458 research outputs found
Targeted Limb Heating Augments the Actions of IGF1 in the Growth Plate and Increases Bone Elongation in Growing Mice
Bone elongation disorders can lead to painful musculoskeletal disabilities in adulthood. Existing treatment options to correct left-right asymmetry in limb length include invasive surgeries and/or drug regimens. These are often only partially effective. Previous studies in weanling mice have shown that a daily application of mild heat (40°C) to limbs on one side of the body could be used to noninvasively enhance bone elongation. However, the impact of heat-treatment on bone at the cellular level remains elusive. The epiphyseal growth plate, the band of cartilage located at each end of long bones, is the main site of longitudinal growth and is regulated by local and systemic growth factors. Insulin-like growth factor 1 (IGF1) is the major regulator of growth and controls bone elongation by promoting chondrocyte proliferation and hypertrophy. The objective of this study was to build upon an established method of targeted limb heating to determine how heat-treatment influences IGF1 action in the growth plate. This study tests the hypothesis that exposure to warm temperature augments the actions of IGF1 in the growth plate and permanently increases length of the extremities. This dissertation demonstrates that differences of less than 1.5% are functionally significant measured by a nearly 20% increase in hindlimb weight bearing on heat-treated sides. Heat-enhanced bone elongation is documented in female C57BL/6 mice after 7 days of heat-treatment during the most active period of growth from 3-4 weeks of age. This increase in bone elongation is accompanied by increased chondrocyte proliferation and hypertrophy in the proximal tibial growth plate. Moreover, this study is the first to show that targeted limb-heating impacts local action of IGF1 in growth plate chondrocytes. Results suggest that heat-induced limb length is IGF1 dependent since the growth effects are attenuated when IGF1 activity is blocked. Administration of a low dose of IGF1 (2.5mg/kg) was found to augment heat enhanced bone elongation and effects were sustained to skeletal maturity (12 weeks of age). These studies help contribute to the ultimate goal of developing a noninvasive method for lengthening bones that may translate in a clinical setting to treat linear growth disorders in children
The ever-expanding conundrum of primary osteoporosis: aetiopathogenesis, diagnosis, and treatment.
In recent years, as knowledge regarding the etiopathogenetic mechanisms of bone involvement characterizing many diseases has increased and diagnostic techniques evaluating bone health have progressively improved, the problem of low bone mass/quality in children and adolescents has attracted more and more attention, and the body evidence that there are groups of children who may be at risk of osteoporosis has grown. This interest is linked to an increased understanding that a higher peak bone mass (PBM) may be one of the most important determinants affecting the age of onset of osteoporosis in adulthood. This review provides an updated picture of bone pathophysiology and characteristics in children and adolescents with paediatric osteoporosis, taking into account the major causes of primary osteoporosis (PO) and evaluating the major aspects of bone densitometry in these patients. Finally, some options for the treatment of PO will be briefly discussed
Effects of dance therapy on balance, gait and neuro-psychological performances in patients with Parkinson's disease and postural instability
Postural Instability (PI) is a core feature of
Parkinsonâs Disease (PD) and a major cause of falls and disabilities. Impairment of executive functions has been called as an aggravating factor on motor performances. Dance therapy has been shown effective for improving gait and has been suggested as an alternative rehabilitative method.
To evaluate gait performance, spatial-temporal (S-T) gait
parameters and cognitive performances in a cohort of patients with PD and PI modifications in balance after a cycle of dance therapy
Ătude des dĂ©terminants gĂ©nĂ©tiques et molĂ©culaires de la scoliose idiopathique
La scoliose idiopathique (SI) est une maladie complexe de la colonne vertébrale. Elle
survient principalement Ă l'adolescence et affecte ~ 1-4% de la population mondiale
pédiatrique avec une prévalence plus élevée chez les femmes. Dans la plupart des cas, la cause
sous-jacente de la SI est inconnue, bien qu'une composante génétique soit bien reconnue. Un
certain nombre de gÚnes et de loci candidats ont été proposés, mais peu ont été répliqués avec
succÚs dans de multiples études. Les études d'association génomique (GWAS) ont identifié
plusieurs gÚnes candidats prédisposant à la SI. Parmi ceux-ci, le locus LBX1 est de loin celui
qui a Ă©tĂ© rĂ©pliquĂ© avec le plus de succĂšs dans diffĂ©rentes populations, bien que jusqu'Ă
maintenant il n'ait pas été testé dans la population québécoise.
Lâobjectif principal de cette thĂšse, a Ă©tĂ© l'Ă©tude des dĂ©terminants gĂ©nĂ©tiques de la SI
par des approches complémentaires. Dans la premiÚre étude, de nouveaux gÚnes enrichis de
variants rares, pouvant contribuer Ă la maladie ont Ă©tĂ© identifiĂ©s par sĂ©quençage dâexomes
entiers (WES) dans une cohorte de patients québécois atteints de SI, suivi d'une deuxiÚme
phase de séquençage ciblé des 24 meilleurs gÚnes candidats dans une seconde cohorte
indépendante. ParallÚlement, nous avons effectué une approche par WES dans une famille
multiplex unique constituée de trois soeurs affectées avec des parents sains. Nos résultats
impliquent un nouveau gÚne, FAT3, non précédemment associé à la SI, comme un gÚne
candidat dâimportance pour cette condition. Dans la deuxiĂšme Ă©tude, nous avons effectuĂ© un
GWAS pour tester les marqueurs génétiques associés à la dans la population québécoise. Les
résultats complets de l'analyse GWAS dépassent le cadre de cette thÚse. L'objectif principal de l'analyse actuelle était de tester l'association du locus LBX1. Nos résultats appuient une
association avec la région proche du gÚne LBX1 dans notre population. Dans la troisiÚme
étude, notre approche est une approche de gÚne candidat. Nous avons essayé d'élucider les
corrélations génétiques et biochimiques des niveaux circulants en YKL-40 avec le risque de
progression de la maladie. Une étude antérieure dans notre laboratoire a démontré que les
patients atteints de SI ont un dysfonctionnement distinctif de la signalisation des récepteurs
couplés aux protéines Gi inhibitrices (Gi), permettant leur classification en trois
endophénotypes (FG1, FG2 et FG3). L'étude des profils d'expression de ces endophénotypes a
montré une élévation significative de l'expression du gÚne CHI3L1, codant pour la
glycoprotĂ©ine sĂ©crĂ©tĂ©e YKL-40, chez les patients classĂ©s dans lâendophĂ©notype FG1 par
rapport à ceux classés dans les endophénotypes FG2 et FG3 qui sont plus enclins à développer
une scoliose sévÚre. Nous avons démontré que les garçons du groupe FG1 présentent des
niveaux plasmatiques significativement plus élevés en YKL-40 que les autres groupes tout
comme les patients présentant une scoliose non-sévÚre. Les SNP dans le gÚne CH13L1 ont
montrĂ© une association significative avec les niveaux plasmatiques dâYKL-40 dans des cas
non-sévÚre. L'analyse fonctionnelle in vitro a révélé que la glycoprotéine YKL-40 pourrait
jouer un rÎle protecteur dans le contexte de la SI en bloquant le défaut de signalisation Gi
induit par l'élévation de l'ostéopontine.
En résumé, nous proposons une nouvelle association du gÚne FAT3 avec la SI,
répliquons l'association du gÚne LBX1 dans une nouvelle cohorte et proposons un nouveau
marqueur biochimique, YKL-40, associé à des formes non sévÚres de la SI.Idiopathic Scoliosis (IS) is a complex disorder of the spine. It mostly occurs between
10 and 15 years old and affects ~1-4% of the global pediatric population with a much higher
prevalence in females. In most cases the underlying cause of IS is unknown, although a
genetic component is well recognized. A number of candidate genes and loci have been
suggested, but few have been successfully replicated in multiple studies. Genome wide
association studies (GWAS) have identified several candidate genes for IS susceptibility.
Among these the LBX1 locus is by far the most successfully replicated locus in different
populations, although until now it has not been tested in the French-Canadian population. Few
studies have attempted to detect rare causal variants in IS and this field of research is still in its
infancy.
The primary goal of this thesis was to investigate the genetic component of IS through
complementary approaches. In the first study, we aimed to find new genes enriched with rare
variants, which might contribute to the disease. Hence, we performed whole exome
sequencing (WES) in a French-Canadian IS cohort, followed by a second phase of targeted
sequencing of the 24 best candidate genes in a replication cohort. In parallel, we performed
WES in a unique multiplex family of three affected sisters with healthy parents. Our results
implicate a novel gene, FAT3, not previously associated with IS, as a strong candidate for this
condition. In the second study we performed a GWAS to test for genetic markers associated
with IS in our French-Canadian cohort. The complete results of the GWAS analysis are
beyond the scope of this thesis. The main objective of the current analysis was to test
association of the LBX1 locus. Our results support an association with the region near the LBX1 gene in our French-Canadian population. In the third study, our approach is a candidate
gene approach. We attempted to elucidate the genetic and biochemical correlates of circulating
YKL-40 levels with the risk of spinal deformity progression in the context of IS. Our prior
works have demonstrated that IS patients exhibit a distinctive G inhibitory (Gi) proteincoupled
receptor signaling dysfunction, which enabled their classification into three distinct
biological endophenotypes (FG1, FG2 or FG3). Previous microarray analysis revealed a
significant elevation in the expression of CHI3L1 gene, encoding for the secreted glycoprotein
YKL-40, in IS patients classified in FG1 endophenotype when compared to FG2 and FG3
ones, which are more prone to develop a severe scoliosis. In this study, we demonstrated that
IS males classified in FG1 endophenotype exhibit significant higher plasma YKL-40 levels
than controls and other IS endophenotypes. Furthermore, the non-severe scoliosis group
showed significant higher levels of YKL-40 than controls. SNPs in CHI3L1 gene showed
significant association with YKL-40 plasma levels in non-severe cases. Functional in vitro
analysis showed that YKL-40 could play a protective role in the context of IS by altering the
Gi-signaling dysfunction induced by the elevation of osteopontin in IS.
In sum, we propose a novel association of FAT3 gene with IS, replicated the
association of LBX1 gene with IS in a new cohort, and propose a new biochemical marker,
YKL-40, associated with non-severe forms of IS and has a plausible protective role in IS
Effets de l'estradiol et du chargement mécanique sur la régulation de la POC5 et du récepteur ADGRG7 dans la scoliose idiopathique
La scoliose est une dĂ©formation complexe en 3D de la colonne vertĂ©brale ayant une prĂ©valence de 1.5-3% dans la population gĂ©nĂ©rale. La forme la plus commune est la scoliose idiopathique (SI) qui inclue la scoliose idiopathique de lâadolescent (SIA) affectant principalement les filles au cours de la pubertĂ©. LâĂ©tiologie est largement inconnue, mais les observations cliniques rĂ©vĂšlent un rĂŽle de lâhĂ©rĂ©ditĂ© ainsi que dâune croissance rapide dans le dĂ©veloppement de la SIA. Il existe une forte Ă©vidence quâune composante gĂ©nĂ©tique entre en jeu dans cette pathologie. RĂ©cemment, de nombreux gĂšnes ont Ă©tĂ© suspectĂ©s dâĂȘtre responsables ou de contribuer Ă la SI. Notre Ă©quipe a identifiĂ© des variantes du gĂšne POC5, codant pour une protĂ©ine centriolaire, dans une large famille française dont plusieurs membres sont atteints de SI. Dans cette mĂȘme famille, nous avons suspectĂ© lâimplication dâune mutation du gĂšne ADGRG7 (rĂ©cepteur orphelin appartenant aux rĂ©cepteurs dâadhĂ©sion couplĂ©s aux protĂ©ines G) dans la pathogĂ©nicitĂ© de la SI. Au travers de nos travaux, nous nous sommes concentrĂ©s sur lâĂ©lucidation du rĂŽle des protĂ©ines POC5 sauvages et mutantes (in vitro et in vivo) ainsi que sur la rĂ©gulation de lâexpression de POC5 et ADGRG7 par lâestradiol (E2), dans le but de tester si ces gĂšnes pourraient ĂȘtre fonctionnellement liĂ©s Ă la scoliose.
Afin dâinvestiguer le rĂŽle du gĂšne POC5 dans la SI, nous avons surexprimĂ© la protĂ©ine POC5 mutante dans des lignĂ©es cellulaires par transfection transitoire (in vitro) et nous avons induit une perte de fonction du gĂšne POC5 dans un modĂšle animal, le poisson zĂšbre (in vivo). Le rĂŽle de POC5 a Ă©tĂ© Ă©tudiĂ© par : 1) Analyses de spectroscopie de masse et co-immunoprĂ©cipitation afin dâidentifier les diffĂ©rents partenaires de liaisons entre la protĂ©ine sauvage (wt POC5) et mutante (mut POC5); 2) immunolocalisation de la protĂ©ine sauvage et mutante au niveau cellulaire; 3) histologie et immunohistochimie rĂ©alisĂ©s sur des tissus issus de poissons zĂšbres contrĂŽles (wt POC5) et scoliotiques (mut POC5). Notre travail a permis dâidentifier plusieurs protĂ©ines partenaires de la POC5, et nous avons trouvĂ© des interactions fonctionnelles entre ces protĂ©ines et la POC5 reliĂ©es aux cils et centrosome. Un certain nombre de protĂ©ines ciliaires ont Ă©tĂ© identifiĂ©es comme interagissant avec wt POC5 et non avec mut POC5 comme CEP290, RAB11, CKAP5, Annexine 2 et Septine 9. Au niveau cellulaire, la localisation et la colocalisation des protĂ©ines wt POC5 et POC5 mutĂ©e avec la tubuline alpha acĂ©tylĂ©e (marqueur ciliaire), confirme la consĂ©quence de la mutation sur la localisation subcellulaire en relation avec la longueur et lâintensitĂ© de la coloration du cil. In vivo, nous avons identifiĂ© de nombreux dĂ©fauts de la rĂ©tine et de lâoreille interne chez le poisson zĂšbre POC5 mutant par rapport au contrĂŽle. Enfin, en utilisant diffĂ©rents marqueurs des couches rĂ©tiniennes et la tubuline acĂ©tylĂ©e, nous avons localisĂ© ces dĂ©fauts dans le segment externe et les cĂŽnes de la rĂ©tine.
Afin dâĂ©tudier le rĂŽle possible de POC5 et ADGRG7 dans la SI, nous avons examinĂ© comment POC5 et ADGRG7 est rĂ©gulĂ© au niveau transcriptionnel. Nous avons utilisĂ© des modĂšles cellulaires, des ostĂ©oblastes humains dĂ©rivĂ©es de contrĂŽles et de SIA patient, et nous avons Ă©tudiĂ© lâexpression de la protĂ©ine POC5 et ADGRG7 en rĂ©ponse Ă une stimulation par lâE2. La rĂ©gion promotrice du gĂšne ADGRG7 a Ă©tĂ© clonĂ©e et analysĂ©e pour les Ă©lĂ©ments cis mĂ©diant les effets de lâE2. Les analyses de dĂ©lĂ©tion du promoteur indiquent que le site SP1 dans le fragment 474bp est requis pour une activitĂ© basale ainsi que pour une activation hormone-dĂ©pendante, et des mutations dans les sites de liaison au sein de cette rĂ©gion rĂ©sultent en la perte de transactivation. Les rĂ©sultats dâimmunoprĂ©cipitation de la chromatine (ChIP) ont montrĂ© que le site SP1 ESRα lie le promoteur dâADGRG7. Nos rĂ©sultats suggĂšrent que la rĂ©gulation de lâexpression dâADGRG7 par lâE2 est due Ă lâassociation des protĂ©ines ESRα et SP1 au promoteur dâADGRG7.
La mĂȘme stratĂ©gie expĂ©rimentale a Ă©tĂ© appliquĂ©e pour l'Ă©tude de la rĂ©gulation de POC5 par E2. L'analyse de dĂ©lĂ©tion et ChIP ont confirmĂ© la rĂ©gulation de POC5 Ă travers ESRα. BasĂ© sur des Ă©tudes de promoteur, qPCR, Western blot et ChIP, cette Ă©tude clarifie comment POC5 et ADGRG7 sont rĂ©gulĂ©es par lâE2.
Un autre aspect de ce projet Ă©tait dâĂ©tudier les diffĂ©rents effets du chargement mĂ©canique sur des cellules, incluant des ostĂ©oblastes humains, exprimant POC5 sauvage ou mutĂ©e. Les cellules ont Ă©tĂ© soumises Ă un stress mĂ©canique Ă diffĂ©rents temps, et diffĂ©rentes voies de signalisations (incluant ERK, p38, NFÎșB) ont Ă©tĂ© testĂ©es. Nos rĂ©sultats montrent une diffĂ©rence dans la rĂ©ponse des cellules surexprimant POC5 mutĂ©e par rapport aux cellules contrĂŽles. Les effets du chargement comprenant les diffĂ©rentes voies de signalisations ont Ă©tĂ© montrĂ©s comme Ă©tant initiĂ©s via TRPV4, un canal calcique permĂ©able activĂ© par lâĂ©tirement localisĂ© dans le cil primaire et la membrane plasmique.Scoliosis is a complex three-dimensional deformity of the spine, with 1.5â3% prevalence in the general population. The most commonly known type of scoliosis is idiopathic scoliosis (IS), including adolescent idiopathic scoliosis (AIS) affecting principally girls during puberty. The etiology is largely unknown, but clinical observations revealed the role of hereditary and rapid growth in the development of this condition. There exists strong evidence that there is a genetic component to the disease. More recently, several genes were suspected to cause or contribute to IS. Our group identified gene variants of POC5 centriolar protein in a large French family with multiple members affected with IS. In the same family, we suspected the involvement of ADGRG7 (an orphan receptor that belongs to the Adhesion G protein-coupled receptors) gene mutation in the pathogenicity of IS. In the present work, we focused on elucidating the role of wild type (wt) and mutant (mut) POC5 proteins (in vitro and in vivo) as well as the regulation of POC5 and ADGRG7 by estradiol (E2), with the goal to test whether these genes could be functionally connected with scoliosis.
To investigate the role of POC5 gene in IS, we overexpressed mutant POC5 in cell lines by transient transfection (in vitro study) and created a loss-of-function model in zebrafish (in vivo study). The role of POC5 was investigated by: 1) mass spectroscopy analysis and co-immunoprecipitation to identify differences in binding partners between the wt POC5 and mut POC5 proteins; 2) immunolocalization of wt and mut POC5 proteins at the cellular level; 3) histology and immunohistochemistry performed on tissues from wt (control) and scoliotic (poc5 mut) zebrafish. Our work identified several interacting partners with POC5, and documented functional connections with respect to cilia and centrosome dysfunction. A number of ciliary proteins were identified to be interacting with wt POC5 but not mut POC5 like CEP290, RAB11, CKAP5, Annexin 2 and Septin 9. At the cellular level, localization and co-localisation of wt POC5 and mut POC5 protein with alpha acetylated tubulin (cilia marker), confirmed the consequence of the mutation on subcellular location with respect to cilium length and staining intensity. In vivo, several defects in the retina of zebrafish and inner ear were identified in mutpoc5 zebrafish compared to wt zebrafish. Finally, using different markers for retinal layers and alpha acetylated tubulin, the defects were localized in the outer segment layer and cones of the retina.
To further investigate the possible roles of POC5 and ADGRG7 in IS, we examined how POC5 and ADGRG7 are regulated at the transcriptional level. Human osteoblasts derived from control and AIS patients were used as a cell model, and the expression of POC5 and ADGRG7 protein was monitored upon E2 stimulation. The promoter region of the human POC5 and ADGRG7 gene was then cloned and analyzed for functional cis-elements mediating effects of E2. Deletion analysis of the ADGRG7 promoter indicates that the SP1 site in the 474bp fragment is required for both basal activity and hormone-induced activation, and mutations of the binding sites within this region result in the loss of transactivation. Further results from chromatin immunoprecipitation (ChIP) assay showed that SP1 and ESRα bind to ADGRG7 promoter. Our results suggest that the regulation of ADGRG7 expression by E2 is due to the association of ESRα and SP1 proteins to ADGRG7 promoter. The same experimental strategy was applied for studying the POC5 regulation by E2. Deletion analysis and ChIP confirmed the regulation of POC5 through ESRα. Through promoter studies, qPCR, and western blot and (ChIP) assay, this study clarifies how POC5 and ADGRG7 are regulated by E2.
Another aspect of this project was to study the differential effects of mechanical stress on wt and mut POC5 expressing cells, including human osteoblasts. Cells were exposed to mechanical stress for different time points and then different signalling pathways (including ERK, p38, NFÎșB) were tested. Our results show that there is difference in the response of control normal cells and cells overexpressing the mut POC5. The effects of loading including the signalling pathways were found to be initiated through TRPV4 which is a stretch-activated Ca2+- permeable channel and localizes to the primary cilium and plasma membrane.
The importance of this work is that it covers several factors that contribute to the pathogenesis of AIS. Based on our findings, AIS is a complex multifactorial disease where POC5, cilia, E2 and mechanical load intreplay in the pathogenesis of the disease. The significance of this work is that it puts the basics for understanding the molecular mechanisms that are implicated in AIS
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