published_or_final_version / Biochemistry / Master / Master of Philosophy

Cette thèse étudie l'adverbe autrement, au travers de ses trois emplois principaux : adverbe de manière, connecteur d'hypothèse négative, et rupteur de topique. L'accent est mis sur son fonctionnement anaphorique et son rôle dans la structure du discours. Après avoir passé en revue les théories du discours et la littérature sur l'adverbe, on dégage les propriétés des trois emplois grâce à des énoncés tirés de corpus oraux et écrits, en montrant comment le contexte sert à la récupération de l'antécédent et comment l'adverbe s'appuie sur le discours et le construit en même temps. Dès l'adverbe de manière, anaphore et portée droite sont essentielles à la construction du sens. Avec le connecteur, les relations référentielles laissent place aux relations logiques de proposition à proposition, tandis que le rupteur de topique est un emploi métalinguistique portant sur des constituants abstraits du discours. Un noyau de sens [l'anaphore et la négation] est dégagé, commun aux trois emplois et permettant d'envisager des points de passage entre eux. Cette étude synchronique est ensuite mise à profit pour reconstruire la grammaticalisation de l'adverbe, le détail des observations présentes contrebalançant la rareté des données historiques. On montre que c'est à travers la notion de construction, c'est-à-dire l'emploi de l'adverbe dans certains contextes, que l'évolution a pu avoir lieu : en particulier, l'ordre des mots en ancien français a été crucial, permettant à l'adverbe de manière d'occuper la position initiale propice à la réanalyse ; l'emploi de conjonctions a aussi favorisé l'émergence de certaines valeurs de l'adverbe. / This dissertation studies the French adverb autrement, through its three main uses : adverb of manner, connective denoting negative hypothesis, and topic shifter. The importance of anaphora resolution and discourse structure is stressed. After a review of the literature on discourse structure and on the adverb, the characteristics of the three uses are defined thanks to spoken and written corpora, showing how context is instrumental in retrieving the antecedent and how the adverb relies on discourse and builds it at the same time. Already in the adverb of manner, anaphor and right scope are crucial in the construction of meaning. With the connective, referential relations leave room for logical relations holding from proposition to proposition, whereas the topic shifter is a metalinguistic use handling abstract discourse entities. A core of meaning [anaphor and negation] is identified, common to the three uses and accounting for bridges between them. This synchronic study is then used to reconstruct the adverb's grammaticalization, detailed observation in the present counterbalancing sparse historical data. It is shown that the notion of a construction, i.e. the use of the adverb in some context, has made evolution possible : in particular, word order in Old French was crucial, allowing the adverb of manner to occupy the initial position where reanalysis could occur ; the use of conjunctions also favored the emergence of some of the adverb's meanings.

Injuries to connective tissues such as ligaments and tendons are common, and rather than healing, repair typically results in fibrosis, or the formation of mechanically inferior and disorganized scar tissue. This fibrotic repair response is due in part to inflammation, during which the injury site is invaded by a number of cell types, including macrophages, neighboring fibroblasts, and homed stem cells or progenitor cells. Activation of macrophages is believed to be modulated by communications with fibroblasts and stem cells, prompting either a pro-fibrotic or a pro-regenerative response. Beyond changes to the cellular microenvironment, fibrosis also results in changes to the organization and mechanical properties of the matrix microenvironment. For healthy fibrous connective tissues, the matrix is comprised of aligned collagen fibers, while scar tissue is disorganized and exhibits weaker mechanical properties than healthy tissue. To date, the nature of the cell-cell and cell-matrix interactions and their relevance in tissue healing or repair remain understudied. To better understand the cellular and matrix-based cues that direct scar formation versus tissue regeneration, and using anterior cruciate ligament (ACL) injuries as a model, Aim 1 of this thesis tests the hypothesis that in vitro models of cellular communications between fibroblasts, macrophages, and mesenchymal stem cells (MSC) can be used to determine the effects of cellular interactions on macrophage activation and fibrosis. In Aim 2, the contribution of matrix-based cues (alignment and mechanical properties) to the inflammatory and fibrotic response, as well as their modulation of cellular interactions, were examined. Findings from these two aims reveal that 1) communications between native tissue fibroblasts and macrophages drive inflammation and fibrosis, while stem cells modulate the repair process through a combination of trophic signaling and immunomodulatory roles, and 2) matrix alignment and mechanical properties exert combined regulation on cell response during inflammation. From a clinical application perspective, stem cells delivered in conjunction with an engineered matrix that provides the critical cues for driving stem cell immunomodulation and trophic signaling will be essential for promoting tissue regeneration and minimizing fibrosis. In particular, an aligned matrix with an elastic modulus similar to that of developing connective tissue may serve to further minimize inflammation and scar formation, and activate stem cell-guided regeneration of mechanically functional connective tissue.

Cystein rich protein 61 (CYR61/CCN1) und Connective tissue growth factor (CTGF/CCN2) stellen aufgrund ihrer Multifunktionalität zwei sehr interessante Vertreter aus der derzeit sechs Mitglieder umfassenden Familie der CCN-Proteine (CCN- CYR61/CCN1, CTGF/CCN2, NOV/CCN3, WISP1-3/CCN4-6) dar. Seit der Entdeckung von CYR61 und CTGF konnten die überlappenden, aber meist nicht redundanten zellspezifischen Effekte in verschiedenen Zellsystemen nachgewiesen werden. Die Einflüsse auf zahlreiche Prozesse wie Proliferation und Migration, aber auch Angiogenese und das Überleben von Zellen lassen eine weitreichende Bedeutung im Zusammenhang mit vielen Entwicklungsprozessen vermuten, so auch der des muskuloskelettalen Systems und der Entwicklung der Lunge. In der vorliegenden Arbeit wurden für die nähere Charakterisierung von CYR61 und CTGF humane mesenchymale Stammzellen (hMSC) und die humane primäre Lungenendothelzelllinie HPMEC-ST1.6R (human pulmonary microvascular endothelial cells) gewählt. Beide Zellsysteme sind für die Untersuchung der Funktionsfähigkeit in den verschiedenen Kompartimenten bestens geeignet. So ist die Zelllinie HPMEC-ST1.6R den primären Endothelzellen, im Vergleich mit anderen in der Forschung eingesetzten Zelllinien, in Bezug auf spezifische Oberflächenmarker am nächsten. Mesenchymale Stammzellen bilden als multipotente Zellen das Rückrat des muskuloskelettalen Systems und sind an der Homöostase des menschlichen Stütz- und Bewegungsapparates maßgeblich beteiligt. Um experimentell nutzbare Konzentrationen an rekombinanten Proteinen zu erhalten, wurde ein Baculovirus-Expressionsystems gewählt. Nach der erfolgreichen Klonierung der CTGF/Fc-Tag Sequenz in einen Expressionsvektor konnte dies auch durch Produktion in SF21-Insektenzellen erreicht und erstmalig rekombinantes CTGF/Fc von hoher Reinheit gewonnen werden. Allerdings konnte eine beständige Funktionsfähigkeit der aufgereinigten Proteine mittels eines Proliferationstestes nachfolgend nur bedingt bestätigt werden. Für die weitere Versuchsplanung, einer Untersuchung der Auswirkung von rekombinantem CTGF (rCTGF) bzw. CYR61 (rCYR61) auf die Zielzellen, musste zunächst die zelleigene ctgf bzw. cyr61 Expression herunterreguliert werden, um einen endogenen Störeffekt auszuschließen. Durch den Einsatz spezifischer shRNAs konnte ctgf/CTGF sowohl in den hMSC-, wie auch den HPMEC-ST1.6R-Zielzellen deutlich herunterreguliert und nachfolgend eine markant reduzierte Proliferation beobachtet werden. Ein Effekt für die Regulation von cyr61 blieb aus. In dieser Arbeit wurden anschließend erstmals mittels Microarray-Analysen Veränderungen im Genexpressionsmuster der ctgf herunterregulierten hMSC- bzw. Lungenendothelzellen gegenüber Kontrollzellen untersucht. Des Weiteren war die Auswirkung einer Behandlung von ctgf herunterregulierten Zielzellen mit rCTGF gegenüber unbehandelten Kontrollzellen von Interesse. Für beide Zellsysteme konnten signifikante Genregulationen nach der Behandlung mit CTGF spezifischen shRNAs gegenüber den Kontrollzellen detektiert werden, mit interessanten Genclustern im Bereich der TGF-beta (transforming growth factor ß) Signalgebung, sowie der fokalen Adhäsion (z.B. VEGF). Eine Behandlung mit rCTGF hingegen zeigte gegenüber den unbehandelten Kontrollzellen in der Auswertung der Microarray-Analyse keine signifikante Veränderung im Genexpressionsmuster. In dieser Arbeit wurden, neben einer effektiven Gewinnung von rekombinantem CTGF und der Herunterregulation der endogenen ctgf Expression, wichtige Erkenntnisse zur Biologie von CTGF (und CYR61) in mesenchymalen Stammzellen hMSC und der Lungenendothelzelllinie HPMEC-ST1.6R erlangt. Die erhaltenen Microarray-Daten bieten eine fundierte Grundlage für zahlreiche fortführende Untersuchungen. / Cystein rich protein 61 (CYR61/CCN1) and connective tissue growth factor (CTGF/CCN2) are two very interesting members of the CNN family (CCN- CYR61/CCN1, CTGF/CCN2, NOV/CCN3, WISP1-3/CCN4-6) consisting of six members so far. Since its discovery the overlapping, but mostly non-redundant effects of CYR61 and CTGF were shown in different cell systems. Both proteins are linked to many different processes like proliferation and migration, but also angiogenesis and survival. They seem to be involved in very fundamental biological processes, amongst other the development of the musculoskeletal system and the lung and were analyzed in this study. To distinguish the two proteins CYR61 and CTGF, primary human mesenchymal stem cells (hMSC) and a human pulmonary endothelial cell line (HPMEC-ST1.6R) were chosen. Both cell systems are suited very well for getting more information about the function in these different compartments. So the cell line HPMEC-ST1.6R is more related to primary endothelial cells in reference to the cell surface markers, compared to other cell lines used for experimental research. Mesenchymal stem cells form the backbone of the musculoskeletal system and are involved in the homeostasis of this complex system. Getting adequate concentrations of recombinant proteins for the upcoming experiments a baculovirus expression system was chosen. After successful cloning of the CTGF/Fc-Tag sequence into an expression vector, recombinant CTGF/Fc of high purity was obtained for the first time, produced in SF21 insect cells. However the stable functioning of the proteins was partly confirmed by proliferation tests. To study the effect of recombinant CTGF or CYR61 in further experiments, the endogenous ctgf or cyr61expression had to be downregulated to avoid negative effects. By using specific shRNAs ctgf/CTGF has been downregulated in hMSC as well as HPMEC-ST1.6R cells and subsequently a reduced proliferation was observed. No effect was detected for the regulation of cyr61. In this study for the first time changes in regulation of gene expression after downregulation of ctgf in hMSC and HPMEC-ST1.6R cells were studied by microarray analyses. Furthermore to discover the effect of treating ctgf downregulated cells with recombinant CTGF compared to control cells was another aim of this experimental series. For both cell systems, significant gene regulations were detected after treatment with CTGF specific shRNAs with interesting gene cluster for TGFß-signaling as well as focal adhesion (e.g. VEGF). In contrast, no significant change in gene regulation was detected by microarray analysis after treating the target cells with rCTGF compared to non-treated cells. In summary, besides the effective preparation of rCTGF and the marked downregulation of ctgf gene expression, this study provides fundamental information about CTGF and its biology in hMSC and HPMEC-ST1.6R cells, as well. Based on the numerous detected gene regulations in the microarray analyses the study provides a basis for further experiments.

This research was conducted to investigate the connective tissue contribution to toughness of cow beef and to find means to decrease it. Intra muscular connective tissue (IMCT) content of meat from cows (~6 years) and heifers (~16 months) varied significantly among muscles (P<0.0001) and maturity groups (P<0.05). Amount of total collagen in IMCT was a constant (37.3-46.3 %) among muscles and between maturity groups. Shear force of <i>biceps femoris</i>, <i>semimembranosus</i> and <i>longissimus</i> muscles had increased significantly with animal maturity (P<0.0001). Shear stress of <i>gluteus medius</i> was similar between maturity groups. Collagen solubility decreased with animal maturity, except for <i>biceps femoris</i>. <p> The impact of the temperature of aqueous heating (55 to 95 ºC) and time on thermolabile proteins, amorphous proteins, Ehrlich chromogen, pyridinoline, thickness change, shrinkage, weight gain, shear force, amide bands and morphology of epimysium was studied. Collagen contributed to 90% (w/w) of epimysial proteins. At 55 ºC, epimysial properties were changed only after exposure to long heating times. Shear stress values of raw cow (39.6 N/mm2) and heifer (30.8 N/mm2) epimysium decreased significantly to 11.6 and 2.1 N/mm2, respectively, at 70 ºC. Amount of epimysial amorphous collagen (14-16% w/w) detected after heating at 70 ºC and above was not related to shear stress decrease. Before and after heating, cow epimysium contained more pyridinoline cross-links than heifer epimysium.<p> The effects of strong and weak acids and alkalis on epimysial properties were studied following heating at 55 and 70 ºC for 15 min. As the concentration of HCl (0.1-0.5 M) and pre-equilibration time were increased at 70 ºC, shear stress decreased to <2 N/mm2. Increasing concentration of CH3COOH (0.1-0.5 M) and pre-equilibration times had decreased shear stress to ~5 N/mm2. At 55 ºC, HCl was not superior to CH3COOH in its ability to decrease epimysial shear stress. Increasing concentration of NaOH (0.01-0.05 M) and high temperature decreased shear stress to ~3 N/mm2. Lack of a shear stress decrease at 55 ºC and increased thermal denaturation temperature (66 ºC compared to 63 ºC in water), indicated that NH4OH had an epimysial stabilization effect, which was not eliminated at 55 ºC.

This research was conducted to investigate the connective tissue contribution to toughness of cow beef and to find means to decrease it. Intra muscular connective tissue (IMCT) content of meat from cows (~6 years) and heifers (~16 months) varied significantly among muscles (P<0.0001) and maturity groups (P<0.05). Amount of total collagen in IMCT was a constant (37.3-46.3 %) among muscles and between maturity groups. Shear force of <i>biceps femoris</i>, <i>semimembranosus</i> and <i>longissimus</i> muscles had increased significantly with animal maturity (P<0.0001). Shear stress of <i>gluteus medius</i> was similar between maturity groups. Collagen solubility decreased with animal maturity, except for <i>biceps femoris</i>. <p> The impact of the temperature of aqueous heating (55 to 95 ºC) and time on thermolabile proteins, amorphous proteins, Ehrlich chromogen, pyridinoline, thickness change, shrinkage, weight gain, shear force, amide bands and morphology of epimysium was studied. Collagen contributed to 90% (w/w) of epimysial proteins. At 55 ºC, epimysial properties were changed only after exposure to long heating times. Shear stress values of raw cow (39.6 N/mm2) and heifer (30.8 N/mm2) epimysium decreased significantly to 11.6 and 2.1 N/mm2, respectively, at 70 ºC. Amount of epimysial amorphous collagen (14-16% w/w) detected after heating at 70 ºC and above was not related to shear stress decrease. Before and after heating, cow epimysium contained more pyridinoline cross-links than heifer epimysium.<p> The effects of strong and weak acids and alkalis on epimysial properties were studied following heating at 55 and 70 ºC for 15 min. As the concentration of HCl (0.1-0.5 M) and pre-equilibration time were increased at 70 ºC, shear stress decreased to <2 N/mm2. Increasing concentration of CH3COOH (0.1-0.5 M) and pre-equilibration times had decreased shear stress to ~5 N/mm2. At 55 ºC, HCl was not superior to CH3COOH in its ability to decrease epimysial shear stress. Increasing concentration of NaOH (0.01-0.05 M) and high temperature decreased shear stress to ~3 N/mm2. Lack of a shear stress decrease at 55 ºC and increased thermal denaturation temperature (66 ºC compared to 63 ºC in water), indicated that NH4OH had an epimysial stabilization effect, which was not eliminated at 55 ºC.

Intramuscular connective tissue is critical in maintaining muscle structure and the transfer of force from contractile elements to the bone. We examined intramuscular connective tissue characteristics in young and old men and women, as well as men and women subjected to simulated microgravity. We hypothesized that intramuscular collagen content, collagen cross-linking and formation of advanced glycation endproducts of old individuals would be greater than young, and that intramuscular collagen content would be elevated following prolonged periods of unloading spanning 35, 60 and 90 days. Vastus lateralis muscle biopsies revealed that intramuscular collagen (Young: 9.6±1.1, Old: 10.2±1.2 ug•mg muscle wet wf-') and collagen cross-links (hydroxylysylpyridinoline, HP) (Young: 395±65, Old: 351±45 mmol HP•mol collagen-1) were unchanged (p>0.05) with aging. The advanced glycation endproduct, pentosidine, was increased (p<0.05) by 203% (Young: 5.2±1.3, Old: 15.9±4.5 mmol pentosidine•mol collagen"') with aging. With unloading, collagen content of the vastus lateralis was unchanged (p>0.05) following all time periods but was found to be elevated (p<0.05) in the soleus following 90 days of unloading. Furthermore, baseline collagen content was found to greater (p<0.05) in the soleus compared to the vastus lateralis. These results suggest the age related decline in whole muscle function is not related to increases in intramuscular collagen content or cross-linking but may be related to the accumulation of advanced glycation endproducts. Muscle function following unloading does not appear to be impacted by collagen content in the vastus lateralis but may play a role in the soleus. / School of Physical Education, Sport, and Exercise Science

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Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1990. / Includes bibliographical references (leaf 80) / Microfiche. / viii, 80 leaves, bound ill. 29 cm