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

Keloid scars (KS) are a type of abnormal scarring which is unique to humans. They extend beyond the confines of the original wound margins, do not regress over time and invade the surrounding unaffected skin. The mechanisms involved in the formation of KS remain largely unknown. Clinical observation has shown that in areas where increased tension occurs, such as the sternum, there is a greater propensity for developing KS. However, the precise relationship between skin tension and KS development is yet to be identified. In view of this, I hypothesize that skin tension plays a significant role in KS development by affecting tension-related biomarkers that may alter the phenotype of KS. Therefore, the objective of this research was to investigate the effect of skin tension in the formation of KS. To this end, the first aim was to identify possible targets among biomarkers that might contribute to the differentiation between KS and hypertrophic scars in tissue and cells obtained from diverse anatomical locations. The second aim was to investigate the effect of tension-related biomarkers on extracellular matrix (ECM) steady-state synthesis in keloid fibroblasts (KF) extracted from a highly tensioned body region (the sternum). The third aim was to develop a 3D in-vitro model to mimic in-vivo tension and to evaluate KF behaviour and ECM synthesis under tension. To achieve these aims 21 biomarkers were selected from published microarray and in-house microarray studies, the inclusion criteria was based on up-regulation of the genes in KS in relation to fibrosis, apoptosis and tension. For this purpose, samples from normal skin and KS were used to perform qRT-PCR screening in tissue and cells, as well as protein analysis by Western and In-cell Western blot. The siRNA knockdown technique was employed to evaluate the functional role of the tension-related markers in keloid fibroblasts. Finally, a photogrammetry technique was employed to evaluate skin tension in-vivo; the results from this evaluation were used in the development and design of a novel in-vitro 3D-model. The first biomarker screening in tissue showed convincing up-regulation of five tension-related targets (Hsp27, PAI-2 and α2β1-integrin, MMP-19 and CPRP). In addition, the expression of the above-mentioned targets was significantly higher in samples from the sternum compared to samples from other anatomical locations. To further validate these findings, the screening of the 21 biomarkers was assessed in KS and KF taken from the sternum. The results demonstrated over expression of 3 of the 5 tension-related targets (Hsp27, PAI-2 and α2β1-Integrin). It was also demonstrated that Hsp27, PAI-2 and α2β1-Integrin performed a functional role in terms of regulation of extracellular matrix production and deposition in KF when their expression was down-regulated by siRNA knockdown. Using the newly created 3D model, it was shown that mechanical tension significantly induced the expression of Hsp27, PAI-2 and α2β1-Integrin as well as ECM components such as Collagen I. Furthermore, the results showed that the knockdown of the expression of Hsp27, PAI-2 and α2β1-integrin in fibroblast populated collagen lattices subjected to tension influenced not only the ECM synthesis but also adhesion and spreading genes in keloid and normal fibroblasts. In summary, this research convincingly shows that skin tension alters keloid fibroblast behaviour, morphology, mechano-responsive gene expression and extracellular matrix production. The findings from my thesis offer insight into keloid pathobiology and provide options for targeted treatment of specific genes affected in keloids by biomechanical stress.

abstract: Brain micromotion is a phenomenon that arises from basic physiological functions such as respiration (breathing) and vascular pulsation (pumping blood or heart rate). These physiological processes cause small micro displacements of 2-4µm for vascular pulsation and 10-30µm for respiration, in rat models. One problem related to micromotion is the instability of the probe and its ability to acquire stable neural recordings in chronic studies. It has long been thought the membrane potential (MP) changes due to micromotion in the presence of brain implants were an artefact caused by the implant. Here is shown that intracellular membrane potential changes are a consequence of the activation of mechanosensitive ion channels at the neural interface. A combination of aplysia and rat animal models were used to show activation of mechanosensitive ion channels is occurring during a neural recording. During simulated micromotion of displacements of 50μm and 100μm at a frequency of 1 Hz, showed a change of 8 and 10mV respectively and that the addition of Ethylenediaminetetraacetic acid (EDTA) inhibited the membrane potential changes. The application of EDTA showed a 71% decrease in changes in membrane potential changes due to micromotion. Simulation of breathing using periodic motion of a probe in an Aplysia model showed that there were no membrane potential changes for <1.5kPa and action potentials were observed at >3.1kPa. Drug studies utilizing 5-HT showed an 80% reduction in membrane potentials. To validate the electrophysiological changes due to micromotion in a rat model, a double barrel pipette for simultaneous recording and drug delivery was designed, the drug delivery tip was recessed from the recording tip no greater than 50μm on average. The double barrel pipette using iontophoresis was used to deliver 30 μM of Gadolinium Chloride (Gd3+) into the microenvironment of the cell. Here is shown a significant reduction in membrane potential for n = 13 cells across 4 different rats tested using Gd3+. Membrane potential changes related to breathing and vascular pulsation were reduced between approximately 0.25-2.5 mV for both breathing and heart rate after the addition of Gd3+, a known mechanosensitive ion channel blocker. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2020

Résumé : Le cytosquelette d’actine génère et mécanotransduit des forces. Dans cette étude, nous montrons que l’actine branchée corticale, qui dépend de RAC1, WAVE et des complexes Arp2/3 contenant ARPC1B, est spécifiquement détectée par le senseur Coronin1B, qui signale, via WISp39 et l’inhibiteur de cycline/CDK p21, à la cellule, de progresser dans le cycle cellulaire. En conséquence, la formation d’un lamellipode et la migration persistante des cellules qui en découle, est corrélée à la durée de la phase G1. L’actine branchée corticale détermine l’entrée en phase S des cellules, en intégrant les stimuli solubles des facteurs de croissance et la mécanotransduction des adhérences à la matrice extracellulaire et aux cellules voisines. Le complexe Arp2/3 est globalement sur-exprimé dans le cancer du sein. Parmi ses sous-unités, la sur-expression de l’isoforme ARPC1B est le plus fort facteur prognostique pour les patientes. En outre, l’inhibition du complexe Arp2/3 bloque la prolifération de lignées de carcinomes mammaires et de mélanomes transformées par l’oncogène RAC1, contre laquelle il n’existe pas de thérapie ciblée. La découverte du checkpoint de l’actine branchée corticale apporte ainsi de nouvelles options pronostiques, diagnostiques et thérapeutiques dans les cancers. / The actin cytoskeleton generates and mechanotransducts forces. Here we report that the cortical branched actin that depends on RAC1, WAVE and ARPC1B-containing Arp2/3 complexes is specifically monitored by the Coronin1B sensor, WISp39 and the cyclin-CDK inhibitory protein p21, to control cell cycle progression. Accordingly, the duration of the G1 phase scales with the persistence of single cell migration, ensuing from branched actin and lamellipodium protrusion. Cortical branched actin determines the cell decision to enter into S phase by integrating soluble stimuli from growth factors and mechanotransduced signals, such as substratum rigidity and cell density. The Arp2/3 complex is overall overexpressed in brest cancer. Among its subunits, The ARPC1B isoform overexpression is the strongest prognostic factor for patients. Furthermore, Arp2/3 inhibition prevents the growth of mammary carcinoma and melanoma cell lines transformed by the RAC1 oncogene, for which no targeted therapy is available. The discovery of the cortical branched actin checkpoint thus provides diagnostic and therapeutic opportunities in cancer.

Le modèle cellulaire de magnétostimulation (MTS) développé dans l'équipe a permis d'obtenir des résultats préliminaires sur les essais de perméabilité endothéliale ainsi que sur les reconstructions et modélisation des fibres d'actine. Les premiers résultats en Magnétocytométrie (MTC) montrent une augmentation de rigidité cellulaire suite à une stimulation mécanique du tapis cellulaire. Les images confocales ont permis de mettre en évidence une restructuration des filaments d'actine dans les cellules endothéliales microvasculaires pulmonaires (HPMEC) soumises au stress mécanique, ainsi qu'une relocalisation des VE-Cadhérines nécessaires aux jonctions intercellulaires. Ces deux résultats concordent avec l'apparition de 'gaps' ou trous inter cellulaires qui permettent d'expliquer l'augmentation de perméabilité mesurée entre les cellules soumises au stress mécanique et la situation contrôle. Des difficultés techniques retardent le travail et l'obtention de résultats en nombre important. Par exemple l'emploi de certaines techniques de marquages difficilement compatibles avec certains supports de culture. Cela empêche dans certains cas la confirmation visuelle de résultats obtenus par MTC. La viabilité cellulaire lors des expérimentation ne permet pas d’allonger les temps d’étude d’une même population cellulaire, ce qui limite certains résultats. Les premiers résultats sont à compéter par une étude plus approfondie des niveaux d'expression de facteurs pro-inflammatoires ainsi que des voies de signalisation et de régulation des VE-Cadhérines et des intégrines AlphaV-Beta3. L’effet de différentes molécules utilisées en clinique devrait être étudié sur le modèle de stress mécanique. / The cellular model of magnétostimulation ( MTS) developed in the team allowed to obtain preliminary results(profits) on the tries(essays) of endothéliale permeability as well as on the reconstructions and the modelling of the fibers of actine. The first results(profits) in Magnétocytométrie ( MTC) show an increase of cellular rigidity further to a mechanical stimulation of the cellular carpet(mat). The confocal images allowed to highlight a restructuring of the strands of actine in cells(units) microvascular lung endothéliales ( HPMEC) subjected(submitted) to the mechanical stress, as well as a relocation of the VE-Cadhérines necessary for the intercellular junctions. These two results(profits) suit to the appearance of ' gaps ' or holes inter cellular which allow to explain the increase.

Cytoskeletal tension plays an important role in numerous biological functions of adherent cells, including mechanosensing of the cell’s microenvironment, mechanotransduction, cell spreading and migration, cell shape stability, and in stem cell lineage. It is believed that for normal biological functions the cell must maintain its cytoskeletal tension stable, at a preferred set-point level, under external perturbations. This is known as tensional homeostasis. Any breakdown of tensional homeostasis is closely associated with disease progression, including cancer, atherosclerosis, and thrombosis. The exact mechanism and the relevant environmental conditions for the maintenance of tensional homeostasis are not yet fully understood. This thesis investigates the impacts of substrate stiffness, availability of functional cadherin junctions and steady shear stress on tensional homeostasis of cells and cell clusters. We define tensional homeostasis as the ability of cells to maintain a consistent level of tension with low temporal traction field fluctuations. Traction forces of isolated cells, multicellular clusters, and monolayer are measured using micropattern traction microscopy. Temporal fluctuations of the traction field are calculated from time-lapsed traction measurements. Results demonstrated that substrate stiffness, cadherin cell-cell junctions and shear stress all impact tensional homeostasis. In particular, we found that stiffer substrates promoted tensional homeostasis in endothelial cells, but were detrimental to tensional homeostasis in vascular smooth muscle cells. We also found that E-cadherins were essential for tensional homeostasis of gastric cancer cells and that extracellular and intracellular mutations of E-cadherin had domain-specific effects on tensional homeostasis. Finally, laminar flow-induced shear stress led to increased traction field fluctuations in endothelial cell monolayers, contrary to reports of physiological shear promoting vascular homeostasis. A possible reason for this discrepancy might be the limitation of our approach which could not account for mechanical balance of traction forces in the monolayers. Through the exploration of these environmental factors, we also found that tensional homeostasis was a length scale-dependent and cell type-dependent phenomenon. These insights suggest that future studies need to take a more comprehensive approach and aim to make observations of different cell types on multiple length scales, in order decipher the mechanism of tensional homeostasis and its role in (patho)physiology. / 2021-08-30T00:00:00Z

La forme du noyau peut varier significativement au cours du développement ou lors de processus pathologiques en raison des forces mécaniques émanant du microenvironnement ou générées par le cytosquelette. L’impact de la morphologie nucléaire sur la machinerie transcriptionnelle n’est cependant pas connu. En utilisant plusieurs approches afin de manipuler la morphologie nucléaire, nous avons observé que des changements de forme de l’enveloppe nucléaire régulent l’activité de AP1 et TEAD. Nous avons montré que l’aplatissement du noyau augmente la phosphorylation de c-Jun et la translocation de YAP, conduisant à une augmentation de la transcription des gènes cibles de AP1 et TEAD. Nous avons également observé que l’aplatissement du noyau se produit au cours du cycle cellulaire et favorise la prolifération via l’activation de TEAD et AP1 qui stimulent la progression de la phase G1 à la phase S. / .The shape of the cell nucleus can vary considerably during developmental and pathological processes as a consequence of the mechanical forces emanating from the microenvironment or generated by the cytoskeleton. However the impact of nuclear morphology on the transcriptional machinery is not known. Using a combination of tools to manipulate the nuclear morphology, we observed that changes in nuclear shape regulate the activity of AP1 and TEAD. We showed that nuclear flattening increases c-Jun phosphorylation and YAP nuclear translocation, leading to transcriptional induction of AP1 and TEAD-target genes. Surprisingly, we found that nuclear compression is necessary and sufficient to mediate c-Jun and YAP activation in response to cell- generated contractility or cell spreading. We additionally observed that nuclear flattening occurs during the cell cycle and promotes proliferation via TEAD and AP1- dependent G1 to S progression.

Indiana University-Purdue University Indianapolis (IUPUI) / Mesenchymal stem cells (MSCs) are responsive to mechanical stimuli that play an essential role in directing their differentiation to the chondrogenic lineage. A better understanding of the mechanisms that allow MSCs to respond to mechanical stimuli is important to improving cartilage tissue engineering and regenerative medicine. Hydrostatic pressure (HP) in particular is known to be a primary mechanical force in joints. However, little is known about the underlying mechanisms that facilitate HP mechanotransduction. Understanding the signaling pathways in MSCs in transducing HP to a beneficial biologic response and their interrelationship were the focus of this thesis. Studies used porcine marrow-derived MSCs seeded in agarose gel. Calcium ion Ca++ signaling, focal adhesion kinase (FAK) involvement, and sirtuin1 activity were investigated in conjunction with HP application. Intracellular Ca++ concentration was previously shown to be changed with HP application. In our study a bioreactor was used to apply a single application of HP to the MSC-seeded gel structures and observe Ca++ signaling via live imaging of a fluorescent calcium indicator in cells. However, no fluctuations in Ca++ concentrations were observed with 10 minutes loading of HP. Additionally a problem with the biore actor design was discovered. First the gel was floating around in the bioreactor even without loading. After stabilizing the gel and stopping it from floating, there were still about 16 µm of movement and deformation in the system. The movement and deformation was analyzed for the gel structure and different parts of the bioreactor. Furthermore, we investigated the role of FAK in early and late chondrogenesis and also its involvement in HP mechanotransduction. A FAK inhibitor was used on MSCs from day 1 to 21 and showed a dose-dependent suppression of chondrogenesis. However, when low doses of FAK inhibitor added to the MSC culture from day 21 to 42, chondrogenesis was not inhibited. With 4 hour cyclic HP, FAK phosphorylation increased. The beneficial effect of HP was suppressed with overnight addition of the FAK inhibitor to MSC medium, suggesting FAK involvement in HP mechanotransd ucation by MSCs. Moreover, sirtuin1 participation in MSC chondrogenesis and mechanotransduc tion was also explored. The results indicated that overnight sirtuin1 inhibition in creased chondrogenic gene expression (Agc, Col2, and Sox9) in MSCs. Additionally, the activity of sirtuin1 was decreased with both 4 hour cyclic hydrostatic pressure and inhibitor application. These two together demonstrated that sirtuin1 inhibition enhances chondrogenesis. In this research we have investigated the role of Ca++ signaling, FAK involvement, and sirtuin1 activity in the mechanotransduction of HP in MSCs. These understand ings about the mechanisms regulating the chondrogenesis with respect to HP could have important implications for cartilage tissue engineering and regenerative studies.

Indiana University-Purdue University Indianapolis (IUPUI) / The Keap1-Nrf2 pathway regulates a wide range of cytoprotective genes, and has been found to serve a protective and beneficial role in many body systems. There is limited information available, however, about its role in bone homeostasis. While Nrf2 activation has been suggested as an effective method of increasing bone mass and quality, there have been conflicting reports which associate Keap1 deficiency with detrimental phenotypes. As Keap1 deletion is a common method of Nrf2 activation, further study should address the impacts of various methods of regulating Nrf2 expression. Also, little research has been conducted on the specific pathways by which Nrf2 activation improves bone quality. In this study, the effects of alterations to Nrf2 activation levels were explored in two specific and varied scenarios. In the first experiment, moderate Nrf2 activation was achieved via partial deletion of its sequestering protein, Keap1, in an aging mouse model. The hypothesis tested here is that moderate Nrf2 activation improves bone quality by affecting bone metabolism and response to mechanical loading. The results of this first experiment suggest a subtle, sex-specific effect of moderate Nrf2 activation in aging mice which improves specific indices of bone quality to varying degrees, but does not affect loading-induced bone formation. It is likely that the overwhelming phenotypic impacts associated with aging or the systemic effects of global Keap1 deficiency may increase the difficulty in parsing out significant effects that can be attributed solely to Nrf2 activation. In the second experiment, a cell-specific knockout of Nrf2 in the osteocytes was achieved using a Cre/Lox breeding system. The hypothesis tested here is that osteocyte-specific deletion of Nrf2 impairs bone quality by affecting bone metabolism and response to mechanical loading. The results of this experiment suggest an important role of Nrf2 in osteocyte function which improves certain indices of bone quality, which impacts male and female bones in different 7 ways, but did not significantly impact loading-induced bone formation. Further studies should modify the method of Nrf2 activation in an effort to refine the animal model, allowing the effects of Nrf2 to be isolated from the potential systemic effects of Keap1 deletion. Future studies should also utilize other conditional knockout models to elucidate the effects of Nrf2 in other specific cell types.

<p>Bone loss diseases, including osteoporosis affect millions of people worldwide. Understanding the underlying mechanisms behind bone homeostasis and adaptation is essential to uncovering new therapeutic targets for the prevention and treatment of bone loss diseases. Primary cilia have been implicated in the development and mechanosensation of various tissue types, including bone. The goal of the studies outlined in this thesis is to determine the mechanosensory role of primary cilia in bone cell function, bone growth, and adaptation. This goal was achieved by exploring two specific scenarios. In the first study, mice models with conditional knockouts of MKS5, a ciliary protein, in osteocytes were utilized to demonstrate that dysfunctional primary cilia in those cells result in impaired loading-induced bone formation. The hypothesis tested is that the existence of functioning primary cilia on osteocytes is crucial for proper bone adaptation following stress. The results of this study support the hypothesis, with the conditional knockout mice showing significantly lower loading-induced bone formation compared to controls. The second study highlighted the importance of the osteoblast primary cilia in bone growth by using mice models with osteoblast-specific deletion of the cilia. The hypothesis tested is that the presence of the primary cilia is crucial for proper bone growth. The results show that conditional knockout mice have lower body weights, decreased femur length, and a significantly lower rate of bone formation, confirming that the primary cilia play a great role in bone growth and development. This study has highlighted the role of primary cilia in bone health and this topic merits further investigation. </p>