Rheumatoid arthritis (RA) which affects 0.5-1% of the world population and is characterised by joint erosions and presence of the autoantibodies anti-citrullinated protein antibodies (ACPA) and rheumatoid factor. Collagen II (CII) is a joint-specific antigen and we have shown that antibodies against CII (anti-CII) are present in around 8% of RA patients. RA patients with anti-CII are characterized by acute RA onset with elevated CRP and early joint erosions at the time of RA onset. Polymorphonuclear granulocytes (PMN) and peripheral blood mononuclear cells (PBMC) are abundant in RA synovial fluids, where they can interact with anti-CII, thus forming immune complexes (IC) with CII. In my thesis I have shown that PMN upregulated the cell surface markers CD66b and CD11b and downregulated CD16 and CD32 after stimulation with anti-CII IC. These changes in CD66b and CD16 associated to joint erosions to a larger extent than did PBMC responses to anti-CII IC. PMN cocultured with PBMC and stimulated with anti-CII IC showed augmented chemokine production that was dependent on TLR4 and functionally active PMN enzymes. This mechanism can lead to accumulation of inflammatory cells in joints of RA patients who are anti-CII positive around the time of RA diagnosis, and may thus help explain the acute onset RA phenotype associated with anti-CII. In a large Swedish RA cohort, anti-CII associated with elevations in clinical and laboratory measures of disease activity at diagnosis and until 6 months, whereas ACPA associated with late inflammation. Anti-CII seropositive RA was associated with improvements in clinical measurements and was negatively associated with smoking in contrast to ACPA that was associated with worseneing of clinical symptoms and associated positively with smoking. Anti-CII levels associated to  HLADRB1*03 and  HLADRB1*01 whereas ACPA showed negative association to HLA-DRB1*03. In a Malaysian RA cohort anti-CII also associated to elevated CRP at the time of diagnosis. Anti-CII seropositive RA represents a distinct phenotype, in many respects representing the converse  to the clinical, genetic and smoking associations described for ACPA. Early determinations of anti-CII in parallel to ACPA predict the inflammatory outcome in RA.

The study seeks to determine the degree of accumulation of Type II error rates, while violating the assumptions of normality, for different specified levels of power among sample means. The study employs a Monte Carlo simulation procedure with three different specified levels of power, methodologies, and population distributions. On the basis of the comparisons of actual and observed error rates, the following conclusions appear to be appropriate. 1. Under the strict criteria for evaluation of the hypotheses, Type II experimentwise error does accumulate at a rate that the probability of accepting at least one null hypothesis in a family of tests, when in theory all of the alternate hypotheses are true, is high, precluding valid tests at the beginning of the study. 2. The Dunn-Bonferroni procedure of setting the critical value based on the beta value per contrast did not significantly reduce the probability of committing a Type II error in a family of tests. 3. The use of an adequate sample size and orthogonal contrasts, or limiting the number of pairwise comparisons to the number of means, is the best method to control for the accumulation of Type II errors. 4. The accumulation of Type II error is irrespective of distributions.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

The large numbers of neurons that comprise the adult brain display an immense diversity. Repeated divisions of a relatively small pool of neural stem cells generate this neuronal diversity during development. To increase progress towards medical treatments for neurodegenerative diseases, it is of interest to understand both how neural stem cells generate the assortment of neurons and how these neurons come together to form a functional brain. Brain assembly occurs sequentially across time with early events laying the foundation for later events. Drosophila neural stem cells, neuroblasts (NBs), are an excellent model for investigating how neural diversity is generated and what roles early and late born neurons have in shaping the stereotypical adult brain structure. Generation of neural diversity, begins with specifying the diverse population of stem cells, called spatial patterning, and continues with diversifying neurons made from the diverse stem cells, called temporal patterning. Drosophila NBs exhibit both spatial and temporal patterning. Drosophila NBs have three types of division modes: type 0, type I and type II. Type II NBs expand the number of neurons made with progeny that exhibit a transit-amplifying division pattern, similar to that of mammalian outer subventricular zone (OSVZ) progenitors. Additionally, type II NBs exhibit temporal patterning across both the NB and their progeny to generate a large diversity of neurons that populate a conserved region of the brain responsible for many sensory and motor functions, called the central complex. Type II NBs have only been identified and studied during later stages in development, with nothing known about their origin or early divisions. In this dissertation, I describe the early lineages of the type II NBs within the Drosophila embryo. I show that type II NBs and lineages originate early in development, exhibit temporal patterning across both the NB and transit-amplifying progeny, and produce neurons that survive into the adult brain to innervate and potentially serve as a foundation within the adult central complex. Additionally, I explain how live imaging of the developing Drosophila brain can answer questions not easily addressed through other methods.

Abstract   Introduction Failure in the mechanism of self-tolerance in T or B cells can lead to autoimmunity. One of the autoimmune diseases is rheumatoid arthritis (RA), which is a chronic inflammatory disease of unknown cause and is characterized by systemic inflammation, autoantibodies and joint destruction. Serology is crucial for the classification of this disease. The first autoantibody found in RA patients was Rheumatoid factor (RF). However, anti-citrullinated peptide antibodies (ACPAs), a relatively new group of autoantibodies found in 70-90% of RA patients, are diagnostically more specific than RF. Type II collagen (CII) is the most abundant protein in human cartilage. In RA patients, immunity against CII leads to cartilage degradation and loss of joint function. Already from the 1970s, antibodies to CII (anti-CII) were found in RA sera, suggesting that CII autoimmunity might be pathogenetically important. Previous studies from our group show that a subgroup of patients with high levels of anti-CII at the time of diagnosis at the same time have high levels of inflammation in the joints. This is probably caused by anti-CII immune complexes (IC) inducing pro-inflammatory cytokines from macrophages. Although anti-CII positive patients have high inflammatory activity early on, as anti-CII levels decrease during the first year, the associated inflammation also diminishes. Thus, anti-CII positive patients have a rather good prognosis. Moreover, it is assumed that since anti-CII positive patients have a better prognosis than ACPA positive, patients with elevated anti-CII at the time of diagnosis might benefit from different and milder treatment. Previous studies from the group were performed on stored patient samples from the time before modern treatments with biologic agents (1995-2005). In this study, we aimed to investigate patients belonging to a more recent RA cohort, diagnosed between 2005-2014, with the aim to investigate whether patients with the anti-CII-associated RA phenotype would respond differently depending on the use of different modern RA therapies. Patients and Methods The primary cohort consisted of 2335 RA patients and 480 non-RA controls from the Epidermiological Investigations in Rheumatoid Arthritis (EIRA) case-control study. As we run into methodological problems two subgroups with 62 and 40 RA patients from the previous anti-CII studies were investigated when modifying the ELISA procedure, as well as a group of earlier investigated patients with non-specific ELISA reactivity. Totally 2776 RA patients were investigated. All investigated patients fulfilled the American College of Rheumatology classification criteria. To measure the anti-CII levels in RA patients and healthy controls anti-CII ELISA was performed. During the experiments, several different sources of CII from human, rat and bovine origin, and two different alternative coating buffers were used. The optical density (OD) was measured at 450 nm and anti-CII concentrations were calculated against the standard curve from an RA patient with high anti-CII levels.   Results My first analysis of the EIRA cohort showed that anti-CII are higher in RA patients than in controls, but could not confirm the association with acute onset RA. This was an unexpected finding and changed the focus of this master thesis project, to modify the measurement of anti-CII. Re-investigation of EIRA I showed that a proprietary coating buffer is important in the assay. Moreover, when different samples from RA patients were tested with bovine, rat and three different lots of human CII, correlation tests with clinical measures showed that bovine collagen and a new lot of human CII- prepared by the supplying company solely for this project- showed the strongest associations. Thereafter the EIRA cohort was re-investigated with two ELISAs, using bovine and human CII coated with the proprietary buffer. At the time of thesis writing almost all of the EIRA samples have been re-analysed, and results from both the modified ELISAs show the awaited clinical associations to early inflammation.   Conclusion Keeping the integrity of triple helical collagen is very important for the identification anti-native CII in RA patients. Our results show that the use of the proprietary coating buffer appears to be instrumental in this assay, irrespective of what source of CII was used. The new lot of human CII shows significant associations with the clinical measures, but associations are somewhat stronger with bovine CII. After finalising the re-investigations, we will be able to conclude which of the two analyses is most appropriate, and the corresponding dataset will then be merged with data from the first part of the EIRA study investigated previously by other group members. As anti-CII analysis shows the association to disease activity and prognosis, it can be used for predicting prognosis of RA and choosing the appropriate therapy in newly diagnosed RA patients, which might be clinically useful for rheumatologists. Our hypothesis is that as anti-CII positive patients have strong early inflammatory response but good long-term prognosis, they might benefit from other and perhaps short-term treatment compared to other RA patients.  If this is correct, our finding can have impact on the economy as it can define the patients who will not need expensive long-term medications. As modern anti-rheumatic therapies carry the risk of infections, such individualized therapies might also benefit anti-CII positive patients.

Les objectifs de ce travail étaient l’évaluation de stades précoces et tardifs de l’arthrose grâce à une étude histologique et biologique de l’arthrose sur un modèle de rupture de ligament croisé crânial chez le lapin (RLCC) et l’évaluation de l’effet de l’âge sur l’évolution de la maladie. L’étude biologique a été réalisée par le dosage sérique d’un marqueur de la dégradation du collagène de type II, le CTX-II, jusqu’à 20 semaines post-chirurgie. Chez les animaux adultes, les concentrations du CTX-II étaient influencées par la chirurgie et par le développement de la pathologie. Chez les jeunes animaux, les niveaux de CTX-II étaient plus élevés en début d’étude et diminuaient au cours du temps. Chez ce groupe d'animaux, les taux de CTX-II n’étaient pas modifiés par l’intervention chirurgicale. L’étude histologique a été réalisée avec une analyse histomorphologique du cartilage et des analyses histomorphométriques du cartilage et de l’os sous-chondral. L’évaluation histologique a permis d’observer des changements liés à l’arthrose chez tous les animaux opérés. Les altérations au niveau du cartilage étaient plus sévères chez les animaux adultes que chez les jeunes, ces derniers présentant une meilleure capacité de compensation à l’instabilité articulaire. Il a été démontré que l’analyse de la plaque osseuse sous-chondrale a permis de distinguer les animaux opérés des animaux non-opérés dans les 2 groupes d’âge, mais les surfaces articulaires affectées n’étaient pas toujours les mêmes. En conclusion, ce travail suggère l’intérêt du CTX-II dans l’évaluation de l’arthrose, mais aussi la pertinence d’effectuer plusieurs temps d’analyse pour mieux connaître l’évolution de la maladie. Les analyses histologiques ont permis de mettre en évidence des changements au niveau du cartilage et de l’os sous-chondral. Les différences observées entre les lapins adultes et les jeunes remettent en question l'utilisation d’animaux trop jeunes dans les études portant sur l’arthrose / The goals of this work were the evaluation of early and late stages of osteoarthritis through a histological and biological study of osteoarthritis using a rabbit model of the anterior cruciate ligament transaction (ACLT) and the evaluation of the effect of age on the evolution of the disease. Biological study was performed with longitudinal analysis of serum level of CTX-II, a marker a type II collagen degradation. In adult animals, serum concentration of CTX-II was influenced by the ACLT surgery and varied with time. In the young rabbits, the serum levels of CTX-II were more elevated at the beginning of study and decrease after. In this animal group, the rates of CTX-II were not influenced by surgical operation. Histological study was accomplished with both histomorphological analysis of the cartilage and with histomorphometric study of both cartilage and sub-chondral bone. Histological evaluation showed osteoarthritis changes in all operated animals. Changes of the cartilage appeared more severe in the adult group compared to the young rabbits, suggesting that these last have a better compensation capacity during articular instability. Bony changes allowed to differentiate operated animals from the unoperated, but the affected articular surfaces were not always the same. In conclusion, this study suggest the interest of the CTX-II biomarker in the evaluation of osteoarthritis, but also the pertinence to perform several time points of analysis to know better the disease evolution. The histological analysis allowed to put in an obvious place changes at the levels of the cartilage and of the sub-chondral bone. Difference noticed between the adult animals and the young rabbits offers a new look for the use of too young animals in osteoarthritis studies

Les NAD(P)H déshydrogénases de type II (Ndh-II) sont des enzymes de faible poids moléculaire capables doxyder le NAD(P)H et de transférer les électrons à un groupement quinone (plastoquinone ou ubiquinone). On les appelle « de type II » par opposition aux déshydrogénases de type I qui correspondent au complexe I mitochondrial. Chez Arabidopsis thaliana, des protéines Ndh-II ont été identifiées sur les faces interne et externe de la membrane interne mitochondriale, sur la membrane des peroxysomes, et au niveau de la membrane thylacoïdale du chloroplaste. Au niveau de la chaîne de transport délectrons mitochondriale, les protéines Ndh-II constituent une voie alternative aux complexes I et II pour lapport des électrons au pool dubiquinones. Cette voie alternative permettrait une adaptation de la chaîne de transport délectrons en fonction du métabolisme de lalgue. Au niveau de la chaîne de transport délectrons chloroplastique, les protéines Ndh-II participeraient à plusieurs mécanismes dadaptation de la chaîne à la quantité et à la qualité de la lumière disponible : transitions détats, transport cyclique délectrons autour du photosystème II. Leur fonction serait de catalyser la réduction non-photochimique du pool de plastoquinones. En 2005, sept open reading frame correspondant à des NAD(P)H déshydrogénases de type II hypothétiques (NDA1 à NDA7) ont été identifiées dans le génome nucléaire de Chlamydomonas. Ces séquences étaient cependant largement incomplètes du fait de régions non séquencées dans le génome de Chlamydomonas. Les données récoltées au cours de ce travail ont permis lobtention dune version complète de la séquence codante des gènes NDA de Chlamydomonas. Ces analyses ont démontré que le gène putatif NDA4 correspondait, en fait, à des régions internes non attribuées au gène NDA2. Chez Arabidopsis thaliana et Solanum tuberosum, une corrélation entre le positionnement phylogénétique des gènes NDH-II et la localisation subcellulaire de la protéine correspondante a été mise en évidence. Lanalyse phylogénétique des séquences des protéines Nda de Chlamydomonas montre que les gènes NDA1, 2 et 3 seraient proches phylogénétiquement et seraient à positionner dans le clade des protéines Ndh-II mitochondriales des plantes supérieures. A linverse, la protéine Nda5 serait dorigine cyanobactérienne et se positionne dans le même clade que les protéines identifiées dans le chloroplaste des plantes supérieures. Les protéines Nda6 et 7 sont très proches du point de vue de la séquence, suggérant une duplication récente des gènes NDA6 et 7. Ces deux protéines se positionnent dans un nouveau clade, apparemment intermédiaire entre le domaine eucaryote et le domaine procaryote. Une étude dexpression des gènes NDA de Chlamydomonas a permis de mettre en évidence lexpression apparemment majoritaire du gène NDA2. Pour étudier la fonction spécifique de NDA2, nous avons inactivé lexpression de ce gène par RNA interférence afin détudier le phénotype des mutants obtenus. Contrairement aux prédictions in silico, il est apparu que la protéine Nda2 se localise au niveau du chloroplaste. Létude de la fluorescence chlorophyllienne de deux mutants montre que la capacité de ces mutants à réduire de manière non-photochimique le pool de plastoquinones est largement diminuée. Dautre part, les mutants sont largement affectés dans leur capacité à modifier la distribution de lénergie dexcitation entre les deux photosystèmes (transition détat) lorsque la respiration mitochondriale est inhibée. Il est connu que les transitions détat sont initiées par des changements de létat rédox du pool de plastoquinones, qui est lui-même dépendant de létat rédox de la cellule. Dans ce cadre, nous proposons que la protéine Nda2 pourrait servir de « senseur » du métabolisme cellulaire de lalgue et permettrait dadapter les flux délectrons chloroplastiques en réponse aux changements du contexte énergétique cellulaire.

The exeC gene, found in the gram-negative bacteria Aeromonas hydrophila codes for a 31 kDa, three domain, bitopic inner membrane protein. The components of the ExeC protein include an amino-terminal cytoplasmic domain, a trans-membrane helix and two periplasmic domains. The two periplasmic domains are involved in recognition and selection of protein substrates which are subsequently transported across the outer membrane and free of the cell. This study focuses exclusively on the two periplasmic domains referred to hereafter as the HR and the PDZ domains. Three constructs were used throughout the course of this study. Two of them were designed, cloned and expressed for this study. The third is a result of previous work. Two constructs contained both the HR and PDZ domains while the other consists of the amino-terminal periplasmic HR domain. Only one construct was used to grow single crystals for analysis by X-ray crystallography. Crystals comprised of the PDZ domain from a degraded construct grew in a hexagonal space group with a hexagonal bi-pyramidal morphology. Crystals diffracted anisotropically to a maximum resolutions of 2 Å along the c axis and 3 Å in the a/b plane. Anisotropy in combination with twinning drastically complicated structure solution. Efforts toward elucidating the crystal structure will be discussed.

As awareness of the costs of workplace injury and illness continues to grow, there has been an increased demand for effective ergonomic interventions to reduce the prevalence of musculoskeletal disorders (MSDs). The goal of ergonomic interventions is to reduce exposures (mechanical and psychosocial); however there is conflicting evidence about the impact of these interventions as many studies produce inconclusive or conflicting results. In order to provide a clearer picture of the effectiveness of these interventions, we must find out if methodological issues, particularly statistical power, are limiting this research. The purpose of this study was to review and examine factors influencing statistical power in ergonomic intervention papers from five peer reviewed journals in 2008. A standardized review was performed by two reviewers. Twenty eight ergonomic intervention papers met the inclusion criteria and were fully reviewed. Data and trends from the reviewed papers were summarized specifically looking at the research designs used, the outcome measures used, if statistical power was mentioned, if a rationale for sample size was reported, if standardized and un-standardized effect sizes were reported, if confidence intervals were reported, the alpha levels used, if pair-wise correlation values were provided, if mean values and standard deviations were provided for all measures and the location of the studies. Also, the studies were rated based on the outcomes of their intervention into one of three categories (shown to be effective, inconclusive and not shown to be effective). Between these three groupings comparisons of post hoc power, standardized effect sizes, un-standardized effect sizes and coefficients of variation were made. The results indicate that in general, a lack of statistical power is indeed a concern and may be due to the sample sizes used, effect sizes produced, extremely high variability in some of the measures, the lack of attention paid to statistical power during research design and the lack of appropriate statistical reporting guidelines in journals where ergonomic intervention research may be published. A total of 69.6% of studies reviewed had a majority of measures with less than .50 power and 71.4% of all measures used had CVs of > .20.

As awareness of the costs of workplace injury and illness continues to grow, there has been an increased demand for effective ergonomic interventions to reduce the prevalence of musculoskeletal disorders (MSDs). The goal of ergonomic interventions is to reduce exposures (mechanical and psychosocial); however there is conflicting evidence about the impact of these interventions as many studies produce inconclusive or conflicting results. In order to provide a clearer picture of the effectiveness of these interventions, we must find out if methodological issues, particularly statistical power, are limiting this research. The purpose of this study was to review and examine factors influencing statistical power in ergonomic intervention papers from five peer reviewed journals in 2008. A standardized review was performed by two reviewers. Twenty eight ergonomic intervention papers met the inclusion criteria and were fully reviewed. Data and trends from the reviewed papers were summarized specifically looking at the research designs used, the outcome measures used, if statistical power was mentioned, if a rationale for sample size was reported, if standardized and un-standardized effect sizes were reported, if confidence intervals were reported, the alpha levels used, if pair-wise correlation values were provided, if mean values and standard deviations were provided for all measures and the location of the studies. Also, the studies were rated based on the outcomes of their intervention into one of three categories (shown to be effective, inconclusive and not shown to be effective). Between these three groupings comparisons of post hoc power, standardized effect sizes, un-standardized effect sizes and coefficients of variation were made. The results indicate that in general, a lack of statistical power is indeed a concern and may be due to the sample sizes used, effect sizes produced, extremely high variability in some of the measures, the lack of attention paid to statistical power during research design and the lack of appropriate statistical reporting guidelines in journals where ergonomic intervention research may be published. A total of 69.6% of studies reviewed had a majority of measures with less than .50 power and 71.4% of all measures used had CVs of > .20.