Every fall, Northeastern America monarch butterflies (Danaus plexippus) undergo an extraordinary migration to their overwintering site in Central Mexico. During their long migration, monarch migrants use sun compass to navigate. To maintain a southward flying direction, monarch migrants compensate for the continuously changing position of the sun by providing timing information to the compass using their circadian clock. Animal circadian clocks depend primarily on a negative transcriptional feedback loop to track time. I started my work to re-construct the monarch butterfly circadian clock negative feedback loop in cell culture, focusing on homologs of Drosophila clock genes. It turned out that in addition to a Drosophila-like cryptochrome (cry1) gene, a second mammalian-like cry2 gene exists in monarch butterflies and many other insects, except in Drosophila. The two CRYs showed distinct functions in our initial assays in cultured Drosophila Schneider 2 (S2) cells. CRY2 functions as a potent transcriptional repressor, while CRY1 is light sensitive but shows no obvious transcriptional activity. The existence of two cry genes in insects changed the Drosophila-centric view of insect circadian clock. During the course of my study, our lab obtained a monarch cell line called DpN1 cells. These cells possess a light-driven clock and contributed tremendously to the research on monarch circadian clock. Using this cell line, I provided strong evidence supporting monarch CRY2’s role as a major circadian clock repressor and identified a protein-protein protective interaction cascade underlying the CRY1-mediated resetting of the molecular oscillator in DpN1 cells. I continued my work trying to understand how insect CRY2 inhibits transcription. I provided evidence suggesting the involvement of monarch PER in promoting CRY2 nuclear entry in both S2 cells and DpN1 cells. Finally, I mapped CRY2’s transcriptional inhibitory activity onto its N-terminal domain. Collectively, my research helped to change our view of insect clocks from a Drosophila-centric standpoint to a much more diverse picture. My studies also advanced the understanding of monarch circadian clock mechanism, and provides a foundation for further studies.

No description available.

No description available.

No description available.

Smith-Magenis syndrome (SMS) is a multiple congenital abnormalities intellectual disability syndrome that results from a deletion of chromosome 17p11.2 or mutation of the retinoic acid inducted one gene (RAI1). SMS is characterized by a multitude of phenotypic features including craniofacial defects, short stature, obesity, intellectual disability, self-abusive behavior, sleep disturbance and behavioral abnormalities. Interestingly, although SMS is a clearly defined syndrome with a known molecular change at its foundation, ~40% of all candidate cases sent to the Elsea lab for evaluation do not have a mutation or deletion of RAI1. We hypothesize that at least one other locus must be responsible for this Smith-Magenis-like (SMS-like) phenotype. To address this hypothesis, we first compiled a cohort of 52 subjects who had been referred to the Elsea lab for a clinical diagnosis of SMS. Once these individuals were confirmed to not have an RAI1 mutation or deletion, their phenotypes were compiled and statically analyzed to distinguish whether SMS and SMS-like cohorts are different in the prevalence of the core phenotypes of SMS such as, but not limited to, sleep disturbance, self-abusive behavior and developmental delay. SMS-like and SMS cohorts are not different in prevalence for these core features. Next, all SMS-like subjects were sent for whole genome array comparative genomic hybridization (aCGH) to identify duplications or deletions of each individual’s genome which contribute to the phenotype observed. We identified 6 pathogenic copy number variants (CNVs) in six individuals which contribute directly to the clinical phenotype, including two del(2)(q37). This study enabled us to draw relationships between SMS and other syndromes that had never been appreciated before and helped to identify pathways in which RAI1 may function. Using the data from our SMS-like study we were able to further characterize two known syndromes; Deletion 2q37 syndrome (brachydactyly mental retardation syndrome) and deletion 2q23 syndrome. With regard to deletion 2q37, syndrome we used genomic data from known and new deletion 2q37 subjects to refine the critical region to one gene: the histone deacetylase 4 gene (HDAC4). Using both clinical and molecular clues, we were able to identify one subject from our SMS-like cohort who has an insertion in HDAC4 which results in a premature stop codon. We conclude from this study that mutation of HDAC4 results in brachydactyly mental retardation syndrome. With regard to deletion 2q23 syndrome there were only five known cases in the published literature to which we were able to add two more. Using as similar approach to our del2q37 study we refined the critical region for this syndrome to one gene, the methyl binding domain 5 gene (MBD5). Using a molecular and clinical approach we were able to conclude that haploinsufficiency of MBD5 results in the core phenotypes seen in del2q23 syndrome including microcephaly, intellectual disabilities, severe speech impairment, and seizures. Using all the data generated from the three previous studies we set out to characterize the molecular function of RAI1. We hypothesize that RAI1 is a transcription factor that regulates gene expression of core genes involved in development, neurological function, and circadian rhythm. Using a ChIP-chip based approach we identified 257 transcripts we believe RAI1 regulates. Following up on these transcripts, using in vitro and in vivo methods, we have been able to conclude that RAI1 is a positive regulator of CLOCK, the master regulator of the central circadian cycle. Taken together, these studies have given us insight into the specific molecular changes that contribute to SMS and SMS-like syndromes. We have unveiled pathways and genes which are important to normal human development and behavior and identified novel functions of RAI1. These studies will provide the foundation for the future discovery of the pathways affected.

As operárias de Melipona quadrifasciata exercem diferentes funções na colônia, segundo sua faixa etária. No interior da colônia, os indivíduos mais jovens, nutrizes, ficam localizados na região mais interna e aproximam-se progressivamente das regiões periféricas à medida que envelhecem. Os indivíduos mais velhos, forrageiras, saem da colônia diariamente. Ritmos biológicos estão presentes nas operárias de todas as idades, mas apenas os ritmos das forrageiras estão bem caracterizados. Neste trabalho, o objetivo foi detectar mudanças no sistema temporal que ocorrem ao longo do desenvolvimento ontogenético de abelhas operárias. O ritmo monitorado foi aquele de consumo de O2, empregando um ciclo claro/escuro como agente sincronizador. O consumo de O2 foi registrado em nutrizes com 24 horas de vida e em forrageiras, sendo que as nutrizes foram divididas em dois grupos: Nutri_BOD, indivíduos colocados em condições constantes de temperatura e iluminação assim que emergiam do favo, e NUTRI_Col, indivíduos devolvidos à colônia por 24 horas antes dos testes. O protocolo experimental foi dividido em três fases: Fase1, forrageiras e Nutriz_BOD eram mantidos em condição constante de iluminação e temperatura durante 24 horas. Nutriz_Col era devolvida a colônia de origem durante esse mesmo intervalo; Fase2, exposição a ciclo claro/escuro 12:12h durante 48 horas; Fase3, todos os indivíduos voltavam às condições ambientais constantes. Em Fase2 e Fase3 foram feitas as medidas de consumo de O2. Os resultados da Fase2 não mostraram arrastamento, período significativo do ritmo difere de 24h. A média de consumo na fotofase apresenta diferença significativa da média de consumo na escotofase, para o grupo das forrageiras e Nutri_Col, sendo o consumo maior na fotofase. Ritmos em livre curso foram encontrados em todas as forrageiras e em 25% das nutrizes de ambos os grupos. Os resultados indicam a existência de um processo de maturação do sistema circadiano, bem como na capacidade de lidar com a luz, que representa uma situação desfavorável à sobrevivência das nutrizes. Como os dois grupos de nutrizes apresentaram ritmos endógenos, o surgimento deles independe da influência da colônia. A colônia, porém, parece modular a velocidade do estabelecimento do ritmo e sua capacidade de sincronizar-se com o ciclo claro/escuro ambiental. / The workers of the stingless bee, Melipona quadrifasciata, assume different tasks during their adult life. Newly emerged individuals - nurses - remain inside the nest, without contact with the external environment. As the workers mature, they migrate to more peripheral regions but only the oldest - foragers - leave the nest. Foragers have strong daily rhythms. Biological rhythms have already been detected in all the workers, but only the rhythms of foragers are well characterized. This work aimed to detect changes in temporal biological system during post-embryonic development. For this, the rhythm of oxygen consumption was measured and light/dark cycle was used as the synchronizing agent. Oxygen consumption was monitored in foragers and in nurses 24 hours after the emergence. The nurses were divided into two groups: 1-Nutri_BOD: the bees were kept in constant conditions of temperature and illumination. 2- NUTRI_Col: newly emerged workers were transferred back to the colony for 24 hours before the experiment. The experimental protocol was divided into three parts: 1- Forager and Nutriz_BOD were kept in constant conditions of illumination and temperature for 24 hours. Nutriz_Col were returned to the colony during the same interval. 2- Animals were exposed to a light/dark cycle, 12:12h, for 48 hours. 3- All the bees returned to constant conditions, for 4 days. Mesurements of O2 consumption were taken during the second and third parts of the experiment. There was not entrainment of the individual\'s rhythms. The period was different from 24h. In foragers and Nutri_Col there were significant differences between the average consumption in the photophase and the average consumption in the scotophase. A free running rhythm was present in all foragers and in 25% of Nutri_Col and Nutri_BOD. Results indicate the existence of a process of maturation running in the circadian system, and an increasing ability to respond to light. Presence of light is a non-permissive condition for the nurses\' survival. Endogenous rhythms are present in both groups of nurses. The presence of the colony is not necessary to the development of the rhythm. However, the colony seems to modulate the rate of to the development and the ability to synchronize with the light/dark cycle.

Fatores ambientais podem modificar o desenvolvimento dos ritmos biológicos em neonatos, como já demonstrado em ratos. Neste contexto insere-se o estudo de recém-nascidos pré-termo mantidos em unidades de cuidado neonatal. Descrevemos neste trabalho a evolução da ritmicidade no ciclo vigília/sono, atividade/repouso, temperatura do punho e alimentação na fase neonatal. Paralelamente, caracterizamos o desenvolvimento dos ritmos biológicos em ratos mantidos sob luz constante durante a lactação e a atuação da melatonina e do exercício físico nessa evolução. Em um estudo longitudinal utilizando actímetros e termistores com memória, identificamos precocemente ritmo circadiano na temperatura do punho, enquanto na atividade motora há predomínio de ritmos ultradianos, bem como no ciclo vigília/sono e no comportamento alimentar, padrão este que se modifica logo após a alta hospitalar. Em ratos sob o paradigma de luz constante, oferecemos melatonina e uma roda durante a lactação e após o desmame, encontrando modificações na emergência do ritmo circadiano em ambos os grupos. / Environmental factors can change the development of biological rhythms in neonates, as has already been demonstrated in rats. In this context is the study of preterm newborns maintained in neonatal care units. We describe in the present work the evolution of rhythmicity in sleep/wake cycle, activity/rest, wrist temperature and feeding behavior along the neonatal phase. Simultaneously, we characterize the development of biological rhythms in rats maintained under constant light during lactation and the action of melatonin and physical exercise in this evolution. Through a longitudinal study using actimeters and thermistors with memory we identified precociously a circadian rhythm in wrist temperature, while in motor activity we found a dominant ultradian rhythm, as well as, in sleep/wake cycle and feeding behavior, with changes in this pattern just after hospital discharge. In rats reared under constant light, we offered melatonin and a wheel during lactation and after weaning, finding differences in the emergency of the circadian rhythm for both groups.

INTRODUÇÃO: As relações entre sono e epilepsia são complexas e de grande importância clínica. A melhor compreensão das inúmeras lacunas que permeiam essa relação reforçaria os alicerces para o desenvolvimento de abordagens terapêuticas mais eficazes que pudessem contribuir para o bem-estar do paciente portador de epilepsia e transtornos do sono. OBJETIVO: O presente estudo teve como principal objetivo o estudo comportamental e a caracterização eletrofisiológica do ciclo vigília-sono (CVS) de ratos adultos tornados epilépticos por pilocarpina. MÉTODO: Ratos Wistar machos (N=6), tornados epilépticos após status epilepticus (SE) induzido por pilocarpina e não epilépticos (N=6) foram submetidos à cirurgia extereotáxica para implante de elétrodos bipolares nas áreas corticais (A3, somatosensorial) e hipocampais (CA1) de ambos os hemisférios. Registros contínuos de 24 horas foram submetidos à minuciosa análise visual e os seguintes parâmetros foram analisados: identificação e quantificação dos padrões eletrofisiológicos das fases do ciclo CVS; duração dos episódios oníricos ocorridos durante o sono dessincronizado (SD); padrão de ocorrência do CVS assim como do ciclo de sono (CS), e análise do volume do núcleo supraquiasmático. Os estudos da distribuição do CVS e comportamento onírico foram submetidos à Análise de Variância Multivariada - MANOVA, ao passo que as análises da ocorrência dos ciclos (CVS e CS) e volume do núcleo supraquiasmático foram submetidas ao teste da Análise de Variância (ANOVA) de dois fatores e ao teste de Mann- Whitney, respectivamente. RESULTADOS: Todas as fases do CVS foram identificadas nos ratos epilépticos. As fases da vigília e do sono eram permeadas por espículas e outros grafoelementos epileptiformes, como ondas delta espiculadas no SS e potenciais de alta frequência e baixa voltagem durante VA e o SD. Ao contrário do padrão de ocorrência típico das fases de vigília e sono em ratos não epilépticos, o grupo epiléptico apresentou diferenças significativas quanto à distribuição dessas fases em função do período. Foi observada redução significativa de VA (p<0,002) com concomitante aumento de SS (p<0,005) e vigília relaxada (VR) (p=0,021) no escuro, sendo que a VR era preponderante apenas na primeira metade da noite. Durante o dia, a quantidade de SS era maior no período da manhã (p<0,001), ao passo que houve redução do SD (p=0,002) concomitante com aumento de VA (p<0,001) no período da tarde.Os animais tornados epilépticos por pilocarpina apresentaram redução no padrão de ocorrência do CVS e CS (p=0,004 e p=0,003, respectivamente). Não houve diferença estatística na duração dos episódios oníricos, assim como no volume do núcleo supraquiasmático entre os grupos analisados (p>0,63 e p=0,47, respectivamente). CONCLUSÃO: Os animais epilépticos apresentaram alterações na arquitetura do CVS, bem como nos padrões de ciclicidade evidenciado pelas alterações de comportamento, especialmente no ciclo escuro. Esses fatos sugerem possível comprometimento estrutural e/ou funcional das circuitarias responsáveis pela geração e manutenção das fases de vigília e sono, assim como dos sistemas de temporização do CVS. Tomados em conjunto, os dados reproduziram anormalidades do CVS observadas em pacientes epilépticos, sugerindo que o presente modelo pode ser uma importante ferramenta para o estudo de mecanismos subjacentes à epilepsia do lobo temporal e sono. / INTRODUCTION: Relationships between sleep and epilepsy are complex and have great clinical importance as well. The full understanding of the various gaps present in this relationship would pave the ground for new studies that could generate new clinical approaches aiming to contribute to the well-being of the patient suffering from epilepsy and sleep disorders. OBJECTIVE: The present study aimed to carry out a behavioral analysis and electro-oscillographic characterization of the phases of sleep-wake cycle (SWC) of pilocarpine- induced epilepsy in adult rats. METHODS: Male Wistar rats that became epileptic after 60 days of pilocarpine-induced status epilepticus (SE) (N=6) and non epileptic ones (N=6) were submitted to extereotaxic surgery for implantation of bipolar electrodes in cortical (A3, somestesic) and hippocampal (CA1) areas in both hemispheres. Twenty-four hour continuous registers were submitted to detailed visual analysis and the following parameters were studied: identification and quantification of electrophysiological parameters of phases of SWC, duration of oniric episodes during desynchronized sleep (DS), the pattern of occurrence of SWC and cycles of sleep (CS). In addition, the volume of suprachiasmatic nuclei was investigated. To analyze the architecture of sleep-wake phases and oniric behavior, Multivariate Analysis of Variance-MANOVA was utilized, whereas the pattern of cycles (SWC and CS) and volume of suprachiasmatic were submitted to Analysis of Variance with 2 factors-Two-way ANOVA and Mann-Whitney test, respectively. RESULTS: In the epileptic rats all phases of SWC were identified. The phases of wake and sleep were permeated by spikes and graph elements epileptiforms such as spiked delta waves in SS and low frequency waves with high voltage during AW and SD phases. In contrast to the pattern of normal rhythmic activity evident in non-epileptic rats the epileptic group presented significant differences concerning distribution of the phases of SWC according to the period. In the dark cycle significant reduction of AW (p<0.002) was observed concomitantly with an increase of SS (p<0.005), while the relaxed wakefulness (RW) showed an increase during the first half of the night (p=0.021). In the light cycle, the SS was more prominent in the morning period (p<0.001), following by a reduction of DS (p=0.002) concomitantly with an increase of AW (p<0.001) during the afternoon in the epileptic group. The number of cycles with a regular sequence of each phase from awake to sleep (SWC) was significantly decreased (p=0.004), as was the number of cycles of sleep (p=0.003) in epileptic rats. No significant differences were found in duration of oniric episodes and volume of suprachiasmastic nuclei (p>0.63 e p=0.47, respectively) between non epileptic and epileptic groups. CONCLUSION: The data obtained revealed that after SE the epileptic animals presented some alterations in the SWC architecture as well as in the cyclicity patterns mainly in dark cycle. Such facts suggest a possible functional and/or structural impairment in the circuitry responsible for the generation of sleep and wake phases and in the SWC timing system. Taken together the data reproduced the abnormalities observed in patients, suggesting that the pilocarpine model is a suitable one to study sleep dysfunctions in temporal lobe epilepsy.

Posto que a Saúde do Trabalhador tenha se consolidado como uma política pública de saúde nas últimas décadas, mas ainda em fase de estruturação de sua rede, o tensionamento de uma discussão acerca da legitimidade da integralidade nas práticas de saúde do trabalhador, momento oportuno à aproximação com a cronobiologia, à luz da saúde coletiva. Esta dissertação visa analisar a presença da integralidade nas práticas de cuidado e de vigilância na Saúde do Trabalhador e a possíveis associações com o campo de conhecimento das propriedades temporais e interseccionalidades com a saúde coletiva e saúde pública. Foi realizada uma pesquisa documental de publicações oficiais do Ministério da Saúde brasileiro referentes à Política de Saúde do Trabalhador, fichas de notificações de acidentes e de agravos relacionados ao trabalho e pesquisa bibliográfica das revisões sistemáticas existentes sobre saúde do trabalhador e cronobiologia, indexadas nas bases de dados Foram pesquisadas as bases de dados eletrônicas (de janeiro de 1975 até dezembro de 2016): MEDLINE/PubMed, Lilacs, Scielo, Cochrane Library e Handsearch via Scirius. No desenvolvimento desta pesquisa a concepção da integralidade se afirmou como conceito entrelaçador a fim de aproximar o tema da cronobiologia com as prática em Saúde do Trabalhador - e não somente da racionalidade médica da saúde ocupacional. Os principais domínios analisados, devido a repercussões da dessincronização foram: integralidade da política de Saúde do Trabalhador brasileira; legislação e jornada de trabalho; implicações neoplásicas e cardiovasculares; classe de trabalhadores; agravos da saúde mental e associações com a desregulação circadiana; alterações no sono, estado de alerta e ocorrência de acidentes. Como resultado, verificou-se que a interação dos fatores do processo de adaptação/dessincronização ao trabalho noturno/rotativo repercute na vida social, familiar e na saúde do trabalhador. Não foi encontrado consenso quanto ao tempo de exposição para aparecimento de sinais de alterações circadianas. Tanto as fichas de notificação de acidente relacionado ao trabalho quanto documentos oficiais da Política de Saúde do Trabalhador não consideram os tópicos acima relacionados. Sugere-se a inclusão do tema nas discussões da Rede Nacional de Atenção Integral à Saúde do Trabalhador e nas práticas de cuidado e de vigilância à saúde do trabalhador, assim como a criação de linha de cuidado que atenda a assistência multiprofissional, acolhimento, vinculação e responsabilização da equipe pelo cuidado do usuário em todos os níveis. Esta pesquisa não busca cessar essa discussão, mas sim iniciá-la, a fim de suscitar outras pesquisas e práticas sobre o tema em prol da Saúde Coletiva. É importante ressaltar que o processo de desenvolvimento de uma política nacional de saúde do trabalhador deve considerar os preceitos da gestão do cuidado em saúde e estar pautado na autonomia do usuário. / Since Worker's Health has consolidated as a public health policy and structured its network in the last decades, the tension of a discussion about the legitimacy of integrality in the health practices of the worker and its appropriation with chronobiology and collective health It is necessary. This dissertation goals to analyze the presence of integrality in care and vigilance practices in Worker 's Health and associations with the field of knowledge of temporal properties and intersectionalities with collective and public health, by means of documentary and empirical analysis. The main areas analyzed were the integrality of the Brazilian Worker's Health policy, legislation; neoplastic and cardiovascular implications to work activity; specificities in the class of workers; mental health associations with circadian dysregulation; changes in sleep, in the alert state and occurrence of accidents. It was verified that the interaction of the factors of the adaptation / desynchronization process to the nocturnal / rotating work has repercussions on the social, family life and health of the worker. The main repercussions of circadian desynchronization on the worker's life were: alterations in sleep and alertness, work accidents, metabolic and cardiovascular alterations, socio-temporal disorders, stress, behavioral and mood changes, cardiometabolic diseases, eating disorders, neoplastic diseases, accidents of work and of route. No consensus was found regarding the time of exposure for signs of circadian changes. Both the work-related accident notification sheets and official Worker Health Policy documents do not consider the above topics. It is suggested that the topic be included in the discussions of the National Network of Integral Attention to Worker's Health and in Brazilian care and surveillance practices, as well as the creation of a care line that addresses multiprofessional assistance, reception, liaison and accountability of the team through user care at all levels. This research does not seek to stop this discussion, but to initiate it, in order to elicit other research and practices on the subject in favor of Collective Health. It is important to emphasize that the process of developing a national worker health policy must consider the precepts of health care management and be guided by the autonomy of the user. In the development of this research, the concept of integrality was affirmed as an interlacing concept in order to bring the subject of chronobiology closer to practices in Worker's Health - and not only the medical rationality of occupational health.

Personnaliser les traitements anticancéreux sur base moléculaire consiste à optimiser la thérapie en fonction des profils d'expression de gènes des cellules saines et tumorales du patient. Les différences au niveau moléculaire entre tissus normaux et cancéreux sont exploitées afin de maximiser l'efficacité du traitement et minimiser sa toxicité. Cette thèse propose une approche pluridisciplinaire expérimentale et mathématique ayant pur but la détermination de stratégies anticancéreuses optimales sur base moléculaire. Cette approche est, tout d'abord, mise en œuvre pour la personnalisation de la chronothérapeutique des cancers, puis pour l'optimisation de la thérapie anticancéreuse dans le cas d'une mutation de l'oncogène SRC. La plupart des fonctions physiologiques chez les mammifères présentent une rythmicité circadienne, c'est-à-dire de période environ égale à 24 h. C'est par exemple le cas de l'alternance activité-repos, de la température corporelle, et de la concentration intracellulaire d'enzymes du métabolisme. Ces rythmes ont pour conséquence une variation de la toxicité et de l'efficacité d'un grand nombre de médicaments anticancéreux selon leur heure circadienne d'administration. De récentes études soulignent la nécessité d'adapter les schémas d'injection chronomodulés au profil moléculaire du patient. La première partie de cette thèse propose une approche combinée expérimentale et modélisatrice pour personnaliser sur base moléculaire la chronothérapie. La première étape a consisté en une preuve de concept impliquant des expérimentations in vitro sur cultures de cellules humaines et des travaux de modélisation in silico. Nous nous sommes focalisés sur l'étude de l'irinotecan (CPT11), un médicament anticancéreux actuellement utilisé en clinique dans le traitement des cancers colorectaux, et présentant des rythmes de chronotoxicité et de chronoefficacité chez la souris et chez l'homme. Sa pharmacocinétique (PK) et pharmacodynamie (PD) moléculaires ont été étudiées dans la lignée de cellules d'adénocarcinome colorectal humain Caco-2. Un modèle mathématique de la PK-PD moléculaire du CPT11, à base d'équations différentielles ordinaires, a été conçu. Il a guidé l'expérimentation qui a été réalisée dans le but de d'évaluer les paramètres du modèle. L'utilisation de procédures d'optimisation, appliquées au modèle calibré aux données biologiques, a permis la conception de schéma d'exposition au CPT11 théoriquement optimaux dans le cas particulier des cellules Caco-2. Le CPT11 s'est accumulé dans les cellules Caco-2 où il a été biotransformé en son métabolite actif le SN38, sous l'action des carboxylestérases (CES). La pré-incubation des cellules avec du verapamil, un inhibiteur non spécifique des transporteurs ABC (ATP-Binding Cassette) a permis la mise en évidence du rôle de ces pompes d'efflux ABC dans le transport du CPT11. Après synchronisation des cellules par choc sérique, qui définit le temps circadien (CT) 0, des rythmes circadiens d'une période de 26 h 50 (SD 63 min) ont été mis en évidence pour l'expression de trois gènes de l'horloge circadienne: REV-ERBα, PER2, et BMAL1; et six gènes de la pharmacologie du CPT11: la cible du médicament la topoisomerase 1 (TOP1), l'enzyme d'activation CES2, l'enzyme de désactivation UGT1A1, et les quatre transporteurs ABCB1, ABCC1, ABCC2, ABCG2. Au contraire, l'expression protéique et l'activité de la TOP1 sont restées constantes. Enfin, la quantité de TOP1 liée à l'ADN en présence de CPT11, un marqueur de la PD du médicament, a été plus importante pour une exposition à CT14 (47±5.2% de la quantité totale de TOP1), que pour une exposition à CT28 (35.5±1.8%). Les paramètres du modèle mathématique de la PK-PD du CPT11 ont ensuite été estimés par une approche de bootstrap, en utilisant des résultats expérimentaux obtenus sur les cellules Caco-2, combinés aux données de la littérature. Ensuite, des algorithmes d'optimisation ont été utilisés pour concevoir les schémas d'exposition théoriquement optimaux des cellules Caco-2 à l'irinotecan. Les cellules synchronisées par un choc sérique ont été considérées comme les cellules saines et les cellules non-synchronisées ont joué le rôle de cellules cancéreuses puisque l'organisation circadienne est souvent perturbée dans les tissus tumoraux. La stratégie thérapeutique optimale a été définie comme celle qui maximise l'efficacité sur les cellules cancéreuses, sous une contrainte de toxicité maximale sur les cellules saines. Les schémas d'administration considérés ont pris la forme d'une exposition à une concentration donnée de CPT11, débutant à un CT particulier, sur une durée comprise entre 0 et 27 h. Les simulations numériques prédisent que toute dose de CPT11 devrait être optimalement administrée sur une durée de 3h40 à 7h10, débutant entre CT2h10 et CT2h30, un intervalle de temps correspondant à 1h30 à 1h50 avant le minimum des rythmes de bioactivation du CPT11 par les CES. Une interprétation clinique peut être établie en ramenant à 24 h ces résultats pour les cellules Caco-2 qui présentent une période de 27 h. Ainsi, une administration optimale du CPT11 chez le patient cancéreux résulterait en une présence du médicament dans le sang pendant 3h30 à 6h30, débutant de 1h30 à 1h40 avant le minimum des rythmes d'activité des CES chez le patient. La deuxième étape de nos travaux a consisté à adapter l'approche mise en œuvre pour optimiser l'exposition des cellules Caco-2 au CPT11, pour l'optimisation de l'administration du médicament chez la souris. Des études récentes mettent en évidence trois classes de chronotoxicité à l'irinotecan chez la souris. La classe 1, les souris de la lignée B6D2F1 femelles, présentent la pire tolérabilité au CPT11 après une administration à ZT3, et la meilleure pour une administration à ZT15, où ZT est le temps de Zeitgeber, ZT0 définissant le début de la phase de lumière. La classe 2, les souris B6D2F1 mâles, montrent une pire heure d'administration à ZT23 et une meilleure à ZT11. Enfin, la classe 3, les B6CBAF1 femelles présentent la pire tolérabilité pour une injection à ZT7, et la meilleure pour ZT15. Nous avons entrepris une approche pluridisciplinaire in vivo et in silico dont le but est la caractérisation moléculaire des trois classes de chronotoxicité, ainsi que la conception de schémas optimaux d'administration pour chacune d'elles. Le modèle mathématique mis au point pour une population de cellules en culture a été adapté pour la construction d'un modèle "corps entier" à base physiologique de la PK-PD de l'irinotecan. Un ensemble de paramètres a été estimé pour la classe 2, en utilisant deux sortes de résultats expérimentaux: d'une part, les concentrations sanguines et tissulaires (foie, colon, moelle osseuse, tumeur) du CPT11 administré aux pire et meilleure heures circadiennes de tolérabilité, et d'autre part, les variations circadiennes des protéines de la pharmacologie du médicament dans le foie et l'intestin. Le modèle ainsi calibré reproduit de façon satisfaisante les données biologiques. Cette étude est en cours pour les classes 1 et 3. Une fois les ensembles de paramètres validés pour chacune des trois classes, ils seront comparés entre eux pour mettre en évidence de possibles différences moléculaires. L'étape suivante consiste en l'application d'algorithmes d'optimisation sur le modèle corps entier pour définir des schémas d'administration chronomodulés optimaux pour chaque classe. La deuxième partie de cette thèse s'intéresse à l'étude de la tyrosine kinase SRC, dont l'expression est dérégulée dans de nombreux cancers. Des études récentes montrent un contrôle de SRC sur la voie mitochondriale de l'apoptose dans des fibroblastes de souris NIH-3T3 transfectés avec l'oncogène v-src, cette régulation étant inexistante dans les cellules parentales. L'oncogène SRC active la voie RAS / RAK / MEK1/2 / ERK1/2 qui augmente la vitesse de phosphorylation de la protéine pro-apoptotique BIK, menant ainsi à sa dégradation par le protéasome. La faible expression de BIK résultant de ce mécanisme rendrait ainsi les cellules v-src résistantes à la plupart des stress apoptotiques. Notre étude a consisté à déterminer, par une approche combinée mathématique et expérimentale, les stratégies thérapeutiques optimales lorsque les cellules NIH-3T3 parentales jouent le rôle de cellules saines, et les fibroblastes transformés celui de cellules cancéreuses. Pour cela, nous avons, tout d'abord, construit un modèle mathématique de la cinétique de BIK en conditions non-apoptotiques. L'estimation des paramètres de ce modèle, en utilisant des données expérimentales existantes, confirme que la phosphorylation de BIK sous le contrôle de SRC est inactive dans les cellules normales. L'étude exprimentale de l'évolution de BIK après le signal apoptotique que constitue une exposition à la staurosporine, démontre une relocalisation de BIK aux mitochondries, la concentration totale de la protéine restant constante durant le stress. Nous avons ensuite conçu un modèle mathématique de la voie mitochondriale de l'apoptose mettant en jeu les protéines anti-apoptotiques de type Bcl2, les protéines effectrices de type BAX, les protéines BH3-only activatrices et les BH3-only sensibilisatrices. Un ensemble de paramètres a été déterminé pour les cellules NIH-3T3 parentales, et celles transformées v-src, en comparant le modèle aux données expérimentales. Le modèle reproduit le fait expérimentalement démontré que la préincubation des cellules v-src avec un inhibiteur de la tyrosine kinase SRC, avant l'exposition à la staurosporine, annihile la résistance des fibroblastes transformés au stress apoptotique. Le modèle prédit que l'administration de l'ABT-737, un inhibiteur de protéines anti-apoptotiques, avant l'exposition à la staurosporine, ne devrait pas être entreprise dans notre systéme biologique, ce qui a été expérimentalement validé. Enfin, le modèle a été utilisé dans des procédures d'optimisation pour déterminer la stratégie thérapeutique théoriquement optimale, lorsque les cellules normales et transformées sont exposées aux mêmes médicaments. Les combinaisons médicamenteuses considérées consiste en une exposition à la staurosporine, précédée d'une exposition à des répresseurs ou activateurs d'expression des protéines de la famille Bcl2. La stratégie optimale est définie comme celle qui maximise le pourcentage de cellules apoptotiques dans les fibroblastes v-src, sous la contrainte que celui dans les cellules normales reste au-dessous d'un seuil de tolérabilité. Les simulations numériques nous permettent de conclure à une combinaison médicamenteuse optimale constituée d'une exposition à la staurosporine, précédée d'une exposition à un répresseur de l'expression de BAX (de manière à diminuer sa concentration en-dessous du seuil apoptotique dans les cellules normales, mais pas dans les cellules cancéreuses), combinée à un répresseur de BCL2 ou un inhibiteur de tyrosines kinases SRC. Cette stratégie optimale aboutit à moins d'1% de cellules apoptotiques dans les cellules saines et plus de 98% dans les cellules cancéreuses. / Personalizing anticancer treatment on molecular basis consists in optimizing the therapy according to the gene expression profiles of healthy and cancer cells of the patient. The molecular differences between normal and tumor tissues are exploited in order to maximize the treatment efficacy and minimize its toxicities. This PhD thesis presents a combined mathematical and experimental approach to optimize anticancer therapeutic strategies on molecular basis. This approach has been undertaken at first for the personalization of cancer chronotherapeutics, and secondly for the optimization of anticancer therapy in the case of mutated SRC oncogene. Most of the physiological functions in mammals display rhythms of period around 24, also called circadian rhythms. For instance, rest-activity rhythm, core temperature, or intracellular concentrations of metabolic enzymes show variations over the 24 h span. Those rhythms result in variations in the toxicity and efficacy of many anticancer drug with respect to their circadian time of administration. Recent studies highlight the need for chronomodulated injection scheme which would be tailored to the patient's molecular profile. Thus the first sections of this thesis propose a combined experimental and mathematical approach for personalizing cancer chronotherapeutics on molecular basis. The first step consisted in a proof of concept which involved in vitro experiments on human cell culture and in silico mathematical modeling. We focused on the anticancer drug irinotecan (CPT11), which is currently used in the treatment of colorectal cancer and displays chronotoxicity and chronoefficacy rhythms both in mice and in cancer patients. Its molecular pharmacokinetics (PK) and pharmacodynamics (PD) were studied in human colorectal adenocarcinoma Caco-2 cells. An ODE-based mathematical model of its PK-PD was built. It guided the design of experiments which were performed in order to estimate parameter values of the model. Optimization procedures were then applied to this data-calibrated model in order to compute theoretically optimal exposure schemes for the Caco-2 cell line. CPT11 accumulated in Caco-2 cells where it was bioactivated into SN38 under the catalytic activity of carboxylesterases (CES). The pre-incubation of cells with verapamil, a non-specific inhibitor of ATP-Binding Cassette (ABC) transporters, increased CPT11 intracellular accumulation, thus demonstrating the involvement of those efflux pumps in CPT11 transport. After cell synchronization by a seric shock which defines the circadian time (CT) 0, circadian rhythm of period 26 h 50 (SD 63 min) were observed for the expression of the three clock genes REV-ERBα, PER2, and BMAL1; and of six metabolic genes: the drug target topoisomerase 1 (TOP1), the activation enzyme CES2, the deactivation enzyme UGT1A1, and the four ABC transporters ABCB1, ABCC1, ABCC2, ABCG2. On the contrary, TOP1 proteic level and activity remained constant. The amount of DNA-bound TOP1 in the presence of CPT11 is a PD marker of the drug and displayed circadian rhythms as it was equal to 47±5.2% of the total amount of TOP1 protein after an exposure at CT14, as compared to 35.5±1.8% after an exposure at CT28. Parameters of CPT11 PK-PD model were estimated from experimental data in Caco-2 cells combined to information from literature by a bootstrap approach. Optimization procedures were then applied to the data-calibrated model in order to compute theoretically optimal exposure schemes for Caco-2 cells. Synchronized cells were considered as healthy cells and non-synchronized cells as cancer ones as the circadian organization is often disrupted in tumor tissues. The adopted therapeutics strategy consisted in maximizing DNA damage in cancer cells under the constraint that DNA damage in the healthy population remained under a tolerability threshold. We considered administration schemes in the form of a cell exposure to an initial extracellular concentration of CPT11, over 1 to 27 h, starting at a particular CT. Numerical procedures predicted that, for all considered doses of CPT11, the optimal exposure lasted from 3h40 to 7h10, and started between CT2h10 and CT2h30, a time interval corresponding to 1h30 to 1h50 before the minimum value of CES activity. A clinical interpretation can be obtained by rescaling to 24 h those results for Caco-2 cells which displayed a period of 26 h 50. Thus, an optimal administration of CPT11 to cancer patients should result in the presence of the drug in the blood during 3h30 to 6h30, starting 1h30 to 1h40 before the minimum value of CES activity in the patient. The second section of our research works has consisted in adapting the pluridisciplinary approach we have undertaken for optimizing CPT11 exposure in Caco-2 cells, for the optimization of CPT11 administration in mice. Recent experimental studies demonstrated the existence of three classes of mice regarding CPT11 chronotoxicity. Female mice of the strain B6D2F1 represented the first class and showed worst tolerability after an injection of CPT11 at ZT3 and best tolerability at ZT15, where ZT is Zeitgeber Time, ZT0 defining the beginning of the light phase. Class 2 was constituted by B6D2F1 male mice and displayed worst toxicity at ZT23 and best toxicity at ZT11. Finally, class 3 was B6CBAF1 female mice and showed worst tolerability at ZT7 and best tolerability at ZT15. Our combined in vivo and in silico approach aimed at characterizing the three chronotoxicity classes at the molecular level and at designing optimal administration schemes for each of them. The mathematical model which was built for Caco-2 cell culture was adapted to design a whole body physiologically based model of CPT11 PK-PD. Parameters were estimated for class 2 by fitting both blood and tissular pharmacokinetics data and measurements of circadian rhythms of proteins involved in CPT11 pharmacology. The calibrated model fitted the biological data. Similar parameter estimations are ongoing for classes 1 and 3. Once three parameter sets are estimated, their comparaison will allow the determination of differences in protein activities and therefore the molecular characterization of the three classes. The next step consists in applying optimization procedures to the calibrated whole body model in order to design theoretically optimal administration schemes for each class. The second project presented in this thesis focuses on the tyrosine kinase SRC, which is deregulated in numerous cancer diseases. Recent studies showed that SRC exerted a control on the mitochondrial pathway of apoptosis in NIH-3T3 mice fibroblasts which were transfected with the oncogene v-src, whereas this control was not observed in parental NIH-3T3 cells. SRC activated the RAS / RAK / MEK1/2 / ERK1/2 pathway which enhanced the phosphorylation of the pro-apoptotic enzyme BIK, thus leading to the protein degradation by the proteasome. As a result, BIK protein expression was low in v-src transformed fibroblasts which possibly contributed to the resistance of those cells to most apoptotic stresses. Our work consisted in designing optimal therapeutics strategies in which parental NIH-3T3 fibroblasts stands for healthy cells and v-src transformed ones for cancer cells. Once again, a combined experimental and mathematical approach was undertaken. First, we built a model for BIK kinetics in non-apoptotic conditions and fitted its parameters to existing experimental data. Estimated parameter values confirmed that SRC-dependent phosphorylation of BIK was inactive in normal cells. Then, we biologically and mathematically investigated BIK kinetics in response to a death signal. We showed a relocalization of BIK to the mitochondria in the first hours of staurosporine exposure, whereas BIK protein total amount remained constant during the apoptotic stress. We then conceived a mathematical model of the mitochondrial pathway of apoptosis in NIH-3T3 cells. It modeled molecular interactions between the anti-apoptotic proteins as BCL2, and the pro-apoptotic enzymes which were divided into three subgroups: the BAX-like effector proteins, the BH3-only activator proteins and the BH3-only sensitizer proteins. Parameters were estimated by fitting experimental results in normal and v-src transformed fibroblasts. The model reproduced the experimentally-demonstrated fact that pre-incubating v-src fibroblasts with an inhibitor of SRC before staurosporine exposure annihilated the cell resistance. The model predicted that an administration of ABT-737, an inhibitor of anti-apoptotic proteins, before staurosporine exposure, should not be undertaken in our particular biological systems, which was experimentally validated. Finally, optimization procedures were applied to the model in order to design optimal therapeutics strategies when both normal and cancer cells were exposed to the same drugs. Considered exposure scheme consisted in the administration of staurosporine after an exposure to activator or of proteins of the mitochondrial pathway of apoptosis. Optimal strategies were defined as the ones which maximized the percentage of apoptotic cells in the cancer cell population, under the constraint that that in the healthy population remained below a toxicity threshold. Optimization procedures allowed to conclude that the optimal drug combinaison consisted in exposing cells to staurosporine after an exposure to a chemical able to repress BAX expression such that its concentration in normal cells was below the needed amount to trigger apoptosis, combined either to a repressor of anti-apoptotic proteins, or an inhibitor of SRC. These optimal strategies led to less than 1% of apoptotic cells in healthy cells, and more than 98% in cancer cells.