Étude du rôle de la membrane basale spécialisée dans la maturation de l'émail

Les cellules épithéliales qui produisent l’émail, les améloblastes, sont séparées de l'émail au niveau de la zone de maturation par une membrane basale spécialisée (MBS) enrichie en laminine 332 (LM-332). Cette protéine hétérotrimérique (composée des chaînes α3, ß3 et γ2) assure l'intégrité structurelle des membranes basales (MB) et influence divers processus cellulaires épithéliaux tels que l'adhésion et la différenciation cellulaire. Des modèles de souris « knockout » (KO), où les gènes codant pour LM-332 ont été supprimés, meurent peu après la naissance. Néanmoins, ce phénotype létal peut être contourné en substituant chez la souris le gène produisant la chaîne γ2 de la laminine (LAMC2) par sa forme humaine, sous le contrôle de l’expression du promoteur-rtTA, de la cytokératine 14, inductible par la prise de doxycycline (Dox) - (Tet-on). Le but de ce projet est d’examiner si l’utilisation de cette protéine humaine chez la souris a un effet sur la structuration de la MBS ainsi que sur la maturation de l'émail. La phase de maturation de l’organe de l’émail chez la souris transgénique a été sévèrement altérée par rapport à une souris normale (WT). La MBS n’est plus visible, une matrice dystrophique s’est formée dans la couche d'émail dans la phase de maturation, et la présence d’une matrice résiduelle de l'émail est observée durant la phase tardive de maturation. Des micro-analyses tomographiques ont révélé une usure excessive des surfaces occlusales des molaires, un écroulement de l'émail sur les pointes des incisives et une hypominéralisation de l'émail. Cependant, aucune altération structurale due à cette recombinaison transgénique n’a été observée dans d'autres sites épithéliaux, tels que la peau, le palais et la langue. Ces résultats indiquent que, bien que ce modèle de souris humanisée soit capable de rétablir ses fonctions dans divers tissus épithéliaux, il est incapable de soutenir la structuration d'une MBS à l'interface entre les améloblastes et l’émail en maturation. Cet échec peut être lié à la composition spécifique de la MBS dans la phase de maturation et supporte l’hypothèse que la MBS est essentielle pour la maturation adéquate de l'émail.

Development of 3D Cancer Cell Models to Test Metabolic Interventions as an Anticancer Strategy

In recent years, it has become evident that the role of mitochondria in the metabolic rewiring is essential for cancer development and progression. The metabolic profile during tumorigenesis has been performed mainly in traditional 2D cell models, including cell lines of various lineages and phenotypes. Although useful in many ways, their relevance can be often debatable, as they lack the interactions between different cells of the tumour microenvironment and/or interaction with the extracellular matrix 1,2. Improved models are now being developed using 3D cell culture technology, contributing with increased physiological relevance 3,4. In this work, we improved a method for the generation of 3D models from healthy and tumour colon tissue, based on organoid technology, and performed their molecular and biochemical characterization and validation. Further, in-plate cryopreservation was applied to these models, and optimal results were obtained in terms of cell viability and functionality of the cryopreserved models. We also cryopreserved colon fibroblasts with the aim to introduce them in a co-culture cryopreserved model with organoids. This technology allows the conversion of cell models into “plug and play” formats. Therefore, cryopreservation in-plate facilitates the accessibility of specialized cell models to cell-based research and application, in cases where otherwise such specialized models would be out of reach. Finally, we briefly explored the field of bioprinting, by testing a new matrix to support the growth of colon tumour organoids, which revealed promising preliminary results. To facilitate the reader, we organized this thesis into chapters, divided by the main points of work which include development, characterization and validation of the model, commercial output, and associated applications. Each chapter has a brief introduction, followed by results and discussion and a final conclusion. The thesis has also a general discussion and conclusion section in the end, which covers the main results obtained during this work.

The Cratylia Mollis Seed Lectin Induces Membrane Permeability Transition In Isolated Rat Liver Mitochondria And A Cyclosporine A-insensitive Permeability Transition In Trypanosoma Cruzi Mitochondria.

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca(2+) and mitochondrial dysfunction due to matrix Ca(2+) overload. In order to investigate the mechanism of Ca(2+) -induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (∆Ψm ) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 μM Ca(2+) was significantly decreased by 50 μg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 μM Ca(2+) this lectin, at 50 μg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ∆Ψm disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca(2+) dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA-insensitive MPT in T. cruzi mitochondria.

Impaired Compensatory Beta-cell Function And Growth In Response To High-fat Diet In Ldl Receptor Knockout Mice.

In this study, we investigated the effect of low density lipoprotein receptor (LDLr) deficiency on gap junctional connexin 36 (Cx36) islet content and on the functional and growth response of pancreatic beta-cells in C57BL/6 mice fed a high-fat (HF) diet. After 60 days on regular or HF diet, the metabolic state and morphometric islet parameters of wild-type (WT) and LDLr-/- mice were assessed. HF diet-fed WT animals became obese and hypercholesterolaemic as well as hyperglycaemic, hyperinsulinaemic, glucose intolerant and insulin resistant, characterizing them as prediabetic. Also they showed a significant decrease in beta-cell secretory response to glucose. Overall, LDLr-/- mice displayed greater susceptibility to HF diet as judged by their marked cholesterolaemia, intolerance to glucose and pronounced decrease in glucose-stimulated insulin secretion. HF diet induced similarly in WT and LDLr-/- mice, a significant decrease in Cx36 beta-cell content as revealed by immunoblotting. Prediabetic WT mice displayed marked increase in beta-cell mass mainly due to beta-cell hypertrophy/replication. Nevertheless, HF diet-fed LDLr-/- mice showed no significant changes in beta-cell mass, but lower islet-duct association (neogenesis) and higher beta-cell apoptosis index were seen as compared to controls. The higher metabolic susceptibility to HF diet of LDLr-/- mice may be explained by a deficiency in insulin secretory response to glucose associated with lack of compensatory beta-cell expansion.

Reduced Plasma Angiotensin Ii Levels Are Reversed By Hydroxyurea Treatment In Mice With Sickle Cell Disease.

Sickle cell disease (SCD) pathogenesis leads to recurrent vaso-occlusive and hemolytic processes, causing numerous clinical complications including renal damage. As vasoconstrictive mechanisms may be enhanced in SCD, due to endothelial dysfunction and vasoactive protein production, we aimed to determine whether the expression of proteins of the renin-angiotensin system (RAS) may be altered in an animal model of SCD. Plasma angiotensin II (Ang II) was measured in C57BL/6 (WT) mice and mice with SCD by ELISA, while quantitative PCR was used to compare the expressions of the genes encoding the angiotensin-II-receptors 1 and 2 (AT1R and AT2R) and the angiotensin-converting enzymes (ACE1 and ACE2) in the kidneys, hearts, livers and brains of mice. The effects of hydroxyurea (HU; 50-75mg/kg/day, 4weeks) treatment on these parameters were also determined. Plasma Ang II was significantly diminished in SCD mice, compared with WT mice, in association with decreased AT1R and ACE1 expressions in SCD mice kidneys. Treatment of SCD mice with HU reduced leukocyte and platelet counts and increased plasma Ang II to levels similar to those of WT mice. HU also increased AT1R and ACE2 gene expression in the kidney and heart. Results indicate an imbalanced RAS in an SCD mouse model; HU therapy may be able to restore some RAS parameters in these mice. Further investigations regarding Ang II production and the RAS in human SCD may be warranted, as such changes may reflect or contribute to renal damage and alterations in blood pressure.

Agrin And Perlecan Mediate Tumorigenic Processes In Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma is the most common type of cancer in the oral cavity, representing more than 90% of all oral cancers. The characterization of altered molecules in oral cancer is essential to understand molecular mechanisms underlying tumor progression as well as to contribute to cancer biomarker and therapeutic target discovery. Proteoglycans are key molecular effectors of cell surface and pericellular microenvironments, performing multiple functions in cancer. Two of the major basement membrane proteoglycans, agrin and perlecan, were investigated in this study regarding their role in oral cancer. Using real time quantitative PCR (qRT-PCR), we showed that agrin and perlecan are highly expressed in oral squamous cell carcinoma. Interestingly, cell lines originated from distinct sites showed different expression of agrin and perlecan. Enzymatically targeting chondroitin sulfate modification by chondroitinase, oral squamous carcinoma cell line had a reduced ability to adhere to extracellular matrix proteins and increased sensibility to cisplatin. Additionally, knockdown of agrin and perlecan promoted a decrease on cell migration and adhesion, and on resistance of cells to cisplatin. Our study showed, for the first time, a negative regulation on oral cancer-associated events by either targeting chondroitin sulfate content or agrin and perlecan levels.

Ankhd1 Silencing Inhibits Stathmin 1 Activity, Cell Proliferation And Migration Of Leukemia Cells.

ANKHD1 is highly expressed in human acute leukemia cells and potentially regulates multiple cellular functions through its ankyrin-repeat domains. In order to identify interaction partners of the ANKHD1 protein and its role in leukemia cells, we performed a yeast two-hybrid system screen and identified SIVA, a cellular protein known to be involved in proapoptotic signaling pathways. The interaction between ANKHD1 and SIVA was confirmed by co-imunoprecipitation assays. Using human leukemia cell models and lentivirus-mediated shRNA approaches, we showed that ANKHD1 and SIVA proteins have opposing effects. While it is known that SIVA silencing promotes Stathmin 1 activation, increased cell migration and xenograft tumor growth, we showed that ANKHD1 silencing leads to Stathmin 1 inactivation, reduced cell migration and xenograft tumor growth, likely through the inhibition of SIVA/Stathmin 1 association. In addition, we observed that ANKHD1 knockdown decreases cell proliferation, without modulating apoptosis of leukemia cells, while SIVA has a proapoptotic function in U937 cells, but does not modulate proliferation in vitro. Results indicate that ANKHD1 binds to SIVA and has an important role in inducing leukemia cell proliferation and migration via the Stathmin 1 pathway. ANKHD1 may be an oncogene and participate in the leukemia cell phenotype.

Enhanced Glucose-induced Intracellular Signaling Promotes Insulin Hypersecretion: Pancreatic Beta-cell Functional Adaptations In A Model Of Genetic Obesity And Prediabetes.

Obesity is associated with insulin resistance and is known to be a risk factor for type-2 diabetes. In obese individuals, pancreatic beta-cells try to compensate for the increased insulin demand in order to maintain euglycemia. Most studies have reported that this adaptation is due to morphological changes. However, the involvement of beta-cell functional adaptations in this process needs to be clarified. For this purpose, we evaluated different key steps in the glucose-stimulated insulin secretion (GSIS) in intact islets from female ob/ob obese mice and lean controls. Obese mice showed increased body weight, insulin resistance, hyperinsulinemia, glucose intolerance and fed hyperglycemia. Islets from ob/ob mice exhibited increased glucose-induced mitochondrial activity, reflected by enhanced NAD(P)H production and mitochondrial membrane potential hyperpolarization. Perforated patch-clamp examination of beta-cells within intact islets revealed several alterations in the electrical activity such as increased firing frequency and higher sensitivity to low glucose concentrations. A higher intracellular Ca(2+) mobilization in response to glucose was also found in ob/ob islets. Additionally, they displayed a change in the oscillatory pattern and Ca(2+) signals at low glucose levels. Capacitance experiments in intact islets revealed increased exocytosis in individual ob/ob beta-cells. All these up-regulated processes led to increased GSIS. In contrast, we found a lack of beta-cell Ca(2+) signal coupling, which could be a manifestation of early defects that lead to beta-cell malfunction in the progression to diabetes. These findings indicate that beta-cell functional adaptations are an important process in the compensatory response to obesity.

Ankhd1 Represses P21 (waf1/cip1) Promoter And Promotes Multiple Myeloma Cell Growth.

ANKHD1 (Ankyrin repeat and KH domain-containing protein 1) is highly expressed and plays an important role in the proliferation and cell cycle progression of multiple myeloma (MM) cells. ANKHD1 downregulation modulates cell cycle gene expression and upregulates p21 irrespective of the TP53 mutational status of MM cell lines. The present study was aimed to investigate the role of ANKHD1 in MM in vitro clonogenicity and in vivo tumourigenicity, as well as the role of ANKHD1 in p21 transcriptional regulation. ANKHD1 silencing in MM cells resulted in significantly low no. of colonies formed and in slow migration as compared to control cells (p < 0.05). Furthermore, in xenograft MM mice models, tumour growth was visibly suppressed in mice injected with ANKHD1 silenced cells compared to the control group. There was a significant decrease in tumour volume (p = 0.006) as well as in weight (p = 0.02) in the group injected with silenced cells compared to those of the control group. Co-immunoprecipitation and chromatin immunoprecipitation (ChIP) assays confirmed the interaction between p21 and ANKHD1. Moreover, overexpression of ANKHD1 downregulated the activity of a p21 promoter in luciferase assays. Decrease in luciferase activity suggests a direct role of ANKHD1 in p21 transcriptional regulation. In addition confocal analysis after U266 cells were treated with Leptomycin B (LMB) for 24 h showed accumulation of ANKHD1 inside the nucleus as compared to untreated cells where ANKHD1 was found to be predominantly in cytoplasm. This suggests ANKHD1 might be shuttling between cytoplasm and nucleus. In conclusion, ANKHD1 promotes MM growth by repressing p21 a potent cell cycle regulator.

Electrochemical degradation of the dye reactive orange 16 using electrochemical flow-cell

Electrochemical removals of color and organic load from solutions containing the dye reactive orange 16 (RO16) were performed in an electrochemical flow-cell, using a platinum working electrode. The influence of the process variables flow-rate, such as NaCl concentration, applied potential and solution pH, were studied. The best color removal achieved was 93% (λ = 493 nm) after 60 min at 2.2 V vs. RHE electrolysis, using 1.00 g L-1 NaCl as supporting electrolyte. The rises in the concentration of NaCl and applied potential increased the color removal rate. The best total organic carbon removal (57%) was obtained at 1.8 V, without the separating membrane, indicating that the ideal conditions for the color removal are not necessarily the same as those to remove the total organic carbon. The degradation efficiency decreased with the solution pH decrease.

The full Bayesian significance test for mixture models: results in gene expression clustering

Gene clustering is a useful exploratory technique to group together genes with similar expression levels under distinct cell cycle phases or distinct conditions. It helps the biologist to identify potentially meaningful relationships between genes. In this study, we propose a clustering method based on multivariate normal mixture models, where the number of clusters is predicted via sequential hypothesis tests: at each step, the method considers a mixture model of m components (m = 2 in the first step) and tests if in fact it should be m - 1. If the hypothesis is rejected, m is increased and a new test is carried out. The method continues (increasing m) until the hypothesis is accepted. The theoretical core of the method is the full Bayesian significance test, an intuitive Bayesian approach, which needs no model complexity penalization nor positive probabilities for sharp hypotheses. Numerical experiments were based on a cDNA microarray dataset consisting of expression levels of 205 genes belonging to four functional categories, for 10 distinct strains of Saccharomyces cerevisiae. To analyze the method's sensitivity to data dimension, we performed principal components analysis on the original dataset and predicted the number of classes using 2 to 10 principal components. Compared to Mclust (model-based clustering), our method shows more consistent results.

Evaluation of Antiproliferative, Anti-Type 2 Diabetes, and Antihypertension Potentials of Ellagitannins from Strawberries (Fragaria x ananassa Duch.) Using In Vitro Models

Strawberries represent the main source of ellagic acid derivatives in the Brazilian diet, corresponding to more than 50% of all phenolic compounds found in the fruit. There is a particular interest in the determination of the ellagic acid content in fruits because of possible chemopreventive benefits. In the present study, the potential health benefits of purified ellagitannins from strawberries were evaluated in relation to the antiproliferative activity and in vitro inhibition of alpha-amylase, alpha-glucosidase, and angiotensin I-converting enzyme (ACE) relevant for potential management of hyperglycemia and hypertension. Therefore, a comparison among ellagic acid, purified ellagitannins, and a strawberry extract was done to evaluate the possible synergistic effects of phenolics. In relation to the antiproliferative activity, it was observed that ellagic acid had the highest percentage inhibition of cell proliferation. The strawberry extract had lower efficacy in inhibiting the cell proliferation, indicating that in the case of this fruit there is no synergism. Purified ellagitannins had high alpha-amylase and ACE inhibitory activities. However, these compounds had low alpha-glucosidase inhibitory activity. These results suggested that the ellagitannins and ellagic acid have good potential for the management of hyperglycemia and hypertension linked to type 2 diabetes. However, further studies with animal and human models are needed to advance the in vitro assay-based biochemical rationale from this study.

Galectin-1 Induces Reversible Phosphatidylserine Exposure at the Plasma Membrane

Cells normally undergo physiological turnover through the induction of apoptosis and phagocytic removal, partly through exposure of cell surface phosphatidylserine (PS). In contrast, neutrophils appear to possess apoptosis-independent mechanisms of removal. Here we show that Galectin-1 (Gal-1) induces PS exposure independent of alterations in mitochondrial potential, caspase activation, or cell death. Furthermore, Gal-1-induced PS exposure reverts after Gal-1 removal without altering cell viability. Gal-1-induced PS exposure is uniquely microdomain restricted, yet cells exposing PS do not display evident alterations in membrane morphology nor do they exhibit bleb formation, typically seen in apoptotic cells. Long-term exposure to Gal-1 prolongs PS exposure with no alteration in cell cycle progression or cell growth. These results demonstrate that Gal-1-induced PS exposure and subsequent phagocytic removal of living cells represents a new paradigm in cellular turnover.

Effect of zinc insertion and hydrophobicity on the membrane interactions and PDT activity of porphyrin photosensitizers

A series of photosensitizers (PS), which are meso-substituted tetra-cationic porphyrins, was synthesized in order to study the role of amphiphilicity and zinc insertion in photodynamic therapy (PDT) efficacy. Several properties of the PS were evaluated and compared within the series including photophysical properties (absorption spectra, fluorescence quantum yield Phi(f), and singlet oxygen quantum yield Phi(Delta)), uptake by vesicles, mitochondria and HeLa cells, dark and phototoxicity in HeLa cells. The photophysical properties of all compounds are quite similar (Phi(f) <= 0.02; Phi(Delta) similar to 0.8). An increase in lipophilicity and the presence of zinc in the porphyrin ring result in higher vesicle and cell uptake. Binding in mitochondria is dependent on the PS lipophilicity and on the electrochemical membrane potential, i.e., in uncoupled mitochondria PS binding decreases by up to 53%. The porphyrin substituted with octyl groups (TC8PyP) is the compound that is most enriched in mitochondria, and its zinc derivative (ZnTC8PyP) has the highest global uptake. The stronger membrane interaction of the zinc-substituted porphyrins is attributed to a complexing effect with phosphate groups of the phospholipids. Zinc insertion was also shown to decrease the interaction with isolated mitochondria and with the mitochondria of HeLa cells, an effect that has been explained by the particular characteristics of the mitochondrial internal membrane. Phototoxicity was shown to increase proportionally with membrane binding efficiency, which is attributed to favorable membrane interactions which allow more efficient membrane photooxidation. For this series of compounds, photodynamic efficiency is directly proportional to the membrane binding and cell uptake, but it is not totally related to mitochondrial targeting.

Lineage relationship of prostate cancer cell types based on gene expression

Background: Prostate tumor heterogeneity is a major factor in disease management. Heterogeneity could be due to multiple cancer cell types with distinct gene expression. Of clinical importance is the so-called cancer stem cell type. Cell type-specific transcriptomes are used to examine lineage relationship among cancer cell types and their expression similarity to normal cell types including stem/progenitor cells. Methods: Transcriptomes were determined by Affymetrix DNA array analysis for the following cell types. Putative prostate progenitor cell populations were characterized and isolated by expression of the membrane transporter ABCG2. Stem cells were represented by embryonic stem and embryonal carcinoma cells. The cancer cell types were Gleason pattern 3 (glandular histomorphology) and pattern 4 (aglandular) sorted from primary tumors, cultured prostate cancer cell lines originally established from metastatic lesions, xenografts LuCaP 35 (adenocarcinoma phenotype) and LuCaP 49 (neuroendocrine/small cell carcinoma) grown in mice. No detectable gene expression differences were detected among serial passages of the LuCaP xenografts. Results: Based on transcriptomes, the different cancer cell types could be clustered into a luminal-like grouping and a non-luminal-like (also not basal-like) grouping. The non-luminal-like types showed expression more similar to that of stem/progenitor cells than the luminal-like types. However, none showed expression of stem cell genes known to maintain stemness. Conclusions: Non-luminal-like types are all representatives of aggressive disease, and this could be attributed to the similarity in overall gene expression to stem and progenitor cell types.