Development of a bovine adenovirus type 2-based gene delivery vector /

ABSTRACT Recombinant adenoviruses are currently under intense investigation as potential gene delivery and gene expression vectors with applications in human and veterinary medicine. As part of our efforts to develop a bovine adenovirus type 2 (BAV2) based vector system, the nucleotide sequence of BAV2 was determined. Sixty-six open reading frames (ORFs) were found with the potential to encode polypeptides that were at least 50 amino acid (aa) residue long. Thirty-one of the BAV2 polypeptide sequences were found to share homology to already identified adenovirus proteins. The arrangement of the genes revealed that the BAV2 genomic organization closely resembles that of well-characterized human adenoviruses. In the course of this study, continuous propagation of BAV2 over many generations in cell culture resulted in the isolation of a BAV2 spontaneous mutant in which the E3 region was deleted. Restriction enzyme, sequencing and PCR analyses produced concordant results that precisely located the deletion and revealed that its size was exactly 1299 bp. The E3-deleted virus was plaque-purified and further propagated in cell culture. It appeared that the replication of such a virus lacking a portion of the E3 region was not affected, at least in cell culture. Attempts to rescue a recombinant BAV2 virus with the bacterial kanamycin resistance gene in the E3 region yielded a candidate as verified with extensive Southern blotting and PCR analyses. Attempts to purify the recombinant virus were not successful, suggesting that such recombinant BAV2 was helper-dependent. Ten clones containing full-length BAV2 genomes in a pWE15 cosmid vector were constructed. The infectivity of these constructs was tested by using different transfection methods. The BAV2 genomic clones did appear to be infectious only after extended incubation period. This may be due to limitations of various transfection methods tested, or biological differences between virus- and E. co//-derived BAV2 DNA.

Development of techniques for testing the effect of the E1 region on adenovirus integration /

Adenoviral vectors are currently the most widely used gene therapeutic vectors, but their inability to integrate into host chromosomal DNA shortened their transgene expression and limited their use in clinical trials. In this project, we initially planned to develop a technique to test the effect of the early region 1 (E1) on adenovirus integration by comparing the integration efficiencies between an E1-deleted adenoviral vector (SubE1) and an Elcontaining vector (SubE3). However, we did not harvest any SubE3 virus, even if we repeated the transfection and successfully rescued the SubE1 virus (2/4 transfections generated viruses) and positive control virus (6/6). The failure of rescuing SubE3 could be caused by the instability of the genomic plasmid pFG173, as it had frequent intemal deletions when we were purifying It. Therefore, we developed techniques to test the effect of E1 on homologous recombination (HR) since literature suggested that adenovirus integration is initiated by HR. We attempted to silence the E1 in 293 cells by transfecting E1A/B-specific small interfering RNA (siRNA). However, no silenced phenotype was observed, even if we varied the concentrations of E1A/B siRNA (from 30 nM to 270 nM) and checked the silencing effects at different time points (48, 72, 96 h). One possible explanation would be that the E1A/B siRNA sequences are not potent enough to Induce the silenced phenotype. For evaluating HR efficiencies, an HR assay system based on bacterial transfonmatJon was designed. We constmcted two plasmids ( designated as pUC19-dl1 and pUC19-dl2) containing different defective lacZa cassettes (forming white colonies after transformation) that can generate a functional lacZa cassette (forming blue colonies) through HR after transfecting into 293 cells. The HR efficiencies would be expressed as the percentages of the blue colonies among all the colonies. Unfortunately, after transfonnation of plasmid isolated from 293 cells, no colony was found, even at a transformation efficiency of 1.8x10^ colonies/pg pUC19, suggesting the sensitivity of this system was low. To enhance the sensitivity, PCR was used. We designed a set of primers that can only amplify the recombinant plasmid fomied through HR. Therefore, the HR efficiencies among different treatments can be evaluated by the amplification results, and this system could be used to test the effect of E1 region on adenovirus integration. In addition, to our knowledge there was no previous studies using PCR/ Realtime PCR to evaluate HR efficiency, so this system also provides a PCR-based method to carry out the HR assays.

Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for ��1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of ��1 or ��3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the ��1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the ��3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the ��3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.

Factors affecting DNA uptake by mammalian cells

The ability to introduce DNA and express custom DNA sequences in bacteria opened the door for improvements in a large number of fields including agriculture, pharmacology, medicine, nutrition, etc. The ability to introduce foreign DNA sequences into mammalian cells in an efficient manner would have a large impact on therapeutic applications especially gene therapy. The methods in use today suffer from low efficiencies and sometimes toxicity. In this work a number of factors were evaluated for their effect onONA uptake efficiency. The factors studied included exposure to sublethal concentration of hydrogen peroxide which have been show to lead to destabilisation ofthe lysosomes. These exposures have proven to be very toxic to cells when combined with either the calcium phosphate or the lipofectAMINE�� transfection methods. Another factor evaluated was exposure to Electro-Magnetic Fields (EMF). This was fuelled by the fact that EMF have been shown to mediate a number of effects on cell structure and/or physiology. EMF exposure by itself was not sufficient to induce the cells to pick up the DNA, therefore its effect on calcium phosphate and lipofectAMINE�� was tested. Although some positive results were obtained, the variability of these results exceeded by far any observed enhancements which discouraged any further work on EMF. Also tested was the possible effect the presence of the cytomegalovirus (CMV) sequence might have on DNA uptake (based on previous results in this lab). It was found that the presence ofCMV in the DNA sequence does not enhance uptake or slow down degradation of the internalised DNA. The final factor tested was the effect of basic amino acids on transfection efficiency. It was found that arginine can enhance DNA uptake by about 170% v/ith calcium phosphate and about 200% with LipofectAMINE��. A model was proposed to explain the effect of arginine as well as the lack of effect from other amino acids.

Permissivity of human HeLa cells to bovine adenovirus type 2 (BaV2 infection

Infection of hUlnan cells by bovine adenovirlls type 2 (BAV2) is abortive. To obtain a better understanding of this pllenomel1011, and in particular to identify Wllich steps in the viral replicative cycles are altered dllring this virlls-host cells interaction, we have llndertaken a detailed study of BAV2 infections of the nonpennissive hUlnan IIeLa cells. Using autoradiography and 3H-thymidine-labeled vvhole virus particles for infection of HeLa cells, vve determined that viral attachluent appears normal. Furthermore, Southern analysis revealed that internalization and transport to the nuclells occurs in BAV2 infected HeLa cells. To investigate viral DNi\ synthesis, infectivity assays involving hydroxyllrea, a viral DN-A synthesis inhibitor, were carried out. The results revealed that Bft:LV2 DNA synthesis does not occur in HeLa cells. Fllrtller investigations into viral early gene expression by northern blotting analyses indicated that HeLa cells fail to support expression of EIA. This suggested that abortive infection by BAV2 could be attributed to faiiure of EIA to express. To test the possibility that the failure to express ElA was due to the inability of the host cell to recognize the E lA prOlTIoter, ,ve carried out transient expression transfection experiments using plaslnids \vith the bacterial lacZ linder the control of either BAV2 or i\d5 EIA promoter. X-gal histochelIlical assays sho\ved expression of lacZ from the Ad5 ElA prOlnoter but no expression of lacZ [rOln the BAV2 EIA prOlTIoter. This further suggests that the abortive infection b:y BAV2 could be attributed to failure of EIA to express dlle to a nonfllnctional prOlTIoter in hlunan cells. Thus we speClllated that abortive infection of HeLa cells by adenoviruses may be averted by providing EtA functions in trans. To demonstrate this, we coinfected HeLa cells with Ad5 and BAV2, reasoning that Ad5 could cOlnpensate for EIA deficiency in BAV2. OUf results showed that BAV2 DNA synthesis was indeed Sllpported in HeLa cells coinfected with Ad5dlE3 as revealed by Southern analysis. In contrast, coinfection of HeLa cells \vith BAV2 and Ad5dlElE3 mutallt did not support BLt\V2 DNA synthesis. Interestingly, BAV2 failed to replicate in 293 cells which are constitlltively expressing the El genes. This could ilnply that El is necessary but not sufficient to avert the failllre ofBAV2 to undergo productive infection ofhulnan cells.

Adenovirus-based exogenous gene expression in mammalian cells

Thesis (Ph.D.)--Brock University, 2010.

Investigating the role of apoptosis regulator EndoG on exogenous DNA uptake, stability, replication and recombination

Endonuclease G (EndoG) is a well conserved mitochondrial nuclease with dual lethal and vital roles in the cell. It non-specifically cleaves endogenous DNA following apoptosis induction, but is also active in non-apoptotic cells for mitochondrial DNA (mtDNA) replication and may also be important for replication, repair and recombination of genomic DNA. The aim of our study was to examine whether EndoG exerts similar activities on exogenous DNA substrates such as plasmid DNA (pDNA) and viral DNA vectors, considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA and nuclease activity in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four day time-course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus (a cationic liposome transfection reagent), targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances it may non-specifically cleave intracellular DNA regardless of its origin. To investigate possible effects of EndoG on viral DNA vectors, we constructed and evaluated AdsiEndoG, a first generation adenovirus (Ad5 ��E1) vector encoding a shRNA directed against EndoG mRNA, along with appropriate Ad5 ��E1 controls. Infection of HeLa cells with AdsiEndoG at a multiplicity of infection (MOI) of 10 p.f.u./cell resulted in an early cell proliferation defect, absent from cells infected at equivalent MOI with control Ad5 ��E1 vectors. Replication of Ad5 ��E1 DNA was detected for all vectors, but AdsiEndoG DNA accumulated to levels that were 50 fold higher than initially, four days after infection, compared to 14 fold for the next highest control Ad5 ��E1 vector. Deregulation of the cell cycle by EndoG depletion, which is characterized by an accumulation of cells in the G2/M transition, is the most likely reason for the observed cell proliferation defect. The enhanced replication of AdsiEndoG is consistent with this conclusion, as Ad5 ��E1 DNA replication is intimately related to cell cycling and prolongation or delay in G2/M greatly enhances this process. Furthermore, infection of HeLa with AdsiEndoG at MOI of 50 p.f.u./cell resulted in an almost complete disappearance of viable, adherent tumour cells from culture, whereas almost a third of the cells were still adherent after infection with control Ad5 ��E1 vectors, relative to the non-infected control. Therefore, targeting of EndoG by RNAi is a viable strategy for improving the oncolytic properties of first generation adenovirus vectors. In addition, AdsiEndoG-mediated knockdown of EndoG reduced homologous recombination between pDNA substrates in HeLa cells. The effect was modest but, nevertheless demonstrated that the proposed role of EndoG in homologous recombination of cellular DNA also extends to exogenous DNA substrates.

Melanosomal targeting sequences from gp100 are essential for MHC class II-restricted endogenous epitope presentation and mobilization to endosomal compartments

CD4+ T lymphocytes play an important role in CD8+ T cell-mediated responses against tumors. Considering that about 20% of melanomas express major histocompatibility complex (MHC) class II, it is plausible that concomitant antigenic presentation by MHC class I and class II complexes shapes positive (helper T cells) or negative (regulatory T cells) anti-tumor responses. Interestingly, gp100, a melanoma antigen, can be presented by both MHC class I and class II when expressed endogenously, suggesting that it can reach endosomal/MHC class II compartments (MIIC). Here, we demonstrated that the gp100 putative amino-terminal signal sequence and the last 70 residues in carboxy-terminus, are essential for MIIC localization and MHC class II presentation. Confocal microscopy analyses confirmed that gp100 was localized in LAMP-1+ endosomal/MIIC. Gp100-targeting sequences were characterized by deleting different sections in the carboxy-terminus (residues 590 to 661). Transfection in 293T cells, expressing MHC class I and class II molecules, revealed that specific deletions in carboxy-terminus resulted in decreased MHC class II presentation, without effects on MHC class I presentation, suggesting a role in MIIC trafficking for these deleted sections. Then, we used these gp100-targeting sequences to mobilize the green fluorescent protein (GFP) to endosomal compartments, and to allow MHC class II and class I presentation of minimal endogenous epitopes. Thus, we concluded that these specific sequences are MIIC targeting motifs. Consequently, these sequences could be included in expression cassettes for endogenously expressed tumor or viral antigens to promote MHC class II and class I presentation and optimize in vivo T cell responses, or as an in vitro tool for characterization of new MHC class II epitopes.

Sampling Interval and estimated Betas : Implications for the Presence of Transitory Components in Stock Prices

We provide a theoretical framework to explain the empirical finding that the estimated betas are sensitive to the sampling interval even when using continuously compounded returns. We suppose that stock prices have both permanent and transitory components. The permanent component is a standard geometric Brownian motion while the transitory component is a stationary Ornstein-Uhlenbeck process. The discrete time representation of the beta depends on the sampling interval and two components labelled \"permanent and transitory betas\". We show that if no transitory component is present in stock prices, then no sampling interval effect occurs. However, the presence of a transitory component implies that the beta is an increasing (decreasing) function of the sampling interval for more (less) risky assets. In our framework, assets are labelled risky if their \"permanent beta\" is greater than their \"transitory beta\" and vice versa for less risky assets. Simulations show that our theoretical results provide good approximations for the means and standard deviations of estimated betas in small samples. Our results can be perceived as indirect evidence for the presence of a transitory component in stock prices, as proposed by Fama and French (1988) and Poterba and Summers (1988).

Identification des canaux TRPC impliqu��s dans la potentialisation �� long terme des interneurones de la r��gion CA1 de l'hippocampe chez le rat

Le r��seau neuronal de l���hippocampe joue un r��le central dans la m��moire en modifiant de fa��on durable l���efficacit�� de ses synapses. Dans les interneurones de la couche oriens/alveus (O/A), l���induction de la potentialisation �� long terme (PLT) requiert les courants postsynaptiques excitateurs ��voqu��s par les r��cepteurs m��tabotropes du glutamate de sous-type 1a (CPSEmGluR1a) et l���entr��e subs��quente de Ca2+ via des canaux de la famille des transient receptor potential (TRP). Le but de ce projet ��tait d���identifier les canaux TRP responsables des CPSEmGluR1a et d���explorer les m��canismes mol��culaires r��gulant leur ouverture. Nous avons d��termin�� par des enregistrements ��lectrophysiologiques que les CPSEmGluR1a ��taient sp��cifiques aux interneurones O/A et qu���ils ��taient ind��pendants de la phospholipase C. Nous avons ensuite examin�� l���expression des TRPC et leur interaction avec mGluR1a par les techniques de RT-PCR, d���immunofluorescence et de co-immunopr��cipitation. Nos r��sultats montrent que TRPC1 et mGluR1a s���associent dans l���hippocampe et que ces deux prot��ines sont pr��sentes dans les dendrites des interneurones O/A. En revanche, TRPC4 ne semble s���associer �� mGluR1a qu���en syst��me recombinant et leur colocalisation para��t limit��e au corps cellulaire. Finalement, nous avons proc��d�� �� des enregistrements d���interneurones dans lesquels l���expression des TRPC a ��t�� s��lectivement supprim��e par la transfection d���ARN interf��rant et avons ainsi d��montr�� que TRPC1, mais non TRPC4, est une sous-unit�� obligatoire du canal responsable des CPSEmGluR1a. Ces travaux ont permis de mieux comprendre les m��canismes mol��culaires �� la base de la transmission synaptique des interneurones O/A et de mettre en ��vidence un r��le potentiel de TRPC1 dans la PLT.

Profil de pratique des m��decins omnipraticiens en sant�� mentale au Qu��bec

Au Qu��bec, face �� la pr��valence ��lev��e des probl��mes de sant�� mentale et �� la p��nurie de m��decins psychiatres, le m��decin omnipraticien (MO) occupe une place primordiale dans la prise en charge et le suivi des soins de sant�� mentale. Dans le contexte de r��forme du syst��me de sant�� mentale ax��e sur un renforcement de la collaboration entre les MO, les psychiatres et les ��quipes de sant�� mentale, notre ��tude vise �� mieux comprendre la pratique clinique et la pratique collaborative d��velopp��e par les MO, leur appr��ciation des outils de travail et de la qualit�� des services de sant�� mentale, dans le but d���am��liorer la compl��mentarit�� des soins au niveau primaire. Cette ��tude transversale impliquait 1415 MO de neuf territoires de centre de sant�� et de services sociaux (CSSS) du Qu��bec. L�����chantillon final ��tait constitu�� de 398 MO repr��sentatifs de lieux de pratique diversifi��s et le taux de r��ponse ��tait de 41%. Nos r��sultats mettent en ��vidence que la pratique clinique et la pratique collaborative des MO diff��re selon le degr�� de gravit�� des probl��mes de sant�� mentale des patients rencontr��s, c���est �� dire, trouble transitoire/mod��r�� de sant�� mentale (TTM.SM) ou trouble grave de sant�� mentale (TG.SM), et que les MO sont favorables au fait de travailler en collaboration avec les autres professionnels de la sant�� mentale. Ainsi, il appara��t important de renforcer l���accessibilit�� des MO aux professionnels de la sant�� mentale, particuli��rement les psychiatres, et de les informer de l���existence des autres acteurs en sant�� mentale sur leur territoire, pour renforcer la collaboration et la qualit�� des soins primaires de sant�� mentale.

Optimisation de la domiciliation des cellules CD34+ de sang de cordon ombilical: ��lucider les m��canismes en cause d��pendant du CXCR4.

Le sang provenant d���un cordon ombilical (SCO) repr��sente une bonne source de cellules souches h��matopo����tiques (CSH) pour des transplantations. Cependant, le nombre de cellules souches contenues dans ce sang est souvent insuffisant pour greffer un adulte. Le m��canisme intervenant dans la domiciliation de ces cellules au sein de la moelle osseuse (MO) est encore mal compris. On sait que l���interaction entre la chimiokine SDF-1 et le r��cepteur CXCR4, pr��sent sur les cellules CD34+ de SCO, m��ne �� la migration de ces cellules en direction de la MO. Nous pensons que l���augmentation de la proportion de cellules qui r��ussit �� se greffer pourra pallier au probl��me du nombre. Les produits de d��gradation, C3a et le C3desarg,, issus du syst��me du compl��ment, sont connus pour favoriser la r��ponse de cellules exprimant CXCR4 vers SDF-1. Nous avons analys�� l���effet du C3adesarg, mol��cule non anaphylatoxique, sur la migration cellulaire vers SDF-1, de m��me que sur la prise de greffe des cellules CD34+ issues de SCO suite �� une transplantation sur des souris NOD/SCIDyC-. Nos exp��riences ont d��montr�� que le C3a ainsi que le C3adesarg augmentaient tous les deux la r��ponse des cellules CD34+ vers SDF-1. Toutefois, nous n���avons pas pu d��montrer que ces mol��cules liaient directement le r��cepteur CXCR4. Par contre, le compos�� C3adesarg favorise la prise de greffe des cellules CD34+ de SCO. Il serait donc un bon candidat pour poursuivre une optimisation de ses propri��t��s. Nous avons ��galement constat�� que suite �� une transplantation chez la souris, les cellules CD34+ de SCO subissent une hausse d���expression transitoire de leur CXCR4 environ quatre jours apr��s la greffe. Cette hausse d���expression co��ncide avec la multiplication des cellules CD34+ dans la MO. Nous avons ��galement confirm�� qu���une cellule CD34+ avec une forte expression de CXCR4 ��tait dans un ��tat prolif��ratif. Nos donn��es sugg��rent que l���interaction directe avec les cellules stromales soit responsable de cette hausse d���expression de CXCR4.

Les R��seaux de la P��nombre:Typologie de l'aide re��ue par les Personnes ��g��es

Nous nous sommes int��ress��s �� l���analyse et �� la mise �� jour d���une typologie de l���aide re��ue par les personnes ��g��es de 65 ans et plus vivant �� domicile. Cette ��tude secondaire s���est bas��e sur les donn��es recueuillies dans deux milieux francophones, Hochelaga-Maisonneuve (HM) et Moncton (MCT). La collecte de donn��es avait ��t�� faite par l���entremise d���un questionnaire administr�� par entrevue face �� face. Les deux objectifs, de cette th��se sont : 1) ��tablir une typologie des r��seaux d���aide, r��sultant de la combinaison des sources d���aide et des t��ches accomplies ; 2) Identifier les principaux d��terminants d���appartenance aux r��seaux. La typologie obtenue met en relation les ressources, formelles ou informelles, utilis��es par les personnes ��g��es et l���aide instrumentale re��ue. La capacit�� ou l���incapacit�� �� effectuer neuf activit��s de la vie quotidienne et huit de la vie domestique ont servi �� ��valuer l���aide re��ue. Six ressources formelles et dix informelles ont ��t�� examin��es selon qu���elles ��taient les 1��res, 2i��mes ou 3i��mes sources d���aide utilis��es par les personnes ��g��es. L���approche privil��gi��e s���est inspir��e de celle des r��seaux sociaux et du mod��le de Pescosolido. C���est l���influence des caract��ristiques sociod��mographiques des personnes ��g��es, de leurs ��tats de sant��, de leurs habitudes de vie sur leurs r��seaux qui nous ont int��ress��s. Les r��sultats sont pr��sent��s �� chaque fois pour nos deux milieux s��par��ment. Nous commen��ons par un descriptif des sources d���aide utilis��es et des aides re��ues. Puis les profils des sources d���aide utilis��es et des activit��s accomplies sont expos��s pour l���ensemble des personnes ��g��es. Ces profils servent de base pour obtenir notre typologie. Elle comprend cinq cat��gories. Ces cat��gories sont toutes compos��es de personnes ��g��es faisant appel �� de l���aide formelle, informelle ou mixte pour accomplir des t��ches uniques ou multiples. La premi��re cat��gorie �� Transitoire ��, comprend 39% (HM) et 46% (MCT) des personnes ��g��es qui d��bute un processus d���incapacit��. Elles font appel �� des ressources informelles pour accomplir une t��che unique. La deuxi��me cat��gorie �� Personnes ��g��es seules �� en rassemble 14% (HM et MCT), majoritairement des femmes, avec peu d���incapacit��s. Ces derni��res utilisent de l���aide formelle pour une t��che unique. La troisi��me cat��gorie �� Familiale �� regroupe 12% (HM et MCT) des personnes ��g��es bien entour��es qui ont plusieurs incapacit��s. Ces gens font appel �� des sources d���aide informelles pour r��aliser des t��ches multiples. La quatri��me cat��gorie �� Tr��s fragile �� rassemble 30% (HM) et 25% (MCT) des personnes ��g��es peu entour��es ayant beaucoup d���incapacit��s. Elles utilisent des ressources d���aide mixtes pour effectuer des t��ches multiples. La cinqui��me cat��gorie �� Pr�� institutionnel �� comprend 4% (HM et MCT) des personnes ��g��es qui ont le plus d���incapacit��s et qui sont seules. Ces gens font appel �� de l���aide formelle pour des t��ches multiples. Les d��terminants d���appartenance �� ces cat��gories proviennent des blocs sociod��mographiques, ��tat de sant�� et r��seaux sociaux de notre mod��le th��orique. Une des contributions importantes de cette th��se a ��t�� de pouvoir identifier cinq cat��gories bien distinctes composant une typologie de l���aide re��ue, ind��pendamment du milieu, par des personnes ��g��es vivant �� domicile. MOTS CL��S : Typologie, r��seaux sociaux, personnes ��g��es, services de soins, formels, informels, aides re��ues, sources d���aide, incapacit��s, d��terminants d���appartenance, fragilit��

La stigmatisation des aidants familiaux de personnes atteintes par la maladie d���Alzheimer

Le vieillissement de la population entra��ne une hausse des maladies chroniques telle que la maladie d���Alzheimer dans nos soci��t��s occidentales. L���enjeu du vieillissement se r��percute aussi dans les r��formes de nos politiques sociales, et plus g��n��ralement dans la gestion des services publics. Dans ce contexte, le r��gime de sant�� publique qu��b��cois conna��t diverses modifications concernant la prestation de soins de premi��re ligne. De nouveaux acteurs acqui��rent des r��les et des responsabilit��s d��finissant des enjeux particuliers. Nous ��tudierons l���un de ces enjeux. Ce m��moire vise �� sp��cifier les processus sociaux �� la base de l���isolement des aidants familiaux de personnes atteintes par la maladie d���Alzheimer. La stigmatisation des aidants et les microprocessus aff��rents sont les principaux m��canismes analys��s. Les donn��es sont extraites d���entrevues semi-structur��es r��alis��es avec une cohorte d���aidants familiaux (N=60) suivie longitudinalement depuis le d��but de leur trajectoire de soins. Une d��marche qualitative soutient ce projet. Nous avons analys�� un ��chantillon de douze participants au moyen d���une approche s��quentielle. Trois processus typiques ont ��t�� identifi��s : le stigma de forme en ruptures (s��paration sociale), le stigma de forme transitoire (stigma transitoire) et le stigma de forme anomique (anomie sociale). Les r��sultats sugg��rent que les r��seaux sociaux des aidants sont soumis �� un ensemble de conditions favorisant la structuration du stigma social, la principale condition ��tant un enjeu de pouvoir concernant le contr��le de la personne malade. Les aidants conjoints de personnes atteintes sont plus enclins �� la stigmatisation en d��but de trajectoire.

Nanoparticules Chitosane-PEG-FA-ADN pour la th��rapie g��nique non virale et application du g��ne de l���IL-1Ra dans un mod��le exp��rimental d���arthrite rhumato��de

La th��rapie g��nique repr��sente l'un des d��fis de la m��decine des prochaines d��cennies dont la r��ussite d��pend de la capacit�� d'acheminer l'ADN th��rapeutique jusqu'�� sa cible. Des structures non virales ont ��t�� envisag��es, dont le chitosane, polym��re cationique qui se combine facilement �� l���ADN. Une fois le complexe form��, l���ADN est prot��g�� des nucl��ases qui le d��gradent. Le premier objectif de l'��tude est de synth��tiser et ensuite ��valuer diff��rentes nanoparticules de chitosane et choisir la mieux adapt��e pour une efficacit�� de transfection s��lective in vitro dans les cellules carcinomes ��pidermo��des (KB). Le deuxi��me objectif de l'��tude est d'examiner in vivo les effets protecteurs du g��ne de l'IL-1Ra (bloqueur naturel de la cytokine inflammatoire, l���Interleukine-1��) complex�� aux nanoparticules de chitosane s��lectionn��es dans un mod��le d'arthrite induite par un adjuvant (AIA) chez le rat. Les nanoparticules varient par le poids mol��culaire du chitosane (5, 25 et 50 kDa), et la pr��sence ou l���absence de l���acide folique (FA). Des mesures macroscopiques de l���inflammation seront ��valu��es ainsi que des mesures de concentrations de l���Interleukine-1��, Prostaglandine E2 et IL-1Ra humaine secr��t��s dans le s��rum. Les nanoparticules Chitosane-ADN en pr��sence de l���acide folique et avec du chitosane de poids mol��culaire de 25 kDa, permettent une meilleure transfection in vitro. Les effets protecteurs des nanoparticules contenant le g��ne th��rapeutique ��taient ��vidents suite �� la d��tection de l���IL-1Ra dans le s��rum, la baisse d'expressions des facteurs inflammatoires, l���Interleukine-1��� et la Prostaglandine-E2 ainsi que la diminution macroscopique de l���inflammation. Le but de cette ��tude est de d��velopper notre m��thode de th��rapie g��nique non virale pour des applications cliniques pour traiter l���arthrite rhumato��de et d���autres maladies humaines.