Matrix Extracellular Phosphoglycoprotein (MEPE) is a member of a family of proteins called small integrin-binding ligand, N-linked glycoproteins (SIBLINGs) which play key roles in biomineralisation. Altered MEPE expression is associated with several phosphate and bone-mineral metabolic disorders such as oncogenic osteomalacia and hypophosphatemic rickets. Despite this, it remains undetermined what impact MEPE has on the growth plate; the cartilage anlagen from which endochondral ossification, the process responsible for linear bone growth, occurs. The work of this thesis has characterised the ATDC5 cell line and the metatarsal organ culture as useful in vitro models of endochondral ossification. These will prove vital in the pursuit of underpinning the molecular mechanisms involved in endochondral bone growth. These models form the basis of the further studies in this thesis examining the role of MEPE within this highly orchestrated process. Before such role can be defined, this thesis details the spatial and temporal localisation patterns of MEPE in 10-day- and 4-week-old murine growth plates. More specifically, MEPE protein and mRNA were preferentially expressed by the hypertrophic chondrocytes as shown by immunohistochemistry and in situ hybridisation respectively. Microdissection of the murine growth plate confirmed this. Localisation of the cleavage product of MEPE, a 2.2kDa acidic serine- and aspirate-rich motif (ASARM) peptide, followed a similar pattern of expression. The localisation of MEPE to sites of mineralisation serves to strengthen its potential role in chondrocyte matrix mineralisation. This thesis identified this role in both mineralising ATDC5 cells and the metatarsal organ culture. The ASARM peptide was found to be the functional component of MEPE and this function was dependent upon its post-translational phosphorylation. Phosphorylated (p)ASARM peptides significantly inhibited chondrocyte matrix mineralisation without altering the proliferation or differentiation of the chondrocyte cells, or their ability to produce an extracellular matrix. mRNA analysis by qPCR indicted a feedback system by which the pASARM peptide functions to allow the release of further ASARM peptides. Moreover, the pASARM peptide inhibited mRNA expression of markers of vascular angiogenesis highlighting a novel mechanism by which they may inhibit chondrocyte matrix mineralisation. This thesis also determines the regulatory cross-talk between the chondrocytes of the murine growth plate, with the most abundant bone cell type, the osteocyte. This cross-talk inhibits chondrocyte matrix mineralisation and is attributed to sclerostin, an osteocyte-specific secretory protein. Furthermore, it is shown that sclerostin acts through the MEPE-ASARM axis to regulate chondrocyte matrix mineralisation and thus endochondral ossification. The work described herein has characterised and validated in vitro models of growth plate chondrocyte matrix mineralisation and has used these to identify the role of MEPE within chondrocyte matrix mineralisation.

Fibrillin-1 is the main component of the 10-12 nm microfibrils, which are found in several elastic and non-elastic tissues. Human fibrillin-1 contains multiple calcium-binding epidermal growth factor-like (cbEGF) domains interspersed with transforming growth factor β-binding protein-like (TB) domains. TB4 domain contains a flexible RGD loop which mediates cell adhesion via αVβ3, α5β1 and αVβ6 integrins. Mutations which introduce amino acid substitutions into TB4 are associated with a wide spectrum of diseases such as Marfan syndrome (MFS), ectopia lentis, Stiff skin syndrome (SSS). Amino acid substitutions such as W1570C, C1564S and C1577G in the TB4 domain have been found to cause SSS. The upstream TB5 domain has been predicted to modulate integrin binding and a deletion in the domain has been found in Weill-Marchesani syndrome (WMS), phenotype of which is similar to SSS (skin fibrosis and short stature), thereby suggesting that the underlying pathogenic mechanism might be similar. This study has used cellular, biochemical and biophysical methods to investigate the effects of SSS substitutions C1564S and W1570C on domain structure and function and compared it to a MFS substitution C1564Y in the TB4 domain and WMS deletion in the TB5 domain. Effects of the SSS mutations on structure of the domains were studied using limited proteolysis, nuclear magnetic resonance spectroscopy and calcium chelation experiments. Subsequently, the ability of human fibroblasts to secrete wild-type and mutant fibrillin-1 was examined to identify the effect of the mutations on the trafficking of the protein. Finally, cell binding assays and SPR was employed to investigate the effect of disease-causing mutations on fibrillin-1/integrin interactions. The results demonstrate that the SSS mutations affect TB4-cbEGF23 interface and calcium-binding to cbEGF23 but do not alter secretion of recombinant fibrillin-1 mutant fragments from the cell. On the other hand, intracellular retention was observed for MFS substitution C1564Y which was shown to be more susceptible to proteolysis than SSS substitution C1564S. WMS deletion also gives rise to partial retention of the recombinant fragment, suggesting a different pathogenic mechanism for these disorders. Cell binding assays and surface plasmon resonance (SPR) experiments show that SSS mutations affect binding to αvβ3 integrin, but not αvβ6 integrin suggesting that selectively impaired integrin interactions may contribute to pathogenesis of SSS.

Naegleria fowleri (N. fowleri) and Naegleria lovaniensis (N. lovaniensis) are closely related amebae found in the environment. N. fowleri causes Primary Amebic Meningoencephalitis (PAM), a fatal disease of the central nervous system, while N. lovaniensis is nonpathogenic. N. fowleri infection occurs when amebae enter the nasal passages, and migrate to the brain. The molecular mechanisms involved in the pathogenesis of PAM are not well-defined. Therefore, the purpose of this study was to define phenotypic characteristics that may be functionally linked to the pathogenicity associated with N. fowleri. Studies revealed that N. fowleri has a faster growth rate and is more resistant to complement-mediated lysis when compared to N. lovaniensis. Additionally, contact-independent cytotoxicity was observed only for N. fowleri. The ability to invade tissues can be a characteristic that distinguishes pathogens from nonpathogens. Therefore, adhesion to extracellular matrix components (ECM), laminin-1, fibronectin, and collagen I, was assessed. N. fowleri exhibited a higher level of adhesion to ECM components and was shown to invade tri-dimensional ECM scaffolds (matrigel and collagen I) to a greater extent than N. lovaniensis. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Attachment of N. fowleri to ECM components was decreased significantly when amebae were pretreated with trypsin, suggesting a role for a surface protein in this process. Pretreatment of N. fowleri amebae with periodate, a sugar oxidant, led to a decrease in attachment to laminin-1 and fibronectin suggesting that the surface component contained a sugar moiety. Western immunoblotting revealed two integrin-like proteins for both species. However, one with a molecular mass of approximately 70 kDa, was detected at a higher level for N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. An anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Zymographic analysis demonstrated differential expression of proteases occurs when N. fowleri and N. lovaniensis invade ECM components using an in vitro invasion assay. These results indicate a distinction in adhesion to, and invasion of, extracellular matrix proteins between N. fowleri and N. lovaniensis.

No description available.

Atherosclerosis, the pathophysiology of many cardiovascular diseases (CVD), is a chronic inflammatory process caused by the sustained accumulation of cholesterol, followed by endothelial dysfunction, and the resulting vascular inflammation. The established treatment for atherosclerosis, to date, involves the use of statins. These medications are hydroxymethylglutaryl coenzyme A reductase (HMG-CoA) inhibitors and lower the levels of by inhibiting HMG-CoA, a rate limiting step in the biosynthesis of cholesterol. Statin therapy varies in effectiveness based on dosage and individual differences, making effective treatment of patients challenging. More recently, extracellular vesicles (EVs) have emerged as a promising field in cardiovascular research. Once thought of as “platelet dust,” EVs are now recognized for their potential as therapeutic targets and tools. In this review, a comprehensive characterization of EVs is provided to explain how EVs are involved in normal physiological function and pathological processes of atherosclerosis. Evidence supports a model where EVs participate in the initiation and progression of atherosclerosis and may also be used as a delivery tool in disease therapy. Currently, cell-derived EVs can be therapeutic agents in animal models, an effective tool in gene therapy, or a drug delivery vehicle. Future experiments enhancing the therapeutic potential of EVs promise to deepen our understanding of EV-based therapy for atherosclerosis precision medicine.

Doctor of Philosophy / Department of Food, Nutrition, Dietetics and Health / Tonatiuh Melgarejo / Mark Haub / Human umbilical cord-derived mesenchymal stromal cells (HUC-MSCs) have an enormous therapeutic potential because of their immunomodulatory and anti-inflammatory properties. However, there are limitations for their therapeutic use due to low cell survival after implantation, the risk of culture-borne pathogens, and the risk of embolism and thrombosis after intravenous infusion. Exosomes, on the other hand, constitute an important part of the MSCs secretome and may play a role in their therapeutic effects. Here, it was demonstrated that HUC-MSC-derived exosomes accumulate in human and mouse osteosarcoma cell lines in vitro and reduce their proliferation. The distribution of HUC-MSCs exosomes was shown in osteosarcoma tumor- bearing mice. Exosome distribution in vivo was observed using Magnetic Resonance Imaging (MRI) of gadolinium-labeled exosomes and by fluorescent imaging after infusion of near infrared dye-labeled exosomes. HUC-MSC exosomes accumulated in the tumor throughout the 48 hours ours post-injection period. In contrast, synthetic lipid nanoparticle accumulate in tumor only for the first 3ours post-injection. These results suggest that labeling with gadolinium or near-infrared dye may affect exosome accumulation within the spleen. In summary, this study showed that HUC-MSCs exosomes can accumulate to osteosarcoma cells in vitro and in vivo, and thus they may be useful for detecting cancer metastasis.

Cartilage has limited intrinsic healing potential upon injury, due to the low cell density and the lack of blood supply. Degenerative disease of the cartilage, such as osteoarthritis (OA), is challenging to treat without clear mechanistic understandings of cartilage development. With over 90% of the cartilage tissue occupied by extracellular matrix (ECM), understanding the cellular and molecular effects of cartilage ECM on chondrogenesis and chondrocyte behavior is crucial for therapeutic development. The focus of this work is to study the regulation of chondrogenesis and hypertrophic maturation of human mesenchymal stem cells (MSCs) by cartilage ECM in the context of potential therapeutic applications. To study the cartilage ECM, we created a decellularized ECM digest from native porcine cartilage and examined its effects on MSCs. Since native cartilage ECM maintains chondrocyte homeostasis without progressing to hypertrophic degeneration, we hypothesized that the decellularized ECM would promote MSC chondrogenesis and inhibit hypertrophy. Indeed, we showed that ECM promoted MSC chondrogenesis and matrix production, and inhibited hypertrophy and endochondral ossification. The chondrogenic effect was shown to potentially involve the PI3K-Akt-Foxo1 and Hif1 pathways. By recapitulating the activated Hif1 pathway, roxadustat, a small molecule stabilizer of Hif, was able to reproduce the chondrogenic and anti-hypertrophic effects of the cartilage ECM. It also reduced the expression of matrix metalloproteases (MMPs) in MSCs, healthy chondrocytes, and OA chondrocytes, and alleviated matrix degradation in bovine cartilage explants. We also attempted to identify ECM components that display chondrogenic properties. Collagen XI, a minor component of articular cartilage, was shown to promote cartilage matrix formation in MSCs and healthy chondrocytes, and to reduce matrix degradation in bovine cartilage explants. Taken together, this study reveals the dual roles of cartilage ECM in promoting chondrogenesis and matrix production and inhibiting cartilage hypertrophy. Importantly, small molecule drugs that recapitulate the signaling pathways of ECM regulation, and collagen XI, a component of the ECM, may serve as leads for further therapeutic development for cartilage injury and degenerative disease.

The infectious first-stage larvae of the nematode Trichinella spiralis is an intracellular parasite of altered skeletal muscle. Invasion of the muscle cell initiates a series of morphological changes in the host muscle cell which ultimately results in a specialized unit called the nurse cell. The completed nurse cell consists of a collagenous capsule, matrix of altered sarcoplasm, and a circulatory rete. The purpose of this study was to determine the types of collagen present in the nurse cell capsule. Additionally, the presence of the gl ycoproteins, laminin and tenascin was determined. This study also sought to demonstrate the location of the selected extracellular matrix proteins within the capsule. Nurse cells were isolated from infected host muscle by sequential protease treatment with pronase, collagenase, and hyaluronidase. Nurse cells were digested with pepsin to produce characteristic pepsin-resistant triple helical fragments of collagen. The nurse cellpepsin digest was characterized by SDS-page, under reduced and nonreduced conditions, with type VI collagen and the ala2a3 chains of type XI collagen. Frozen tissue sections of infected and non-infected rat diaphragms were screened with specific polyclonal antibodies against types I, m, IV, V/Xl, and VI collagen, laminin, and tenascin. Indirect immunofluorescence using FITC secondary antibodies was used to locate the protein in the capsule and host tissue. SDS-page of the nurse cell-pepsin digest produced an electrophoretic pattern of resistant fragments characteristic for types I, III, IV, V, and VI collagen. Additionally, fragments migrated with an apparent molecular weight expected for pepsin resistant fragments of laminin. Indirect immunofluorescence showed types I, III, IV, and VI collagen, and laminin were distributed throughout the capsule. Serum No. 4876, which recognizes type V /XJ collagen, localized to the larvae. Tenascin failed to stain the nurse cell or host tissue. The results show that the capsule is a heterogenic structure with types I, III, IV, V, and VI collagens, and laminin distributed throughout the structure. The immunolocalization of Serum No. 4876 to the larvae suggests that a nematode collagen shares an amino acid sequence in common with mammalian type V /XI collagen.

Psychotrophic bacteria produce heat-stable lipase, protease, and phospholipase. Pervious studies indicate the production of multiple enzymes in several strains of Pseudomonas fluorescens, but conclusive evidence is lacking. The influence of culture conditions on the production and thermostability of phospholipase, protease, and lipase was investigated in 17 raw milk and environmental isolates. Production and thermostability of the enzymes were influenced by strain, stage of growth, and the culture medium. Cross-reactivity of antibodies raised to a purified protease and a commercial lipase indicated the immunological diversity of the enzymes. Protease purification was undertaken to investigate the production of multiple proteases within a single strain. A single monomeric protease with a molecular weight of 52 kDa was purified from P. fluorescens RO98. Biochemical characterization of the enzyme revealed that it was a zinc-metallo acidprotease with pH and temperature optima of 5.0 35°C, respectively. The enzyme was thermostable with a D55 of 41 min and a D62.5 of 18 h. Although sensitive assays exist for proteases, they are not suitable for detection of protease activity in milk in the presence of milk proteins. Existing immunoassays approach the required sensitivity but take about 6 h and cannot distinguish between active and inactive enzyme. An immunoassay that can be completed within 2 h and that can detect and distinguish both total and active enzyme was explored. The ratio of these two forms gives insight into the history of the milk. The ability of the purified protease to hydrolyze hydrophobic peptides associated with bitterness in Cheddar cheese was also investigated. Results demonstrated that the protease had the potential to debitter Cheddar cheese because it was able to hydrolyze the bitter peptides commonly found during aging. Two lipolytic enzymes with molecular weights of 50 (Pf-lip1) and 12 kDa (Pf-lip2) were purified from P. fluorescens RO98. Differences were observed in their biochemical properties. D62.5-values of 12.7 and 29.9 h were determined for Pf-lip1 and Pf-lip2, respectively. Pf-lip1 preferred longer chain length fatty acids, and Pf-lip2 preferred shorter chain length substrates. Pf-lip1 hydrolyzed milk fat and emulsified triolein, but Pf-lip2 did not, indicating that the latter was an esterase. This information is of significance to the dairy industry because activity tests that assay both the lipolytic enzymes need to be used in order to direct raw milk to short shelf-life products during processing and ensure quality of long shelf-life products

No description available.