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.

The replacement of missing teeth by osseointegrated dental implants is a commonly utilised treatment option in dentistry. However, successful treatment outcomes are dependent on sufficient bone quantity in the proposed surgical site for implant placement (Buser et al., 2004). Surgical augmentation of bone defects is commonly performed prior to or during implant placement. Bone augmentation procedures of the maxillary sinus or guided bone regeneration (GBR) procedures of alveolar ridge defects have utilised a variety of bone graft materials in block or particulate form, either alone or in combination with resorbable or non-resorbable barrier membranes. Objective: The aim of this study was to determine whether Hydroxyapatite β-Tricalcium Phosphate (HA-TCP) either alone or combined with Enamel Matrix Derivative (EMD) or recombinant human Platelet Derived Growth Factor-BB (rhPDGF-BB) is osteoinductive when implanted into a nonosseous site. Methods: Twenty CD-1 adult male mice underwent intramuscular implantation into both hindlimbs of an empty gelatine capsule or a gelatine capsule containing one of the following: 10 mg of uncoated particulate HA-TCP, (Straumann Bone Ceramic®, HA-TCP), EMD coated HA-TCP, (Emdogain®, HATCP + EMD) or rhPDGF-BB coated HA-TCP (HA-TCP + PDGF). Ten animals were sacrificed at four and eight weeks with five specimens from each group retrieved at each time point. The area of graft placement was radiographed and after graft retrieval, a semi-quantitative histological examination was performed with the aim of assessing the inflammatory changes, reparative processes and osteoinduction within the graft site. Results: At both 4 and 8 weeks, histological analysis failed to demonstrate any osteoinductive activity in any of the specimens from the three experimental groups. A minimal chronic inflammatory response and foreign body reaction was seen in the experimental groups which reduced over time. The particles were embedded within fibrous connective tissue and were encapsulated by a dense cellular layer consisting of active fibroblasts and occasional macrophages with the thickness of this layer decreasing over time. At 4 weeks, a greater density of the fibrous connective tissue was demonstrated in the HA-TCP + EMD group (P<0.001) while a greater thickness in the capsule thickness was seen in the HA-TCP group (P=0.022) although no differences were seen after 8 weeks. Greater neovascularisation was seen in the HA-TCP + PDGF group after 8 weeks (P=0.043) while greater amounts of adipose tissue surrounding the particles were detected in the HA-TCP + PDGF group at 4 weeks (P=0.002) and in the HA-TCP + EMD group at eight weeks (P=0.002). Conclusions: The results of this study suggest that the use of commercially available HA-TCP alone or in combination with EMD or rhPDGF-BB is biocompatible but not osteoinductive in the murine model. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1522641 / Thesis (D.Clin.Dent.) -- University of Adelaide, School of Dentistry, 2010

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) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

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

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 3 uppsatser.