Tesis sobre el tema "Heterogeneity and plasticity"

Vascular smooth muscle cell (VSMC) accumulation is a hallmark of atherosclerosis and vascular injury. However, fundamental aspects of proliferation and the phenotypic changes within individual VSMCs, which underlie vascular disease remain unresolved. In particular, it is not known if all VSMCs proliferate and display plasticity, or whether individual cells can switch to multiple phenotypes. To assess whether proliferation and plasticity in disease is a general characteristic of VSMCs or a feature of a subset of cells, multi-colour lineage labelling is used to demonstrate that VSMCs in injury-induced neointimal lesions and in atherosclerotic plaques are oligo-clonal, derived from few expanding cells, within mice. Lineage tracing also revealed that the progeny of individual VSMCs contribute to both alpha Smooth muscle actin (aSma)-positive fibrous cap and Mac-3-expressing macrophage-like plaque core cells. Co-staining for phenotypic markers further identified a double-positive aSma+ Mac3+ cell population, which is specific to VSMC-derived plaque cells. In contrast, VSMC-derived cells generating the neointima after vascular injury generally retained expression of VSMC markers and upregulation of Mac3 was less pronounced. Monochromatic regions in atherosclerotic plaques and injury-induced neointima did not contain VSMC-derived cells expressing a different fluorescent reporter protein, suggesting that proliferation-independent VSMC migration does not make a major contribution to VSMC accumulation in vascular disease. Similarly, VSMC proliferation was examined in an Angiotensin II perfusion model of aortic aneurysm in mice, oligo-clonal proliferation was observed in remodelling regions of the vasculature, however phenotypic changes were observed in a large proportion of VSMCs, suggesting that the majority of VSMCs have some potential to modulate their phenotype. To understand the mechanisms behind the inherent VSMC heterogeneity and observed functionality, the single cell transcriptomic techniques Smart-seq2 and the Chromium 10X system were optimized for use on VSMCs. The work within this thesis suggests that extensive proliferation of a low proportion of highly plastic VSMCs results in the observed VSMC accumulation after injury, and the atherosclerotic and aortic aneurysm models of cardiovascular disease.

En aquesta tesi doctoral, el nostre objectiu principal és entendre la importància de la regulació epigenètica en la determinació del destí cel lular i de les seves possibles tran- sicions cap a altres estats. Per tal d’estudiar-ho, en primer lloc, formulem un model es- tocàstic de regulació epigenètica. En aquest model, ens centrem en l’anàlisi de la reprogra- mació cel·lular, és a dir, la situació on el sistema es mou de l’epi-fenotip diferenciat, carac- teritzat per tenir el sistema de regulació epigenètica pel gen de diferenciació(pluripotència) obert(tancat), cap a l’epi-fenotip pluripotent, definit en aquest cas per tenir el sistema de regulació epigenètica pel gen de diferenciació (pluripotència) tancat(obert). En particular, dins de la heterogeneïtat intrínsica dels sistemes de regulació epigenètica, nosaltres identifiquem l’existència de dos possibles escenaris: l’escenari resistent, on la reprogramació no pot tenir lloc, i l’escenari plàstic, que és el qual permet el canvi de l’epi-fenotip diferenciat a l’epi-fenotip pluripotent. Aquest darrer escenari, relacionat amb l’existència de plasticitat epigenètica, ha estat associat amb envelliment. De fet, quan al model de regulació epigenètica només s’hi consideren efectes d’envelliment, el sistema representa un estat plàstic saludable, on les propietats de cèl·lula mare són adquirides de forma temporal, ja que el sistema de regulació epigenètica pot retornar a l’epi-fenotip diferenciat. Aquesta situació és la que probablement és responsable de regenerar i rejuvenir els teixits i, per tant, és la situació desitjada, ja que permetria un envelliment ‘saludable’. No obstant, quan als efectes de l’envelliment se li sumen alteracions de l’activitat epigenètica, que són freqüents a mesura que s’envelleix, l’estat plàstic esdevé un estat plàstic patològic, on en aquest cas, les propietats de cèl lula mare són adquirides de forma irreversible, és a dir, són permanents. Aquest escenari és el que probablement predisposa el sistema al càncer, ja que implica l’acumulació d’epi-fenotips indecisos que tenen el sistema de regulació epigenètica pel gen de pluripotècia obert, és a dir, que aquest gen es pot expressar. Per tal d’estudiar aquesta situació més en detall, formulem un context general per a l’estudi d’un model estocàstic d’escales múltiples conjunt per a la regulació epigenètica i la xarxa de regulació genètica. En particular, nosaltres ens centrem en una xarxa de regulació genètica formada per 2 gens, un gen que promou la diferenciació i un gen que promou la pluripotència. Quan analitzem aquest model conjunt, veiem que el paper que juga la regulació epigènetica és cabdal ja que permet a la xarxa de regulació epigenètica canviar d’estat, en altres paraules, permet un canvi del destí cel·lular de l’epi-fenotip diferenciat a l’epi-fenotip pluripotent (reprogramació) o el canvi invers (diferenciació). Aquest model conjunt ens permet identificar els sistemes de regulació epigenètica responsables d’atrapar la cèl·lula en un estat de cèl·lula mare, impedint-ne la seva diferenciació. La nostra formulació ens permet disenyar estratègies epigenètiques amb les quals podem aconseguir cèl·lules amb alta probabilitat de diferenciació, partint de cèl lules que inicialment eren resistents a la diferenciació. Com hom pot imaginar, aquestes estratègies són molt rellevants per a l’estudi i el tractament del càncer i altres malalties associades amb l’envelliment.
In this thesis, we aim at understanding the importance of epigenetic regulation(ER) in cell fate decisions and transitions. In order to address this issue, we first formulate a stochastic model of epigenetic regulation. Within this model, we focus our discus- sion in cell reprogramming, i.e. the system moves from the differentiated epi-phenotype, characterised by differentiation(pluripotency) ER system open(closed), to the pluripotent epi-phenotype, where the ER system for differentiation(pluripotency) is closed(open). In particular, within the intrinsic heterogeneity existing in ER systems, we identify the appearance of two relevant scenarios: the resilient scenario, where reprogramming cannot occur, and the plastic one, which is the one allowing for the switch from the di erentiated epi-phenotype to the pluripotent epi-phenotype. The latter, which is characterised by ex- hibiting epigenetic plasticity, has been linked to ageing. In fact, when just ageing e ects are considered in the ER model, the system displays a `healthy' plasticity, where the stem-cell like properties can be acquired, but then, the ER system can go back to the dif- ferentiated epi-phenotype. This scenario may be related to regeneration and rejuvenation of tissues. Nevertheless, when ageing is considered along with epigenetic dis-regulations, which are likely to occur withing ageing cells/tissues, the plastic state leads to a patholog- ical plasticity, where stem cell features are acquired irreversibly. This scenario is the one which may predispose the system to cancer, as it implies the accumulation of undecided epi-phenotypes with the pluripotency ER system sustained in its on state. In order to further analyse this issue, we formulate a general framework for the study of a combined epigenetic regulation-gene regulatory network (ER-GRN)stochastic multi- scale model, which we later focus on our particular case of interest, i.e. a 2 gene regulatory network with one gene promoting differentiation and one gene promoting pluripotency. When analysing the ER-GRN model formulated, we show that the role played by ER is central since it allows the GRN to switch state, i.e. cell fate transitions from the differ- entiated phenotype to the pluripotent one (reprogramming) or vice versa (differentiation). The ER-GRN model allows to identify which ER systems are responsible for locking the cell in a stem cell like state and our formulation allows us to design epigenetic-based strategies able to obtain differentiation-primed cells from differentiation-resilient cells. Such strategies are of key relevance in the treatment of cancer and other age-associated diseases.

Plasticity, heterogeneity and modelling approach constitute the three pillars on the top of which this thesis investigates the complexity of cell metabolism. The multiple sides of metabolic plasticity have been explored as cell adaptive response to varying conditions, demand and perturbations under both physiological and pathological conditions. By investigating cell populations as homogeneous and heterogeneous systems, new in silico predictive models and novel computational constraint-based methodologies have been defined. This work started from the investigation of cell populations as homogeneous systems, where the average behaviour is described and cell-to-cell differences are temporarily hidden. Reconstructing high-quality genome-scale metabolic models is crucial to computationally address cell metabolism and organize all the available metabolic knowledge of given cells or organisms. Although multiple tools for performing this task already exist, a pipeline for the semi-automatic reconstruction of genome-scale networks has been proposed to solve some current critical issues and generate higher quality models. The application of this approach for the genome-wide metabolic reconstruction of yeast Zygosaccharomyces parabailii showed adherence of in silico simulations to experimental data and literature findings. Moreover, metabolic plasticity in response to different metabolic regimes has been explored through constraint-based modelling. The potentialities of genome-scale reconstructions in mirroring the systemic perspective coexist with difficulty in their management. In this work, greater control is achieved by switching to smaller-scale core networks. In particular, core modelling has been exploited as an effective mean to investigate intertumoural heterogeneity, and plasticity of the implemented tumour metabolic programs as adaptation to different environmental scenarios. The effectiveness of homogeneous systems to lower overall system complexity level without compromising biological validity of in silico outcomes goes along with the need to address cell-to-cell variations of cell populations. In this regard, classic constraint-based modelling has been extended to deal with heterogeneous systems. A new strategy, called popFBA, has been developed to reconstruct and simulate cell populations metabolism, by putting emphasis on the relationships established among their components. Using as case study the ecosystemic view of cancer populations, popFBA highlighted that the achievement of optimal biomass is consistent with metabolic plasticity of population components under different scenarios together with a cooperative behaviour. At the same time, countless combinations of flux distributions for the individual population components prompted to develop a novel methodology called single-cell Flux Balance Analysis (scFBA). This metodology integrates single-cell transcriptomics data as further constraints on the individual components through the computation for each reaction of a Reaction Activity Score, which we implemented in a previous computational framework called MaREA. In this way, scFBA efficiently reduced the amount of allowable individual flux distributions, and captured complex networks of interactions between cells of a specific population. In view of the findings of this research, a deep characterization of metabolic plasticity within cell populations and of the intricate dialogue between cells and their environment can assist the formulation of more rational and personalized strategies. Their devising could enable to hamper disease progression, or to exploit metabolism of given microorganisms for producing relevant chemical compounds.
Plasticity, heterogeneity and modelling approach constitute the three pillars on the top of which this thesis investigates the complexity of cell metabolism. The multiple sides of metabolic plasticity have been explored as cell adaptive response to varying conditions, demand and perturbations under both physiological and pathological conditions. By investigating cell populations as homogeneous and heterogeneous systems, new in silico predictive models and novel computational constraint-based methodologies have been defined. This work started from the investigation of cell populations as homogeneous systems, where the average behaviour is described and cell-to-cell differences are temporarily hidden. Reconstructing high-quality genome-scale metabolic models is crucial to computationally address cell metabolism and organize all the available metabolic knowledge of given cells or organisms. Although multiple tools for performing this task already exist, a pipeline for the semi-automatic reconstruction of genome-scale networks has been proposed to solve some current critical issues and generate higher quality models. The application of this approach for the genome-wide metabolic reconstruction of yeast Zygosaccharomyces parabailii showed adherence of in silico simulations to experimental data and literature findings. Moreover, metabolic plasticity in response to different metabolic regimes has been explored through constraint-based modelling. The potentialities of genome-scale reconstructions in mirroring the systemic perspective coexist with difficulty in their management. In this work, greater control is achieved by switching to smaller-scale core networks. In particular, core modelling has been exploited as an effective mean to investigate intertumoural heterogeneity, and plasticity of the implemented tumour metabolic programs as adaptation to different environmental scenarios. The effectiveness of homogeneous systems to lower overall system complexity level without compromising biological validity of in silico outcomes goes along with the need to address cell-to-cell variations of cell populations. In this regard, classic constraint-based modelling has been extended to deal with heterogeneous systems. A new strategy, called popFBA, has been developed to reconstruct and simulate cell populations metabolism, by putting emphasis on the relationships established among their components. Using as case study the ecosystemic view of cancer populations, popFBA highlighted that the achievement of optimal biomass is consistent with metabolic plasticity of population components under different scenarios together with a cooperative behaviour. At the same time, countless combinations of flux distributions for the individual population components prompted to develop a novel methodology called single-cell Flux Balance Analysis (scFBA). This metodology integrates single-cell transcriptomics data as further constraints on the individual components through the computation for each reaction of a Reaction Activity Score, which we implemented in a previous computational framework called MaREA. In this way, scFBA efficiently reduced the amount of allowable individual flux distributions, and captured complex networks of interactions between cells of a specific population. In view of the findings of this research, a deep characterization of metabolic plasticity within cell populations and of the intricate dialogue between cells and their environment can assist the formulation of more rational and personalized strategies. Their devising could enable to hamper disease progression, or to exploit metabolism of given microorganisms for producing relevant chemical compounds.

Les gliomes diffus de bas grade (DLGG) sont des tumeurs gliales de grade II qui affectent principalement les jeunes adultes. Elles sont caractérisées par une croissance lente et une activité mitotique réduite. Cependant, ces tumeurs diffusent et envahissent le cerveau sain via les vaisseaux sanguins et les fibres nerveuses. Après plusieurs années de croissance lente, ces tumeurs peuvent évoluer vers des glioblastomes, des tumeurs cérébrales très agressives dont la survie médiane moyenne est alors de 12 à 15 mois après le diagnostic. La caractérisation cellulaire des DLGG est encore limitée ce qui nuit à la recherche d’un traitement à un stade précoce. Dans ma thèse de doctorat, je me suis focalisée sur la caractérisation de 'hétérogénéité cellulaire des DLGG mutés pour IDH1. En effectuant une analyse d'immunofluorescence sur des astrocytomes et oligodendrogliomes de grade II, j'ai identifié deux sous-populations cellulaires largement non chevauchantes et exprimant respectivement les facteurs de transcription SOX9 et OLIG1. Ces cellules s’apparentent à des cellules de type astrocytaire et oligodendrocytaire et expriment des marqueurs moléculaires distincts. Les cellules SOX9 expriment APOE, KCNN3, CRYAB et ID4, tandis que les cellules OLIG1 expriment préfentiellement PDGFRA, SOX8, MASH1 et SOX4. Par ailleurs, j’ai montré que les cellules SOX9 présentent une activation particulière des voies de signalisation, notamment Notch, BMP et leurs cibles en aval. Pour étudier le rôle de la voie de signalisation Notch dans la formation de ces 2 sous-populations tumorales, j'ai purifié par tri magnétique les cellules tumorales à partir d'échantillons de gliomes fraîchement réséqués et j'ai surexprimé le domaine intracellulaire Notch (NICD), une forme active de Notch. J’ai ainsi montré que cette activation augmentait l’expression des marqueurs cellulaires associés aux cellules SOX9+ et une baisse de ceux associés aux cellules OLIG1+. J'ai ensuite étendu ces analyses à une lignée cellulaire anaplasique dérivée d'un patient et mutée pour IDH1. Ces résultats indiquent un rôle clé de la signalisation Notch dans la régulation de la plasticité des cellules tumorales. Des expériences similaires pour étudier l'activation de la signalisation BMP (bone morphogenetic protein) n'ont pas montré d'effet notable sur la plasticité. Néanmoins, le traitement des cellules par des membres de la famille BMP a fortement augmenté l’expression de CRYAB, un marqueur associé à SOX9, et a diminué l’expression de OLIG1 et OLIG2. En conclusion, j'ai identifié deux sous-populations tumorales non chevauchantes dans des gliomes diffus de bas grade et j'ai démontré le rôle déterminant de la voie de signalisation Notch dans leur formation. Ces résultats permettront de mieux comprendre l'hétérogénéité tumorale dans les DLGG et de concevoir de nouvelles stratégies thérapeutiques contre ces tumeurs
Diffuse Low-Grade Gliomas (DLGG) are WHO grade II glial tumors affecting younger adults. They are characterized as silent, slow growing tumors with fewer mitotic activities. However, they diffuse and invade the healthy brain via blood vessels and nerve fibers. These, over a period of years develop to malignant Glioblastoma, aggressive brain tumors where patients have an average medial survival of 12-15 months after diagnosis. Ill-defined phenotypic and cellular diversity of DLGG poses serious limitation to treatment and prevention at the early stage.In my PhD thesis, I aimed to address this limitation by characterizing the cellular heterogeneity in IDH1-mutated DLGG. By performing immunofluorescence analysis on grade II astrocytoma and oligodendroglioma, I have identified two largely non-overlapping cellular subpopulations expressing SOX9 and OLIG1 transcription factors, which represent astrocyte-like and oligodendrocyte-like cells, respectively. Upon further investigation, I have shown that these subpopulations express distinct molecular markers. Sox9 cells are associated with APOE, KCNN3, CRYAB and ID4, while Olig1 cells showed strong correlation with the expression of PDGFRA, SOX8, MASH1, and SOX4. In addition, the sox9 cells show a particular activation of signaling pathways including Notch, BMP and their downstream targets.To ascertain the role of Notch signaling in regulating the formation of these tumoral subpopulations, I used magnetic sorting of tumor cells from freshly resected glioma samples and overexpressed Notch Intracellular Domain (NICD), an active form of Notch. Increased Notch activation resulted in an upregulation of Sox9- and downregulation of Olig1-associated cell markers. I have then extended these analyses on one anaplastic IDH1 mutated patient derived cell line which reproduced similar gene expression profile confirming the robustness of the role of Notch signaling in regulating the plasticity of the cells. Parallel experiments performed by activation of Bone Morphogenetic Protein (BMP) signaling on IDH1 mutated cell line did not show a prominent effect on the plasticity. Nevertheless, BMP signal activation highly upregulated CRYAB, a SOX9 related marker and downregulated OLIG1 and OLIG2.In conclusion, I have identified two non-overlapping tumor subpopulations in diffuse low-grade gliomas and demonstrated the deterministic role of Notch signaling pathway in their formation. I believe that these findings would aid in better understanding tumoral heterogeneity in DLGG and be extended in designing new therapeutic strategies against these tumors

The role of predation as a structuring agent of communities has been a focal area of research in marine ecology. Conversely, studies focusing on foraging plasticity, particularly by shorebirds, and consequences for benthic community structure, heterogeneity and ecosystem functioning have been limited in marine soft-sediments. Temporal interactions with foraging plasticity is an even greater knowledge gap. The primary goal of this dissertation was to expand on current understanding of foraging plasticity and the broader ecological role of Greater Flamingos as predators in marine sedimentary ecosystems. The specific aim was to test the long-term effects of different foraging structure sizes (which is a subtle form of foraging plasticity) on sedimentary assemblages following foraging disturbances. In situ comparisons of large and small foraging structures created by the flamingos with adjacent non-foraged sediments (controls) indicated that small channels had greater concentrations of sediment organic matter relative to large channels following feeding. Additionally, small feeding structures supported greater abundances of the burrowing amphipod Urothoe grimaldii and the grazing gastropod Assiminea globulus along with elevated size-specific sediment deposition rates. These results shed light on the differential timedependent impacts flamingo foraging plasticity can generate on basal trophic resources and higher consumers. Results also showed that irrespective of size, feeding channels made by flamingos generally became enriched with microalgae following foraging, with this enrichment effects spilling over into non-foraged controls sediments. This indicates that foraging by flamingos has the potential to create resource islands that become enriched with microalgae over time, with effects spilling over into adjacent non-foraged areas. Importantly, these positive effects may oppose the negative effects of bioturbation by burrowing sandprawns on productivity, which is a dominant process in the study area. This study emphasizes the importance of Greater Flamingos in regulating basal resource supply, community structure and spatio-temporal heterogeneity and has highlighted the need for predation models within sedimentary environments to incorporate time-dependent effects of foraging plasticity into current thinking.

Plant root system response to nutrient heterogeneity was tested in ten plant species of varying life form and successional status. All plants tested are native to the South Carolina coastal plain. Morphological responses of the root system (scale, precision and discrimination) and overall plant response (sensitivity) to increasing nutrient heterogeneity were tested. Ten individuals of each species were placed into four treatments which had varying nutrient distribution but the same overall nutrient addition. Plants were harvested when roots reached pot edge. I observed high variation in scale (mass and extent of a root system), precision (the ability to proliferate roots in nutrient patches) and sensitivity (growth benefits gained as nutrient heterogeneity increases; measured as total biomass). No significant discrimination responses were observed, although greatest mean root density occurred at intermediate fertility levels for all species. I tested the hypothesis that scale and precision would be negatively correlated, and I did not observe this relationship in these plant species. However, in herbaceous species scale and precision were positively correlated. Sensitivity was not closely related to precision indicating that proliferating roots in fertile patches does not always yield growth benefits in heterogeneous soils. Further, some sensitive species had very low precision suggesting that other characteristics lead to positive growth response in heterogeneous environments. Plasticity of root uptake rates and demography of roots are proposed as two other mechanisms which may play important roles in plant sensitivity responses. Scale was negatively correlated to sensitivity for herbaceous plants suggesting that plants that monopolize the most soil space are not able to gain benefits from nutrient patches within the soil matrix. There was no trend observed to suggest that plant life form was correlated with precision or sensitivity. However, scale was greater in herbs than in woody plants, possibly because the two life forms develop at different times.
Master of Science

Melanoma is the deadliest form of skin cancer due to its high propensity to metastasize and resistance to current therapies. We have created a spontaneous mouse model of metastatic melanoma (Dct-Grm1/K5-Edn3) where metastasis to the lungs is 80% penetrant. The primary tumors of these mice present cellular heterogeneity with cells at varying levels of differentiation. The main goal of this study was to determine the metastatic potential of the primary tumor resident Tyrosinase positive cells and evaluate the dynamic phenotypic changes as those cells move from the primary tumors to the sites of metastasis. To accomplish this aim I crossed the Dct-Grm1/K5-Edn3 mice to CreERT2/mT/mG mice to indelibly label Tyrosinase cell populations within the primary tumor with Green Fluorescent Protein (GFP) by topical application of 4-hydroxytamoxifen (4HT) at the tumor site. In vivo lineage tracing and characterization of GFP+ cells were performed in the metastatic lesions. In the 4HT treated Dct-Grm1/ K5-Edn3/Tyr-CreERT2/mT/mG mice, primary tumor derived Tyrosinase positive cells or their progeny (GFP+) established successful metastases in the distant organs indicating the tumorigenic capacity of the differentiated cell populations. Numerous metastatic melanoma cells were identified in the vasculature of the metastatic organs and established close association with the vascular endothelium. The intravascular cells lost pigmentation and did not express melanocytic markers; however, they mimicked endothelial cell properties and gained the expression of CD31 (also known as platelet endothelial cell adhesion molecule PECAM-1) and vascular endothelial (VE)-Cadherin. In the lung metastatic foci, GFP+ cells resumed pigmentation production and lost the expression of endothelial cell markers. Evidence from other metastatic organs in the mice further supported the phenotypic plasticity of metastatic melanoma cells. The in vivo lineage tracing system established in the melanoma mouse model revealed tumor phenotypic plasticity and will be a powerful model to evaluate and help us understand the etiology and pathogenesis of melanoma metastasis. Further characterization of those more aggressive cells in melanoma will allow for the development of new prognostic tests and novel therapeutic strategies to eliminate metastasis.

Cette thèse porte sur l'analyse statistique et mathématique de la croissance cellulaire à l'échelle individuelle sous l'effet d'un stress. À partir de l'analyse des données recueillies par Sebastián Jaramillo et James Broughton sous la direction de Meriem El Karoui, nous avons construit différents modèles permettant une compréhension à différents niveaux de l'impact que la réponse hétérogène au stress génotoxique (réponse SOS) a sur la croissance d'une population de bactéries Escherichia coli. Pour modéliser la dynamique de ces populations on utilise des processus stochastiques à valeurs mesures.Nous construisons tout d'abord on construit un modèle stochastique basé sur le modèle "adder" de contrôle de la taille, étendu pour incorporer la dynamique de la réponse SOS et son effet sur la division cellulaire. Le cadre choisi est paramétrique et le modèle est ajusté par maximum de vraisemblance aux données de lignées individuelles obtenues en mother machine. Cela nous permet de comparer quantitativement les paramètres inférés dans différents environnements.Nous nous intéressons ensuite aux propriétés ergodiques d'un modèle plus général que "adder", répondant à des questions ouvertes sur son comportement en temps long. On considère un flot déterministe général et un noyau de fragmentation non nécessairement auto-similaire. Nous montrons l'existence des éléments propres. Ensuite, une dollar_h_dollar-transformée de Doob avec la fonction propre nous ramène à l'étude d'un processus conservatif. Enfin, en montrant une propriété de petite set pour les compacts de l'espace d'états, nous obtenons alors la convergence exponentielle du modèle.Finalement, nous considérons un modèle bitype structuré en âge modélisant la plasticité phénotypique observée dans la réponse au stress. Nous étudions la probabilité de survie et le taux de croissance de la population en environnement constant et périodique. Nous mettons en lumière un trade-off pour avoir la survie de la population, ainsi qu'une sensibilité par rapport aux paramètres du modèle qui n'est pas la même pour la probabilité de survie et pour le taux de croissance.Nous concluons avec une section indépendante, initiée durant le CEMRACS 2022. Nous étudions numériquement la propagation spatiale des populations structurés en taille modélisant le mouvement collectif de clusters de Myxobactéries à travers de systèmes d'équations de réaction-diffusion
This thesis focuses on understanding individual-scale cell growth under stress. Starting from the analysis of the data collected by Sebastián Jaramillo and James Broughton under the supervision of Meriem El Karoui, we have developed various models to comprehend the impact of the heterogeneous response to genotoxic stress (SOS response) on the growth of a Escherichia coli populations. We employ measure-values stochastic processes to model the dynamics of these populations.Firstly, we construct a stochastic model based on the "adder" size-control model, extended to incorporate the dynamics of the SOS response and its effect on cell division. The chosen framework is parametric, and the model is fitted by maximum likelihood to individual lineage data obtained in mother machine. This allows us to quantitatively compare inferred parameters in different environments.Next, we explore the ergodic properties of a more general model than the "adder," addressing open questions about its long-time behaviour. We consider a general deterministic flow and a fragmentation kernel that is not necessarily self-similar. We demonstrate the existence of eigenelements. Then, a Doob dollar_h_dollar-transform with the found eigenfunction reduces the problem to the study of a conservative process. Finally, by proving a "petite set" property for the compact sets of the state space, we obtain the exponential convergence.Finally, we consider a bitype age-structured model capturing the phenotypic plasticity observed in the stress response. We study the survival probability of the population and the population growth rate in constant and periodic environments. We evince a trade-off for population establishment, as well as a sensitivity with respect to the model parameters that differs for survival probability and growth rate.We conclude with an independent section, collaborative work initiated during CEMRACS 2022. We investigate numerically the spatial propagation of size-structured populations modeling the collective movement of Myxobacteria clusters via a system of reaction-diffusion equations

Epithelial mesenchymal plasticity in cancer generally refers to the ability of a cancer cell to transform into a different cell form, which facilitates the metastatic spread of a cancer. This thesis explores the roles of four cancer-associated genes that affect the transition of the cell state during cancer metastasis, and includes extensive research on two of the four gene targets, namely TRIM28 and TGFBI. The effects of these genes in breast cancer systems indicated great potential for improving therapeutic responses towards cancer drugs, which would alleviate the suffering of breast cancer patients.

Les cancers pédiatriques représentent un défi thérapeutique, et une compréhension des mécanismes d’échappement aux traitements est nécessaire pour pouvoir développer de nouvelles approches thérapeutiques. L'ADN circulant peut être libéré par une tumeur dans les fluides corporels et permet de détecter et suivre des altérations génétiques tumorales par des prélèvements successifs peu invasifs tels qu'une prise de sang. Dans ce travail, une technique de séquençage de type « whole exome » (séquençage de l’ensemble des exons) de l’ADN circulant a été mis au point pour permettre l’étude des altérations génétiques tumorales dans le plasma chez des enfants atteints de cancer.Ces analyses soulignent la grande hétérogénéité génétique spatiale et temporelle des cancers pédiatriques. De plus, un rôle important de l’évolution clonale dans la progression de la maladie a ainsi pu être mis en évidence. Des approches utilisant les caractéristiques particuliers de l'ADN circulant ont également permis d’inférer le profil d’expression, basées sur l’empreinte des sites de début de transcription, ou le profil épigénétique de la tumeur. En plus d'une aide à une classification des tumeurs, ces particularités pourront aider à l'observation d'un changement d'identité cellulaire sous traitement. L'ADN circulant est donc un formidable outil pour mieux comprendre l'échappement aux traitements d'une tumeur par son hétérogénéité spatiale et temporelle et sa plasticité
Pediatric cancers represent a therapeutic challenge, and an understanding of the mechanisms of escape from treatment is necessary in order to be able to develop new therapeutic approaches. Circulating DNA can be released by a tumor in bodily fluids and enable to detect and follow genetic alterations in tumors by successive minimally invasive samples such as a blood test. In this work, a “whole exome” sequencing technique for circulating DNA was developed to allow the study of genetic tumor alterations in plasma in children with cancer. These analyzes highlight the frequent spatial and temporal genetic heterogeneity of pediatric cancers. In addition, an important role of clonal evolution in the progression of the disease was thus highlighted. Approaches using the particular characteristics of circulating DNA have also made it possible to infer the expression profile, based on the imprint of the transcription start sites, or the epigenetic profile of the tumor. In addition to an aid in the classification of tumors, these features may help to observe a change in cellular identity under treatment. Circulating DNA is therefore an important tool for better understanding mechanisms of escape from treatment of a tumor based on spatial and temporal heterogeneity and cellular plasticity

Le cancer ovarien se distingue par une forte mortalité, liée entre autre à un diagnostic tardif. Une chimiorésistance importante est observée en même temps qu'un fort taux de récidive. La Transition Epithélio-Mésenchymateuse (TEM) est un des facteurs observés dans la chimiorésistance des cellules et de la progression du cancer. Elle implique une perte de l'adhérence à la Matrice Extra Cellulaire (MEC) et des jonctions intercellulaires, au profit d'un gain en motilité ou au potentiel invasif des cellules. La TEM est un phénomène physiopathologique qui présente une forte plasticité. Ces cellules, dites intermédiaires, présentent des caractéristiques à la fois épithéliales et mésenchymateuses et définissent un spectre de la TEM. Ces cellules sont souvent plus corrélées à un faible pronostic vital. Des méthodes omiques, notamment transcriptomiques, existent dans la caractérisation de ces cellules intermédiaires. Toutefois, ces méthodes sont souvent coûteuses, laborieuses, ne prennent pas en compte la morphologie à l'échelle de la cellule ou l'agencement cellulaire dans l'espace, qui sont des éléments importants dans la TEM. Dans ce but, nous avons développé une approche phénomique basée exclusivement sur la microscopie.Cette approche fonctionne sur FIJI au moyen d'un algorithme de type macro, prêt à l'emploi ou modifiable suivant l'expertise ou le besoin de l'utilisateur. Dans le cadre de l'étude de la TEM dans le cancer ovarien, nous avons étudié quatre lignées représentatives du spectre de la TEM. La TEM est induite par l'utilisation de deux cytokines, le TNFα et le TGFβ1, dont l'effet associatif reste dépendant du contexte et n'a pas ou peu été décrit dans le cancer ovarien. Grâce à notre pipeline d'analyse, nous caractérisons la plasticité cellulaire par une analyse en cellule unique, incluant la morphologie et les différents marquages moléculaires liés à la TEM effectués. Nous avons analysé plus de soixante-dix mille cellules et combiné plus de cinquante paramètres via FIJI, Celltool et des méthodes créées. Parmi celles-ci on retrouve un outil de topographie cellulaire pour normaliser la distance entre le noyau et la membrane cellulaire, ou de mesure de l'isolement cellulaire au sein de son environnement. Cet ensemble de paramètres a ensuite été ramené à une seule mesure statistique pour comparer de façon interlignées et intralignées. Nous avons pu alors distinguer efficacement les quatre lignées entre elles, ainsi que l'impact des cytokines par lignée. Nous démontrons ainsi l'additivité des cytokines dans l'induction de la TEM dans chaque lignée le long du spectre de la TEM. Parallèlement à cette étude, notre méthodologie a déjà permis de démontrer l'importance de la MEC sur la dissémination du cancer ovarien en présence de flux ascitique. Ces résultats montrent qu'une analyse phénomique en cellule unique est particulièrement prometteuse pour caractériser l'hétérogénéité ainsi que la plasticité en réponse à un microenvironnement complexe
Ovarian cancer is characterized by a high mortality rate, linked among other things to a late diagnosis. Significant chemoresistance is observed at the same time as a high rate of recurrence. Epithelial to Mesenchymal Transition (EMT) is one of the factors observed in cell chemoresistance and cancer progression. It involves a loss of adhesion to the Extracellular Matrix (ECM) and intercellular junctions, in favor of a gain in motility or the invasive potential of the cells. EMT is a pathophysiological phenomenon that has a high degree of plasticity. These cells, known as intermediates, may present both epithelial and mesenchymal characteristics and define a spectrum of EMT. These cells are often correlated with a worse prognosis for life. Omics methods, particularly transcriptomic methods, exist in the characterization of these intermediate cells. However, these methods are often expensive, laborious, and do not take into account morphology at scale of the cell, or cell arrangement in space, which are important elements in EMT. To this end, we have developed a phenomics approach based exclusively on microscopy.This approach works on FIJI using a macro algorithm, ready to use or modifiable according to the user's expertise or needs. In the study of EMT in ovarian cancer, we studied four representative lines of the EMT spectrum. EMT is induced by the use of two cytokines, TNFα and TGFβ1, whose associative effect remains context-dependent and has not been described or only slightly described in ovarian cancer. Thanks to our analysis pipeline, we characterize cell plasticity by a single-cell analysis, including the morphology and the different molecular labelling performed related to the EMT. We analysed more than seventy thousand cells and combined over fifty parameters via FIJI, Celltool, and designed methods. These include a cell topography tool to normalize the distance between the nucleus and the cell membrane, or to measure cell isolation within its environment. This set of parameters was then reduced to a single statistical measure for comparison between and within the different cell lines. We were then able to effectively distinguish the four lineages from each other, as well as the impact of cytokines by line. We thus demonstrate the additivity of cytokines in the induction of EMT in each lineage along the EMT spectrum. In parallel with this study, our methodology has already demonstrated the importance of ECM on the dissemination of ovarian cancer in the presence of ascitic flow. These results show that a single-cell phenomics analysis is particularly promising for characterizing heterogeneity as well as plasticity in response to a microenvironment

This dissertation aims to identify the functional characteristics and genetic factors present within breast cancers that contribute to intratumoural heterogeneity and therapy resistance. The study utilises breast cancer cell line model systems to address epithelial-mesenchymal plasticity (EMP) at the cellular and functional level and underpins its role in cancer biology and chemoresistance. This research also interrogates the EMP programme in single cell-generated clones and through shRNA mediated functional drug screening assay identifies inhibitors that provides significant synergistic drug combinations. A comprehensive review of the drugs that can clinically target EMP was also consolidated.

On admet communément que les tumeurs proviennent de cellules échappant aux contrôles homéostatiques qui sous-tendent les structures histologiques saines et que le phénotype d’une cellule n’est pas le résultat de processus génétiques et biochimiques déterministes mais la conséquence stochastique de réseaux de régulation intra- et intercellulaires. Ce doctorat vise à étudier quantitativement l’homéostasie phénotypique de populations cellulaires et à présenter une approche à la question fondamentale, mais jusqu’alors jamais étudiée, concernant l’autonomie versus le contrôle collectif du devenir des cellules. Nous avons étudié sur le long terme, par cytométrie de flux et dans des conditions 2D puis 3D, le niveau d’expression de CD24 et CD44 de deux lignées cellulaires de cancer du sein (SUM149-PT et SUM159-PT). Trois phénotypes ont été isolés (CD24-/CD44+, CD24+/CD44+, CD24-/CD44-), ce dernier n’avait pour le moment pas été documenté dans la littérature. Le comportement phénotypique des sous-populations CD44-low et CD44-high a été caractérisé en évaluant leur proportion et en analysant leur spectre de fluorescence. Ainsi nous avons observé des comportements périodiques d’apparition et de disparition de pool de cellules caractéristiques des lignées et une re-diversification des phénotypes pour chacune des sous-population. Seule la population issue de CD24-/CD44- re-diversifiée présente le même équilibre que la population initiale non triée. En 3D, le processus de re-diversification a été observé dans les tumorsphères issues de CD24-/CD44+ et CD24+/CD44+. Les cellules CD24-/CD44- n’ont pas ce potentiel mais survivent néanmoins à l’anoïkis. Ces comportements laissent penser qu’il existe une coordination intercellulaire régulant l’équilibre des proportions phénotypiques. Pour découvrir les règles sociales régissant l’organisation spatiale inter-phénotypique, nous avons mis en place un rapporteur des variations du niveau d’expression endogène des marqueurs d’intérêt et élaboré un modèle théorique d’interactions cellulaires. Ce travail a conforté notre hypothèse selon laquelle il s’établit des règles sociales inter-cellulaires déterminant l’expression phénotypique à l’échelle uni- et pluricellulaire
It is commonly accepted that tumors arise from cells that escape the homeostatic controls which underlie the healthy histological structure and that cell phenotype is not the result of deterministic biochemical and genetic processes, but rather the stochastic and dynamic outcome of multiple intra- and intercellular regulation networks. This PhD aims to quantitatively study the phenotypic homeostasis of the cell populations and to present an approach to the fundamental question, never heretofore studied, regarding the autonomy versus collective control of cell fate. We studied in the long run, using flow cytometry and in 2D and 3D conditions, the level of expression of CD24 and CD44 of two breast cancer cell lines (SUM149-PT and SUM159-PT). Three phenotypes were isolated (CD24-/CD44+, CD24+/CD44+, CD24-/CD44-), the latter had not previously been documented in the literature. The phenotypic behavior of CD44-low and CD44-high subpopulations has been characterized by assessing their proportion and analyzing the fluorescence map. Thereby, we observed both a periodic behavior of appearance and disappearance of pool of cells characteristics of each cell lines and a phenotypic re-diversification for each subpopulation. Only the resulting population derived from CD24-/CD44- provided the same balance as the original unsorted population. 3D re-diversification process was observed in tumorspheres from CD24-/CD44+ and CD24+/CD44+. The cells CD24-/CD44did not have that potential but nonetheless outlived anoikis. These behaviors suggest that there is an inter-cell coordination regulating the balance of phenotypic proportions. To discover the social rules regulating inter-phenotypic spatial organization, we have set up a reporter of the endogenous variations of CD24 and CD44 and developed a theoretical model of cell interactions. This work has confirmed our hypothesis that inter-cellular social rules are determining the phenotypic expression at both the uni- and multicellular scales

[Truncated abstract] This study focussed on how isolation and environmental heterogeneity in salt lakes has influenced intraspecific variation in the calanoid copepod Calamoecia clitellata. Calamoecia clitellata relies on passive vectors for dispersal, and this, coupled with the insular nature of salt lakes, may promote genetic divergence at a molecular level. When contrasting environments are involved, genetic divergence may also occur at the life history level, possibly due to local adaptation. I examined the distribution of genetic variation among 14 populations in Western Australia using molecular genetic markers, and examined variation in life history traits among contrasting environments. To ascertain how isolation had influenced molecular genetic variation, I determined population genetic structure and used a phylogeographic approach to infer the impact of historical events. Environmentally induced variation was also evident in the field, with a switch from subitaneous egg production to resting egg production coinciding with changing environmental conditions. It is proposed that plasticity in life history traits has evolved in response to temporal environmental heterogeneity … It can be concluded that isolation in salt lakes in Western Australia has influenced molecular and phenotypic variation in C. clitellata in contrasting ways. At the molecular level, contemporary and historical isolation have promoted genetic divergence of populations, yet when coupled with environmental heterogeneity, marked phenotype plasticity has arisen. This study raises questions as to whether phenotype plasticity is a widespread phenomena in zooplankton found in temporary saline waters and an adaptive strategy to tolerate marked temporal environmental heterogeneity

Cette thèse se concentre sur la caractérisation de l’hétérogénéité moléculaire et métabolique des glioblastomes (GB). Nous avons identifié sept sous-classes de GB se distinguant par des activités régulatrices propres, ainsi que par des caractéristiques moléculaires et biologiques spécifiques. En intégrant des analyses transcriptomiques, métabolomiques, de flux extracellulaire, et l’évaluation de l’activité de régulateurs spécifiques, nous avons caractérisé l’hétérogénéité des cellules souches de GB (CSG) et leur plasticité en fonction de leur état (souche, différencié) ou du microenvironnement (normoxie, hypoxie, chimiothérapie). L’évaluation de leur sensibilité au témozolomide a révélé une résistance intrinsèque dans les CSG glycolytiques et l’apparition d’une résistance acquise en réponse à des stimuli environnementaux. Nos découvertes soulignent l’importance de cibler l’autophagie et le stress oxydatif pour surmonter la résistance à la chimiothérapie, et renforcent la nécessité d’approches thérapeutiques personnalisées adaptées à l’hétérogénéité et à la plasticité des GB
This thesis focuses on characterizing the molecular and metabolic heterogeneity of glioblastomas (GB). We identified seven distinct subclasses of GB, each characterized by unique regulatory activities, as well as by specific molecular and biological characteristics. Integrating transcriptomic, metabolomic and extracellular flux analyses, along with the evaluation of specific regulatory activities, we characterized the heterogeneity of GB stem cells (GSC) and their plasticity depending on their cellular state (stem, differentiated) or the microenvironment (normoxia, hypoxia, chemotherapy). The assessment of their sensitivity to temozolomide revealed an intrinsic resistance in glycolytic GSC and the emergence of acquired resistance in response to environmental stimuli. Our findings highlight the importance of targeting autophagy and oxidative stress to overcome chemotherapy resistance, and reinforce the need for personalized therapeutic approaches adapted to the heterogeneity and plasticity of GB

Microscopic dynamics and mechanical response of polymer glasses are studied in four projects using molecular dynamics simulations of a simple bead-spring model. The first project studies the interplay between physical aging and mechanical perturbation. Structural, dynamical and energetic quantities are monitored in the recovery regime following aging and uniaxial tensile deformation periods. The total engineering strain is found to control a continuous transition from transient to permanent mechanical rejuvenation: After deformation in the pre-yield regime all quantities quickly reset to pre-deformation values, while deformation around the yield point results in the erasure of aging history. Deformation in the post-yield regime, however, drives the system into a distinct thermodynamic state. In the second project, I introduce an efficient algorithm that detects microscopic relaxation events, which are the basis of aging dynamics and plasticity. I use this technique to calculate the density-density correlations from the spatio-temporal distribution of so called hops in quiescent polymer glasses at different temperatures and ages. Correlation ranges are extracted and I analyze the size distributions of collaboratively rearranging groups of particles. Furthermore, I spatially resolve dynamical heterogeneity (DH) as hop-clusters, and I compare cluster growth, as well as volume distribution during aging with the four-point dynamical susceptibility Χ₄ as the established measure of DH. The third and fourth project use the hop detection technique to investigate the link between relaxation events and local structure. Quasi-localized low-energy vibrational modes, called soft modes, are found to correlate with the location and direction of hops. In the third project, I analyze the temperature- and age-dependence of this correlation in quiescent polymer glasses, and I show that the soft modes are long lived structural features. The fourth project extends the analysis to mechanically deformed polymer glasses. I find that the spatial correlation of hops and soft modes is reduced to pre-aging values after deformation in the strain softening regime. This reveals an additional perspective on mechanical rejuvenation and substantiates the findings from the first project. In the strain hardening regime the correlation increases, and this novel effect is linked to a growing localization of the soft modes.
Science, Faculty of
Physics and Astronomy, Department of
Graduate

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
While grasses have a large ecological and economical relevance all over the world, they also may be aggressive invaders. Many African grasses are invaders in other Neotropical seasonally dry ecosystems. Positive response to disturbance and superior competitive ability had been associated with this success, but other species attributes and community features are expected to be important. Here we compare the effect of abiotic stress in the dominance of exotic over native grasses between a reserve of cerrado in the Southeast Brazil and in seasonally dry and early sucessional sites in the Hawai’i Island. Because of the low richness of native grasses in Hawai’i, mostly caused by geographical isolation and short evolutionary history, stressful sites should be more susceptible to invasion there than in the cerrado. We sampled the abundance and performance of these species in multiple sites in both ecosystems, where we also measured major abiotic variables that could cause plant stress. Moreover, we followed the reproductive phenology of species in the cerrado for two years. We estimated the effect of environmental heterogeneity on occurrence and performance of grasses with Chi-square tests and Generalized Additive Mixed Models, and described and compared the phenology of grasses with circular analysis and Pianka’s niche overlap coefficient. In addition to water and disturbance regime, local variation in soil availability and canopy cover were important in Hawaii and in the cerrado, respectively. In both sites exotic grasses were either species that perform very well in a small range of conditions (Urochloa decumbens in the cerrado, and Schyzachyrium condensatum and Hypaerrhaenia rufa in Hawaii), or more generalist species tolerant to intermediate stress (Melinis minutiflora and Melinis repens). Phenological plasticity in U. decumbens and in lesser extent M. minutiflora may contribute with their success in the cerrado, especially because native grasses had high phenological overlap with each other and 1 with two African grasses, and were weakly responsive to environmental variation. While lower rainfall and elevation and newer soils in Hawai’i limited the occurrence of M. minutiflora, they favored M. repens and a native grass. One stress tolerant exotic grass (Andropogon virginicus) was favored in wetter and undisturbed sites with young soils. In contrast, the grasses from cerrado were highly tolerant (Andropogon bicornis), intolerant (T. leiostachya) or generalist species (Loudetipsis chrisothrix) across the gradient of soil waterlogging, but in general were less affected by higher canopy cover than African grasses. Fire could indirectly favor African grasses in both ecosystems, either by increasing N soil content or decreasing canopy cover. Our findings highlight the superior performance of African grasses in mesic condition, but suggest that plastic species as M. minutiflora could succeed at intermediate levels of abiotic stress, especially in Hawai’i. Further studies should address physiological mechanisms of African versus native grasses under stress, and compare ecological and genetic attributes of African grasses in introduced sites and in the native range. Climatic change may favor or constrain different African grasses depending on the region, but the current scenarios of increasing global mean temperatures and spread of arid zones should favor these species. Local predictive models could help to prevent further spread of these species, altogether with fire avoidance and suppression of early invasions.
Apesar da grande importância ecológica e econômica das gramíneas em todo o mundo, muitas espécies são invasores agressivos. Gramíneas africanas causam impactos intensos em outros ecossistemas sazonais neotropicais, principalmente por terem grande habilidade competitiva. Nesse estudo comparamos a influência do estresse abiótico no sucesso dessas espécies em relação a gramíneas nativas em uma reserva de cerrado no sudeste do Brasil e em áreas em sucessão primária na ilha do Hawaii. Amostramos a abundância e desempenho das gramíneas nativas e exóticas mais comuns nesses dois ambientes, bem como as variáveis abióticas que poderiam causar estresse nessas espécies. No cerrado também acompanhamos a fenologia dessas espécies por dois anos. Estimamos o efeito da heterogeneidade ambiental por meio de teste de Chi-quadrado e modelos generalizados aditivos mistos, e descrevemos e comparamos os dados fenológicos com estatística circular e o coeficiente de sobreposição de nicho de Pianka. Além do regime hídrico e histórico de perturbações, a variação local em profundidade do solo e cobertura do dossel foi importante respectivamente no Havaí e no cerrado. Nos dois ambientes as gramíneas exóticas foram espécies competitivas em um espectro menor de condições (Urochloa decumbens no cerrado, e Schyzachyrium condensatum e Hypaerrhaenia rufa no Havaí), ou espécies generalistas tolerantes a estresse intermediário (Melinis minutiflora e Melinis repens). No cerrado, enquanto a fenologia aparentemente é importante para o sucesso de M. minutiflora, e principalmente U. decumbens, ela pode limitar a presença de M. repens. No Havaí, a única gramínea nativa encontrada se restringiu a áreas com intenso déficit hídrico, que também favoreceu M. repens e restringiu M. minutiflora, enquanto gramíneas exóticas tolerantes ao estresse (Andropogon virginicus) poderiam ser favorecidas em áreas mais úmidas, não perturbadas e com solos mais recentes. Por outro lado, no cerrado as gramíneas 3 nativas foram altamente tolerantes ao estresse (Andropogon bicornis), intolerantes (T. leiostachya) ou generalistas (Loudetipsis chrisothrix) ao longo do gradiente de encharcamento do solo, mas foram menos afetas pelo dossel mais fechado em relação a gramíneas africanas. Entretanto, a fenologia foi muito similar entre essas espécies, e respondeu fracamente à variação ambiental. O fogo pode favorecer indiretamente as gramíneas Africanas nos dois ecossistemas, seja por meio do aumento do teor de N no solo ou pela diminuição da cobertura do dossel. Nossos resultados reiteram o desempenho superior de gramíneas africanas em condições abióticas amenas, e também sugerem que espécies plásticas como M. minutiflora podem ser bem sucedidas mesmo em estresse intermediário, especialmente no Havaí. Estudos futuros deveriam comparar mecanismos fisiológicos e outros atributos ecológicos e genéticos de gramíneas africanas em áreas introduzidas e no habitat original. Mudanças climáticas podem favorecer ou restringir diferentes gramíneas africanas dependendo da região, mas os cenários de aumento das temperaturas médias e surgimento de mais zonas áridas devem favorecer a expansão dessas espécies. Modelos preditivos locais, aliados à prevenção de incêndios e supressão de invasões iniciais, podem a ajudar a prevenir ou limitar essa expansão no futuro.

La dynamique d’une population résulte de la combinaison de plusieurs traits d’histoire de vie qui sont façonnés par l’histoire évolutive de cette population. L’altération d’un de ces traits par des contraintes environnementales peut donc avoir des effets sur la persistance de la population. Les ajustements individuels de certains traits phénotypiques pourraient permettre à cette population de répondre rapidement à ces contraintes sans la nécessité immédiate d’adaptations génétiques. Durant cette thèse, la variabilité de certains traits a été identifiée chez 3 espèces sentinelles des écosystèmes polaires. Ces résultats permettent de mieux comprendre les associations entre ces traits et les pressions évolutives qui en sont à l’origine, ainsi que l’importance de traits tels que la personnalité dans la part inexpliquée de la variabilité de la valeur sélective des individus. A terme, nous pourrions ainsi mieux évaluer la capacité d’adaptation des populations face aux changements globaux
Population dynamics is driven by several life history traits shaped by the evolutionary history of the population. The alteration of one of these traits by environmental constraints may thus have effects on the population persistence. Individual adjustments of some phenotypic traits could then enable this population to rapidly respond to these constraints without the immediate necessity of genetic adaptations. During this PhD project, we identified variability in some of these traits in 3 sentinel species of polar ecosystems. These results allowed us to better understand the associations between these traits and the evolutionary pressures underlying these associations, as well as the importance of traits such as personality in the amount of variability in individuals’ fitness that remains unexplained. In the long term, we should then be able to better gauge the adaptive capacity of populations to face global changes

La déformation plastique du alpha-titane est fortement anisotrope. Elle met en jeu des familles de systèmes de glissement aux propriétés diverses et différents types de macles. Dans cette étude, des essais de traction sur des échantillons de alpha-titane de pureté commerciale sont couplés avec des mesures d'émission acoustique et d'extensométrie locale à haute résolution. Ces essais révèlent la présence d'un puits sur la courbe d'évolution du taux d'écrouissage. Un effet inverse de la vitesse de déformation sur la profondeur de ce puits est trouvé selon que les échantillons sont déformés suivant le sens long ou le sens travers de la tôle laminée initiale. Des analyses statistiques des lignes de glissement montrent une prédominance initiale du glissement prismatique, particulièrement prononcée dans les échantillons prélevés suivant le sens long. Une diminution de l'activité relative du glissement prismatique est observée au cours de la déformation des deux types d'éprouvettes. Les fractions volumiques de macles sont plus élevées dans les essais réalisés en sens travers mais restent néanmoins très faibles (< 5 %), en particulier au niveau du puits (< 2 %). Ces résultats fournissent une base physique pour l'élaboration d'un modèle capable d'expliquer ce comportement particulier de l'écrouissage. Le modèle s'appuie sur un schéma auto-cohérent en élastoviscoplasticité, basé sur la méthode des champs translatés et utilisant une linéarisation affine de la relation constitutive viscoplastique. Le modèle considère la plasticité cristalline et traite séparément la densité de dislocations mobiles et la vitesse moyenne des dislocations. Il suppose une plus faible sensibilité à la vitesse de déformation ainsi qu'une multiplication plus rapide des dislocations sur les systèmes prismatiques. A partir de ces différentes hypothèses, les courbes de traction sont correctement reproduites et des estimations raisonnables des coefficients de Lankford, de l'activité relative du glissement prismatique et de l'évolution des textures sont obtenues. Plus important encore, l'effet inverse de la vitesse de déformation sur la profondeur du puits du taux d'écrouissage selon l'orientation de l'axe de traction est retrouvé de manière qualitative, ce qui permet d'avancer une explication aux phénomènes observés. Par ailleurs, les mesures d'émission acoustique et d'extensométrie locale à haute résolution permettent d'analyser le caractère intermittent et ondulatoire du alpha-titane à une échelle mésoscopique. Ces données sont confrontées aux prédictions du modèle actuel et serviront de base pour le développement futur d’un modèle plus complexe
The plasticity of alpha-titanium is strongly anisotropic. It involves slip systems families with various properties and different kinds of twins. In this study, tensile tests on commercially pur alpha-titanium samples are coupled with acoustic emission and high-resolution extensometry measurements. These tests show the presence of a well on the strain dependence of the work hardening. An opposite strain rate effect on the well depth is found whether specimens are elongated along the rolling or the transverse direction of the initially laminated sheet. Slip lines analysis reveals an initial predominance of prismatic slip, particularly pronounced in specimens strained along the rolling direction. The relative activity of prismatic slip is then observed to decrease with the deformation of both kinds of samples. The twin volume fractions are higher in the tests performed in the transverse direction but still remain very low (< 5 %), especially around the well (< 2 %). These results provide grounds for elaboration of a model capable of explaining such peculiar work hardening behavior. The model relies on a self-consistent scheme in elastoviscoplasticity, based on the translated field method and an affine linearization of the viscoplastic flow rule. The model considers crystal plasticity and deals separately with mobile dislocation density and dislocation velocity. It assumes lower strain rate sensitivity as well as higher dislocation multiplication rate for prismatic systems. Based on these assumptions, the model reproduces correctly the stress-strain curves and gives sound estimates of Lankford coefficients, prismatic slip activity and textures evolution. Most importantly, the opposite effect of strain rate on the well depth with regard to the orientation of the tensile axis is qualitatively retrieved, which allows putting forward an explanation of the observed phenomena. Besides, acoustic emission and high-resolution extensometry measurements allow analyzing the intermittent and wave nature of alpha-titanium at a mesoscopic scale. These data are confronted with the predictions of the present model and will be used as grounds for the future development of a more complex model

Les cellules cancéreuses ayant des propriétés souches, appelées cellules souches cancéreuses (CSCs), sont au sommet de l'organisation hiérarchique des tumeurs et sont soupçonnées d'être responsable des rechutes et de la formation de métastases. Cependant, nous nous sommes demandé comment ces cellules sont-elles capables de supporter le stress métabolique lié à l'oncogène et de croitre dans un environnement nutritif défavorable distinct de celui d'une tumeur établie ? Nous avons mis en évidence dans ce travail le rôle de la réponse aux glucocorticoïdes dans le contrôle de la plasticité mammaire et l'induction d'une plasticité métabolique nécessaire au maintien du potentiel tumorigène des CSCs. Nous avons montré qu'en régulant la reprogrammation cellulaire via l'induction du gène MAFB, les glucocorticoïdes contrôlent le caractère souche des cellules épithéliales malignes en activant la voie de biosynthèse des hexosamines. L'efficacité anabolique de ces cellules est alors augmentée, favorisant le développement de la tumeur et des métastases. Ensemble, nos résultats suggèrent que l'inhibition de l'activité métabolique des glucocorticoïdes pourrait être une stratégie pour éliminer les CSCs et traiter les tumeurs mammaires
Cancer cells with stemness properties - generally designated cancer stem cells (CSCs) - are at the apex of the hierarchical organisation of tumours and are believed to drive tumour recurrence and metastasis formation. However, how they perform these neoplastic activities in a nutritive environment that is distinct from the one in an established tumour is unknown. Here, we unveil the prominent role of glucocorticoid activity in the control of mammary cancer cell plasticity and the induction of metabolic pliancy necessary for the tumorigenic potential of CSCs. By regulating MAFB-dependent cell reprogramming, glucocorticoids control stemness traits in malignant epithelial cells. As an integral part of this regulation, glucocorticoids activate the hexosamine biosynthetic pathway and rewire the metabolism of CSCs. The anabolic efficiency of these cells increases then, fostering tumour and metastasis development. Together, our findings suggest that inhibition of glucocorticoid metabolic activity could be an original strategy for CSC eradication and tumour treatment

Enquanto em regiões temperadas o conhecimento sobre a relação solo-vegetação é consolidado, nos trópicos e subtrópicos é preliminar. É urgente a necessidade de se determinar os fatores abióticos que controlam padrões vegetacionais visando dar suporte a estudos de recuperação e conservação. O presente estudo analisa a relação entre fatores abióticos e vegetação campestre na forma de três artigos científicos (capítulos I, II e III) e um artigo de revisão (capítulo IV). Nos primeiros três artigos, analisou-se a variância da vegetação em diferentes escalas ecológicas; e no artigo de revisão, discutiu-se de forma aplicada a variância de fatores bióticos e abióticos em resposta à degradação. Dessa forma, a presente tese objetivou responder as seguintes questões: (I) Que diferenças podem ser observadas dentro de uma mesma espécie quanto à funcionalidade e suas estratégias de alocação sob diferentes graus de disponibilidade de recursos?; (II) Quão variáveis são as propriedades físicas e químicas do solo em diferentes escalas espaciais; e existem propriedades dos solos que podem explicar com maior precisão a distribuição das espécies em diferentes escalas espaciais? (III) Que porcentagem de variância da vegetação pode ser explicada por propriedades pedológicas e climáticas; e quais características de solo e clima melhor explicam esses padrões de vegetação? No capítulo IV é apresentado um modelo conceitual sobre degradação dos campos e sua aplicação aos campos do Rio Grande do Sul (RS). Para responder as questões acima usei dados ao nível de espécie de campos calcáreos da Alemanha (capítulo I); dados ao nível de comunidade em seis áreas campestres do Rio Grande do Sul, sul do Brasil (capítulos II e III); e através da revisão de literatura relacionada à degradação, quanto à capacidade de recuperação dos campos do RS (capítulo IV). Os resultados evidenciaram que: (I) dentro de espécies ficaram evidentes duas estratégias frente à limitação de recursos, enquanto a resposta dos atributos aos diferentes tratamentos se mostrou constante; (II) a variação dos parâmetros do solo relaciona-se à escala espacial aplicada e a variância da vegetação geralmente responde a diferentes parâmetros de solo em diferentes escalas; (III) 45% da variância da vegetação entre biomas nos campos do RS foi explicada por características pedológicas e climáticas, sendo em grande parte governada pela precipitação anual e a porcentagem de saturação por alumínio do solo; e (IV) o modelo conceitual apresenta variações ao longo de dois eixos (biótico e abiótico) e poderá servir de suporte a estudos de conservação e recuperação de campos tropicais e suptropicais, bem como facilitar a tomada de decisões quanto ao manejo e conservação. Como conclusão geral, verificou-se que a vegetação campestre responde a variações ambientais em diferentes escalas espaciais e pode adotar diferentes estratégias para sobrepor filtros ambientais e processos de degradação. O entendimento da relação entre a vegetação e o meio abiótico é de grande importância para tomada de decisões quanto ao emprego de formas alternativas de manejo e conservação.
Whereas in temperate regions the abiotic-biotic relationship is well-known, in the tropics and subtropics our understanding is still preliminary. There is an urgent need to determine abiotic factors that control vegetation patterns in order to give support to restoration and conservation approaches. The present thesis analyses the relationship between abiotic factors and grassland vegetation in three original research papers (chapters I, II and III) and a review paper (chapter IV). In the first three papers, vegetation variance in response to abiotic factors was analyzed at different ecological scales; and in the fourth, the variance in biotic and abiotic factors in response to degradation process was discussed with a more applied view. Thus in this thesis the aim is to answer the following questions: (I) Which differences can be found in functional plant traits and allocation strategies within species at different levels of water and nutrient availability?; (II) How variable are physical and chemical parameters in different spatial scales; and are there soil parameters that can more accurately explain plant distribution in different spatial scales? (III) How much of RS grassland vegetation variance can be explained by soil and climatic properties; and which climatic and soil properties better explain these vegetation patterns? In chapter IV a conceptual model of grassland degradation is presented and applied to Rio Grande do Sul (RS) grasslands. To address these questions I used species-level data in a calcareous grassland in Germany (chapter I); community-level data in six sites in RS, South Brazilian grasslands (chapter II and III); and a review of literature studies concerning RS grassland degradation and restorability (chapter IV). The results showed that: (I) at a intraspecific level, the study species showed two allocation strategies in relation to resource stress, while the responses of individual traits to the soil treatments were consistent across species; (II) soil parameters variation are related to the measurement scale applied and the vegetation variance often responds to different soil parameters at different scales; (III) climatic and soil properties explained 45% of vegetation variance between biomes in RS grasslands and the main factors controlling its variance are annual precipitation and percent aluminum saturation; and (IV) the conceptual model is displayed as biotic and abiotic changes along the axes and can serve as a general framework to study degradation and restorability of tropical and subtropical grasslands, and further it may facilitate decisions on alternative management and conservation. As a general conclusion, the grassland vegetation responds to changes in the environment in different scales and may use different strategies to overcome environmental selective forces and degradation process. The understanding of this relationship is of high importance to facilitate decisions on alternative management and conservation.

Malgré les nombreux progrés réalisés ces dernières années dans la prise en charge thérapeutique du cancer broncho-pulmonaire, cette pathologie reste la première cause de décès lié au cancer dans le monde. L’enjeu majeur pour cette maladie est donc de développer de nouveaux traitements et d’optimiser l’utilisation des drogues existantes, en particulier les sels de platine. Les protocoles combinant des inhibiteurs de points de contrôle immunitaire avec des sels de platine est actuellement en plein essor malgré un certain manque en études précliniques. Dans ce travail, j’ai cherché à évaluer l'impact du cisplatine sur l'expression de PD-L1 en analysant des patients ayant reçu une chimiothérapie néo-adjuvante à base de cisplatine. Le traitement d'induction augmentait considérablement le marquage PD-L1 des cellules tumorales et immunitaires du microenvironnement. Vingt-deux patients présentaient une variation positive du pourcentage de cellules tumorales PD-L1+ après chimiothérapie néoadjuvante; dont 9 (23,1%) passant de <50% à ≥50% des cellules tumorales marquées. Nous avons également confirmé la régulation positive de PD-L1 par le cisplatine, tant au niveau de l'ARNm qu’au niveau protéique, in-vitro et in-vivo sur des souris nude et des souris immunocompétentes greffées par des tumeurs expérimentales issues de lignées cellulaires de cancer du poumon A549, LNM-R ou LLC1. L’up-régulation de PD-L1 par le cisplatine fait intervenir la voie de signalisation PI3K/AKT. De plus, l'administration combinée d'anticorps anti-PD-L1 (3 mg / kg) et de cisplatine (1 mg / kg) à des souris portant un carcinome pulmonaire réduisait significativement la croissance tumorale par rapport aux traitements en monothérapie et par rapport aux contrôles. Le cisplatine augmente donc précocément et durablement l'expression de PD-L1 et pourrait donc agir de manière synergique avec un blocage de PD-1 / PD-L1 pour améliorer la réponse tumorale aux traitements. En parallèle, nous avons pu développer une thérapie ciblée anti-neurotensine permettant de bloquer ses effets paracrines stimulants la prolifération, la croissance, et les capacités d’invasion des cellules de tumeurs pulmonaires. Les anticorps anti-neurotensine amélioraient également la sensibilité au cisplatine de tumeurs préalablement résistantes par des mécanismes qui impliquent probablement l’augmentation de l’influx et la diminution de l’efflux de platine au niveau de sa cible intra-nucléaire qu’est l’ADN. L’ensemble de ces résultats apportent du rationnel à la réalisation d’essais cliniques impliquant le cisplatine et visant par différents biais à améliorer l’efficacité de traitements systémiques de cancers broncho-pulmonaires non à petites cellules
Despite many advances in the recent years in the therapeutic management of bronchopulmonary cancer, it remains the leading cause of death linked to cancer in the world. The major challenge for this disease is therefore to develop new treatments and optimize the use of existing drugs, in particular platinum salts. The number of clinical protocols testing combined therapies including immune check-point inhibitors and platinum salts is currently increasing in lung cancer treatment, however preclinical studies and rationale are often lacking. Here, we evaluated the impact of cisplatin treatment on PD-L1 expression analyzing the clinicopathological characteristics of patients who received cisplatin-based neoadjuvant chemotherapy followed by surgery and showed that cisplatin-based induction treatment significantly increased PD-L1 staining in both tumor and immune cells from the microenvironment. Twenty-two patients exhibited positive PD-L1 staining variation after neoadjuvant chemotherapy; including 9 (23.1%) patients switching from <50% to ≥50% of stained tumor-cells. We also confirmed the up-regulation of PD-L1 by cisplatin, at both RNA and protein levels, in nude and immunocompetent mice bearing tumors grafted with A549, LNM-R, or LLC1 lung cancer cell lines. Up-regulation of PD-L1 by cisplatin involved the PI3K / AKT signaling pathway.The combined administration of anti-PD-L1 antibodies (3mg/kg) and cisplatin (1mg/kg) to mice harboring lung carcinoma significantly reduced tumor growth compared to single agent treatments and controls. Overall, these results suggest that cisplatin treatment could synergize with PD-1/PD-L1 blockade to increase the clinical response, in particular through early and sustainable enhancement of PD-L1 expression. Simultaneously, we were able to develop a targeted anti-neurotensin therapy to block its paracrine effects which stimulate proliferation, growth, and metastatic potential of lung tumor cells. Anti-neurotensin antibodies also improved the sensitivity to cisplatin of previously resistant tumors by mechanisms which probably involve increased influx and decreased efflux of platinum at the intra-nuclear level where resides its target DNA. All of these results provide a rationale for carrying out clinical trials involving cisplatin and aiming, by various ways, to improve the effectiveness of systemic treatments for non-small cell broncho-pulmonary cancers

Le tissu adipeux constitue la plus grande reserve d'energie de l'organisme. Son metabolisme est controle essentiellement par les hormones: l'insuline et les catecholamines. Un certain nombre de pathologies sont associees a une modification de la masse adipeuse, par l'intermediaire de variations du nombre et/ou du volume des adipocytes. L'obesite est un modele de choix permettant d'etudier les facteurs de regulation controlant les phenomenes d'hypertrophie (augmentation du volume) et d'hyperplasie (augmentation du nombre) adipocytaire. Nos resultats mettent en evidence le role preponderant de l'insuline sur l'hypertrophie des adipocytes blancs. En revanche, la proliferation du tissu adipeux blanc in vivo semble etre controlee de facon majoritaire par l'innervation sympathique. Par ailleurs, nous avons montre l'existence d'adipocytes bruns au sein de depots blancs. Chaque depot adipeux est caracterise par l'expression plus ou moins importante de certaines proteines impliquees dans le metabolisme adipocytaire. L'ensemble de ces resultats permet, d'une part, de souligner le role majeur de l'insuline et de l'innervation sympathique sur le developpement du tissu adipeux blanc, et, d'autre part, de mettre en evidence les differences de regulation entre les tissus adipeux blanc et brun. Enfin, ce travail remet en cause la classification initiale de ces tissus, et pose un certain nombre de questions concernant l'heterogeneite de la population adipocytaire et la nature des cellules precurseurs des deux types de tissus

Les performances du végétal et des arthropodes dont il constitue le microhabitat dépendent des températures de surface foliaire. Celles-ci peuvent dévier fortement de la température de l’air et présenter des niveaux d’hétérogénéité différents selon l'échelle spatiale considérée. La feuille atténue les températures extrêmes en rapprochant son amplitude de variation journalière de celle de la température de l’air. Cependant, cette réponse diminue l’hétérogénéité des températures de surface foliaire et les capacités de thermorégulation comportementale des arthropodes à l'échelle de la feuille. Les températures moyennes de surface foliaire atténuent peu le réchauffement, et déterminent localement la performance photosynthétique du végétal. De l’échelle de la feuille à celle de la canopée, les plantes montrent des réponses différentes au réchauffement. Ce type de changement d'échelle est primordial pour améliorer notre compréhension de l'impact des changements climatiques
Plant performance and leaf-dwelling arthropods are impacted by leaf surface temperatures. Leaf surface temperatures can show important deviation from air temperature and present different levels of heterogeneity depending on the spatial scale. The leaf buffers temperature extremes by getting closer in amplitude to air temperature. However, this physiological response decreases the heterogeneity of temperatures at the leaf surface and the opportunities for arthropods to behavioraly thermoregulate in this microclimate. Mean temperatures at the leaf surface show low buffering abilities in response to warming and locally determine photosynthetic performance. From the leaf to the canopy scale, plants show different responses to warming and scaling is crucial to increase our understanding of the impact of global warming

Nutrients are usually patchily distributed in natural soils. Plants are often able to respond to nutrient heterogeneity in artificial conditions by active plastic changes of root system morphology. The occurrence or magnitude of a foraging response can be altered by the presence of competition. However, it is unclear to what extent root foraging takes place in the field. I conducted a field experiment in order to determine the effect of an artificial nutrient patch on fine belowground biomass of (a) an established community and (b) model plants. The study array consisted of a grid of 30×30 cm plots with model plants located in the centre. Half of the plots contained the artificial patch located 5.5 cm from the model plant. Fertilizer patch treatment did not increase mean plot fine underground biomass. Instead, fine underground biomass was higher in places of greater soil moisture estimated from mean plot EIVs. Neither total model plant root biomass nor proportion of roots in the enriched quarter increased in the fertilizer treatment. Competition was probably higher in fertilized than in control plots judging by a 2-fold increase in death rate of model plants. However, greater proportion of model plants flowered in the treatment plots. Possible causes include a plastic response to the patch as well...

Clonal plants may be able to cope with spatial heterogeneity due to the physiological integration of ramets. Previous studies demonstrated that benefits of clonal integration increase with patch contrast between individual ramets. However, the same magnitude of contrast may be perceived differently in rich and poor environments. According to the theoretical work of Caraco and Kelly (1991), I expected these benefits to be the greatest in overall poor conditions and high between-patch contrast. To test this hypothesis, I conducted experiments with pairs of ramets of a stoloniferous grass, Agrostis stolonifera, grown in variously nutrient rich conditions. The experiment with pairs of ramet of similar developmental age showed only very weak effect of integration on growth of ramets, although integration significantly improved survival of ramets and also affected root-shoot ratio of ramets. Nevertheless, there were considerable benefits of integration in the experiment with developmentally older mother ramets and their daughter ramets. Contrary to the predictions, the benefits of integration were bigger in rich conditions and they decreased with increasing between-patch contrast. In addition, effect of integration on root-shoot ratio of ramets was opposite to the expected specialization for acquisition...

Size-selective harvesting (e.g. trophy hunting) involves removing specific individuals that exhibit desirable phenotypes. The ecological and evolutionary consequences of selective harvesting are controversial because it remains unclear how environmental conditions dilute or strengthen the effects of this activity. In Spain, the trophy hunting of male Iberian ibexes (Capra pyrenaica Schinz, 1838), associated with the ongoing environmental changes of the Mediterranean basin, may have undesirable consequences on the phenotypic traits and population dynamics of this species. By analysing a comprehensive hunting inventory of individual body condition, and correspondent individual metrics, gathered from wild Iberian ibex populations, this thesis is aimed at investigating the direct and indirect pathways by which hunting strategy, environmental conditions, density-dependence factors and diseases (e.g. sarcoptic mange) influences the phenotypic traits of Iberian ibex. The first study (chapter 2), carried out in the Els Ports de Tortosa-Beseit National Hunting Reserve, northeast Spain, showed that ibexes diet includes a wide range of plant species, that both diet quality and composition follows a seasonal pattern driven by landscape heterogeneity and that the diversity of ibex diet, together with the heterogeneity of Mediterranean ecosystems, hamper the use of remotely sensed products (e.g. Normalized Difference Vegetation Index) to predict dietary features, i.e. systematic faecal samples are necessary to update the nutritional condition of ibex individuals. Then, two long-term observational studies highlighted the importance of habitat characteristics on the horn growth patterns of male ibexes. The first study (chapter 3) showed that habitat fostered significant horn growth differences among males and confirmed that compensatory horn growth does not occurs in male ibexes. The second study (chapter 4) demonstrated that pine forest encroachment accounted for the decrease of annual segments length over the last two decades and suggested that the selective removal of slow-growing, small-horned males could be important to mitigate the undesirable consequences of trophy hunting. The last study (chapter 5), carried out in the Sierra Nevada National Park, south Spain, indicated that the bottom-up regulation of body condition is disrupted by highly infectious parasitic diseases, such as the sarcoptic mange. Apart from representing an important scientific contribution towards a better understanding of Iberian ibex ecology, the results of this thesis have great implications for the management of Mediterranean ungulates and open a direct venue for scientific cooperation and collaborative planning. The synergies between hunters, game managers and researchers are of utmost importance to achieve the long-term sustainability of Iberian ibex populations.
A caça seletiva (e.g. caça troféu) consiste na remoção de indivíduos com determinadas características fenotípicas de uma população selvagem. Apesar de ser uma prática comum, as suas consequências ecológicas e evolutivas são controversas, pois não é claro se as condições ambientais diluem ou intensificam os efeitos desta atividade. Em Espanha, a exploração cinegética do macho-montês (Capra pyrenaica Schinz, 1838), através da caça troféu, juntamente com as alterações ambientais registadas na bacia Mediterrânica, podem traduzir-se num efeito indesejado nas características fenotípicas e dinâmica populacional da espécie. Mediante a análise de um extenso repositório de informação individual sobre métricas de crescimento e condição corporal, esta tese tem como principal objetivo determinar os efeitos diretos e indiretos da estratégia de caça, das condições ambientais, da densidade populacional e da sarna sarcótica nas características fenotípicas da cabra-montês. O primeiro estudo (capítulo 2), realizado na Reserva Nacional de Caça de Els Ports de Tortosa-Beseit, nordeste de Espanha, confirmou o quão sazonal e diversa é a dieta da cabra-montês em ambientes Mediterrânicos e demonstrou que a heterogeneidade dos ecossistemas Mediterrânicos torna necessária a realização de amostragens sistemáticas para a atualização dos dados relativos à dieta da espécie, um aspeto crucial para o desenvolvimento de diversos caracteres sexuais secundários. Posteriormente, dois estudos observacionais revelaram a importância do habitat nos padrões de crescimento dos segmentos córneos do macho montês. O primeiro (capítulo 3) demonstrou que a heterogeneidade individual da longitude dos anéis de crescimento é potenciada por diferenças no habitat e que os machos não são capazes de recuperar do crescimento deficitário dos anéis após um eventual período de défices nutricionais. O segundo (capítulo 4) evidenciou que o aumento da área de pinhal, e consequente diminuição da área de pastagens naturais, é um fator decisivo para o decréscimo contínuo da longitude dos anéis de crescimento ao longo das últimas duas décadas. Este último, sugere também que a extração seletiva de animais com reduzidas taxas de crescimento pode contribuir para a mitigação dos efeitos da caça troféu. O último estudo (capítulo 5), realizado no Parque Nacional de Sierra Nevada, sul de Espanha, revelou que a regulação da condição corporal exercida por aspetos ambientais e populacionais é quebrada por infeções parasitárias contagiosas, como a sarna sarcóptica. Além da sua importância ecológica, os resultados aqui reportados traduzem-se num conjunto de medidas de ampla aplicabilidade para a gestão das populações de cabra-montês. A presente tese reforça a importância das sinergias entre caçadores, gestores de caça e investigadores tendo em vista a sustentabilidade da atividade cinegética e das populações ibéricas de cabra-montês.
Programa Doutoral em Biologia

Tese de doutoramento do Programa Interuniversitário de Doutoramento em Envelhecimento e Doenças Crónicas, apresentada à Faculdade de Medicina da Universidade de Coimbra
Glioblastoma (GBM) is the most malignant primary tumor of the central nervous system. Despite all efforts, the median survival time for GBM patients remains approximately between 12 to 15 months under therapy. GBM is a diffuse astrocytoma, highly proliferative, angiogenic, and locally invasive, that develops resistance to the alkylating agents used in chemotherapy, such as temozolomide (TMZ), which is considered part of the gold standard treatment. This limited success appears to be related with several mechanisms, namely: 1) the occurrence of gene mutations, that cause permanent activation and/or inhibition of several molecular signalling pathways involved in tumor growth and proliferation, such as protein kinase C (PKC) activation, cell survival, tumor suppressor genes and apoptosis; 2) the presence of a population of cells known to be chemo and radioresistant, the glioma stem-like cells (GSCs), that are responsible for generating tumor heterogeneity and recurrence after therapy, and; 3) the inexistence of a specific therapeutic target for non-GSCs and GSCs that would permit the development of more specific therapeutic approaches for this neoplasia. Therefore, in this work we aimed to: 1) study the PKC activation contribution to the aggressiveness of GBM, emphasizing the importance of combined therapeutic protocols, including TMZ with PKC inhibitors, namely tamoxifen (TMX); 2) characterize the GSCs and study their plasticity to understand glioma stem-like cells state and its differentiation properties, in order to contribute to the prevention of tumor recurrence; and 3) evaluate the potential of specific cell surface markers as therapeutic targets to non-GSCs and GSCs, allowing the accessibility of therapeutic agents most exclusively to the tumor niche, by a liposome-mediated drug delivery approach. First, using two GBM cell lines, the U87 and U118 cells, we observed that the combination of TMX and TMZ alters the phosphorylation status of PKC, by western blot. We found that TMX is an inhibitor of the p-PKC and that this combination is more effective in the reduction of proliferation and in the increase of apoptosis than each drug alone, by flow cytometry, which presents a new therapeutic strategy in GBM treatment. We then concluded that the combination of TMX and TMZ seems to potentiate the effect of each other in GBM cell lines. In order to study the heterogeneity between GBM cells and further understand the variability in the chemotherapeutic response, we next isolated and characterized a human GBM cell line, termed GBM11, obtained by surgical biopsy from a patient bearing a recurrent GBM, and compared the effect of TMX in monotherapy and in combination with TMZ on this GBM cell line with that observed in U87 and U118 cell lines. We observed that the effect of TMX plus TMZ or with TMX alone on GBM11 cells proliferation, death or migration capability, by flow cytometry and scratch assays, was similar, suggesting that, for recurrent tumors, the best choice of second-line treatment may be TMX alone, which may also reduce putative side effects of combined treatment with TMZ. The chemo- and radioresistance of GBM are also due to GSCs which contribute to tumor growth and relapse, highlighting this cell population as a main focus for GBM therapeutic research. We considered that the understanding of GBM stem state plasticity is of utmost importance to identify the mechanisms involved in GSCs resistance to therapy, which may justify tumor recurrence and so, constitute a step forward to the identification of new approaches to treat GBM. Our results demonstrated that, in four GBM cell lines and in the respectively GSC lines, the plasticity of the GBM stem-like cell state is based on the modulation of specific markers expression associated with this state, such as SOX2 or as Connexin 46 and 43, through immunofluorescence, western blot and PCR real time assays. Moreover, by immunohistochemistry analyses, we observed that this dynamic expression is in accordance with the upregulation of these stem-like cell markers in human samples of higher glioma grades, namely GBM, compared to lower grades, suggesting a direct correlation with the poor prognosis of GBM patients. As so, due to the plasticity of the stem-like cells status, the strategy of targeting both GSCs and non-GSCs may represent a promising approach in order to overcome tumor aggressiveness, and eventually to avoid the known chemotherapeutic side effects, which could improve the survival time and quality of life of GBM patients. In this regard, we next evaluate the potential of the cell surface nucleolin (NCL), described as overexpressed in cancer cells, as a target to specifically recognize non-GSCs and GSCs, taunting a possible therapeutic target for drug delivery in two different GBM cell lines. For that, we used a previously designed F3-peptide-targeted sterically stabilized pH-sensitive liposome (SLpH), which specifically recognizes nucleolin, as a tool to target overexpressed-nucleolin cells. Overall, we showed that NCL overexpression ensures an efficient drug delivery in both cells with stem-like and non-stem-like phenotypic characteristics, by flow cytometry assays, which could validate NCL as a potential therapeutic target in GBM. Altogether, our results showed: 1) a synergistic effect of TMX and TMZ in GBM cell lines and a more efficient effect of TMX alone in recurrent GBM compared to the combined therapy; 2) the plasticity of stem-like cell state through the reversibility of stem-like cell markers expression, and the identification of putative markers associated with this reversibility, the SOX2 and Cx46 and 43, which constitutes a step closer to the understanding of stem cell behaviour; and 3) that the success of targeting both non-GSCs and GSCs, through the nucleolin target, may be the basis for developing a specific treatment for GBM.
O Glioblastoma (GBM) é o tumor primário mais maligno do sistema nervoso central. Apesar de todos os esforços, o tempo médio de sobrevivência para doentes com GBM permanece aproximadamente entre os 12 a 15 meses sob terapia. O GBM é um astrocitoma difuso, altamente proliferativo, angiogénico e localmente invasivo, que desenvolve resistência aos agentes alquilantes utilizados na quimioterapia, como a temozolomida (TMZ), que é considerada parte do tratamento padrão. Este sucesso limitado parece estar relacionado com vários mecanismos, tais como: 1) a ocorrência de mutações genéticas que causam ativação permanente e / ou inibição de várias vias de sinalização molecular envolvidas no crescimento e proliferação de tumores, como a ativação da proteína cínase C (PKC), na sobrevivência celular, na inibição de genes supressores de tumores e apoptose; 2) a presença de uma população de células conhecidas como quimio- e radiorresistentes, as células de glioma do tipo estaminal (GSCs), que são responsáveis ​​pela heterogeneidade tumoral e recorrência após a terapia e; 3) a inexistência de um alvo terapêutico para não-GSCs e GSCs que permita o desenvolvimento de abordagens terapêuticas mais específicas para esta neoplasia. Assim, neste trabalho, objetivámos: 1) estudar a contribuição da ativação da PKC para a agressividade do GBM, enfatizando a importância de protocolos terapêuticos combinados, incluindo a TMZ com inibidores de PKC, nomeadamente o tamoxifeno (TMX); 2) caraterizar as GSCs e estudar a plasticidade das propriedades destas células estaminais do GBM, no sentido de compreender o estado estaminal do glioma e, consequentemente, entender as propriedades de diferenciação, contribuindo para a recorrência do tumor; e 3) avaliar o potencial de marcadores de superfície celular específicos, como alvos terapêuticos para as não-GSCs e GSCs, a fim de permitir a acessibilidade de agentes terapêuticos mais exclusivamente ao nicho do tumor, por meio de uma abordagem de administração de fármacos mediada por lipossomas. Inicialmente, usando duas linhas celulares de GBM, a U87 e a U118, observámos que a combinação de TMX e TMZ altera o estado de fosforilação da PKC, por western blot. Descobrimos que o TMX é um inibidor da p-PKC e que esta combinação é mais eficaz na redução da proliferação e no aumento da apoptose do que cada fármaco em monoterapia, através de ensaios de citometria de fluxo, o que pode representar uma nova estratégia terapêutica no tratamento do GBM. Concluímos, então, que a combinação de TMX e TMZ potencializa o efeito entre si nas linhas celulares de GBM. No sentido de estudar a heterogeneidade entre células de GBM e compreender melhor a variabilidade da resposta à quimioterapia, isolámos e caracterizámos uma linha celular de GBM humana, denominada GBM11, obtida através de uma biópsia cirúrgica de um doente com glioblastoma recorrente, e comparámos o efeito do TMX em monoterapia e em combinação com a TMZ, nesta linha celular, com o observado nas linhas celulares U87 e U118. Na verdade, observámos que o efeito do TMX e TMZ ou do TMX sozinho nas células de GBM11 sobre a proliferação celular, morte ou capacidade de migração, através de ensaios de citometria de fluxo e migração, era semelhante, o que pode sugerir que, para os tumores recorrentes, como o caso do GBM11 previamente tratado com TMZ, a melhor escolha do tratamento de segunda linha pode ser apenas TMX, a fim de reduzir os efeitos secundários putativos do tratamento combinado com TMZ. A quimio- e a radiorresistência do GBM devem-se, também, à existência de GSCs, que contribuem para o crescimento tumoral e recorrência destacando-se, assim, esta população celular como o foco principal da investigação terapêutica no GBM. Consideramos que a compreensão da plasticidade do estado estaminal no GBM é de extrema importância para identificar os mecanismos e fatores envolvidos na resistência das GSCs à terapia, o que pode justificar a recorrência do tumor e, portanto, constituir um progresso na identificação de novas abordagens terapêuticas. Os nossos resultados demonstraram, em quatro linhas celulares de GBM e nas respetivas linhas de GSCs, a plasticidade do estado estaminal com base na modulação da expressão de marcadores específicos associados, tais como o SOX2 e outros marcadores como a Conexina 46 e 43, através de ensaios de imunofluorescência, western blot e PCR em tempo real. Além disso, através de ensaios de imunohistoquímica, verificámos que essa expressão dinâmica está de acordo com a regulação positiva destes marcadores celulares em graus superiores de amostras humanas de glioma, nomeadamente no GBM, comparativamente a graus inferiores, sugerindo uma correlação direta com o mau prognóstico de doentes com GBM. Assim, devido à plasticidade do estado estaminal, a estratégia de atingir designadamente ambas as GSCs e não-GSCs pode representar uma abordagem importante no sentido de diminuir a agressividade do tumor e, eventualmente, evitar os efeitos colaterais quimioterapêuticos conhecidos, o que pode melhorar o tempo e a qualidade de vida de doentes com GBM. Neste sentido, avaliámos o potencial da nucleolina (NCL) de superfície celular, descrita como estando sobre-expressa nas células tumorais, como um alvo terapêutico para o reconhecimento específico de ambas as não-GSCs e GSCs, contribuindo para a entrega direcionada de fármacos encapsulados em nanopartículas, em duas linhas celulares de GBM. Para isso, utilizámos um lipossoma previamente desenhado, sensível ao pH e estericamente estabilizado, contendo na sua constituição um péptido F3, capaz de reconhecer especificamente a nucleolina constituindo, assim, uma ferramenta- alvo para as células com sobre-expressão de nucleolina. Em suma, demostrámos que a sobre-expressão de nucleolina per se pode identificar ambas as não-GSCs e GSCs, através de ensaios de citometria de fluxo, mediando a entrega direcionada intracelular, o que pode validar a NCL como um potencial alvo terapêutico no GBM. Em conclusão, o presente estudo demonstrou: 1) um efeito sinergístico do TMX e TMZ em linhas celulares de GBM e um efeito mais eficiente do TMX em monoterapia numa situação de GBM recorrente em comparação com a terapia combinada; 2) a plasticidade do estado estaminal através da reversibilidade da expressão dos marcadores de células do tipo estaminal e a identificação de dois marcadores putativos associados a essa reversibilidade, o SOX2 e a Cx46 e 43, constituindo um passo mais próximo na compreensão do comportamento das células estaminais; e 3) que o sucesso em atingir especificamente células não-GSCs e GSCs, através da sobre-expressão de nucleolina, poderá ser a base de desenvolvimento de um tratamento específico para o GBM.
Conselho Nacional de Desenvolvimento Tecnológico (CNPq), Brasil; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasil; Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ); Pró-Saúde - Associação Beneficente de Assistência Social e Hospitalar, Brasil; FEDER/COMPETE/ FCT PTDC/EBB-EBI/120634/2010 e PDTC/QUI-BIQ/120652/2010