Adhesion, which includes cell-to-cell and cell-to-extracellular-matrix adhesion, plays an important role in cancer invasion and metastasis. After undergoing morphological changes malignant and invasive tumour cells, i.e., cancer cells, break away from the primary tumour by loss of cell-cell adhesion, degrade their basement membrane and migrate through the extracellular matrix by enhancement of cell-matrix adhesion. These processes require interactions and signalling cross-talks between proteins and cellular components facilitating the cell adhesion. Although such processes are very complex, the necessity to fully understand the mechanism of cell adhesion is crucial for cancer studies, which may contribute to improving cancer treatment strategies. We consider mathematical models in an attempt to understand better the roles of cell adhesion involved in cancer invasion. Using mathematical models and computational simulations, the underlying complex biological processes can be better understood and their properties can be predicted that might not be evident in laboratory experiments. Cancer cell migration and invasion of the extracellular matrix involving adhesive interactions between cells mediated by cadherins and between cell and matrix mediated by integrins, are modelled by employing two types of mathematical models: a continuum approach and an individual-based approach. In the continuum approach, we use Partial Differential Equations in which cell adhesion is treated as non-local and formulated by integral terms. In the individual-based approach, we first develop pathways for cell-cell and cell-matrix adhesion using Ordinary Differential Equations and later incorporate the pathways in a simulation environment for multiscale computational modelling. The computational simulation results from the two different mathematical models show that we can predict invasive behaviour of cancer cells from cell adhesion properties. Invasion occurs if we reduce cell-cell adhesion and increase cell-matrix adhesion and vice versa. Changing the cell adhesion properties can affect the spatio-temporal behaviour of cancer cell invasion. These results may lead to broadening our understanding of cancer cell invasion and in the long term, contributing to methods of patient treatment.

O telômero é uma sequência repetitiva da dupla cadeia do DNA que protege as pontas dos cromossomos. Seu comprimento é mantido pela telomerase, cuja expressão ocorre em células de câncer, mas não em células somáticas. A célula apresenta um número definido de divisões antes do telômero sofrer erosão. A quebra do DNA ativa a p53, supressor tumoral que induz senescência e respostas de pontos de checagem. O desenvolvimento de inibidores de telomerase tem importância clínica para o câncer. Estudamos os efeitos dos inibidores de telomerase. Duas linhagens celulares LC-HK2 (NSCLC) e hTERT RPE-1 foram tratadas com os inibidores TMPyP4 (5µM) e Thymoquinone (10 e 40 µM) durante 72 e 120 h. TMPyP4 aumentou a porcentagem de células com dano na membrana, induziu mudança na morfologia da célula e diminuiu a expressão do mRNA da vimentina e vinculina. Thymoquinone aumentou a frequência de células senescentes, células com dano na membrana e induziu morte celular. Ambos os inibidores diminuíram a atividade da telomerase, afetando a proliferação e induzindo morte celular. / The telomere is a repetitive double-strand sequence of DNA that protects the chromosomes ends. Its length is maintained by telomerase, whose expression occurs in cancer cells, but not in somatic cells. The cell has a defined number of divisions before the telomere undergoes erosion. DNA break activates p53, tumor suppressor and induces senescence and checkpoint responses. The development of telomerase inhibitors is of clinical importance for cancer. We studied the effects of telomerase inhibitors. Two cell lines LC-HK2 (NSCLC) and hTERT RPE-1 were treated with inhibitors TMPyP4 (5 M) and Thymoquinone (10 and 40 M) for 72 and 120 h. TMPyP4 increased the percentage of cells with membrane damage, induced change in cell morphology, and decreased mRNA expression of vimentin and vinculin. Thymoquinone increased the frequency of senescent cells, cells with membrane damage and induced cell death. Both inhibitors decreased telomerase activity, affecting proliferation and inducing cell death.

The alpha-, beta-, and gamma-Protocadherins (gamma-Pcdhs) are cadherin superfamily adhesion molecules encoded by clustered gene families. The 22 gamma-Pcdhs are combinatorially expressed in the central nervous system (CNS) by neurons and astrocytes, and play critical roles in synaptogenesis, dendrite arborization, and the survival of subsets of neurons. The gamma-Pcdhs promiscuously form cis-multimers that interact strictly homophilically in trans (Molumby et al., 2016; Schreiner and Weiner, 2010); the alpha- and beta-Pcdhs were subsequently shown to interact in a similar homophilic manner (Rubinstein et al., 2015; Thu et al., 2014). The Pcdh gene clusters thus have the potential to generate millions of distinct adhesive interfaces, providing CNS cells with molecular identities that shape neuronal morphology. We demonstrated previously that, in mice lacking the gamma-Pcdhs in the cerebral cortex, pyramidal neurons exhibit severely reduced dendrite arborization (Garrett et al., 2012a). This, combined with many studies of gamma-Pcdh interactions in vitro, suggests that homophilic, adhesive gamma-Pcdh interactions between neurons, and between neurons and glia, provide a positive signal for dendrite growth. However, in retinal starburst amacrine cells and cerebellar Purkinje cells, loss of the gamma-Pcdhs resulted in aberrant dendrite fasciculation and self-crossing (Lefebvre et al., 2012), suggesting that these molecules can mediate repulsive self-avoidance between a neuron’s own dendrites. In Chapter I of this thesis I utilized transgenic mice to manipulate expression in vivo, to show that the complexity of a cortical neuron’s dendritic arbor is determined by homophilic gamma-Pcdh isoform matching with other cells. Expression of the same single isoform in a neuron can result in either exuberant, or minimal, dendrite complexity depending on whether surrounding cells express the same isoform. Additionally, loss of gamma-Pcdh in astrocytes, or induced astrocyte-neuron mis-matching, reduces dendrite complexity cell non-autonomously. This indicates a neuron’s pattern of connectivity is indeed regulated by specific interactions between cells that are distinct from the repulsive self-avoidance seen in isoneuronal processes of planar cell types. In addition to modulating dendrite branch development, the gamma-Pcdhs have been shown to regulate the progression of spinal cord synaptogenesis (Garrett and Weiner, 2009). A role for these molecules in cortical dendritic spines and synapses, however, had yet not been examined. In Chapter II of this thesis, I provide evidence that the gamma-Pcdhs negatively regulate synapse formation and spine morphogenesis in forebrain neurons. Mice lacking all gamma-Pcdhs in the cortex exhibit significantly increased spine and synapse density in vivo, while spine density is significantly decreased in mice overexpressing one of the 22 gamma-Pcdh isoforms. To explain this functional result, we present in vitro evidence to show that gamma-Pcdhs physically and functionally interact with the synaptic cell adhesion molecule neuroligin-1. This work suggests a potential new mechanism by which gamma-Pcdhs regulate the “choice” between dendrite arbor growth and formation and/or stabilization of dendritic spines and synapses in the developing brain. Given that disruptions in the pattern and density of dendritic arbors and spines are a hallmark of neurodevelopmental disorders such as autism and Down, Rett, and fragile X syndromes, my work may provide the basic science foundation for future therapeutic approaches focused on Pcdhs and their associated signaling pathways.

Copies of author's previously published articles inserted. / Includes bibliographical references. / vii, 39 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Shows that the cytokine tumour necrosis factor [alpha] (TNF-[alpha]) enhances the adhesion of neutrophils to the endothelium by an action both on the neutrophil and on the endothelial cell. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995?

Spatial and temporal tracking of passively diffusing functionalized colloids continues to be an improving and auspicious approach to measuring weak specific and non-specific biomolecular interactions. Evidence of this is given by the recent increase in published studies involving the development and implementation of these methods. The primary aim of the work presented in this dissertation was to modify and optimize video microscopy (VM) and total internal reflection microscopy (TIRM) methods to permit the collection of equilibrium binding and sampling data from interaction of surface-immobilized biomolecules. Supported lipid bilayers were utilized as model systems for functionalizing colloid and wall surfaces. Preliminary results measuring calcium-specific protein-protein interactions between surface immobilized cadherin fragments demonstrate the potential utility of this experimental system and these methods. Additionally, quantum dot-modified colloids were synthesized and evanescent wave-excited luminescence from these particles was used to construct potential energy profiles. Results from this work demonstrate that colloids can be used as ultra-sensitive probes of equilibrium interactions between biomolecules, and specialized probes, such as those modified with quantum dots, could be used in a spectral multiplexing mode to simultaneously monitor multiple interactions.

Moderne Methoden für die Einzelzellanalyse werden dank der fortschreitenden Weiterentwicklung immer sensitiver. Dabei steigen jedoch auch die Anforderungen an das Probenmaterial. Viele Aufbereitungsprotokolle adhärenter Zellen beinhalten eine enzymatische Spaltung der Oberflächenproteine, um die Ablösung vom Zellkultursubstrat zu ermöglichen. Verschiedene Methoden, wie die Patch-Clamp-Technik oder eine auf der Markierung extrazellulärer Domänen von Membranproteinen basierende Durchflusszytometrie können dann nur noch eingeschränkt eingesetzt werden. Daher ist die Etablierung neuer Zellablösemethoden dringend notwendig. In der vorliegenden Arbeit werden erstmals PEG-basierte thermo-responsive Oberflächen erfolgreich für die Zellkultur eingesetzt. Dabei wird das zerstörungsfreie Ablösen verschiedener Zelllinien von den Oberflächen durch Temperatursenkung realisiert. Die Funktionalität der Oberflächen wird durch Variation der Polymerstruktur, sowie der Konzentration der Beschichtungslösung, durch Beschichtung der Oberflächen mit einem zelladhäsionsfördernden Protein (Fibronektin) und durch Adsorption zelladhäsionsvermittelnder Peptide (RGD) optimiert. Um den Zellablösungsprozess detaillierter zu untersuchen, wird hier zum ersten Mal der direkte Zellkontakt mit thermo-responsiven Oberflächen mittels oberflächensensitiver Mikroskopie (TIRAF) sichtbar gemacht. Mit dieser Technik sind die exakte Quantifizierung und die Analyse der Reduktion der Zelladhäsionsfläche während des Abkühlens möglich. Hierbei werden in Abhängigkeit von der Zelllinie Unterschiede im Zellverhalten während des Ablösens festgestellt: Zellen, wie eine Brustkrebszelllinie und eine Ovarzelllinie, die bekanntermaßen stärker mit ihrer Umgebung in Kontakt treten, vergrößern im Verlauf des Beobachtungszeitraumes den Abstand zwischen Zellmembran und Oberfläche, reduzieren jedoch ihre Zell-Substratkontaktfläche kaum. Mausfibroblasten hingegen verkleinern drastisch die Zelladhäsionsfläche. Der Ablösungsprozess wird vermutlich aktiv von den Zellen gesteuert. Diese Annahme wird durch zwei Beobachtungen gestützt: Erstens verläuft die Reduktion der Zelladhäsionsfläche bei Einschränkung des Zellmetabolismus durch eine Temperatursenkung auf 4 °C verzögert. Zweitens hinterlassen die Zellen Spuren, die nach dem Ablösen der Zellen auf den Oberflächen zurückbleiben. Mittels Kombination von TIRAF- und TIRF-Mikroskopie werden die Zelladhäsionsfläche und die Aktinstruktur gleichzeitig beobachtet. Die Verknüpfung beider Methoden stellt eine neue Möglichkeit dar, intrazelluläre Prozesse mit der Zellablösung von thermo-responsiven Oberflächen zu korrelieren. / Modern methods for single-cell analysis are becoming increasingly sensitive. At the same time, requirements for the sample material are on the rise. Today, sample preparation of adherent cells usually includes steps of enzymatic treatment to digest surface proteins thus, inducing cell detachment from culture substrates. This strongly limits the application of different techniques like patch clamp or labelling of extracellular domains of membrane proteins for flow cytometry. Therefore, a new cell detachment method is urgently required. In the present work, new PEG-based thermo-responsive polymers are used for cell culture for the first time. Here, non-destructive detachment of different cell lines from polymer-coated surfaces is realised by controlled temperature reduction. The surface functionality is systematically optimised by varying the concentration of the coating solutions, by artificial surface coating of a cell adhesion-mediating protein (fibronectin) and by co-adsorption of a cell adhesion-mediating peptide (RGD). For detailed analysis of the cell detachment process, TIRF microscopy is used to directly visualise the cell contacts on the thermo-responsive surfaces. Using this technique allows both the quantification and analysis of the reduction of the cell adhesion area during sample cooling. Furthermore, for several cell lines, different behaviours in cell detachment are observed. Cells that have close contact to their substrate like MCF-7 breast cancer cell line and CHO-K1 ovary cells increase the distance between cell membrane and surface, but there is only little decrease of cell-substrate adhesion area. In contrast, L929 fibroblasts reduce the cell adhesion area drastically. Furthermore, the hypothesis that the cell detachment is an active process is shown by lowering the cell metabolism by temperature reduction to 4 °C and by the cell traces that are left behind after rinsing the surfaces. A combination of TIRAF and TIRF enables visualising the cell adhesion area and actin structures. Measuring both parameters simultaneously opens up new possibilities to correlate intracellular and cell detachment processes on thermo-responsive surfaces.

Cell adhesion is the first step of cell response to materials and the extracellular matrix (ECM), and is essential to all cell behaviours such as cell proliferation, differentiation, migration and apoptosis for anchor-dependent cells. Therefore, studies of cell attachment have important implications to control and study cell behaviours. During many developed techniques for cell attachment, the manipulation of surface chemistry is a very important method to control initial cell attachment. To control cell adhesion on a two-dimensional surface is a simple model to study cell behaviours, and is a fundamental topic for cell biology, tissue engineering, and the development of biosensors. From the engineering point of view, the preparation of a material with controllable surface chemistry can help studies of cell behaviours and help scientists understand how surface features and chemistry influence cell behaviours. During the fabrication, the challenge is to create a surface with heterogeneous surface properties in the micro scale and subsequently to guide cell initial adhesion. In order to control cell adhesion in a spatial and temporal manner, a photochemical method to control surface chemistry was employed to control the surface property for cell adhesion in this project. Two photocleavable derivatives of the nitrobenzyl group were tried on two types of surfaces: a model self-assembled monolayer (SAM) with alkanethiol-gold surface and biodegradable chitosan. Reactive functional groups on two different surfaces can be inactivated by covalent binding with these photocleavable molecules, and light can be further introduced into the system as a stimulus to recover their reactivity. By simply applying a photomask with diffe

Various genes have evolved to protect the cell against stressor-induced damage or death including the heat shock proteins (HSPs). Stressor-induced HSP gene expression involves the activation of heat shock factor (HSF), which binds to the heat shock element (HSE) found in the promoter region of <em>hsp</em> genes. Previously, our laboratory has examined the expression and function of <em>hsp</em> genes in the South African clawed frog, <em>Xenopus laevis</em>. Amphibians are particularly susceptible to adverse environmental conditions, including high temperatures and toxicants. In contrast to the many known inducers of HSF activation in poikilothermic vertebrates, few inhibitors have been either discovered or described in the literature. The present study has compared for the first time the effect of two heat shock response (HSR) inhibitors, quercetin and KNK437, on <em>hsp</em> gene expression in <em>Xenopus</em> A6 cells, demonstrating their efficacy in poikilotherms. Northern blot and densitometric analysis showed that cells treated with either quercetin or KNK437 decreased the heat shock-induced accumulation of <em>hsp70</em>, <em>hsp47</em>, and <em>hsp30</em> mRNAs. Additionally, constitutive levels of <em>hsp47</em> and <em>hsc70</em> mRNAs were reduced. In comparison, neither quercetin nor KNK437 affected the levels of constitutively expressed <em>ef1&alpha;</em> mRNAs under control or heat shock conditions. Western blot and densitometric analysis in this study showed that under heat shock conditions, exposure to quercetin or KNK437 significantly decreased the accumulation of HSP30, and that KNK437 was more effective in doing so than quercetin. In comparison, levels of actin were not significantly affected by either heat shock or exposure to DMSO, quercetin, or KNK437. These findings suggest that one mechanism by which quercetin and KNK437 inhibits the HSR in <em>Xenopus</em> is through the inhibition of HSF activity. <br /><br /> Results of this study also suggest that KNK437 inhibits the acquisition of thermotolerance in poikilotherms, similar to observations in mammalian systems. In the presence of KNK437, cells given a 2 h heat pretreatment at 33ºC followed by a thermal challenge for 1 h at 37ºC, showed numerous ruffled membrane edges and some aggregates of disrupted stress fibers. In comparison, cells directly challenged for 1 h at 37ºC, showed a marked decrease in HSP30, which was located predominantly at the cellular periphery in conjunction with actin aggregates. These cells showed virtually no intact stress fibers spanning cells and no coherent cell-cell connections. A 3-D analysis of cells given a 1 h thermal challenge at 37ºC (after a prior 2 h heat shock at 33ºC) in the absence of KNK437, showed numerous linear actin bundles transversing the entire cell, even extending into areas of cell-cell contact, and abundant HSP30 concentrated in the perinuclear region surrounding an intact nucleus. However, in the presence of KNK437, there was a significant emergence of membrane ruffles indicating global instability of cellular adhesion. This study has demonstrated that KNK437, which is the more specific and efficient HSR inhibitor, will be an important inhibitor to compare with the well-documented quercetin for future investigations.

Various genes have evolved to protect the cell against stressor-induced damage or death including the heat shock proteins (HSPs). Stressor-induced HSP gene expression involves the activation of heat shock factor (HSF), which binds to the heat shock element (HSE) found in the promoter region of <em>hsp</em> genes. Previously, our laboratory has examined the expression and function of <em>hsp</em> genes in the South African clawed frog, <em>Xenopus laevis</em>. Amphibians are particularly susceptible to adverse environmental conditions, including high temperatures and toxicants. In contrast to the many known inducers of HSF activation in poikilothermic vertebrates, few inhibitors have been either discovered or described in the literature. The present study has compared for the first time the effect of two heat shock response (HSR) inhibitors, quercetin and KNK437, on <em>hsp</em> gene expression in <em>Xenopus</em> A6 cells, demonstrating their efficacy in poikilotherms. Northern blot and densitometric analysis showed that cells treated with either quercetin or KNK437 decreased the heat shock-induced accumulation of <em>hsp70</em>, <em>hsp47</em>, and <em>hsp30</em> mRNAs. Additionally, constitutive levels of <em>hsp47</em> and <em>hsc70</em> mRNAs were reduced. In comparison, neither quercetin nor KNK437 affected the levels of constitutively expressed <em>ef1&alpha;</em> mRNAs under control or heat shock conditions. Western blot and densitometric analysis in this study showed that under heat shock conditions, exposure to quercetin or KNK437 significantly decreased the accumulation of HSP30, and that KNK437 was more effective in doing so than quercetin. In comparison, levels of actin were not significantly affected by either heat shock or exposure to DMSO, quercetin, or KNK437. These findings suggest that one mechanism by which quercetin and KNK437 inhibits the HSR in <em>Xenopus</em> is through the inhibition of HSF activity. <br /><br /> Results of this study also suggest that KNK437 inhibits the acquisition of thermotolerance in poikilotherms, similar to observations in mammalian systems. In the presence of KNK437, cells given a 2 h heat pretreatment at 33ºC followed by a thermal challenge for 1 h at 37ºC, showed numerous ruffled membrane edges and some aggregates of disrupted stress fibers. In comparison, cells directly challenged for 1 h at 37ºC, showed a marked decrease in HSP30, which was located predominantly at the cellular periphery in conjunction with actin aggregates. These cells showed virtually no intact stress fibers spanning cells and no coherent cell-cell connections. A 3-D analysis of cells given a 1 h thermal challenge at 37ºC (after a prior 2 h heat shock at 33ºC) in the absence of KNK437, showed numerous linear actin bundles transversing the entire cell, even extending into areas of cell-cell contact, and abundant HSP30 concentrated in the perinuclear region surrounding an intact nucleus. However, in the presence of KNK437, there was a significant emergence of membrane ruffles indicating global instability of cellular adhesion. This study has demonstrated that KNK437, which is the more specific and efficient HSR inhibitor, will be an important inhibitor to compare with the well-documented quercetin for future investigations.

No description available.