Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration

Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.

To divide or differentiate? : nestin-mediated regulation of Cdk5 in cell fate decisions

The molecular functions of the non-cell cycle-related Cyclin-dependent kinase 5 (Cdk5) have been of primary interest within the neuroscience field, but novel undertakings are constantly emerging for the kinase in tissue homeostasis, as well as in diseases such as diabetes and cancer. Although Cdk5 activation is predominantly regulated by specific non-cyclin activator protein binding, additional mechanisms have proved to orchestrate Cdk5 signaling in cells. For example, the interaction between the intermediate filament protein nestin and Cdk5 has been proposed to determine cellular fate during neuronal apoptosis through nestin-dependent adjustment of the sensitive balance and turnover of Cdk5 activators. While nestin constitutes a crucial regulatory scaffold for appropriate Cdk5 activation in apoptosis, Cdk5 itself phosphorylates nestin with the consequence of filament reorganization in both neuronal progenitors and differentiating muscle cells. Interestingly, the two proteins are often found coexpressed in various tissues and cell types, proposing that nestin-mediated scaffolding of Cdk5 and its activators may be applicable to other tissue systems as well. In the literature, the molecular functions of nestin have remained in the shade, as it is mostly exploited as a marker protein for progenitor cells. In light of these studies, the aim of this thesis was to assess the importance of the nestin scaffold in regulation of Cdk5 actions in cell fate decisions. This thesis can be subdivided into two major projects: one that studied the nature of the Cdk5-nestin interplay in muscle, and one that assessed their role in prostate cancer. During differentiation of a myoblast cell line, the filament formation properties of nestin was found to be crucial in directing Cdk5 activity, with direct consequences on the process of differentiation. Also the genetic knockout of nestin was found to influence Cdk5 activity, although differentiation per se was not affected. Instead, the genetic ablation of nestin had broad consequences on muscle homeostasis and regeneration. While the nestin-mediated regulation of Cdk5 in muscle was found to act in multiple ways, the connection remained more elusive in cancer models. Cdk5 was, however, established as a significant determinant of prostate cancer proliferation; a behavior uncharacteristic for this differentiation-associated kinase. Through complex and simultaneous regulation of two major prostate cancer pathways, Cdk5 was placed upstream of both Akt kinase and the androgen receptor. Its action on proliferation was nonetheless mainly exerted through the Akt signaling pathway in various cancer models. In summary, this thesis contributed to the knowledge of Cdk5 regulation and functions in two atypical settings; proliferation (in a cancer framework) and muscle differentiation, which is a poorly understood model system in the Cdk5 field. This balance between proliferation and differentiation implemented by Cdk5 is ultimately regulated (where present) by the dynamics of the cytoskeletal nestin scaffold.

Supraorganized collagen enhances Schwann cell reactivity and organization in vitro

We investigated the reactivity and expression of basal lamina collagen by Schwann cells (SCs) cultivated on a supraorganized bovine-derived collagen substrate. SC cultures were obtained from sciatic nerves of neonatal Sprague-Dawley rats and seeded on 24-well culture plates containing collagen substrate. The homogeneity of the cultures was evaluated with an SC marker antibody (anti-S-100). After 1 week, the cultures were fixed and processed for immunocytochemistry by using antibodies against type IV collagen, S-100 and p75NTR (pan neurotrophin receptor) and for scanning electron microscopy (SEM). Positive labeling with antibodies to the cited molecules was observed, indicating that the collagen substrate stimulates SC alignment and adhesion (collagen IV labeling - organized collagen substrate: 706.33 ± 370.86, non-organized collagen substrate: 744.00 ± 262.09; S-100 labeling - organized collagen: 3809.00 ± 120.28, non-organized collagen: 3026.00 ± 144.63, P < 0.05) and reactivity (p75NTR labeling - organized collagen: 2156.33 ± 561.78, non-organized collagen: 1424.00 ± 405.90, P < 0.05; means ± standard error of the mean in absorbance units). Cell alignment and adhesion to the substrate were confirmed by SEM analysis. The present results indicate that the collagen substrate with an aligned suprastructure, as seen by polarized light microscopy, provides an adequate scaffold for SCs, which in turn may increase the efficiency of the nerve regenerative process after in vivo repair.

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)-based scaffolds for tissue engineering

Development and selection of an ideal scaffold is of importance for tissue engineering. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is a biocompatible bioresorbable copolymer that belongs to the polyhydroxyalkanoate family. Because of its good biocompatibility, PHBHHx has been widely used as a cell scaffold for tissue engineering. This review focuses on the utilization of PHBHHx-based scaffolds in tissue engineering. Advances in the preparation, modification, and application of PHBHHx scaffolds are discussed.

Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery.

A sequential analysis of therapist scaffolding and child concept formation in narrative therapy

Narrative therapy is a postmodern therapy that takes the position that people create self-narratives to make sense of their experiences. To date, narrative therapy has compiled virtually no quantitative and very little qualitative research, leaving gaps in almost all areas of process and outcome. White (2006a), one of the therapy's founders, has recently utilized Vygotsky's (1934/1987) theories of the zone of proximal development (ZPD) and concept formation to describe the process of change in narrative therapy with children. In collaboration with the child client, the narrative therapist formalizes therapeutic concepts and submits them to increasing levels of generalization to create a ZPD. This study sought to determine whether the child's development proceeds through the stages of concept formation over the course of a session, and whether therapists' utterances scaffold this movement. A sequential analysis was used due to its unique ability to measure dynamic processes in social interactions. Stages of concept formation and scaffolding were coded over time. A hierarchical log-linear analysis was performed on the sequential data to develop a model of therapist scaffolding and child concept development. This was intended to determine what patterns occur and whether the stated intent of narrative therapy matches its actual process. In accordance with narrative therapy theory, the log-linear analysis produced a final model with interactions between therapist and child utterances, and between both therapist and child utterances and time. Specifically, the child and youth participants in therapy tended to respond to therapist scaffolding at the corresponding level of concept formation. Both children and youth and therapists also tended to move away from earlier and toward later stages of White's scaffolding conversations map as the therapy session advanced. These findings provide support for White's contention that narrative therapists promote child development by scaffolding child concept formation in therapy.

Isolation and characterization of genomic DNA sequences that enhance the stability of plasmid DNA in mammalian cells /

The ease of production and manipulation has made plasmid DNA a prime target for its use in gene transfer technologies such as gene therapy and DNA vaccines. The major drawback of plasmid however is its stability within mammalian cells. Plasmid DNA is usually lost by cellular mechanisms or as a result of mitosis by simple dilution. This study set out to search for mammalian genomic DNA sequences that would enhance the stability of plasmid DNA in mammalian cells.Creating a plasmid based genomic DNA library, we were able to screen the human genome by transfecting the library into Human Embryonic Kidney (HEK 293) Cells. Cells that contained plasmid DNA were selected, using G418 for 14 days. The resulting population was then screened for the presence of biologically active plasmid DNA using the process of transformation as a detector.A commercially available plasmid DNA isolation kit was modified to extract plasmid DNA from mammalian cells. The standardized protocol had a detection limit of -0.6 plasmids per cell in one million cells. This allowed for the detection of 45 plasmids that were maintained for 32 days in the HEK 293 cells. Sequencing of selected inserts revealed a significantly higher thymine content in comparison to the human genome. Sequences with high A/T content have been associated with Scaffold/Matrix Attachment Region (S/MAR) sequences in mammalian cells. Therefore, association with the nuclear matrix might be required for the stability of plasmids in mammalian cells.

Show me, help me, let me : supporting teachers' changing conceptions of reading assessment and reading instruction

This qualitative inquiry used case study methodology to explore the change processes of 3 primary-grade teachers throughout their participation in 7 -month professional learning initiative focused on reading assessment and instruction. Participants took part in semimonthly inquiry-based professional learning community sessions, as well as concurrent individualized classroom-based literacy coaching. Each participant's experiences were first analyzed as a single case study, followed by cross-case analyses. While their patterns of professional growth differed, findings documented how all participants altered their understandings of the roles and relevancy of individual components of reading instruction (e.g., comprehension, decoding) and instructional approaches to scaffold students' growth (e.g., levelled text, strategy instruction), and experienced some form of conceptual change. Factors identified as affecting their change processes included; motivation, professional knowledge, professional beliefs (self-efficacy and theoretical orientation), resources (e.g., time, support), differentiated professional learning with associated goal-setting, and uncontrollable influences, with the affect of each factor compounded by interaction with the others. Comparison of participants' experiences to the Cognitive-Affective Model of Conceptual Change (CAMCC) and the Interconnected Model of Teacher Professional Growth (IMTPG) demonstrated the applicability of using both conceptual models, with the IMTPG providing macrolevel insights over time and the CAMCC microlevel insights at each change intervaL Recommendations include the provision of differentiated teacher professional learning opportunities, as well as research documenting the effects of teacher mentorship programs and the professional growth of teacher educators. ii

Closing the Summer Learning Gap for Vulnerable Children: An Examination of a Summer Family Literacy Program for Junior Kindergarten Children At-Risk for Reading Difficulties

The learning gap created by summer vacation creates a significant breach in the learning cycle, where student achievement levels decrease over the course ofthe summer (Cooper et aI., 2000). In a review of 39 studies, Cooper and colleagues (1996) specified that the summer learning shortfall equals at least one month loss of instruction as measured by grade level equivalents on standardized test scores. Specifically, the achievement gap has a more profound effect on children as they grow older, where there is a steady deterioration in knowledge and skills sustained during the summer months (Cooper et aI., 1996; Kerry & Davies, 1998). While some stakeholders believe that the benefits of a summer vacation overshadow the reversing effect on achievement, it is the impact of the summer learning gap on vulnerable children, including children who are disadvantaged as a result of requiring special educational needs, children from low socioeconomic backgrounds, and children learning English as a second language, that is most problematic. More specifically, research has demonstrated that it is children's literacy-based skills that are most affected during the summer months. Children from high socioeconomic backgrounds recurrently showed gains in reading achievement over the summer whereas disadvantaged children repeatedly illustrate having significant losses. Consequently, the summer learning gap was deemed to exaggerate the inequality experienced by children from low socioeconomic backgrounds. Ultimately, the summer learning gap was found to have the most profound on vulnerable children, placing these children at an increased chance for academic failure. A primary feature of this research project was to include primary caregivers as authentic partners in a summer family literacy program fabricated to scaffold their children's literacy-based needs. This feature led to the research team adapting and implementing a published study entitled, Learning Begins at Home (LBH): A Research-Based Family Literacy Program Curriculum. Researchers at the Ontario Institute designed this program for the Study of Education, University of Toronto. The LBH program capitalized on incorporating the flexibility required to make the program adaptable to meet the needs of each participating child and his or her primary caregiver. As it has been well documented in research, the role primary caregivers have in an intervention program are the most influential on a child's future literacy success or failure (Timmons, 2008). Subsequently, a requirement for participating in the summer family literacy program required the commitment of one child and one of his or her primary caregivers. The primary caregiver played a fundamental role in the intervention program through their participation in workshop activities prior to and following hands on work with their child. The purpose of including the primary caregiver as an authentic partner in the program was to encourage a definitive shift in the family, whereby caregivers would begin to implement literacy activities in their home on a daily basis. The intervention program was socially constructed through the collaboration of knowledge. The role ofthe author in the study was as the researcher, in charge of analyzing and interpreting the results of the study. There were a total of thirty-six (36) participants in the study; there were nineteen (19) participants in the intervention group and seventeen (17) participants in the control group. All of the children who participated in the study were enrolled in junior kindergarten classrooms within the Niagara Catholic District School Board. Once children were referred to the program, a Speech and Language Pathologist assessed each individual child to identify if they met the eligibility requirements for participation in the summer family literacy intervention program. To be eligible to participate, children were required to demonstrate having significant literacy needs (i.e., below 25%ile on the Test of Preschool Early Literacy described below). Children with low incident disabilities (such as Autism or Intellectual Disabilities) and children with significant English as a Second Language difficulties were excluded from the study. The research team utilized a standard pre-test-post-test comparison group design whereby all participating children were assessed with the Test of Preschool Early Literacy (Lonigan et aI., 2007), and a standard measure of letter identification and letter sound understanding. Pre-intervention assessments were conducted two weeks prior to the intervention program commencing, and the first set of the post-intervention assessments were administered immediately following the completion of the intervention program. The follow-up post-intervention assessments took place in December 2010 to measure the sustainability of the gains obtained from the intervention program. As a result of the program, all of the children in the intervention program scored statistically significantly higher on their literacy scores for Print Knowledge, Letter Identification, and Letter Sound Understanding scores than the control group at the postintervention assessment point (immediately following the completion of the program) and at the December post-intervention assessment point. For Phonological Awareness, there was no statistically significant difference between the intervention group and the control at the postintervention assessment point, however, there was a statistically significant difference found between the intervention group and the control group at the December post-intervention assessment point. In general, these results indicate that the summer family literacy intervention program made an immediate impact on the emergent literacy skills of the participating children. Moreover, these results indicate that the summer family literacy intervention program has the ability to foster the emergent literacy skills of vulnerable children, potentially reversing the negative effect the summer learning gap has on these children.

Ligand design for molecule-based magnetic and/or conducting materials

Work in the area of molecule-based magnetic and/or conducting materials is presented in two projects. The first project describes the use of 4,4’-bipyridine as a scaffold for the preparation of a new family of tetracarboxamide ligands. Four new ligands I-III have been prepared and characterized and the coordination chemistry of these ligands is presented. This project was then extended to exploit 4,4’-bipyridine as a covalent linker between two N3O2 macrocyles. In this respect, three dimeric macrocycles have been prepared IV-VI. Substitution of the labile axial ligands of the Co(II) complex IV by [Fe(CN)6]4- afforded the self-assembly of the 1-D polymeric chain {[Co(N3O2)H2O]2Fe(CN)6}n•3H2O that has been structurally and magnetically characterized. Magnetic studies on the Fe(II) complexes V and VI indicate that they undergo incomplete spin crossover transitions in the solid state. Strategies for the preparation of chiral spin crossover N3O2 macrocycles are discussed and the synthesis of the novel chiral Fe(II) macrocyclic complex VII is reported. Magnetic susceptibility and Mössbauer studies reveal that this complex undergoes a gradual spin crossover in the solid state with no thermal hysteresis. Variable temperature X-ray diffraction studies on single crystals of VII reveal interesting structural changes in the coordination geometry of the macrocycle accompanying its SCO transition. The second project reports the synthesis and characterization of a new family of tetrathiafulvalene derivatives VIII – XII, where a heterocyclic chelating ligand is appended to a TTF donor via an imine linker. The coordination chemistries of these ligands with M(hfac)2.H2O (M( = Co, Ni, Mn, Cu) have been explored and the structural and magnetic properties of these complexes are described.

Towards reverse engineering of Photosystem II: Synergistic Computational and Experimental Approaches

ABSTRACT Photosystem II (PSII) of oxygenic photosynthesis has the unique ability to photochemically oxidize water, extracting electrons from water to result in the evolution of oxygen gas while depositing these electrons to the rest of the photosynthetic machinery which in turn reduces CO2 to carbohydrate molecules acting as fuel for the cell. Unfortunately, native PSII is unstable and not suitable to be used in industrial applications. Consequently, there is a need to reverse-engineer the water oxidation photochemical reactions of PSII using solution-stable proteins. But what does it take to reverse-engineer PSII’s reactions? PSII has the pigment with the highest oxidation potential in nature known as P680. The high oxidation of P680 is in fact the driving force for water oxidation. P680 is made up of a chlorophyll a dimer embedded inside the relatively hydrophobic transmembrane environment of PSII. In this thesis, the electrostatic factors contributing to the high oxidation potential of P680 are described. PSII oxidizes water in a specialized metal cluster known as the Oxygen Evolving Complex (OEC). The pathways that water can take to enter the relatively hydrophobic region of PSII are described as well. A previous attempt to reverse engineer PSII’s reactions using the protein scaffold of E. coli’s Bacterioferritin (BFR) existed. The oxidation potential of the pigment used for the BFR ‘reaction centre’ was measured and the protein effects calculated in a similar fashion to how P680 potentials were calculated in PSII. The BFR-RC’s pigment oxidation potential was found to be 0.57 V, too low to oxidize water or tyrosine like PSII. We suggest that the observed tyrosine oxidation in BRF-RC could be driven by the ZnCe6 di-cation. In order to increase the efficiency of iii tyrosine oxidation, and ultimately oxidize water, the first potential of ZnCe6 would have to attain a value in excess of 0.8 V. The results were used to develop a second generation of BFR-RC using a high oxidation pigment. The hypervalent phosphorous porphyrin forms a radical pair that can be observed using Transient Electron Paramagnetic Resonance (TR-EPR). Finally, the results from this thesis are discussed in light of the development of solar fuel producing systems.

3-O-Demethylation of thebaine and the synthesis of a flacourtia aglycone from benzoic acid.

Two synthetic projects were embarked upon, both fraught with protecting group nuance and reaction selectivity. Transformations of the opiate skeleton remain a valuable tool for the development of new medicines. Thebaine, a biosynthetic intermediate in the expression of morphine, was converted in three steps to oripavine through two parallel modes. Through the use of protecting group manipulations, two irreversible scaffold rearrangements were avoided during aryl methyl ether bond cleavage. This chemistry constitutes a new path in manipulations of the morphinan scaffold through protective groups. A new compound family, the flacourtosides, contains an unusual cyclohexenone fragment. The newly described compounds show in preliminary tests antiviral activity against dengue and chikungunya. This aglycone was approached on three pathways, all beginning with the chemoenzymatic dihydroxylation of benzoic acid. A first attempt from a known vinyl epoxide failed to epimerize and cooperate under deprotective conditions. A second and third attempt made use of a diastereoselective dihydroxylation reaction, which was critical in reaching the correct stereochemistry and oxidation state. The methyl ester of the aglycone was prepared, constituting the first synthesis of the non-trivial natural product framework.

Étude des mécanismes moléculaires menant à la migration cellulaire associée à Rac1 et ARF6.

Le facteur de l’ADP-ribosylation 6 (ARF6) et Rac1 sont des petites protéines liant le GTP qui régulent plusieurs voies de signalisation comprenant le trafic de vésicules, la modification des lipides membranaires et la réorganisation du cytosquelette d’actine. Cependant, les mécanismes moléculaires par lesquels ARF6 et Rac1 agissent de concert afin de contrôler ces différents processus cellulaires restent méconnus. Dans cette étude, nous montrons que, dans les cellules HEK293, ARF6 et Rac1 sont retrouvées en complexe suite à la stimulation du récepteur à l’angiotensine. Des expériences réalisées in vitro nous indiquent que ces deux GTPases interagissent ensemble directement, et que ARF6 s’associe préférentiellement avec la forme inactive de Rac1. L’inhibition de l’expression de ARF6 par interférence à l’ARN entraîne une activation marquée en cellule de Rac1 via le facteur PIX, indépendamment de la stimulation d’un récepteur, ce qui provoque la migration non contrôlée des cellules. Les arrestines, protéines de régulation de la désensibilisation des récepteurs couplés aux protéines G, servent de protéines d’échafaudage pour Rac1 et ARF6, en interagissant directement avec les GTPases et en augmentant leur association stimulée par l’angiotensine. De plus, les arrestines permettent l’activation, en s’en dissociant, de la MAP Kinase p38 qui régule l’activité de ARF6 et son interaction précoce avec les arrestines. Mis ensemble, ces résultats montrent que les arrestines contrôlent l’activité de ARF6, en influençant p38. ARF6 joue un rôle inhibiteur sur l’activation basale de Rac1 pour permettre ensuite son recrutement et son activation dépendante de l’angiotensine. Cette étude nous a permis de préciser le mode de régulation mis en jeu dans l’initiation de la migration cellulaire, suite à l’activation d’un récepteur couplé aux protéines G. Par le fait même, nous avons identifié certains des acteurs impliqués dans ce processus, offrant ainsi de nouvelles cibles pour le traitement des déséquilibres pathophysiologiques de la migration cellulaire.

La calnexine: un élément clé dans l'apoptose chez la levure Schizosaccharomyces pombe.

La mort cellulaire programmée (PCD pour Programmed Cell Death) est un processus essentiel aux cellules. Le PCD a d’abord été caractérisé dans le développement cellulaire et peut être divisé en plusieurs groupes selon les caractéristiques observées. L’apoptose, un sous-groupe du PCD, est caractérisé par plusieurs distinctions morphologiques et signalétiques attribué tout d’abord aux organismes complexes pour son rôle dans le développement et dans le maintien de l’intégrité tissulaire. Depuis la dernière décennie, de nombreuses études font état de l’existence d’un programme apoptotique dans des organismes unicellulaires comme les levures. Ce programme apoptotique a surtout été étudié chez les levures Saccharomyces cerevisiae et Schizosaccharomyces pombe et partage certaines caractéristiques avec l’apoptose des mammifères. Par contre, l’apoptose associé aux levures est distinct à certains égards entre autre par l’absence de certains homologues présents chez les mammifères. L’intérêt au niveau de l’étude du phénomène apoptotique chez les levures est sans cesse grandissant par la facilité avec laquelle les levures peuvent être utilisées comme système modèle. L’apoptose peut être induit dans les cellules de différentes façons en réponse à des stimuli internes ou externes. L’accumulation de protéines mal repliées au niveau du réticulum endoplasmique (RE) causant un stress est un inducteur bien caractérisé de la voie apoptotique. La signalisation de l’apoptose dans un cas de stress au RE fait appel aux transducteurs des signaux de la voie du UPR ( Unfolded Protein Response). Récemment, il a été montré que la calnexine, une chaperone transmembranaire du RE connue et caractérisée surtout pour ses fonctions d’aide au repliement des protéines et au contrôle de qualité, joue un rôle dans la transduction du signal apoptotique en réponse au stress du RE chez mammifères. Le rôle de la calnexine dans ce cas consiste principalement en l’échafaudage pour le clivage par la caspase 8 de la protéine apoptotique Bap31. Nous avons tout d’abord démontré que le stress du RE et que la déficience en inositol, un précurseur essentiel de nombreuses molécules signalétiques, sont deux inducteurs de l’apoptose chez la levure S. pombe. Ces deux voies semblent induire l’apoptose par deux voies distinctes puisque seule la voie de la déficience en inositol induit l’apoptose de façon dépendante à la métacaspase Pca1p. La calnexine, essentielle à la viabilité chez la levure S. pombe, est impliquée dans ces deux phénomènes apoptotiques. L’apoptose induit par le stress du RE nécessite une version de la calnexine ancrée à la membrane du RE pour être optimal. De façon opposée, l’apoptose induit par une déficience en inositol nécessite la présence de la queue cytosolique ancrée à la membrane de la calnexine pour être retardé. Ces deux actions différentes imputables à une même protéine laisse croire à une double fonction pro et anti-apoptotique de celle-ci. Suite à la découverte de l’existence d’un clivage endogène de la calnexine en situation normale de croissance, un modèle a été élaboré expliquant les rôles distincts de la calnexine dans ces deux voies apoptotiques. Ce modèle fait état d’un rôle associé au clivage de la calnexine dans l’apoptose.

Le 17B-Estradiol combiné à un biopolymère à base de chitosan accroît la biocompatibilité des cellules progénitrices dérivées de la moelle osseuse

Les cellules dérivées de la moelle osseuse, principalement les cellules endothéliales progénitrices, sont réduites chez les patients souffrant de maladies cardiovasculaires. Leur mobilisation et leur incorporation aux sites de lésion vasculaire sont des évènements prépondérants dans l’accélération des processus de réendothélialisation. Dans un modèle murin, le 17β-estradiol favorise les processus de guérison vasculaire par la mobilisation et le recrutement des cellules endothéliales progénitrices dérivées de la moelle osseuse. Il existe présentement plusieurs stratégies afin d’augmenter la mobilisation des cellules progénitrices ainsi que leur incorporation à la paroi vasculaire. Cependant, peu d’études privilégient la livraison locale d’un nombre élevé de cellules progénitrices fonctionnelles par un véhicule biodégradable et leur maintien au site de lésion afin de favoriser la réendothélialisation ciblée. Un polymère d’intérêt pour cette application s’avère être le chitosan. Ce biopolymère non toxique et biodégradable est couramment utilisé dans l’ingénierie tissulaire et, depuis peu, est utilisé dans la guérison vasculaire. Le chitosan complexé à la phosphorylcholine voit sa solubilité s’accroître dans les solutions aqueuses ainsi que sa biocompatibilité cellulaire en condition physiologique. Le projet de ce mémoire visait donc : 1) à étudier in vitro, la capacité d’un polymère de chitosan complexé à la phosphorylcholine à influencer l’adhésion, la survie, la différenciation et la fonctionnalité cellulaire dans un modèle murin de culture mixte de cellules dérivées de la moelle osseuse et 2) de déterminer l’impact de la présence du 17β-estradiol sur ces mêmes comportements cellulaires. Nos travaux démontrent que la matrice de chitosan-phosphorylcholine s’avère compatible avec notre modèle de culture cellulaire. En effet, ce polymère est capable de promouvoir l’organisation et le développement des cellules dérivées de la moelle osseuse de façon comparable à la matrice normalement utilisée dans la croissance in vitro des cellules endothéliales progénitrices, la fibronectine. De plus, ce polymère n’a nullement compromis l’activité migratoire des cellules, laissant supposer qu’il pourrait éventuellement être un véhicule approprié pour effectuer une livraison cellulaire à un site de lésion. Il s’avère que le 17β-estradiol, lorsqu’ajouté au milieu de culture ou complexé au polymère de chitosan phosphorylcholine, est capable de moduler le comportement cellulaire, et ce, de façon différente. Le 17β-estradiol complexé au polymère de chitosan-phosphorylcholine démontre, par rapport à sa forme soluble, une plus grande aptitude à accroître le nombre de cellules hématopoïétiques ainsi que des cellules endothéliales progénitrices dérivées de la moelle osseuse in vitro. De plus, le 17β-estradiol complexé au polymère de chitosan-phosphorylcholine permet une amplification marquée des cellules endothéliales progénitrices et leur offre un support adéquat afin de favoriser la guérison vasculaire. L’ensemble de nos travaux suggère que le polymère de chitosan complexé à la phosphorylcholine en présence ou non de 17β-estradiol est une matrice compatible avec les cellules progénitrices dérivées de la moelle osseuse in vitro. Le 17β-estradiol complexé au polymère est toutefois plus efficace que sa forme soluble à promouvoir l’amplification du nombre de cellules progénitrices. Ce polymère représente un outil thérapeutique attrayant et une matrice de livraison d’agent bioactif prometteuse pour le recrutement cellulaire dans l’accélération de la guérison vasculaire.