The characterization of the normal pulmonary acinus is a necessary first step in understanding the nature of respiratory physiology and in assessing the etiology of pulmonary pathology. Murine models play a vital role in the advancement of current understanding of the dynamics of gas exchange, particle deposition and the manifestations of diseases such as COPD, Cystic Fibrosis and Asthma. With the advent of interior tomography techniques, high-resolution micro computed tomography (μCT) systems provide the ability to nondestructively assess the pulmonary acinus at micron and sub-micron resolutions. With the application of Systematic Uniform Random Sampling (SURS) principles applied to in-situ fixed, intact, ex-vivo lungs, we seek to characterize the structure of pulmonary acini in mice and study the variations across dimensions of age, location within the lung and strain phenotypes. Lungs from mice of three common research strains were perfusion fixed in-situ, and imaged using a multi-resolution μCT system (Micro XCT 400, Zeiss Inc.). Using lower resolution whole lung images, SURS methods were used for identification of region-specific acini for high-resolution imaging. Acinar morphometric metrics included diameters, lengths and branching angles for each alveolar duct and total path lengths from entrance of the acinus to the terminal alveolar sacs. In addition, other metrics such as acinar volume, alveolar surface area and surface area/volume ratios were assessed. A generation-based analysis demonstrated significant differences in acinar morphometry across young and old age groups and across the three strains. The method was successfully adapted to large animals and the data from one porcine specimen has been presented. The registration framework provides a direct technique to assess acinar deformations and provides critical physiological information about the state of alveolar ducts and individual alveoli at different phases of respiration. The techniques presented here allow us to perform direct assessment of the three-dimensional structure of the pulmonary acinus in previously unavailable detail and present a unique technique for comprehensive quantitative analysis. The acinar morphometric parameters will help develop improved mathematical and near-anatomical models that can accurately represent the geometric structure of acini, leading to improved assessment of flow dynamics in the normal lung.

<p>  </p> <p>Traditionally, the exocrine and endocrine cellular compartments of the pancreas have been considered distinct functional systems. However, recent studies suggest a more intricate relationship between the exocrine and endocrine, which may impact pancreatic growth and health. Additionally, translational control mechanisms have been linked to organ development. Our lab has shown that the mRNA translation factor eukaryotic initiation factor 5A (eIF5A), when in its post-translationally modified “hypusinated” form, plays a role in pancreas development. The hypusination of eIF5A requires the rate-limiting enzyme deoxyhypusine synthase (<em>Dhps</em>) to post-translationally modify a critical lysine residue which in turn produces the active form of eIF5A that functions in mRNA translation. When we generated animals with a deletion of <em>Dhps</em> in the pancreatic progenitor cells, there was no alteration in islet mass but significant exocrine insufficiency at embryonic (E) day 18.5 concomitant with downregulation of proteins required for exocrine pancreas development and function. Resultantly these animals died by 6 weeks-of-age. These observations prompted the question, is the phenotype caused by the absence of hypusinated eIF5A or the increase of unhypusinated eIF5A? To address this, we generated a mouse model wherein <em>Eif5a</em> is deleted in the pancreas (eIF5A∆PANC) and these mutant animals also display exocrine insufficiency. Interestingly, beta cell mass is increased at E18.5, and the mutant animals maintain euglycemia and survive up to 2 years. Ongoing analyses are interrogating the differences between these animal models with the goal to determine if mRNA translation facilitates cellular communication between the exocrine and endocrine pancreas.</p>

No description available.

Las patologías más importantes del páncreas exocrino, como la pancreatitis crónica (PC) o el cáncer de páncreas, representan un gran problema de salud pública en Europa. En la PC, el tejido acinar es substituido por complejos ductales. Además, es difícil mantener el fenotipo diferenciado de las células acinares en cultivo ya que sufren una transdiferenciación acinar-ductal.Las células madre embrionarias (ES) de ratón han sido utilizadas en la última década para generar in vitro células completamente diferenciadas de varios linajes celulares. No obstante, la capacidad de las células ES a diferenciarse a tipos celulares de origen endodérmico es muy limitada. El objetivo principal de este proyecto ha consistido en desarrollar estrategias para diferenciar células ES de ratón a células pancreáticas acinares con una elevada eficiencia mediante 1) la optimización de las condiciones de cultivo con tal de activar vías de señalización implicadas en el desarrollo/diferenciación pancreáticas; 2) la sobreexpresión de factores transcripcionales maestros utilizando vectores virales con el fin de recapitular específicamente un programa de diferenciación acinar; 3) la selección genética de las células comprometidas al linaje acinar con el objetivo de purificar las células acinares diferenciadas.Mediante la integración de estos abordajes, hemos conseguido aislar células que comparten características fenotípicas con células acinares inmaduras según la expresión de marcadores de diferenciación y la respuesta funcional a secretagogos. / Exocrine pancreatic diseases such as chronic pancreatitis (PC) or pancreatic cancer are major health issues in Europe. In CP, the acinar tissue is substituted by ductal complexes. In addition, it is difficult to maintain the differentiated phenotype of the acinar cells in culture as within few days an acinar-ductal transdifferentiation takes place.In the last decade, mouse embryonic stem cells (mES) have been used to generate differentiated cells of a variety of cellular lineages in vitro. However, the ability of ES cells to differentiate into endodermal lineages is limited. The main objective of this project has focused on the development of strategies to differentiate mES to pancreatic acinar cells with high efficiency by means of: 1) Optimization of cell culture conditions to activate signalling pathways involved in pancreatic differentiation/development; 2) the overexpression of master transcription factors involved in pancreas development using viral vectors in order to recapitulate specific acinar differentiation program; 3) the genetic selection of cells committed to the acinar linage in order to purify the differentiated cells.The integration of these different strategies allowed us to isolate cells that share phenotypic features with immature acinar cells according to the expression of differentiation markers and the functional response to acinar secretegogues.

Pancreatic acinar cells display plasticity to acquire distinct differentiation programs, being involved in diseases as chronic pancreatitis and pancreatic ductal adenocarcinoma. This work shows that acinar cells cultured in suspension undergo dedifferentiation, acquiring a pancreatic embryonic progenitor phenotype. Dedifferentiated cells turn on a senescent program, associated with activation of p53 and Ras pathways. A similar progenitor‐like phenotype with activation of senescence is present in experimental chronic pancreatitis. Acinar cultures lacking p53 overcome growth arrest and lose the pancreatic phenotype, undergoing an epithelial‐mesenchymal transition, while maintaining the expression of pre‐pancreatic endoderm and stem cell markers. In experimental acute pancreatitis, absence of p53 results in increased acinar cell proliferation and delayed regeneration. Our findings support a role for acinar cell dedifferentiation in the initiation of pancreatic diseases. A p53‐ dependent control of cell growth and epithelial differentiation constitutes a tumor suppressive mechanism that may limit PDAC development. / Las células pancreáticas acinares poseen plasticidad que les permite adquirir distintos programas de diferenciación, estando implicadas en enfermedades como la pancreatitis crónica y el adenocarcinoma ductal pancreático. En este trabajo hemos demostrado que las células acinares cultivadas en suspensión se desdiferencian, adquiriendo un fenotipo de progenitores pancreáticos embrionarios. En estas células se induce un programa de senescencia asociado con la activación de las vías de p53 y Ras. Un fenotipo similar se evidencia en modelos de pancreatitis crónica experimental. Cultivos acinares en los que se ha inactivado p53 sobrepasan el bloqueo de crecimiento y pierden el fenotipo pancreático, presentando una transición epitelio‐mesenquimal y manteniendo la expresión de marcadores de endodermo pre‐pancreático y de células madre. Durante la inducción de una pancreatitis aguda experimental, la ausencia de p53 resulta en un incremento de la proliferación acinar y en un retraso en la regeneración. Nuestros resultados demuestran que la desdiferenciación de las células acinares participa en el desarrollo de enfermedades pancreáticas. El control del crecimiento celular y de la diferenciación pancreática epitelial dependiente de p53 constituye un mecanismo de supresión tumoral que puede limitar el desarrollo del PDAC.

L’ADKP est la 5e cause de décès par cancer dans le monde occidental, et il est estimé qu’il constituera la 2e cause de mort par cancer d’ici à 2030. Le taux de survie à 5 ans est inférieur à 4%, et la médiane de survie est d’environ 6 mois. Ce pronostic sombre est dû à une pathologie asymptomatique dans les phases précoces du développement tumoral, résultant en un diagnostic tardif. Les tumeurs primaires sont constituées de structures épithéliales néoplasiques entourées par un stroma abondant empêchant l’accès des chimiothérapies aux cellules tumorales. La compréhension des mécanismes d’initiation et de progression tumorale est donc primordiale pour développer de nouvelles stratégies visant à la détection et à la prise en charge thérapeutique optimale des patients atteints d’ADKP. Le TGFbeta est une cytokine assurant de nombreuses fonctions physiologiques comme la régulation de l’immunité, la cicatrisation, le développement ou encore l’angiogenèse. Les résultats présentés dans ce manuscrit mettent en évidence que l’activation de la voie de signalisation TGFbeta perturbe la différenciation des cellules acineuses au cours du développement, et perturbe l’identité acineuse lorsqu’elle est activée dans le pancréas chez l’adulte. L’induction en parallèle de l’apoptose des cellules acineuses et d’une métaplasie acino-canalaire (ou ADM) mène à la disparition quasi-totale du tissu acineux au profit de structures canalaires typiques d’un pancréas en régénération. Lorsque l’oncogène KRASG12D est exprimé en parallèle dans le tissu pancréatique chez l’adulte, ce tissu canalaire régénératif est mis à profit par KRAS pour le développement précoce de lésions pré-néoplasiques. Mes travaux au sein de l’équipe du Dr. Laurent BARTHOLIN permettent donc de démontrer in vivo un nouveau rôle du TGFbeta dans la carcinogénèse pancréatique / PDA is the 5th cause of cancer related death in the western countries, and is estimated to move the second rank by 2020. The 5-year survival rate is less than 4%, and the median survival is around 6 months. The poor prognostic of this tumor is due to asymptomatic early phases of the disease, resulting in a late diagnosis. Primary tumors are composed by ductal neoplastic lesions embedded into a highly abundant stroma that prevents the access of chemotherapeutic drugs to the tumor cells. Thus, understanding tumor initiation and progression mechanisms is needed to develop new strategies aiming at detecting and taking care of patients in the most optimal manner. TGFbeta is a cytokine playing several physiological functions such as immunity regulation, wound healing, development or angiogenesis. Results presented in this manuscript demonstrate that activation of TGFbeta signaling disturb acinar cell differentiation during development, and disrupts acinar cell identity when activated in the adult pancreas. The simultaneous induction of acinar cell apoptosis and ADM leads to the massive loss of acinar cells and the emergence of ductal structures typical of pancreas regeneration. When the KRASG12D oncogene is expressed in combination with the activation of TGFbeta signaling, these regenerative duct structures are harnessed by KRASG12D to develop early neoplastic lesions. Thus, my work in Dr. Laurent BARTHOLIN’s team demonstrates a new function of TGFbeta in pancreatic carcinogenesis in vivo

No description available.

L'adénocarcinome canalaire pancréatique (PDAC) est une maladie agressive dans le monde. En raison du manque d'outils de détection hautement sensibles et spécifiques à un stade précoce de la maladie et de traitements peu curatifs, la plupart des patients sont diagnostiqués à un stade incurable avec une mortalité élevée. Comprendre la pathologie moléculaire, en particulier le stade précoce de cette maladie mortelle, est d’une importance capitale pour améliorer les bons pronostics de ces patients. La première et principale partie de mes travaux reposait sur une analyse de profilage génétique dans laquelle ils mettaient en évidence le rôle de la signalisation TGFβ / Activine et du récepteur ALK4 au cours du développement de la PDAC, en plus des quatre gènes oncogéniques courants (KRAS, CDKN2A, TP53 et SMAD4). ). D'autre part, INHBA (codant pour ActivinA) en tant que membre clé des ligands de la superfamille TGFβ est une cible de l'activation oncogénique de Kras dans les cellules du canal pancréatique et a été désigné comme composant potentiel de la SASP par une analyse au sécrétome de cellules subissant une sénescence induite par Kras. . Le but de mes travaux est donc d’étudier l’impact de la signalisation ActivinA lors de l’initiation de la PDAC. Dans la première partie de mon projet, j'ai démontré qu'ActivinA était un facteur SASP protecteur de la tumeur, produit par les cellules sénescentes d'ADM / PanIN, qui limitent leur prolifération et leur expansion dans les lésions hautement prolifératives via son récepteur ALK4 lors de l'initiation du PDAC. De plus, nos travaux ont révélé que les cellules sénescentes Dclk1 + limitent l'expansion des lésions prénéoplasiques et ont un impact partiel sur le composant stromal soutenu par la signalisation ActivinA. En outre, j'ai également exploré les fonctions autocrines et paracrines de la signalisation ActivinA au dernier stade de la PDAC. Nous avons constaté que le manque de signalisation ActivinA-ALK4 chez la souris entraînait une augmentation de la capacité de prolifération des cellules tumorales et une accumulation réduite de cellules stromales, ce qui pourrait aider à expliquer leurs métastases variées et un temps de survie relativement plus long. En conclusion, mon travail donne un autre aperçu de la signalisation ActivinA et Dclk1 dans l'initiation de la PDAC et leur existence dans des cellules sénescentes au sein de lésions précurseurs pancréatiques de bas grade devrait être considérée comme une étape cruciale pour développer de nouvelles stratégies diagnostiques et thérapeutiques pour les patients atteints de PDAC / Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease worldwide and remains the ranking of 7th leading cause of cancer related death in the past 5 years. Because of the lack of highly sensitive and specific detective tools in an early stage of this disease and low curative treatments, most of the patients are diagnosed in an incurable stage and end up with a high mortality. Understanding the molecular pathology especially the early stage of this lethal disease is of great importance to improve the outcome of these patients. The first and major part of my work was based on gene profiling analysis in which they emphasized the role of TGFβ/Activin signalling and ALK4-receptor during the development of PDAC in addition to the four common oncogenic genes (KRAS, CDKN2A, TP53 and SMAD4). On the other hand, INHBA(encoding for ActivinA) as a key member of TGFβ-superfamily ligands is a target of Kras oncogenic activation in pancreatic duct cell and has been pointed a potential SASP candidate component through a secretome analysis of cells undergoing Kras induced senescence. Therefore, the aim of my work is to investigate the impact of ActivinA-signalling during PDAC initiation. In the first part of my project, I demonstrated that ActivinA as a tumour-protecting SASP factor, produced by ADM/PanINs senescent cells, that limit their proliferation and expansion into highly proliferative lesions through its ALK4 receptor in the initiation of PDAC. Furthermore, our work revealed that senescent Dclk1+ cells limit the expansion of preneoplastic lesions and partly impact the stromal component which is sustained by ActivinA-signalling. Besides, I also explored the autocrine and paracrine functions of ActivinA-signalling in the late stage of PDAC. We found that lack of ActivinA-ALK4 signalling in mice leads to an increased proliferative capacity in tumor cells and a reduced accumulation of stromal cells, which might help to explain their diverse metastasis and a relative longer survival time. To conclude, my work gives another insight of ActivinA-signalling and Dclk1 in the onset of pancreatic cancer and their existence in senescent cells within low grade pancreatic precursor lesions should be considered a crucial step for developing novel diagnostic and therapeutic strategies for PDAC patients

Tot i que es coneix l’involucrament de les cèl·lules pancreàtiques acinars en patologies exocrines (pancreatitis i càncer de pàncrees), la manca de models normals basats en cèl·lules ha limitat l’estudi de les alteracions que succeeixen en el programa de diferenciació pancreàtica. Hem demostrat prèviament que les cèl·lules mare embrionàries murines, que són pluripotents, poden adquirir un fenotip acinar in vitro. Això es va aconseguir, en part, amb una combinació de senyals que provenien del cultiu de pàncrees fetals que no era, però, específic del llinatge pancreàtic. L’objectiu d’aquest treball ha estat el de desenvolupar un protocol selectiu pel llinatge acinar basat en l’activació seqüencial de vies de senyalització que recapitulin el desenvolupament pancreàtic in vivo, a través de la formació definitiva de l’endoderm, l’especificació pancreàtica i acinar i l’expansió/diferenciació de progenitors acinars. El tractament de cossos embrionaris amb Activina A va promoure l’expressió de gens d’endoderm com està prèviament descrit. El tractament subsegüent amb àcid Retinoic, FGF10 i Ciclopamina, un inhibidor de la via de Hedgehog, va resutar en la inducció dels marcadors de progenitors pancreàtics Pdx1, Ptf1a i Cpa1 però també d’aquells expressats en el llinatge pancreàtic, que van ser reduïts amb la inhibició de BMPs. Les cèl·lules van ser a continuació cultivades en Matrigel utilitzant un sistema de cultiu en 3D en presència de fol·listatina, dexametasona i KGF comportant una inducció significativa dels nivells de mRNA i proteïna de marcadors acinars i una disminució de l’expressió dels de marcadors acinars. A més, es va veure que Amyl es secretava en el medi. Aquestes dades indiquen que l’activació selectiva del programa de diferenciació acinar en cèl·lules mare embrionàries es pot dur a terme mitjançant una inducció esgraonada de vies de senyalització involucrades en el desenvolupament pancreàtic exocrí proporcionant una eina potencial per estudiar la diferenciació pancreàtica i malalties relacionades amb el pàncrees. / Despite known involvement of pancreatic acinar cells in exocrine pathologies (pancreatitis and pancreatic cancer), the lack of normal cell-based models has limited the study of the alterations that occur in the acinar differentiation program. We have previously shown that mESC (murine embryonic stem cells), which are pluripotent, can acquire an acinar phenotype in vitro. This was achieved, in part, by a combination of signals provided by the culture of foetal pancreases which was, however, no specific for the acinar lineage. The aim of this work was to develop a protocol selective for the acinar lineage based on the sequential activation of signaling pathways that recapitulate pancreatic development in vivo, through the definitive endoderm formation, the pancreatic and acinar specification and the expansion/differentiation of acinar progenitors. Treatment of embryoid bodies with Activin A enhanced the expression of endodermal genes as previously described. Subsequent treatment with Retinoic acid, FGF10 and Cyclopamine, an inhibitor of the Hedgehog pathway, resulted in the enhancement of pancreatic progenitor markers Pdx1, Ptf1a and Cpa1 but also of those expressed in the hepatic lineage, which were reduced by BMPs inhibition. Cells were further cultured in Matrigel using a 3D culture system in the presence of follistatin, dexamethasone, and KGF leading to a significant enhancement of the mRNA and protein levels of acinar markers while decreasing the expression of endocrine ones. Moreover, active Amyl was released into the medium. These data indicate that the selective activation of the acinar differentiation program in ES cells can be achieved by stepwise induction of signaling pathways involved in pancreatic exocrine development providing a potential tool for studying pancreatic differentiation and pancreas-related diseases.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.