Tubulointerstitial fibrosis is the key histological predictor of the progression of declining renal function and the final common pathway of progressive kidney disease, regardless of aetiology. Despite its significance, there are currently no treatments available to abrogate this process and those that suffer with this burden eventually succumb to renal failure. Tubulointerstitial fibrosis is largely mediated by fibroblasts and myofibroblasts present in the interstitium. In response to injury, activated fibroblasts differentiate into myofibroblasts which serves as a histological hallmark of fibrosis. Myofibroblasts are characterised as the key contributors to interstitial volume and their presence ultimately leads to loss of renal function. The pathological entities leading to fibrosis inextricably depend on complex signalling pathways. Whilst many of the well-known growth factors that exert effects on renal fibroblasts (such as FGF, EGF and PDGF) involve the activation of receptor tyrosine kinases, the intracellular signalling events dictating the response of fibroblasts remain undefined. The kinase mTOR, responsible for integrating stress and amino acids and controlling cell growth, is increasingly recognised for its ability to integrate growth factor signals mediated through the upstream serine/threonine kinase PI3K. A number of recent studies have highlighted the role of PI3K and mTOR in the regulation of key events relevant to fibrosis, serving as a basis for Chapter 3: The role of PI3K and mTOR in the regulation of fibroblast proliferation and collagen synthesis, and the first part of Chapter 5: The role of PI3K and mTOR in the regulation of myofibroblast differentiation. These studies have identified a key role for PI3K and mTOR in the regulation of fibroblast proliferation, differentiation and collagen synthesis. The work described within has also attempted to examine the derivation of myofibroblasts via EMT. EMT is a process that is integral to embryogenesis and may act as an important source of myofibroblasts during fibrosis. This process is examined in Chapter 4: Development and validation of an ex vivo model of EMT. This model aims to better represent the in vivo environment and has also been used to identify novel regulators involved in EMT being utilised in the second part of Chapter 5: The role of PI3K and mTOR in EMT. Although cytokines and growth factors are thought to be chiefly responsible for tubulointerstitial fibrosis, we now know that serine proteases of the coagulation cascade may also play roles in renal disease. However, unlike their role in glomerular diseases, the role of coagulation in tubulointerstitial fibrosis is less well-known. The work described in Chapter 6: Constituents of the coagulation cascade are spatially and functionally related to experimental tubulointerstitial fibrosis has examined temporal and spatial in vivo relationships of coagulation factors and markers of fibrosis that aid our understanding of mechanisms of fibrosis. The aim of this thesis was to examine those facets of renal fibroblast function that are most devastating to renal function and culminate in an expansion of the renal interstitium during fibrosis. This work hopes to provide useful information to aid the understanding of the multifaceted mechanisms involved in renal tubulointerstitial fibrosis.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed Dec. 12, 2007). Vita. Includes bibliographical references.

<p>All metazoans possess an epithelial barrier that protects them from their environment and prevents loss off body fluid. Insects, which have an open circulatory system, depend on fast mechanism to seal wounds to avoid excessive loss of body fluids. As in vertebrates, and non-insect arthropods such as horseshoe crab and crustaceans, insects form a clot as the first response to tissue damage. Insect hemolymph coagulation has not been characterized extensively at the molecular level before, and the aim of my studies was to gain more knowledge on this topic. Morphological characterization of the<i> Drosophila </i>hemolymph clot showed that it resembles the clots previously described in other larger bodied insects, such as <i>Galleria mellonella</i>. The <i>Drosophila</i> clot is a fibrous network of cross-linked proteins and incorporated blood cells. The proteins building up the clot are soluble in the hemolymph or released from hemocytes upon activation. Since bacteria are caught in the clot matrix and thereby prevented from spreading it is likely that the clot serves as a first line of defense against microbial intruders. The bacteria are not killed by the clot. What actually kills the bacteria is not known at this point, although the phenoloxidase cascade does not seem to be of major importance since bacteria died in the absence of phenoloxidase. We identified and characterized a new clot protein which we named gp150 (Eig71Ee). Eig71Ee is an ecdysone-regulated mucin-like protein that is expressed in salivary glands, the perithophic membrane of the gut and in hemocytes, and can be labeled with the lectin peanut agglutinin (PNA). Eig71Ee was found to interact with another clot protein (Fondue), and the reaction was catalyzed by the enzyme transglutaminase. This is the first direct functional confirmation that transglutaminase acts in <i>Drosophila </i>coagulation. A protein fusion construct containing Fondue tagged with GFP was created. The fusion construct labeled the cuticle and the clot, and will be a valuable tool in future studies. Functional characterization of the previously identified clotting factor Hemolectin (Hml) revealed redundancy in the clotting mechanism. Loss of Hml had strong effects on larval hemolymph clotting ex vivo, but only minor effects, such as larges scabs, <i>in vivo</i> when larvae were wounded. An immunological role of Hml was demonstrated only after sensitizing the genetic background of Hml mutant flies confirming the difficulty of studying such processes in a living system. Hemolectin was previously considered to contain C-type lectin domains. We reassessed the domain structure and did not find any Ctype lectin domains; instead we found two discoidin domains which we propose are responsible for the protein’s lectin activity. We also showed that lepidopterans, such as<i> Galleria</i> <i>mellonella</i> and <i>Ephestia kuehniella</i>, use silk proteins to form clots. This finding suggests that the formation of a clot matrix evolved in insects by the co-option of proteins already participated in the formation of extracellular formations.</p>

The role of the nonionic surfactant Tween 80 on the behavior of the therapeutic recombinant protein Factor VIII (rFVIII) was investigated at solid/liquid and air/water interfaces. In order to provide a model system to compare results obtained for the complicated rFVIII-Tween system, a well-characterized globular protein lysozyme was used. The experimental scheme involved the introduction of the protein and Tween to the adsorption substrate in different manners, either lysozyme Tween together or in sequence as lysozyme followed by Tween or vice versa. It was observed that the addition of Tween together with lysozyme reduced the amounts adsorbed at hydrophobic surfaces, while no such reduction was observed on hydrophilic surfaces. A high Tween concentration was required to effect the removal of the lysozyme molecules from the hydrophobic surface and Tween was not effective in removing lysozyme from the hydrophilic surface at any concentration. These results suggest that the Tween-surface interaction is important in determining lysozyme adsorption. Similar observations were made for the rFVIII-Tween system at hydrophobic and hydrophilic silica interfaces. In this case, the presence of interfacial and solution Tween together resulted in complete prevention of rFVIII adsorption. Electrostatic forces were observed to be play an important role in rFVIII adsorption. The rFVIII-Tween interactions at solid interfaces were also evaluated using intrinsic fluorescence and biological activity measurements. Results obtained with respect rFVIII adsorbed mass, and structure or biological activity change upon adsorption, were evaluated in parallel. This parallel evaluation suggested that rFVIII adsorption on hydrophilic, negatively charged surfaces is likely to be highly ordered and oriented in a manner that retains the solvent accessibility of the active sites in rFVIII. On the other hand, rFVIII may adsorb to hydrophobic surfaces in different orientations, with a likelihood of surface induced unfolding. rFVIII-Tween interaction at the air/water interface was investigated separately. Surface tension data recorded for rFVIII-Tween mixtures suggested that Tween dominated the air/water interface as the Tween concentration was increased. Reduced interface-induced unfolding was observed at high Tween concentrations. These results were also thought to contribute to the reduction in rFVIII aggregation typically observed as a result of exposure to the air/water interface. / Graduation date: 2006

To achieve predictive capability for complex environmental systems with coagulation and arsenic sorption, a unified improved coagulation model coupled with arsenic sorption was developed. A unified coagulation model coupled with arsenic sorption was achieved by the following steps: (1) an improved discretized population balance equation (PBE) was developed to obtain the exact solution of conventional coagulation, (2) the improved PBE was extended to an adjustable geometric size interval having higher numerical stability, accuracy, and computational efficiency than existing models for fractal aggregate coagulation that includes agglomeration and fragmentation, (3) a surface complexation equilibrium model and a sorption kinetic model was introduced to predict arsenic sorption behavior onto hydrous metal oxide surfaces, and (4) an improved discretized PBE was coupled with arsenic sorption kinetics and equilibrium models by aid of collision efficiency ?? depending on surface charge (potential) on the hydrous metal oxide particles, colliding particle size ratio, and fluid strain-rate in applied flow system. The collision efficiency ?? into the improved (r,r)ij(r,r)ijdiscretized coagulation model for fractal aggregate yielded a unified improved coagulation model coupled with arsenic sorption kinetics and the equilibrium model. Thus, an improved unified coagulation model could provide high statistical accuracy, numerical stability, and computational efficiency to enhance predictive capability for behavior of arsenic sorption and fractal colloid particle aggregation and break-up, simultaneously. From the investigation, it is anticipated that the unified coagulation model coupled with arsenic sorption kinetics and equilibrium will provide a more complete understanding of the arsenic removal mechanism and its application to water/wastewater treatment. Further, this coupled model can be applied to other water and wastewater treatment systems combined with sorption and filtration processes. These combined processes can be optimized by the coupled model that was developed in this study. By simulating the arsenic sorption and particle size distribution as a pretreatment before filtration (sand filtration or membrane filtration), the overall arsenic removal efficiency and operation cost can be estimated.

Des résultats d’expériences cliniques de reconstruction de l’os maxillaire faites à partir de la greffe d’une "pâte osseuse" gélifiée par l’ajout de facteur tissulaire ont été le primum movens de ce travail. Cette "pâte osseuse", faite d’os en poudre et de plasma enrichi en plaquette (PRP) à laquelle on ajoute du facteur tissulaire, est un modèle à la fois de la coagulation et de la régénération osseuse. Pour analyser des effets de la coagulation, nous avons utilisé un modèle connu : la culture primaire de cellules endothéliales (HUVEC). Les effets in vitro des facteurs de coagulation, dénommés protéases de la coagulation, pris séparément, ont été bien étudiés dans ces cellules, néanmoins aucune information sur l’effet combiné de ces protéases ou du plasma en coagulation n’était connue. Nous avons mesuré la "signalisation calcium" comme réponse cellulaire aux différents agents et ces mesures de la signalisation calcium ont été complétées par la mesure d’une autre réponse biologique, à savoir la sécrétion de cytokines pro-inflammatoires (IL-6 et IL-8). Pour l’étude de la régénération osseuse, la signalisation calcium a été mesurée sur une lignée d’ostéosarcomes humains (SaOS-2), stimulée par des protéases de la voie extrinsèque de la coagulation (facteur VIIa, facteur Xa et thrombine). Comme réponse biologique complémentaire, nous avons évalué l’effet des protéases d’intérêt sur l’apoptose induite par l’absence de sérum dans le milieu de culture. Les premiers travaux, réalisés sur les HUVEC, nous ont permis de montrer que le facteur Xa et la thrombine induisaient des signaux calcium différents sur ces cellules en mono couches, alors que le complexe facteur tissulaire – facteur VIIa ne provoquait aucune signalisation calcium. Nous avons également pu montrer une addition des signaux calcium induits par le facteur Xa et la thrombine. L’activation in situ du facteur Xa et de la thrombine à partir de leur zymogène a permis à la fois de confirmer les résultats précédents et de se rapprocher de l’in vivo. Finalement, au plus proche de l’in vivo, les expériences faites avec du plasma en coagulation ont également permis de détecter un signal calcium. La réponse biologique (sécrétion d’IL-6 et d’IL-8) en aval du signal calcium a confirmé les résultats calcium. En ce qui concerne la régénération osseuse étudiée à partir de SaOS-2, nous avons démontré l’expression du facteur tissulaire sur la lignée SaOS-2 et nous avons montré que le facteur VIIa, le facteur Xa et la thrombine induisaient tous des signaux calcium. Ces signaux présentaient des caractéristiques propres suivant la ou les protéase(s) utilisée(s) pour la stimulation. Les mesures ont également permis de caractériser, sur ces cellules, les récepteurs activés par les protéases d’intérêt, à savoir les "protease-activated receptors" 1 et 2 (PAR-1 et PAR-2). Comme réponse biologique, nous avons mesuré la diminution de l’apoptose induite par les protéases en absence de sérum dans le milieu de culture. Il a ainsi été montré que seule l’activation du récepteur PAR-1 permettait de diminuer l’apoptose. Finalement, nous avons caractérisé la voie suivie, qui passait par la phosphoinositide 3-kinase et la voie des MAPK Raf/MEK/ERK 1/2. En conclusion, cette thèse a permis de montrer, d’une part, que le facteur Xa et la thrombine provoquent des réponses calciques et proinflammatoires additifs dans les cellules endothéliales et, d’autre part, que le complexe facteur tissulaire – facteur VIIa, le facteur Xa et la thrombine induisent des signaux calcium caractéristiques dans les ostéosarcomes par l’activation des récepteurs PAR, l’activation de PAR-1 diminuant l’apoptose induite par l’absence de sérum dans le milieu de culture.

Many pathogen infections in nature are accompanied by injury and subsequent coagulation. Despite the contribution of hemolymph coagulation to wound sealing, little is known about its immune function. Based on the molecular knowledge of Drosophila innate immunity, this thesis investigated the immune function of clot both in vitro and in vivo, the immune relevant genes involved in a natural infection model, involving entomopathgenic nematodes (EPN) and the factors leading to crystal cell activation. Transglutaminase (TG) and its substrate Fondue (Fon) have been identified as bona fide clot components in Drosophila larvae. By knocking down TG or Fon via RNAi, we observed an increased susceptibility to EPN in larvae. In addition, this increased susceptibility was associated with an impaired ability of hemolymph clots to entrap bacteria. Immunostaining revealed that both clot components (Fon and TG) were able to target microbial surfaces. All these data suggest an immune function for the Drosophila hemolymph clot. Strikingly, similar results were obtained when we ran parallel experiments with human FXIIIa, an ortholog of Drosophila TG, indicating a functional conservation. We also found evidence for the regulation on both clot and immunity by eicosanoids in Drosophila larvae. The combination of EPN infection with the Drosophila model system allowed us to discover an immune function for TEP3 and Glutactin. However the molecular mechanism underlying the involvement of these two proteins in this particular host-pathogen interaction remains to be elucidated. Prophenoloxidase, the proform of enzyme involved in hardening the clot matrix, has been shown to be released by rupture of crystal cells. This cell rupture is dependent on activation of the JNK pathway, Rho GTPases and Eiger. Our work further identified the cytoskeletal component, Moesin, and the cytoskeletal regulator Rac2 as mediators of cell rupture. Despite the possible role of caspases in crystal cell activation, such cell rupture was turned out to be different from apoptosis. The implication of Rab5 in this process indicated that proper endocytosis is required for cell activation and subsequent melanization. Our findings furthered not only our understanding of the release of proPO via cell rupture but also our knowledge on different paths of immune cell activation. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: In press. Paper 4: Manuscript.<strong></strong></p>

All metazoans possess an epithelial barrier that protects them from their environment and prevents loss off body fluid. Insects, which have an open circulatory system, depend on fast mechanism to seal wounds to avoid excessive loss of body fluids. As in vertebrates, and non-insect arthropods such as horseshoe crab and crustaceans, insects form a clot as the first response to tissue damage. Insect hemolymph coagulation has not been characterized extensively at the molecular level before, and the aim of my studies was to gain more knowledge on this topic. Morphological characterization of the Drosophila hemolymph clot showed that it resembles the clots previously described in other larger bodied insects, such as Galleria mellonella. The Drosophila clot is a fibrous network of cross-linked proteins and incorporated blood cells. The proteins building up the clot are soluble in the hemolymph or released from hemocytes upon activation. Since bacteria are caught in the clot matrix and thereby prevented from spreading it is likely that the clot serves as a first line of defense against microbial intruders. The bacteria are not killed by the clot. What actually kills the bacteria is not known at this point, although the phenoloxidase cascade does not seem to be of major importance since bacteria died in the absence of phenoloxidase. We identified and characterized a new clot protein which we named gp150 (Eig71Ee). Eig71Ee is an ecdysone-regulated mucin-like protein that is expressed in salivary glands, the perithophic membrane of the gut and in hemocytes, and can be labeled with the lectin peanut agglutinin (PNA). Eig71Ee was found to interact with another clot protein (Fondue), and the reaction was catalyzed by the enzyme transglutaminase. This is the first direct functional confirmation that transglutaminase acts in Drosophila coagulation. A protein fusion construct containing Fondue tagged with GFP was created. The fusion construct labeled the cuticle and the clot, and will be a valuable tool in future studies. Functional characterization of the previously identified clotting factor Hemolectin (Hml) revealed redundancy in the clotting mechanism. Loss of Hml had strong effects on larval hemolymph clotting ex vivo, but only minor effects, such as larges scabs, in vivo when larvae were wounded. An immunological role of Hml was demonstrated only after sensitizing the genetic background of Hml mutant flies confirming the difficulty of studying such processes in a living system. Hemolectin was previously considered to contain C-type lectin domains. We reassessed the domain structure and did not find any Ctype lectin domains; instead we found two discoidin domains which we propose are responsible for the protein’s lectin activity. We also showed that lepidopterans, such as Galleria mellonella and Ephestia kuehniella, use silk proteins to form clots. This finding suggests that the formation of a clot matrix evolved in insects by the co-option of proteins already participated in the formation of extracellular formations.

<p>Thrombosis, or pathological blood clot formation, is intimately associated with cardiovascular disease and is the leading cause of morbidity and mortality in the western world. Antithrombotics are commonly prescribed as prophylactic medications or as rapid onset anticoagulants in acute care clinical settings. Although a number of antithrombotics are clinically available, their use is limited by immunogenicity, toxicity, and inability to be controlled with an antidote in the event of hemorrhage. Therefore, new antithrombotics that are effective, yet can be rapidly controlled are urgently needed. </p><p>Aptamers are oligonucleotides that form complex secondary and tertiary structures based on intramolecular base pairing and nucleic acid folding that allows them to bind to molecular targets with high affinity and specificity. Aptamers can be isolated that bind to proteins, such as clotting proteins, and modulate protein function. However, unlike most currently used antithrombotics, aptamers can be directly controlled with an antidote and therefore represent a safer class of therapeutic agents. </p><p>To generate a novel anticoagulant, we developed an aptamer-antidote pair against prothrombin. Prothrombin is a blood protein that plays an essential role in clot formation. I truncated, optimized, and studied the mechanism of an aptamer that can bind to prothrombin and inhibit prothrombin function, thereby severely impeding clot formation. Moreover, to increase the safety profile of this anticoagulant aptamer, I developed an antidote that can quickly reverse aptamer function and restore normal clotting. This aptamer and antidote pair is the first antidote reversible anticoagulant that targets prothrombin and may prove to be a valuable clinical anticoagulant.</p><p>A number of anticoagulants are in development, and a wide debate regarding the optimal protein target for anticoagulation is underway. We have previously generated anticoagulant aptamers to human coagulation factor VII, factor IX, factor X, and prothrombin. I compared the effects of these four anticoagulant aptamers to determine their impact on thrombin generation and clot formation. Each aptamer exerts its own unique effect on thrombin generation/clot formation, depending on the role that its protein target plays in coagulation. These studies provide valuable data regarding target validation and the anticoagulant effects of different therapeutic aptamers.</p><p>Robust anticoagulation is required during acute clinical surgical procedures to treat thrombosis. Currently used anticoagulants have several untoward side effects and most are not antidote controllable. I tested the effects of combining two anticoagulant aptamers to assess potential drug synergy. Several combinations of two anticoagulant aptamers were synergistic and severely impaired blood clot formation. One specific pair of aptamers that targeted factor X (FX) and prothrombin in combination was extremely potent and could keep blood fluid in an ex vivo model of extracorporeal circulation. Additionally, this pair of aptamers could be functionally modulated with two different types of antidotes. In conjunction with antidote reversal, this strategy of combining aptamer anticoagulants may prove useful in a variety of highly prothrombotic acute clinical settings. </p><p>Finally, to explore the potential of aptamers to regulate platelet function, I isolated and characterized an aptamer toward platelet glycoprotein VI. Glycoprotein VI is a platelet surface receptor that plays a key role in platelet activation and platelet plug formation. I isolated several aptamers that bind to glycoprotein VI, and show that the lead aptamer binds to platelets with high affinity and causes platelet activation and aggregation. This aptamer could potentially be further developed for topical administration to manage bleeding, or for biomarker detection of soluble glycoprotein VI in patient plasma.</p> / Dissertation

Surgery precipitates a hypercoagulable state and has been shown to increase the development of cancer metastases in animal models, however mechanism(s) responsible for this are largely unknown. We hypothesize that the prometastatic effect of surgery may be secondary to postoperative hypercoagulable state. Surgical stress was induced in mice by partial hepatectomy or nephrectomy, preceded by intravenous injection of CT26-LacZ or B16F10-LacZ cells to establish pulmonary metastases with or without perioperative anticoagulation and their lung tumor cell emboli (TCE) were quantified. Fibrinogen and platelets were fluorescently labeled prior to surgical stress to evaluate TCE-associated fibrin and platelet clots. Surgery significantly increased metastases while anticoagulation with five different agents attenuated this effect. Fibrin and platelet clots were associated with TCE significantly more frequently in surgically stressed mice. Surgery promotes the formation of fibrin and platelet clots around TCE and this appears to be the mechanism for the increase in metastases seen following surgery.