INTRODUÇÃO: A definição de fatores preditivos de lesão morfológica e funcional da tuba uterina poderia colaborar na escolha do tratamento de pacientes com gestação ectópica. O objetivo deste estudo foi verificar o comportamento do tecido trofoblástico em relação à sua penetração na parede da tuba uterina em gestações ampulares, relacionar a profundidade dessa penetração com idade gestacional, concentração de beta-hCG, tipo de imagem ultra-sonográfica e dimensão da massa ectópica à ultra-sonografia e avaliar a possibilidade de predição dessa invasão pelos parâmetros estudados. MÉTODOS: realizou-se estudo retrospectivo, entre 1° de janeiro de 2000 a 31 de março de 2004, com 105 pacientes com gestação tubária ampular submetidas à salpingectomia. As imagens ectópicas foram classificadas pelo aspecto ultra-sonográfico em anel tubário, massa complexa e embrião com atividade cardíaca e sua dimensão foi obtida pela medida do maior eixo. Histologicamente a invasão trofoblástica na parede tubária foi classificada em grau I: quando limitada à mucosa da tuba uterina; grau II: até a camada muscular; grau III: invasão de toda a espessura da tuba uterina. RESULTADOS: 29 pacientes tiveram infiltração tubária grau I, 30 pacientes infiltração grau II e 46 pacientes infiltração grau III. Os graus de invasão trofoblástica não estiveram associados à idade gestacional (p = 0,53) nem ao maior diâmetro da imagem à ultra-sonografia (p = 0,43). Os diferentes graus de invasão trofoblástica apresentaram diferença significativa da beta-hCG (p < 0,001). O grau I apresentou valores menores que os graus II e III (p < 0,05) e o grau II valores menores que o grau III (p < 0,05). Houve associação entre o grau de invasão trofoblástica e a descrição do tipo de imagem identificada à ultra-sonografia (p = 0,001). Embrião com atividade cardíaca foi mais prevalente nos casos de invasão grau III. O valor de 2 400 mUI/ml apresentou sensibilidade de 82,8%, especificidade de 85,5%, valor preditivo positivo de 68,6% e valor preditivo negativo de 92,7% (acurácia de 84,8%) para determinar invasão trofoblástica grau I. beta-hCG de 5 990 mUI/ml foi o melhor ponto de corte para predição de invasão trofoblástica grau III: sensibilidade de 82,6%, especificidade de 74,6%, valor preditivo positivo de 71,7% e valor preditivo negativo de 84,6% (acurácia de 78,1%). CONCLUSÕES: Em gestações ampulares, o tecido trofoblástico se desenvolve a partir de sua penetração na parede tubária, a profundidade da penetração do trofoblasto na tuba uterina relaciona-se às concentrações séricas de beta-hCG e ao tipo de imagem ultra-sonográfica, sendo que a concentração sérica da beta-hCG é a melhor preditora da profundidade da invasão na tuba uterina. / INTRODUCTION: The definition of predictive factors of morphologic and functional damage to the Fallopian tube may help in the choice of treatment for patients with ectopic pregnancy. The objective of the present study was to verify the presence of trophoblastic invasion into the tubal wall in ampular pregnancies, correlate the depth of penetration of trophoblastic tissue into the tubal wall with gestational age, beta-hCG concentration, type of ultrasonographic image and dimension of the ectopic mass upon ultrasound, and to evaluate the possible prediction of this invasion based on the parameters studied. METHODS: A retrospective study was conducted on 105 patients with ampular pregnancy submitted to salpingectomy between January 1, 2000 and March 31, 2004. Ectopic images were classified based on ultrasonographic findings in tubal ring, complex mass and embryonic heart activity. The dimension of the mass was determined by measuring the major axis. Histologically, trophoblastic invasion into the tubal wall was classified as grade I when limited to the tubal mucosa, grade II when reaching the muscle layer, and grade III when comprising the full thickness of the Fallopian tube. RESULTS: Twenty-nine patients had tubal infiltration grade I, 30 had grade II and 46 had grade III. The level of trophoblastic invasion was associated neither with gestational age (p = 0.53) nor with a greater diameter of the ultrasound image (p = 0.43). The different levels of trophoblastic invasion were significantly associated with beta-hCG concentration (p < 0.001), with lower concentrations being observed for grade I compared to grades II and III (p < 0.05) and for grade II compared to grade III (p < 0.05). There was an association between the level of trophoblastic invasion and the type of ultrasonographic image (p = 0.001). Embryos with heart activity were more prevalent in cases of grade III invasion. beta-hCG levels of 2 400 mIU/ml showed 82.8% sensitivity, 85.5% specificity, a positive predictive value of 68.6% and a negative predictive value of 92.7% (84.8% accuracy) for the diagnosis of grade I trophoblastic invasion. A beta-hCG titer of 5990 mIU/ml was the best cut-off for the prediction of grade III trophoblastic invasion: 82.6% sensitivity, 74.6% specificity, positive predictive value of 71.7% and negative predictive value of 84.6% (78.1% accuracy). CONCLUSIONS: trophoblastic tissue penetrate tubal wall in ampular pregnancies, the depth of penetration of trophoblastic tissue is correlated with beta-hCG concentration and type of ultrasonographic image and beta-hCG titer is the best predictor of the depth of penetration into tubal wall.

INTRODUÇÃO: A definição de fatores preditivos de lesão morfológica e funcional da tuba uterina poderia colaborar na escolha do tratamento de pacientes com gestação ectópica. O objetivo deste estudo foi relacionar a penetração do tecido trofoblástico na parede tubária acometida por gestação ampular com a concentração sérica materna de VEGF, avaliar a possibilidade do VEGF predizer a profundidade da invasão do tecido trofoblástico, comparando o desempenho do VEGF com o desempenho das concentrações séricas de beta-hCG na predição da profundidade da invasão do trofoblasto. MÉTODOS: realizou-se estudo prospectivo, entre 21 de dezembro de 2006 a 30 de setembro de 2007, com 30 pacientes com gestação tubária ampular submetidas à salpingectomia. Foram dosadas as concentrações séricas maternas de VEGF após confirmação do diagnóstico de gestação tubária e antes da realização da salpingectomia. Histologicamente a invasão trofoblástica na parede tubária foi classificada em grau I: quando limitada à mucosa da tuba uterina; grau II: até a camada muscular; grau III: invasão de toda a espessura da tuba uterina. RESULTADOS: 10 pacientes tiveram infiltração tubária grau I, 9 pacientes infiltração grau II e 11 pacientes infiltração grau III. Os diferentes graus de invasão trofoblástica apresentaram diferença significativa das concentrações séricas de VEGF (p< 0,001). O título sérico de VEGF de 305 pg/mL apresentou sensibilidade de 100,0%, especificidade de 85,0%, valor preditivo positivo de 76,9% e valor preditivo negativo de 100,0% para determinar invasão trofoblástica grau I. Título sérico de 425,9 pg/mL foi o melhor ponto de corte para predição de invasão trofoblástica grau III: sensibilidade de 81,8%, especificidade de 94,7%, valor preditivo positivo de 90,0% e valor preditivo negativo de 90,0%. Regressão logística selecionou a concentração sérica de VEGF como fator de melhor desempenho na predição da invasão trofoblástica quando comparado com título sérico de beta-hCG. CONCLUSÕES: em gestações ampulares, a profundidade da penetração do tecido trofoblástico na parede tubária acometida por gestação ectópica se relaciona com a concentração sérica de VEGF, a concentração sérica de VEGF é preditora da profundidade da invasão do tecido trofoblasto na parede tubária acometida por GE e a concentração sérica de VEGF apresenta melhor desempenho que a concentração sérica de beta-hCG como preditora da profundidade da invasão do trofoblasto na parede da tuba uterina acometida por gestação ampular / INTRODUCTION: The definition of predictive factors of morphologic and functional damage to the Fallopian tube may help in the choice of treatment for patients with ectopic pregnancy. The objective of the present study was to correlate the depth of penetration of trophoblastic tissue into the tubal wall with maternal serum VEGF concentrations, to evaluate the prediction of this invasion based on these concentrations and to compare the performances of VEGF and beta-hCG as predictors of trophoblastic invasion. METHODS: A prospective study was conducted on 30 patients with ampular pregnancy submitted to salpingectomy between December 21st, 2006 and September 30th, 2007. Maternal serum VEGF concentrations were measured after the diagnosis confirmation and before salpingectomy was performed. Histologically, trophoblastic invasion into the tubal wall was classified as grade I when limited to the tubal mucosa, grade II when reaching the muscle layer, and grade III when comprising the full thickness of the Fallopian tube. RESULTS: ten patients had tubal infiltration grade I, nine had grade II and eleven had grade III. The different levels of trophoblastic invasion were significantly associated with VEGF concentrations (p< 0.001). VEGF levels of 305.0 pg/mL showed 100.0% sensitivity, 85.0% specificity, a positive predictive value of 76.9% and a negative predictive value of 100.0% for the diagnosis of grade I trophoblastic invasion. A VEGF titer of 425.9 pg/mL was the best cut-off for the prediction of grade III trophoblastic invasion: 81.8% sensitivity, 94.7% specificity, positive predictive value of 90.0% and negative predictive value of 90.0%. Logistic regression showed that VEGF presented higher performance as a predictor of trophoblastic invasion than beta-hCG. CONCLUSIONS: in ampullary pregnancies, the depth of penetration of trophoblastic tissue into the tubal wall is correlated with serum VEGF concentrations, serum VEGF titer is predictor of the depth of penetration into tubal wall and VEGF concentrations present higher performance than beta-hCG as predictor of the trophoblastic invasion

RUNX3是RUNX轉錄因子家族的其中一位成員。RUNX轉錄因子家族是負責調控細胞的增殖和分化。最近研究表明RUNX3可能在造血過程中扮演其中一個角色。可是，它在髓系細胞中的調節角色依然未明。此前，我們發現在核心結合因子急性骨髓性白血病中的融合蛋白RUNX1-ETO和CBFB-MYH11會抑制RUNX3基因表達，並且RUNX3表達水平對兒童急性骨髓性白血病的預後有顯著影響。本研究的目的是要調查RUNX3在成人急性骨髓性白血病的預後價值，並透過闡明RUNX3的轉錄調節去了解其在髓系細胞分化扮演的角色。 / 首先，我們透過實時定量聚合鏈反應去量化在174個成人急性骨髓性白血病的患者骨髓中的RUNX3表達，從而調查RUNX3表達與成人急性骨髓性白血病預後的關係。我們發現低RUNX3表達與較好預後的核型(P=0.045)，NPM1基因突變(P=0.014) 和較年青患者(P=0.084) 有關聯。在存活分析中，我們把有完整生存數據的非急性前骨髓性白血病病人分成高RUNX3表達和低RUNX3表達兩組。在成人急性骨髓性白血病中，高RUNX3表達和較差整體存活率(OS) (P=0.011)和無事件存活率(EFS) (P=0.003)有顯著的關聯，這和我們在兒童急性骨髓性白血病所觀察的一致。高RUNX3表達和較差存活率的關係在有野生型FLT3基因的病人中更為明顯(OS, P=0.004; EFS, P=0.001)。由於低RUNX3表達和較好預後核型有關聯，我們進一步只對擁有較差預後核型的病人作將存活分析，發現RUNX3表達仍是影響EFS的一個顯著因素(P=0.017)。在多元分析中，高RUNX3表達在所有病人(EFS, P=0.026, HR=2.433, 95%CI = 1.114-5.356)，野生v 型FLT3基因的病人(OS, P=0.016, HR=4.830, 95%CI = 1.335-17.481; EFS, P=0.007, HR=4.103, 95%CI = 1.480-11.372)和較差預後核型的病人(EFS, P=0.024,HR=2.339, 95%CI = 1.117-4.896) 中都是一個獨立的不利預後因素。 / 接著，我們研究RUNX3基因的表達調控。我們鑒定出一個最小啟動子區對於在髓系細胞的基因表達有關鍵作用。透過預測啟動子區和轉錄因子結合位點的分析，顯示這個活性區域含有PU.1，AP-1和Sp1轉錄因子結合位點。我們透過報告基因系統研究，染色質免疫沈澱技術及電泳遷移率改變分析去闡明PU.1，c-Jun及Sp1和相對的轉錄因子結合位點參與RUNX3基因的表達調控。我們進一步透過PU.1基因剔除去證實RUNX3是PU.1的直接下遊靶基因並發現PU.1與RUNX3表達在急性骨髓性白血病人中呈正相關性。 / 由於RUNX3基因表達受到PU.1, c-Jun及Sp1的控制，我們繼續研究RUNX3在髓系細胞分化的功用。我們透過實時定量聚合鏈反應及流式細胞儀檢測發現RUNX3過度表達誘導K562細胞株作單核細胞及粒細胞分化。RUNX3能激活髓系基因的啟動子。它在成熟髓系細胞的表達水平明顯比血幹細胞為高。根據以上結果，RUNX3也許在單核細胞及粒細胞分化中有一定功能。但是，有別於其他癌細胞，RUNNX3不能在髓系細胞誘導細胞凋亡和周期阻滯。 / 總括而言，RUNX3表達在成人急性骨髓性白血病中是一個獨立的預後因素。除此之外，本研究表明RUNX3受到PU.1，c-Jun及Sp1的表達調控並在單核細胞及粒細胞分化中有一定功能。 / RUNX3 is a member of Runt-related domain (RUNX) transcription factor family, which regulates cell proliferation and differentiation. Recent studies have suggested a role of RUNX3 in hematopoiesis. However, its regulatory function in myeloid cells remains unclear. Our group previously showed that RUNX3 expression was repressed by the fusion proteins RUNX1-ETO and CBFB-MYH11 in core-binding factor acute myeloid leukemia (CBF-AML) and had prognostic implication in childhood AML patients. The aim of this study is to investigate the prognostic value of RUNX3 in adult AML patients and its role in myeloid differentiation by elucidating its transcriptional control. / To investigate the relationship between RUNX3 expression and prognosis of adult AML, RUNX3 expression in the diagnostic bone marrow samples from 174 adult AML patients were quantified by real time quantitative PCR (RQ-PCR). Low RUNX3 expression was found to be associated with favorable cytogenetic group (P=0.045), NPM1 mutations (P=0.014) and younger age (P=0.084). For the survival analysis, 110 non-acute promyelocytic leukemia (non-APL) patients with complete survival data were dichotomized into high and low expression groups. Concordant with our previous observation in childhood AML, a significant association between high RUNX3 expression and poorer overall survival (OS) (P=0.011) and event-free survival (EFS) (P=0.003) was observed. The association between high RUNX3 expression and poorer survival was further strengthened in patients with wild-type FLT3 (P=0.004 and 0.001 for OS and EFS respectively). Since low RUNX3 expression was associated with favorable cytogenetics, the analysis was next restricted to patients with non-favorable cytogenetics and RUNX3 expression remained as a significant factor for EFS (P=0.017). In multivariate analysis, high RUNX3 expression was an independent adverse prognostic factor in the whole cohort (EFS, P=0.026, HR=2.433, 95%CI = 1.114-5.356), patients with wild-type FLT3 (OS, P=0.016, HR=4.830, 95%CI = 1.335-17.481; EFS, P=0.007, HR=4.103, 95%CI = 1.480-11.372) and patients with non-favorable genetics (EFS, P=0.024,HR=2.339, 95%CI = 1.117-4.896). / Next, the transcriptional regulation of RUNX3 in myeloid cells was investigated. A minimal promoter region was identified to be critical for myeloid-specific promoter activity. Sequence analysis of the fragment revealed potential transcription factor binding sites for PU.1, AP-1 and Sp1.The involvement of these putative binding sites and corresponding transcription factors in transcriptional regulation of RUNX3 was demonstrated by promoter reporter assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA).Furthermore, PU.1 knockdown in U937 cells confirmed RUNX3 was a direct downstream target of PU.1 and a positive correlation between PU.1 and RUNX3 expression was observed in AML patient samples. / As RUNX3 was shown to be transcriptionally regulated by PU.1, c-Jun and Sp1, a role of RUNX3 in myeloid differentiation was postulated. Overexpression of RUNX3 induced both monocytic and granulocytic markers in K562 myeloid cells as detected by flow cytometry and RQ-PCR. RUNX3 was also found to activate myeloid-specific gene promoters and its expression was significantly higher in mature myeloid cells than in hematopoietic stem cells. This suggested a role of RUNX3 in both monocytic and granulocytic differentiation. However, unlike in other solid tumors, RUNX3 did not induce apoptosis and cell cycle arrest in myeloid cells. / In conclusion, RUNX3 expression was an independent prognostic factor in adult AML. Furthermore, our findings showed that RUNX3 was transcriptionally regulated by the master myeloid regulator PU.1 along with c-Jun and Sp1 and implicated a role in monocytic and granulocytic differentiation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Kwan, Tsz Ki. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 171-202). / Abstracts also in Chinese.

INTRODUÇÃO: A definição de fatores preditivos de lesão morfológica e funcional da tuba uterina poderia colaborar na escolha do tratamento de pacientes com gestação ectópica. O objetivo deste estudo foi relacionar a penetração do tecido trofoblástico na parede tubária acometida por gestação ampular com a concentração sérica materna de VEGF, avaliar a possibilidade do VEGF predizer a profundidade da invasão do tecido trofoblástico, comparando o desempenho do VEGF com o desempenho das concentrações séricas de beta-hCG na predição da profundidade da invasão do trofoblasto. MÉTODOS: realizou-se estudo prospectivo, entre 21 de dezembro de 2006 a 30 de setembro de 2007, com 30 pacientes com gestação tubária ampular submetidas à salpingectomia. Foram dosadas as concentrações séricas maternas de VEGF após confirmação do diagnóstico de gestação tubária e antes da realização da salpingectomia. Histologicamente a invasão trofoblástica na parede tubária foi classificada em grau I: quando limitada à mucosa da tuba uterina; grau II: até a camada muscular; grau III: invasão de toda a espessura da tuba uterina. RESULTADOS: 10 pacientes tiveram infiltração tubária grau I, 9 pacientes infiltração grau II e 11 pacientes infiltração grau III. Os diferentes graus de invasão trofoblástica apresentaram diferença significativa das concentrações séricas de VEGF (p< 0,001). O título sérico de VEGF de 305 pg/mL apresentou sensibilidade de 100,0%, especificidade de 85,0%, valor preditivo positivo de 76,9% e valor preditivo negativo de 100,0% para determinar invasão trofoblástica grau I. Título sérico de 425,9 pg/mL foi o melhor ponto de corte para predição de invasão trofoblástica grau III: sensibilidade de 81,8%, especificidade de 94,7%, valor preditivo positivo de 90,0% e valor preditivo negativo de 90,0%. Regressão logística selecionou a concentração sérica de VEGF como fator de melhor desempenho na predição da invasão trofoblástica quando comparado com título sérico de beta-hCG. CONCLUSÕES: em gestações ampulares, a profundidade da penetração do tecido trofoblástico na parede tubária acometida por gestação ectópica se relaciona com a concentração sérica de VEGF, a concentração sérica de VEGF é preditora da profundidade da invasão do tecido trofoblasto na parede tubária acometida por GE e a concentração sérica de VEGF apresenta melhor desempenho que a concentração sérica de beta-hCG como preditora da profundidade da invasão do trofoblasto na parede da tuba uterina acometida por gestação ampular / INTRODUCTION: The definition of predictive factors of morphologic and functional damage to the Fallopian tube may help in the choice of treatment for patients with ectopic pregnancy. The objective of the present study was to correlate the depth of penetration of trophoblastic tissue into the tubal wall with maternal serum VEGF concentrations, to evaluate the prediction of this invasion based on these concentrations and to compare the performances of VEGF and beta-hCG as predictors of trophoblastic invasion. METHODS: A prospective study was conducted on 30 patients with ampular pregnancy submitted to salpingectomy between December 21st, 2006 and September 30th, 2007. Maternal serum VEGF concentrations were measured after the diagnosis confirmation and before salpingectomy was performed. Histologically, trophoblastic invasion into the tubal wall was classified as grade I when limited to the tubal mucosa, grade II when reaching the muscle layer, and grade III when comprising the full thickness of the Fallopian tube. RESULTS: ten patients had tubal infiltration grade I, nine had grade II and eleven had grade III. The different levels of trophoblastic invasion were significantly associated with VEGF concentrations (p< 0.001). VEGF levels of 305.0 pg/mL showed 100.0% sensitivity, 85.0% specificity, a positive predictive value of 76.9% and a negative predictive value of 100.0% for the diagnosis of grade I trophoblastic invasion. A VEGF titer of 425.9 pg/mL was the best cut-off for the prediction of grade III trophoblastic invasion: 81.8% sensitivity, 94.7% specificity, positive predictive value of 90.0% and negative predictive value of 90.0%. Logistic regression showed that VEGF presented higher performance as a predictor of trophoblastic invasion than beta-hCG. CONCLUSIONS: in ampullary pregnancies, the depth of penetration of trophoblastic tissue into the tubal wall is correlated with serum VEGF concentrations, serum VEGF titer is predictor of the depth of penetration into tubal wall and VEGF concentrations present higher performance than beta-hCG as predictor of the trophoblastic invasion

Um método de cromatografia líquida de alta eficiência por fase reversa (RP-HPLC) para análise qualitativa e quantitativa do hormônio folículo estimulante humano íntegro (hFSH), foi estabelecido e validado quanto à exatidão, precisão e sensibilidade. O FSH humano é um hormônio glicoprotéico dimérico largamente utilizado em medicina reprodutiva tanto para diagnóstico quanto para terapia. A metodologia desenvolvida preserva a integridade da proteína, permitindo a análise da forma heterodimérica intacta, e não somente de suas subunidades, como é normalmente obtida na maioria das condições geralmente empregadas. Esta técnica foi também utilizada para a comparação da hidrofobicidade relativa de preparações de hFSH hipofisária, urinária e derivadas de células de ovário de hamster chinês (CHO) bem como de outros dois hormônios glicoprotéicos, sintetizados na hipófise anterior: hormônio humano estimulante da tireóide (hTSH) e hormônio luteinizante humano (hLH). O menos hidrofóbico dos três hormônios analisados foi o hFSH, seguido do hTSH e do hLH. Uma diferença significativa (p<0,005) foi observada entre o tempo de retenção (tR) das preparações hipofisária e recombinante de hFSH, refletindo diferenças estruturais nas suas cadeias de carboidratos. Duas isoformas principais foram detectadas no hFSH urinário, incluindo uma forma que foi significativamente diferente (p<0,005) das preparações hipofisária e recombinante. Foram demonstradas linearidade da curva dose-resposta (r=0,9965, n=15) para esta metodologia de RP-HPLC, bem como uma precisão inter-ensaio, cujo coeficiente de variação é menor que 4%, para a quantificação de diferentes preparações de hFSH e uma sensibilidade da ordem de 40 ng. Foram também analisados o comportamento cromatográfico e a hidrofobicidade relativa das subunidades individuais das preparações recombinantes e hipofisária de hFSH. Além disso, a exata massa molecular das subunidades individuais de hFSH e do heterodímero foram simultaneamente determinadas por espectrometria de massa MALDI-TOF. A presente metodologia representa, em nossa opinião, uma ferramenta essencial para a caracterização e controle de qualidade deste hormônio, que ainda não consta das principais farmacopéias. / A reversed-phase high-performance liquid chromatography (RP-HPLC) method for the qualitative and quantitative analysis of intact human follicle-stimulating hormone (hFSH) was established and validated for accuracy, precision and sensitivity. Human FSH is a dimeric glycoprotein hormone widely used as a diagnostic analyte and as therapeutic product in reproductive medicine. The technique developed preserves the protein integrity, allowing the analysis of the intact heterodimeric form rather than just of its subunits, as it is the case for the majority of the conditions currently employed. This methodology has also been employed for comparing the relative hydrophobicity of pituitary, urinary and two Chinese hamster ovary (CHO)-derived hFSH preparations, as well as of two other related glycoprotein hormones of the anterior pituitary: human thyroid-stimulating hormone (hTSH) and human luteinizing hormone (hLH). The least hydrophobic of the three glycohormones analyzed was hFSH, followed by hTSH and hLH. A significant difference (p<0.005) was observed in tR between the pituitary and recombinant hFSH preparations, reflecting structural differences in their carbohydrate moieties. Two main isoforms were detected in urinary hFSH, including a form which was significantly different (p<0.005) for the pituitary and recombinant preparations. The linearity of the dose-response curve (r = 0.9965, n = 15) for this RP-HPLC methodology, as well as an inter-assay precision with relative standard deviation less than 4% for the quantification of different hFSH preparations and a sensitivity of the order of 40 ng, were demonstrated. The chromatographic behavior and relative hydrophobicity of the individual subunits of the pituitary and recombinant preparations were also analyzed. Furthermore, the accurate molecular mass of the individual hFSH subunits and of the heterodimer were simultaneously determined by matrix-assisted laser desorption ionization time-of-flight mass spectral analysis (MALDI-TOF-MS). The present methodology represents, in our opinion, an essential tool for characterization and quality control of this hormone that is not yet described in the main pharmacopoeias.

Le synaptic scaling est une forme de plasticité homéostatique par lequel les synapses ajustent leur efficacité pour compenser des variations normales ou pathologiques de l'activité neuronale notamment lors des maladies neurodégeneratives ou suite à la perte d’afférences sensorielles après une lésion. Dans un modèle expérimental classique, le traitement chronique des neurones primaires avec la tétrodotoxine (TTX) pour bloquer la propagation des potentiels d'action présynaptiques induit une augmentation significative de l'amplitude des courants miniatures excitateurs transmis par les récepteurs du glutamate AMPA postsynaptiques. Plusieurs voies de signalisation ont été proposées, dont celle impliquant les microARNs (miRs), de petits ARN non-codants qui inhibent la traduction des protéines en se liant aux ARN messagers cibles. Dans ce contexte, nous avons exploré l'hypothèse que le microARN, miR-124, fortement exprimé dans le cerveau, pourrait être un régulateur important de l'homéostasie synaptique en contrôlant l'expression de la protéine synaptopodine, une protéine structurante des épines dendritiques et indispensable à l'expression du synaptic scaling.En combinant des approches de RTq-PCR, d'immunocytochimie et d'électrophysiologie in vitro, nous avons montré dans un premier temps que la privation globale de l'activité des neurones primaires d’hippocampe diminuait le niveau d'expression de miR-124 et augmentait celui de la synaptopodine et des récepteurs AMPA dont la sous-unité GluA2 est une autre cible de miR-124. Par ailleurs, en rendant des synapses individuelles inactives via l’expression présynaptique de la toxine tétanique, nous avons observé que le recrutement synaptique des récepteurs AMPA et de la synaptopodine était spécifique de ces synapses, suggérant une régulation homéostatique locale. Dans un deuxième temps, nous avons trouvé que la surexpression de miR-124 ou l’inhibition de son interaction avec l’ARNm de la synaptopodine ou de GluA2 bloquaient la réponse synaptique homéostatique induite par le traitement TTX. Enfin, des expériences de FRAP ont suggéré que la synaptopodine influençait le trafic des récepteurs AMPA à la membrane probablement en les stabilisant à la synapse, ce qui expliquerait ainsi son rôle pendant la plasticité homéostatique. / Synaptic scaling is a form of homeostatic plasticity where synapses adjust their own efficacy to compensate for normal or pathological variations in neuronal activity such as neurodegenerative disorders or sensory deprivation after a lesion. In a well-established paradigm, the chronic application of tetrodotoxin (TTX) in primary neurons, to block presynaptic action potential propagation, induces a significant upscaling of miniature excitatory postsynaptic currents mediated-AMPA receptors. Numerous regulators of this plasticity have been identified including microRNAs (miR), which are small endogenous non-coding RNAs, inhibiting protein translation by binding to mRNA targets. This led us to hypothesize that the most highly expressed microRNA in the brain, miR-124, could be an important regulator of homeostatic scaling by controlling the expression of synaptopodin, a structural protein of dendritic spines playing a crucial role in homeostatic plasticity.By combining qRT-PCR, immunocytochemistry and in vitro electrophysiology approaches, first we showed that a global 48hrs TTX treatment in hippocampal primary neurons led to a decrease in miR-124 level and an increase in the expression of synaptopodin and synaptic AMPA receptors containing the GluA2 subunit which is another miR-124 target. Moreover, we observed that the synaptic accumulation of AMPA receptors and synaptopodin could be synapse-specific by expressing the tetanus toxin to block the activity of individual presynapses, which suggested a local homeostatic regulation. Importantly, we found that overexpressing miR-124 or inhibiting its interaction with synaptopodin or GluA2 mRNAs blocked the synaptic homeostatic response. In addition, FRAP experiments suggested that synaptopodin controlled AMPA receptor trafficking at the membrane by probably retaining them in dendritic spines, which could explain its role during homeostatic plasticity.

La biogenèse du ribosome est un processus complexe et dynamique qui nécessite de nombreuses étapes de maturation et de modification des ARNr ainsi que l’assemblage et le transport des RNPs précurseurs. Un ribosome mature contient une centaine de pièces, ARN et protéines confondus, mais son assemblage requiert l’intervention de plus de 400 facteurs de synthèse. De part le coût énergétique important de ce processus, plusieurs voies de régulation interviennent pour contrôler la biogenèse des ribosomes en fonction des conditions nutritives. L’une des voies les plus connue est la voie TOR (Target of rapamycin). Cette voie de régulation agît principalement au niveau de la transcription des différents intervenants de la biogenèse : les ARNr, les protéines ribosomiques mais aussi les facteurs de synthèse. Ces facteurs, ayant une action transitoire dans la maturation des ribosomes, sont, par économie, recyclés pour la synthèse de nouveaux ribosomes. Nous nous sommes donc intéressés au devenir de ces facteurs, plus particulièrement de ceux intervenants dans la biogenèse de la petite sous unité, lorsque les conditions environnementales sont inadaptées à la croissance cellulaire. Ainsi, nous avons pu montré, pour quatre facteurs particuliers : Dim2, Rrp12, Hrr25 et Fap7, que leur localisation est dépendante de la synthèse ribosomique. Ainsi, lors de carence en sources nutritives, l’inhibition de la synthèse et de l’activité ribosomique entraîne un confinement de ces facteurs ribosomiques dans le nucléole ou dans des corps cytoplasmiques. En outre, la localisation particulière des facteurs ribosomiques Hrr25 et Fap7 dans les P-bodies en phase de croissance saturée laisse penser que ces corps cytoplasmiques sont le lieu de dégradation des pré-ribosomes lorsque les carences nutritives perdurent.

Das Lektin aus <i>Pisum sativum</i>, der Gartenerbse, ist Teil der Familie der Leguminosenlektine. Diese Proteine haben untereinander eine hohe Sequenzhomologie, und die Struktur ihrer Monomere, ein all-ß-Motiv, ist hoch konserviert. Dagegen gibt es innerhalb der Familie eine große Vielfalt an unterschiedlichen Quartärstrukturen, die Gegenstand kristallographischer und theoretischer Arbeiten waren. Das Erbsenlektin ist ein dimeres Leguminosenlektin mit einer Besonderheit in seiner Struktur: Nach der Faltung in der Zelle wird aus einem Loop eine kurze Aminosäuresequenz herausgeschnitten, so dass sich in jeder Untereinheit zwei unabhängige Polypeptidketten befinden. Beide Ketten sind aber stark miteinander verschränkt und bilden eine gemeinsame strukturelle Domäne. Wie alle Lektine bindet Erbsenlektin komplexe Oligosaccharide, doch sind seine physiologische Rolle und der natürliche Ligand unbekannt. In dieser Arbeit wurden Versuche zur Entwicklung eines Funktionstests für Erbsenlektin durchgeführt und seine Faltung, Stabilität und Monomer-Dimer-Gleichgewicht charakterisiert. Um die spezifische Rolle der Prozessierung für Stabilität und Faltung zu untersuchen, wurde ein unprozessiertes Konstrukt in <i>E. coli</i> exprimiert und mit der prozessierten Form verglichen. <br /> <br /> Beide Proteine zeigen die gleiche kinetische Stabilität gegenüber chemischer Denaturierung. Sie denaturieren extrem langsam, weil nur die isolierten Untereinheiten entfalten können und das Monomer-Dimer-Gleichgewicht bei mittleren Konzentrationen an Denaturierungsmittel auf der Seite der Dimere liegt. Durch die extrem langsame Entfaltung zeigen beide Proteine eine apparente Hysterese im Gleichgewichtsübergang, und es ist nicht möglich, die thermodynamische Stabilität zu bestimmen. Die Stabilität und die Geschwindigkeit der Assoziation und Dissoziation in die prozessierten bzw. nichtprozessierten Untereinheiten sind für beide Proteine gleich. Darüber hinaus konnte gezeigt werden, dass auch unter nicht-denaturierenden Bedingungen die Untereinheiten zwischen den Dimeren ausgetauscht werden.<br /> <br /> Die Renaturierung der unprozessierten Variante ist unter stark nativen Bedingungen zu 100 % möglich. Das prozessierte Protein dagegen renaturiert nur zu etwa 50 %, und durch die Prozessierung ist die Faltung stark verlangsamt, der Faltungsprozess ist erst nach mehreren Tagen abgeschlossen. Im Laufe der Renaturierung wird ein Intermediat populiert, in dem die längere der beiden Polypeptidketten ein Homodimer mit nativähnlicher Untereinheitenkontaktfläche bildet. Der geschwindigkeitsbestimmende Schritt der Renaturierung ist die Assoziation der entfalteten kürzeren Kette mit diesem Dimer. / The lectin from <i>Pisum sativum</i> (garden pea) is a member of the family of legume lectins. These proteins share a high sequence homology, and the structure of their monomers, an all-ß-motif, is highly conserved. Their quaternary structures, however, show a great diversity which has been subject to cristallographic and theoretical studies. Pea lectin is a dimeric legume lectin with a special structural feature: After folding is completed in the cell, a short amino acid sequence is cut out of a loop, resulting in two independent polypeptide chains in each subunit. Both chains are closely intertwined and form one contiguous structural domain. Like all lectins, pea lectin binds to complex oligosaccharides, but its physiological role and its natural ligand are unknown. In this study, experiments to establish a functional assay for pea lectin have been conducted, and its folding, stability and monomer-dimer-equilibrium have been characterized. To investigate the specific role of the processing for stability and folding, an unprocessed construct was expressed in <i>E. coli</i> and compared to the processed form.<br /> <br /> Both proteins have the same kinetic stability against chemical denaturant. They denature extremely slowly, because only the isolated subunits can unfold, and the monomer-dimer-equilibrium favors the dimer at moderate concentrations of denaturant. Due to the slow unfolding, both proteins exhibit an apparent hysteresis in the denaturation transition. Therefore it has not been possible to determine their thermodynamic stability. For both proteins, the stability and the rates of association and dissociation into processed or unprocessed subunits, respectively, are equal. Furthermore it could be shown that even under non-denaturing conditions the subunits are exchanged between dimers.<br /> <br /> Renaturation of the unprocessed variants is possible under strongly native conditions with 100 % yield. The processed protein, however, can be renatured with yields of about 50 %, and its refolding is strongly decelerated. The folding process is finished only after several days. During renaturation, an intermediate is populated, in which the longer of the two polypeptide chains forms a homodimer with a native-like subunit interface. The rate limiting step of renaturation is the association of the unfolded short chain with this dimer.

Contagious bovine pleuropneumonia (CBPP) is a severe infectious disease caused by Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) and is a vast problem in Africa. Current CBPP prevention is based on attenuated live strain vaccines, but these are limited by factors such as short-term immunity, cold-chain dependence and retained virulence. CBPP can be diagnosed using post-mortem examination, identification of the agent using culture and PCR based methods as well as serological diagnostic methods, but the latter are generally not sensitive enough and there is also demand for an inexpensive, pen side field test.The research presented in this thesis was focused on using recombinantly expressed surface proteins from M. mycoides SC to characterize humoral immune responses to CBPP. Thereby candidate proteins to be used in development of serological diagnostic methods and possibly subunit vaccines could be identified. As a first step, five putative variable surface proteins of M. mycoides SC were expressed and purified from E. coli in Paper I. These proteins were analyzed using immunoblotting techniques and results showed that one protein, MSC_0364, was variably expressed on the surface of M. mycoides SC in vitro. Paper II presents expanded efforts including cloning and expression of 64 recombinant surface proteins and an assay for high throughput analysis of protein-specific IgG, IgA and IgM titers in hundreds of sera using a bead-based screening assay. The assay was evaluated by protein-specific inhibition experiments, comparisons to Western blotting and monitoring of immune responses over time in a study with sera taken from eight animals over 293 days from a previous vaccine trial.Papers III and IV present applications using the recombinant proteins and bead-based screening assay wherein proteins for diagnostic and vaccine development were identified. In Paper III, the assay was used to screen 61 proteins using well-characterized serum samples from cattle with CBPP and healthy controls, resulting in selection of eight proteins suitable for diagnostic use. These proteins were combined and evaluated in a proof-of-concept ELISA with a discriminative power that enabled 96% correct classification of sera from CBPP-affected and CBPP-free bovines. Paper IV reports the results and protein-specific analyses of a vaccine trial using the recombinant putative variable surface proteins presented in Paper I as a subunit vaccine. The vaccine conferred no protection, but a weak vaccine response could not be excluded as the cause of failure. In an effort to identity other protein candidates to be used in a subunit vaccine, protein-specific analysis of humoral immune responses elicited by the currently approved live strain vaccine, T1/44, were investigated. Here, five proteins with high IgG titers associated to immunity were identified: LppQ, MSC_02714, MSC_0136, MSC_0079 and MSC_0431. These proteins may be important in the development of a novel subunit vaccine against CBPP. / QC 20100719

Homotrimeric P2X3 and heteromeric P2X2/3 receptors are present in sensory ganglia and participate in pain perception. In order to develop pharmacological antagonists for these receptors, it is important to clarify which amino acid (AA) residues constitute the agonist binding pouch as well as to learn the stoichiometry of the receptor subunits forming a heteromeric receptor. We expressed the homomeric human (h)P2X3 receptor or its mutants in HEK293 cells and measured the ATP-induced responses by the whole-cell patch-clamp method. For the binding-site mutants, all conserved and some non-conserved AAs in the four nucleotide binding segments (NBSs) of the P2X3 subunit were sequentially replaced by alanine. Especially the positively charged AAs Lys and Arg appeared to be of critical importance for the agonist effects. We concluded that groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the P2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor. We were also interested to find out, whether two heteromeric receptors (P2X2/3 and P2X2/6), where P2X2 combines with two different partners, have an obligatory subunit stoichiometry of 1:2 or whether the subunit stoichiometry may be variable. For this purpose we used non-functional P2X2, P2X3 and P2X6 subunit-mutants to investigate the composition of heteromeric P2X2/3 and P2X2/6 receptors. The subunit stoichiometry of P2X2/3 and P2X2/6 was found to be 1:2 and 2:1, respectively. Thus, recognitions sites between P2X2 and its partners rather than random association may govern the subunit compositions of the receptor trimers.