Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis in cells. It has been implicated as a therapeutic target for various metabolic diseases like type II diabetes and obesity. However, its role in cancer is context-dependent and therefore warrants further studies to explore its possible use as a therapeutic target. AMPK can either promote or retard the growth of cancer cells depending on other cues and stresses in the milieu of the cancer cells. This study aims to understand AMPK signalling in response to extracellular cues of matrix deprivation and matrix stiffness that are important determinants of metastasis. 1) Calcium-Oxidant Signalling Network Regulates AMPK Activation upon Matrix Deprivation. Recent work from our lab, as well as others, has identified a novel role for the cellular energy sensor AMP-activated protein kinase in epithelial cancer cell survival under matrix deprivation. However, the molecular mechanisms that activate AMPK upon matrix-detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, while re-attachment to the matrix leads to its dephosphorylating and inactivation. Since matrix-detachment leads to loss of integrin signalling, we investigate whether integrin signalling negatively regulates AMPK activation. However, modulation of FAK or Src, the major downstream components of integrin signalling, fails to cause a corresponding change in AMPK signalling. Further investigations reveal that the upstream AMPK kinases, LKB1 and CaMKKβ, contribute to AMPK activation upon detachment. Additionally, we show LKB1 phosphorylation and cytosolic translocation upon matrix deprivation, which might also contribute to AMPK activation. In LKB1-deficient cells, we find AMPK activation to be predominantly dependent on Caskβ. We observe no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment is not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signalling and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that ER calcium release induced store-operated calcium entry (SOCE) contributes to intracellular calcium increase, leading to ROS production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. We find a significant increase in LKB1 as well as pACC levels in breast tumour tissues in comparison to normal tissues. Further, we observe a significant correlation between LKB1 and pACC levels in breast tumour tissues suggesting that LKB1-AMPK signaling pathway is active in vivo in breast cancers. Thus, the Ca2+/ROS triggered LKB1/CaMKK-AMPK signalling cascade may provide a quick, adaptable switch to promote survival of metastasising cancer cells. 2) Extracellular Matrix Stiffness Regulates Stemless through AMPK. Cancer cells experience changes in extracellular matrix stiffness during cancer progression. However, the signalling pathways utilised in sensing matrix stiffness are poorly understood. In this study, we identify AMPK pathway as a possible mechanosensory pathway in response to matrix stiffness. AMPK activity, as measured by downstream target phosphorylation, is found to be higher in soft matrix conditions. We additionally show that compared to stiff matrices, soft matrices increase stemless properties, as evidenced by the increased expression of stemless markers, which is dependent on AMPK activity. Thus, we elucidate a novel mechanotransduction pathway triggered by matrix stiffness that contributes to stemness of cancer cells by regulating AMPK activity. Taken together, our study identifies a novel calcium-oxidant signaling network in the rapid modulation of AMPK signaling in the context of matrix detachment. This pathway is especially relevant in the context of metastasising cancer cells that may not face energy stress in the blood stream but are matrix-deprived. Inhibition of AMPK might compromise the viability of these circulating cells thereby reducing the metastatic spread of cancer. Our study further suggests that varying stiffnesses experienced by cancer cells can modulate AMPK activity and this, in turn, regulates stem-like properties. Thus our study provides novel insights into various extracellular cues that regulate this kinase and contribute to cell survival and metastasis. This knowledge can be utilised in the stage-specific use of AMPK inhibitors in the treatment of breast cancer patients.

Orientador: William Dias Belangero / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-28T23:43:39Z (GMT). No. of bitstreams: 1 Faria_LidiaDornelasde_M.pdf: 3066212 bytes, checksum: 6cb5506682740e2fcb2de4b1694998a3 (MD5) Previous issue date: 2015 / Resumo: A Terapia por ondas de choque (TOC) é uma alternativa não invasiva utilizada como método de indução a formação óssea que consiste em pulsos sonoros de alta energia transmitidas de modo focal a um tecido específico. Artigos demonstram aumento de vascularização, que ativação de proteínas como BMP (bone morphogenic protein) e Erk (extracellular signal-regulated kinase) induzindo diferenciação osteogênica após sua utilização no tecido ósseo. O presente estudo visou avaliar os níveis das proteínas Erk e Akt (akutely transforming), envolvidas na cascata protéica responsiva a deformação celular gerada por estímulo mecânico e consequente transformação em estímulo bioquímico induzindo a osteogênese. Os animais selecionados para o estudo foram anestesiados e divididos em dois diferentes grupos, onde no dia 1, o primeiro grupo foi submetido a TOC em sessão única de 500 impulsos gerados por aparelho eletrohidráulico a 0,12mJ/mm²na tíbia intacta e o segundo não recebeu TOC. Na sequência, os animais foram divididos em 3 subgrupos para cada tempo de segmento de 7, 14 e 21 dias A determinação dos níveis das proteínas propostas foi realizada por meio de immunoblotting. A fosforilação das proteínas Erk e Akt dos tecidos ósseos das tíbias extraídas dos ratos aumentou nos grupos submetidos a TOC após 7, 14 e se manteve elevado até o 21° dia quando comparado ao controle / Abstract: Extracorporeal shock wave therapy (ESWT) is a non-invasive alternative used as a method for inducing bone formation that consists of high-energy acoustic pulses transmitted in a focal way to a specific tissue. Studies show increase in vascularization which activate proteins such as BMP and Erk inducing osteogenic differentiation after its use in the bone tissue. The present study aimed at evaluating the levels of Erk and AKT proteins involved in the protein cascade responsive to cell deformation in biochemical stimulus inducing osteogenesis. The animals selected for the study were under anesthesia and divided in two different groups where on day 1 the first group was submitted to ESWT in one 500 pulse-session generated by an electrohydraulic device at 0,12mJ/mm² in intact tibia and fibula and the second did not receive ESWT. Then the animals were divided into 3 sub-groups, one for each segment times of 7, 14 and 21 days. Immunoblotting analysis was performed to determine the levels of the proposed proteins. The Erk and Akt protein phosphorylation of the bone tissues of extracted tibia from the animals increased in the groups submitted to ESWT and kept elevated until the 21st day when compared to the control group / Mestrado / Fisiopatologia Cirúrgica / Mestra em Ciências

La thérapie cellulaire se positionne comme une stratégie prometteuse pour inciter le cœur infarci à se régénérer. A cet effet, des études récentes placent des espoirs considérables dans l’utilisation des cellules souches embryonnaires et notre laboratoire a déjà démontré comment les différencier en progéniteurs cardiovasculaires, un type de précurseurs cellulaires qui ne peut aboutir qu’à la formation de cardiomyocytes, de cellules endothéliales ou de cellules de muscles lisses. Cet engagement précoce réduit leur capacité de prolifération anarchique et en même temps leur permet de rester suffisamment plastiques pour éventuellement s’intégrer plus facilement avec le tissue hôte. Cependant, les études précliniques et cliniques d’injection de ces cellules s’avérèrent décevantes. Malgré de légères améliorations de la fonction cardiaque, on observa une trop faible survie cellulaire ainsi qu’un taux de rétention des cellules dans le myocarde remarquablement bas. Afin d’étudier ce problème, mes travaux de thèse ont porté non seulement sur la conception de nouveaux biomatériaux pouvant servir de moyen de transport et d’intégration des cellules dans la zone infarcie, mais aussi sur la conception de biomatériaux permettant de contrôler précisément l’environnement cellulaire au cours du processus de différenciation de cellules souches pluripotentes humaines en cardiomyocytes. Grâce aux importantes interactions entre nos laboratoires de recherche fondamentale et de recherche clinique, nous avons tout d’abord développé de nouvelles techniques de fabrication et de caractérisation de patches de fibrine cellularisés qui sont récemment entrés dans un essai clinique de phase I. A partir de cette formulation clinique approuvée par les autorités de régulation, nous avons élaboré toute une gamme de matériaux composites uniquement à base de matières premières pertinentes dans ce cadre clinique, dans le but d’améliorer la maturation des progéniteurs cardiovasculaires une fois greffés sur le cœur défaillant. Dans cette optique, nous avons également développé un modèle in vitro permettant d’étudier précisément l’influence combinée de la rigidité du substrat et du confinement spatial sur la différenciation des cellules souches en cardiomyocytes. Grâce à des techniques de microfabrication sur substrat mou, il a été possible de positionner précisément les cellules souches pluripotentes dans des espaces restreints d’élasticité variable. Ainsi, nous avons pu observer que même en utilisant des protocoles chimiques éprouvés basés sur la modulation de cascades de signalisation impliquées dans le développement cardiaque, une très forte hétérogénéité pouvait apparaître en fonction de l’environnement physique des cellules. Nous avons ainsi pu extraire les caractéristiques principales permettant une différenciation cardiaque efficace, reproductible et standardisée et les avons appliquées à la fabrication d’une nouvelle génération de patches composés de matériaux cliniques et de couches multiples de bandes synchrones de cardiomyocytes. De fait, ces travaux ouvrent de nouvelles voies dans l’utilisation de biomatériaux pour la production industrielle de cardiomyocytes et pour la fabrication de patches cliniques, cellularisés ou non, dans le traitement de l’insuffisance cardiaque. / Cell therapy is a promising strategy to help regenerate the damaged heart. Recent studies have placed a lot of hopes in embryonic stem cells and our lab had previously found a way to differentiate them into cardiac progenitors, cells that can only differentiate into cardiomyocyte, endothelial cells or smooth muscle cells. This early commitment decreases their proliferative capabilities, yet maintains their plasticity for better integration inside the host tissue. However, clinical and pre-clinical injection studies did not really meet the expectations. Even though slight improvements in cardiac function were demonstrated, very low cell viability has been observed, as well as a very low retention of the cells inside the myocardium. To address this problem, my PhD projects not only focus on the design of new biomaterials to act as a vehicle for cell delivery and retention in the infarcted area, but also on the design of biomaterials that control the cellular environment during the differentiation of pluripotent stem cells into cardiomyocytes. Going back and forth between the labs and the clinics, we first developed new techniques for the fabrication and the characterization of a cell-laden fibrin patch that is now undergoing phase I clinical trial. From the approved clinical formulation, we then propose new blends of clinical materials that will eventually improve the maturation of the cardiac progenitors once grafted onto the failing heart. In this perspective, we developed an in vitro model to investigate the combined influence of matrix elasticity and topographical confinement on stem cell differentiation into cardiomyocytes. By using microfabrication techniques to pattern pluripotent stem cells on substrates of controlled stiffness, we demonstrate that even using a widely recognized chemical-based protocol to modulate signaling cascades during differentiation, much heterogeneity emerges depending on the cellular physical environment. We thus extracted the main features that led to controlled and reproducible cardiac differentiation and applied it to the fabrication of next generation of multi-layered anisotropic cardiac patches in compliances with clinical requirements. This work opens new routes to high-scale production of cardiomyocytes and the fabrication of cell-laden or cell-free clinical patches.

Buňka tvoří složitý biologický systém vystavený mnoha mimobuněčným mechanickým podnětům. Hlubší pochopení jejího mechanického chování je důležité pro charakterizaci její odezvy v podmínkách zdraví i nemoci. Výpočtové modelování může rozšířit pochopení mechaniky buňky, která může přispívat k vytvoření vztahů mezi strukturou a funkcí různých typů buněk v různých stavech. Za tímto účelem byly pomocí metody konečných prvků (MKP) vytvořeny dva bendotensegritní modely buňky v různých stavech: model vznášející se buňky pro analýzu její globální mechanické odezvy, jako je protažení nebo stlačení, a model buňky přilnuté k podložce, který vysvětluje odezvu buňky na lokální mechanické zatížení, jako třeba vtlačování hrotu při mikroskopii atomárních sil (AFM). Oba zachovávají základní principy tensegritních struktur jako je jejich předpětí a vzájemné ovlivnění mezi komponentami, ale prvky se mohou nezávisle pohybovat. Zahrnutí nedávno navržené bendotensegritní koncepce umožňuje těmto modelům brát v úvahu jak tahové, tak i ohybové namáhání mikrotubulů (MTs) a také zahrnout vlnitost intermediálních filament (IFs). Modely předpokládají, že jednotlivé složky cytoskeletu mohou měnit svůj tvar a uspořádání, aniž by při jejich odstranění došlo ke kolapsu celé buněčné struktury, a tak umožňují hodnotit mechanický příspěvek jednotlivých složek cytoskeletu k mechanice buňky. Model vznášející se buňky napodobuje realisticky odezvu síla-deformace během protahování a stlačování buňky a obě odezvy ilustrují nelineární nárůst tuhosti s růstem mechanického zatížení. Výsledky simulací ukazují, že aktinová filamenta i mikrotubuly hrají klíčovou úlohu při určování tahové odezvy buňky, zatímco k její tlakové odezvě přispívají podstatně jen aktinová filamenta. Model buňky přilnuté k podložce dává odezvu síla-hloubka vtlačení ve dvou různých místech odpovídající nelineární odezvě zjištěné experimentálně při AFM. Výsledky simulací ukazují, že pro chování buňky je rozhodující místo vtlačení a její tuhost určují aktinová povrchová vrstva, mikrotubuly a cytoplazma. Navržené modely umožňují cenný vhled do vzájemných souvislostí mechanických vlastností buněk, do mechanické úlohy komponent cytoskeletu jak individuálně, tak i ve vzájemné synergii a do deformace jádra buňky za různých podmínek mechanického zatížení. Tudíž tato práce přispívá k lepšímu pochopení mechaniky cytoskeletu zodpovědné za chování buňky, což naopak může napomáhat ve zkoumání různých patologických podmínek jako je rakovina a cévní choroby.

Indiana University-Purdue University Indianapolis (IUPUI) / Articular cartilage is subjected to dynamic mechanical loading during normal daily activities. This complex mechanical loading, including cell deformation and interstitial fluid flow, affects chondrocyte mechano-chemical signaling and subsequent cartilage homeostasis and remodeling. Focal adhesion kinase (FAK) and Src are known to be main mechanotransduction proteins, but little is known about the effect of mechanical loading on FAK and Src under its varying magnitudes and types. In this study, we addressed two questions using C28/I2 chondrocytes subjected to the different types and magnitudes of mechanical loading: Does a magnitude of the mechanical loading affect activities of FAK and Src? Does a type of the mechanical loading also affect their activities? Using fluorescence resonance energy transfer (FRET)-based FAK and Src biosensor in live C28/I2 chondrocytes, we monitored the effects of interstitial fluid flow and combined effects of cell deformation/interstitial fluid flow on FAK and Src activities. The results revealed that both FAK and Src activities in C28/I2 chondrocytes were dependent on the different magnitudes of the applied fluid flow. On the other hand, the type of mechanical loading differently affected FAK and Src activities. Although FAK and Src displayed similar activities in response to interstitial fluid flow only, simultaneous application of cell deformation and interstitial fluid flow induced differential FAK and Src activities possibly due to the additive effects of cell deformation and interstitial fluid flow on Src, but not on FAK. Collectively, the data suggest that the intensities and types of mechanical loading are critical in regulating FAK and Src activities in chondrocytes.

Indiana University-Purdue University Indianapolis (IUPUI) / Bone is a dynamic organ that responds to its external environment. Cell signaling cascades are initiated within bone cells when changes in mechanical loading occur. To describe these molecular signaling networks that sense a mechanical signal and convert it into a transcriptional response, we proposed the mechanosome model. “GO” and “STOP” mechansomes contain an adhesion-associated protein and a nucleocytoplasmic shuttling transcription factor. “GO” mechanosomes functions to promote the anabolic response of bone to mechanical loading, while “STOP” mechanosomes function to suppress the anabolic response of bone to mechanical loading. While much work has been done to describe the molecular mechanisms that enhance the anabolic response of bone to loading, less is known about the signaling mechanisms that suppress bone’s response to loading. We studied two adhesion-associated proteins, Src and Pyk2, which may function as “STOP” mechanosomes. Src kinase is involved in a number of signaling pathways that respond to changes in external loads on bone. An inhibition of Src causes an increase in the expression of the anabolic bone gene osteocalcin. Additionally, mechanical stimulation of osteoblasts and osteocytes by fluid shear stress further enhanced expression of osteocalcin when Src activity was inhibited. Importantly, fluid shear stress stimulated an increase in nuclear Src activation and activity. The mechanism by which Src participates in attenuating anabolic gene transcription remains unknown. The studies described here suggest Src and Pyk2 increase their association in response to fluid shear stress. Pyk2, a protein-tyrosine kinase, exhibits nucleocytoplasmic shuttling, increased association with methyl-CpG-binding protein 2 (MBD2), and suppression of osteopontin expression in response to fluid shear stress. MBD2, known to be involved in DNA methylation and interpretation of DNA methylation patterns, may aid in fluid shear stress-induced suppression of anabolic bone genes. We conclude that both Src and Pyk2 play a role in regulating bone mass, possibly through a complex with MBD2, and function to limit the anabolic response of bone cells to fluid shear stress through the suppression of anabolic bone gene expression. Taken together, these data support the hypothesis that “STOP” mechanosomes exist and their activity is simulated in response to fluid shear stress.

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Der Herzmuskel muss sich einer dynamischen und sich mechanisch verändernden Umgebung anpassen. Die Mechanosignaltransduktion ermöglicht es Zellen mechanischen Kräfte zu erfassen und durch nachgeschaltete biochemische Signalkaskaden darauf zu reagieren. Obwohl verschiedene Gewebestrukturen und Proteine damit in Verbindung gebracht wurden, wie das Herz die mechanischen Kräfte wahrnimmt, ist unser Verständnis der kardialen Mechanosignaltransduktion unvollständig. Durch Dehnung aktivierte Ionenkanäle spielen eine wichtige Rolle bei der mechanosensitiven Autoregulation des Herzens. Um die funktionelle Rolle von PIEZO1 in Kardiomyozyten zu untersuchen, habe ich daher PIEZO1 in induzierten pluripotenten Stammzellen mittels Genomeditierung deletiert. Die PIEZO1-/- Zellen wurden dann in lebensfähige, herzähnlich schlagende Kardiomyozyten differenziert. In phänotypische Analysen der elektrophysiologischer Eigenschaften, Zellmorphologie und der herzähnlichen Schlagaktivität habe ich den Effekt der PIEZO1-deletion in genomeditierten Kardiomyozyten untersucht. Die Deletion von PIEZO1 zeigte zum ersten Mal, dass es PIEZO1-abhängige dehnungsaktivierte und Kalzium-Ströme in vom Menschen stammenden differenzierten Kardiomyozyten gibt. Dies legt nahe, dass PIEZO1 eine Rolle in der Mechanosignaltransduction in Herzzellen spielt. Darüber hinaus zeigte eine RNA-Sequenz Analyse, dass der Verlust von PIEZO1 in vom Menschen stammenden differenzierten Kardiomyozyten mit der Herunterregulation von Proteinen korreliert, die für die extrazellulärer Matrix von Bedeutung sind. Diese Daten unterstreichen die Rolle von PIEZO1 in Kardiomyozyten und legen seine Bedeutung für die Organisation und Struktur der extrazellulären Matrix nahe. / The cardiac muscle has to adapt in a highly dynamic mechanical environment. Mechanotransduction is the process that allows cells to sense the mechanical forces and respond by downstream biochemical signaling cascades. Although different tissue structures and proteins have been implicated in how the heart senses the mechanical forces, yet our understanding in cardiac mechanotransduction is incomplete. Stretch-activated channels (SACs) have been suggested to play an important role in the mechanosensitive autoregulation of the heart. PIEZO1 is a stretch-activated channel and has been involved in vascularization, erythrocyte volume homeostasis and regulation of the baroreceptor reflex, yet its role in cardiac mechanotransduction has not been described. To study the functional role of PIEZO1 in cardiomyocytes I have generated a PIEZO1 knockout (KO) human induced pluripotent cell (hiPSC) line using genome editing technology. The genome edited cells were then differentiated into viable, beating cardiomyocytes. Different phenotypic analyses were conducted, including the evaluation of electrophysiological characteristics, observation of cell morphology and beating activity of the genome edited hiPSC-derived cardiomyocytes. With this approach the aim was to gain more insight into PIEZO1 function in cardiomyocytes using a reliable, efficient and reproducible human cellular model system. For the first time PIEZO1-dependent calcium transients and stretch-activated currents were observed in hiPSC-derived cardiomyocytes (hiPSC-CMs). This proposes a possible role of PIEZO1 as a cardiac mechanotransducer. Furthermore, RNA-seq analysis revealed that loss of PIEZO1 in hiPSC-CMs is associated with downregulation of the expression of extracellular matrix-associated proteins. These data highlight the role of PIEZO1 in cardiomyocytes and suggest its implication in extracellular matrix organization and structure.