Les capacités de cicatrisation de la gencive en font un modèle de régénération tissulaire naturelle. Ces capacités sont liées en grande partie à l’activité des fibroblastes. Composante cellulaire principale du tissu conjonctif gingival, ils sont au cœur de la régulation des réponses inflammatoires et des processus de cicatrisation. Nous avons supposé que ce tissu pouvait contenir des cellules souches, pouvant expliquer en partie, ces capacités de réparation. Au cours de cette thèse, nous avons pu mettre en évidence la présence de cellules souches mésenchymateuses aux propriétés communes avec les cellules souches adultes dérivées des crêtes neurales. Ces cellules expriment des marqueurs spécifiques des cellules souches et des crêtes neurales. Par ailleurs, elles présentent des capacités d’auto-renouvellement et de multipotence. Elles sont, en effet, capables de se différencier en adipocytes, ostéocytes et chondrocytes. Nous nous sommes plus particulièrement intéressés à la différenciation chondro/endochondrale. La culture des cellules, sous forme de sphères en suspension, a permis de mettre en évidence leurs capacités de différenciation en tissus cartilagineux et articulaires. Elles s’organisent spontanément en plusieurs types cellulaires différents, générant notamment des chondrocytes hypertrophiques et des synoviocytes selon leur localisation au sein des sphères et du milieu de culture utilisé. Le comportement de ces cellules soumises à ces conditions a permis de montrer leurs facultés à reproduire, in vitro, des processus proches de ceux retrouvés au cours du développement. Ces résultats permettent une meilleure compréhension des phénomènes de différenciation des cellules souches adultes, ouvrant ainsi de nouvelles perspectives pour des applications en thérapie cellulaire articulaire et osseuse. / The healing capacity of the gingiva makes it a model of natural tissue regeneration. These capabilities are largely related to the fibroblast activity. They are the main cellular component of the gingival connective tissue and they regulate inflammatory responses and healing process. We hypothesized that this tissue could contain stem cells, which could explain, in part, these repair capabilities. In this thesis, we were able to demonstrate the presence of mesenchymal stem cells with properties shared with the neural crest-derived adult stem cells. These cells express specific markers of stem cells and neural crest. Moreover, they do have the capacity to self-renew and multipotency. They are, indeed, able to differentiate into adipocytes, chondrocytes and osteocytes. We have particularly focused on the chondro / endochondral differentiation. When cultivated as micromasses cultures in suspension, cells were able to differentiate into cartilage and joint tissues. They organize themselves spontaneously into several different cell types, including hypertrophic chondrocytes and synoviocytes depending on their location within the micromasses and the culture medium used. The behavior of these cells under these conditions has shown their ability to replicate in vitro, close to those found during the development process. These results allow a better understanding of adult stem cells differentiation, opening new perspectives for applications in joint and bone cell therapy.

La voie de signalisation de l’inflammasome est impliquée dans plusieurs pathologies inflammatoires dont la fibrose pulmonaire et plus récemment, elle a été mise en lumière dans le développement de la fibrose hépatique. L’activation de ce complexe permet la maturation et la libération de la cytokine pro-inflammatoire IL-1β par les cellules immunitaires telles les macrophages, après reconnaissance de motifs bactériens ou de signaux de danger. L’objectif de cette thèse était de montrer les mécanismes moléculaires et cellulaires par lesquelles l’inflammasome est impliqué dans la fibrose hépatique. Par une première approche avec un hepatotoxique, le CCl4, chez la souris, nous avons observé que nos stratégies bloquantes de la signalisation de l’inflammasome ne permettent pas de faire le lien direct avec le développement de la fibrogénèse. Dans une seconde approche in vitro, nous avons montré que les fibroblastes hépatiques répondent à des médiateurs pro-inflammatoires comme l’IL-1β, le TNF-α et l’IL-8 par un déséquilibre de la balance MMP/TIMP favorable à la fibrolyse, une exacerbation de la réponse inflammatoire et la diminution de l’expression d’un marqueur d’activation des fibroblastes, l’α-SMA. Enfin, la mise en co-culture de ces fibroblastes avec différents macrophages a montré des effets similaires après activation de l’inflammasome par le LPS et les cristaux de MSU dans les cellules immunitaires, suggérant ainsi un rôle indirect de l’activation de l’inflammasome sur la réponse des fibroblastes hépatiques activés. / Inflammasome pathway is implicated in several inflammatory diseases such as pulmonary fibrosis. Nowadays, several data exist and suggest the implication of this pathway in liver fibrosis development. Once activated, the inflammasone pathway leads to the production and the release of IL-1β, a pro-inflammatory cytokine, by immune cells such as macrophages. The aim of this thesis was to describe the molecular and the cellular mechanism underlining the implication of the inflammasome pathway in liver fibrosis development. To assess this hypothesis, we have firstly inhibited inflammasome pathway in CCl4 hepatotoxicity mouse model. However, this approach did not clearly establish the implication of this pathway in liver fibrosis development. Thus in a second part, we have used an in vitro approach and demonstrate that liver fibroblasts response to pro-inflammatory mediators such as IL-1β, TNF-α and IL-8, and lead to a change in MMP/TIMP balance. The changes conduce to fibrosolysis, an exacerbation of the inflammatory response and the decrease in the expression of α-SMA, an activation marker of fibroblasts. Finally, by co-culturing the fibroblasts with different macrophages, we showed similar effects after inflammasome activation by LPS and the MSU crystals in immune cells, suggesting an indirect role of the activation of inflammasome on the response of activated liver fibroblasts.

Neurogenesis occurs in exclusive regions in the adult nervous system, the subventricular zone and dentate gyrus in the brain, and olfactory epithelium (OE) in the periphery. Cell replacement after death or injury, occurs to varying degrees in neural tissue, and is thought to be dependent upon the biological responses of stem and/or progenitor cells. Despite the progress made to identify adult OE and central nervous system (CNS) progenitors and lineage trace their progeny, our spatial and temporal understanding of embryonic OE neuroglial progenitors has been stalled by the paucity of identifiable genes able to distinguish individual candidate progenitors. In the developing CNS, radial glia serve as both neural progenitors and scaffolding for migrating neuroblasts and are identified by the expression of a select group of antigens, including nestin. Here, I show that the embryonic OE contains a novel radial glial-like progenitor (RGLP) that is not detected in adult OE. RGLPs express the radial glial antigens nestin, GLAST and RC2, but not brain lipid binding protein (BLBP), which, distinct from CNS radial glia, is instead found in olfactory ensheathing cells, a result confirmed using lineage tracing with BLBP-cre mice. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate the “CNS-specific” nestin regulatory elements, and produce spatially restricted neurons in the OE and vomeronasal organ. The dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, where GFP is found in a subpopulation of GFP+ Mash1+ neuronal progenitors, despite the fact that endogenous nestin expression is found in RGLPs throughout the OE. In vitro, embryonic OE progenitors produce three biologically distinct colony subtypes, that when generated from Nestin-cre/ZEG mice, produce GFP+ neurons, recapitulating their in vivo phenotype, and are enriched for the most neurogenic colony subtype. Neurogenesis in vitro is driven by the proliferation of nestin+ progenitors in response to FGF2. I thus provide evidence for a novel neurogenic precursor, the RGLP of the OE, that can be regulated by FGF2, and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during OE development. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

The discovery that proprotein convertase subtilisin/kexin type 9 (PCSK9) mediates degradation of low-density lipoprotein receptors (LDLR) indicates a critical role in LDL metabolism. PCSK9 is a secreted protein that binds to the epidermal growth factor-like (EGF)-A domain of LDLR and directs the receptor for degradation in lysosomes by an unknown mechanism. A gain-of-function mutation, D374Y, increases binding to LDLR EGF-A >10-fold and is associated with a severe form of hypercholesterolemia in humans. Similar to previous studies, data obtained in my project has established that PCSK9 was capable of promoting robust LDLR degradation in liver-derived cell lines; however, minimal effects on LDLR levels were detected in several lines of fibroblast cells despite normal LDLR-dependent cellular uptake of PCSK9. Importantly, a PCSK9 degradation assay showed that 125I-labeled wild-type PCSK9 was internalized and degraded equally in both hepatic and fibroblast cells, indicating dissociation of wild-type PCSK9 from recycling LDLRs in fibroblasts. Moreover, PCSK9 recycling assays confirmed that no recycling of wild-type PCSK9 to the cell surface could be detected in fibroblast cells. In contrast, more than 60% of internalized PCSK9-D374Y recycled to the cell surface in these cells, and thus had reduced ability to direct the LDLR for lysosomal degradation despite persistent binding. Co-localization studies indicated that PCSK9-D374Y trafficked to both lysosomes and recycling compartments in fibroblast cells, whereas wild-type PCSK9 exclusively trafficked to lysosomes. We conclude that two factors diminish PCSK9 activity in fibroblast cells: i) an increased dissociation from the LDLR in early endosomal compartments, and ii) a decreased ability of bound PCSK9 to direct the LDLR to lysosomes for degradation. Finally, an LDLR variant that binds to PCSK9 in a Ca2+-independent manner could partially restore wild-type PCSK9 activity, but not PCSK9-D374Y activity, in fibroblast cells.

Living organisms are made up of a multitude of individual cells that are surrounded by biomolecules and fluids. It is well known that cells are highly regulated by biochemical signals; however it is now becoming clear that cells are also influenced by the mechanical forces and mechanical properties of the local microenvironment. Extracellular forces causing cellular deformation can originate from many sources, such as fluid shear stresses arising from interstitial or blood flow, mechanical stretching during breathing or compression during muscle contraction. Cells are able to sense variations in the mechanical properties (elasticity) of their microenvironment by actively probing their surroundings by utilizing specialized proteins that are involved in sensing and transmitting mechanical information. The actin cytoskeleton and myosin-II motor proteins form a contractile (actomyosin) network inside the cell that is connected to the extracellular microenvironment through focal adhesion and integrin sites. The transmission of internal actomyosin strain to the microenvironment via focal adhesion sites generates mechanical traction forces. Importantly, cells generate traction forces in response to extracellular forces and also to actively probe the elasticity of the microenvironment. Many studies have demonstrated that extracellular forces can lead to rapid cytoskeletal remodeling, focal adhesion regulation, and intracellular signalling which can alter traction force dynamics. As well, cell migration, proliferation and stem cell fate are regulated by the ability of cells to sense the elasticity of their microenvironment through the generation of traction forces. In vitro studies have largely explored the influence of substrate elasticity and extracellular forces in isolation, however, in vivo cells are exposed to both mechanical cues simultaneously and their combined effect remains largely unexplored. Therefore, a series of experiments were performed in which cells were subjected to controlled extracellular forces as on substrates of increasing elasticity. The cellular response was quantified by measuring the resulting traction force magnitude dynamics. Two cell types were shown to increase their traction forces in response to extracellular forces only on substrates of specific elasticities. Therefore, cellular traction forces are regulated by an ability to sense and integrate at least two pieces of mechanical information - elasticity and deformation. Finally, this ability is shown to be dependent on the microtubule network and regulators of myosin-II activity.

Orientador: Valeria Helena Alves Cagnon Quitete / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T09:05:18Z (GMT). No. of bitstreams: 1 Hetzl_AmandaCia_D.pdf: 39910159 bytes, checksum: 9f69d8623d48f1fccdc9aebc181573a3 (MD5) Previous issue date: 2013 / Resumo: A senescência é fator determinante para a ocorrência de alterações morfofuncionais da próstata. O objetivo desse estudo foi caracterizar e correlacionar as interações entre os receptores dos fatores de crescimento fibroblásticos (FGFR2, FGFR7, FGFR8), fator de crescimento epidermal (EGFR), ?-actina e vimentina e os receptores androgênicos (AR), estrogênicos ? e ? (ER?, ER?) e de prolactina (PR) nos compartimentos epiteliais e estromal frente à condição de senilidade e variações hormonais. Além disso, caracterizar e correlacionar o AR, ER?, ER? e PR com os FGFs nos compartimentos epitelial e estromal de amostras humanas com adenocarcinoma de alto grau e baixo grau. 50 ratos machos senis (10 meses de idade) e 10 ratos machos jovens (4 meses de idade) foram divididos em grupos: Jovem (JOV) e Senil (SE): óleo de amendoim por 30 dias; Castrado (CAS): castração cirúrgica e química; Tamoxifeno-Letrozol (TAM): tamoxifeno e de letrozol por 30 dias; Castrado+estrógeno (REEST): tratamento similar ao CAS, e posteriormente recebeu injeções de 17?-estradiol por 30 dias; Tamoxifeno-Letrozol+Andrógeno (RETEST): após tratamento similar ao grupo TAM, os animais receberam injeções de Cipionato de Testosterona por 30 dias. Os animais foram sacrificados e amostras do lobo ventral foram coletadas e submetidas às análises de Microscopia de Luz, imunohistoquímicas, western blotting e dosagem hormonal. 30 amostras prostáticas humanas foram divididas em grupos: Adenocarcinoma de alto grau e Adenocarcinoma de baixo grau. As amostras foram submetidas às análises de Microscopia de luz e imunohistoquímicas. Após a administração estrogênica, presença de microácinos, células inflamatórias e hipertrofia do estroma prostático foram observados. A hiperandrogenização levou à recuperação epitelial. No SE houve aumento de vimentina, ER? e PR em relação ao JOV. No CAS observou-se localização diferencial da prolactina e ?-actina em relação ao SE. No RETEST, observou-se recuperação do padrão de distribuição de reatividade da ?-actina e da prolactina em relação ao SE. No REEST foi observado aumento de ER? e ER? e localização diferencial destes, somando-se a diminuição da ?-actina e vimentina em relação ao SE. No TAM foi observada diminuição de ER? e ?-actina, e aumento de prolactina no compartimento estromal, em relação ao SE. Em humanos, os FGFR2 e FGFR8 apresentaram-se aumentados no estágio inicial do câncer prostático, sugerindo essas moléculas como bons alvos terapêuticos. Pode-se concluir que o envolvimento do ER? na ativação do estroma reativo tornou o microambiente favorável à progressão do câncer, devido à potencialização do desequilíbrio estromal, e o ER? contribuíram para a inibição das lesões précancerosas em homens na senescência. Já, o desequilíbrio causado pela ablação e/ou reposição hormonal não somente alterou o feedback entre os hormônios esteróides como modificou a localização da reatividade das moléculas nos compartimentos prostáticos, provavelmente interferindo nas sinalizações autócrinas e parácrinas dos estrógenos, EGF e prolactina, apontando esses como deflagradores da formação do estroma reativo. A ablação hormonal nos animais senis levou ao aumento da reatividade dos FGFs, sugerindo interações entre os hormônios e suas vias de sinalização e o microambiente prostático senil. As vias dos FGFs podem ser ativadas também de maneira andrógeno-independente, uma vez que os FGFs apresentaram níveis de detecção aumentados mesmo diante da intensa depleção androgênica imposta pela castração / Abstract: Senescence is a determining factor for morphological and functional prostatic alterations. The objective of this study was to characterize and correlate the interactions among fibroblast growth factor receptors (FGFR2, FGFR7, FGFR8), epidermal growth factor (EGFR), ?-actin and vimentin and the androgen receptor (AR), estrogen ? and ? (ER?, ER?) and prolactin (PR) in epithelial and stromal prostatic compartments in elderly rats on hormonal variation. Also, the objective was to characterize and correlate the AR, ER?, ER? and PR with the FGFs in the human prostatic samples, presenting high grade and low grade adenocarcinoma. Fifty male rats (10 months old) and 10 young male rats (4 months old) were divided into groups: Young (JOV) and Senile Groups (SE)- peanut oil injections for 30 days, Castrated Group (CAS)- surgical and chemical castration; Tamoxifen-Letrozole Group (TAM)- tamoxifen and letrozole injections in period of 48 hours for 30 days; Castrated + estrogen Group (REEST)- surgical and chemical castration and subsequently the animals received 17?-estradiol injections for 30 days; Tamoxifen- Letrozole + Androgen Group (RETEST): after treatment similar to the TAM group, the animals received testosterone cypionate injections for 30 days. After the treatment, the animals were sacrificed and the ventral lobe samples were collected and analyzed for the Light Microscopy, immunohistochemistry and Western blotting. Thirty human prostatic samples were collected from elderly men and divided into High-grade and Low-grade Adenocarcinoma Groups. The samples were submitted to light microscopy and immunohistochemical analyses. After estrogen administration, epithelial atrophy, microacini, inflammatory cells and stromal hypertrophy were observed. The hyperandrogenization led to the recovery of epithelium. The vimentin, ER? and PR increase was verified in the SE group in relation to JOV one. Differential localization of PR and ?-actin was seen in the CAS group in relation to SE one. Recovery of the distribution pattern of ?-actin and prolactin reactivities was observed in the RETEST group in relation to SE. In the REEST group, it was observed the ER? and ER? increase and differential localization of these receptors, and the ?-actin and vimentin decrease in relation to SE. In the group TAM, it was observed the ER? and ?-actin decrease and the prolactin increase in the stromal compartment in relation to SE group. Regarding to human samples, increased FGFR2 and FGFR8 were observed in the early stages of prostate cancer, suggesting these molecules as good therapeutic targets. Thus, it can be concluded that the involvement of ER? in activation of reactive stromal led to the favorable microenvironment to cancer progression considering the strong stromal imbalance, and the ER? contributed to the inhibition of precancerous lesions in elderly men. The imbalance caused by ablation and/or hormone therapy not only changed the feedback between steroid hormones but also changed the reactivity localization of molecules in prostatic compartments, probably interfering in the autocrine and paracrine signaling of estrogen, prolactin and EGF, and pointing these molecules as possible triggers of the formation of reactive stroma. The present results demonstrated that hormone ablation in senile rats led to increased reactivities of the FGFs, suggesting interactions among hormones and their signaling pathways and senile prostatic microenvironment. Furthermore, it can be concluded that the ways of FGFs can be activated also androgen-independent manner, considering that the FGFs showed increased levels in the severe androgen depletion characterized by castration / Doutorado / Anatomia / Doutora em Biologia Celular e Estrutural

Transforming growth factor-β (TGF-β) superfamily of cytokines comprises of several members, which can broadly be sub-divided into three classes [TGF-βs, Activin/Nodal, and Bone morphogenetic proteins (BMPs)]. Most members of this family play critical roles during embryo development differentiation and regulation of homeostasis. In mammals there are three TGF-β isoforms, TGF-β1, 2 and 3. All the three TGF-β isoforms have important roles in embryo development as revealed by mouse knock-out models. TGF-β has also been associated with several pathological conditions such as inflammation, Fibrosis, and cancer. In cancers, TGF-β plays both tumor suppressive and tumor promoting roles depending upon the context. TGF-β has growth inhibitory effect on epithelial cells which is essential to maintain tissue homeostasis. TGF-β induces the expression of several cyclin dependent kinase inhibitors such as p21Cip1, p15Ink4b while down-regulating the expression of cMYC in the epithelial cells. In lieu of its tumor suppressive role, several cancers harbor mutations in the components of the TGF-β signaling axis such as receptors and effector molecules called SMADs. Interestingly various cancers also show hyper activation of TGF-β signaling. It has been suggested that cancer cells become unresponsive to the growth inhibitory effects of TGF-β by losing the expression of p21Cip1, and p15INK4b. Oncogenic transformation of cancer cells can override the growth inhibitory effects of TGF-β. While the loss of growth inhibitory effects by TGF-β are seen in the tumor cells, several tumor promoting actions are also observed in these cells such as induction of EMT. TGF-β activates mesenchymal cells leading to the formation of a reactive stroma in tumors and TGF-β suppresses almost all types of cells of the immune system causing a local immune-suppressive environment. TGF-β also recruits mesenchymal stem cells into the stroma which secrete several cytokines. The sum total of all these effects is pro-angiogenic, pro-infiltrative and pro-metastatic. In the canonical TGF-β signaling pathway, ligands bind to the hetero-tetrameric receptor complex of TGFβR1 and TGFβR2 leading to activation of the TGFβR1 by TGFβR2. Activated TGFβR1 then phosphorylates and activates R-SMAD molecules (SMAD2, SMAD3) which complexes with the co-SMAD (SMAD4) and translocate into the nucleus to effect transcriptional changes. Non-canonical TGF-β signals are many and almost all the known signaling pathways like MAPK, WNT, PI3K-AKT, NOTCH, Integrin, Hedgehog, Hippo etc. have been shown to be activated by TGF-β in different contexts. The canonical TGF-β/SMAD pathway has been shown to be essential for both tumor suppressive and tumor promoting actions of TGF-β. Although the non-canonical signalling pathways have been shown to be context dependent, the exact mechanisms have not been elucidated. In previous studies, we have shown the importance of non-canonical TGF-β signaling in normal vs. carcinoma cells. However, there has been no study that addressed the differential effects of TGF-β on cells of connective tissue origin. To throw light on such questions we have undertaken this study with the following objectives: 1) Whole genome expression profiling of TGF-β targets in normal fibroblasts, transformed fibroblasts and sarcoma cells 2) Elucidation of non-canonical signaling pathways differentially regulated by TGF-β 3) Identification and characterization of novel TGF-β targets The cell-lines chosen for the study are: 1) hFhTERT (human foreskin fibroblasts immortalized with human terminal telomerase); 2) hFhTERT-LTgRAS (hFhTERT transformed with SV40 large T antigen and activated RAS); and 3) HT1080 (fibrosarcoma). We performed whole genome expression profiling using 4×44K Agilent Human Whole Genome Oligonucleotide Arrays. Analysis of the microarray results revealed that TGF-β regulated a large number of genes in all the three cell-lines but few targets were found to be commonly regulated between any two or all the three cell-lines. 5291 genes were differentially regulated by TGF-β between hFhTERT and hFhTERT-LTgRAS and 2274 genes were differentially regulated by TGF-β between hFhTERT and HT1080 cells. Gene set enrichment analysis (GSEA) of these two gene lists revealed enrichment of similar gene sets in the HT1080 and hFhTERT-LTgRAS cells compared to the hFhTERT cells. MAPK signaling pathway components were enriched in the hFhTERT cells. Closer inspection revealed that several upstream regulators of the MAPK pathway were in fact down-regulated by TGF-β in these cells compared to both hFhTERT-LTgRAS and HT1080 cells suggesting a depression of the MAPK pathway by TGF-β in the hFhTERT cells. Assessment of the phosphorylation status of ERK1/2 and p38 MAPK proteins after TGF-β treatment showed that both ERK1/2 and p38 MAPK pathways were not activated in response to TGF-β in the hFhTERT cells. On the other hand in hFhTERT-LTgRAS and HT1080 cells, both ERK1/2 and p38 MAPK were activated post TGF-β treatment. Activity of the AP1 and SMAD responsive p3TP-lux reporter plasmid was dependent on only the SMAD pathway in hFhTERT cells while in the hFhTERT-LTgRAS and HT1080 cells both MAPK and SMAD pathway were found to regulate the expression of the p3TP-lux reporter. This suggests activation of MAPK and SMAD pathways in transformed and tumor cells while there is no activation of MAPK in normal cells of mesenchymal origin. Components of the WNT signaling pathway such as WNT ligands WNT4, and WNT11, frizzled receptors, FZD4, FZD8 and FZD9, regulators like SFRP1, SFRP2, AXIN2 and several targets of the WNT-β-catenin pathway were regulated by TGF-β in the hFhTERT cells but not in the hFhTERT-LTgRAS and HT1080 cells suggesting a positive regulation of the pathway by TGF-β in the hFhTERT cells. Indeed, TGF-β induced the activity of the WNT responsive reporter, pTOP-FLASH in the hFhTERT cells but not in the hFhTERTLTgRAS and HT1080 cells. WNT4 and WNT11 were two of the novel targets of TGF-β identified in hFhTERT cells. Further experiments suggested that TGF-β conferred regulation of these genes was specific to the fibroblast cells since induction of these genes by TGF-β was not observed in any of the cancer cell lines or in HaCaT cells. Some recent studies have demonstrated remodelling of cytoskeleton in epithelial cells by the non-canonical WNT ligands such as WNT5a, WNT4 and WNT11. WNT4 has also been shown to be required for the maintenance of α-SMA levels in smooth muscle cells. In this study we have shown that WNT4 can induce α-SMA in the hFhTERT cells leading to their activation. TGF-β conferred activation of these cells was also found to be dependent on the presence of WNT4. In brief, our study identified differentially activated pathways by TGF-β in immortal and transformed fibroblasts. WNT4 was identified as a crucial molecule required for the TGF-β conferred activation of fibroblasts.

[EN] Heart failure (HF) constitutes a major public health problem worldwide. Operationally it is defined as a clinical syndrome characterized by the marked and progressive inability of the ventricles to fill and generate adequate cardiac output to meet the demands of cellular metabolism that may have significant variability in its etiology and it is the final common pathway of various cardiac pathologies. Much attention has been paid to the understanding of the arrhythmogenic mechanisms induced by the structural, electrical, and metabolic remodeling of the failing heart. Due to the complexity of the electrophysiological changes that may occur during heart failure, the scientific literature is complex and sometimes equivocal. Nevertheless, a number of common features of failing hearts have been documented. At the cellular level, prolongation of the action potential (AP) involving ion channel remodeling and alterations in calcium handling have been established as the hallmark characteristics of myocytes isolated from failing hearts. At the tissue level, intercellular uncoupling and fibrosis are identified as major arrhythmogenic factors. In this Thesis a computational model for cellular heart failure was proposed using a modified version of Grandi et al. model for human ventricular action potential that incorporates the formulation of the late sodium current (INaL) in order to study the arrhythmogenic processes due to failing phenotype. Experimental data from several sources were used to validate the model. Due to extensive literature in the subject a sensitivity analysis was performed to assess the influence of main ionic currents and parameters upon most related biomarkers. In addition, multiscale simulations were carried out to characterize this pathology (transmural cardiac fibres and tissues). The proposed model for the human INaL and the electrophysiological remodeling of myocytes from failing hearts accurately reproduce experimental observations. An enhanced INaL appears to be an important contributor to the electrophysiological phenotype and to the dysregulation of calcium homeostasis of failing myocytes. Our strand simulation results illustrate how the presence of M cells and heterogeneous electrophysiological remodeling in the human failing ventricle modulate the dispersion of action potential duration (APD) and repolarization time (RT). Conduction velocity (CV) and the safety factor for conduction (SF) were also reduced by the progressive structural remodeling during heart failure. In our transmural ventricular tissue simulations, no reentry was observed in normal conditions or in the presence of HF ionic remodeling. However, defined amount of fibrosis and/or cellular uncoupling were sufficient to elicit reentrant activity. Under conditions where reentry was generated, HF electrophysiological remodeling did not alter the width of the vulnerable window (VW). However, intermediate fibrosis and cellular uncoupling significantly widened the VW. In conclusion, enhanced fibrosis in failing hearts, as well as reduced intercellular coupling, combine to increase electrophysiological gradients and reduce electrical propagation. In that sense, structural remodeling is a key factor in the genesis of vulnerability to reentry, mainly at intermediates levels of fibrosis and intercellular uncoupling. / [ES] La insuficiencia cardíaca (IC) constituye un importante problema de salud pública en todo el mundo. Operacionalmente se define como un síndrome clínico caracterizado por la incapacidad marcada y progresiva de los ventrículos para llenar y generar gasto cardíaco adecuado para satisfacer las demandas del metabolismo celular, que puede tener una variabilidad significativa en su etiología y es la vía final común de varias patologías cardíacas. Se ha prestado mucha atención a la comprensión de los mecanismos arritmogénicos inducidos por la remodelación estructural, eléctrica, y metabólica del corazón afectado de IC. Debido a la complejidad de los cambios electrofisiológicos que pueden ocurrir durante la IC, la literatura científica es compleja y, a veces equívoca. Sin embargo, se han documentado una serie de características comunes en corazones afectados de IC. A nivel celular, se han establecido como las características distintivas de los miocitos aislados de corazones afectados de IC la prolongación del potencial de acción (PA), que implica la remodelación de los canales iónicos y las alteraciones en la dinámica del calcio. A nivel de los tejidos, el desacoplamiento intercelular y la fibrosis se identifican como los principales factores arritmogénicos. En esta tesis se propuso un modelo celular computacional para la insuficiencia cardíaca utilizando una versión modificada del modelo de potencial de acción ventricular humano de Grandi y colaboradores que incorpora la formulación de la corriente tardía de sodio (INaL) con el fin de estudiar los procesos arritmogénicas debido al fenotipo de la IC. Los datos experimentales de varias fuentes se utilizaron para validar el modelo. Debido a la extensa literatura en la temática se realizó un análisis de sensibilidad para evaluar la influencia de las principales corrientes iónicas y los parámetros sobre los biomarcadores relacionados. Además, se llevaron a cabo simulaciones multiescala para caracterizar esta patología (en fibras y tejidos transmurales). El modelo propuesto para la corriente tardía de sodio y la remodelación electrofisiológica de los miocitos de corazones afectados de IC reprodujeron con precisión las observaciones experimentales. Una INaL incrementada parece ser un importante contribuyente al fenotipo electrofisiológico y la desregulación de la homeostasis del calcio de los miocitos afectados de IC. Nuestros resultados de la simulaciones en fibra ilustran cómo la presencia de células M y el remodelado electrofisiológico heterogéneo en el ventrículo humano afectado de IC modulan la dispersión de la duración potencial de acción (DPA) y el tiempo de repolarización (TR). La velocidad de conducción (VC) y el factor de seguridad para la conducción (FS) también se redujeron en la remodelación estructural progresiva durante la insuficiencia cardíaca. En nuestras simulaciones transmurales de tejido ventricular, no se observó reentrada en condiciones normales o en presencia de la remodelación iónica de la IC. Sin embargo, determinadas cantidades de fibrosis y / o desacoplamiento celular eran suficientes para provocar la actividad reentrante. En condiciones donde se había generado la reentrada, el remodelado electrofisiológico de la IC no alteró la anchura de la ventana vulnerable (VV). Sin embargo, niveles intermedios de fibrosis y el desacoplamiento celular ampliaron significativamente la VV. En conclusión, niveles elevados de fibrosis en corazones afectados de IC, así como la reducción de acoplamiento intercelular, se combinan para aumentar los gradientes electrofisiológicos y reducir la propagación eléctrica. En ese sentido, la remodelación estructural es un factor clave en la génesis de la vulnerabilidad a las reentradas, principalmente en niveles intermedios de fibrosis y desacoplamiento intercelular. El remodelado electrofisiológico promueve la arritmogénesis y puede ser alterado dependi / [CAT] La insuficiència cardíaca (IC) constitueix un important problema de salut pública arreu del món. A efectes pràctics, es defineix com una síndrome clínica caracteritzada per la incapacitat marcada i progressiva dels ventricles per omplir i generar el cabal cardíac adequat, per tal de satisfer les demandes del metabolisme cel·lular, el qual pot tenir una variabilitat significativa en la seua etiologia i és la via final comuna de diverses patologies cardíaques. S'ha prestat molta atenció a la comprensió dels mecanismes aritmogènics induïts per la remodelació estructural, elèctrica, i metabòlica del cor afectat d'IC. A causa de la complexitat dels canvis electrofisiològics que poden ocórrer durant la IC, trobem que la literatura científica és complexa i, de vegades, equívoca. No obstant això, s'han documentat una sèrie de característiques comunes en cors afectats d'IC. A nivell cel·lular, com característiques distintives dels miòcits aïllats de cors afectats d'IC, s'han establert la prolongació del potencial d'acció (PA), que implica la remodelació dels canals iònics, i les alteracions en la dinàmica del calci. A nivell dels teixits, el desacoblament intercel·lular i la fibrosi s'identifiquen com els principals factors aritmogènics. Per tal d'estudiar els processos aritmogènics a causa del fenotip de la IC, es va proposar un model cel·lular computacional d'IC utilitzant una versió modificada del model de potencial d'acció ventricular humà de Grandi i els seus col·laboradors, el qual incorpora la formulació del corrent de sodi tardà (INaL). Amb l'objectiu de validar el model es van utilitzar dades experimentals de diverses fonts. A causa de l'extensa literatura en la temàtica, es va realitzar una anàlisi de sensibilitat per tal d'avaluar la influència de les principals corrents iòniques i els paràmetres sobre els biomarcadors relacionats. A més, es van dur a terme simulacions multiescala per a la caracterització d'aquesta patología (fibres i teixits transmurals). El model proposat per al corrent de sodi tardà i la remodelació electrofisiològica dels miòcits de cors afectats d'IC van reproduir amb precisió les observacions experimentals. Una INaL incrementada sembla contribuir de manera important al fenotip electrofisiològic i a la desregulació de l'homeòstasi del calci dels miòcits afectats d'IC. Els resultats de les nostres simulacions en fibra indiquen que la presència de cèl·lules M i el remodelat electrofisiològic heterogeni en el ventricle humà afectat d'IC modulen la dispersió de la durada del potencial d'acció (DPA) i el temps de repolarització (TR). La velocitat de conducció (VC) i el factor de seguretat per a la conducció (FS) també es van reduir en la remodelació estructural progressiva durant la IC. A les nostres simulacions transmurals de teixit ventricular, no s'observà cap reentrada ni en condicions normals ni en presència de la remodelació iònica de la IC. No obstant això, amb determinades quantitats de fibrosi i/o desacoblament cel·lular sí que es provocà l'activitat reentrant. I amb les condicions que produïren la reentrada, el remodelat electrofisiològic de la IC no va alterar l'amplada de la finestra vulnerable (FV). Tanmateix, nivells intermedis de fibrosi i el desacoblament cel·lular sí que ampliaren significativament la FV. En conclusió, nivells elevats de fibrosi en cors afectats d'IC, així com la reducció d'acoblament intercel·lular, es combinen per augmentar els gradients electrofisiològics i reduir la propagació elèctrica. Per tant, la remodelació estructural és un factor clau en la gènesi de la vulnerabilitat a les reentrades, principalment en nivells intermedis de fibrosi i desacoblament intercel·lular. / Gómez García, JF. (2015). Multiscale Modeling and Simulation of Human Heart Failure [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/52389 / TESIS

Úlcera de membros inferiores é uma das complicações do diabetes melito que acomete aproximadamente 15% dos portadores da doença e resultam em elevada taxa de mortalidade. São feridas de difícil resolução, em geral, refratárias aos diversos tipos de tratamentos. Um dos principais aspectos que contribuem para sua cronicidade é a modificação no comportamento de fibroblastos. O objetivo do estudo foi comparar in vitro modificações no comportamento de fibroblastos derivados de ferida de membro inferior de indivíduos diabéticos com fibroblastos derivados de pele normal, quanto a proliferação celular e a capacidade de contração de modelo tridimensional de matriz de colágeno povoado por células, além de investigar o perfil de expressão gênica diferencial dessas células. Para tanto, foram cultivados fibroblastos provenientes de tecido de granulação de feridas de membro inferior de pacientes com diabetes do tipo 2 (FFD) e de pele normal (FC). Foram verificadas anormalidades morfológicas no grupo FFD compatíveis com senescência celular; menor contração da matriz de colágeno povoado por fibroblastos do grupo FFD (redução de 23% da área original) comparado ao grupo FC (redução de 30% da área original p<0,001). Pela técnica de microarray foram identificados 143 genes diferencialmente expressos, em sua maioria hipoexpressos, no grupo FFD, dentre os quais, destacam-se aqueles relacionados a processos de senescência e apoptose celular, bem como déficit na síntese e contração de fibras colágenas / Lower limbs ulcer is one of the complications of diabetes mellitus that affects approximately 15% of the patients and results in high mortality. Wounds that is difficult to heal, generally refractory to many treatments. One of the main aspects that contribute to disease chronicity is the change in the fibroblast behavior. The aim of this study was to compare changes in the in vitro fibroblasts behavior from diabetic leg ulcer with fibroblasts derived from normal skin as to cell proliferation and contraction capacity of a threedimensional fibroblasts populated collagen lattice, as well as to investigate the pattern of differential gene expression in these cells. For this purpose, fibroblasts were cultured from granulation tissue of lower limb ulcer in patients with type 2 diabetes (FFD) and from normal skin (FC). Morphological abnormalities compatible with cellular senescence were observed in the FFD. Reduced matrix contraction in fibroblasts populated group (FFD reduction of 23% from the original area) compared to FC (30% reduction from the original area) - (p <0.001). Using DNA microarray 143 differentially expressed genes were identified, most of them were underexpressed in FFD group, among which are those related to senescence and apoptosis, as well as deficits in the synthesis and contraction of collagen fibers

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13290号 / 論医博第2188号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 木村 剛, 教授 浅野 雅秀 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM