Hyaluronan (HA) is a negatively charged extracellular matrix (ECM) component with water-attracting properties. It is the dominating ECM component in the renal medullary interstitium, where the amount changes in relation to hydration status: it increases during hydration and decreases during dehydration. It has, therefore, been suggested that HA participates in the regulation of renal fluid handling by changing the permeability properties of the interstitial space. This thesis investigates potential mechanisms for such a role in renal fluid regulation. The results demonstrate that the high renal HA content of late nephrogenesis decreases during the completion of kidney development in the rat, which takes place in the neonatal period. The heterogenous distribution of HA is mainly established during the first three weeks after birth. On day 21, the HA content is similar to that in the adult rat. The process is dependent on normal Ang II function. It primarily involves a reduction of HA synthase 2 expression and an increase of medullary hyaluronidase 1.  The cortical accumulation of HA that results from neonatal ACE inhibition can partly explain the pathological condition of the adult kidney, which causes reduced urinary concentration ability and tubulointerstitial inflammation. It is possible to reduce renomedullary HA with the HA synthesis inhibitor 4-MU, and the kidney’s ability to respond to a hydration challenge will then be suppressed, without affecting GFR.  The investigation of renomedullary interstitial cells (RMIC) in culture, shows that media osmolality and hormones of central importance for body fluid homeostasis, such as angiotensin II, ADH and endothelin, affect HA turnover through their effect on the RMICs, in a manner comparable to that found in vivo during changes in hydration status.  In established streptozotocin-induced diabetes, HA is regionally accumulated in the kidney, proteinuria and polyuria, reduced urine osmolality, and reduced response to ADH V2 activation will occur. As opposed to the proteinuria, the HA accumulation is not sensitive to mTOR inhibition, suggesting an alternate pathway compared to other ECM components  Taken together, the data suggest that during normal physiological conditions, renomedullary interstitial HA participates in renal fluid handling by affecting the interstitial prerequisites for fluid flux across the interstitial space. This is possible due to the water-attracting and physicochemical properties of this glycosaminoglycan. During pathological conditions, such as diabetes, the elevated interstitial HA can contribute to the defective kidney function, due to the proinflammatory and water-attracting properties of HA.

Current feeder-free culture systems employing undefined Matrigel are still more effective in maintaining human embryonic stem (ES) cells than defined surfaces using extracellular matrix (ECM) proteins. While the role of substrate stiffness in stem cell fate is becoming increasingly evident, all previous culture systems use ECM proteins on rigid polystyrene surfaces. Here, we used factorial designs to screen and evaluate combinations ECM proteins and substrate stiffness for their effect on short-term pluripotency and self-renewal. Using optimal conditions determined from our screening experiments, defined and near xeno-free culture systems maintained CA1 human ES cells for over 10 passages in Essential 8 (E8) medium. Under these conditions, we found that human ES cell self-renewal was greater on soft polydimethylsiloxane (PDMS) substrates than on rigid polystyrene dishes. The culture systems and screening tools developed in this project will help develop robust and defined xeno-free culture systems that incorporate both biochemical and biomechanical factors.

Current feeder-free culture systems employing undefined Matrigel are still more effective in maintaining human embryonic stem (ES) cells than defined surfaces using extracellular matrix (ECM) proteins. While the role of substrate stiffness in stem cell fate is becoming increasingly evident, all previous culture systems use ECM proteins on rigid polystyrene surfaces. Here, we used factorial designs to screen and evaluate combinations ECM proteins and substrate stiffness for their effect on short-term pluripotency and self-renewal. Using optimal conditions determined from our screening experiments, defined and near xeno-free culture systems maintained CA1 human ES cells for over 10 passages in Essential 8 (E8) medium. Under these conditions, we found that human ES cell self-renewal was greater on soft polydimethylsiloxane (PDMS) substrates than on rigid polystyrene dishes. The culture systems and screening tools developed in this project will help develop robust and defined xeno-free culture systems that incorporate both biochemical and biomechanical factors.

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Les gliomes sont les tumeurs primitives les plus fréquentes du système nerveux central. Cette dernière décennie, l’apport de la biologie moléculaire a permis de mieux appréhender leurs comportements et de mieux préciser leur origine. Nous avons montré que le profil moléculaire des glioblastomes (grade IV dans la classification de l’OMS) et celui des astrocytomes pilocytiques (grade I dans la classification de l’OMS) différait notamment par l’expression d’un gène nommé KIAA 0510. La caractérisation de sa séquence nous a mené à nous intéresser à la Ténascine R, une glycoprotéine de la matrice extracellulaire impliquée dans les processus de migration et de différenciation cellulaire. Par ailleurs, l’expression de la Ténascine R pendant le développement, suggère son implication au cours de la corticogenèse.Dans le but de mieux comprendre l’origine des astrocytomes pilocytiques, notamment ceux de la région des voies optiques, nous avons mis en évidence au niveau du chiasma optique en développement des cellules de la glie radiaire à partir desquelles les astrocytomes pilocytiques des voies optiques pourraient dériver. / Gliomas are the most frequently occurring primary tumors in the central nervous system. These last years, molecular biology technics allowed a better understanding of the gliomagenesis as well as behaviour of these tumors. We have previously shown that molecular profiling of glioblastomes (WHO grade IV) and pilocytic astrocytomas (WHO grade I) differed for KIAA 0510 gene expression. This sequence was fully characterized and shown to be part of the tenascin R gene encoding for an extracellular matrix glycoprotein involved in migration and cell differentiation. In addition, during development, Tenascin-R may be involved in corticogenesis.In parallel, in the developing optic chiasm, we evidenced cells with radial glial characteristics from which the hypothalamo-chiasmatic pilocytic astrocytomas could derive.

In the developing visual system, the axon of a pre-synaptic cell must be guided to a post-synaptic partner. Retinal ganglion cells (RGCs) in the eye are an excellent model to study this process. Multiple classes exist that respond to specific types of light input, and these project to different destinations in the brain that process distinct types of information. The RGC axons that navigate to the lateral geniculate nucleus (LGN) do so in a class-specific manner. Axons from RGCs that mediate non-image forming functions innervate the ventral LGN (vLGN) and the intergeniculate leaflet (IGL). Axons from RGCs that process image-forming information bypass these regions to innervate the dorsal LGN (dLGN). The extracellular protein reelin was identified as a potential factor in RGC axonal targeting of the vLGN and IGL, and the reeler mutant mouse used to study the effects of its functional absence. Anterograde labeling of RGCs and their axons with Cholera toxin B (CTB) revealed reduced patterns of retinal innervation to the vLGN and IGL in mutant mice. Moreover, the absence of functional reelin resulted in axons incorrectly growing into inappropriate regions of the thalamus. We identified these misrouted axons as those of the intrinsically photosensitive RGCs (ipRGCS), a class of RGCs known to project to the affected subnuclei. In contrast to defects in ipRGC targeting, no deficits were seen in retinogeniculate or corticothalamic projections in classes of axons that normally target the dLGN. Immunohistochemistry did not reveal any effects of the absence of the functional reelin on the LGN cytoarchitecture, which is unlike many other brain regions altered in the reeler. In summary, results suggest that intact reelin is required for class-specific retinogeniculate targeting to the vLGN and IGL. The defects are likely to be in targeting and not in neuronal positioning.

Acquisition of invasiveness through extracellular matrix is a crucial characteristic of transition to malignancy in the breast. It was previously shown that Polo-like kinase 1 (PLK-1), a mitotic kinase and genome stability regulator, is involved in acquisition of invasiveness in a breast cell model (HMT-3522 cell line) of pre-invasive to invasive transition. This and other data led to the suggestion that a new class of genes called GISEM for Genome Instability and Extracellular Matrix Invasiveness may exist. Previous lab data show that XRCC3 is found downregulated in progression from preinvasive to invasive phenotype. This led to the hypothesis that XRCC3 may be a negative regulator of invasion. To support this hypothesis, overexpression of XRCC3 in the invasive T4-2 cells downregulated invasion, but also growth. In order to verify the role of XRCC3 in invasiveness, and determine whether it is independent from any effects on growth, we tested the effect of downregulating XRCC3 on the invasiveness of T4-2 cells. Short-term downregulation of XRCC3 using siRNAs produced a significant increase in invasiveness, suggesting a role for XRCC3 as a negative regulator of invasion. During the invasion assay time course, XRCC3 downregulation had no effect on growth or apoptosis supporting the idea that this is a direct effect on invasion and not an artifact of the assay. XRCC3 is one amongst the five members of the RAD51 paralog family, consisting of accessory proteins or RAD51 cofactors (namely RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) which interact with each other to form complexes (BCDX2, BC, DX2 and CX3) that collaboratively assist RAD51 in homologous recombinational repair (HRR) of DNA double-strand breaks. To see if these interactions are important in terms of invasion, as they have been demonstrated for DNA repair, we studied the effect of XRCC3 downregulation on the levels of RAD51 paralogs. We found lowered levels of RAD51C, but not RAD51B or RAD51D, when XRCC3 was downregulated. Since XRCC3 forms the CX3 complex with RAD51C, we downregulated RAD51C using siRNAs in T4-2 cells and found this to significantly increase invasiveness. Consistent with previous findings by other groups, downregulating RAD51C also lead to decreased levels of XRCC3 in invasive T4-2 cells. These results suggest that the XRCC3-RAD51C interaction is important for invasion as well as the previously studied DNA repair function. In delineating the mechanism by which XRCC3 acts as a negative regulator of invasion, we further questioned if XRCC3 alters secreted factors that are important for the invasiveness of T4-2 cells and tested the effects of conditioned medium (CM) from XRCC3 altered T4-2 cells on parental T4-2 cells’ ability to invade. Results show a significant increase in invading T4-2 cells when suspended in CM from XRCC3 siRNA transfected T4-2 cells, suggesting a direct effect of XRCC3 siRNAs on the ability of T4-2 CM to induce invasiveness in T4-2 cells. Furthermore, we investigated the effects of XRCC3 inhibition on cell surface integrins and focal adhesion kinase (FAK). Indirect immunofluorescence results show increased formation of focal adhesions containing two phosphorylated FAK residues- autophosphorylated FAK-Y397 and FAK-Y861 (previously implicated in increased migration and invasion of tumor cells) in XRCC3 siRNA transfected T4-2 cells. Overall, these results support a new role of XRCC3 in invasion, in addition to its previously reported role in DNA repair. These findings imply that loss of XRCC3 function in cancer progression would upregulate invasion as well as downregulate DNA repair and genome stability. Therefore, stabilization of XRCC3 function could provide a promising therapeutic against breast cancer progression. The dual role of XRCC3 in invasion and DNA repair also renders it an attractive candidate risk biomarker of breast pre-cancer to invasive cancer progression.

Chronic obstructive pulmonary disease (COPD) including emphysema is a devastating condition, increasing in prevalence in the US and worldwide. There remains no cure for COPD, rather only symptomatic treatments. Due to unique challenges of the lung, translation of therapies for acute lung injury to target chronic lung diseases like COPD has not been successful. We have been investigating lung derived extracellular matrix (ECM) hydrogels as a novel approach for delivery of cellular therapies to the pulmonary system. During the course of this work we have developed and characterized a lug derived ECM hydrogel that exhibits “injectability,” allowing cells or dugs to be delivered in a liquid and encapsulated at body temperature. The hydrogel self assembles in <5 minutes and achieves mechanical stiffness similar to other soft tissue ECM hydrogels. The hydrogel can support 3D cell growth and encapsulated cell viability. Encapsulated hMSCs can also still be activated by simulated inflammatory environments. Naïve mouse macrophages exposed to the fully formed gel were not significantly induced to express markers for pro or anti-inflammatory polarized phenotypes, but increased expression for several secreted inflammatory mediators was observed. We also investigated a novel approach for preparing and solubilizing the isolated ECM proteins, using digestion time as a variable for controlling hydrogel density (interconnectivity), mechanical stiffness, component protein size distribution, and cell behavior on fully formed gels. The potential future impact for the presented research includes optimization for future animal studies, expansion to additional applications, and the development of new derivative materials.

Adhesion of hematopoietic cells to the bone marrow microenvironment is important for their proper development. It is proven that Src-family kinases (SFK) regulate cell adhesion, although their exact role in the regulation of adhesion signaling remains unclear. Since adhesion processes are investigated mainly in adherent cell types, far less is known about hematopoietic cells. However, defects in the cell adhesion accompany a number of hematological diseases, like chronic myeloid leukaemia (CML). SFK overexpression is one of the proposed mechanisms of resistance to the first-line CML treatment, imatinib mesylate. Second generation drugs (e. g. dasatinib) inhibit SFK together with Bcr-Abl. Additionally, SFK-specific inhibitors (PP2, Src inhibitor-1) are also available, but there are no studies about effects of these drugs on cellular adhesivity of hematopoietic precursors. To explore the dynamics of hematopoietic cell adhesion to the extracellular matrix, we introduced a new approach using the RTCA xCELLigence DP system along with the well-established method of fluorimetric detection of adherent cell fraction. Our general observation is that various drugs (dasatinib, imatinib, PP2, Src inhibitor-1) induce pro-adhesive effects in several leukemic cell lines. Direct comparison of the kinetics of...

Nous étudions la migration et la protéolyse de la matrice extracellulaire dans le cancer du sein. Pour cela, nous avons mis en place deux systèmes modèles. Le premier, se base sur une lame basale reconstituée et permet d'évaluer le potentiel invasif de lignées cellulaires tumorales. Nous montrons que les cellules cancéreuses migrent différemment à travers un gel pour former des amas de taille variable directement corrélé à leur pouvoir invasif. Dans notre système, seule la migration de type mésenchymateuse est utilisée par les cellules. Ce type de mouvement est directement dépendant de protéases sécrétées par les cellules. Nous avons, donc mesurée la synthèse au niveau transcriptionnel de la classe d'enzyme majoritairement impliquée dans la dissémination tumorale, les matrice métalloprotéases (MMPs). Nous avons ainsi pu montrer que l'expression de 3 MMPs est corrélée aux capacités migratoires des cellules donc à leur potentiel invasif. Le processus physique par lequel les enzymes dégradent les matrices est très peu étudié au niveau expérimental. Le second système que nous utilisons se base sur un modèle de matrice conjonctive majoritairement composer de collagène de type I. Nous utilisons la gélatine, pour étudier la protéolyse de gels protéiques par différentes classes de protéases. A partirdes études sur la solubilisation enzymatique des gels par l'-chymotrypsine, la protéinase K et la papaïne, nous montrons qu'il existe des mécanismes de dégradation distincts. Le premier est un mécanisme anormal dont la cinétique est limitée par la diffusion de l'enzyme, le second est brownien et la cinétique est limitée par la réaction. Ce second mécanisme dépend directement d'interactions eléctrostatiques entre l'enzyme et le gel. Nous observons pour deux des enzymes que l'évolution des temps de dégradation mais également la cinétique dépendent de la concentration en protéine dans les gels. / We study the migration and proteolysis of the extracellular matrix in breast cancer. For this, we set up two model systems. The first is based on a reconstituted basement membrane and allows the evaluation of invasive potential tumor cell lines. We show that cancer cells migrate differently across the gel to form clusters of variable size directly correlates with their invasiveness. In our system, only the migration of mesenchymal type is used by the cells. This type of movement is directly dependent proteases secreted by the cells. We therefore measured the synthesis at the transcriptional level of the enzyme class mainly involved in tumor dissemination, the matrix metalloproteases (MMPs). We were able to show that the expression of 3 MMPs is correlated with migratory capacity of cells, therefore their invasive potential. The physical process by which enzymes degrade the matrix is very little studied at the experimental level. The second system we use is based on a model of connective matrix mainly composed of collagen type I. We use gelatin for the study of protein gels proteolysis by different classes of proteases. Based on the study of gels enzymatic solubilization by a- chymotrypsin, proteinase K and papain, we show that there are distinct mechanisms of degradation. The first mechanism is abnormal whose kinetic is limited by enzyme diffusion, and the second is Brownian and the kinetic is reaction limited. The second mechanism depends directly on electrostatic interactions between enzyme and gel. We observe for two enzymes that the evolution of degradation time but also the degradation kinetics depend on the concentration of protein in gels.