Purpose. In the genetically defective Royal College of Surgeons (RCS) rat model for retinal degeneration, a breakdown occurs in the retinal pigment epithelial (RPE) cell tight junctions just as the photoreceptors begin to degenerate. These experiments sought to determine the impact of the RPE genetic defect on this alteration in the RPE cell tight junctions. Methods. Retinal pigment epithelial cell cultures prepared from RCS and control rats were treated with hormonally defined medium (HDM), base medium conditioned by RCS or control retinas, or unconditioned base medium. The tight junctions formed by these cultures were assayed functionally by measuring transepithelial electrical resistance and permeability. Junction structure was evaluated by immunolocalization of the tight junction protein zonula occludens I and of the junction-associated actin microfilaments. Results. Retinal pigment epithelial cultures from dystrophic rats formed structurally and functionally normal tight junctions when maintained in hormonally defined medium. The junctions remained stable when the medium bathing the apical surface was switched to base medium preconditioned by normal retinas. In contrast, cultures treated with medium preconditioned by degenerating dystrophic retinas or with unconditioned medium exhibited a breakdown in their tight junctions. Conclusions. Retinal pigment epithelial cells isolated from dystrophic RCS rats can form tight junctions normally in vitro. Normal, but not dystrophic, retinas release factors that support RPE tight junctions. Therefore, the junctional abnormality seen in dystrophic rat RPE cells in vivo is probably caused by the loss of trophic factors normally provided by the healthy neural retina rather than by a direct effect of the genetic defect on the tight junctions.

No description available.

Development is punctuated by morphogenetic rearrangements of epithelial tissues, including complete detachment of individual motile cells during epithelial-mesenchymal transition (EMT). Dramatic actin rearrangements occur as cell-cell junctions are dismantled and cells become independently motile during EMT. Characterizing dynamic actin rearrangements and identifying actin machinery driving these rearrangements is essential for understanding basic mechanisms of cell-cell junction remodeling; yet, neither the precise series of actin rearrangements at cell-cell junctions that accompany EMT, nor the machinery that controls actin rearrangement during EMT, have been identified. This work represents a detailed study of junctional actin reorganization in cells undergoing EMT, identifies actin regulatory proteins that control this actin reorganization, and defines the specific function of one regulatory protein, zyxin, in EMT. Using immunofluorescence and live cell imaging of HGF induced scattering of MDCK cells, dynamic actin rearrangement events occurring during EMT are characterized. Junctional actin characteristic of cell-cell adherent cells is rearranged into contractile medial actin networks linked to the junctional membrane in the initial steps of cell scattering. This actin rearrangement is accompanied by dynamic redistribution of specific actin regulatory proteins, namely α-actinin and zyxin-VASP complexes. α-Actinin mediates higher order structure of junctional actin. Zyxin-VASP complexes mediate linkage of dynamic medial actin networks to adherens junction membranes. Zyxin regulation of actin-membrane linkage controls whether cell migration during EMT occurs independently in solitary cells or is coordinated through tissues. The functional analysis employed here uses novel, quantitative methods that define specific cellular EMT ‘phenotypes’ to reveal the precise role of zyxin in EMT. Constitutive active zyxin mutants exhibit persistent actin-membrane linkages and a scattering phenotype in which cells migrate without loss of cell-cell adhesion. Zyxin is proposed to regulate EMT progression by regulating disruption or maintenance of actin membrane linkages at cell-cell junctions. Zyxin alters the ability of cells to fully detach and migrate independently during EMT and may be an important regulator of morphogenetic plasticity.

2-ω-carboxyethylpyrrole (CEP) is a product of Docosahexaenoic acid (DHA) oxidation, which forms covalent adducts with different proteins. CEP-modified proteins can interact with macrophage receptor, integrin αDβ2. This study aims to compare αDβ2 binding to its physiological ligand, fibrinogen, and CEP-modified fibrinogen, which is formed during inflammation. We hypothesize that modification of fibrinogen changes its ligand-binding properties to integrin αDβ2 which can affect macrophage migration and retention. Recombinant αD I-domain and αDβ2-transfected HEK293 cells were used for the experiments. In biolayer interferometry (BLI) assays, fibrinogen was immobilized at pH 5.0 and fibrinogen-CEP at pH 3.5. Our results showed that the affinity of αD I-domain binding to fibrinogen-CEP was higher than fibrinogen, and the binding was inhibited by the anti-CEP antibody. The optimal expression of αD I-domain was found at 25°C. In cell adhesion assays, optimal concentrations were used for the inhibition assay; fibrinogen at 2µg/ml and fibrinogen-CEP at 8µg/ml. αDβ2-transfected cells demonstrated stronger adhesion to fibrinogen-CEP, and this adhesion was significantly inhibited by polyglutamic acid, which mimics CEP-mediated binding. These findings suggest that αDβ2's interaction with DHA-modified extracellular matrix (ECM) proteins significantly increases macrophage adhesion and may serve for macrophage retention during chronic inflammation. Developing small molecules that can inhibit αDβ2-CEP interaction could be a breakthrough in treating inflammatory diseases. The main conclusions drawn from the study are that CEP-modified fibrinogen has a higher affinity for αD I-domain binding than native fibrinogen, and this interaction can be inhibited by targeting CEP. The study provides insights into the potential therapeutic applications of inhibiting αDβ2-CEP interactions in inflammatory diseases.

Polycystic kidney disease is a disorder that leads to fluid filled cysts that replace normal renal tubes. During the process of cellular development and in the progression of the diseases, fibrocystin can lead to impaired organ formation and even cause organ defects. Besides cellular polarity, mechanical properties play major roles in providing the optimal apical-basal or anterior–posterior symmetry within epithelial cells. A breakdown of the cell symmetry that is usually associated with mechanical property changes and it is known to be essential in many biological processes such as cell migration, polarity and pattern formation especially during development and diseases such as the autosomal recessive cystic kidney disease. Since the breakdown of the cell symmetry can be evoked by several proteins including fibrocystin, we hypothesized that cell mechanics are altered by fibrocystin. However, the effect of fibrocystin on cell migration and cellular mechanical properties is still unclear. In order to explore the function of fibrocystin on cell migration and mechanics, we analyzed fibrocystin knockdown epithelial cells in comparison to fibrocystin control cells. We found that invasiveness of fibrocystin knockdown cells into dense 3D matrices was increased and more efficient compared to control cells. Using optical cell stretching and atomic force microscopy, fibrocystin knockdown cells were more deformable and exhibited weaker cell–matrix as well as cell–cell adhesion forces, respectively. In summary, these findings show that fibrocystin knockdown cells displayed increased 3D matrix invasion through providing increased cellular deformability, decreased cell–matrix and reduced cell–cell adhesion forces

Polysialylated NCAM (polySia-NCAM) is re-expressed in a number of tumours, including small cell lung carcinoma and neuroblastoma and is strongly associated with aggressive, invasive and metastatic tumours in the clinic. SiRNA knockdown of the polysialyltransferases (polySTs), the enzymes responsible for polysialylation of neural cell adhesion molecule (NCAM), has been shown to abolish cell migration. PolySia-NCAM is thus a highly attractive novel therapeutic target. A library of potential polyST inhibitors has been synthesised, using substrate-based design and computational chemistry. Compounds synthesised include N-acylmannosamine analogues, thio-linked CMP-sialic acid analogues, N-acyl modified sialic acids and compounds incorporating elements of both approaches. Novel methodology development in the synthesis of many of the compounds is described, notably a novel route to N-acyl sialosides. In addition, compounds identified from in silico screening were considered. Routes to synthesis and isolation of analogues of biologically active compounds are described. Using an enzyme assay, compounds were evaluated for their ability to reduce polySia synthesis through polyST inhibition. Effects of agents on polySia expression in cells, and the ability of compounds to reduce cell migration in vitro was studied using a wound healing ‘scratch assay’. The data from these experiments revealed a number of potent modulators of polySia assembly and their efficacy in reducing cell migration, as well as the limits of the biosynthetic pathway to accept unnatural sialic acid precursors. This is the first example of polyST inhibition modulating tumour cell migration, and points to the potential of the polysialyltransferases as a therapeutic target in metastatic tumours. / EPSRC and BACR / The full text will be available at the end of the extended embargo: 5th March 2027

Neuroblastoma is a highly metastatic and invasive tumour with poor prognosis. Despite recent advances in the treatment of neuroblastoma, mortality is still high due to uncontrolled metastatic disease, and novel therapeutic approaches for the treatment of neuroblastoma are therefore desperately needed. A potential novel approach for therapy of neuroblastoma relates to the polysialic acid decoration of the neural cell adhesion molecule (PSANCAM). PSA-NCAM is selectively re-expressed in a number of tumours including neuroblastoma, where it is thought to modulate tumour dissemination. Expression is strongly associated with poor clinical prognosis and an aggressive tumour phenotype. Inhibition of the enzymes responsible for synthesis of PSA, the polysialyltransferases (polySTs) presents a novel and selective therapeutic opportunity. The aims of the studies described in this thesis are to evaluate PSANCAM expression and function in neuroblastoma, and to develop and utilise cell-based models to pharmacologically investigate novel polyST inhibitors. PSA-NCAM was seen to be highly expressed in neuroblastoma clinical specimens and associated with phenotypes of tumour aggressiveness. A screening panel consisting of cell lines with a range of PSA-NCAM expression types was established and utilised to develop assays for pharmacologically assessing novel polyST inhibitors. Using cytidine monophosphate (CMP), a naturally-occurring inhibitor of polySTs, the robustness of the assays was confirmed before progression to evaluate novel molecules. From 16 compounds identified in an in vitro screen of polyST inhibition, three promising polyST inhibitors were identified. These promising polyST inhibitors modulated PSA-NCAM expression on the tumour cell surface and led to a significant reduction in cell migration. Therefore the work presented in this thesis suggests that targeting polySTs is a promising novel therapeutic strategy for neuroblastoma and further research in this area is warranted. / Mu'tah University and Jordan Armed Forces, Hashemite Kingdom of Jordan. / The full text will be available at the end of the extended embargo period: 5th March 2027

La sclérose en plaques (SP) est une maladie neuro-inflammatoire caractérisée par l’invasion de cellules immunitaires périphériques dans le système nerveux central (SNC), entraînant une perte de myéline à des endroits bien délimités appelés « plaques » ou lésions. Les processus neuroinflammatoires sont associés au dommage des neurones et oligodendrocytes (OLs) en SP. Les mécanismes sous-tendant cette dégradation des OLs par les cellules immunitaires en SP sont toutefois encore mal compris. Les lymphocytes T CD4 activés, notamment les sous-types proinflammatoires Th1 et Th17, jouent un rôle clé dans la pathobiologie de la SP et de son modèle murin l’encéphalite auto-immune expérimentale (EAE). Nous avons donc choisi d’investiguer leur contribution à l’endommagement des OLs en neuroinflammation. Pour ce faire, nous avons premièrement caractérisé les interactions entre les lymphocytes Th17 et les OLs matures in vivo à l’aide de l’imagerie intravitale chez la souris EAE (microscopie deux photons) et in vitro en utilisant des cultures primaires humaines. Ceci nous a permis de mettre en évidence que les lymphocytes pro-inflammatoires Th17 adhèrent de façon prolongée aux OLs et leur causent plus de dommage que les lymphocytes anti-inflammatoires Th2. Après avoir établi que le contact avec les lymphocytes Th17 entraîne tout d’abord la perte des prolongements cellulaires puis la mort des OLs, nous avons identifié deux mécanismes à l’origine de ces dommages. En effet, tandis que la sécrétion de glutamate par les lymphocytes Th17 à proximité des OLs entraîne une perte des prolongements cellulaires de ces derniers et une diminution de leur capacité à myéliniser, la sécrétion de granzyme B mène à la mort des OLs. Dans le but de comprendre comment prévenir les dommages causés par les lymphocytes Th17 aux OLs en SP, nous avons par la suite étudié les mécanismes sous-tendant le contact entre les deux types cellulaires. Comme nous avons confirmé que les OLs matures n’expriment pas le MHC II au niveau protéique, nous avons caractérisé l’expression par les OLs de molécules d’adhérence cellulaire (CAMs) qui seraient susceptibles de sous-tendre l’adhérence des lymphocytes Th17. Nous avons découvert que cette interaction est notamment médiée par ALCAM, et que bloquer cette molécule permet de diminuer le dommage aux OLs médié par les Th17 in vitro. A l’inverse, l’expression et/ou la sécrétion d’ICAM-1 par les OLs semble avoir un effet protecteur face aux lymphocytes Th17. En résumé, nous avons distingué de nouveaux mécanismes impliqués dans le dommage aux OLs en neuroinflammation et identifié de nouvelles cibles thérapeutiques prometteuses pour la protection des OLs en SP. / Multiple Sclerosis (MS) is a neuroinflammatory disease characterized by infiltration of immune cells into the central nervous system (CNS), demyelination in multifocal areas called “plaques” or lesions, and damage to neurons and oligodendrocytes (OLs). The mechanisms underlying immune-mediated injury to OLs in MS remains only partially understood. Activated CD4 T cells, in particular pro-inflammatory subsets Th1 and Th17, play an important role in the pathobiology of MS and its animal model experimental autoimmune encephalitis (EAE). We set out to investigate their contribution to immune-mediated oligodendrocytic damage in neuroinflammation. We first characterized the interactions between Th17 cells and mature OLs in vivo using live imaging of EAE mice (two photon microscopy) and in vitro using human primary cell cultures. We found that pro-inflammatory Th17 cells form prolonged contacts with OLs and cause greater harm compared to anti-inflammatory Th2 cells. After demonstrating that contact with Th17 cells leads first to destruction of cell processes and then death of OLs, we identified two mechanisms underlying these deleterious impacts. Indeed, while secretion of glutamate by Th17 cells in contact with OLs is associated with damage to OLs cell processes and impairment of their myelinating capacity, secretion of granzyme B leads to OLs death. To better understand how to prevent Th17-mediated OLs injury in MS, we next studied mechanisms involved in the interaction between these two cell types. As we confirmed that mature OLs do not express MHC II at the protein level, we characterized expression of cell adhesion molecules (CAMs) by OLs that could mediate Th17 cell adhesion. We discovered that ALCAM contributes to OLs and Th17 cells interactions, and that blocking this olecule reduces Th17-mediated OL damage in vitro. Inversely, ICAM-1 expression and/or secretion by OLs seems to have a protective effect in neuroinflammatory conditions. In summary, we have uncovered new mechanisms implicated in OLs njury in neuroinflammation and have identified potential novel therapeutic targets for neuroprotection in MS.

Homocysteine (Hcy) has been identified as a primary risk factor for atherosclerosis as it induces endothelial cell (EC) activation/dysfunction and thus potentially initiating atherosclerotic plaque formation. There is accumulating evidence indicating a key role for oxidative stress in mediating Hcy atherogenic effects. The aim of this study was to evaluate the effects of chronic treatment with Hcy on EC activation and to explore the role of oxidative stress in these effects. Human umbilical vein endothelial cells (HUVEC) were cultured and treated chronically with DL-Hcy for 5-9 days. An in vitro flow system was also used to characterize the different types of interactions between DL-Hcy-treated HUVEC and neutrophils under physiological flow conditions. EC activation was studied by characterizing the activation of the JNK pathway and the up-regulation of different cell adhesion molecules (CAM) and cytokines, using different techniques including western blot, immunohistochemical staining, enzyme-linked immunosorbent assay and polymerase chain reaction. The role of oxidative stress was investigated by measuring the production of ROS and evaluating the efficiency of antioxidants. Furthermore, the role of nitric oxide and nitric oxide synthase in modulating Hcy effects was investigated. Chronic treatment with DL-Hcy did not kill the EC however, it inhibited cell proliferation. Furthermore, this treatment induced EC activation/dysfunction which was characterized by sustained activation of the JNK pathway, which in turn mediated up-regulation of E-selectin, ICAM-1 and to lesser extent P-selectin. Furthermore, DL-Hcy induced production of IL-8 protein. These CAM and chemokines collectively mediated different interactions between DL-Hcy-treated HUVEC and neutrophils under flow conditions including tethering, rolling, adherence and transmigration. DL-Hcy was also shown to induce significant ROS generation which mediated activation of the JNK pathway. Antioxidants restored DL-Hcy-induced interactions under flow to the basal level. DL-Hcy was shown to induce eNOS uncoupling which mediated, at least in part, the DL-Hcy-induced ROS production. Furthermore, short term treatment with NO inhibited DL-Hcy-induced HUVEC:neutrophil interactions in a cGMP-independent manner. In summary, this research showed that DL-Hcy has several proatherogenic effects, mediated at least in part by the JNK pathway, and induces EC activation/dysfunction priming for atherosclerosis initiation. The data supports that oxidative stress mediates the majority of Hcy atherosclerotic effects. Antioxidants tested, JNK inhibitors and NO showed promising results in reversing all DL-Hcy effects and restoring EC normal status. ¿

At present there is an extraordinary need to overcome barriers in regards to discovering novel and enhanced biomaterials for various tissue engineering applications. The need for durable orthopaedic implants is on the rise to limit issues such as revision surgery. A promising pathway to enhance fixation is to accelerate the onset and rate of early cellular adhesion and bone growth through nanoscale surface topography at the implant surface. The main aim of this research project was to investigate cellular response to altered physical and mechanical characteristics of materials suitable for orthopaedic applications. Four injection moulded polymeric substrates were produced, each with varied compositional and topographical characteristics. The four materials fabricated are Polyether-ether-ketone (PEEK), PEEK with 30% glass fibre (GL/PEEK) composite, PEEK and GL/PEEK with grooved topography. SEM and AFM analysis was used to investigate the groove dimensions and surface roughness of all samples followed by mechanical testing using a nano indenter to detect the Young’s modulus, stiffness and hardness of all four substrates. These tests were performed to determine which material has similar characteristics to cortical bone. These tests were followed by wettability and surface energy testing. Cell-substrate adhesion was examined using a cell viability assay to identify if there is a significant difference (p<0.05) between the percentage of viable cells on all four PEEK based materials. Imaging of MG-63 osteosarcoma cells using immunohistochemistry staining kits was conducted to observe the relationship between cell length and surface topography followed by a comparison between HaCaT (skin) cells and MG-63 (bone) cells. Following experimental testing mechanical variations between PEEK and GL/PEEK were identified alongside physical characterization differences. The grooved topography increased the surface roughness of PEEK and GL/PEEK in comparison to the planar surface. After 72 hours a correlation between the increased surface roughness and the percentage of viable MG-63 cells could be identified. When assessing the effect surface topography has on the water contact angles and surface energy, all four substrates showed no correlation. However, the grooved topography did increase the water contact angle and reduced the surface energy of PEEK in comparison to planar PEEK. Images of the four substrates after cell culture observed the grooved topography to affect the cellular orientation of both MG-63 and HaCaT cells. Polycaprolactone (PCL) scaffolds with a concentration of 1, 3, and 5% triclosan (an antimicrobial and antifungal agent) were fabricated using electrospinning. In addition to PCL + Triclosan scaffolds PCL with a concentration of 1% silver (an antimicrobial agent that can reduce the risk of infection) and 1, 3, and 5% triclosan were also electrospun. The pore size and fibre diameters of the scaffolds were investigated using SEM and Image J software followed by wettability and surface energy testing. MG-63 cells were cultured on all PCL scaffolds to study cellular viability percentage after 24 and 72 hours. The findings obtained showed the physical characteristics of PCL scaffolds to affect cellular viability of MG-63 cells. The output from these findings aim to provide data at a proof of concept level in understanding the relationship between the mechanical and physical characteristics of biomaterials and cellular behaviour.