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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Corneal endothelial cells have been shown to possess a uniform polygonal and complex multipolar shape at their apical and basolateral surface respectively. We established a morphological timetable to study how this complex shape arises postnatally. Corneas were collected from mice between postnatal day 8 to postnatal day 35 and labelled with antibodies specific for ZO-1 and NCAM at apical and basolateral region, respectively. Images were collected using wide-field fluorescence microscopy and morphometrically analyzed. Results showed that apical cell sizes increase linearly over the first 3 weeks, plateauing at 4-5 weeks postnatally with increased regularity. Basolateral membrane surfaces remained relatively smooth prior to eyelid opening and thereafter begins developing showing differences in development from periphery to the center until about 4 weeks postnatally when the wavy processes get vivid. Results indicate concurrent and independent development at both poles of the corneal endothelium, with more complexity seen at the basolateral surface.

Cell-cell junctions are essential for tissue homeostasis. Prominent among these junctions are adherens junctions and tight junctions. Adherens junctions mediate adhesion between adjacent cells while tight junctions are responsible for establishing apical-basolateral polarity and limiting paracellular permeability. Loss or disruption of either adherens junctions or tight junctions leads to a myriad of disease states, thus these junctions need to be tightly regulated to prevent dysfunction. A unique property of tight junctions is their dependence on adherens junctions for proper assembly and maintenance. Loss or disruption of adherens junction leads to abnormal tight junctions. Understanding the mechanisms that mediate tight junction coupling to adherens junctions is important for treating diseases that arise from disrupted cell-cell junctions. Currently, two controversial models exist for how tight junctions are coupled to adherens junctions. In the first model, the adherens junction protein α-catenin is critical for tight junction assembly. The second model suggests that a second adherens junction protein, nectin is critical for tight junction assembly through binding the tight junction protein ZO-1, and disruption of tight junction assembly is independent of E-cadherin. α-catenin also binds ZO-1, but the consequences of this interaction are unknown. I hypothesized that α-catenin binding to ZO-1 plays a critical role in coupling tight junctions to adherens junctions. To test this, I mapped the ZO-1 binding site on α-catenin and engineered a point mutant of α-catenin that failed to bind ZO-1. Expression of this point mutant in epithelial cells showed that ZO-1 binding to α-catenin is essential for tight junction assembly and maintenance, while adherens junctions were unaffected. These findings established a role for ZO-1 binding to α-catenin in coupling tight junctions to adherens junctions during junction assembly, as well as at steady-state conditions. After discovering the importance of ZO-1 binding to α-catenin in coupling tight junctions to adherens junctions, I wanted to study whether this interaction is critical in a physiological setting. Tight junctions and adherens junctions are both strengthened in response to mechanical force; however the mechanisms responsible for tight junction strengthening were unknown. Using the system I previously developed, I show that ZO-1 binding to α-catenin is essential for increased tight junction integrity in response to mechanical force, coupling changes in tight junctions to increased stability of adherens junctions. Together, these findings identify a novel interaction that is critical for coupling tight junctions to adherens junctions under several conditions, and provide mechanistic insight into the cellular response to mechanical force.

Die Fähigkeit zur Analyse großer Datenmengen sowie das extrahieren wichtiger Erkenntnisse daraus, sind in der modernen Unternehmenswelt ein entscheidender Wettbewerbsvorteil geworden. Umso wichtiger ist es, dabei vor allem nachvollziehbar, reproduzierbar und effizient vorzugehen. Der Beitrag stellt mit dem Instrument der skriptbasierten Datenanalyse eine Möglichkeit vor, um diesen Anforderungen gerecht zu werden.

Abstract The expression intensities of cytokeratins and tight junction proteins were determined on re-epithelization. Experimental blister wound healing was studied in patients with diabetes mellitus and in patients with obstructive jaundice. Suction blisters were induced on healthy volunteers, and the healing blisters were biopsied at different time points. Cytokeratin expression and the tight junction proteins ZO-1 and occludin were studied immunohistochemically. Blisters were induced on 17 patients with diabetes and 11 control subjects, and the healing process was followed indirectly by measuring water evaporation and blood flow in the wounds. Microvascular reactivity in the diabetic patients was also studied by using non-immunologic contact irritants. Wound healing, skin collagen synthesis and serum levels of procollagen propeptides were studied in 24 patients with obstructive jaundice caused by neoplastic pancreaticobiliary obstruction and in 17 control patients with the corresponding condition without jaundice. Cytokeratin expression was altered in healing epidermis. In the suprabasal layer, K10 was replaced by K14 and, most likely, by K16. K18 keratin, which is not present in normal epidermis, was found in the basal and suprabasal layers. Thus, there was a shift towards lower molecular weight cytokeratins, which is a reflection of immaturity, and probably towards motility. The tight junction proteins ZO-1 and occludin were expressed in the migrating epidermal sheet, where they apparently form an early barrier. Enhanced expression was seen in the hyperproliferative zone of the wound edge. The diabetic patients showed slower restoration of the epidermal barrier and a weaker initial inflammatory response. Obstructive jaundice and its resolution had no effect on healing. Skin collagen synthesis was decreased in jaundiced patients, and it increased slightly after drainage. Serum type III collagen propeptide levels were elevated in patients with biliary obstruction and dropped after drainage. The elevated levels may be related to the increased synthesis due to fibrosis. As a conclusion, diabetes mellitus impairs epidermal wound healing, while obstructive jaundice does not.

Les échanges entre les cellules tumorales et le microenvironnement jouent un rôle essentiel dans le développement des tumeurs. Dans ce contexte, la nouvelle famille de métalloprotéases, les protéines ADAM, constituent aujourd’hui des régulateurs majeurs de la progression tumorale en agissant sur la biodisponibilité des médiateurs de la communication cellulaire que sont les cytokines, chimiokines et facteurs de croissance. Au sein de cette famille, ADAM12 est la plus associée au cancer. Elle possède la particularité de jouer un rôle dans la signalisation cellulaire, de façon indépendante de son activité métalloprotéase, notamment dans les voies de signalisation du TGFβ. Notre étude montre pour la première fois un rôle pour la forme membranaire d'ADAM12 dans l'induction de la transition épithélio-mésenchymateuse (EMT), un processus essentiel à l'invasion tumorale dont le TGFβ est un inducteur majeur. Cet effet est médié par l'activation des voies de signalisation du TGFβ, impliquant les protéines SMAD3, AKT et ERK et requiert le domaine cytoplasmique d'ADAM12L mais pas son domaine catalytique. L'activation de ces voies de signalisation pourrait impliquer une relocalisation d'ADAM12L au sein de plates-formes de signalisation dans les radeaux lipidiques. Par ailleurs, nous avons montré qu'ADAM12L interagit avec les protéines ZO-1 et ZO-2, des protéines des jonctions serrées, et pourrait favoriser leur désassemblage au cours de l'EMT. Nos travaux ont permis de mettre en évidence une nouvelle fonction pour ADAM12L dans l'EMT, par un mécanisme impliquant une modulation des signaux régulant ce processus. Une meilleure compréhension de la dynamique de ces mécanismes moléculaires pourrait permettre de développer de nouvelles thérapies ciblées pour lutter contre la progression tumorale. / Communication between tumoral cells and the microenvironnement plays an essential role in the developpement of tumors. In that context, the new family of metalloproteases, the ADAM proteins, are major regulators of the tumoral progression by acting on the bioavaibility of importants mediators of cellular communication as cytokines and growth factors. Among this family, ADAM12 is the most associated with cancer. It has been shown to mediate signaling pathways by a process independant of its metalloproteasis activity, in particular for TGFβ signaling. This study show for the first time a role for the membrane form of ADAM12 in the induction of epithelial to mesenchymal transition (EMT), a essential process involved in tumor invasion, whom TGFβ is a main inducer. This effect is mediated by the activation of TGFβ signaling pathways, SMAD3, AKT and ERK and require the cytoplasmic tail of ADAM12L but not its catalytic activity. Activation of these pathways could involve a relocalisation of ADAM12L in special signaling platform in lipid rafts. Moreover, we have shown that ADAM12L interact with ZO-1 and ZO-2, two proteins of tight junctions, and could facilitate their desassembling during EMT. This work underscore for the first time a new function of ADAM12L in EMT, by a mecanism invovlving a modulation of signals regulating this process. A better understanding of the dynamic of these molecular mecanisms could allow the developpement of new targeted therapies to fight against tumoral progression.

Bröstcancer är den vanligaste formen av cancer som drabbar kvinnor. Det är en heterogen och komplex sjukdom som består av flera undergrupper, var och en med distinkt morfologi och kliniska implikationer [1]. För att modellera och studera cellbiologi, vävnadsmorfologi, molekylära mekanismer och läkemedels effekter används cellkulturer [2]. Idag är tvådimensionella (2D) modeller fortfarande den mest använda metoden för att odla celler in vitro [3]. En nackdel med 2D-modeller är att mikromiljön i dessa modeller inte imiterar in vivo strukturen av tumörer och vävnader, då de saknar tre dimensionella (3D) cell-cell och cellextracellulär matrix (ECM) interaktioner [2]. På grund av nackdelarna med 2D-modeller, har 3D-modeller blivit mer intressanta som alternativ för att lösa behovet av en pålitlig preklinisk modell för läkemedelstestning och för studier av cancerbiologi. För att utveckla ett redskap som är relevant för cancerforskning etablerar professor My Hedhammars laboratorium en 3D-modell av bröstcancer. I en sådan ny modell används Biosilk som byggnadsställning för att odla odödliga cellinjer som är representativa för de tre huvudklasserna av bröstcancer (i.e. MCF-7 (luminal-lik), SKBR-3 (HER2-överuttryckt) och MDAMB- 231 (trippel-negativ)). Eftersom transkriptions signaturer kan användas för att klassificera och studera bröstcancer är det viktigt att undersöka om och hur tillväxt i 3D-Biosilk kan påverka genuttrycksprofiler. Hypotesen som testades i denna studie var om cellkulturer i 3DBiosilk kan ha signifikanta skillnader i uttryck av biomarkörer, relevanta för bröstcancerbiologi, vid jämförelse av samma cellinje kultiverad i 2D. För att testa detta utvärderades kvalitén och reproducerbarheten av 3D-Biosilk konstruktionen med hjälp av olika kvalitetstester. Strukturen granskades med brightfield mikroskopi, arean av konstruktionen mättes med ImageJ, infärgning med phalloidin bekräftade cellnärvaro och cellvidhäftning till modellen. Alamar blue utfördes för att bedöma den cellulära metaboliska aktiviteten i modellen. Förändringarna av målgenernas genuttryck undersöktes med kvantitativ omvänd transkription PCR (RT-qPCR) och detta påvisade en statistiskt signifikant skillnad i genuttrycket beroende på om cellerna odlats i 2D- eller 3D-Biosilk modeller. I cellinje MDA-MB-231 hittades tre gener, i cellinje SKBR-3 hittades två gener och i cellinje MCF-7 hittades fyra gener. Genuttrycket för en av dessa gener i cellinje MCF-7, som var kultiverad i 3D-Biosilk, var nedreglerad (i.e. ZO-1). Detta kunde valideras på proteinnivå med immunofluorescens. Sammanfattningsvis, celler odlade i 3D-Biosilk visar på en mer aggressiv fenotyp. / Breast cancer is the most common cancer among women. It is a heterogenous and complex disease composed of several subtypes, each with distinct morphological and clinical implications [1]. To model and study cell biology, tissue morphology, molecular mechanisms and drug actions, cell cultures are canonically used [2]. Today two-dimensional (2D) models are still widely the preferred method for culturing cells in vitro [3]. A drawback with 2D models is that the microenvironment in these models does not mimic the in vivo structure of tumors and tissues, lacking three-dimensional (3D) cell-cell and cell-extracellular matrix (ECM) interactions [2]. Due to the disadvantages of 2D models, 3D cultures have become an increasingly interesting alternative to solve the need for a reliable preclinical model for drug testing and the study of cancer biology. To develop a relevant tool for cancer research, the laboratory of professor My Hedhammar is currently establishing a 3D model of breast cancer. In such novel model, Biosilk is used as scaffold to grow immortalized cell lines representative of the three major classes of breast cancer (i.e. MCF-7 (luminal-like), SKBR-3 (HER2-overexpression) and MDA-MB-231 (triplenegative)). Since transcriptional signatures can be used to classify and study breast cancers, it is important to investigate if and how growth in 3D-Biosilk can impact gene expression profiles. The hypothesis tested in this study was that cells cultured in 3D-Biosilk have differences in expression of biomarkers relevant to breast cancer biology, when compared to the same cell lines cultured in 2D. To examine this, 3D-Biosilk models were created and evaluated to ensure their quality and reproducibility, for instance, the scaffold structure was monitored by brightfield microscopy, the construct’s area was measured with ImageJ, staining with phalloidin confirmed the presence of cells as well as their attachment to the construct, and Alamar blue was used to assess the cellular metabolic activity. Differences in gene expression of target genes were investigated using reverse transcription quantitative PCR (RTqPCR), which revealed statistically significant changes depending on whether the cells were cultivated in 2D or a 3D-Biosilk model. For cell line MDA-MB-231 three genes were found, for SKBR-3 two genes were found and for MCF-7 four genes were found. The expression of one gene which was found downregulated in MCF-7 cultured in 3D-Biosilk (i.e. ZO-1) was validated at protein level by immunofluorescence. In conclusion, cultivating cells in 3D-Biosilk indicates a more aggressive phenotype.

Master of Science / Department of Biochemistry / Bruce D. Schultz / John M. Tomich / Peptides based on the second transmembrane segment of the glycine receptor (M2GlyR) were made to provide a potential therapeutic treatment for cystic fibrosis (CF) and a latent absorption enhancer for drug delivery. For similarity of presentation, unique synthetic peptide sequences have been given alpha-numeric designations. Results are presented from studies focusing on four peptides. In the first study, the contributions of synthetic peptides p1171, p1172 and p1173 to net transepithelial ion transport were measured as a first step toward the goal of testing whether pore length or electrostatics of pore lining residues will affect anion transport. Peptide p1130 exhibits many attributes that make it an ideal synthetic peptide for CF treatment, but has low permselectivity for anions. Therefore, it is used as a platform for modification. Peptide p1171 is doubly substituted with diaminopropionic acid at positions T13 and T17. Peptide p1172 and p1173 are separately one and two helical turn(s) inserted into the p1130 backbone. Apical exposure of MDCK monolayers to these peptides caused a rapid increase in short circuit current (Isc), an indicator of net ion transport. The increase in Isc caused by p1172 or p1173 was accompanied by increase in transepithelial electrical conductance (gte). The electrophysiological results suggested that these modified peptides can assemble in the apical membrane of epithelial cells to form functional ion-conducting pores. Peptide NC-1059, which provides for ion transport across epithelial cells derived from many sources, was studied further to assess cellular changes that account for increased gte. NC-1059 increased Isc, gte and enhanced permeation of dextrans in a concentration dependent manner. Results from previous and current studies show that NC-1059 modulated the epithelial paracellular pathway by altering the distribution and abundance of junctional proteins. Immunoblotting and immunolabeling with confocal microscopy showed that NC-1059 induces reorganization of actin and causes a reduction in F-actin abundance in epithelial cells. The distributions were changed and cellular abundances were reduced of tight junction proteins occludin and ZO-1 and adherens junction proteins E-cadherin and β-catenin by NC-1059. These effects were largely reversed in 24 hr and fully recovered in 48 hr. Therefore, NC-1059 has the therapeutic potential to increase the efficiency of drug delivery across barrier membranes.

The disappearance of the wetlands of Taiwan have reflected all kinds of biological drifting about aimlessly. We have to take action for protecting the wetlands, due to there are less than before. In the face of the wetlands that there are not much left, we must treasure and offer protection. The action that a lot of wetlands protection at present is going on. We need to invest more research and monitoring, and should investigate the state of wetland health that file, expect that can utilize limited resources more effectively during the process of protecting. This research expects to build the index system constructing out a set of Taiwan wetlands and assessing to the characteristic of the wetland of Taiwan. After consulting the domestic and international relevant index system, this research drafts preliminary wetland health assessment system. Through Delphi method, gathering opinions and suggestions from experts and scholars this research could set up the health assessment indicators of Taiwan artificial wetlands. Index system is divided into environment and management two general parts, and is categorized into six major indexes groups, including organism, water, soil, environment, management and social economy, amounts to 18 indexes. In the case of Zo-Zai Wetland Park, in Kaohsiung, Taiwan, we applied this index system to inspect the health degree of Zo-Zai Wetland Park and its index results. Assessing the result for being 'good' in health, and cause the unhealthy projects of wetland include restoration species grow up degree, the distribution of the invasive species, the water quality situation, the hydrology situation and the maintenance funds of the wetland . The whole but the speech, the continent Zo-Zai Wetland Park still belongs the health, but influence the wetland healthy factors still need to monitor continuously in order to maintain wetland health quality. This research hopes to diagnose the health state of the wetlands, through health assessment indicators system and regular wetland monitoring, Then designing the wetland follow-up planning and management strategies to result the wetlands¡¦ problems earlier. Finally, it hopes to help restoring the wetlands in Taiwan.