No description available.

INTRODUCTION: Following damaging stimuli, skeletal muscle exhibits coordinated interplay between intra- and extra-cellular processes resulting in satellite cell (SC) recruitment. SCs are known to play a central role in muscle plasticity post-injury by differentiating into myoblasts (MBL) and fusing with damaged tissue to donate myonuclei. Yet, their role within skeletal muscle remodeling through paracrine signaling remains to be fully elucidated. Thus, the purpose of this project was two-fold: 1) develop an in vitro model of MBL intercellular communication following myotube damage and 2) to determine if MBL proximity alone is adequate for improving tissue repair and reducing cellular stress during recovery. METHODS: C2C12 myotubes were exposed to 1 hour of electrical pulse stimulation (EPS) with 15Hz pulse for 5s and 5Hz pulse for 5s, separated by a 5s break. Myotubes were then introduced to non-electrically stimulated (NS) MBL adhered to a porous cell insert to allow paracrine signaling and samples were collected at varying timepoints post-EPS. RESULTS: EPS induced Z line sarcomeric disorganization and creatine kinase release into the cell culture media, which was mitigated in MBL+ groups (p<0.05). A significant main effect of MBL exposure was observed in EPS myotubes where MBL+ myotubes had greater Hsp70 gene expression, calpain 3 protein and gene expression, and t-ACC, p-ACCSer79, t-ULK, p-ULKSer555 protein expression than MBL- myotubes when recovering from EPS (p<0.05). A main effect of time was observed where B-dystroglycan and p-mTORSer2448 protein expression decreased in the EPS myotubes, and myotube diameter only decreased in the MBL+ condition (p<0.05). CONCLUSION: MBL signaling to damaged myotubes is evident and may increase catabolic processes through upregulating contraction-mediated protease activity and autophagy, as well as increase ATP generation through oxidative phosphorylation during regeneration. / Thesis / Master of Science (MSc) / When muscle damage occurs, whether through rigorous exercise or physical trauma, the muscle relies on a specific group of stem cells to help repair itself. These stem cells, termed satellite cells, can migrate to specific sites of muscle damage, differentiate into myoblasts, and donate nuclei and genetic material to the injured muscle. This increase in nuclear content helps the muscle synthesize more protein to rebuild and regenerate and promotes muscle growth. However, when the satellite cell becomes dysfunctional, as seen in aging muscle and certain genetic conditions, the muscle struggles to repair itself in response to damage and cannot grow in response to exercise. Satellite cell biology has clearly defined the role of nuclear donation in muscle function, however very little is known about how this stem cell ‘talks’ to the muscle through signaling molecules. As such, this thesis elucidates the effect of myoblast signaling on electrically stimulated damaged immature muscle fibers, otherwise known as myotubes, by preventing myoblast-myotube physical interactions in cell culture experimentation. Interestingly, the data presented here demonstrate that myoblast exposure to damaged myotubes may increase muscle protein breakdown as myotube diameters are reduced in size acutely post-damage, likely resulting from the increase in protease and autophagy protein expression markers. Additionally, myoblast exposure to damaged myotubes may increase mitochondrial fatty acid oxidation to generate energy, which is the fuel of choice during muscle regeneration.

No description available.

No description available.

Inflammasome signaling during infections results in cell death and processing and secretion of cytokines from the IL-1 family, which facilitates control over an infection. Pseudomonas aeruginosa and Salmonella Typhimurium are opportunistic bacterial pathogens which may induce acute infections and activate various innate immune signaling pathways, including inflammasomes. However, under favourable conditions these pathogens may evade immune clearance resulting in the establishment of a chronic infection. In this study, I evaluated the modulation of host inflammasome signaling induced by P. aeruginosa and S. Typhimurium during chronic infections. I used a collection of P. aeruginosa clinical isolates obtained from the sputum of cystic fibrosis patients collected during stable and exacerbation periods of disease. I demonstrated that the majority of isolates displayed poor inflammasome signaling and only a small proportion of isolates retained their ability to induce inflammasome activation, which may be associated with pulmonary exacerbations in cystic fibrosis. Sequencing and bioinformatics revealed genetic variations within the type III and type VI secretion systems of P. aeruginosa. While an inactivation of the type III secretion system is expected to impair inflammasome signaling, my results indicate that the type VI secretion system inhibits inflammasome signaling in eukaryotic cells. Due to the lack of chronic animal models for P. aeruginosa, I utilized a murine model of chronic S. Typhimurium infection to assess the modulation of inflammasome signaling throughout the course of a chronic infection. I observed that S. Typhimurium isolated during the acute phase of infection displayed an increased potential to activate inflammasome signaling and this ability progressively declined during the chronic phase of infection. This reduction in inflammasome activation was associated with reduced expression of bacterial virulence factors, such as flagella and the type III secretion system, and was dependent on the NLRP3 inflammasome. Overall, these results reveal that the expression of virulence factors is modulated during chronic bacterial infections, which results in a reduction of inflammasome activation leading to co-survival of the pathogen and host.

Problem Statement: The use of pulsed electric fields (PEFs) as a tumor treatment modality is receiving increased traction. A typical clinical procedure involves insertion of a pair of electrodes into the tumor and administration of PEFs (amplitude: ~1 kV/cm; pulse-width: 100 μs). This leaves a zone of complete cell death and a sub-lethal zone where a fraction of the cells survive. There is substantial evidence of an anti-tumor systemic immune profile in animal patients treated with PEFs. However, the mechanism behind such immune profile alterations remains unknown, and the effect of PEFs on cell signaling within sub-lethal zones remains largely unexplored. Moreover, different values of a PEF pulse parameter, for e.g. the pulse-widths of 100 μs and 100 ns, may have different effects on cell signaling. Thus, the challenge of answering the mechanistic questions is compounded by the large PEF parameter space consisting of different combinations of pulse-widths, amplitudes, and exposure times. Intellectual merit: This Ph.D. research provides proof that sub-lethal PEFs can enhance anti-tumor signaling in triple negative breast cancer cells by abrogating thymic stromal lymphopoietin signaling and enhancing stimulatory proteins such as the tumor necrosis factor. Furthermore, experimental evidence produced during this Ph.D. research demonstrates that PEFs may not directly impact the intracellular mitochondrial membrane at clinically relevant field amplitudes. As demonstrated in this work, PEFs may influence the mitochondria via an indirect route such as disruption of the actin cytoskeleton and/or alteration of ionic environment in the cytoplasm due to cell membrane permeabilization. Thus, a reductionist approach to understanding the influence of PEFs on cell signaling is proposed by limiting the study to membrane dynamics. To overcome the problem of investigating the entire PEF parameter space, this Ph.D. research proposes a first-principle thermodynamic approach of scaling the PEF parameter space such that an understanding developed in one regime of PEF pulse parameter values can be used to understand other regimes of the parameter space. Demonstration of the validity of this scaling model is provided by coupling Monte-Carlo methods for density-of-states with the steepest-entropy-ascent quantum thermodynamic framework for the non-equilibrium prediction of the lipid membrane dynamics. / Ph. D. / A complete cure for cancer is still far from being realized despite very promising developments on the front of molecular drug therapy. One promising conceptual approach would be to achieve the ability to re-tune the cancerous signals that drive disease progression. To overcome current challenges in tuning cancerous signaling a paradigm change in cancer treatment is necessary. For example, a treatment strategy to alter cell signaling which leverages both the physical and chemical properties that accompany malignancy may be required. Electric fields, be it in the form of low-amplitude steady state fields or high-amplitude pulsed electric fields (PEFs), can induce distinct physical and chemical effects on cells. Hence, the use of electric fields as a clinical tumor treatment modality is receiving increased traction. However, the effect of these electric fields on cell signaling and cell behavior remains largely unexplored. This Ph.D. work provides experimental evidence that PEFs can directly impact cancerous cell signaling towards a less inflammatory and possibly less cancerous state. Although a noteworthy finding, the data poses another challenging question, i.e., how does the electric field impact cell behavior? Answering this mechanistic question is essential for FDA approval and a broader clinical use of the electric field modalities. An impediment to answering this question is the vast parameter space of electric fields (e.g., amplitude, pulse width, and number of pulses), which makes performing experimental mechanistic studies untenable. It is argued via experimental evidence gathered during this work that applying scaling laws applicable to lipid membranes may provide a solution to reducing the candidate PEF parameters to a manageable number. A non-equilibrium thermodynamic model is proposed that allows studying the behavior of lipid species using scaled electric field parameters. Thus, the v understanding gained via the proposed model can direct the next level of extensive biological assays and animal studies and eventually lead to effective cancer treatments.

Osteoarthritis (OA) ist eine degenerative Erkrankung des hyalinen Knorpels. Knorpel ist ein avaskuläres Gewebe. Wenn dieser beschädigt wird, ist es schwierig, ihn zu reparieren. Der Gelenkknorpel ist verantwortlich für die glatte, reibungs- und schmerzlose Bewegung des Kniegelenks. Schwere Verluste oder die komplette Zerstörung des Gelenkknorpels führen zu hoher Reibung und Schmerzen bei der Bewegung des Kniegelenks, wie es oft in den späten Stadien der OA der Fall ist. Der komplette Gelenkersatz bleibt die ultimative Lösung. Jedoch gibt es viele andere Möglichkeitenden Knorpel über die Implantation von Stammzellen zu reparieren oder zu regenerieren, jedoch oft mit schwerwiegenden Folgen. Die Transplantation embryonaler Stammzellen kann beispielsweise zu Teratombildung führen. Die Nutzung von induzierten pluripotenten Stammzellen ermöglicht die Virusintegration in das Genom. Alternativ entstand das Konzept der Vorläufer- oder Reparaturzellen in situ. Beispielsweise fand man in späten Stadien der Osteoarthritis im menschlichen Knorpel chondrogene Vorläuferzellen mit migratorischen Fähigkeiten (CPCs). Bei Knorpelregeneration mit diesen Zellen sind bisher keine Risokofaktoren bekannt. Sie haben eine enorme Fähigkeit für die Knorpelreparatur ohne das schwerwiegende Risikofaktoren bisher bekannt waren. Allerdings bestehen noch Fragen zum Beispiel wie man CPCs in situ induziert, um das Gewebe auf physiologische Weise zu reparieren. Zweitens haben CPCs eine begrenzte Lebensdauer, zumindest in vitro. Darüber hinaus gibt es keine verfügbare optimierte Methode, um eine vollständige chondrogene Differenzierung von Stammzellen zu erreichen. Vor kurzem wurden primäre Zilien gefunden, die hilfreich für die Stammzelldifferenzierung sein könnten. Diese Zilien arbeiten als Dual-Sensor für mechanochemische Signale. Dieser Sensor wurde auch auf CPCs gefunden, bei Chondrozyten gewonnen aus Kiefergelenken (TMJ) von Discoidin-Domänen- Rezeptor-1- Knockout- Mäusen (DDR- 1 KO). OA ist nicht nur auf die großen Gelenke beschränkt, sondern wirkt sich auch auf die kleinen Gelenke wie das Kiefergelenk aus. Es ist gut bekannt, dass Chondrozyten im Gelenkknorpel keinen direkten Zell-zu-Zell-Kontakt besitzen vielmehr beruht die Kommunikation auf Zell-Matrix - Wechselwirkungen über Zellrezeptoren z.B Integrine oder DDRs. DDR -1- KO-Mäuse zeigen typische Symptome der Arthrose des Kiefergelenkknorpels. Die aus dem Kiefergelenknorpel der DDR - 1 KO Mäuse abgeleiteten Chondrozyten behielten ihre Arthroseeigenschaften. Einerseits wiesen sie eine hohe Expression von runx2 und Kollagen Typ I als typische osteogene Signaturen auf sowie andererseits eine geringe Expression von sox9, Kollagen Typ II und Aggrecan, welche relevant für die chondrogene Differenzierung sind. Die osteoarthritischen Charakteristika könnten zu einem normalen Chondrozyten- Typ umgekehrt werden über den Knockdown von runx2 oder der Exposition dieser Zellen in einer dreidimensionalen Umgebung und in Gegenwart von extrazellulärer Matrix (ECM) -Komponenten wie Laminin und Nidogen. Die Umkehr in Richtung des chondrogenen Phänotyps ist auch für die Pathogenese der Meniskusdegeneration von großer Bedeutung. Der Meniskus ist in den meisten Fällen der Ausgangspunkt für die Entwicklung von OA des Kniegelenks. Der Meniskus ist ein Bindegewebsknorpel, der als Stoßdämpfer wirkt. Hierbei verschlimmert eine Menikusschädigung die OA Pathogenese durch verstärkten Knorpelabbau. Der innere Teil des Meniskus ist avaskulärer Natur und hat eine sehr begrenzte Eigenreparaturfähigkeit. Es gibt jedoch andere Möglichkeiten, wie die teilweise Entfernung des Meniskus, die zu einer kurzfristigen Entlastung führt. Dies verhindert jedoch nicht die langfristigen Folgen, die letztlich zur Entwicklung von OA führen. Der innere Teil des menschlichen Meniskus birgt einzigartige Vorläuferzellen (MPC) und kann zur Meniskus-Regeneration über den TGFß -Signalweg aktiviert werden. Darüber hinaus wurden Maus-Meniskus-Progenitorzellen (MMPCs) in gesundem Meniskusgewebe untersucht. Diese Zellen wurden mittels immunohistochemischen Techniken ex vivo charakterisiert und behielten ihre Stammzelleigenschaften auch in vitro. Mit der Anwendung verschiedener Stammzellen zur Therapie der Knorpelregeneration sind viele kritische Konsequenzen assoziiert. Im Fokus standen deshalb gewebespezifische Zellen auch in situ Vorläuferzellen genannt, die bereits in erkranktem Knorpel vorhanden sind. Diese Zellen können sich in chondrogener Richtung entwickeln. Hierfür benötigen sie möglicherweise nur geringe Manipulationen, um daraufhin hyalinen Knorpel zu produzieren.

Technological limitations have prevented interrogation and manipulation of many signaling pathways in model and living systems required for the development of diagnostic and therapeutic modalities in diseases, such as cancer. Liposome-supported plasmon resonant gold nanoshells are biologically inspired composite structures, in which the liposome allows for the encapsulation of substances, and the plasmon resonant structure facilitates rapid release of encapsulated contents upon laser light illumination. As shown in this work, we overcome current limitations in cellular manipulation using plasmon resonant gold-coated liposomes in conjunction with light-activated release to achieve accurate probing of complex cellular responses. Development toward this goal was demonstrated with four specific aims. The first specific aim was to develop a computational model of heat diffusion to investigate the light-induced heating of gold-coated liposomes. This model was used to optimize the photothermal process for release of an encapsulated payload. The second aim was to demonstrate encapsulation and on-demand release of molecules in a spectrally-controlled manner, where plasmon resonant nanoparticles only release content upon illumination with a wavelength of light matching their plasmon resonance band. The third specific aim was to demonstrate that this release mechanism can be used in a biological setting to deliver a peptide and extracellularly activate surface membrane receptors with single-cell spatial and high temporal resolution. The fourth specific aim further refined the level of spatial and temporal control of payload release using gold-coated liposomes with optical trapping to demonstrate mirco-manipulation of liposome movement and rapid content release to enable accurate perturbation of cellular functions in response to released compounds. Through this work, we have developed an experimental system with the potential for the delivery and localized release of an encapsulated agent with high spatial and temporal resolution. This on-demand release system is compatible with a broad range of molecules and uses biologically safe near-infrared light. In combination with the spectral tunability of these plasmon resonant nanoshells and spectrally-selective release, this technology may allow for interrogation of complex and diverse signaling pathways in living tissues or their models with unprecedented spatial and temporal control.

The adaptor protein Shb has been implicated in the signaling of several tyrosine kinase receptors and previous studies have suggested a role for Shb in the signal transduction of T cells. Shb associates with the T cell receptor (TCR) and partakes in the signal propagation of activated T lymphocytes. In order to explore Shb’s influence on TCR signaling in vivo, T cell development and function was studied in a Shb knockout mouse. The loss of Shb led to aberrant TCR signaling in both thymocytes and peripheral CD4+ TH cells, with elevated basal phosphorylation of key components in the signal cascade. Shb was found to be dispensable for thymocyte development, but its absence resulted in a TH2 bias in in vitro stimulated peripheral CD4+ TH cells. As imbalances in TH2 responses are linked to allergic diseases, we further explored Shb’s role in immune regulation in a mouse model of atopic dermatitis. Shb knockout mice exhibit more aggravated signs of atopic dermatitis, including increased immune cell recruitment to the affected areas and elevated mRNA levels of typical TH2 cytokines. The effect of Shb on hematopoiesis in general was determined by examining populations of long-term hematopoietic stem cells (LT-HSCs) and hematopoietic progenitor cells in bone marrow of Shb knockout and wild type mice. Shb deficient bone marrow was found to contain significantly fewer relative numbers of LT-HSCs due to a proliferative defect. The reduced cell cycle activity of Shb LT-HSCs could further be linked to an abnormal regulation of the focal adhesion kinase/Rac1/p21-activated kinase pathway. Since alterations in LT-HSC proliferative abilities may have implications for leukemia development, BCR-Abl induced myeloid neoplasia was investigated in the absence of Shb. Shb deficiency confers a more aggressive progression of BCR-Abl induced myeloid neoplasia characterized by an increased peripheral blood neutrophilia and a deregulated cytokine profile. In addition, focal adhesion kinase and STAT3 signaling is hyperactivated in Shb knockout leukemic cells. In conclusion, Shb appears to be a multifunctional signaling mediator that controls several responses in hematopoietic cells, under homeostatic as well as disease conditions.

Résumé : La variation de la [Ca2+] intracellulaire participe à nombreux de processus biologiques. Les cellules eucaryotes expriment à la membrane plasmique une variété de canaux par lesquelles le calcium peut entrer. Dans les cellules non excitables, deux mécanismes principaux permettent l'entrée calcique; l'entrée capacitative de Ca2+ via Orai1 (SOCE) et l'entrée calcique activé par un récepteur (ROCE). Plusieurs protéines clés sont impliquées dans la régulation de ces voies d'entrée calcique, ainsi que dans l'homéostasie calcique. TRPC6 est un canal calcique impliquée dans l'entrée calcique dans les cellules à la suite d’une stimulation d’un récepteur hormonal. TRPC6 transloque à la membrane cellulaire et il y demeure jusqu'à ce que le stimulus soit retiré. Les mécanismes qui régulent le trafic et l'activation de TRPC6 sont cependant encore peu connus. Des découvertes récentes ont démontré qu'il y a un rôle potentiel de Rho kinase dans l'activité de TRPC6. Rho kinase est activée par la petite protéine G RhoA qui peut être activée par les protéines G hétérotrimériques Gα12 et Gα13. En plus de Gα12 et Gα13, les protéines de désensibilisation des GPCR β -arrestin 1 et / ou β-arrestin 2 peuvent aussi activer RhoA. Le but de notre étude est d'examiner la participation des protéines Gα12/13 et β-arrestin 1/ β-arrestin 2 dans l'activation de TRPC6 et de la protéine Orai1. Nous avons utilisé des ARN interférant (siRNA) spécifiques pour induire une réduction de l'expression de Gα12/13 ou β-arrestin 1/β-arrestin 2. La conséquence sur l’entrée de Ca2+ dans les cellules a été ensuite déterminée par imagerie calcique en temps réel suite à une stimulation par la vasopressine (AVP), thapsigargin ou carbachol. Nous avons donc identifié que dans des cellules A7r5, une lignée cellulaire de musculaires lisses vasculaires où le canal TRPC6 exprimé de manière endogène, la diminution de l’expression des protéines Gα12 ou Gα13 ne semble pas modifier l’entrée Ca2+ induit par l’AVP par rapport aux cellules témoins. D'autre part, la diminution de l’expression β-arrestin 1 ou β-arrestin 2 dans des cellules HEK 293 ainsi que des cellules HEK 293 exprimant de façon stable TRPC6 (cellules T6.11) ont augmenté l’entrée de Ca2+ induite par thapsigargin, un activateur pharmacologique de SOCE. Des études de co-immunoprécipitation démontrent une interaction entre la β-arrestin 1 et STIM1, alors qu'aucune interaction n'a été observée entre les β-arrestin 1 et Orai1. Nous avons de plus montré à l'aide d'analyse en microscopie confocale que la diminution de l’expression β-arrestin 1 ou β-arrestin 2 n’influence pas la quantité d’Orai1 à la périphérie cellulaire. Cependant, des résultats préliminaires indiquent que la diminution de l’expression β-arrestin 1 ou β-arrestin 2 augmente la quantité de STIM1-YFP dans l'espace intracellulaire et diminue sa quantité à la périphérie cellulaire. En conclusion, nous avons montré que les β-arrestin 1 ou β-arrestin 2 sont impliquées dans l'entrée capacitative de Ca2+ (SOCE) et contrôlent la quantité de STIM1 dans le réticulum endoplasmique. / Abstract : In an organism, intracellular [Ca2+] takes part in many biological processes. Eukaryotic cells express a variety of channels in the plasma membrane through which calcium can enter. In non-excitable cells, two main mechanisms allow calcium entry; the store-operated calcium entry via Orai1 (SOCE) and receptor-operated calcium entry (ROCE). Several key proteins are involved in the regulation of these calcium entry pathways as well as in calcium homeostasis. TRPC6 is a calcium channel implied in calcium entrance into the cells following hormonal stimulation and translocates to the plasma membrane. TRPC6 channel appear to the plasma membrane until the stimulus is present. Although, the mechanisms that regulate the trafficking and activation of TRPC6 are still little known. Recent findings have demonstrated that there is a potential role of Rho kinase in activity of TRPC6. Rho kinase is activated by the small G protein RhoA that itself can be activated by the heterotrimeric G proteins Gα12 and Gα13. In addition to Gα12 and Gα13 proteins, cytosolic GPCR desensitizing proteins β-arrestin 1 and/or β-arrestin 2 could also activate RhoA. The purpose of our study is to investigate the involvement of the proteins Gα12/13 and β-arrestin 1/β-arrestin 2 in the activation of TRPC6 and Orai1 protein. We used siRNA specific to Gα12/13 or β-arrestin 1/β-arrestin 2 to knockdown their endogenous expression. Then, calcium imaging in real time was performed in order to see the quantity of calcium entered into the cell following stimulation by vasopressin (AVP), thapsigargin, or carbachol. We hence identified that in A7r5 cell, vascular smooth muscle cell where TRPC6 channel expressed endogenously; reduced expression of Gα12 or Gα13 proteins does not seem to modify the AVP-induced Ca2+ entry compared to control cells. On the other hand, calcium imaging experiment in knocked down β-arrestin 1 or β-arrestin 2 in HEK 293 cells as well as HEK 293 cells stably transfected with TRPC6 (T6.11 cells) resulted in an increased thapsigargin-induced calcium entry. The co-immunoprecipitation studies demonstrate an interaction between β-arrestin 1 and STIM1, a calcium sensor in SOCE influx, while no interaction was observed between β-arrestin 1 and Orai1.We moreover showed by confocal microscopy that reduced expression of β-arrestin 1/ β-arrestin 2 does not influence the quantity of Orai1 at the cell periphery. Preliminary results showed that reduced expression of β-arrestin 1 or β-arrestin 2 increases the quantity of STIM1-YFP in the intracellular space and less it’s in peri-membrane space. In conclusion, we showed that β-arrestin 1 or β-arrestin 2 are involved in the store-operated calcium entry (SOCE) and control the quantity of STIM1 in the endoplasmic reticulum.