A cysteine-containing elastin-like polypeptide (ELP2cys) was successfully synthesized and purified, and was shown to behave in a similar fashion to other well-characterized ELPs. Incorporating the ELP2cys as a crosslinking agent into a solution of sulphated hyaluronan (CMHA-S) not only decreased the gelation time of the solution but also increased the crosslinking density of the resultant hydrogel, in turn increasing both the resiliency and stiffness of the construct. Preliminary in vitro work involved culture of human disc cells, followed by their encapsulation within the hydrogel. Unfortunately the results were inconclusive, although it appeared as though the addition of ELP2cys to the matrix did not negatively affect the viability of the cells, as compared to hydrogels with CMHA-S only. This study showed that ELP2cys is a valuable addition to the family of recombinant elastin-like polypeptides, and shows promise as a crosslinking agent in the formation of hyaluronan hydrogels.

Hyaluronidase 2 (HYAL2) is a GPI-linked protein that is proposed to initiate the degradation of hyaluronan (HA), a major extracellular matrix component of many vertebrate tissues. Hyal2 knockout (KO) mice displayed craniofacial abnormalities and severe preweaning lethality. 54% of the surviving KOs developed a grossly dilated left or right atrium, requiring euthanasia, by 3 months of age. We hypothesize that the absence of HYAL2 leads to the accumulation of HA in organs/tissues where HA is normally abundant resulting in developmental defects and organs dysfunction. Molecular and histological analysis of HYAL2 KO hearts demonstrated extracellular accumulation of high molecular mass (HMM) HA in the heart valves, myocardium, serum and lungs which was associated with severe cardiopulmonary dysfunction. Further, structural and functional analyses of Hyal2 KO mouse hearts using high-frequency ultrasound revealed atrial dilation accompanied by diastolic dysfunction that was evident as early as 4 weeks of age, and progressed with age. Further, 50% of HYAL2 KO mice exhibited a triatrial heart (cor-triatriatum). Histological analyses revealed that the atrial dilation was the result of excess tissue, and did not correlate with the presence of cor triatrium. Hyal2 KO mice were found to have increased numbers of mesenchymal cells at early stages of development, presumably due to the presence of excess HA, that lead to cardiac dysfunction. Further examination of HYAL2 distribution in a broad range of mouse tissues, and accumulation of HA in its absence demonstrated that HYAL2 is mainly localized to endothelial cells and some specialized epithelial cells, and plays a major role in HMM-HA degradation. These studies demonstrated that HYAL2 is important for HA degradation and organ development. In the longer term, our findings will be valuable for understanding pathologies associated with the disruption of HA catabolism, and potentially in the identification of HYAL2-deficient patients. / May 2016

This thesis presents the preparation of hyaluronan-based biomaterials with imaging capability and their application as scaffolds in tissue engineering. First, we have synthesized HA derivatives functionalized with different chemoselective groups. Then, functional ligands with capacities for hydrophobic drug loading, imaging, and metal ion coordination were chemically conjugated to HA by chemoselective reactions with these groups. An injectable in situ forming HA hydrogel was prepared by hydrazone cross-linking between hybrid iron-oxide nanogel and HA-aldehyde (paper-I). The degradation of this hydrogel could be monitored by MRI and UV-vis spectroscopy since it contained iron oxide as a contrast agent and pyrene as a fluorescent probe. Additionally, this hydrogel has a potential for a delivery of hydrophobic drugs due to its pyrene hydrophobic domains. The degradation study showed that degradability of the hydrogel was correlated with its structure. Based on the obtained results, disulfide cross-linked and fluorescently labeled hydrogels with different HA concentration were established as a model to study the relationship between the structure of the hydrogel and its degradability (paper-II). We demonstrated that disulfide cross-linked HA hydrogel could be tracked non-invasively by fluorescence imaging. It was proved that the in vivo degradation behavior of the hydrogel is predictable basing on its in vitro degradation study. In paper-III, we developed a disulfide cross-linked HA hydrogel for three-dimensional (3D) cell culture. In order to improve cell viability and adhesion to the matrix, HA derivatives were cross-linked in the presence of fibrinogen undergoing polymerization upon the action of thrombin. It led to the formation of an interpenetrating double network (IPN) of HA and fibrin. The results of 3D cell culture experiments revealed that the IPN hydrogel provides the cells with a more stable environment for proliferation. The results of the cellular studies were also supported by in vitro degradation of IPN monitored by fluorescence measurements of the degraded products. In paper-IV, the effect of biomineralization on hydrogel degradation was evaluated in a non-invasive manner in vitro. For this purpose, two types of fluorescently labeled hydrogels with the different ability for biomineralization were prepared. Fluorescence spectroscopy was applied to monitor degradation of the hydrogels in vitro under two different conditions in longitudinal studies. Under the supply of Ca2+ ions, the BP-modified hydrogel showed the tendency to bio-mineralization and reduction of the rate of degradation. Altogether, the performed studies showed the importance of imaging of hydrogel biomaterials in the design of optimized scaffolds for tissue engineering.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Ácido Hialurônico (HA) é um componente da matriz extracelular, responsável pela hidratação e manutenção do equilíbrio osmótico tecidual. Concentrações de HA estão elevadas em vários tipos de cânceres, incluindo próstata. Hialuronidases (HAases), são uma família de enzimas relacionadas com a propagação de infecções bacterianas, toxinas de venenos e progressão tumoral. A quebra do HA em pequenos fragmentos (3-25 dissacarídeos) promovidos pela ação das HAases tipo Hyal1, Hyal2 e Hyal3, está relacionada à promoção do câncer através da indução da angiogênese e estímulo a proliferação através de ativação da via tirosina quinase. Algumas isoformas de HAases, descritas como produto de splicing alternativo, possuem atividade enzimática diversificada. A heterogeneidade de expressão das HAases foi identificada em alguns tipos de câncer e pode ser correlacionada com o comportamento diferenciado dos tumores. Para este trabalho estudamos amostras de 55 pacientes submetidos a prostatectomia radical por carcinoma de próstata (CP) . A média de idade foi 66 anos e o tempo médio de seguimento 73,7 meses. Os pacientes foram divididos em dois grupos para análise dos resultados: 1- Escore de Gleason (EG) >=7 (30) e EG <=6 (25). 2- Comportamento tumoral (recidiva-19, e não recidiva-36), considerando o nível sérico de Antígeno Específico da Próstata (PSA) 0,2 ng/mL. O grupo controle foi representado por 11 pacientes com hiperplasia prostática benigna, submetidos à ressecção retropúbica. As HYAL foram identificadas por PCR, com uso de primers específicos para as variantes 1, 2, 3, 4 e 5 e wt da HYAL1, wt da HYAL2, e wt e variantes 1, 2 e 3 da HYAL3. As HYAL mais freqüentemente expressas pelo CP foram HYAL2-wt (65,4%), HYAL1-v1 (63,3%) e HYAL3-wt (47,2%). Em tecidos prostáticos benignos, a HYAL3-v1 foi expressa em 90,9% dos casos, estando presente em 36% dos tumores com EG baixo, e não expressa em tumores com EG alto (p=<0,001). Nos tumores sem recidiva HYAL1-v3 foi expressa em 30,5% dos casos versus 5,2% em casos que recorreram (p=0,041). HYAL3 v2, foi expressa por 33,3% dos tumores que não recorreram e não expressa em tumores que recorreram (p=0,002). Concluímos que a expressão de HYAL1-v3, HYAL3-v1 e HYAL3-v2 está relacionada a tumores mais diferenciados e com menores taxas de recidiva, podendo ser utilizadas como marcadores na prática clínica identificando candidatos a terapias mais conservadoras. / Hyaluronic acid (HA) is a component of the extracellular matrix that hydrates and maintains the osmotic balance of tissues. HA concentration is elevated in several cancers including prostate. Hyaluronidases (HAases) are a family of enzymes related to the spread of bacterial infections, toxins of venoms and probably cancer progression. Small fragments of HA are generated by HAase Hyal1, Hyal2 and Hyal3, stimulating endothelial proliferation and activating mitogen-activated protein kinase pathway. Several isoforms of HAases have been described as a product of alternative splicing, and are responsible for differences in the enzyme activity. The heterogeneity of HAses expression has been identified in tumors and could be related to the differences in their biological behavior. Fifty-five patients submitted to radical prostatectomy for prostate cancer (PC) were the subject of this study. The mean age was 66 years old and the mean follow-up was 73,7 months. Patients were divided into two groups for the analyses: 1- High Gleason score (GE) >=7 (30) and low Gleason score <=6 (25). 2- Tumor behavior; recurrence - 19 and nonrecurrence - 36. Biochemical recurrence was considered when PSA was higher than 0.2 ng/mL. The control was represented by 11 patients submitted to retropubic prostate resection for benign prostatic hyperplasia. The alternative splicing forms of HYAL were identified by PCR, and the primer sequences identified variants 1, 2, 3, 4, 5 e wt of HYAL1, wt of HYAL2, wt and variants 1, 2 and 3 of HYAL3. The HYAL more frequently expressed by PC was HYAL2-wt (65.4%), HYAL1-v1 (63.3%) and HYAL3-wt (47.2%). In benign prostate tissue the main expressed HAase was HYAL3-v1 in 90.9%, being present in 36% of low Gleason score tumors and not expressed by tumors with high Gleason score (p=<0.001). For tumors that not recurred there was expression of HYAL1-v3 in 30.5% of the cases vs. 5.2% in cases that recurred (p=0.041). The same difference was noted regarding the expression of HYAL3-v2, that was expressed by 33.3% of tumors that not recurred and not expressed by tumors that recurred (p=0.002). We conclude that there is a profile of HAase related to low Gleason score and non-recurrent PC that is characterized by expression of HYAL1-v3, HYAL3-v1 and HYAL3-v2 that could be used in clinical practice to choose a better treatment.

<div>Vocal fold scarring is the fibrotic manifestation of most common pathological voice disorders. Voice disorders lead to direct healthcare costs of over $200 million annually and significantly reduce quality of life for patients. Despite advances in understanding the pathophysiology of vocal fold scarring, effective treatments for scarring and fibrosis remain elusive. The wound-healing cascade associated with vocal fold injury involves complex signaling interactions between cells and their extracellular matrix (ECM), which remain largely unexplored due to the lack of a physiologically relevant preclinical model to study them. Traditional preclinical models do not capture the complex 3D microenvironment of the vocal folds, and the use of stem cells or fibroblasts alone in models has resulted in poor reproducibility and predictability of in vitro models. Toward this end, this work describes the development of a preclinical model that strives to take into account cellular interactions between fibroblasts and epithelial cells and achieve a balance in the native vocal fold 3D environment to function as an in vitro model.</div><div><br></div><div>Since a major shortcoming of current in vitro models is the lack of a standardized epithelial fibroblast coculture, initial work focused on developing a coculture system between commercially available tracheal epithelial cells and vocal fold fibroblasts in an in vitro setting that would provide more accurate information about the disease pathophysiology and help design better targeted treatments. We designed a healthy and disease state coculture model that can be induced into a fibroplastic state to overexpress stress fibers using TGFβ1. We also demonstrated that both cell types maintained phenotype in the healthy and disease state coculture models.</div><div><br></div><div>To further transfer this model in a physiologically relevant 3D system, follow-up research characterized 3D matrices to mimic the native ECM of the vocal folds by using natural biomimetic materials found in the vocal folds such as hyaluronic acid, type I collagen, and type III collagen. We hypothesized that the ability to control the viscoelastic and structuralcharacteristics of the scaffold in combination with presenting relevant biological cues to cells will result in a better biomimetic scaffold. This research is expected to lay effective groundwork for developing a functional tissue engineered 3D coculture model that retains the reproducibility necessary to serve as a viable diagnostic and therapeutic screening platform.</div>

Osteoarthritis (OA) is a debilitating condition affecting over 21 million persons in the United States. This number is expected to rise in the coming decades. Treatment approaches for OA focus on symptom modifying measures (i.e., pain relief) as disease modifying interventions do not currently exist. However, some of the interventions used to alleviate the symptoms of OA are also thought to have disease-modifying benefits. Two such non-surgical interventions for OA are intra-articular hyaluronan (HA) injections and physical exercise. In order to effectively study their effects in human OA, animal models that are amenable for studying intervention outcomes are needed. The research focused on developing and characterizing a progressive non-surgical model of knee OA in adult mice. This model was used to firstly, examine the capacity of intra-articular HA injections to prevent knee joint degeneration, and secondly to examine the capacity of moderate exercise to prevent onset and progression of joint degeneration. Intra-articular injections of TGF--β1 into murine knees produce synovial hyperplasia, osteophyte formation, and fibrotic changes on cartilage surfaces and joint capsules. However, additional exposure of the joints to high intensity treadmill running (biomechanical overuse) results in more widespread and focal OA-like cartilage erosions of both the tibial and femoral surfaces, similar to that described for the pathological appearance of late human knee OA. Taken together, these data support that synovitis and soft-tissue activation in early OA joints may precede and/or accelerate the process cartilage degeneration characteristic of progressive and late stage osteoarthritis. Intra-articular injections of high molecular weight HA one day following TGF--β1 injections resulted in decreased synovial hyperplasia, minimized osteophyte formation, and significantly decreased severity of cartilage lesions. A four week, alternate day, low intensity aerobic treadmill running program prior to TGF--β1 injections and overuse also resulted in decreased severity of cartilage lesions.

<p>One main hurdle to xenotransplantation, i.e. transplantation between different species, is the immunological barrier that the organ meets in the recipient. The aim of this thesis was to characterise xenogeneic rejection mechanisms by using the concordant mouse-to-rat heart transplantation model.</p><p>Graft-infiltrating immune cells could be isolated from both rejecting and non-rejecting grafts using ex vivo propagation, a technique based on incubation of graft biopsies in culture medium for 48 hours. The numbers of recovered T lymphocytes were considerably higher in grafts undergoing cell-mediated rejection than in grafts undergoing acute vascular rejection (AVR) or in non-rejecting transplants. Thus, ex vivo propagation should be a valuable tool for further studies of cell-mediated rejection.</p><p>Cytokine patterns in the grafts, as measured by a quantitative real-time RT-PCR method, showed that AVR and cell-mediated rejection are associated with an increase of both pro-inflammatory cytokines (IL-1β and TNF-α) and more specific cytokines (IL-2, IL-10, IL-12p40 and IFN-γ). These data differed considerably from the patterns seen in the spleens of the recipients. Cell-mediated xenograft rejection was also found to be associated with a local accumulation of hyaluronan.</p><p>Oral administration of xenogeneic cells stimulated a production of antibodies that could induce hyperacute rejection of cardiac xenografts when passively transferred to graft recipients. This is in contrast to several models for autoimmune diseases and allogeneic transplantation where oral administration of antigens is an effective way to induce unresponsiveness. Hence, future attempts to induce oral tolerance in xenotransplantation should be done with caution.</p>

<p>The glycosaminoglycan hyaluronan is an essential component of the extracellular matrix in all higher organisms, affecting cellular processes such as migration, proliferation and differentiation. Hyaluronan is synthesized by a plasma membrane bound hyaluronan synthase (HAS) which exists in three genetic isoforms. This thesis focuses on the understanding of the hyaluronan biosynthesis by studies on the expression and regulation of the HAS proteins.</p><p>In order to characterize the structural and functional properties of the HAS isoforms we developed a method to solubilize HAS protein(s) while retaining enzymatic activity. The partially purified HAS protein is, most likely, not asscociated covalently with other components. Cells transfected with cDNAs for HAS1, HAS2 and HAS3 were studied and all three HAS isozymes were able to synthesize high molecular weight hyaluronan chains in intact cells. The regulation of the hyaluronan chain length involves cell specific elements as well as external stimulatory factors. HAS3 transfected cells with high hyaluronan production exhibit reduced migration capacity and reduced amounts of a cell surface hyaluronan receptor molecule (CD44) compared to wild-type cells.</p><p>The three HAS isoforms were studied and shown to be differentially expressed and regulated in response to external stimuli. Platelet derived growth factor (PDGF-BB) and transforming growth factor (TGF-<i>β</i>1) are important regulators of HAS at both the transcriptional and translational level. The HAS2 isoform is the isoform most susceptible to external regulation.</p><p>The role of the UDP-glucose dehydrogenase in mammalian glycosaminoglycan biosynthesis was assessed. The enzyme is essential for hyaluronan, heparan sulfate and chondroitin sulfate biosynthesis, but does not exert a rate-limiting effect.</p>

<p>Hyaluronan (HA), a structural component in the extracellular matrix (ECM), has been recognized as a signaling molecule. It is important during various biological activities such as embryogenesis, angiogenesis, wound healing and tumor progression. Increased amount of hyaluronan during embryonic development is necessary for cell migration and differentiation, but the increased production of hyaluronan by tumor cells or tissue fibroblasts is correlated to poor prognosis for tumor progression and chronic inflammation, respectively. Therefore, understanding the mechanisms regulating HA-enriched matrices and the roles of HA in the biological functions is of fundamental biological importance.</p><p>Four novel findings are described in this thesis: (1) HA fragments (HA12) and the known angiogenic factor FGF-2 promote endothelial cell differentiation by induction of common but also distinct sets of genes, particularly, upregulation of the chemokine <i>CXCL1/GRO1</i> gene is necessary for HA12-induced angiogenesis and this effect is dependent on CD44 activation. (2) High concentrations of hyaluronan suppress PDGF-BB-induced fibroblasts migration and PDGFRβ tyrosine phosphorylation upon activation of hyaluronan receptor CD44, probably by recruiting a CD44-associated phosphatase to the PDGFRβ. (3) PDGF-BB stimulates <i>HAS2</i> transcriptional activity and HA synthesis through upregulation of MAP kinase and PI3 kinase signaling pathways in human dermal fibroblasts. (4) Specific suppression of <i>HAS2</i> gene in the invasive breast cancer cell line Hs578T by RNA interference (RNAi) leads to a less aggressive phenotype of breast tumor cells. This suppressive effect can be reversed by exogenously added hyaluronan.</p><p>In conclusion, binding of hyaluronan to CD44 plays an important role in cell signaling, inflammation and tumor progression. Further studies are required to elucidate the molecular mechanisms through which hyaluronan levels are regulated under physiological or pathological conditions, and to explore compounds involved in hyaluronan accumulation and activity as targets for therapies of chronic inflammation and tumors.</p>