The genus Bartonella comprises emerging pathogens that are causative agents of a wide range of clinical manifestations such as cat scratch disease, bacillary angiomatosis, and Carrion’s disease. All species are transmitted by blood-sucking arthropods and infect erythrocytes and endothelial cells of hosts. Carrion’s disease is a bi-phasic infection caused by Bartonella bacilliformis which is characterized by hemolysis of infected erythrocytes followed by invasion of the vascular endothelium. This provokes pronounced cellular proliferation, angiogenesis and skin eruptions called verruga peruana. Endothelial cells are thought to be the primary niche wherein bacteria reside between inoculation and erythrocyte infection. This study aims to elucidate some of the endothelial factors involved during the verruga peruana phase of Carrion’s disease. In order to adhere to and invade human microvascular endothelial cells (HMEC-1), B. bacilliformis engages a family of cell receptors called integrins. We used anti-integrin antibodies to show that the primary integrin involved is the fibronectin receptor á5â1, although the vitronectin receptor áVâ3 also plays a minor role. We show B. bacilliformis invasion is also dependent on integrin ligands, fibronectin and vitronectin as antibodies against these proteins decreased invasion and attachment, whereas pre-treatment of the bacteria with these molecules enhanced infection of endothelial cells. Bacterial uptake requires various host cytoplasmic signaling pathways to work in tandem, and our study identified three mitogen activated protein kinases involved. Apart from MAPKs, phosphotidylinositol 3 kinase plays a role during invasion and cell survival. PI3K inhibitors blocked bacterial internalization and B. bacilliformis infected cells showed accelerated apoptosis. Lastly, microarray analysis was performed to study the gene expression profile of B. bacilliformis infected HMEC-1 cells. Numerous molecules of the integrin signaling pathways are involved, suggesting integrins as the major receptor recruited for the successful infection by B. bacilliformis. In summary this is the first study to demonstrate the role of integrins as B. bacilliformis receptors and integrin ligands as facilitators of infection. Gene expression analysis suggests the possibility that integrin mediated signaling pathways are the key modulators of cellular alterations during B. bacilliformis infection. This hypothesis is supported by the identification of some members of the integrin signaling pathway necessary for B. bacilliformis entry into endothelial cells.

The evolution of biomechanical behavior of arteries plays a key role in the onset and progression of cardiovascular disease. Biomechanical behavior is governed by the content and organization of the key structural constituents (e.g., collagen, elastin, and smooth muscle) and vessel geometry. The evolution of biomechanical behavior of arteries is governed by biologically-mediated synthesis, degradation, and reorganization of these key structural constituents. A hallmark goal in biomechanics is quantifying the relationship between the microstructure of tissues and their mechanical response throughout tissue growth and remodeling; this will provide a crucial link in understanding the tissue level effects of biological processes involved in disease and normal growth Fibulin-5 (fbln5) is an ECM protein that binds tropoelastin and interacts with integrins. Arteries from fbln5 knockout mice lack functional elastic fibers and provide a system for investigating the link between an artery's microstructure and its mechanical response. The overall goal of this project was to develop multi-scaled theoretical and experimental frameworks to quantify the relationship between microstructural content and organization and tissue level material properties of arteries from fbln5 null mice and littermate controls and to quantify the effects of fbln5 on the in vivo maturation of mouse carotid arteries. We found significant differences in the mechanical properties of carotid arteries of fbln5 null mice, and these differences were correlated with altered extracellular matrix organization. We also developed a microstructurally-motivated 3-dimensional constrained mixture model for vascular growth and remodeling. Using physiological rates of constituent growth and turnover, the model captured the salient findings found in the literature. Incorporating experimentally measured fiber angle data into constitutive relations yielded greater predictive accuracy. This dissertation incorporates experimental data quantified at the micro (microstructural-level fiber distributions) and macro (tissue-level mechanical response) scale and incorporates these data into microstructurally motivated constitutive relations. The use of structurally motivated constitutive relations and experimentally measured microstructural data provides a foundation for future work in further understanding the relationship between processes governing microstructure and the tissue level effects of disease and normal growth.

Epithelial to mesenchymal transition (EMT), the transdifferentation of an epithelial cell into a mesenchymal fibroblast, is a cellular process necessary for embryonic development and wound healing. However, uncontrolled EMT can result in accumulation of myofibroblasts and excessive deposition of ECM, contributing to the pathological progression of fibrotic diseases such as pulmonary fibrosis. The ability to control EMT is important for development of novel therapeutics for fibrotic pathologies and for designing novel biomaterials for tissue engineering applications seeking to promote EMT for development of complex tissues. EMT is a highly orchestrated process involving the integration of biochemical signals from specific integrin-mediated interactions with extracellular matrix (ECM) proteins and soluble growth factors such as TGFβ. TGFβ, a potent inducer of EMT, is activated via cell contraction-mediated mechanical release of the growth factor from a macromolecular latency complex. Thus TGFβ activity and subsequent EMT may be influenced by the biochemical and biophysical state of the surrounding ECM. Based on these knowns, it was hypothesized that both changes in integrin engagement and increases in substrate rigidity would modulate EMT due to changes in epithelial cell contraction and TGFβ activation. Here we show that integrin-specific interactions with fibronectin (Fn) fragments displaying both the RGD and PHSRN binding sites facilitate cell binding through α5β1 and α3β1 integrins, and lead to maintenance of epithelial phenotype, while Fn fragments displaying only the RGD site facilitate cell binding through αv integrins and lead to EMT. An in depth investigation into α3β1 binding to Fn fragments indicates that binding is dependent on both the presence and orientation of the PHSRN site. Studies investigating the contribution of ECM stiffening on EMT responses show that increasingly rigid Fn substrates are sufficient to induce spontaneous EMT. Analysis of TGFβ-responsive genes implicate TGFβ-expression, activation or signaling as a mechanism for the observed EMT responses. Together these results suggest that the ECM micromechanical environment is a significant contributor to the onset of EMT responses and provide insights into the design of biomaterial-based microenvironments for the control of epithelial cell phenotype.

The Drosophila dorsal group gene pipe provides the crucial link that transmits dorsal-ventral (DV) polarity information from the ovary to the embryo. Females homozygous for mutations in pipe produce dorsalized embryos. pipe encodes ten protein isoforms with amino acid sequence similarity to vertebrate glycosaminoglycan 2-O-sulfotransferases, suggesting that Pipe functions by modifying a carbohydrate-bearing molecule that controls embryonic DV patterning. Two major components of my project have been to examine the functional specificities of different Pipe isoforms and to identify Pipe's enzymatic substrate and learn how it participates in DV pattern formation. I have used two approaches to investigate whether the various Pipe isoforms share the same functional specificities. In one approach, I expressed each isoform in the follicle cells and found that the expression of only one of them was able to rescue the pipe mutant phenotype or ventralize progeny embryos. In a second set of transgenic studies, three of the other isoforms were individually shown to restore the production of a pipe-dependent sulfated epitope when expressed in the salivary glands of otherwise pipe null mutant embryos. These data indicate that distinct functional specificities are associated with the various Pipe protein isoforms. In addition, these studies allowed me to determine that embryos from females lacking endogenous pipe expression nevertheless retain polarity along their dorsal-ventral axis, suggesting the existence of a second polarizing signal in addition to the ventral transcription of pipe. To identify Pipe’s substrate, I developed a technique for metabolic labeling which enabled me to identify a molecule exhibiting Pipe-dependent sulfation. This molecule was identified as the protein Vitelline Membrane-Like (VML), a putative component of the vitelline membrane layer of the eggshell. The involvement of VML in dorsalventral patterning was demonstrated on the basis of the enhancing effects of a vml mutation on the severity of dorsalization of embryos from females of a sensitized genetic background. Thus, VML represents a bona fide substrate of Pipe that participates in the establishment of dorsal-ventral polarity. In these studies I was also able to show Pipedependent sulfation of other vitelline membrane components which may also influence embryonic dorsal-ventral patterning. / text

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The total annual incidence of arthritis in this prospective cross-sectional study on adults was 115/100 000. The annual incidence of rheumatoid arthritis (RA) was 24/100 000, 29/100 000 for women, and 18/100 000 for men. For reactive arthritis (ReA) the annual incidence was slightly higher, 28/100 000, and for undifferentiated arthritis 41/100 000. The annual incidence of Lyme disease and sarcoid arthritis was low. The annual incidence of arthritis in this study compares well with findings in earlier reports from both registers and case review studies. Almost 50% of the patients in the series of 71 patients with arthritis of less than 3 months’ duration had a preceding infection. Campylobacter jejuni ReA dominated the enteric ReA group. We found only a few patients with preceding Chl. trachomatis, Chl. pneumoniae, Borrelia burgdorferi or parvovirus B19 infections. The arthritis patients with a preceding infection went into remission more often than the patients without a preceding infection. The disease specificity of anti-CCP antibodies for RA was high, 96%, confirming earlier results. Anti-CCP antibodies differentiated RA from other arthritides. Several patients in the different diagnosis groups had raised serum COMP levels, indicating cartilage involvement very early in the disease, even in mild and self-limiting disease with good prognosis. The economic burden of early joint inflammation was found to be considerable already during the first few months of the arthritis irrespective of diagnosis. Surprisingly, patients with ReA generated almost as high costs as patients with RA during thefirst few months of the disease, even though most of the ReA patients had a relatively mild disease. Sick leave accounted for about 50% of the costs. The distribution of costs in the different patient groups was skewed. The median cost per patient for the group of patients with RA was US$4385, for ReA US$4085, for other types of specified arthritis US$3361, and for undifferentiated arthritis US$1482. This underlines the necessity of quick referral and therapy, not only to decrease the inflammation and prevent functional impairment, but also to decrease the costs of early arthritis.

Aggrecan is one of the most important structural components of the extracellular matrix (ECM) of articular cartilage, where it contributes to the hydration of the tissue and its ability to resist compressive loads during joint movement. Increased aggrecan degradation and loss occurs in joint diseases and is thought to be mediated by enzymes such as the matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS). It has also been proposed that aggrecan release from the cartilage can be mediated by a non-proteolytic mechanism which involves the degradation of hyaluronan (RA) to which the aggrecan is bound. As aggrecan degradation and loss is known to be induced by pro-inflammatory cytokines, IL-1, TNFalpha, IL-6, IL-17 and OSM were used to investigate the mechanisms involved in proteoglycan catabolism in organ cultures of bovine articular cartilage. Irrespective of the cytokine, all aggrecan fragments generated were characteristic of aggrecanase action, and no additional aggrecan-degrading enzymatic activity was detected. In the presence of OSM, more rapid aggrecan release was observed, due to both proteolysis and fragmentation of HA by hyaluronidase activity. Moreover, addition of OSM resulted in the cleavage of aggrecan at a non-canonical aggrecanase site near its carboxy-terminal globular domain. Such cleavage could be reproduced in vitro by the action of either ADAMTS-4 or ADAMTS-5. Gene expression analysis revealed that both aggrecanases were highly induced by the cytokines, and while ADAMTS-4 was the major aggrecanase to be stimulated in all conditions, ADAMTS-5 remains the predominant aggrecanase to be expressed in cartilage. Thus, the present study shows that aggrecanase activity is primarily responsible for aggrecan degradation in the early stages of cytokine stimulation, and that in the presence of OSM, aggrecanase substrate specificity can be differentially modulated and hyaluronidase-mediated RA degradation can be induced.

Includes bibliographical references (leaves 292-352) / xvi, 352 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Determines the role of matrix metalloproteinases, MMP-2 and MMP-9 in reperfusion injury following skeletal muscle ischaemia; and, whether inhibition of MMPs by doxycycline protects against tissue damage. / Thesis (M.D.)--University of Adelaide, Dept. of Surgery, 2002

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 202-229). Also available in print.