Skeletal muscle function is important to the human body for daily activities. Mechanical signals are critical to the maintenance of that function. Muscle diseases, such as the muscular dystrophies, in which the force transmission apparatus is compromised, have devastating effects on muscle function and quality of life. Mechanical signals activate intracellular signaling to maintain function. ERK2 has been shown to be quickly and strongly upregulated following stretch, leading to cell proliferation. Stretch has been shown to cause deformation of caveolae, invaginations of the plasma membrane that inhibit ERK signaling. This leads to the hypothesis that stretch induced deformation of caveolae may initiate mechanotransduction by activating ERK2. Reducing caveolin-3 expression via siRNA knockdown eradicated the stretch-induced effect on ERK2 activation, indicating that caveolin is required for the stretch response. Stabilizing caveolae structure by temperature reduction or destabilizing caveolae by cholesterol depletion resulted in changes consistent with the hypothesis that proper caveolae structure plays an important role in inhibition of signaling molecules and that deformation mediates mechanotransduction, resulting in changes in activation of ERK2.

The aim of this study was to understand the cellular mechanisms in enterocytes, which may decrease glucose uptake by viscous oat β-glucan. An in-vitro cell model examined the effect of diffusion limitation, fluid shear stimulation, and increased intestinal stretching. Mechanical stimulation of IEC-6 was assessed. A Flexcell Cell Streamer device applied different fluid flow stresses on cells. Flexcell FX-4000 was used for biaxial stretching of the cells. Following the confirmation of appropriate use of the cell model, the results indicated that high viscosity oat β-glucan might provide a physical barrier limiting diffusion of nutrients to the cells apical surfaces. Western blot analysis confirmed weak mechanical stimulation on the cells. Mechanical stimulation did not influence glucose uptake. Strain-induced cells showed lower activities in their glucose uptake. In conclusion, there may be a significant contribution of direct effects of the viscosity of oat β-glucan on cellular mechanisms of uptake in enterocytes.

Osteoarthritis is the end result of damage to articular cartilage, which lacks the ability to self-repair. Tissue engineering of cartilage is a promising field of study that aims to promote healing of cartilage in vivo by manipulation of the chondrocytes that maintain the tissue, or through in vitro production of new cartilage for implantation into cartilage defects. Tissue-engineered cartilage constructs require mechanical stimulation to produce matrix components in quantities and proportions similar to native cartilage tissue, and adenosine triphosphate (ATP) is thought to be an autocrine/paracrine biochemical mediator of these mechanical forces on the cell, after its release from chondrocytes under mechanical stress. This study determined culture conditions for chondrocytes in 3D agarose scaffolds from mature donors undergoing total joint arthroplasty for the treatment of osteoarthritis, then supplemented these cells in vitro with exogenous ATP in concentrations varying from 50 nM to 1 mM in the presence of the radioisotopes [35S] and [3H]-proline, with radioisotope incorporation acting as markers of proteoglycan and collagen synthesis respectively. The basal concentrations of ATP in the chondrocyte cultures as well as the ATP half-life in the cultures were determined by lucifer/luciferase assay and luminometry. The P2Y receptor expression on the populations of chondrocytes from 8 donors was determined by flow cytometry, with largely varied individual expression and heterogeneity of P2Y1 and P2Y2 receptors. Exogenous ATP was found to increase synthesis of matrix components by 200% of the control cultures at doses of 100 nM to 1 µM. Patients with worse arthritis patterns, who were on chronic narcotic medications and who smoked were more likely to have a negative response to the exogenous ATP supplementation. The basal concentration of ATP in the cultures was less than 1 nM, and the ATP half-life varied from 1-2 hours, depending on the expression of P2Y1 receptors expressed by the donor’s chondrocyte population (R2 = 0.99). Supplementation of exogenous ATP to tissue-engineered cartilage in vitro appears to be a promising technique for improving the matrix synthesis of these constructs. / Thesis (Master, Mechanical and Materials Engineering) -- Queen's University, 2011-01-28 10:49:47.118

The binding between the 45 kDa N-terminal domain of the a subunit of the GPIb-IX-V complex (GPIbαN) on the platelet membrane and the A1 domain of von Willebrand factor (VWF-A1), a multimeric protein circulating in the plasma, plays a key role in platelet adhesion and thrombus initiation at sites of cut-injury and atherosclerotic plaque rupture where blood vessels are subjected to high haemodynamic shear. A fundamental yet unresolved issue is how haemodynamic force upregulates this interaction (binding kinetics) and how a mechanical stimulus is translated into a biochemical signal (mechanotransduction). In order to address above issues, we setup a new biomembrane force probe (BFP) with the drifting reduction, temperature control and concurrent fluorescence imaging. My research findings are summarized into three aims: 1. VWF regions surrounding A1 hinder A1-GPIbα interaction at zero force, which is relieved by increasing force that stabilizes the interaction, giving rise to a VWF-GPIbα catch bond. 2. Three transport-related physical factors: receptor-ligand separation distance, Brownian motion and diffusivity govern the VWF-GPIbα association. 3. Mechanical force and structural variation regulate platelet signaling via the engagement duration of GPIbα mechanosensor. My thesis study advances our understanding of the biophysical and structural basis of how the VWF activation, its interaction with GPIbα and signal transduction are regulated by force when platelets' haemostatic functions are most needed.

It is becoming clear that mechanical stimuli are critical in regulating cell biology; however, the short-term structural response of a cell to mechanical forces remains relatively poorly understood. We mechanically stimulated cells expressing actin-EGFP with controlled forces (0-20nN) in order to investigate the cell’s structural response. Two clear force dependent responses were observed: a short-term local deformation of actin stress fibres and a long-term force-induced remodelling of stress fibres at cell edges, far from the point of contact. We were also able to quantify strain dynamics occurring along stress fibres. The cell exhibits complex heterogeneous negative and positive strain fluctuations along stress fibres, indicating localized dynamic contraction and expansion. A ~50% increase in myosin contractile activity is apparent following application of 20nN force. Directly visualizing force-propagation and stress fibre strain dynamics has revealed new information about the pathways involved in mechanotransduction which ultimately govern the downstream response of a cell.

It is becoming clear that mechanical stimuli are critical in regulating cell biology; however, the short-term structural response of a cell to mechanical forces remains relatively poorly understood. We mechanically stimulated cells expressing actin-EGFP with controlled forces (0-20nN) in order to investigate the cell’s structural response. Two clear force dependent responses were observed: a short-term local deformation of actin stress fibres and a long-term force-induced remodelling of stress fibres at cell edges, far from the point of contact. We were also able to quantify strain dynamics occurring along stress fibres. The cell exhibits complex heterogeneous negative and positive strain fluctuations along stress fibres, indicating localized dynamic contraction and expansion. A ~50% increase in myosin contractile activity is apparent following application of 20nN force. Directly visualizing force-propagation and stress fibre strain dynamics has revealed new information about the pathways involved in mechanotransduction which ultimately govern the downstream response of a cell.

Nous cherchons à comprendre le mode de fonctionnement d’HACE1, un suppresseur de tumeur majeur, dont l’expression est altérée dans 50% des tumeurs humaines. Notre équipe a démontré que cette ubiquitine-ligase cible et provoque la dégradation de Rac1 au niveau du protéasome. Rac1 est une protéine oncogénique promouvant la croissance tumorale. En ciblant Rac1, HACE1 permet de restreindre le stress oxydatif des cellules, diminuant ainsi leurs dommages à l’ADN. Pour comprendre les voies de signalisation cellulaires ciblées par HACE1, nous avons ensuite recherché des protéines interagissant avec elle. Mes travaux révèlent que l'optineurine (OPTN), une protéine jusqu’alors connue dans des désordres neuro-dégénératifs, forme un complexe avec HACE1. Nous avons montré que ce complexe régule transcriptionnellement et traductionnellement la Cycline-D1. Nous avons également montré que l’OPTN se localise dans les points d'ancrage des cellules à la matrice extracellulaire (MEC) et régule leur formation. En réponse à l’élasticité de la MEC, nous avons montré que le complexe HACE1-OPTN réprime, au travers du métabolisme, la division cellulaire. Une étude que nous avons effectuée sur des données cliniques indiquerait l'importance d’une perte d’expression d’OPTN dans le cancer du sein, associée à des dérégulations des tensions de la MEC tumorale / We have established a novel regulatory mechanism that restricts Rac1 activity through ubiquitylation and targeting to the proteasome of the active form of the GTPase for degradation and signal termination. This regulation is dominant over the classical GEF/GAP cycle of regulation. We identified the E3 ubiquitin ligase (E3L) HACE1 as the main enzyme that catalyzes Rac1 ubiquitylation. HACE1 is a major tumor suppressor that limits Rac-dependent NADPH oxidase complex activity, S phase entry, cell migration, mammary cell transformation and tumor growth in animal models. In order to determine how HACE1 activity is controlled, we conducted a whole genome two-hybrid screen and identified Optineurin (OPTN) as a primary partner of the E3L HACE1. We report that OPTN is a new Extracellular matrix (ECM) stiffness sensor that activates HACE1 E3L activity. OPTN controls adhesion-mediated ubiquitin-proteasome degradation of Rac1 to tune Rac1 signaling with tissue stiffness. Loss of OPTN is associated with a gain of cell-ECM adhesive properties and enhanced integrin-mediated proliferative signaling and metabolic activity. Interestingly, cells that loose OPTN display atypical mechanical properties and apparent uncoupling of Focal Adhesions growth from acto-myosin contractile activity. Together, our findings establish the first link between the Rac1 ubiquitylation pathway and ECM compliance sensing and define OPTN as a previously unknown mechanical sensor. OPTN and HACE1 would then act as a tumor-suppressor complex that adapts cell proliferative response to ECM mechanical properties in order to insure both Tensional integrity and Redox homeostasis of cells

Current cancer therapies are highly cytotoxic and their delivery to exclusively the affected site is poorly controlled, resulting in unavoidable and often severe side effects. In an effort to overcome such issues, magnetic nanoparticles have been recently gaining relevance in the areas of biomedical applications and therapeutics, opening pathways to alternative methods. This led to the concept of magnetic particle hyperthermia in which magnetic nano beads are heated by a high power magnetic field. The increase in temperature kills the cancer cells, which are more susceptible to heat in comparison to healthy cells. In this dissertation, the possibility to kill cancer cells with magnetic nanowires is evaluated. The idea is to exploit a magnetomechanical effect, where nanowires cause cancer cell death through vibrating in a low power magnetic field. Specifically, the magnetic nanowires effects to cells in culture and their ability to induce cancer cell death, when combined with an alternating magnetic field, was investigated. Nickel and iron nanowires of 35 nm diameter and 1 to 5 μm long were synthesized by electrodeposition into nanoporous alumina templates, which were prepared using a two-step anodization process on highly pure aluminum substrates. For the cytotoxicity studies, the nanowires were added to cancer cells in culture, varying the incubation time and the concentration. The cell-nanowire interaction was thoroughly studied at the cellular level (mitochondrial metabolic activity, cell membrane integrity and, apoptosis/necrosis assay), and optical level (transmission electron and confocal microscopy). Furthermore, to investigate their therapeutic potential, an alternating magnetic field was applied varying its intensity and frequency. After the magnetic field application, cells health was measured at the mitochondrial activity level. Cytotoxicity results shed light onto the cellular tolerance to the nanowires, which helped in establishing the appropriate nanowire concentrations to use the nanowires + alternating magnetic field combination as a cancer treatment. Different levels of cancer cell death were achieved by changing the incubation time of the nanowires with the cells and the alternating magnetic field parameters. Cell viability was significantly affected in terms of mitochondrial activity and cell membrane integrity after applying the treatment (nanowires + alternating magnetic field) using a low-frequency alternating magnetic. Theoretical calculations considering the magnetic and viscous torques showed that the nanowires vibrate as a consequence of the applied magnetic field. This, alongside the fact that no temperature increase was measured during the treatment, makes the magnetomechanical effect the most probable action mechanism in the applied treatment that is inducing cell death. Inducing cancer cell death via magnetomechanical action using magnetic nanowires resulted in killing up to 60% of cancer cells with only 10 minutes of treatment. The required magnetic field for treatment is in a low power regime, which is safe, does not cause any discomfort to the patients, and can be generated with compact and cheap instruments.

Opioids are gold-standard analgesics for pain relief in chronic pain conditions. Paradoxically, chronic opioid use causes an enhanced pain sensitivity termed ‘Opioid-induced hyperalgesia’ (OIH). OIH is a clinically relevant problem associated with the use of opioids. In addition to decreasing quality of life, increased pain from OIH necessitates increasing dosages of analgesics to effectively control the pain, resulting in an increased risk of opioid epidemics, addiction, and overdose. To prevent this clinically important effect, it is necessary to understand how chronic opioid use causes hyperalgesia. Our preliminary studies revealed that synaptic plasticity in the spinal dorsal horn (SDH) is dependent on neuron type in the OIH model and occurs concurrently with hyperalgesia, suggesting central sensitization as a mechanism of OIH. We found that astrocyte ablation blocked mechanical hyperalgesia and neuron type-dependent synaptic plasticity, indicating that astrocytes are critically involved in OIH. Additionally, morphine treatment upregulated IL-1β expression in the SDH in our preliminary experiments. Inhibition of IL-1β prevented OIH and blocked the repeated morphine-induced synaptic plasticity in the SDH, suggesting IL-1β is a key player in the pathogenesis of OIH. Astrocytes and other glial cells are critical in the development and maintenance of neuroinflammatory conditions, such as OIH, through the release of proinflammatory cytokines (PICs), including IL-1β. The mechanosensitive ion channel, Piezo1, was recently found to be upregulated in astrocytes and microglia under LPS-induced inflammatory conditions, and activation of Piezo1 was found to reduce IL-1β expression in LPS-inflamed primary mouse astrocytes. The goal of this study was to investigate the function of Piezo1 as a potential treatment for neuroinflammatory diseases of the CNS in a model of LPS-induced inflammation. In this study, we created a culture cell model of LPS-induced astrocytic neuroinflammation using the C8-S type II astrocyte culture cell line. We used a multi-disciplinary approach of electrophysiology and imaging to assess changes in calcium flux induced by the selective Piezo1 agonist, Yoda1, and mechanosensitive ion channel activity in the LPS-stimulated C8-S culture astrocytes. We found that calcium flux is increased in LPS stimulation and augmented by additional Yoda1 treatment. We also found that LPS stimulation increases mechanosensitive ion currents and stiffens cell membranes using patch-clamp electrophysiology techniques. These results indicate that Piezo1 is likely upregulated in the LPS model of cultured astrocytes, thus mechanosensitive responses are increased. Results from these experiments reveal key information about the mechanical properties of Piezo1 and poise Piezo1 as a promising therapeutic for OIH and other neuroinflammatory diseases caused by astrocytic IL-1β release.

Aims Angiotensin II (Ang II) stimulates cardiac remodelling and fibrosis in the mechanically overloaded myocardium. Although Rho GTPases regulate several cellular processes, including myocardial remodelling, involvement in mediating mechanical stretch-induced regulation of angiotensinogen (Ao), the precursor to Ang II, remains to be determined. We, therefore, examined the role and associated signalling mechanisms of Rho GTPases (Rac1 and RhoA) in regulation of Ao gene expression in a stretch model of neonatal rat cardiac fibroblasts (CFs). Methods and resultsCFs were plated on deformable stretch membranes. Equiaxial mechanical stretch caused significant activation of both Rac1 and RhoA within 25 min. Rac1 activity returned to control levels after 4 h, whereas RhoA remained at a high level of activity until the end of the stretch period (24 h). Mechanical stretch initially caused a moderate decrease in Ao gene expression, but was significantly increased at 824 h. RhoA had a major role in mediating both the stretch-induced inhibition of Ao at 4 h and the subsequent upregulation of Ao expression at 24 h. β1 integrin receptor blockade by Tac β1 expression impaired acute (2 and 15 min) stretch-induced Rac1 activation, but increased RhoA activity. Molecular experiments revealed that Ao gene expression was inhibited by Rac1 through both JNK-dependent and independent mechanisms, and stimulated by RhoA through a p38-dependent mechanism. Conclusion These results indicate that stretch-induced activation of Rac1 and RhoA differentially regulates Ao gene expression by modulating p38 and JNK activation.