O presente trabalho teve como objetivo o estudo da dinâmica folicular da ovelha Santa Inês no protocolo de nove dias de sincronização de cios, com e sem a administração do eCG, utilizando um novo dispositivo intravaginal de silicone Primer-PR® (Tecnopec Brasil). No experimento 1, dez ovelhas receberam o dispositivo vaginal no D0, retirada no D9 e aplicação de 0,4mL de d-cloprostenol. Os exames ultrassonográficos foram diários, desde D-3 da colocação do dispositivo vaginal até o D9 e a partir deste, de 12 em 12 horas até a ovulação. Concomitantemente ao experimento 1, as coletas de sangue foram realizadas entre as ovelhas que receberam o dispositivo (grupo GP, n=10) e àquelas em estado fisiológico (grupo GF, n=12). No experimento 2, nove ovelhas receberam igual tratamento do experimento 1 e 250UI de eCG na retirada do dispositivo. Os exames ultrassonográficos para o acompanhamento da dinâmica folicular ocorreram de 12 em 12 horas do D9 até a ovulação. Neste experimento, o sangue foi coletado em dias intervalados desde D-1 até D13. No experimento um, a emergência folicular ocorreu em 8,5 dias ± 16 horas após a colocação do dispositivo vaginal. As ovelhas entraram e saíram do cio, respectivamente, 45,6 ± 12,71h e 72 ± 12,96h após a retirada do mesmo e a ovulação ocorreu em 73 ± 14,38 h. O diâmetro inicial médio do folículo foi de 3,48 ± 0,28 mm e do ovulatório de 5,22 ± 0,72 mm, diferindo significativamente (p=0,006) entre o primeiro e o segundo folículo ovulatório e entre o primeiro folículo de animais com simples (5,77 ± 0,34mm) ou dupla (4,83 ± 0,18mm) ovulação (P=0,003). A taxa de crescimento no período foi 0,73 ± 0,43 (mm/dia). No experimento 2, o estro iniciou-se às 36 ± 6,4h, finalizou às 81 ± 8,46h com a ovulação em 74,3 ± 7,56 h após a retirada do dispositivo vaginal. O diâmetro inicial médio do folículo foi de 4,04 ± 0,78 mm e do ovulatório de 5,40 ± 0,59 mm, diferindo significativamente entre o primeiro folículo de animais com simples (5,91± 0,27mm) ou dupla (5,2 ± 0,22mm) ovulação (P=0,007). A taxa de crescimento folicular no período foi de 0,66 ± 0,43 mm ao dia. No experimento 1, o dispositivo Primer-PR® elevou a concentração plasmática de progesterona do grupo GP a 6,56 ± 2,32 ng/mL, enquanto o grupo fisiológico (GF) ficou em 3,8 ± 2,69 ng/mL no mesmo período. No experimento 2 a concentração plasmática de progesterona durante a permanência do dispositivo vaginal foi de 11,50 ± 3,11ng/mL. Conclui-se que foi possível realizar o acompanhamento da dinâmica folicular dentro do protocolo de nove dias de sincronização de cios na ovelha Santa Inês e que o novo dispositivo utilizado foi eficiente em sincronizar a emergência da onda folicular ovulatória. / The present study aimed the assessment of follicular dynamics in Santa Inês ewes during the nine day estrus synchronization protocol, with or without the administration of eCG, using a new intravaginal silicon device, Primer-PR® (Tecnopec Brasil). In Experiment 1, ten ewes received the vaginal device on D0, which was removed on D9 and received 0,4mL of d-cloprostenol. Ultrasound exams were performed daily, since D-3 of the device placement until D9 and every 12 hours thereafter, until ovulation. During Experiment 1, blood samples were taken from the ewes that received the device (grupo GP, n=10) and the ewes that were in physiological state (grupo GF, n=12). In Experiment 2, nine ewes received the same treatment in Experiment 1 and 250UI of eCG on the day of the device removal. Ultrasound exams for follicular development were performed every 12 hours starting on D9 until ovulation. Blood samples were taken every other day from D-1 until D13. In Experiment 1, follicular emergence occurred in 8,5 days ± 16 hours after the vaginal device placement. The ewes started and ended estrus at, respectively, 45,6 ± 12,71h and 72 ± 12,96h after device removal and ovulation was detected in 73 ± 14,38 h. The mean initial follicle diameter was 3,48 ± 0,28 mm and the mean ovulatory follicle diameter was 5,22 ± 0,72 mm, being statistically different (p=0,006) between the first and second ovulatory follicle and between the first follicle of single ovulation (5,77 ± 0,34mm) or double ovulation individuals (4,83 ± 0,18mm) (P=0,003). The follicular growth rate during this period was 0,73 ± 0,43 (mm/day). In Experiment 2, estrus started at 36 ± 6,4h, ended at 81 ± 8,46h with ovulation occurring in 74,3 ± 7,56 h after the vaginal device removal. The mean initial follicle diameter was 4,04 ± 0,78 mm and the mean diameter ovulatory follicle was 5,40 ± 0,59 mm, differing statistically between the first follicle of single ovulation individuals (5,91± 0,27mm) or double ovulation individuals (5,2 ± 0,22mm) (P=0,007). Follicular growth rate during the period was 0,66 ± 0,43 mm per day. In Experiment 1, Primer-PR® device increased plasmatic progesterone concentration in group GP to 6,56 ± 2,32 ng/mL, whereas physiological group (GF) had 3,8 ± 2,69 ng/mL during the same period. In Experiment 2 plasmatic progesterone concentration throughout the vaginal device use was 11,50 ± 3,11ng/mL. It can be concluded that it was possible to perform ultrasound evaluation of follicular dynamics during the nine day estrus synchronization protocol in Santa Inês ewes and that the new device used was efficient in synchronizing the emergence of ovulatory follicular wave.

O objetivo do presente estudo foi avaliar as características de dinâmica ovariana, aspectos do processo de involução uterina e características da hemodinâmica uterina e ovariana em éguas durante o período pós-parto (n = 10). Para isso, o conteúdo apresentado nesta dissertação está dividido em dois capítulos. No primeiro capítulo, os aspectos do processo de involução uterina e características de vascularização endometrial e mesometrial foram avaliados. Uma drástica redução (P<0,05) no diâmetro uterino foi observada durante os primeiros 7 dias pós-parto, com subsequente diminuição nas taxas de involução uterina, sendo que este processo se completou em torno de 21 dias para o corno anteriormente não gravídico (CNG) e 24 dias para o corno anteriormente gravídico (CG). Em relação às quantidades de fluido intrauterino, foi observado aumento nas quantidades entre os dias 1 e 2 pós-parto (d1 e d2), com significativa redução entre o d4 e d7, sendo que nenhuma quantidade de fluido foi observada em nenhum animal após o d16, ou após o terceiro dia pós-ovulação (D3). Nenhuma diferença (P>0,05) foi observada entre os cornos uterinos (CG e CNG) em relação às características de vascularização. Neste contexto, foi observado aumento (P<0,05) nos escores de vascularização endometrial entre d1 e d4, permanecendo semelhante até o d13, assim como aumento (P<0,05) de vascularização mesometrial entre o d1 e d2. No entanto, houve redução (P<0,05) nos escores de vascularização mesometrial do d2 ao d10. Após a ovulação, foi observado aumento de vascularização endometrial do D0 ao D5, com redução dos escores do D5 ao D11 e um novo aumento entre o D11 e o D14. De forma semelhante, o padrão de vascularização mesometrial foi similar ao observado no endométrio. No segundo capítulo, foram avaliados os aspectos de desenvolvimento e vascularização folicular durante o primeiro estro pós-parto, assim como as características de desenvolvimento e vascularização do corpo lúteo (CL) e níveis séricos de progesterona (P4) durante os 16 dias após a primeira ovulação pós-parto. Adicionalmente, os animais foram divididos em dois grupos, sendo o grupo precoce (ovulação antes do d10) ou tardia (ovulação após o d10). Em relação ao desenvolvimento folicular, nenhuma diferença foi observada entre os grupos nos dias relativos à primeira ovulação pós-parto, no entanto, quando avaliados os dias relativos ao parto, diferenças foram observadas entre os grupos. Neste contexto, o grupo precoce apresentou maior diâmetro (P<0,05) do folículo dominante (F1) em todos os dias no período pós-parto inicial (d1-d7), assim como apresentou um maior número de folículos (P<0,05) na classe > 25 mm durante os primeiros dias pós-parto (d1-d3). Adicionalmente, os animais do grupo tardia apresentaram maior número de folículos (P<0,05) na classe de 20-25 mm durante período pós-parto inicial (d4-d7). Em relação às características de vascularização, nenhuma diferença (P>0,05) foi observada entre os grupos quanto a vascularização do F1, pedículo ovariano ipsilateral ao F1, área e vascularização do CL ou níveis séricos de P4. Portanto, no período pós-parto inicial, padrões distintos nas características de vascularização uterina foram observados, porém, após a primeira ovulação pós-parto, os padrões observados assemelham-se aos de éguas não prenhes, possivelmente refletindo um retorno às características uterinas pré-gestacionais. Em relação às características de desenvolvimento folicular, não foram observadas diferenças entre os grupos em relação aos dias precedendo a primeira ovulação pós-parto, assim como as características de dinâmica folicular se assemelham aos resultados obtidos em ciclos de éguas não gestantes. No entanto, quando avaliadas as características em relação ao pós-parto imediato, diferenças no perfil de desenvolvimento folicular foram observadas entre os grupos, devido ao maior desenvolvimento folicular no grupo precoce durante a primeira semana pós-parto. Portanto, apesar do número reduzido de animais, os resultados observados sugerem que eventos cruciais ocorrem antes do parto, levando ao maior desenvolvimento folicular em alguns animais, principalmente os que apresentam rápido desenvolvimento folicular e ovulação pós-parto. / The aim of this study was to evaluate the characteristics of ovarian dynamics, aspects of the uterine involution process and characteristics of uterine and ovarian hemodynamic during the postpartum period in mares (n = 10). Thus, the content presented in this thesis is divided into two chapters. In the first chapter, the aspects of the uterine involution process and characteristics of endometrial and mesometrial vascularization were evaluated. A rapid reduction (P<0,05) in uterine diameter was observed during the first 7 days postpartum, with subsequent decrease in the rate of uterine involution, and this process was completed in about 21 days for formerly nongravid horn (CNG) and 24 days for the formerly gravid horn (CG). For intrauterine fluid accumulations, an increase (P<0,05) was observed between days 1 and 2 postpartum (d1 and d2), with a significant reduction between d4 and d7, and no amount of fluid collections was observed in the animals after d16 or after the third day post-ovulation (D3). No differences (P>0,05) was observed between the uterine horns (CG and CNG) in relation to vascular perfusion characteristics. In this context, an increase (P<0,05) in the endometrial vascularity scores was observed between d1 and d4, remaining similar until d13, as well as an increase (P<0,05) in mesometrial vascularization occurred between d1 and d2. However, a reduction (P<0,05) in scores of mesometrial vasculature was observed from d2 to d10. After ovulation, an increase of endometrial vascularization was observed from D0 to D5, with reduction in scores from D5 to D11 and a further increase between D11 and D14. Similarly, the pattern of mesometrial vascularization was similar to that observed in the endometrium. In the second chapter, follicular development and vascularization were assessed during the first postpartum estrus, as well the characteristics of development and vascular perfusion of the corpus luteum (CL) and serum progesterone (P4) during the 16 days after the first postpartum ovulation. Additionally, the animals were divided into two groups: the group early (ovulation before the d10) or late (ovulation after d10). With regard to follicular development, no difference was observed between groups in days relative to the first postpartum ovulation, however, when evaluated on the day of parturition, differences were observed between groups. In this respect, the early group had a larger diameter (P<0,05) of the dominant follicle (F1) each day in the early postpartum period (d1-d7) and had a greater number of follicles (P<0,05) in the > 25 mm class during the first days postpartum (d1-d3). Additionally, the animals in the late group showed a greater number of follicles (P<0,05) in the 20-25 mm class during early postpartum period (d4-d7). Regarding the characteristics of vascular perfusion, no difference (P>0,05) was observed between groups regarding the vascularization of F1, ipsilateral ovarian pedicle blood flow, area and vascularization of the CL or serum P4. Therefore, in the initial postpartum period, distinct patterns in the characteristics of uterine vascularization was observed, however, after the first ovulation postpartum, the observed patterns resemble those observed in non-pregnant mares, possibly reflecting a return to nonpregnant uterine characteristics. Regarding the characteristics of follicular development, no differences were observed between groups in relation to the days prior to the first postpartum ovulation, as well the characteristics of follicular dynamics are similar to the results obtained in cycles of non-pregnant mares. However, when the characteristics were evaluated concerning the immediate postpartum period, differences in the profile of follicular development were observed between groups, due to increased follicular development in the early group during the first week postpartum. Therefore, although a small number of animals, the results suggest that critical events occur before foaling, leading to increased follicle development in some animals, particularly those with rapid follicular development and postpartum ovulation.

Diante da hipótese de que micropartículas poliméricas podem atravessar a barreira epidérmica através da rota transfolicular, e baseado na evidência de que a iontoforese é um método que consegue direcionar a liberação de fármacos para os folículos pilosos, este trabalho teve como objetivo estudar in vitro a permeação cutânea do minoxidil sulfato (MXS), fármaco utilizado no tratamento da alopecia androgênica, tanto em sua forma microencapsulada como não encapsulada utilizando ou não a iontoforese, na tentativa de aumentar, controlar e direcionar a sua liberação tópica para o folículo piloso. O MXS foi primeiramente incorporado em um gel hidrofílico contendo 2,0% (m/m) do ativo e sua permeação e retenção cutânea in vitro verificada com e sem a presença de corrente elétrica durante 6 h, utilizando células de difusão e pele de orelha de porco. A quantidade de MXS retida no EC da pele foi determinada e diferenciada daquela retida nos folículos pilosos utilizando-se a técnica denominada tape stripping diferencial. Foi observado que o fluxo passivo de fármaco através da pele aumentou 150 vezes com aplicação de iontoforese anódica e que o aumento do pH da formulação de 3,5 para 5,5 restringiu 3 vezes essa permeação iontoforética e aumentou a retenção do MXS no EC e folículos pilosos. Estes resultados mostram que a iontoforese do MXS nestas condições é capaz de promover a liberação folicular do fármaco de maneira bastante significativa. Uma série de micropartículas de quitosana contendo MXS foi obtida por spray drying modificando quantidades e proporções de polímero e fármaco. O sistema selecionado para estudo foi obtido a partir de 1,50 g de polímero e 0,75 g de MXS, e apresentou alta eficiência de encapsulação (~82%), diâmetro médio igual a 3,05 µm, morfologia esférica e sem porosidades, e potencial zeta igual a + 5,87 mV. Quando incorporadas a uma formulação hidroalcoólica, essas micropartículas sofreram intumescimento, aumentando 1,5 vezes o seu diâmetro médio, mas não tiveram sua morfologia esférica alterada. Experimentos de liberação in vitro mostraram que as micropartículas obtidas foram capazes de sustentar 3,5 vezes a liberação do MXS. As micropartículas ainda restringiram a permeação passiva do fármaco, reduzindo 2 vezes seu fluxo de permeação e aumentando em 5 vezes a retenção de fármaco na região folicular, apesar das partículas em si não penetrarem a pele após administração passiva. Assim, este sistema foi capaz de promover uma liberação mais sustentada do fármaco, o que deve reduzir o número de aplicações do produto pelo paciente ao longo do dia, e garantiu a entrada de grandes quantidades do fármaco nos folículos pilosos, seu alvo de ação. A iontoforese dessas micropartículas, apesar de também não fazê-las penetrar a pele, conseguiu direcioná-las mais rapidamente para as aberturas foliculares, como mostrou os estudos de microscopia confocal de varredura a laser das micropartículas marcadas. Adicionalmente, a iontoforese aumentou 6 vezes a quantidade de MXS retida nos folículos já nas primeiras 3 h de aplicação, garantindo assim que grandes quantidades do fármaco atingissem seu local de ação mais rapidamente que quando as partículas foram aplicadas passivamente sobre a pele. / Given the hypothesis that polymeric microparticles can penetrate the skin barrier along the transfollicular route, and based on the evidence that iontophoresis is a method that can direct the delivery of drugs to the hair follicles, this work aimed to study the in vitro skin permeation of minoxidil sulfate (MXS), a drug used to treat androgenic alopecia, both in its micro-encapsulated and non-encapsulated form, using or not iontophoresis, in an attempt to increase, control and direct its topical delivery to the hair follicle. The MXS was first incorporated in a hydrophilic gel containing 2.0% (w/w) MXS and its skin permeation and retention was in vitro observed with and without the presence of electric current for 6 h, using diffusion cells and skin of porcine\'s ears. The amount of MXS retained in EC was determined and differentiated from that retained in the hair follicles using the technique called differential tape stripping. It was observed that the passive flux of drug through the skin was increased 150-fold with the application of anodal iontophoresis and, by increasing the pH of the formulation from 3.5 to 5.5, iontophoretic permeation of MXS was 3-fold restricted, whereas it increased its retention in stratum corneum and hair follicles. These results show that iontophoresis of MXS in these conditions can promote the follicular delivery of the drug quite significantly. A series of chitosan microparticles containing MXS was obtained by spray drying, modifying quantities and proportions of polymer and drug. The system selected for study was obtained from 1.50 g of polymer and 0.75 g of MXS, and showed high encapsulation efficiency (~ 82%), mean diameter of 3.05 µm, spherical morphology without porosities, and zeta potential equal to + 5.87 mV. When incorporated into a hydro ethanolic formulation, these microparticles suffered swelling, increasing 1.5 times its diameter, but their spherical morphology was not modified. Permeation experiments showed in vitro that the microparticles obtained were able to sustain 3.5 times the release of MXS. The microparticles also restricted the passive permeation of the drug, reducing 2-fold its permeation flux and increasing by 5-fold the retention of drug in the follicular region, although the microparticles themselves did not penetrate the skin after passive administration. Thus, this system was able to promote a more sustained release of the drug, which must reduce the number of product applications by the patient throughout the day, and ensured the entry of large amounts of drug in hair follicles, their target. Iontophoresis of microparticles, although not making them penetrate the skin either, was able to direct them quickly to the follicular openings, as shown by laser confocal scanning microscopy studies of the labeled microparticles. In addition, iontophoresis increased 6-fold the amount of MXS retained in the follicles within the first 3 h of application, thereby ensuring that large quantities of the drug achieved its site of action more quickly than when the particles were applied passively to the skin.

O linfoma folicular é entidade clinicamente heterogênea, com carência de marcadores prognósticos que estratifiquem grupos de risco para otimização do manejo. É relativamente raro em pacientes abaixo de 40 anos. Os aspectos clínicos e patológicos desse tipo de linfoma, nessa faixa etária, assim como o comportamento biológico, são pouco conhecidos. No presente estudo, uma série de 208 pacientes entre 19-40 anos de idade foi, retrospectivamente, avaliada quanto aos achados anatomoclínicos e moleculares. Essas variáveis foram, então, correlacionadas com seguimento e sobrevida. A mediana de idade na apresentação foi de 35 anos, com leve predomínio no sexo feminino (56%). A maioria dos casos se manifestou como doença nodal (87%). Concomitância de linfoma folicular com linfoma difuso de grandes células B foi encontrada em 7 (3%) pacientes. Estudos imuno-histoquímicos revelaram expressão de CD10 (91%), BCL6 (97%), BCL2 (95%), MUM1/IRF4 (17%) e CD23 (25%). Rearranjos envolvendo os genes BCL2 e BCL6 foram encontrados em 74% e 20%, respectivamente. A sobrevida média geral estimada dos pacientes, em que foi possível o seguimento, foi de 13 anos. Presença de anemia, elevação da desidrogenase lática, acometimento de medula óssea, índice prognóstico internacional do linfoma folicular na faixa de alto risco, padrão de \"céu estrelado\", Ki-67 >= 50% e ausência do rearranjo do gene BCL2 e presença do BCL6 relacionaram-se diretamente com pior sobrevida geral. O estadiamento de Ann Arbor III/IV e MDM2 >=20% têm fortes indícios desta associação negativa com sobrevida geral. A combinação BCL2+ e BCL6- correlacionou-se com maior sobrevida média. O grau histológico determinou redução gradual da sobrevida, com semelhanças nas curvas de sobrevida entre os graus 1, 2 e 3A. Não houve diferença significativa para as curvas de sobrevida relacionando faixa etária, persistência da zona do manto, presença de áreas difusas, fibrose, expressão de CD10, BCL6 ou CD23, padrão de trama de células foliculares dendríticas ou clonalidade para cadeia leve de imunoglobulina. Esses achados revelaram que o linfoma folicular em adultos jovens apresenta similaridades com o linfoma folicular que ocorre em adultos mais velhos, incluindo a frequência de apresentação nos diversos sítios anatômicos, grau histológico e fatores prognósticos adversos / Follicular lymphoma is a clinically heterogeneous group of disease and therefore with a need of characterization of prognostic markers to stratify risk groups and to optimize clinical management. It is relatively rare in patients younger than 40 years, and the clinicopathologic characteristics and biological behavior in this age group are poorly understood. In the current study, samples from a cohort of 208 patients between 19-40 years of age were evaluated retrospectively with respect to clinical, histologic and molecular characteristics. These findings were then correlated with the follow up and the clinical outcome. The median age at presentation was 35 years with a slight female preponderance (56%). Most of the cases presented with nodal disease (87%). Concomitant follicular lymphoma and diffuse large B-cell lymphoma was observed in 7 (3%) patients. Immunohistological studies showed the expression in the following frequency: CD10 (91%), BCL6 (97%), BCL2 (95%), MUM1/IRF4 (12%), MDM2 (17%) and CD23 (25%). BCL2 and BCL6 rearrangements were present in 74%, and 20%, respectively. The estimated overall survival of patients was 13 years (mean). The presence of anemia, elevated lactose dehydrogenase, bone marrow involvement, high-risk follicular lymphoma international prognostic index, \"starry sky\" appearance, proliferative index >= 50%, absence of BCL2 rearrangement and presence of BCL6 rearrangement correlated with adverse overall outcome. Ann Arbor stage III/IV and MDM2 >= 20% correlated with high trend toward worse overall survival. The combination BCL2+ and BCL6- was associated with better overall survival. No impact on overall survival was observed related to age, persistence of mantle zone, presence of diffuse areas, fibrosis, expression. of CD10, BCL6 or CD23, follicular dendritic cells meshwork or clonality. These findings revealed that follicular lymphoma in young adults demonstrate similarities with that of older adults, including the frequency of presentation at various anatomic sites, grade, and adverse prognostic factors

Ce travail a eu pour objectif de proposer des modèles conjoints d'intensités de processus d'événements récurrents et d'un événement terminal dépendant. Nous montrons que l'analyse séparée de ces événements conduit à des biais d'estimation importants. C'est pourquoi il est nécessaire de prendre en compte les dépendances entre les différents événements d'intérêt. Nous avons choisi de modéliser ces dépendances en introduisant des effets aléatoires (ou fragilités) et de travailler sur la structure de dépendance. Ces effets aléatoires prennent en compte les dépendances entre événements, les dépendances inter-récurrences et l'hétérogénéité non-observée. Nous avons, en premier lieu, développé un modèle conjoint à fragilités pour un type d'événement récurrent et un événement terminal dépendant en introduisant deux effets aléatoires indépendants pour prendre en compte et distinguer la dépendance inter-récurrences et celle entre les risques d'événements récurrents et terminal. Ce modèle a été ajusté pour des données de patients atteints de lymphome folliculaire où les événements d'intérêt sont les rechutes et le décès. Le second modèle développé permet de modéliser conjointement deux types d'événements récurrents et un événement terminal dépendant en introduisant deux effets aléatoires corrélés et deux paramètres de flexibilités. Ce modèle s'avère adapté pour l'analyse des risques de récidives locorégionales, de récidives métastatiques et de décès chez des patientes atteintes de cancer du sein. Nous confirmons ainsi que le décès est lié aux récidives métastatiques mais pas aux récidives locorégionales tandis que les deux types de récidives sont liés. Cependant ces approches font l'hypothèse de proportionnalité des intensités conditionnellement aux fragilités, que nous allons tenter d'assouplir. Dans un troisième travail, nous proposons de modéliser un effet potentiellement dépendant du temps des covariables en utilisant des fonctions B-Splines. / This work aimed to propose joint models for recurrent events and a dependent terminal event. We show how separate analyses of these events could lead to important biases. That is why it seems necessary to take into account the dependencies between events of interest. We choose to model these dependencies through random effects (or frailties) and work on the dependence structure. These random effects account for dependencies between events, inter-dependence recurrences and unobserved heterogeneity. We first have developed a joint frailty model for one type of recurrent events and a dependent terminal event with two independent random effects to take into account and distinguish the inter-recurrence dependence and between recurrent events and terminal event. This model was applied to follicular lymphoma patient’s data where events of interest are relapses and death. The second proposed model is used to model jointly two types of recurrent events and a dependent terminal event by introducing two correlated random effects and two flexible parameters. This model is suitable for analysis of locoregional recurrences, metastatic recurrences and death for breast cancer patients. It confirms that the death is related to metastatic recurrence but not locoregional recurrence while both types of recurrences are related. However, these approaches do the assumption of proportional intensities conditionally on frailties, which we want to relax. In a third study, we propose to model potentially time-dependent regression coefficient using B-splines functions.

Breast cancer (BC) is the most common cancer in women. It is a highly heterogeneous disease in terms of histology, therapeutic response and patient outcomes. Early and accurate detection of breast cancer is crucial as the patient prognosis varies greatly depending on the diagnosis of the disease. Patient outcomes have been linked to the presence of tumor infiltrating lymphocytes (TIL) in solid tumors. In human BC, higher TIL infiltration is associated with a better prognosis and also predicts relevant responses to pre-operative chemotherapy. TIL are primarily composed of T cells, albeit around 20% of BC patients (pts) show significant B cell infiltration, and can organize in tertiary lymphoid structures (TLS) located in the peritumoral stroma, which are associated with survival in HER2+ and triple negative BC patients. Further, these studies revealed that CD4+ follicular helper T (Tfh) cells producing CXCL13 were specifically associated with peritumoral TLS. CXCL13 is an important B cell chemoattractant whose function is to recruit B cells to the germinal center (GC) in secondary lymphoid organs and TLS, where they can mature and differentiate into memory or antibody-producing B cells. The principal objective of this thesis project was to investigate the role of CXCL13 and Tfh cells play in the development and/or maintenance of GC-like structures in BC-associated TLS.Further understanding of the factors that promote TLS formation in vivo could provide important insight for treatment decisions in BC. CXCL13 expression was originally identified as an important signal associated with TLS that was predictive for patient outcomes. We investigated factors capable of inducing CXCL13 expression in CD4+ T cells isolated from peripheral blood, using flow cytometry analysis. Treatment with TGFβ1 alone, or together with several cytokines (IL12, IL21, and in particular IL2 blockade), increased CXCL13 expression in activated CD4+ T cells. Similar to our characterization of Tfh TIL in fresh tumor tissues, these CXCL13-producing CD4+ T cells were CXCR5 negative and expressed the Tfh markers PD-1 and ICOS. The positive correlation, in treated cells and fresh TIL, between CXCL13-producing CD4+ T cells and FoxP3-expressing regulatory CD4+ T cells, and the diminished chemokine production upon depletion of the latter population, suggest a possible positive relationship between regulatory CD4+ T cells and CXCL13-producing CD4+ T cells.We then derived a GC-associated B cell gene signature for integration in our previously published Tfh cell gene signature, including CXCL13 gene. The combined GC gene signature was tested for its ability to sensitively detect BC-associated TLS using a qRT-PCR-based assay on two different cohorts, a primary BC set (n=83) and a retrospective series (n=52) of formalin-fixed paraffin-embedded (FFPE) BC tissues. These data revealed a correlation between gene signature expression and the extent of TLS scored by trained pathologists on dual-immunohistochemistry stained (CD3+CD20 for T and B cells, respectively) FFPE tissue sections. In addition, the high GC signature expression predicted better overall and disease-free survival of BC pts in our retrospective BC cohort, as well as in public microarray data.This thesis research has demonstrated that CXCL13-producing CD4+ T cells lacking CXCR5 differentiate and exert their function in IL-2-limited but TGF-β1-rich conditions. Furthermore, we developed a GC-associated gene signature able to detect TLS in BC and predict BC pts better survival. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Le lymphome folliculaire (LF) représente le 2ème lymphome par ordre de fréquence et reste considéré à l’heure actuelle comme incurable. De nombreuses questions sur le processus de lymphomagénèse sont encore non résolues et il n’existe aucun marqueur génomique ou moléculaire unanimement reconnu permettant de prédire l’évolution des patients. Nos travaux de recherche s’inscrivent dans l’objectif de mieux comprendre l’impact des altérations moléculaires identifiées dans ces tumeurs, grâce à une approche intégrative visant à combiner des données génomiques, transcriptomiques et mutationnelles. Ce travail a permis de construire un score, basé sur l’expression d’un panel de gènes, prédictif du risque de progression de la maladie. Ce score a été confirmé sur une seconde cohorte de patients, validant son utilité potentielle en pratique clinique. Par ailleurs, nos résultats suggèrent que les cellules tumorales peuvent acquérir des propriétés évocatrices d’un profil de cellules souches et associées à un pronostic particulièrement défavorable. Une 2ème partie de notre travail a porté sur les altérations touchant le gène EZH2, muté chez 25% des patients. Nous avons démontré qu’un gain génomique au niveau du locus EZH2 pouvait également avoir des conséquences sur le profil transcriptomique et un impact pronostique, soulignant l’importance de prendre en compte l’ensemble des anomalies touchant ce gène. Enfin, nous rapportons qu’un polymorphisme constitutionnel situé dans ce gène est associé au risque de progression des patients traités par un anticorps anti-CD20. L’ensemble de ces résultats apporte un éclairage nouveau sur la biologie du LF et peut contribuer à améliorer la prise en charge des patients / Follicular Lymphoma (FL) is the 2nd most frequent lymphoma subtype and is usually considered incurable with current strategies. Several questions regarding the lymphomagenesis process are still pending, and no molecular or genomic marker has been unanimously recognized yet to predict outcome. We performed an integrative analysis combining genomic, transcriptomic and mutational data in the view to bringing new highlights in the molecular alterations acting in FL. Based on gene-expression profiling data we developed a model able to predict progression-free survival in FL patients. We confirmed its predictive value in another cohort of patients, thereby allowing its potential use in clinical practice. Furthermore, our results highlight that some tumors show a stem-cell-like gene-expression profile that was associated with highly unfavorable outcome. In the second part of our work, we focused on alterations of the gene EZH2. Although mutations have been reported in 25% of FL patients, we questioned whether genomic gains at EZH2 locus could also contribute to lymphomagenesis. We showed that such gain may impact the transcriptional profile and have a prognostic significance, thus highlighting the crucial interest of determining both kinds of alterations. Finally, we report that a germ-line polyporphism in the EZH2 gene was significantly associated with progression-free survival in patients treated by anti-CD20 therapy. Taken together, these results bring new highlights on FL biology and may help to improve the clinical management of FL patients

Les LT CD4 folliculaires (TFH) forment un lignage distinct de LT contrôlant spécifiquement les lymphocytes B (LB) et la mise en place de la mémoire B. Alors que ces cellules étaient considérées comme des cellules effectrices uniquement, récemment il a été identifié, chez l'Homme et la souris, l'existence de TFH mémoires. Les TFH mémoires en tant que LT CD4 mémoires sont nécessaires, en cas de nouvelle rencontre avec l'antigène (Ag), à la mise en place d'une réponse Anticorps (Ac) rapide, efficace et de forte affinité. En effet, leur présence est corrélée à la génération et le maintien à long terme d'Ac de forte affinité lors d'infections virales. De plus, des études récentes montrent que l'analyse des TFH mémoires dans le sang périphérique peut fournir des indices pour comprendre le mode d'action des vaccins ainsi que la pathogenèse de maladies auto-immunes. Par ailleurs, dans le contexte de nombreuses maladies, de récents travaux suggèrent que l'évaluation de la fréquence et du phénotype des TFH mémoires dans le sang périphérique pourrait servir de bio-marqueur à l'établissement de diagnostique. Tout comme les cellules B mémoires qui sont subdivisées en différentes sous-populations en fonction de leur localisation et de la nature de leur Ac, différentes populations de TFH mémoires ont été récemment identifiées. Certaines se situent dans les organes lymphoïdes secondaires (OLS) drainants le site d'immunisation, de vaccination ou d'infection, ou circulantes dans les OLS non-drainants ou à proximité des plasmocytes à longue durée de vie dans la MO. Ces observations soulèvent donc la question majeure de leurs phénotypes, différences fonctionnelles et interactions face aux différentes populations de cellules B mémoires. L'objectif de mes travaux de Thèse a consisté à étudier l'hétérogénéité phénotypique et fonctionnelle présente entre ces différentes populations de TFH mémoires aux localisations diverses. De plus au vu de l'hétérogénéité existante au sein des LB mémoires (nœuds lymphatiques ou rate) et plasmocytes à longue durée de vie (MO), nous avons aussi évalué l'interaction cellulaire et fonctionnelle qui a lieu entre ces populations mémoires. Dans ce contexte, nous avons développé un modèle expérimental unique de vaccination protéique chez la souris sauvage non modifiée. / T Helper Follicular (TFH) cells form a distinct lineage of helper T cells and they specifically control B cells and memory B cell generation. While these cells were considered as effector cells, recently it was identified in Human and in mouse, the existence of memory TFH cells. Memory TFH cells, as CD4 memory T cells, are necessary in case of antigen (Ag) rechallenge to establish a fast, efficient and high affinity Antibody (Ab) response. Indeed, their presence is correlated with the generation and the long-term maintenance of high affinity Ac during viral infections. Moreover, recent studies have shown that analysis of memory TFH cells in the blood may provide clues to understanding the mode of action of vaccines and the pathogenesis of autoimmune diseases. In addition, in the context of many diseases, recent works have also suggested that the frequency and phenotype of memory TFH cells in the blood could serve as a biomarker for diagnosis. Likewise to memory B cells that are subdivided into different cell populations based on their location and the nature of their Ab, different populations of memory TFH cells have recently been identified. Some are in secondary lymphoid organs (SLO) draining the site of immunization, vaccination or infection, or circulating in the non-draining SLO or near the long-lived plasma cells (PC) in bone marrow (BM). These observations raise the question of their phenotypes, functional differences and interactions with the different subsets of memory B cells. The aim of my thesis was to study the phenotypic and functional heterogeneity between the different subsets of memory TFH cells. Due to the heterogeneity of memory B cells (draining lymph nodes or non-draining spleen) and long-lived PCs (BM), we also evaluated the cellular and functional interaction that occurs between these different memories populations. In this context, we have developed a unique experimental model of protein vaccination in unmodified wild-type mice. Specifically, after immunization, we evaluated the development of memory TFH cells and memory B cells specific for the same Ag in the draining SLO and circulating in the spleen and BM. We demonstrated that local memory TFH cells (that reside in the draining SLO) exhibit a more polarized phenotype than their circulating counterparts (present in non-draining SLO).

Un microenvironnement riche en IL-4 a été mis en évidence dans le lymphome folliculaire (FL). Cette IL-4 impliquée dans la croissance tumorale a été démontrée comme principalement secrétée par les lymphocytes T follicular helper (Tfh). Dans cette étude, nous étudions l’interaction bidirectionnelle entre les cellules fibroblastiques réticulaires (FRC) dont le réseau est augmenté dans le FL et les lymphocytes Tfh par analyse des profils d’expression génique, et co-culture in vitro des lymphocytes Tfh primaires avec des cellules fibroblastiques humaines de type FRC-like. Nous démontrons que les cellules FRC-like augmentent in vitro la croissance des sous-populations de Tfh. De plus, nous avons mis en évidence une augmentation spécifique de la sécrétion d’IL-4 par les précurseurs Tfh (pre-Tfh) co-cultivés avec les cellules FRC-like, augmentation d’IL-4 impliquant les voies Notch et ICAM1/LFA1. Cette observation est particulièrement intéressante dans le contexte du FL car les lymphocytes pre-Tfh de FL comparés à des lymphocytes pre-Tfh d’amygdales non tumorales sont caractérisés par un profil d’expression génique enrichi en gènes des voies Notch et des intégrines en plus d’une surexpression d’IL-4. En conclusion, notre description de l’interaction entre les cellules stromales et les sous-populations Tfh démontrent une modification du profil cytokinique des Tfh au stade précurseur qui pourrait expliquer le profil cytokinique retrouvé dans le microenvironnement du FL, et apporter de nouveaux éléments pour la mise en évidence de nouvelles cibles thérapeutiques. / The enrichment of the microenvironment with tumor-promoting interleukin 4 (IL4) has been implicated in the pathogenesis of follicular lymphoma (FL) and was found to be conferred mainly by T follicular helper (Tfh) cells. In this study, we investigated the bidirectional crosstalk of fibroblastic reticular cells that are expanded in FL and Tfh cells with the analysis of gene expression profiles of the respective, and an in-vitro co-culture model of human induced FRC-like cells. We demonstrated that FRC-like cells enhance the growth of Tfh cell subsets in vitro. Crucially, we uncovered a specific upregulation of IL-4 secretion by precursor Tfh (pre-Tfh) cells co-cultured with FRC-like cells. Additionally, we demonstrated that Notch and ICAM1/LFA1 are two pathways involved in IL-4 secretion following FRClike cell / Tfh cell crosstalk. This observation was particularly interesting in FL context, because FL pre-Tfh cells display an enriched Notch and integrin gene expression profile as well as an overexpression of IL-4, compared to their tonsil counterpart. Altogether, we described new interactions between stromal cells and Tfh subsets and uncovered a specific cytokine profile modification at pre-Tfh stage after contact with FRC-like cells that could explain the high levels of IL-4 in FL and provide a novel target for therapy.

Le lymphome folliculaire (FL) est le lymphome B indolent le plus fréquent. Outre des altérations géniques récurrentes, le micro-environnement tumoral, et notamment les cellules stromales lymphoides,joue un rôle majeur dans le développement de ce cancer. Cependant, la caractérisation in-situ des cellules stromales lymphoïdes chez l'homme tout comme les facteurs menant à la polarisation du stroma en un stroma protumoral ont été peu étudiés. Dans cette thèse, nous avons montré, que les cellules stromales présentes dans les ganglions et la moelle osseuse envahis des patients atteints de FL surexpriment fortement la chimiokine CXCL12. Nous avons ensuite tenté de comprendre les mécanismes responsables de cette induction. Alors que les cellules B tumorales induisent une surexpression de la chimiokine CCL2 dans les cellules stromales de façon dépendante de leur synthèse de TNF, elles ne contribuent pas à l'induction de CXCL12. A l'inverse, le principal compartiment TCD4 impliqué dans la croissance tumorale du FL, les cellules T follicular helper (TFH), augmentent l'expression de CXCL12 dans les cellules stromales. Le taux d'IL-4, la principale cytokine produite par les TFH de FL, est d'ailleurs corrélé à celui de CXCL12 au sein de ma niche tumorale du FL. De plus, à l’aide d'un modèle de différenciation en stroma lymphoide, nous avons démontré que l’IL4 induit l’expression de CXCL12 par les cellules stromale in vitro. Cette production est augmentée quand les cellules stromales sont déjà engagées vers la voie de différentiation lymphoide par un traitement TNF/LT qui favorise l'activation de STAT6 par l'IL-4. Nous avons validé ces résultats dans un modèle de formation d'organe lymphoide ectopique chez la souris. Enfin, CXCL12 induit la migration et l'adhésion au stroma des B de FL via l'activation de cascades de signalisations qui peuvent être abrogées par l'utilisation d'un inhibiteur de Btk utilisé en clinique, l'Ibrutinib. Ces résultats sont en faveur de l'intérêt de considérer la boucle IL-4/CXCL12 pour développer de nouvelles stratégies thérapeutiques dans cette pathologie constamment fatale. / Follicular lymphoma (FL) is the most frequent indolent B-cell lymphoma. Beside recurrent genetic alterations, tumor microenvironment, including lymphoid stromal cells, has been shown to play a key role in FL development. However, in situ characterization of lymphoid stromal cells is still lacking in humans and there are very few studies focusing on the factors that could lead to stroma polarization in normal and pathological context. In this thesis, we showed first that in FL, lymph node (LN) and bone marrow (BM) infiltrating stromal cells highly express the chemokine CXCL12. We next focused on the mechanisms underlying this upregulation. Interestingly, whereas malignant FL B cells induced overexpression of CCL2 in stromal cells in a TNF-dependent manner, they did not contribute to CXCL12 induction. Conversely, FL-infiltrating follicular helper T cells (FL-TFH), the key FL-supportive T-cell subset could trigger CXCL12 expression in stromal cells. IL-4 is the main FL-TFH-derived cytokine and showed a positive correlation with CXCL12 expression inside FL cell niches. Moreover, based on our in vitro lymphoid stroma differentiation model, we demonstrated that IL-4 promoted CXCL12 expression in stromal cells, together with a phenotype close to that identified in situ within FL cell niche. Such IL4 dependent CXCL12 regulation is more pronounced in stromal cells already committed towards lymphoid stromal cells by a prestimulation by TNF/LT in association with an increased STAT6 activation. These data were validated in a model of ectopic lymphoid organ formation in mice. Finally, CXCL12 induced FL B-cell migration, and adhesion to stromal cells through the activation of a signaling pathway that could be abrogated by the Btk inhibitor Ibrutinib. These data argue for considering IL-4/CXCL12 axis as a potential therapeutic target to disrupt FL protective cell niche in this still fatal malignancy.