A maior parte dos casos de doença renal policística autossômica dominante (DRPAD) é causada por mutações no gene PKD1 (Polycystic Kidney Disease 1). O insulto por isquemia/reperfusão (IR) constitui-se em uma causa freqüente de lesão renal aguda, incluindo a população de pacientes com DRPAD, mas a relação entre policistina-1 e IR é essencialmente desconhecida. Uma vez que a policistina-1 modula proliferação, diferenciação celular e apoptose em sistemas de cultura de células, sua menor atividade biológica na DRPAD poderia favorecer um maior grau de lesão renal. Utilizamos uma linhagem endogâmica de camundongos 129Sv com uma mutação nula em Pkd1 para testar esta hipótese. Camundongos Pkd1+/- não apresentam cistos renais até 12 semanas de vida, constituindo-se em um modelo puro de haploinsuficiência para este gene. Um insulto IR bilateral de 32 min foi induzido em camundongos machos de 10-12 semanas de idade, heterozigotos e selvagens, por meio do clampeamento reversível de ambos os pedículos renais. Os animais foram analisados 48 h, 7 dias (d) e 14 d após o insulto. Camundongos Pkd1+/- apresentaram FENa, FEK e SCr mais elevadas que animais Pkd1+/+ 48 h após IR. O dano cortical residual foi mais severo em heterozigotos que em selvagens em todos os tempos avaliados. A marcação para PCNA também foi mais alta em camundongos Pkd1+/- que Pkd1+/+ 48 h e 7 d pós-IR, enquanto a taxa de apoptose e a infiltração inflamatória intersticial foram maiores em heterozigotos que em selvagens nos seguimentos de 48 h, 7 d e 14 d pós-IR. A expressão renal de p21 foi menor nos camundongos Pkd1+/- que Pkd1+/+ no tempo de 48 h pós-insulto, tanto no nível transcricional como traducional. Análises adicionais realizadas 6 semanas após o insulto IR revelaram dilatação tubular e formação de microcistos nos camundongos haploinsuficientes para Pkd1, assim como fibrose renal aumentada nesses animais, comparados aos camundongos selvagens. Por fim, um insulto de 35 min de isquemia/reperfusão acompanhou-se de uma mortalidade precoce substancialmente maior nos animais Pkd1+/-. Esses achados sugerem que isquemia/reperfusão induza uma lesão mais severa em rins de camundongos haploinsuficientes para Pkd1, um processo aparentemente dependente de uma deficiência relativa da atividade de p21, assim como dilatação tubular e formação de microcistos. Em conjunto, nossos resultados sugerem que a heterozigose para mutação nula em Pkd1 em camundongo (e talvez em humanos) esteja associada a um risco aumentado para lesão renal por isquemia/reperfusão e a um pior impacto desse insulto sobre a progressão da doença renal. / The majority of autosomal dominant polycystic kidney disease (ADPKD) cases are caused by mutations in the PKD1 gene. Ischemia/reperfusion is a frequent cause of acute kidney injury, including the ADPKD patient population, but the relationship between polycystin-1 and ischemia/reperfusion is essentially unknown. Since polycystin-1 modulates cell proliferation, cell differentiation and apoptosis in cell culture systems, its lower biological activity in ADPKD might amplify the degree of renal injury. Using an inbred 129Sv mouse line with a Pkd1-null mutation, 32-min renal ischemia/reperfusion was induced in 10-12 week-old male non-cystic mice, heterozygotes and wild types. The animals were analyzed at 48h, 7 days (d) and 14d after the insult. Pkd1+/- mice showed higher FENa, FEK and SCr than Pkd1+/+ animals at 48h of follow-up. The residual cortical damage was more severe in heterozygotes than wild types at all evaluated time points. The PCNA staining was also higher in Pkd1+/- than Pkd1+/+ mice at 48h and 7d, while cell apoptotic rates and the interstitial inflammatory infiltration were higher in heterozygotes than wild types at 48h, 7d and 14d postischemia/ reperfusion. The expression of p21 was lower in Pkd1+/- than Pkd1+/+ kidneys at 48h, both at the transcriptional and translational levels. Additional analyses performed 6 weeks after the insult showed tubular dilatation and microcyst formation in the haploinsufficient mice, and increased renal fibrosis in these animals compared to wild types. Thirty-fivemin ischemia/reperfusion, at last, was accompanied by a substantially higher early mortality of Pkd1+/- animals. These findings suggest that ischemia/reperfusion induces a more severe injury in kidneys of Pkd1- haploinsufficient mice, a process that is apparently dependent on a relative deficiency of p21 activity, as well as tubular dilatation and microcyst formation. Altogether, our results suggest that mouse Pkd1-null heterozygosity (and maybe human) is associated with a higher risk for renal ischemia/reperfusion injury and with a worse impact of this insult upon renal disease progression.

INTRODUÇÃO: O papilomavírus humano (HPV) encontra-se amplamente distribuído na população mundial. Apesar da grande maioria das infecções serem transientes, assintomáticas e passíveis de regressão espontânea, a infecção persistente por tipos de alto risco de HPV é necessária para o desenvolvimento de neoplasias intraepiteliais cervicais. Uma vez que apenas uma pequena parcela das infecções progride à lesões malignas após um longo período desde o diagnóstico inicial de lesões precursoras, tem-se iniciado a busca por fatores que possam influenciar na progressão ou na eliminação destas manifestações iniciais. A variabilidade genética viral tem sido apontada como um dos fatores que interagem neste processo. Embora virtualmente todos os tumores da cérvice uterina apresentem o DNA viral, neoplasias em outros sítios anatômicos têm sido apenas em parte correlacionadas com a presença viral, sendo o HPV proposto como um dos agentes causadores de tumores em sítios de cabeça e pecoço. MÉTODOS: Espécimens clínicos de tumores de cabeça e pescoço, fixados em formalina e contidos em parafina (FFPE), provenientes do Instituto do Câncer do Estado de São Paulo (n=79) e da Santa Casa de Misericórida de São Paulo (n=94), tiveram seu DNA extraído, seguido de diagnóstico e genotipagem de HPV pela metodologia de Inno-LiPA. Análises de linhagens moleculares foram realizadas nas amostras HPV-16 positivas. Análise imunohistoquímica de P16INK4a foi realizada em todas as amostras. RESULTADOS: A presença do DNA viral foi encontrada em 24,1% (19/79) dentre a série de tumores provenientes do ICESP, sendo a cavidade oral o sítio em que foi observada a maior proporção de DNA viral (27,1%), enquanto que 13,8% (13/94) dentre os espécimens provenientes da Santa Casa apresentaram-se positivos para HPV, sendo a cavidade oral o sítio em que foi observada a maior proporção do DNA viral (18,1%). O HPV-16 foi o tipo mais prevalente, detectado em 73,4% das amostras HPV positivas provenientes do ICESP e 61,5% das amostras provenientes da Santa Casa. Independente da Instituição, as amostras foram alocadas no clado das linhagens Asiático-Americana e Europeia em 50%, cada uma, entre os 18 tumores HPV-16 positivos em que as análises de linhagem foram possíveis. Não foi observada, nestas séries, correlação entre a superexpressão de P16INK4a e a presença do DNA viral. CONCLUSÃO: Nas amostras analisadas, o DNA de HPV foi detectado em 18,5% dos 173 espécimens. O HPV-16 foi o tipo mais prevalente. Isolados da linhagem Europeia e da linhagem Asiatico-Americano foram detectados em 50% dos casos, cada uma, dentre as amostras HPV-16 analisadas por este estudo / INTRODUCTION: Human papillomaviruses (HPV) are widely distributed worldwide. Although the majority of infections are usually transient, asymptomatic and frequently regress spontaneously, persistent infections by high-risk HPVs are necessary for the development of cervical intraepithelial neoplasia. Once only a small proportion of infections progress to malignant lesions after a long period of time since the initial diagnosis of precursor lesions, the search for factors that might influence the progression or clearence of these early manifestations are currently under way. Viral genetic variability has been proposed as one of the factors interacting in this process. Although virtually all cervix tumors present the viral DNA, neoplasias from other anatomical sites have been only in part correlated with viral presence, and HPV has been proposed as one causative agent in tumors from head and neck sites. METHODS: Clinical specimens of formalin-fixed paraffin embedded head and neck tumors, provided by the Cancer Institute of São Paulo (n=79) and also by the Santa Casa de Misericórdia de São Paulo (n=94), were submitted to DNA extraction and further HPV diagnostic and genotyping by the Inno-LiPA methodology. Molecular lineages analyses were performed in all HPV-16 positive samples. P16INK4a immunohistochemical analyses were conducted in all samples. RESULTS: HPV DNA was detected among 24.1% (19/79) of samples provided by ICESP, tumors from oral cavity presented the highest viral positivity (27.1%), whereas 13,8% (13/94) of the samples from Santa Casa presented HPV DNA, tumors from the oral cavity also presented the highest HPV positivity with 18.1% of viral DNA presence. HPV-16 was the most prevalent type detected in 73.4% and 61.5% of HPV positive ICESP and Santa Casa samples, respectively. Irrespective of the Institution, samples submitted to lineage analyzes were allocated in the Asiatic-American and European phylogenetic branches in 50%, each one, among the 18 tumors HPV-16 positive for which lineage analysis was possible. No correlation between P16INK4a overexpression and HPV DNA presence was observed. CONCLUSION: In this study, HPV DNA was detected in 18.5% among 173 head and neck tumor specimens. HPV-16 was the most prevalent type. The European and the Asiatic-American lineage were detected in 50% of the cases, each one, among the cases HPV-16 positive analyzed

Le travail s’est articulé autour de la synthèse et de l’évaluation biologique de nouveaux inhibiteurs de protéines kinases dont essentiellement de kinases dépendantes de cyclines (CDKs). La dérégulation de ces kinases est mise en cause dans l’apparition de nombreuses pathologies prolifératives telles que le cancer ou non prolifératives telles que les maladies neurodégénératives. Deux types d’inhibiteurs ont été préparés. La première famille de composés étudiée est la famille des purines, sur laquelle a été introduite des substructures de type aminodiols en position C-2. Ces aminodiols ont été préparés de manière stéréosélective via la réaction de dihydroxylation asymétrique décrite par Sharpless ou à partir de dérivés d’acides aminés. Parmi les inhibiteurs réalisés, plusieurs ont montré une inhibition envers les protéines CDK5, CDK9 et CK1 très supérieure (IC50 de l’ordre de 100 nM) à celle de la (R)-Roscovitine, molécule actuellement en phase clinique II dans plusieurs cancers. Ces inhibitions des cibles enzymatiques s’accompagnent d’un effet antiprolifératif sur plusieurs lignées tumorales. Enfin l’étude d’une des molécules a été complétée par des tests très favorables sur des enzymes de microsomes hépatiques (IC50 > 5 μM). Dans une seconde partie, des imidazo[1,2-b]pyridazines ont été préparées. Une synthèse originale conduisant aux imidazo[1,2-b]pyridazines 3,6,8-trisubstituées a été mise au point. Les produits présentent une inhibition forte envers les protéines kinases telles que CDK5 et CK1 (IC50 < 50 nM). Ils montrent également des propriétés antiprolifératives sur les cellules tumorales SH-SY5Y. Enfin, des imidazo[1,2-b]pyridazines 3,6-disubstituées se révèlent être des inhibiteurs sélectifs de la protéine CLK1 (IC50 < 100 nM). / This thesis focuses on the synthesis and biological evaluation of new kinase inhibitors. Kinase deregulation is associated with numerous diseases such as cancer or neurodegenerative diseases. In the first part of this work, 2,6,9-trisubstituted purines bearing at C-2 position aminodiols derivatives were prepared. Aminodiols were obtained either via Sharpless asymmetric dihydroxylation or by reduction of amino esters. The compounds appeared to be more potent against kinases than (R)-roscovitine which is presently undergoing phase II clinical tests. In particular, inhibition of CK1, CDK5 and CDK9 were observed with IC50 < 200 nM. The compounds prepared showed an antiproliferative effect against tumor cell-lines (SH-SY5Y). Eventually, one of the most promising compounds was assayed against a series of cyt P450 enzymes and did not showed any inhibition (IC50 > 5 μM). The second family of compounds prepared in this work is imidazo[1,2-b]pyridazines. A new route to 3,6,8-trisubstituted imidazo[1,2-b]pyridazines was first developed. These products were shown to be highly potent inhibitors of several kinases such as CDK5 and CK1 (IC50 < 50 nM). The kinase inhibitions were accompanied by antiproliferative activities against tumor cell-lines. Finally, a series of 3,6-disubtituted imidazo[1,2-b]pyridazines was also prepared and appeared to be selective inhibitors of CLK1 (IC50 < 100 nM).

INTRODUÇÃO: Na Neoplasia Endócrina Múltipla tipo 2 (NEM2), o desenvolvimento do Carcinoma Medular de Tireoide (CMT), Feocromocitoma (FEO) e Hiperparatireoidismo primário (HPT) está associado à mutações germinativas ativadoras no proto-oncogene RET. Casos de CMT esporádico podem apresentar mutações somáticas no RET (~40%). A variabilidade fenotípica observada em casos de CMT e FEO familiais associados à NEM2 indica o envolvimento de eventos genéticos adicionais que seriam responsáveis pelas diferenças clínicas observadas nos indivíduos afetados (idade de desenvolvimento, progressão e agressividade do tumor). Outras alterações genéticas no RET como duplas mutações, SNPs e haplótipos específicos podem influenciar na susceptibilidade, agressividade e modulação do fenótipo NEM2. Entretanto, os estudos de outros genes envolvidos no processo da tumorigênese NEM2 ainda estão em andamento. Recentemente foi mostrado que RET ativado controla a expressão de proteínas inibidoras do ciclo celular (p18 e p27). Mutações germinativas no gene p27 foram recentemente associadas à susceptibilidade de tumores neuroendócrinos e estão associadas à síndrome NEM4 (Neoplasia endócrina múltipla tipo 4). Mutações somáticas, inativadoras de p27, são raramente encontradas em vários tipos de tumores. Entretanto, diversos estudos documentaram que a redução na expressão e a sublocalização citoplamática de p27 são controladas por alterações pós-transducionais e/ou epigenéticas. OBJETIVOS: o estudo teve como objetivos avaliar a participação de genes, recentemente associados ao RET ativado, em tumores de pacientes com NEM2 e também verificar se polimorfismos no gene p27 estariam atuando como moduladores de fenótipo em uma grande família com NEM2. CASUÍTICA: foram analisadas 66 amostras tumorais advindas de 36 pacientes com diagnóstico clínico e genético de NEM2 e 28 indivíduos pertencentes a uma grande família com NEM2A-CMTF e mutação C620R no gene RET. MÉTODOS: As análises somáticas do p27 e também de p15, p18 e RET foram realizadas por PCR e sequenciamento direto de DNA e análise de microssatélites para p27 foi realizada por PCR e eletroforese capilar. Análises de expressão e localização da proteína p27 celular foram realizadas por Western blot e imunohistoquímica. A análise da modulação de fenótipo na família com NEM2A foi realizada por meio da amplificação do éxon 1 do gene p27 na amostra de sangue total. RESULTADOS: Não foram encontradas mutações somáticas no gene p27 e também nos genes p15 e p18. Entretanto, verificamos baixa expressão proteica de p27 em tumores CMT e FEO, a qual se encontrava relacionada com o tipo e agressividade do códon mutado no RET, principalmente em tumores que apresentavam mutação RET no códon 634 (controle x 634 p=0,05; controle x 634/791 p= 0,032; 620 x 634 p=0,045; 620 x 634/791 p= 0,002; 620 x 634 + 634/791 p=0,036). Notou-se também correlação positiva entre os níveis de expressão de p27 na localização nuclear, analisada por imunohistoquímica, e o genótipo TT do SNP p27 p.V109G (p=0,03). CONCLUSÕES: Alterações moleculares somáticas no gene p27 nos tumores NEM2 não são frequentes. Entretanto, a redução na expressão e a localização citoplasmática de p27 provavelmente estão associadas a alterações somáticas em outros genes que controlam os processos de fosforilação da proteína p27 (eventos pós-transducionais) / INTRODUCTION: In Multiple Endocrine Neoplasia type 2 (MEN2) the development of medullary thyroid carcinoma (MTC), pheochromocytoma (PHEO) and primary hyperparathyroidism (HPT) are associated with activating germline mutations in RET proto-oncogene. Cases of sporadic MTC may have somatic RET mutations (~ 40%). The phenotypic variability observed in cases with familial MTC/MEN2 and PHEO/MEN2 indicates the probable involvement of additional genetic events that could be responsible for the clinical differences observed in the affected individuals (age development, progression and aggressiveness of the tumor). Other genetic alterations such as RET double mutations, SNPs and specific haplotypes may influence susceptibility, aggressiveness and MEN2 phenotype modulation. However, studies of other genes involved in the tumorigenesis of MEN2 are still in progress. Recently, it was shown that the activated RET controls the expression of cell cycle inhibitory proteins (p18 and p27). Germline mutations in the p27 gene have recently been associated with the susceptibility to neuroendocrine tumors and are associated with the MEN4 syndrome (Multiple endocrine neoplasia type 4). Somatic inactivating mutations p27 are rarely found in many types of tumors. However, several studies have documented that reduced expression and subcellular location of p27 is controlled by post-transductional changes and/or epigenetic factors. OBJECTIVES: This study aimed to evaluate the role of genes recently associated with RET activated in tumors from MEN2 patients and also check whether polymorphisms in the p27 gene would be acting as modulators of phenotype in a large MEN2 family. PATIENTS: We analyzed 66 tumor samples from 36 patients with clinical and genetic diagnosis of MEN2 and from 28 individuals belonging to a large family with FMTC/MEN2A and RET C620R mutation. METHODS: The analyses of somatic p27, p15, p18 and RET were performed by PCR and direct sequencing of DNA and microsatellite analysis was performed for p27 by PCR and capillary electrophoresis. Expression analysis and subcellular localization of p27 protein were performed by Western blot and immunohistochemistry. The analysis of phenotype modulation in MEN2A families was performed by the amplification of exon 1 of the p27 gene in a whole blood sample. RESULTS: There were no somatic mutations in the p27 gene and also in the p15 and p18 genes. However, we verified a low p27 protein expression in MTC/MEN2 and PHEO/MEN2 that showed a definite correlation with the type and aggressiveness of the mutated RET codon, mainly in those tumors from cases with germline RET codon 634 mutations (control vs 634, p=0,05; control vs 634/791, p= 0,032; 620 vs 634, p=0,045; 620 vs 634/791, p= 0,002; 620 vs 634 + 634/791, p=0,036). It was also verified a positive correlation between the immunohistochemistry expression of nuclear p27 subcellular location and the p27 p.V109G TT genotype (p=0,03). CONCLUSIONS: The reduction in the expression of p27 and its subcellular localization are likely to be associated with somatic changes in other genes that control the processes of phosphorylation of p27 protein through post-transductional events

INTRODUÇÃO: Na Neoplasia Endócrina Múltipla tipo 2 (NEM2), o desenvolvimento do Carcinoma Medular de Tireoide (CMT), Feocromocitoma (FEO) e Hiperparatireoidismo primário (HPT) está associado à mutações germinativas ativadoras no proto-oncogene RET. Casos de CMT esporádico podem apresentar mutações somáticas no RET (~40%). A variabilidade fenotípica observada em casos de CMT e FEO familiais associados à NEM2 indica o envolvimento de eventos genéticos adicionais que seriam responsáveis pelas diferenças clínicas observadas nos indivíduos afetados (idade de desenvolvimento, progressão e agressividade do tumor). Outras alterações genéticas no RET como duplas mutações, SNPs e haplótipos específicos podem influenciar na susceptibilidade, agressividade e modulação do fenótipo NEM2. Entretanto, os estudos de outros genes envolvidos no processo da tumorigênese NEM2 ainda estão em andamento. Recentemente foi mostrado que RET ativado controla a expressão de proteínas inibidoras do ciclo celular (p18 e p27). Mutações germinativas no gene p27 foram recentemente associadas à susceptibilidade de tumores neuroendócrinos e estão associadas à síndrome NEM4 (Neoplasia endócrina múltipla tipo 4). Mutações somáticas, inativadoras de p27, são raramente encontradas em vários tipos de tumores. Entretanto, diversos estudos documentaram que a redução na expressão e a sublocalização citoplamática de p27 são controladas por alterações pós-transducionais e/ou epigenéticas. OBJETIVOS: o estudo teve como objetivos avaliar a participação de genes, recentemente associados ao RET ativado, em tumores de pacientes com NEM2 e também verificar se polimorfismos no gene p27 estariam atuando como moduladores de fenótipo em uma grande família com NEM2. CASUÍTICA: foram analisadas 66 amostras tumorais advindas de 36 pacientes com diagnóstico clínico e genético de NEM2 e 28 indivíduos pertencentes a uma grande família com NEM2A-CMTF e mutação C620R no gene RET. MÉTODOS: As análises somáticas do p27 e também de p15, p18 e RET foram realizadas por PCR e sequenciamento direto de DNA e análise de microssatélites para p27 foi realizada por PCR e eletroforese capilar. Análises de expressão e localização da proteína p27 celular foram realizadas por Western blot e imunohistoquímica. A análise da modulação de fenótipo na família com NEM2A foi realizada por meio da amplificação do éxon 1 do gene p27 na amostra de sangue total. RESULTADOS: Não foram encontradas mutações somáticas no gene p27 e também nos genes p15 e p18. Entretanto, verificamos baixa expressão proteica de p27 em tumores CMT e FEO, a qual se encontrava relacionada com o tipo e agressividade do códon mutado no RET, principalmente em tumores que apresentavam mutação RET no códon 634 (controle x 634 p=0,05; controle x 634/791 p= 0,032; 620 x 634 p=0,045; 620 x 634/791 p= 0,002; 620 x 634 + 634/791 p=0,036). Notou-se também correlação positiva entre os níveis de expressão de p27 na localização nuclear, analisada por imunohistoquímica, e o genótipo TT do SNP p27 p.V109G (p=0,03). CONCLUSÕES: Alterações moleculares somáticas no gene p27 nos tumores NEM2 não são frequentes. Entretanto, a redução na expressão e a localização citoplasmática de p27 provavelmente estão associadas a alterações somáticas em outros genes que controlam os processos de fosforilação da proteína p27 (eventos pós-transducionais) / INTRODUCTION: In Multiple Endocrine Neoplasia type 2 (MEN2) the development of medullary thyroid carcinoma (MTC), pheochromocytoma (PHEO) and primary hyperparathyroidism (HPT) are associated with activating germline mutations in RET proto-oncogene. Cases of sporadic MTC may have somatic RET mutations (~ 40%). The phenotypic variability observed in cases with familial MTC/MEN2 and PHEO/MEN2 indicates the probable involvement of additional genetic events that could be responsible for the clinical differences observed in the affected individuals (age development, progression and aggressiveness of the tumor). Other genetic alterations such as RET double mutations, SNPs and specific haplotypes may influence susceptibility, aggressiveness and MEN2 phenotype modulation. However, studies of other genes involved in the tumorigenesis of MEN2 are still in progress. Recently, it was shown that the activated RET controls the expression of cell cycle inhibitory proteins (p18 and p27). Germline mutations in the p27 gene have recently been associated with the susceptibility to neuroendocrine tumors and are associated with the MEN4 syndrome (Multiple endocrine neoplasia type 4). Somatic inactivating mutations p27 are rarely found in many types of tumors. However, several studies have documented that reduced expression and subcellular location of p27 is controlled by post-transductional changes and/or epigenetic factors. OBJECTIVES: This study aimed to evaluate the role of genes recently associated with RET activated in tumors from MEN2 patients and also check whether polymorphisms in the p27 gene would be acting as modulators of phenotype in a large MEN2 family. PATIENTS: We analyzed 66 tumor samples from 36 patients with clinical and genetic diagnosis of MEN2 and from 28 individuals belonging to a large family with FMTC/MEN2A and RET C620R mutation. METHODS: The analyses of somatic p27, p15, p18 and RET were performed by PCR and direct sequencing of DNA and microsatellite analysis was performed for p27 by PCR and capillary electrophoresis. Expression analysis and subcellular localization of p27 protein were performed by Western blot and immunohistochemistry. The analysis of phenotype modulation in MEN2A families was performed by the amplification of exon 1 of the p27 gene in a whole blood sample. RESULTS: There were no somatic mutations in the p27 gene and also in the p15 and p18 genes. However, we verified a low p27 protein expression in MTC/MEN2 and PHEO/MEN2 that showed a definite correlation with the type and aggressiveness of the mutated RET codon, mainly in those tumors from cases with germline RET codon 634 mutations (control vs 634, p=0,05; control vs 634/791, p= 0,032; 620 vs 634, p=0,045; 620 vs 634/791, p= 0,002; 620 vs 634 + 634/791, p=0,036). It was also verified a positive correlation between the immunohistochemistry expression of nuclear p27 subcellular location and the p27 p.V109G TT genotype (p=0,03). CONCLUSIONS: The reduction in the expression of p27 and its subcellular localization are likely to be associated with somatic changes in other genes that control the processes of phosphorylation of p27 protein through post-transductional events

Bevor sich eine Zelle teilt muss sie ihr gesamtes genetisches Material verdoppeln. Eukaryotische Genome werden von einer Vielzahl von Replikationsstartpunkten, den sogenannten Origins, aus repliziert, die über das gesamte Genome verteilt sind. In dieser Arbeit wird der zugrundeliegende molekulare Mechanismus quantitativ analysiert, der für die nahezu simultane Initiierung der Origins exakt ein Mal pro Zellzyklus verantwortlich ist. Basierend auf umfangreichen experimentellen Studien, wird zunächst ein molekulares regulatorisches Netzwerk rekonstruiert, welches das Binden von Molekülen an die Origins beschreibt, an denen sich schließlich komplette Replikationskomplexe (RKs) bilden. Die molekularen Reaktionen werden dann in ein Differentialgleichungssystem übersetzt. Um dieses mathematische Modell zu parametrisieren, werden gemessene Proteinkonzentrationen als Anfangswerte verwendet, während kinetische Parametersätze in einen Optimierungsverfahren erzeugt werden, in welchem die Dauer, in der sich eine Mindestanzahl von RKs gebildet hat, minimiert wird. Das Modell identifiziert einen Konflikt zwischen einer schnellen Initiierung der Origins und einer effizienten Verhinderung der DNA Rereplikation. Modellanalysen deuten darauf hin, dass eine zeitlich verzögerte Origininitiierung verursacht durch die multiple Phosphorylierung der Proteine Sic1 und Sld2 durch Cyclin-abhängige Kinasen, G1-Cdk bzw. S-Cdk, essentiell für die Lösung dieses Konfliktes ist. Insbesondere verschafft die Mehrfach-Phosphorylierung von Sld2 durch S-Cdk eine zeitliche Verzögerung, die robust gegenüber Veränderungen in der S-Cdk Aktivierungskinetik ist und außerdem eine nahezu simultane Aktivierung der Origins ermöglicht. Die berechnete Verteilung der Fertigstellungszeiten der RKs, oder die Verteilung der Originaktivierungszeiten, wird auch genutzt, um die Konsequenzen bestimmter Mutationen im Assemblierungsprozess auf das Kopieren des genetischen Materials in der S Phase des Zellzyklus zu simulieren. / Before a cell divides it has to duplicate its entire genetic material. Eukaryotic genomes are replicated from multiple replication origins across the genome. This work is focused on the quantitative analysis of the underlying molecular mechanism that allows these origins to initiate DNA replication almost simultaneously and exactly once per cell cycle. Based on a vast amount of experimental findings, a molecular regulatory network is constructed that describes the assembly of the molecules at the replication origins that finally form complete replication complexes. Using mass–action kinetics, the molecular reactions are translated into a system of differential equations. To parameterize the mathematical model, the initial protein concentrations are taken from experimental data, while kinetic parameter sets are determined using an optimization approach, in particular a minimization of the duration, in which a minimum number of replication complexes has formed. The model identifies a conflict between the rapid initiation of replication origins and the efficient inhibition of DNA rereplication. Analyses of the model suggest that a time delay before the initiation of DNA replication provided by the multiple phosphorylations of the proteins Sic1 and Sld2 by cyclin-dependent kinases in G1 and S phase, G1-Cdk and S-Cdk, respectively, may be essential to solve this conflict. In particular, multisite phosphorylation of Sld2 by S-Cdk creates a time delay that is robust to changes in the S-Cdk activation kinetics and additionally allows the near-simultaneous activation of multiple replication origins. The calculated distribution of the assembly times of replication complexes, that is also the distribution of origin activation times, is then used to simulate the consequences of certain mutations in the assembly process on the copying of the genetic material in S phase of the cell cycle.

La phosphorylation des protéines par les kinases est l’une plus importantes modification post-traductionnelle dans les processus cellulaires tels que la division, la différenciation, la prolifération et l’apoptose. Due à leur rôle clef, un dérèglement des protéines kinases peut entrainer de nombreuses pathologies proliférative telles que le cancer et non prolifératives telles que les maladies neurodégénératives. Le travail de thèse s’est construit autour de 2 séries d’inhibiteurs de protéine kinases comportant les noyaux imidazo[1,2-b]pyridazine et imidazo[4,5-b]pyridine. L’objectif est d’inhiber sélectivement les protéines kinases choisies, pour leurs implications dans les pathologies visées au laboratoire. Les imidazo[1,2-b]pyridazines ont été préparées pour identifier des inhibiteurs de CLK1 et DYRK1A, cibles potentielles dans la maladie d’Alzheimer. Parmi les imidazo[1,2-b]pyridazines synthétisées, plusieurs molécules se sont révélées particulièrement sélectives de DYRKs et CLKs, avec des IC50 < 100 nM. Une relation structure-activité basée sur la synthèse de 70 molécules, a permis de dégager des éléments structuraux de la sélectivité des molécules. L’évaluation des produits a également été portée sur les kinases de parasites. Il a ainsi été possible d’identifier quelques inhibiteurs actifs sur PfCLK1. La seconde partie de cette thèse avait pour objectif l’optimisation du protocole de synthèse imidazo[4,5-b]pyridines, analogue de la roscovitine. Des dérivés s’étaient révélés capables d’inhiber la formation de kystes, dans un modèle cellulaire de polykystose rénale. Une synthèse en sept étapes a conduit à plusieurs grammes d’imidazo[4,5-b]pyridine 3,5,7 trisubstitués, qui sont ainsi disponibles pour l’évaluation in vivo. / Phosphorylation by protein kinases is one of the most important post-translational modification in cellular processes such as division, differentiation, proliferation and apoptosis. Kinase deregulation is associated with numerous diseases such as cancer or neurodegenerative diseases. Imidazo[1,2-b]pyridazine and imidazo[4,5-b]pyridine were prepared to inhibit protein kinases involved in diseases targeted in the laboratory. The imidazo[1,2-b]pyridazines were synthesized to identify inhibitors of CLK1 and DYRK1A, potential targets in Alzheimer's disease. Among the imidazo[1,2-b]pyridazines synthesized, several molecules were found selective of DYRKs and CLKs, with IC50 < 100 nM. A structure-activity relationship based on the synthesis of 70 molecules, led to the identification of the structural bases of the selectivity. Products were also evaluated against parasite kinases. It was possible to identify some highly potent inhibitors on PfCLK1. The aim of second part of this thesis was to optimize the synthetic process to obtain imidazo[4,5-b]pyridines, which are close analogues of roscovitine. Derivatives had proved capable of inhibiting the formation of cysts in a cellular model of polycystic kidney disease. A seven-step synthesis has led to several grams of 3,5,7-trisubstituted imidazo[4,5-b]pyridine which is now available for evaluation in vivo.

Les protéines sont les produits finaux de la machinerie génétique. Elles jouent des rôles essentiels dans la définition de la structure, de l'intégrité et de la dynamique de la cellule afin de promouvoir les diverses transformations chimiques requises dans le métabolisme et dans la transmission des signaux biochimique. Nous savons que la doctrine centrale de la biologie moléculaire: un gène = un ARN messager = une protéine, est une simplification grossière du système biologique. En effet, plusieurs ARN messagers peuvent provenir d’un seul gène grâce à l’épissage alternatif. De plus, une protéine peut adopter plusieurs fonctions au courant de sa vie selon son état de modification post-traductionelle, sa conformation et son interaction avec d’autres protéines. La formation de complexes protéiques peut, en elle-même, être déterminée par l’état de modifications des protéines influencées par le contexte génétique, les compartiments subcellulaires, les conditions environmentales ou être intrinsèque à la croissance et la division cellulaire. Les complexes protéiques impliqués dans la régulation du cycle cellulaire sont particulièrement difficiles à disséquer car ils ne se forment qu’au cours de phases spécifiques du cycle cellulaire, ils sont fortement régulés par les modifications post-traductionnelles et peuvent se produire dans tous les compartiments subcellulaires. À ce jour, aucune méthode générale n’a été développée pour permettre une dissection fine de ces complexes macromoléculaires. L'objectif de cette thèse est d'établir et de démontrer une nouvelle stratégie pour disséquer les complexes protéines formés lors du cycle cellulaire de la levure Saccharomyces cerevisiae (S. cerevisiae). Dans cette thèse, je décris le développement et l'optimisation d'une stratégie simple de sélection basée sur un essai de complémentation de fragments protéiques en utilisant la cytosine déaminase de la levure comme sonde (PCA OyCD). En outre, je décris une série d'études de validation du PCA OyCD afin de l’utiliser pour disséquer les mécanismes d'activation des facteurs de transcription et des interactions protéine-protéines (IPPs) entre les régulateurs du cycle cellulaire. Une caractéristique clé du PCA OyCD est qu'il peut être utilisé pour détecter à la fois la formation et la dissociation des IPPs et émettre un signal détectable (la croissance des cellules) pour les deux types de sélections. J'ai appliqué le PCA OyCD pour disséquer les interactions entre SBF et MBF, deux facteurs de transcription clés régulant la transition de la phase G1 à la phase S. SBF et MBF sont deux facteurs de transcription hétérodimériques composés de deux sous-unités : une protéine qui peut lier directement l’ADN (Swi4 ou Mbp1, respectivement) et une protéine commune contenant un domain d’activation de la transcription appelée Swi6. J'ai appliqué le PCA OyCD afin de générer un mutant de Swi6 qui restreint ses activités transcriptionnelles à SBF, abolissant l’activité MBF. Nous avons isolé des souches portant des mutations dans le domaine C-terminal de Swi6, préalablement identifié comme responsable dans la formation de l’interaction avec Swi4 et Mbp1, et également important pour les activités de SBF et MBF. Nos résultats appuient un modèle où Swi6 subit un changement conformationnel lors de la liaison à Swi4 ou Mbp1. De plus, ce mutant de Swi6 a été utilisé pour disséquer le mécanisme de régulation de l’entrée de la cellule dans un nouveau cycle de division cellulaire appelé « START ». Nous avons constaté que le répresseur de SBF et MBF nommé Whi5 se lie directement au domaine C-terminal de Swi6. Finalement, j'ai appliqué le PCA OyCD afin de disséquer les complexes protéiques de la kinase cycline-dépendante de la levure nommé Cdk1. Cdk1 est la kinase essentielle qui régule la progression du cycle cellulaire et peut phosphoryler un grand nombre de substrats différents en s'associant à l'une des neuf protéines cycline régulatrice (Cln1-3, Clb1-6). Je décris une stratégie à haut débit, voir à une échelle génomique, visant à identifier les partenaires d'interaction de Cdk1 et d’y associer la cycline appropriée(s) requise(s) à l’observation d’une interaction en utilisant le PCA OyCD et des souches délétées pour chacune des cyclines. Mes résultats nous permettent d’identifier la phase(s) du cycle cellulaire où Cdk1 peut phosphoryler un substrat particulier et la fonction potentielle ou connue de Cdk1 pendant cette phase. Par exemple, nous avons identifié que l’interaction entre Cdk1 et la γ-tubuline (Tub4) est dépendante de Clb3. Ce résultat est conforme au rôle de Tub4 dans la nucléation et la croissance des faisceaux mitotiques émanant des centromères. Cette stratégie peut également être appliquée à l’étude d'autres IPPs qui sont contrôlées par des sous-unités régulatrices. / Proteins are the end-products of gene interpretative machinery. Proteins serve essential roles in defining the structure, integrity and dynamics of the cell and mediate most chemical transformations needed for everything from metabolic catalysis to signal transduction. We know that the central dogma of molecular biology, one gene = one mRNA = one protein is a gross simplification and that a protein may do different things depending on the form in which its mRNA was spliced, how and where it is post-translationally modified, what conformational state it may be in or finally, which other proteins it may interact with. Formation of protein complexes may, themselves, be governed by the states in which proteins are expressed in specific cells, cellular compartments or under specific conditions or dynamic phases such has growth or division. Protein complexes involved in mitotic cell cycle regulation are particularly challenging to dissect since they could only form during specific phases of the cell cycle, are highly regulated by post-translational modifications and can be found in any subcellular compartments. To date, no general methods have been developed to allow fine dissection of these protein complexes. The goal of this thesis was to establish and demonstrate a novel strategy for dissecting protein complexes regulating the budding yeast Saccharomyces cerevisiae (S. cerevisiae) mitotic cell cycle. In this thesis, I describe my development and optimization of a simple survival-selection Protein-fragment Complementation Assay using the prodrug-converting enzyme, yeast cytosine deaminase as reporter (OyCD PCA). I further describe, in a series of proof of principle studies, applications of the OyCD PCA to dissect the mechanism of transcriptional activation by key mitotic transcription factors and to dissect protein-protein interactions (PPIs) among regulators of the mitotic cell cycle. A key feature of the OyCD PCA is that it can be used to detect both formation and disruption of PPIs by virtue of having positive readouts for both assays. I applied the OyCD PCA in a strategy to dissect interactions between the key transcription factors of the G1/S phase: SBF and MBF. These two heterodimeric transcription factors are composed of, respectively, two distinct DNA-binding subunits named Swi4 and Mbp1 and a common transcription activation subunit called Swi6. I took advantage of the dual selection by OyCD PCA to engineer a specific mutant of Swi6 in order to demonstrate the rewiring of a transcriptional network. We isolated Swi6 with mutations found in its C-terminal domain previously identified for binding Swi4 and Mbp1 and important for SBF and MBF activities. Our results support a model where Swi6 undergoes a conformational change upon binding to Swi4 or Mbp1. In addition, this Swi6 mutant was used to dissect the regulatory mechanism that governs the entry of S. cerevisiae to a new round of cell division also known as START. We found that the SBF and MBF repressor Whi5 directly binds to the C-terminal domain of Swi6. Finally, I applied the OyCD PCA to dissect the yeast cyclin dependent kinase Cdk1-protein complexes. Cdk1 is the essential kinase that regulates cell cycle progression and can phosphorylate a large number of different substrates by teaming up with one of nine cyclin regulatory proteins (Cln1-3, Clb1-6). I describe a strategy to identify interaction partners of Cdk1 that can easily be scaled up for a genome-wide screen and associate the complexes with the appropriate cyclin(s) required for mediating the interaction using the OyCD PCA and deletion of the cyclin genes. My results allow us to postulate which phase(s) of the mitotic cell cycle Cdk1 may phosphorylate proteins and what function potential or known substrates of Cdk1 may take on during that phase(s). For example, we identified the interaction between Cdk1 and the γ-tubulin (Tub4) to be dependent upon Clb3, consistent with its role in mediating nucleation and growth of mitotic microtubule bundles on the spindle pole body. This strategy can also be applied to study other PPIs that are contingent upon accessory subunits.

Das humane Zytomegalievirus, ist ein ubiquitäres Pathogen, welches akute und persistierende Infektionen verursacht. Bei immunsupprimierten Patienten kann das Virus zu schweren Erkrankungen, wie Hepatitis, Pneumonie und bei kongenitaler Infektion außerdem zu Schädigungen des ZNS führen. HCMV blockiert die Zellproliferation durch einen Arrest am G1/S-Übergang des Zellzyklus, andererseits wird aber gleichzeitig die Expression S-Phase spezifischer Gene aktiviert. Teilweise lässt sich dies durch eine Virus vermittelte spezifische Inhibition der zellulären DNA-Repliaktion sowie durch eine massive Deregulation Zyklin-assozzierter Kinasen erklären. Zellkulturexperimente deuten darauf hin, dass die Zellzyklusalterationen wichtige Voraussetzungen für eine erfolgreiche Virusreplikation darstellen. Es ist hingegen nicht bekannt, welche Relevanz sie für die Virusvermehrung in vivo und das pathologische Erscheinungsbild im erkrankten Organismus besitzen. Diese Frage kann nur in einem Tiermodell sinnvoll angegangen werden. Aufgrund der Wirtsspezifität der Zytomegalieviren, ist man dabei auf die Verwendung der jeweiligen artspezifischen CMV angewiesen. Murines CMV (MCMV) und Ratten-CMV (RCMV) sind dabei die bislang bestuntersuchtesten Systeme. Das Anliegen dieser Arbeit war es zu prüfen, inwieweit die für HCMV beschriebenen Zellzyklusregulationen in MCMV und RCMV auf Zellkulturbasis konserviert sind. Es konnte gezeigt werden, dass sowohl RCMV als auch MCMV einen antiproliferativen Effekt auf infizierte Zellen besitzen und ebenso wie HCMV zu einem Zellzyklusarrest führen. Nager-Zytomegalieviren können Zellen auch in der G2-Phase arretieren und in dieser Zellzyklusphase auch effizient replizieren können. Die Infektion mit Nager-CMV führt außerdem auf breiter Basis zur Veränderung Zyklin-assoziierter Kinaseaktivitäten. Allen Zytomegalieviren ist die Hemmung der zellulären DNA-Synthese am G1/S-Übergang durch die Inhibition des replication licensing, dem Beginn der DNA-Synthese gemein. Durch diese vergleichende Studie wird einerseits deutlich, dass wesentliche funktionelle Schritte der Zellzyklusregulation zwischen den Zytomegalieviren konserviert sind, aber andererseits die zu Grunde liegenden molekularen Mechanismen zum Teil deutlich variieren. / Human Cytomegalovirus (HCMV) is an ubiquitous, species-specific beta-herpesvirus that, like other herpesviruses, can establish lifelong latency following primary infection. HCMV infection becomes virulent only in immunocompromised patients such as premature infants, transplant recipients and AIDS patients where the virus causes severe disease like hepatitis, pneumonitis and retinitis. Congenital infection produces birth defects, most commonly hearing loss. To develop rational-based strategies for prevention and treatment of HCMV infection, it is crucial to understand the interactions between the virus and its host cell that support the establishment and progression of the virus replicative cycle. In general, herpesviruses are known to replicate most efficiently in the absence of cellular DNA synthesis. What is more, they have evolved mechanisms to avoid the cell´s DNA replication phase by blocking cell cycle progression outside S phase. HCMV has been shown to specifically inhibit the onset of cellular DNA synthesis resulting in cells arrested with a G1 DNA content. Towards a better understanding of CMV-mediated cell cycle alterations in vivo, we tested murine and rat CMV (MCMV/RCMV), being common animal models for CMV infection, for their influence on the host cell cycle. It was found that both MCMV and RCMV exhibit a strong anti-proliferative capacity on immortalised and primary embryonic fibroblasts after lytic infection. This results from specific cell cycle blocks in G1 and G2 as demonstrated by flow cytometry analysis. The G1 arrest is at least in part caused by a specific inhibition of cellular DNA synthesis and involves both the formation and activation of the cells’ DNA replication machinery. Interestingly, and in contrast to HCMV, the replicative cycle of rodent CMVs started from G2 as efficiently as from G1. Whilst the cell cycle arrest is accompanied by a broad induction of cyclin-cdk2 and cyclin-cdk1 activity, cyclin D1-cdk4/6 activity is selectively suppressed in MCMV and RCMV infected cells. Thus, given that both rodent and human CMVs are anti-proliferative and arrest cell cycle progression we found a surprising divergence of some of the underlying mechanisms. Therefore, any question put forward to a rodent CMV model involving cell cycle regulation has to be well defined in order to extrapolate meaningful information for the human system.

Les protéines sont les produits finaux de la machinerie génétique. Elles jouent des rôles essentiels dans la définition de la structure, de l'intégrité et de la dynamique de la cellule afin de promouvoir les diverses transformations chimiques requises dans le métabolisme et dans la transmission des signaux biochimique. Nous savons que la doctrine centrale de la biologie moléculaire: un gène = un ARN messager = une protéine, est une simplification grossière du système biologique. En effet, plusieurs ARN messagers peuvent provenir d’un seul gène grâce à l’épissage alternatif. De plus, une protéine peut adopter plusieurs fonctions au courant de sa vie selon son état de modification post-traductionelle, sa conformation et son interaction avec d’autres protéines. La formation de complexes protéiques peut, en elle-même, être déterminée par l’état de modifications des protéines influencées par le contexte génétique, les compartiments subcellulaires, les conditions environmentales ou être intrinsèque à la croissance et la division cellulaire. Les complexes protéiques impliqués dans la régulation du cycle cellulaire sont particulièrement difficiles à disséquer car ils ne se forment qu’au cours de phases spécifiques du cycle cellulaire, ils sont fortement régulés par les modifications post-traductionnelles et peuvent se produire dans tous les compartiments subcellulaires. À ce jour, aucune méthode générale n’a été développée pour permettre une dissection fine de ces complexes macromoléculaires. L'objectif de cette thèse est d'établir et de démontrer une nouvelle stratégie pour disséquer les complexes protéines formés lors du cycle cellulaire de la levure Saccharomyces cerevisiae (S. cerevisiae). Dans cette thèse, je décris le développement et l'optimisation d'une stratégie simple de sélection basée sur un essai de complémentation de fragments protéiques en utilisant la cytosine déaminase de la levure comme sonde (PCA OyCD). En outre, je décris une série d'études de validation du PCA OyCD afin de l’utiliser pour disséquer les mécanismes d'activation des facteurs de transcription et des interactions protéine-protéines (IPPs) entre les régulateurs du cycle cellulaire. Une caractéristique clé du PCA OyCD est qu'il peut être utilisé pour détecter à la fois la formation et la dissociation des IPPs et émettre un signal détectable (la croissance des cellules) pour les deux types de sélections. J'ai appliqué le PCA OyCD pour disséquer les interactions entre SBF et MBF, deux facteurs de transcription clés régulant la transition de la phase G1 à la phase S. SBF et MBF sont deux facteurs de transcription hétérodimériques composés de deux sous-unités : une protéine qui peut lier directement l’ADN (Swi4 ou Mbp1, respectivement) et une protéine commune contenant un domain d’activation de la transcription appelée Swi6. J'ai appliqué le PCA OyCD afin de générer un mutant de Swi6 qui restreint ses activités transcriptionnelles à SBF, abolissant l’activité MBF. Nous avons isolé des souches portant des mutations dans le domaine C-terminal de Swi6, préalablement identifié comme responsable dans la formation de l’interaction avec Swi4 et Mbp1, et également important pour les activités de SBF et MBF. Nos résultats appuient un modèle où Swi6 subit un changement conformationnel lors de la liaison à Swi4 ou Mbp1. De plus, ce mutant de Swi6 a été utilisé pour disséquer le mécanisme de régulation de l’entrée de la cellule dans un nouveau cycle de division cellulaire appelé « START ». Nous avons constaté que le répresseur de SBF et MBF nommé Whi5 se lie directement au domaine C-terminal de Swi6. Finalement, j'ai appliqué le PCA OyCD afin de disséquer les complexes protéiques de la kinase cycline-dépendante de la levure nommé Cdk1. Cdk1 est la kinase essentielle qui régule la progression du cycle cellulaire et peut phosphoryler un grand nombre de substrats différents en s'associant à l'une des neuf protéines cycline régulatrice (Cln1-3, Clb1-6). Je décris une stratégie à haut débit, voir à une échelle génomique, visant à identifier les partenaires d'interaction de Cdk1 et d’y associer la cycline appropriée(s) requise(s) à l’observation d’une interaction en utilisant le PCA OyCD et des souches délétées pour chacune des cyclines. Mes résultats nous permettent d’identifier la phase(s) du cycle cellulaire où Cdk1 peut phosphoryler un substrat particulier et la fonction potentielle ou connue de Cdk1 pendant cette phase. Par exemple, nous avons identifié que l’interaction entre Cdk1 et la γ-tubuline (Tub4) est dépendante de Clb3. Ce résultat est conforme au rôle de Tub4 dans la nucléation et la croissance des faisceaux mitotiques émanant des centromères. Cette stratégie peut également être appliquée à l’étude d'autres IPPs qui sont contrôlées par des sous-unités régulatrices. / Proteins are the end-products of gene interpretative machinery. Proteins serve essential roles in defining the structure, integrity and dynamics of the cell and mediate most chemical transformations needed for everything from metabolic catalysis to signal transduction. We know that the central dogma of molecular biology, one gene = one mRNA = one protein is a gross simplification and that a protein may do different things depending on the form in which its mRNA was spliced, how and where it is post-translationally modified, what conformational state it may be in or finally, which other proteins it may interact with. Formation of protein complexes may, themselves, be governed by the states in which proteins are expressed in specific cells, cellular compartments or under specific conditions or dynamic phases such has growth or division. Protein complexes involved in mitotic cell cycle regulation are particularly challenging to dissect since they could only form during specific phases of the cell cycle, are highly regulated by post-translational modifications and can be found in any subcellular compartments. To date, no general methods have been developed to allow fine dissection of these protein complexes. The goal of this thesis was to establish and demonstrate a novel strategy for dissecting protein complexes regulating the budding yeast Saccharomyces cerevisiae (S. cerevisiae) mitotic cell cycle. In this thesis, I describe my development and optimization of a simple survival-selection Protein-fragment Complementation Assay using the prodrug-converting enzyme, yeast cytosine deaminase as reporter (OyCD PCA). I further describe, in a series of proof of principle studies, applications of the OyCD PCA to dissect the mechanism of transcriptional activation by key mitotic transcription factors and to dissect protein-protein interactions (PPIs) among regulators of the mitotic cell cycle. A key feature of the OyCD PCA is that it can be used to detect both formation and disruption of PPIs by virtue of having positive readouts for both assays. I applied the OyCD PCA in a strategy to dissect interactions between the key transcription factors of the G1/S phase: SBF and MBF. These two heterodimeric transcription factors are composed of, respectively, two distinct DNA-binding subunits named Swi4 and Mbp1 and a common transcription activation subunit called Swi6. I took advantage of the dual selection by OyCD PCA to engineer a specific mutant of Swi6 in order to demonstrate the rewiring of a transcriptional network. We isolated Swi6 with mutations found in its C-terminal domain previously identified for binding Swi4 and Mbp1 and important for SBF and MBF activities. Our results support a model where Swi6 undergoes a conformational change upon binding to Swi4 or Mbp1. In addition, this Swi6 mutant was used to dissect the regulatory mechanism that governs the entry of S. cerevisiae to a new round of cell division also known as START. We found that the SBF and MBF repressor Whi5 directly binds to the C-terminal domain of Swi6. Finally, I applied the OyCD PCA to dissect the yeast cyclin dependent kinase Cdk1-protein complexes. Cdk1 is the essential kinase that regulates cell cycle progression and can phosphorylate a large number of different substrates by teaming up with one of nine cyclin regulatory proteins (Cln1-3, Clb1-6). I describe a strategy to identify interaction partners of Cdk1 that can easily be scaled up for a genome-wide screen and associate the complexes with the appropriate cyclin(s) required for mediating the interaction using the OyCD PCA and deletion of the cyclin genes. My results allow us to postulate which phase(s) of the mitotic cell cycle Cdk1 may phosphorylate proteins and what function potential or known substrates of Cdk1 may take on during that phase(s). For example, we identified the interaction between Cdk1 and the γ-tubulin (Tub4) to be dependent upon Clb3, consistent with its role in mediating nucleation and growth of mitotic microtubule bundles on the spindle pole body. This strategy can also be applied to study other PPIs that are contingent upon accessory subunits.