Tesi sul tema "Cellular Migration"

Migration of myogenic cells is an important step in myogenesis and skeletal muscle repair. Migration is required for the cells to reach the site of damage, for their alignment and subsequent fusion. Limited migration is also one of the limitations of proposed therapies of diseases, such as Duchenne Muscular Dystrophy (DMD). Therefore, revealing the regulators of myogenic cell migration is important for improving our knowledge of myogenesis, but could also be applied in therapies for conditions, associated with loss of muscle mass and muscle weakness. In this thesis, extracellular and intracellular regulation of C2C12 myoblast migration was investigated. It was demonstrated that medium conditioned by myotube cultures in vitro, is capable of inducing the migration and chemotaxis of myoblasts. A model of serially passaged myoblasts was used to reveal potential changes in the migratory behaviour of these cells, in the context of skeletal muscle ageing. PI3K/AKT and MAPK/ERK pathways were investigated and their requirement for the process of myoblast migration was revealed. Further activation of these pathways with phospho-tyrsoine phosphatase and PTEN inhibitor Bpv(Hopic) was associated with larger increases in myoblast migration. Silencing of either PI3K/AKT or MAPK/ERK signalling pathways, in a situation where the other pathway remained activated, resulted in a significant inhibition of myoblast migration. Similarly, inhibition of FAK signalling, using the PF-228 inhibitor did not significantly affect PI3K/AKT and MAPK/ERK pathways, but resulted in reduced myoblast migration, suggesting the indispensability of individual signalling pathways for myoblast migration in response to myotube CM, regardless of the activity of other signalling pathways. Finally, considering the link between myoblast fusion and migration and in an attempt to propose genetic targets for future research, an investigation was made on the expression of Spire and Formin genes, involved in actin polymerisation and intracellular trafficking, in myoblasts undergoing differentiation and fusion. The expression of these genes was revealed in C2C12 myoblasts and it was demonstrated that the expression levels of two of these genes (Spire1 and Formin1) are altered following inhibition of myoblast differentiation/fusion by both 10μM Bpv(Hopic) and serial passaging, suggesting their potential association with these processes. Further investigations to reveal the function of Spire and Formin genes and their protein products in skeletal muscle are proposed.

2011/2012<br>ABSTRACT - The ability to control the cellular microenvironment, such as cell-substrate and cell-cell interactions at the micro- and nanoscale, is important for advances in several fields such as medicine and immunology, biochemistry, biomaterials, and tissue engineering. In order to undergo fundamental biological processes, most mammalian cells must adhere to the underlying extracellular matrix (ECM), eliciting cell adhesion and migration processes that are critical to embryogenesis, angiogenesis, wound healing, tissue repair, and immunity response, to cite few. For instance, upon receiving and responding to complex molecular signals, cells migrate from the epithelial layers to target locations, where they differentiate to form specialised cells that make up various organs and tissues. However, improper cell adhesion and migration have been implicated in disease states such as tumour invasion and cancer cell metastasis. In the past few years, several tailored surfaces that aim to mimic cell-ECM interactions have been developed, including biodevices based on proteins and shorter peptide chains, DNA, RNA, and lipids. Among the different nanomaterials employed in such studies, those resulting from self-assembled monolayers (SAMs) of alkanethiols on gold (Au) probably represent the most useful and flexible model systems of surface engineering for cell biology evaluations. These platforms are promising for tuning surface properties or to introduce novel biofunctionalities via coupling reactions with various alkanethiols tail groups that can be exposed to the solution phase. Deeply involved in this research field, the aim of this doctoral work was to gain a basic understanding and develop chemical strategies towards the controlled multidirectional (i. e. bidirectional) cellular migration on tailored Au surfaces. As already described, several artificial substrates were prepared in the last years to better understand the cellular responses to different mechanical and biochemical surface properties. To date, however, no reports concerning the bidirectional movement of the cells along a defined substrate have been published. The controlled multidirectional migration offers several advantages respect to the monodirectional approach, since the cellular functions can be obtained and, in principle, recycled with spatio-temporal control. In fact, once the cells reach the target position along the surface and perform specific biochemical or physiological cellular functions (repair, growth, movement, immunity, communication, and phago/endocytosis), the reversible movement could allow to recall them back to the starting position. By this way, also studies of dynamic cell-cell interactions can also be exploited allowing for a deeper knowledge about the fundamentals of the cell biology and biochemistry. The multidirectional migration can be determined through the production of dynamic haptotactic chemical gradients along Au surfaces. Specifically, the long-term idea of this project is to use SAMs of thiolated DNA chains (DNA-SH) adsorbed onto Au surfaces as a template for the hybridisation with complementary peptidic nucleic acid (PNA) strands functionalised with peptidic motifs able to stimulate cellular motility. By this way, supramolecular chemical gradients of motogenic motifs can be bound in a directional manner onto Au surfaces and dictate a dynamic bidirectional cell migration. Framed in such research project, this doctoral thesis focused on the production of a static, monodirectional and motogenic gradient along Au surfaces, to prove the efficacy of a specific peptidic motif, and generate modified PNA strands necessary for the production of the corresponding dynamic gradients. Chapter 1 deals with a careful description of the biochemical mechanisms involved in the cellular migration process, focusing on the chemotaxis and haptotaxis phenomena. Through a comprehensive overview on the state of the art concerning the biomimetic approaches for studying the cellular migration, the main strategies towards the engineering of different surfaces, have been thoroughly reviewed by means of key examples reported in the literature. Chapter 2 is centred on the results obtained by producing and using the thiolated peptide isoleucine-glycine-aspartic acid-glutammine-lysine-1-thiol decanoic acid (IGDQK-SH) as a motogenic motif for both cells found in physiologic environment (fibroblasts) and phatological conditions (MDA-MB-231 cancer cells). Upon synthesising IGDQK-SH (1), a systematic approach for the generation of the motogenic chemical gradient along Au surfaces has been developed. Evidences of the success of the preparation of such static chemical gradient were obtaining by engaging specific characterisation methodologies, such as water contact angle (WCA), Atomic Force Microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) analysis, along with computational analysis of peptide’s conformations once bound to the different Au surfaces. This allowed determining the biophysical properties, morphology, chemical composition and possible structure of the resulting Au surfaces, respectively. IGDQK-SH chemical gradient was able to induce and control the cellular migration of the two different cell lines showing interesting differences related to the surface properties and peptide’s conformations after the formation of SAMs in the presence of filler molecules with different hydrophobicity. In particular, the experimental findings suggested a pronounced migration attitude of the cancer cells upon their exposition to the IGDQK-SH-bearing surfaces, compared to the fibroblasts. This result might suggest a role of the IGD motif in the stimulation of the cancer cells towards their enhanced motility and metastatic progression in vivo, and is currently under investigation. Once proved the efficiency of the motogenic peptide, we moved towards the final goal of the project synthesising two functionalised single-stranded PNA dodecamers (ssPNA 12-mers) 30 and 31 bearing the Rhodamine B and the tetrapeptide IGDQ for characterising the chemical gradient through microscopy-based investigations and stimulate cell motility, respectively. Chapter 3 indeed provides a general overview on the different methodologies available for the solid phase peptide synthesis (SPPS) describing the synthetic attempts to produce the desired PNAs. Attention will be focused on the Fmoc/Cbz protecting group strategy, which allowed us to isolate the target PNA oligomers.<br>RIASSUNTO - Lo studio e il controllo dei microambienti cellulari, quali interazioni cellula-superficie e cellula-cellula, assumono particolare rilevanza in diversi campi scientifici come medicina e immunologia, biochimica, ingegneria dei tessuti e dei biomateriali. Al fine di svolgere le funzioni biologiche fondamentali, le cellule dei mammiferi devono poter aderire alla matrice extra-cellulare (ECM) sottostante, provocando adesione e migrazione cellulare che risultano essenziali, ad esempio, nei processi di embriogenesi, angiogenesi e riparazione dei tessuti. Infatti, stimolate da complessi segnali molecolari, le cellule migrano dagli strati epiteliali verso il loro target, raggiunto il quale si differenziano e specializzano formando organi e tessuti. Al contrario, anomalie nell’adesione e migrazione cellulare possono dar luogo al sorgere di diverse malattie, quali tumori e metastasi cancerose. Negli ultimi anni sono state progettate e sviluppate diverse superfici, compresi biodispositivi basati su proteine, DNA, RNA e lipidi, con lo scopo di mimare le interazioni cellula-ECM. Tra i nanomateriali impiegati in questi studi, quelli derivanti dalla formazione di self-assembled monolayers (SAMs) di tioli alchilici su oro (Au) rappresentano probabilmente il modello più adatto e flessibile di superfici ingegnerizzate al fine di valutare i fenomeni biologici. Questi sistemi permettono di modulare le proprietà delle superfici o di introdurre nuovi gruppi funzionali attraverso reazioni di coupling, sfruttando la presenza dei gruppi terminali dei tioli che risultano esposti al solvente. Lo scopo di questo lavoro di dottorato è quello di acquisire le conoscenze di base e di sviluppare metodologie chimiche al fine di indurre e controllare la migrazione cellulare multidirezionale (i.e. bidirezionale) su superfici di Au funzionalizzate. Come già descritto, negli anni sono stati impiegati diversi substrati artificiali con lo scopo di meglio comprendere le reazioni cellulari alle differenti proprietà meccaniche e biochimiche di tali superfici. Tuttavia, ad oggi, non sono stati ancora pubblicati studi riguardanti il movimento bidirezionale di cellule lungo un substrato. Rispetto all’approccio monodirezionale, la migrazione multidirezionale controllata offre diversi vantaggi, poiché in questo modo le funzioni cellulari possono essere indotte e, in principio, replicate attraverso un controllo spazio-temporale. Infatti, una volta raggiunto l’obiettivo sulla superficie e svolte le funzioni cellulari specifiche (riparazione, crescita, movimento, immunità, comunicazione, fagocitosi), il movimento reversibile permette di richiamare le cellule alla posizione iniziale. Pertanto, anche lo studio delle interazioni dinamiche cellula-cellula potrà fornire una più approfondita conoscenza della biologia e della biochimica cellulare. La migrazione multidirezionale può essere determinata attraverso la produzione di gradienti chimici dinamici aptotattici su superfici di Au. Nel dettaglio, l’idea alla base di questo progetto è quella di utilizzare SAMs di catene di DNA aventi un tiolo terminale (ssDNA-SH) per la funzionalizzazione di superfici di Au, e usarle come template nell’ibridizzazione con catene complementari di acido nucleico peptidico (PNA) aventi un peptide in grado di stimolare la migrazione cellulare. In questo modo è possibile generare un gradiente chimico supramolecolare direzionale lungo le superfici di Au al fine di ottenere al migrazione cellulare bidirezionale. Questa tesi di dottarato è focalizzata sulla produzione di un gradiente statico, monodirezionale e motogenico su superfici di Au, per provare l’efficacia di un motivo peptidico specifico, e generare filamenti di PNA modificati, necessari per la produzione di corrispondenti gradienti dinamici. Il Capitolo 1 riporta un’accurata descrizione dei meccanismi biochimici coinvolti nei processi di migrazione cellulare, concentrandosi sui fenomeni di chemiotassi e aptotassi. Dopo un’esauriente studio dello stato dell’arte, le principali strategie di funzionalizzazione di diverse superfici sono state dettagliatamente riviste attraverso gli esempi chiave riportati in letteratura. Il Capitolo 2 è centrato sui risultati ottenuti producendo e utilizzando il pentapeptide composto da isoleucina-glicina-acido aspartico-glutammina-lisina-acido decanoico-1-tiolo (IGDQK-SH) come motivo motogenico per le cellule presenti in ambienti fisiologici (fibroblasti) e in condizioni patologiche (MDA-MB-231 cellule cangerogene). Una volta sintetizzato l’IGDQK-SH(1) è stato sviluppato un approccio sistematico per la produzione del gradiente motogenico sulle superfici di Au. Al fine di verificare l’effettiva presenza di tale gradiente sono state utilizzate differenti tecniche di caratterizzazione, quali water contact angle (WCA), Atomic Force Microscopy (AFM) e X-ray photoelectron spectroscopy (XPS) analysis, oltre all’analisi computazionale per stabilire la conformazione del peptide una volta legato alla superficie di Au. Questo ha permesso di determinare le proprietà biofisiche, la morfologia, la composizione chimica e la possibile struttura delle superfici finali di Au funzionalizzate. Il gradiente chimico di IGDQK-SH ha permesso di indurre e controllare la migrazione di due differenti linee cellulari, mostrando interessanti differenze relative alle proprietà della superficie e alla conformazione del peptide dopo la formazione del SAMs in presenza di molecole filler aventi diversa idrofobicità. In particolare, i risultati sperimentali suggeriscono una maggiore attitudine alla migrazione da parte delle cellule cancerogene su superfici di Au funzionalizzate con il peptide IGDQK-SH rispetto ai fibroblasti. Questo risultato potrebbe suggerire un ruolo del motivo IGD nella stimolazione della mobilità e della progressione metastatica in vivo delle cellule cancerogene, ed è attualmente oggetto di ricerca. Una volta provata l’efficienza del peptide motogenico, obiettivo finale di questo lavoro è stata la sintesi di due singoli filamenti di dodecamero di PNA 30 e 31 funzionalizzati rispettivamente con la Rodammina B e il tetrapeptide IGDKQ al fine di caratterizzare il gradiente chimico utilizzando tecniche microscopiche e stimolare la migrazione cellulare. Il Capitolo 3 offre una visione generale sulle differenti metodologie impiegate nella sintesi peptidica in fase solida (SPPS), descrivendo le strategie sintetiche utilizzate per produrre gli oligomeri di PNA necessari, con particolare attenzione per la strategia dei gruppi protettivi Fmoc/Cbz.<br>RéSUMé - La possibilité de contrôler le microenvironnement cellulaire, telles que les interactions cellule-substrat et cellule-cellule à l’échelle micro et nano, est importante pour les avancées dans certains domaines tels que la médecine et l’immunologie, la biochimie, les biomatériaux, et l’ingénierie tissulaire. Afin d’être soumis aux processus biologiques fondamentaux, la plupart des cellules mammifères doivent adhérer à la matrice extracellulaire sous-jacente (ECM), en induisant des procédés d’adhésion et de migration cellulaires qui sont critiques à l’embryogenèse, l’angiogenèse, la cicatrisation des blessures, la réparation des tissus, et la réponse immunitaire, pour n’en citer que quelques-uns. Par exemple, lorsque les cellules reçoivent et répondent à des signaux moléculaires complexes, elles migrent des couches épithéliales aux emplacements cibles, où elles se différencient afin de former des cellules spécialisées qui constituent divers organes et tissus. Cependant, une adhésion et une migration cellulaire incorrecte ont été impliquées dans des états de maladie tels que l’invasion de tumeur et les métastases de cellules cancéreuses. Au cours des dernières années, plusieurs surfaces confectionnées dans le but d’imiter les interactions cellule-ECM ont été développées, incluant des bio dispositifs basés sur des protéines et des chaines peptidiques courtes, sur l’ADN, l’ARN, et sur des lipides. Parmi les différents nanomatériaux employés dans de telles études, ceux résultants de monocouches auto-assemblées (SAMs) d’alcanethiols sur l’or (Au) représentent probablement les systèmes modèles les plus utiles et flexibles d’ingénierie de surface pour des évaluations biologiques cellulaires. Ces plateformes sont prometteuses pour moduler des propriétés de surface ou pour introduire de nouvelles biofonctionnalités via des réactions de couplage avec divers groupements alcanethiols qui peuvent être exposés à la phase liquide. Fortement impliqué dans ce domaine de recherche, l’objectif de ce travail de doctorat était d’acquérir une compréhension basique et de développer des stratégies chimiques à l’égard de la migration cellulaire multidirectionnelle contrôlée (i.e. bidirectionnelle) sur des surfaces d’Au fonctionnalisées. Comme cela a déjà été décrit, plusieurs substrats artificiels ont été préparés au cours des dernières années afin de mieux comprendre les réponses cellulaires à différentes propriétés mécaniques et biochimiques de surface. Cependant, jusqu’à présent, aucun rapport sur le mouvement bidirectionnel de cellules le long d’un substrat défini n’a été publié. La migration multidirectionnelle contrôlée offre plusieurs avantages par rapport à l’approche monodirectionnelle, puisque les fonctions cellulaires peuvent être obtenues et, en principe, recyclées avec un contrôle spatio-temporel. En fait, une fois que les cellules atteignent la position cible le long de la surface et réalisent des fonctions cellulaires biochimiques ou physiologiques spécifiques (réparation, croissance, mouvement, immunité, communication, et phago/endocytose), le mouvement réversible pourrait permettre de les rappeler à la position de départ. De cette façon, des études d’interactions cellule-cellule dynamiques peuvent également être exploitées, menant à une connaissance plus approfondie des fondamentaux de la biologie et biochimie des cellules. La migration multidirectionnelle peut être établie par la production de gradients dynamiques chimiques haptotactiques le long de surfaces d’Au. Plus précisément, l’idée à long terme de ce projet est d’utiliser des SAMs de chaînes d’ADN thiolées (ADN-SH) adsorbées sur des surfaces d’Au comme modèles pour l’hybridation avec des brins d’acides nucléiques peptidiques (ANP) complémentaires, fonctionnalisés avec des motifs peptidiques capables de stimuler la motilité cellulaire. De cette façon, les gradients chimiques supramoléculaires de motifs motogéniques peuvent être liés d’une manière directionnelle sur des surfaces d’Au et peuvent dicter une migration cellulaire bidirectionnelle dynamique. Cette thèse de doctorat, incluse dans un tel projet de recherche, s’est concentrée sur la production d’un gradient statique, directionnel et motogénique le long de surfaces d’Au, afin de prouver l’efficacité d’un motif peptidique spécifique, et de générer des brins d’ANP modifiés nécessaires à la production des gradients dynamiques correspondant. Le Chapitre 1 donne une description minutieuse des mécanismes biochimiques impliqués dans le procédé de migration cellulaire, se concentrant sur les phénomènes de chimitaxie et haptotaxie. A travers une vue d’ensemble complète sur l’état de l’art des approches biomimétiques pour l’étude de la migration cellulaire, les stratégies principales menant à l’ingénierie de différentes surfaces, ont été revues en détails à l’aide d’exemples clés reportés dans la littérature. Le Chapitre 2 est centré sur les résultats obtenus par la formation et l’utilisation du peptide thiolé isoleucine-glycine-aspartic acid-glutammine-lysine-1-thiol decanoic acid (IGDQK-SH) en tant que motif motogénique pour les cellules à la fois trouvées dans un environnement physiologique (fibroblastes) et dans des conditions pathologiques (cellules cancéreuses MDA-MB-231). Après avoir synthétisé IGDQK-SH (1), une approche systématique pour la génération du gradient chimique motogénique le long de surfaces d’Au a été développée. Des preuves du succès de la préparation de tels gradients chimiques statiques ont été obtenus par des méthodologies de caractérisation spécifiques, telles que des analyses d’angle de contact (WCA), par microscopie à force atomique (AFM) et par spectrométrie photoélectronique X (XPS), accompagné d’analyses informatiques des conformations du peptide une fois lié aux différentes surfaces d’Au. Ceci a permis de déterminer les propriétés biophysiques, la morphologie, la composition chimique et la structure possible des surfaces d’Au résultantes, respectivement. Le gradient chimique de IGDQK-SH a pu induire et contrôler la migration cellulaire de deux différentes lignes cellulaires montrant des différences intéressantes liées aux propriétés de surface et aux conformations du peptide après la formation des SAMs en présence de molécules de remplissage présentant différentes hydrophobicités. En particulier, les résultats expérimentaux ont suggéré une attitude de migration prononcée des cellules cancéreuses, après leur exposition aux surfaces portant l’IGDQK-SH, comparé aux fibroblastes. Ce résultat peut suggérer un rôle du motif IGD dans la stimulation des cellules cancéreuses à l’égard de leur mobilité accrue et progression métastatique in vivo, et est actuellement analysé. Une fois que l’efficacité du peptide motogénique fut prouvée, nous nous sommes penchés sur l’objectif final du projet, en synthétisant deux dodécamères d’ANPs simples brins fonctionnalisés 30 et 31, portant la Rhodamine B et le tétrapeptide IGDQ pour caractériser le gradient chimique par des analyses de microscopie et pour stimuler la motilité de la cellule, respectivement. En effet, le Chapitre 3 donne une vue d’ensemble sur les différentes méthodologies disponibles pour la SPPS décrivant les essais synthétiques afin de synthétiser les ANPs désirés. L’attention sera concentrée sur la stratégie impliquant les groupements protecteurs Fmoc/Cbz, qui nous a permis d’isoler les oligomères d’ANP cibles.<br>XXV Ciclo<br>1984