Thèse de doctorat : Pharmacologie moléculaire et cellulaire : Strasbourg 1 : 2008. / Titre provenant de l'écran-titre. Bibliogr. p. 197-221.

The assembly, form, and function of the heart is regulated by complex mechanical signals originating from intrinsic and extrinsic sources, such as the cytoskeleton and the extracellular matrix. During development, mechanical forces influence the self-assembly of highly organized ventricular myocardium. However, mechanical overload induces maladaptive remodeling of tissue structure and eventual failure. Thus, mechanical forces potentiate physiological or pathological remodeling, depending on factors such as frequency and magnitude. We hypothesized that mechanical stimuli in the form of microenvironmental stiffness, cytoskeletal architecture, or cyclic stretch regulate cell-cell junction formation and cytoskeletal remodeling during development and disease. To test this, we engineered cardiac tissues in vitro and quantified structural and functional remodeling over multiple spatial scales in response to diverse mechanical perturbations mimicking development and disease. We first asked if the mechanical microenvironment impacts tissue assembly. To investigate this, we cultured two-cell cardiac µtissues on flexible substrates with tunable stiffness and monitored cell-cell junction formation over time. As myocytes transitioned from isolated cells to interconnected µtissues, focal adhesions disassembled near cell-cell interfaces and mechanical forces were transmitted almost completely through cell-cell junctions. However, µtissues cultured on stiff substrates mimicking fibrotic microenvironments retained focal adhesions near the cell-cell interface, potentially to reinforce the cell-cell junction in response to excessive forces generated by myofibrils in stiff microenvironments. Intercellular electrical conductance between myocytes was measured as a function of connexin 43 immunosignal and the length-to-width ratio of cell pairs. We observed that conductance was correlated to connexin 43 immunosignal and cell pair length-to-width ratio, indicating that tissue architecture can affect electrical coupling. The impact of mechanical overload was also determined by applying chronic cyclic stretch to engineered cardiac tissues. Stretch activated gene expression patterns characteristic of pathological remodeling, including up-regulation of focal adhesion genes, and impacted sarcomere alignment and myocyte shape. Furthermore, chronic cyclic stretch altered intracellular calcium cycling in a manner similar to heart failure and decreased contractile stress generation, suggestive of maladaptive remodeling. In summary, we show that the assembly, form, and function of cardiac tissue is sensitive to a wide range of mechanical cues that emerge during physiological and pathological growth. / Engineering and Applied Sciences

Ο αυξητικός παράγοντας πλειοτροπίνη (Pleiotrophin, PTN) έχει μοριακή μάζα 18 kDa και ανήκει σε μια διακριτή οικογένεια αυξητικών παραγόντων που δεσμεύονται στην ηπαρίνη και σχετίζονται με αγγειογένεση και καρκινική ανάπτυξη. Στην παρούσα εργασία, μελετήσαμε τη συμμετοχή των ενδοκυτταρικών κινασών FAK και ILK στην επαγόμενη από PTN κυτταρική μετανάστευση σε ανθρώπινα ενδοθηλιακά κύτταρα από φλέβα ομφάλιου λώρου (HUVEC). Εξωγενής χορήγηση ΡΤΝ επάγει τη φωσφορυλίωση της κινάσης FAK στις τυροσίνες 397 και 925, ενώ μειώνει τη φωσφορυλίωση της κινάσης FAK στη τυροσίνη 576. Η κινάση ILK φαίνεται να εμπλέκεται στη διεγερτική δράση της PTN στη μετανάστευση των κυττάρων HUVEC, αφού μείωση της έκφρασης της κινάσης ILK με παρεμβαλλόμενο RNA στα κύτταρα HUVEC, οδήγησε σε αναστολή της επαγόμενης από ΡΤΝ κυτταρικής μετανάστευσης. Επιπλέον, διέγερση των κυττάρων HUVEC με PTN είχε ως αποτέλεσμα την επαγωγή της ενεργοποίησης της κινάσης ILK. Με σκοπό να διερευνηθεί η θέση της κινάσης ILK στο μονοπάτι μεταγωγής σήματος που ενεργοποιείται από τη PTN στα κύτταρα HUVEC, μελετήσαμε την πιθανή αλληλεπίδραση της ILK με μόρια που είναι γνωστό ότι συμμετέχουν σε αυτό το μονοπάτι. Παρατηρήθηκε ότι η κινάση ILK αλληλεπιδρά σε μεγάλο βαθμό με την κινάση FAK, ενώ μικρού βαθμού αλληλεπίδραση φαίνεται και με τις ιντεγκρίνη β3 και κινάση c-Src. Ενδιαφέρον παρουσιάζει το γεγονός ότι η διέγερση με PTN των κυττάρων HUVEC αυξάνει την αλληλεπίδραση μεταξύ των κινασών FAK και ILK. Τέλος, με δεδομένο ότι η β-κατενίνης εμπλέκεται στη κυτταρική μετανάστευση, ερευνήσαμε κατά πόσο η PTN και η κινάση ILK εμπλέκονται στο σηματοδοτικό μονοπάτι της β-κατενίνης στα κύτταρα HUVEC. Η ΡΤΝ αυξάνει με δοσο-εξαρτώμενο και χρονο-εξαρτώμενο τρόπο τη φωσφορυλίωση της β-κατενίνης σε κύτταρα HUVEC, φαινόμενο που αναστέλλεται μετά τη μείωση της έκφρασης της κινάσης ILK με siRNA. Συμπερασματικά, στην παρούσα εργασία καταδεικνύεται ότι η ΡΤΝ έχει διαφορική δράση στη φωσφορυλίωση τυροσινών της κινάσης FAK διαφορετικών θέσεων και ότι η κινάση ILK συμμετέχει στη διεγερτική δράση της PTN στη μετανάστευση των κυττάρων. / Pleiotrophin (PTN) is an 18 kDa secreted growth factor that displays high affinity for heparin. A growing body of evidence indicates that PTN is involved in cell proliferation, migration and differentiation. In the present work, we studied the possible role of two intracellular kinases, focal adhesion kinase (FAK) and integrin-linked kinase (ILK), in the PTN-induced migration of human umbilical vein endothelial cells (HUVEC). Exogenous administration of PTN significantly increased the phosphorylation of FAK kinase in tyrosines 397 and 925 and decreased phosphorylation in tyrosine 576. ILK seems to be involved in PTN-induced migration of HUVEC, since suppression of the ILK kinase using small interfering RNA (siRNA) abolished the stimulatory effect of PTN in migration of HUVEC. In addition, stimulation of HUVEC with PTN increased the ILK kinase activity. In order to determine which other signaling mediators are involved in the PTN signaling pathway, we studied the interaction of ILK with other proteins that have been implicated in the PTN-induced signal transduction. ILK strongly interacted with FAK kinase and to a lesser extent with c-src kinase and integrin ανβ3. Interestingly, PTN increased the degree of interaction between ILK and FAK kinases. Finally, it has been well described that β-catenin is involved in cell migration and that PTN increases β-catenin phosphorylation. We therefore investigated whether PTN affects β-catenin phosphorylation in HUVEC through activation of ILK kinase. PTN significantly increased phosphorylation of β-catenin in a concentration and time dependent manner, which seemed to be abolished after suppression of the ILK kinase using siRNA. Collectively, these results suggest a role of FAK and ILK kinases in the PTN-related signaling cascade which leads to cell migration both human endothelial cells.

No description available.

This thesis presents the design, fabrication, and testing of a micromixing device intended for use in investigating protein dynamics on a microsecond timescale by Fourier transform infrared (FTIR) spectroscopy. Numerical modeling of flow was implemented to predict the influence of flow rates and geometric variations on mixing performance in three passive mixers. The simulation models were validated by experimental measurements using optical and infrared detection. The optimum level of mixing was observed in a multi-lamination mixer that combined thin filaments of differing fluids in an alternating manner. The multi-laminates were transferred onto polished calcium fluoride infrared-transparent optical windows by lithographic processing of an Epon-based polymer, SU-8. A rigid seal between two microchannels was accomplished through thermal bonding of an unexposed resist layer, which acted as a thermal epoxy under the influence of temperature. The multi-lamination mixer was used to study the changes in the secondary structure of beta-Lactoglobulin in deuterated phosphate buffer under varying physicochemical conditions by time-resolved FTIR spectroscopy using focal plane array detection. Upon a pH jump from pH 2 to neutral pH, a gradual loss of alpha-helical content, accompanied by an increase in random coils and turns was observed within 2 ms of mixing. In a second kinetic experiment, mixing of a neutral-pH solution of beta-Lactoglobulin with a 60% trifluoroethanol solution resulted in the formation of an alpha-helical intermediate with an accompanying increase in intramolecular beta-sheet structure within 500 mus of mixing. These results indicate that the multi-lamination mixer designed and fabricated in this study is well suited for investigations of protein dynamics on the micro- to millisecond timescale by time-resolved FTIR spectroscopy.

The toxic metal ion cadmium (Cd2+) is pro-oxidant and specifically disrupts the actin cytoskeleton in renal mesangial cells. This study investigated the role of Cd2+-mediated redox modulation of actin through protein S-glutathionylation and the effects of cytoskeletal changes on focal adhesions (FAs) through a Ca2+/calmodulin dependent-protein kinase II (CaMK-II) pathway. Only at low concentrations of Cd2+ (0.5-2 μM) was there an increase in actin glutathionylation, which was a reactive oxygen species-independent, total glutathione-dependent effect. Immunofluorescence of the cytoskeleton suggests that increases in glutathionylation levels occurring under low [Cd2+] are protective in vivo. Higher concentrations (>= 10 μM) of Cd2+ resulted in loss of vinculin and focal adhesion kinase (FAK) from FAs, concomitant with cytoskeletal disruption. Inhibition of CaMK-II preserved cytoskeletal integrity and focal contacts, while decreasing the migration of FAK-phosphoTyr925 to a membrane-associated compartment. This study adds further insight into the Cd2+-mediated effects on the cytoskeleton and FAs.

A major limitation in developing a successful cancer treatment is the need for a distinction between normal and cancerous tissue. For solid tumors, this distinction can be made on a spatial basis, and successful treatments have been developed accordingly; however, many of these treatments cause pathologies in healthy tissue, much to the detriment of patient health. To address this issue for solid tumours, a conceptual approach would be to administer the chemotherapeutic drug locally, such that the intra-tumour concentration was high, while the systemic exposure to the drug remained low, thus, minimizing side effects. The present research focuses on providing proof-of-concept for the electrochemical generation of a toxicant from a prodrug, and subsequent use to elicit cytotoxicity in cancer cells, in attempts to electrochemically mimic monooxygenase-catalyzed bioactivation. Electro-oxidation of acetaminophen and cyclophosphamide substrates at graphite and Ti/RuO2 anodes was successful in generating their respective toxicants; however, the graphite anode was superior with respect to current efficiency and toxicant yield. Electrolyses conducted in batch and flow reactors produced effluents which reduced EMT-6 cell viability to the IC99 level. This thesis provides proof-of-concept for electrochemical prodrug activation as a viable area for further cancer research.

The toxic metal ion cadmium (Cd2+) is pro-oxidant and specifically disrupts the actin cytoskeleton in renal mesangial cells. This study investigated the role of Cd2+-mediated redox modulation of actin through protein S-glutathionylation and the effects of cytoskeletal changes on focal adhesions (FAs) through a Ca2+/calmodulin dependent-protein kinase II (CaMK-II) pathway. Only at low concentrations of Cd2+ (0.5-2 μM) was there an increase in actin glutathionylation, which was a reactive oxygen species-independent, total glutathione-dependent effect. Immunofluorescence of the cytoskeleton suggests that increases in glutathionylation levels occurring under low [Cd2+] are protective in vivo. Higher concentrations (>= 10 μM) of Cd2+ resulted in loss of vinculin and focal adhesion kinase (FAK) from FAs, concomitant with cytoskeletal disruption. Inhibition of CaMK-II preserved cytoskeletal integrity and focal contacts, while decreasing the migration of FAK-phosphoTyr925 to a membrane-associated compartment. This study adds further insight into the Cd2+-mediated effects on the cytoskeleton and FAs.

Environmental and food spoilage fungi cause billions of dollars in damage in North America alone each year, in the form of rotted wood and crops, spoiled food, and human and animal illness. Each of these threats could be drastically reduced if early and more rapid detection processes are developed to replace the serological methods that are currently in practice. The current North American protocol for establishing identification of contaminating fungi both in environment and food have a time frame of approximately one week to twenty-two days. The use of a Fourier transform infrared (FTIR) spectrometer, coupled with a focal-plan-array (FPA) detector, can theoretically shorten the time (analysis within minutes after obtaining a pure culture) it takes to identify and classify a fungal cell. FPA-FTIR spectroscopy is advantageous as little to no sample preparation is required and results are obtained in less than one minute per sample. The fungal subset chosen for this study includes representatives from five phyla, including Zygomycota (Mucor heimalis), Ascomycota (Neurospora crassa, Ophiostoma minor, Chaetomium globosporum, Alternaria brassicicola), Basidiomycota (Schizophyllum commune, Chaetomium globosporum), Deutromycota (Aspergillus niger, Penicillium notatum, Aureobasidium pullulans) and the Mycetozoa (dictyostelium discoideum, physarum polycephalum). Different variables were tested and evaluated, including variability in growth parameters, wet deposition of fungi versus dry smearing of fungi, optimal absorbance range, and spectral processing parameters as well as discrepancies from one instrument to another, as well as spectral reproducibility from one instrument to another. By following the experimental protocol developed, reproducible spectra were attained, and differentiation of the fungi within the set selected for this study was achieved. The results of this work demonstrate that FPA-FTIR spectroscopy can potentially be employed for the accurate identification of environmental and food spoilage fungi.

Flodpärlmusslan (Margaritifera margaritifera. L) har en obligatorisk fas i sin komplexa livcykel där den lever som parasit på antingen öringens (Salmo trutta, L) eller laxens (Salmo salar, L) gälar. För värdfisken skulle detta kunna innebära en ökad energikostnad när den födosöker i rinnande vatten. Öringens furagering består i att den finner en utgångspunkt (eng. focal point) i strömmen utifrån vilken den fångar sina byten. I denna studie undersöks det om en infektion av flodpärlmusslans glochidielarver på öring påverkar öringens födointag och hantering av födan. Sextio årsungar av öring infekterades med glochidielarver av känd koncentration, och deras födosökning mättes över en 10-veckors period för att bedöma effekten av den växande mussellarven i jämförelse med en icke-infekterad (kontrollgrupp) grupp fiskar. Individuella födoförsök, tio försök per mätningstillfällena, utfördes i ett strömakvarium där en enskild öring matades med drivande föda i fem minuter. Bytet släpptes ned i strömmen med två sekunders intervall, antalet byten räknades och furageringsbeteendet observerades. Födoförsöken genomfördes med två veckors intervall. Vid försöken hittades inga signifikanta skillnader med avseende på: 1) antalet fångade byten, 2) proportionen av fångade byten av försökets första tre minuter, eller 3) de rumsliga aspekterna av öringens utgångspunkt. Vidare uppmättes ingen signifikant effekt av tid på furageringsframgången på den infekterade gruppen jämfört med kontrollgruppen. Däremot var det ett högre antal infekterade individer som spottade ut byten jämfört med kontrollgruppen. Skillnaden i utspottade byten pekar på att infektionen har en negativ påverkan på fiskarnas furageringsförmåga. Dock visade försöken inga resultat som stödjer tidigare studier där signifikant skillnad erhållits med avseende på födointag. Förklaring till detta kan finnas i metodskillnader mellan denna och tidigare studier, inklusive skillnad i typ av byte, strömakvarier, ursprungsström för musslorna och öringen och infektionsgrad hos de infekterade fiskarna samt kontrollgruppen. / The freshwater pearl mussel (Margaritifera margaritifera, L) has a mandatory phase of its complex life cycle where it lives as a parasite on the gills of either trout (Salmo trutta, L) or salmon (Salmo salar, L). For the host fish this could mean an increased cost when it forages in running water. The trout´s foraging consists of finding a focal point in the stream from where it catches its prey. This study examines whether an infection of the freshwater pearl mussel´s glochidium larva on the gills of brown trout affect trout foraging. Sixty juvenile (0+) brown trout were infected with glochidium larva of a known concentration, and trout foraging behaviour was measured over a 10-week period to assess the effects of the growing mussel larvae of the fish compared to non-infected (control) fish.  Individual feeding trials, ten replicates for each session, were conducted in a stream aquarium where a single trout was fed drifting prey for five minutes. The prey were released into the stream every two seconds, the number of prey captured was counted, and the foraging behavior was characterized. Feeding trials were conducted at two-week intervals. No significant differences between the infected and control groups were found for: 1) the number of prey eaten, 2) the proportion of prey eaten during the first three minutes of the trial, or 3) location of the focal points. Furthermore, there was no significant effect of time on foraging success of the infected group compared to the control group. However, there was a higher number of infected individuals who spat out the prey compared with the control group. The difference in rejected prey suggests that the infection has a negative impact on the fish’s foraging ability. However, the results of this experiment do not support previous studies, where a significant difference was obtained with respect to food intake. This might be explained by differences in experimental protocol between this and previous studies, including differences in prey type, stream aquaria, stream origins of mussels and trout, and the infection status of infected and control fish.