%/year, p=0.03) and 5-9 year olds (7.6%/year, p<0.0001), but not in other age groups. Lower income children were more likely to be hospitalized at least once (hazard ratio (HR) 1.17, 95% confidence intervals (CI) 1.05-1.30) or visit the ED (HR 1.21, 95% CI 1.09-1.35) and had more IBD-related physician visits (odds ratio (OR) 3.73, 95% CI 1.05-13.27). Lower income children with Crohn's disease (CD) (not ulcerative colitis [UC]) were more likely to undergo intra-abdominal surgery within 3 years of diagnosis (OR 1.22, 95% CI 1.01-1.49), especially if diagnosed after 2000 (OR 1.79, 95% CI 1.27-2.53). Finally, changes in health services utilization and surgical rates were described, as were changes in specialist care provision and immunomodulator use in children with IBD between 1994-2007. The changes to care included increased outpatient care provided by paediatric gastroenterologists, and increased immunomodulator use. Children diagnosed with CD, but not UC, in recent years had lower surgical rates. In CD patients, intra-abdominal surgical rates within three years of diagnosis decreased from 18.8% in children diagnosed in 1994-1997 to 13.6% in those diagnosed in 2001-2004 (P = 0.035). When stratified by age at diagnosis, this decrease was significant in children diagnosed ≥10 years old (OR 0.67, 95% CI 0.48-0.93). The OCCC will continue to be used to investigate the epidemiology and burden of paediatric IBD and to improve the care received by children with IBD in Ontario.

Inflammatory bowel disease (IBD), characterized by chronic gastrointestinal inflammation, represents a significant childhood chronic disease. In this thesis, a case ascertainment definition of paediatric-onset IBD was validated using administrative data and developed the Ontario Crohn’s and Colitis Cohort (OCCC). The epidemiology of paediatric IBD in Ontario was described, demonstrating that Ontario has one of the highest worldwide incidence rates. Statistically significant increases in incidence were noted in 0-4 year olds (5.0%/year, p=0.03) and 5-9 year olds (7.6%/year, p<0.0001), but not in other age groups. Lower income children were more likely to be hospitalized at least once (hazard ratio (HR) 1.17, 95% confidence intervals (CI) 1.05-1.30) or visit the ED (HR 1.21, 95% CI 1.09-1.35) and had more IBD-related physician visits (odds ratio (OR) 3.73, 95% CI 1.05-13.27). Lower income children with Crohn's disease (CD) (not ulcerative colitis [UC]) were more likely to undergo intra-abdominal surgery within 3 years of diagnosis (OR 1.22, 95% CI 1.01-1.49), especially if diagnosed after 2000 (OR 1.79, 95% CI 1.27-2.53). Finally, changes in health services utilization and surgical rates were described, as were changes in specialist care provision and immunomodulator use in children with IBD between 1994-2007. The changes to care included increased outpatient care provided by paediatric gastroenterologists, and increased immunomodulator use. Children diagnosed with CD, but not UC, in recent years had lower surgical rates. In CD patients, intra-abdominal surgical rates within three years of diagnosis decreased from 18.8% in children diagnosed in 1994-1997 to 13.6% in those diagnosed in 2001-2004 (P = 0.035). When stratified by age at diagnosis, this decrease was significant in children diagnosed ≥10 years old (OR 0.67, 95% CI 0.48-0.93). The OCCC will continue to be used to investigate the epidemiology and burden of paediatric IBD and to improve the care received by children with IBD in Ontario.

No description available.

Περιγράψαμε την Διαταραχή Μεταγωγής Σήματος Αυξητικής Ορμόνης (GHTD), σε παιδιά με σοβαρή καθυστέρηση ανάπτυξης, φυσιολογική έκκριση αυξητικής ορμόνης (GH), χαμηλό IGF-I αλλά φυσιολογική ανταπόκριση IGF-I σε χορήγηση hGH, που παρουσιάζουν ελαττωματική φωσφορυλίωση του STAT3. Η διαταραχή θεραπεύεται με hGH. Οι CIS πρωτεΐνες είναι αρνητικοί ρυθμιστές του σηματοδοτικου μονοπατιού της GH που ανταγωνίζονται STATs για θέση πρόσδεσης με τον υποδοχέα GHR ή συμμετέχουν στην αποδόμηση του JAK2/GHR μέσω ουβυκουιτίνης/προτεασώματος. Η αδυναμία φωσφορυλίωσης του STAT3 και JAK2 φαινεται να προσπερνάται με χρήση εναλακτικής οδου. Με ‘διασυνομιλία’ της GH με το μονοπάτι του EGF. Συγκεκριμένα η GH φωσφωρυλιώνει το EGFR μέσω φωσφορυλίωσης JAK2. Τέλος, η φωσφορυλίωση STAT3 προκαλείται και από την 17β-οιστραδιόλη. Υπάρχει μία κλινική οντότητα της “καθυστέρησης της ήβης και ανάπτυξης” όπου γίνεται σημαντική επιτάχυνση στην ανάπτυξη μετά την εφηβεία όπου υπάρχει φυσιολογικό τελικό ανάστημα που ταιριάζει με την πορεία ενός ασθενή. Σκοπός: Μελετήθηκε η αρνητική ρύθμιση της GH σε ινοβλάστες παιδιών με GHTD και φυσιολογικών. Στη συνέχεια, η πιθανή συσχέτιση του σηματοδοτικού μονοπατιού της GH και EGF στα παιδιά και τέλος η πιθανή συσχέτιση των στερεοειδών του φύλου με το GH άξονα σε ένα GHTDπαιδί . Yλικά/μέθοδοι: Σε πρωτογενείς καλλιέργειες ινοβλαστών ούλων μαρτύρων και GHTD παιδιών μελετήσαμε την έκφραση και ενεργοποίηση της CIS και JAK2, με επαγωγή με hGH, με Western blot. Στη συνέχεια μελετήθηκε η έκφραση του EGF και pEGF στα κύτταρα των παιδιών και μαρτύρων με επαγωγή των κυττάρων με GH και EGF έλεγχος με Western blot και coimmunoprecipitation. Τέλος μελετήθηκε η φωσφoρυλίωση STAT3 και JAK2 σε ινοβλαστες (προεφηβικούς και εφηβικούς) του ασθενή με GH και 17-β οιστραδιόλη με Western immunoblotting. Αποτελέσματα: Η έκφραση του CIS με 200 ng/ml hGH έδειξε μόνο στους ασθενείς αύξηση της συνολικής CIS και της ουβικουτινυλιομενής μορφής της. Η έκφραση και ενεργοποίηση τoυ JAK2 μόνο στους ασθενείς με επαγωγή με hGΗ δείχνει καθυστερημένη ενεργοποίηση του. Το STAT3 φωσφωρυλιώνεται φυσιολογικά με επαγωγή των κυττάρων των ασθενών με EGF όχι με GH. Οι pEGFRs φωσφωρυλιώνουν φυσιολογικά το JAK2 στους ασθενείς, ενώ όχι στους μάρτυρες. Ο ένας ασθενής προεφηβικά δε φωσφωρυλιώνει το STΑT3 με GH. Εμφανίζει την ουβικουτινιλιωμένη μορφή του CIS. Δεν φωσφωρυλιώνει το STAT3 με 17β-οιστραδιόλη προ-εφηβικά, και εμφανίζει την ουβικουτινιλιωμένη μορφή του CIS. Ενώ εφηβικά φωσφορυλιώνει το STAT3 με 17β-οιστραδιόλη και δεν εμφανίζει την ουβικουτινιλιωμένη μορφή του CIS. Συμπεράσματα: Βρέθηκε καθυστερημένη ενεργοποίηση του GH άξονα σε 2 ασθενής με GHTD μαζί με υπερέκφραση της CIS και ουβικουτινυλιομενής CIS. Η σηματοδότηση της GH γίνεται φυσιολογικά στους μάρτυρες άρα δεν υπάρχει λόγος εναλλακτικής οδού, σε αντίθεση με ασθενείς που χρειάζεται να χρησιμοποιήσουν το μονοπάτι του EGF. Ο ένας ασθενής ξεπέρασε την αδυναμία να φωσφορυλιώσει το STAT3 χωρίς hGH αλλά με την έναρξη της εφηβείας. Ένας λόγος που το παιδί αυτό έδειξε σημαντική επιτάχυνση στην ανάπτυξη είναι ότι μετά την εφηβεία δεν υπήρχε υπερλειτουργία του ανασταλτικού μηχανισμού της GH, διαμέσου του Ub CIS. / We have previously described a new disorder (GHTD) in 4 children with growth delay, normal provoked and spontaneous GH secretion, and low IGF-I concentrations but normal IGF-I generation test results who have a defect in the phosphorylation of the signal transducer and activator of transcription STAT-3. These children respond with a significant increase in their growth velocity after administration of hGH. CIS proteins are inhibitors of the GH signal transduction pathway, by distinct mechanisms: by competition with STATs for common tyrosine-binding sites on the cytoplasmic tail of GHR or by a proteasome-dependent mechanism. Monoubiquitinated form of CIS protein was observed in 2 GHTD patients.Also STAT3 phosphorylation defect could be overcomed by using an alternative pathway the one of Epidermal Growth factor (EGF). Also in one patient its STAT3 defect was overcomed when he entered puberty, sex steroids may enhanced his growth. Objective: The purpose of the study was the characterization of the molecular mechanisms involved in GH signal transduction pathway in GHTD patients, as a possible cause of an increased expression of its inhibitors. Also to search if there is a possible crosstalk between GH and EGF growth transduction pathways. And finally the role of sex steroids in GH signalling in one GHTD patient. Patients/Material and Methods: In primary fibroblast cell cultures from gingival biopsies of the GHTD patients and age-matched normal children we studied: expression analysis, in cells inducted with GH, of CIS and JAK2 phosphorylationby western immunoblotting and RT-PCR. Also the cells of the same children and controls were inducted with EGF and STAT3 phosphorylation was studied. Finally the cells of one of the patients were inducted with GH and 17β-estradiole before and after puberty and its STAT3 phosphorylation and CIS expression were studied. Results: Expression analysis in the childrens’ fibroblasts showed an overexpression of CIS in 2 patients as compared to normal children. STAT3 defect was not present in the patients fibroblasts that were inducted with EGF. Also one GHTD patient that he couldn't phosphorylate in his inducted fibroblast with GH and 17-b estardiole STAT3 and ubiquitinated CIS was present to his cells before puberty this defect was overcomed after he entered puberty. Conclusions: The overexpression of CIS may inhibit the activation of STAT3 and may be involved in the pathogenesis of the severe short stature of the GHTD children. Also GH signalling pathway has no defect in control patients so there is no need of using an alternative pathway such as the one of EGF that occurs in GHTD patients. Also one GHTD patient that showed a STAT3 defect before puberty was overcomed after he entered puberty and without GH treatment. There is a clinical status that is called 'growth dealy' that matches this patients profile, that shows rapid growth after puberty. A probable cause could be that ubiquitinated form of CIS was not present after he entered puberty.