Auswahl der wissenschaftlichen Literatur zum Thema „Sh3tc2“

N6-methyladenosine (m6A) modification plays a crucial role in determining the fate and function of RNA transcripts in tumor cells. Nevertheless, how m6A regulates the expression of key molecules and coordinates its involvement in the development of colorectal cancer (CRC) remains largely unclear. Here, we report that the m6A reading protein YTHDF1-mediated up-regulation of SH3TC2 promotes CRC growth both in vitro and in vivo. In a pan-cancer analysis across more than thirty types of cancer, we found that SH3TC2 was dysregulated in nine cancers, including BLCA, CHOL, COAD, LAML, PAAD, READ, SKCM, BRCA, and TGCT, and was closely associated with patient prognosis in four cancers, including COAD, MESO, PAAD, and READ. In particular, SH3TC2 was overexpressed in CRC as confirmed by six independent study cohorts. Clinically, high expression of SH3TC2 predicted worse disease-free survival (DFS) in CRC patients. SH3TC2 showed fascinating diagnostic value and was correlated with immunosuppression in CRC. Functionally, RNA-sequencing combined with experiments revealed that knockdown of SH3TC3 significantly inhibited cell-cycle progress of CRC, impairing cell growth. Mechanistically, YTHDF1 protein directly binds with SH3TC2 mRNA and promotes its elevation in an m6A-dependent manner. Thus, our findings provide a mechanism to target the YTHDF1/SH3TC2 axis for CRC therapy.

SH3 domain and tetrapeptide repeat 2 (SH3TC2) is a protein-encoding gene and has previously been described as a critical signaling hub for neurological disorders. Although increasing evidence supports a vital role of SH3TC2 in the tumorigenesis of various kinds of cancer, no systematic analysis of SH3TC2 is available. The function and mechanism of SH3TC2 in other cancers remain unknown. Thus, this study aimed to analyze SH3TC2 in various kinds of cancer to find its tumorigenic role in one or more specific cancers. In the current study, we analyzed the expression level and prognostic value of SH3TC2 in different tumors in the TCGA-GTEx pan-cancer dataset. Subsequently, the prognostic role and mechanism of SH3TC2 in colorectal cancer (CRC) were further explored via clinical samples and in vitro and in vivo experiments. We observed differential expression of SH3TC2 in colon adenocarcinoma (COAD), acute myeloid leukemia (LAML), READ (rectum adenocarcinoma), SKCM (skin cutaneous melanoma), and TGCT (testicular germ cell tumors). Subsequently, SH3TC2 showed a significant effect on the clinical stage and prognostic value in CRC, LAML, and SKCM. Moreover, we found in the TCGA database and seven GEO datasets that SH3TC2 was significantly highly expressed in tumor tissue. Through enrichment analysis of SH3TC2 and its co-expressed genes, we found that SH3TC2 may play a role in the MAPK signaling pathway. Correlation analysis indicated that SH3TC2 was significantly associated with multiple key factors in the MAPK signaling pathway. Additionally, higher expression of SH3TC2 was found in tumor tissue in our cohort including 40 CRC patients. Overexpression of SH3TC2 may imply poor prognosis. Knockdown of SH3TC2 significantly inhibited tumor invasion, migration, and proliferation. More importantly, knockdown of SH3TC2 inhibited tumor growth in a CRC mouse model. The study preliminarily conducted a pan-cancer study of SH3TC2 and further explored the mechanism of SH3TC2 in CRC. Our research revealed that higher expression of SH3TC2 may promote CRC progression and invasion via the MAPK signaling pathway.

Human induced pluripotent stem cells (hiPSCs) represent a powerful tool to investigate neuropathological disorders in which the cells of interest are inaccessible, such as in the Charcot–Marie–Tooth disease (CMT), the most common inherited peripheral neuropathy. Developing appropriate cellular models becomes crucial in order to both study the disease’s pathophysiology and test new therapeutic approaches. The generation of hiPS cellular models for disorders caused by a single nucleotide variation has been significantly improved following the development of CRISPR-based editing tools. In this study, we efficiently and quickly generated, by CRISPR editing, the two first hiPSCs cellular models carrying alterations involved in CMT4C, also called AR-CMTde-SH3TC2. This subtype of CMT is associated with alterations in the SH3TC2 gene and represents the most prevalent form of autosomal recessive demyelinating CMT. We aimed to develop models for two different SH3TC2 nonsense variants, c.211C>T, p.Gln71* and the most common AR-CMTde-SH3TC2 alteration, c.2860C>T, p.Arg954*. First, in order to determine the best CRISPR strategy to adopt on hiPSCs, we first tested a variety of sgRNAs combined with a selection of recent base editors using the conveniently cultivable and transfectable HEK-293T cell line. The chosen CRISPR base-editing strategy was then applied to hiPSCs derived from healthy individuals to generate isogenic CMT disease models with up to 93% editing efficiency. For point mutation generation, we first recommend to test your strategies on alternative cell line such as HEK-293T before hiPSCs to evaluate a variety of sgRNA-BE combinations, thus boosting the chance of achieving edited cellular clones with the hard-to-culture and to transfect hiPSCs.

The neuromuscular junction (NMJ) appears to be a site of pathology in a number of peripheral nerve diseases. Charcot-Marie-Tooth (CMT) 4C is an autosomal recessive, early onset, demyelinating neuropathy. Numerous mutations in the SH3TC2 gene have been shown to underlie the condition often associated with scoliosis, foot deformities, and reduced nerve conduction velocities. Mice with exon 1 of the Sh3tc2 gene knocked out demonstrate many of the features seen in patients. To determine if NMJ pathology is contributory to the pathomechanisms of CMT4C we examined NMJs in the gastrocnemius muscle of SH3TC2-deficient mice. In addition, we performed proteomic assessment of the sciatic nerve to identify protein factors contributing to the NMJ alterations and the survival of demyelinated axons. Morphological and gene expression analysis of NMJs revealed a lack of continuity between the pre- and post-synaptic apparatus, increases in post-synaptic fragmentation and dispersal, and an increase in expression of the gamma subunit of the acetylcholine receptor. There were no changes in axonal width or the number of axonal inputs to the NMJ. Proteome investigations of the sciatic nerve revealed altered expression of extracellular matrix proteins important for NMJ integrity. Together these observations suggest that CMT4C pathology includes a compromised NMJ even in the absence of changes to the innervating axon.

Next-generation sequencing (NGS) allows the detection of plentiful mutations increasing the rate of patients getting a positive diagnosis. However, while single-nucleotide variants (SNVs) or small indels can be easily detected, structural variations (SVs) such as copy number variants (CNVs) are often not researched. In Charcot–Marie–Tooth disease (CMT), the most common hereditary peripheral neuropathy, the PMP22-duplication was the first variation detected. Since then, more than 90 other genes have been associated with CMT, with point mutations or small indels mostly described. Herein, we present a personalized approach we performed to obtain a positive diagnosis of a patient suffering from demyelinating CMT. His NGS data were aligned to the human reference sequence but also studied using the CovCopCan software, designed to detect large CNVs. This approach allowed the detection of only one mutation in SH3TC2, the frequent p.Arg954*, while SH3TC2 is known to be responsible for autosomal recessive demyelinating CMT forms. Interestingly, by modifying the standard CovCopCan use, we detected the second mutation of this patient corresponding to a 922 bp deletion in SH3TC2 (Chr5:148,390,609-Chr5:148,389,687), including only one exon (exon 14). This highlights that SVs, different from PMP22 duplication, can be responsible for peripheral neuropathy and should be searched systematically. This approach could also be employed to improve the diagnosis of all inherited diseases.

Charcot–Marie–Tooth disease type 4C (CMT4C) is an autosomal recessive neuropathy associated with SH3TC2 mutations, resulting in slow conduction velocity via hypomyelination. The occurrence of CMT4C in demyelinating Charcot–Marie–Tooth (CMT) varies among ethnicities, and several variants have been reported as the founder mutation. In Korea, the incidence of CMT4C was calculated as approximately 2%, and all patients have compound heterozygous mutations, which is partly due to the prohibition of consanguineous marriage. Herein, we describe a 25-year-old male who presented a slowly progressive limb weakness and impaired vibration sensation. Whole-exome sequencing revealed homozygous variants c.929G>A of SH3TC2 after identifying negative multiplex ligation-dependent probe amplification results of PMP22. Based on our literature review, this is the first CMT4C patient with a homozygous variant with each allele inherited from both the parents.

Solitary CNS Lymphomatoid granulomatosis (LG), a rare lymphoproliferative disorder, was discovered in a patient collecting rare neurological disorders.A patient diagnosed with Charcot-Marie-Tooth type 4C (SH3CT2) and Myasthenia Gravis, treated with mycophenolate, presented with musical hallucinosis, described as choral music occurring in short bursts. Investigations deemed this secondary to hearing loss. Neuro-imaging revealed a cerebellar cavernoma and a developmental venous anomaly, thought to be incidental. A rapidly expanding, right frontal lesion was noted on subsequent imaging. Biopsy of this lesion revealed a florid inflammatory infiltrate composed of small lymphocytes, macrophages and non-caseating granulomata. Immunohistochemical staining for CD20 showed moderate numbers of B-lymphocytes, positive for Epstein Barr virus-encoded small RNA, suggesting a diagnosis of LG.Immunosupression is a risk factor for EBV mediated lymphoproliferative disorders. SH3TC2 is involved in myelination and maintenance of the node of Ranvier and mutations disturb interactions with the GTPase Rab11. Outside of the peripheral nervous system mutated Rab11 molecules are known to have neoplastic potential through prevention of the normal function of binding the oncogene Evi5, potentially explaining the development of LG as another rare disorder. Despite tenuous links, these rare neurological disorders appear unrelated in this individual.

The implementation of NGS methods into clinical practice allowed researchers effectively to establish the molecular cause of a disorder in cases of a genetically heterogeneous pathology. In cases of several potentially causative variants, we need additional analysis that can help in choosing a proper causative variant. In the current study, we described a family case of hereditary motor and sensory neuropathy (HMSN) type 1 (Charcot–Marie–Tooth disease). DNA analysis revealed two variants in the SH3TC2 gene (c.279G>A and c.1177+5G>A), as well as a previously described variant c.449−9C>T in the MPZ gene, in a heterozygous state. This family segregation study was incomplete because of the proband’s father's unavailability. To evaluate the variants’ pathogenicity, minigene splicing assay was carried out. This study showed no effect of the MPZ variant on splicing, but the c.1177+5G>A variant in the SH3TC2 gene leads to the retention of 122 nucleotides from intron 10 in the RNA sequence, causing a frameshift and an occurrence of a premature stop codon (NP_078853.2:p.Ala393GlyfsTer2).

Charcot-Marie-Tooth disease type 4C, an autosomal recessive genetic neuropathy, is caused by mutations in the SH3TC2 (SH3 domain and tetratricopeptide repeats 2) gene. Interestingly, although mutations in this gene have been observed in European gypsies, a population that originated in India, there are few publications describing Indian patients. We report our analysis of a 50-year-old woman of Asian Indian descent with onset of progressive distal weakness and sensory loss in childhood. A clinical examination revealed the presence of a neuropathy with pes cavus without spinal abnormalities. Electrophysiological testing confirmed a sensorimotor length-dependent neuropathy with demyelinating features. A genetic analysis revealed she carries 2 novel mutations, c.2488G>T variant (rs879254317) and c.731+5G>A variant (rs879254316), in the SH3TC2 gene. Further genetic testing demonstrated that her son is a carrier of the c.731+5G>A mutation. Our analysis confirms that this patient is a compound heterozygote inheriting these mutations, which are in trans, in an autosomal recessive pattern. Her son developed an episode of sciatic neuropathy with complete resolution. We hypothesize that in his case, haploinsufficiency caused by c.731+5G>A mutation may have predisposed him to the development of this focal neuropathy.

La maladie de Charcot-Marie-Tooth (CMT) est la neuropathie périphérique héréditaire la plus fréquente chez l’humain. Elle touche les motoneurones (MN) et les cellules de Schwann (CS). La majorité des gènes impliqués, dont SH3TC2 et GDAP1, peuvent être affectés par des mutations non-sens. En 2021, peu de modèles cellulaires humains existaient, et aucun traitement curatif n'était disponible pour les patients. Les travaux de cette thèse se centre sur SH3TC2, responsable de la forme démyélinisante autosomique récessive la plus fréquente des CMT, nommée CMT4C ou AR-CMTde-SH3TC2 et sur GDAP1 notamment responsable d’une forme axonale AR-CMTax-GDAP1. Dans un premier temps, nous avons analysé une cohorte de 103 patients mutés sur SH3TC2 et montré que plus de 80 % des patients possédaient au moins un allèle avec une mutation non-sens, associé à une gravité clinique accrue. Nous avons également identifié 22 nouvelles mutations pathogènes sur ce gène. La seconde partie de ce travail a consisté à créer les premiers modèles cellulaires neuronaux humains pour SH3TC2. À partir de cellules souches pluripotentes induites (hiPSCs) issues d’un individu contrôle, nous avons utilisé la technologie CRISPR-Cas9 pour produire, avec plus de 90% d’efficacité, deux modèles humains in vitro contenant des mutations non-sens induisant un codon stop prématuré (PTC) : un modèle homozygote p.(Arg954*) (PTC de type UGA) et un modèle homozygote p.(Gln71*) (PTC de type UAG). Ces hiPSCs contrôle et mutées ont ensuite été différenciées en CS. Nous avons mis en évidence une expression précoce de SH3TC2 dans les CS contrôle. Dans les modèles CS AR-CMTde-SH3TC2, une expression réduite de SH3TC2, un retard de maturation, une capacité réduite à soutenir les MN en coculture, et des anomalies dans le recyclage des récepteurs à la transferrine ont été observées. Enfin, nous avons testé plusieurs molécules thérapeutiques ciblant les mutations non-sens, des agents de translecture et des inhibiteurs du mécanisme de surveillance des ARN non-sens (NMDi). Sur un modèle de progéniteurs neuronaux dérivés d’hiPSCs portant la mutation homozygote non-sens p.(Ser194*) (UGA) sur GDAP1, nous avons testé une de ces molécules et montré qu’elle stabilisait l'ARNm muté GDAP1, restaurait son expression protéique et corrigeait la morphologie mitochondriale. Dans les modèles CS créés dans cette thèse pour SH3TC2, nos premiers résultats suggèrent l’effet positif de deux de ces molécules sur la réexpression de la protéine pour les deux types de codons UGA et UAG. Dans la quatrième partie de ce travail, nous avons développé un modèle 3D de coculture CS/MN permettant d’induire la myélinisation, étape ultime pour étudier les maladies démyélinisantes comme l’AR-CMTde-SH3TC2. Les molécules thérapeutiques identifiées pourront être testées sur ces modèles cellulaires de coculture et potentiellement in vivo pour évaluer leur capacité à induire une remyélinisation. Ce travail de thèse souligne l'importance des modèles cellulaires adaptés pour comprendre les mécanismes physiopathologiques de la CMT et ouvre des perspectives prometteuses pour de nouvelles approches thérapeutiques
Charcot-Marie-Tooth disease (CMT) is the most common hereditary peripheral neuropathy in humans. It affects motor neurons (MNs) and Schwann cells (SCs). Most of the genes involved, such as SH3TC2 and GDAP1, can be affected by nonsense mutations. As of 2021, few human cellular models existed, and no curative treatment was available for patients. This thesis primarily focuses on SH3TC2, responsible for the most common autosomal recessive demyelinating form of CMT, known as CMT4C or AR-CMTde-SH3TC2, and on GDAP1, notably responsible for an axonal form, AR-CMTax-GDAP1. In the first part of this work, we analyzed a cohort of 103 patients with SH3TC2 mutations and demonstrated that more than 80% of the patients carried at least one allele with a nonsense mutation, associated with increased clinical severity. We also identified 22 new pathogenic mutations in this gene. The second part of my work involved creating the first human neuronal cell models for SH3TC2. Using induced pluripotent stem cells (hiPSCs) derived from a control individual, we employed CRISPR-Cas9 technology to generate, with over 90% efficiency, two in vitro human models containing nonsense mutations inducing a premature stop codon (PTC): a homozygous p.(Arg954*) model (UGA-type PTC) and a homozygous p.(Gln71*) model (UAG-type PTC). These controls and mutated hiPSCs were then differentiated into Schwann cells (SCs). We observed early SH3TC2 expression in control SCs. In AR-CMTde-SH3TC2 SC models, reduced SH3TC2 expression, delayed maturation, impaired ability to support MNs in co-culture, and abnormalities in transferrin receptor recycling were noted. Finally, we tested several therapeutic molecules targeting nonsense mutations, including readthrough agents and inhibitors of nonsense-mediated mRNA decay (NMDi). In a model of neuronal progenitors derived from hiPSCs carrying the homozygous nonsense mutation p.(Ser194*) (UGA) on GDAP1, we tested one of these molecules and demonstrated that it stabilizes the mutated GDAP1 mRNA, restores its protein expression, and corrects mitochondrial morphology. In the SC models created in this thesis for SH3TC2, our early results suggest a positive effect of two of these molecules on protein re-expression for both UGA and UAG codons. In the fourth part of this work, we developed a 3D co-culture model of SCs/MNs that enables myelination, the ultimate step to studying demyelinating diseases such as AR-CMTde-SH3TC2. The identified therapeutic molecules can be tested on these co-culture cellular models and potentially in vivo to evaluate their capacity to induce remyelination. This thesis highlights the importance of appropriate cellular models to understand the pathophysiological mechanisms of CMT and opens promising perspectives for new therapeutic approaches

Lymphocyte antigen receptor engagement triggers activation of various signaling cascades, initiated by sequential activation of the Src and Syk families of protein tyrosine kinases, and leading to gene transcription. Although they lack both enzymatic and transcriptional activity, adaptor proteins are crucial for this process. The cytoplasmic adaptor protein 3BP2 contains a PH domain, proline–rich regions, and a SH2 domain, and interacts with Syk kinases, the LAT adaptor and the PLCg. 3BP2 plays a positive role in NK cytotoxicity, in basophilic degranulation, and activation of NFAT and AP-1 transcription factors leading to interleukine-2 gene transcription in T cells. Finally, point mutations in 3bp2 gene leads to cherubism, a human pathology related to bone development. 3BP2 thus seems to have an important function in hematopoietic cells. In order to better understand its role in lymphocytes, we looked for and identified novel 3BP2 interacting partners. Among those are essential proteins for lymphocyte signaling like HIP-55 and CIN85 and nucleotidic exchange factors Vav1 and Vav2. We showed that 3BP2 interacts with SH3 domain of HIP-55 or CIN85 by the different proline rich domains, and that the interaction is required for endocytosis and regulation of actin. Moreover, our data indicate that the basal interaction between 3BP2 and Vav family members, mediated by the first proline-rich domain of 3BP2 and a SH3 domain of Vav, is reinforced in a phosphotyrosine-dependent way upon immunoreceptors stimulation in T and B cells. We show that there is a functional cooperation between 3BP2 and Vav family members in small GTPase Rac and NFAT transcription activation in lymphocytes. Finally, a differential exression of 3BP3 is observed in Th1 type lymphocytes. And also, the retroviral overexpression of 3BP2 in primary T lymphocytes accelerates the Th1 type cytokines synthesis and the activation induced cell death. These results suggest 3BP2’s implication in T lymphocyte differentiation and its role in the regulation of cell survival. These results suggest that 3BP2 allows the formation of various multimolecular complexes, and might take part in the regulation of different signaling pathway activated in lymphocytes during immune response
L’engagement des récepteurs à l’antigène des lymphocytes déclenche l’activation de différentes cascades de signalisation, initiées par l’activation séquentielle des protéines tyrosine kinases des familles Src et Syk, et aboutissant à la transcription de gènes. Les protéines adaptatrices, pourtant dépourvues d’activité enzymatique ou transcriptionnelle, sont cruciales dans ce processus. La protéine adaptatrice cytoplasmique 3BP2 est constituée d’un domaine PH, de régions riches en proline et d’un domaine SH2, et interagit avec les protéines kinases Syk, l’adaptateur LAT et la PLCg. 3BP2 joue un rôle positif dans la cytotoxicité des cellules NK, dans la dégranulation des mastocytes, et dans l’activation des facteurs de transcription NFAT et AP-1 conduisant à la transcription du gène de l’interleukine-2 dans les cellules T. Enfin, des mutations ponctuelles dans le gène 3bp2 sont à l’origine de la pathologie humaine du chérubisme qui a trait au développement osseux. 3BP2 semble donc posséder une fonction importante dans les cellules du système hématopoïétique. Afin de mieux comprendre son rôle dans les lymphocytes, nous avons recherché et identifié de nouveaux partenaires d’interaction de 3BP2. Parmi ceux-ci se trouvent des protéines essentielles pour la signalisation lymphocytaire comme les protéines HIP-55 et CIN85 et les facteurs d’échange nucléotidiques Vav1 et Vav2. Nous avons montré que 3BP2 interagit directement avec le domaine SH3 de CIN85 ou de HIP-55, qui sont impliqués dans l’endocytose et la régulation d’actine, par les différents domaines riches en proline. D’autre part, nos travaux indiquent que l’interaction basale entre 3BP2 et les membres de la famille Vav, médiée par le premier domaine riche en proline de 3BP2 et un domaine SH3 de Vav, est renforcée de façon phosphotyrosine-dépendante après stimulation des immunorécepteurs des cellules T et B. Cette interaction physique se double d’une interaction fonctionnelle, puisque nous avons montré que 3BP2 coopère avec les membres de la famille Vav pour activer la petite GTPase Rac, la transcription de NFAT dans les lymphocytes. Enfin, une expression différentielle de 3BP2 dans les lymphocytes de type Th1 a été observée. Par ailleurs, la surexpression rétrovirale de 3BP2 dans les lymphocytes T primaires accélère la synthèse des cytokines de type Th1, la mort cellulaire induite par l’activation. Ces résultats suggèrent une implication de 3BP2 au cours de différenciation de lymphocyte T et un rôle dans la régulation de la survie. Ces résultats indiquent que 3BP2, via l’assemblage d’édifices multimoléculaires variés, participe à la régulation des voies de signalisation des lymphocytes pour la réponse immune

Les ostéoclastes sont des cellules multinucléées capables de résorber le tissu osseux, elles se différencient à partir de la lignée hématopoïétique en présence de Receptor Activator of Nuclear Factor NF-kB (RANK-L) et M-CSF (Macrophage-Colony Stimulating Factor). Les récepteurs RANK et M-CSFR relaient et amplifient le signal de leurs ligands en activant de multiples voies de signalisation intracellulaire dont l’intégration aboutira à l’intégration de NFATc1, facteur de transcription essentiel pour la différenciation des ostéoclastes. Les protéines adaptatrices, du fait de leur structure, jouent un rôle crucial dans cette signalisation. De nombreuses études ont montré que la protéine adaptatrice 3BP2/ SH3BP2, initialement identifiée comme une protéine interagissant avec la kinase c-Abl puis comme partenaire des kinases de la famille Src et Syk, joue un rôle important dans la signalisation et l’activation des leucocytes. Des études génétiques ont montré que des mutations du gène 3bp2 chez l’homme sont associées à une dysplasie osseuse génétique « Chérubinisme » et à un phénotype ostéopénique inflammatoire chez la souris. Ces observations laissent entrevoir un rôle additionnel de 3BP2 dans la régulation des cellules du système osseux et particulièrement la différenciation des ostéoclastes. Dans le but d’étudier le rôle de 3BP2 dans la différenciation des ostéoclastes, nous avons utilisé la lignée RAW264. 7, une lignée myélo-monocytaire murine capable de se différencier en ostéoclaste en présence de RANK-L. Avec la méthode d’interférence ARN, nous avons développé des modèles cellulaires « perte de fonction » n’exprimant plus 3BP2. Ces modèles nous ont permis de montrer que l’expression de 3BP2 est essentielle pour la différenciation des ostéoclastes et que son absence altère la différenciation de ces cellules en ostéoclastes. Nous avons ainsi montré que l’effet de l’absence de 3BP2 est restreint à la voie de signalisation RANK sans aucune conséquence sur la voie de signalisation GM-CSF et la différenciation des cellules dendritiques. Dans un premier temps, nous avons montré que cet effet « perte de fonction » est lié à un défaut de polymérisation d’actine et d’activation de la protéine tyrosine kinase Src et des voies de signalisation MAPKs (MEK, ERK, JNK) contrôlées par RANK-L. Nous avons observé également une inhibition de l’induction de NFATc1 et AP-1, des facteurs de transcription essentiels à la différenciation des ostéoclastes. Par la suite, l’analyse transcriptionnelle de différentes protéines impliquées dans la différenciation des ostéoclastes (CaR, TRAP, Cathépsine K) a révélée une inhibition significative de leur induction dans les cellules déficients en 3BP2. Nous avons finalement montré que la reconstruction avec des molécules Src et NFATc1 constitutivement actives restaure la différenciation des ostéoclastes dans les cellules déficients en 3BP2. En conclusion, notre étude a montré que la protéine 3BP2, via la protéine tyrosine kinase Src, joue un rôle central au cours de la différenciation des ostéoclastes en contrôlant les voies de signalisation RANK, impliquées dans l’activation de NFATc1, facteur de transcription clé de la différenciation ostéoclastique
Osteoclasts are multinucleated bone-resorbing cells, which derived from hematopoietic cells of the monocyte/macrophage lineage following stimulation with two essential cytokines, RANK-L and M-CSF. The molecular pathways involved in osteoclast formation involve complex network of signaling molecules, including adaptor proteins kinases, which ultimately lead to the activation of a transcriptional program in which NFATc1 plays a pivotal role. The adaptor protein 3BP2, originally identified as a c-Abl binding protein, and a partner of Src and Syk kinases families, has been involved in leucocytes signaling and activation? Genetic studies have further associated mutations of the 3BP2 gene of the human bone disease Cherubism and to inflammation and bone dysfunction in mouse. However, how wild-type 3BP2 exactly functions in osteoclast differentiation has yet been elucidated. In this study, we have investigated the role of endogenous 3BP2 exactly functions in osteoclast differentiation using siRNA-mediated silencing of 3BP2 expression in the RAW264. 7 monocyte/macrophage cell line. We show here that 3BP2 was required for RANK-L-induced differentiation of RAW264. 7 cells was associated with reduced RANK-L-induced actin reorganization and Src, ERK, JNK, IKKα/β, but not p38 phosphorylation. Following RANK-L stimulation, the 3BP2-deficient cells exhibited impaired up-regulation of Src, c-fos and NFATC1 mRNA expression, whereas NFATc2 and NFATc3messengers were not significantly affected. Compared to control cells, 3BP2-knockdown cells induced to osteoclast by RANK-L displayed no up-regulation of Src and NFATc1 proteins? In addition, the introduction of constitutively active mutants of Src and NAFTc1 in 3NP2 deficient cells restored osteoclast differentiation. Finally, we provide evidence that enhanced osteoclast differentiation triggered by a 3BP2 Cherubism mutant also required NFAT activity in RAW264. 7 cells. Together, this study demonstrates that 3BP2 is a key regulator of RANK-mediated osteoclastogenesis through Src and NFATc1 activation

Afin de mieux comprendre les bases physiopathologiques des tumeurs osseuses des mâchoires, nous avons étudié deux modèles de tumeurs associées à des mutations génétiques connues : la tumeur kératokystique odontogène (TKO), liée à la mutation de PTCH1, et le chérubinisme, lié à la mutation de SH3BP2. Au regard des travaux d’oncogénétique, nous formulons l’hypothèse que le développement des tumeurs ostéolytiques bénignes des mâchoires de l’enfant et leur agressivité repose sur un mécanisme génétique. Nous avons montré que la présence d’une mutation de PTCH1 (germinale avec syndrome de Gorlin) dans les TKO était un facteur de mauvais pronostic, stimulant un centre tumoral secondaire, responsable de lésions à distance, mais que cette agressivité pouvait aussi être liée à des mécanismes inflammatoires. Dans le chérubinisme, nous avons montré que la mutation était responsable du phénotype, mais que le type de mutation n’influençait pas le pronostic ni l’agressivité. L’agressivité tumorale est liée au phénotype des cellules géantes multinucléées (cellules myéloïdes à différenciation macrophagique ou ostéoclastique). Nous avons montré, que le modèle murin ne pouvait pas s’appliquer à la pathologie humaine, avec notamment un rôle très secondaire du TNF-α. Enfin nous avons démontré le rôle important de NFATc1 dans la physiopathologie du chérubinisme qui nous a permis de proposer, le tacrolimus, comme le premier agent thérapeutique efficace. Nos résultats suggèrent que les mutations induisent la pathologie et que les changements du microenvironnement (liés à la flore buccale ou à l’éruption dentaire) entretiennent la pathologie
To determine pathophysiological bases of jawbone tumors, we studied two genetic models of jawbone tumors: keratocystic odontogenic tumors (KOT) associated to PTCH1 mutation and cherubism associated to SH3BP2 mutation. From oncogenetic theory, we postulate that genetic background controls the development of benign children jawbone tumors. From our work, we demonstrated that PTCH1 mutation (germline mutation in Gorlin syndrome) was an unfavorable prognosis factor for KOT, leading to distant and independent daughter tumors. Moreover, we showed, that chorionic inflammation was associated with a high recurrence rate. In cherubism, SH3BP2 mutation produced cherubism phenotype, but the type of mutation did not affect the aggressiveness of the disease. Cherubism aggressiveness was determined by the phenotype of giant multinucleated cells (whether osteoclasts or macrophages). Furthermore, we showed that murine model could not be transposed to human pathology; indeed it appeared that TNF- α did not play a critical role in human cherubism. On the other side, we showed that NFATc1 played a crucial role in cherubism pathophysiology; this observation allowed us to propose, the tacrolimus, as an effective treatment for this disease. Our results suggest that genetic background induced tumor development, and that microenvironment changes (due to flora of the oral cavity and to teeth eruptions) are responsible to the maintenance and the progression of the disease

My PhD thesis can be devided into two parts: 1. Hereditary motor-sensory neuropathies (HMSN) 2. Selected muscle disorders The main emphasis was on the first part - hereditary motor and sensory neuropathies. Research was focused on autosomal recessive forms - demyelinating type CMT4C and axonal type CMT2B1. Most of the results obtained are related to these disorders. Data, which were obtained, are unique and were published in international journals with impact factor. Results obtained from CMT4C study are accepted for publication in Clinical Genetics. Results obtained in LMNA study (CMT2B1) were published in Journal of Human Genetics. The author performed and validated these new methods and original results, which are due to be used in genetic molecular testing of patients with hereditary neuropathies and muscle disorders: 1. Sequencing of all coding exons of the SH3TC2 gene. First mutations in the SH3TC2 gene in Czech HMSN I patients were found. 2. The prevalent mutation among Czech CMT4C patients was proven to be p.Arg954Stop. 3. Real-time PCR assay targeted at detection of the prevalent mutation p.Arg954Stop in the SH3TC2 gene was validated and is now used in our lab on a daily basis as a quick and efficient screening. 4. Molecular genetic testing of the SH3TC2 gene was introduced into the routine...

碩士
國立臺北科技大學
土木與防災研究所
100
The step-by-step integration is the most frequently adopted way to obtain the dynamic responses of complex nonlinear structure system. The major topic of this study is to develop a new integration method which can have computational efficiency, unconditional stability, and favorable numerical dissipation, which can accurately integrate the low frequency modes while it can effectively filter out the spurious participation of high frequency modes. Some numerical examples and actual pseudodynamic tests were conducted to confirm the numerical properties of the proposed integration method, especially the characteristics of favorable numerical dissipation.

INTRODUCTION: Bladder cancer (BC) is highly prevalent. It presents as either non-muscle invasive or muscle-invasive disease. The prognosis of muscle invasive disease is poor, with a 5-year survival rate of less than 50%. Treatment approaches for both types of BC have not advanced much in the last few years and new therapies are needed to overcome the large burden of BC. Recently, a large effort has been undertaken to classify BC into molecular subtypes. These analyses have revealed significant alterations in epigenetic modifiers in BC. A previous study from our group revealed that SH3GL2, a negative regulator of receptor tyrosine kinase (RTK) signaling, was lost with high frequency in BC, leading to increased growth of tumor cells in-vitro and in-vivo. Conversely, forced expression of SH3GL2 in BC cell lines attenuated oncogenic behaviors including growth and migration. In addition to genomic deletion, SH3GL2 is subject to methylation-induced silencing, a key epigenetic mechanism. OBJECTIVE: Epigenetic mechanisms of gene regulation are known to be perturbed in BC. The objectives of this study were to investigate methylation of the SH3GL2 promoter and to test whether agents that promote Deoxyribonucleic acid (DNA) demethylation could be used to re-express SH3GL2 thereby restoring regulation of RTK signaling. METHODS: Methylation of a specific CpG island in the SH3GL2 promoter was analyzed using methylation-specific Polymerase Chain Reaction (PCR) in a panel of BC cell lines with known SH3GL2 messenger Ribonucleic Acid (mRNA) status. Selected BC cell lines were treated with a variety of demethylating agents at different doses and for different times to evoke the re-expression of silenced SH3GL2. Demethylation inhibitors were combined with the histone deacetylase inhibitor, trichostatin A (TSA), to determine whether further re-expression could be achieved. RESULTS: The SH3GL2 promoter displayed differing extents of promoter methylation among cell lines examined. In RT4 cells, the only cell line with detectable expression of SH3GL2 mRNA and protein, the promoter was completely unmethylated. In contrast, T24 and 253J cells displayed significant promoter methylation with little to no SH3GL2 mRNA expressed, consistent with methylation-induced silencing. Treatment of T24 and 253J with 5-Aza-2’-deoxycytidine (5-Aza-dC, 20 M), a DNA methyltransferase (DNMT) inhibitor increased gene expression but this was not dose- or time-dependent. Two additional DNMT inhibitors, Zebularine and RG-108 were also tested. A much higher dosage of Zebularine was required to trigger activation (500 M) while RG-108 was unable to trigger gene reactivation at all. Combination treatment with 5-Aza-dC and TSA further increased SH3GL2 expression compared to either agent alone. These results suggest that DNA methyltransferase inhibition is an effective treatment to re-express SH3GL2 in cells with SH3GL2 promoter silencing. CONCLUSION: The present study shows silencing of SH3GL2 in a variety of BC cell lines as a consequence of DNA promoter hypermethylation. Treatment with demethylating agents was able to increase gene expression. Based on prior findings showing attenuation of tumor cell growth and migration with forced expression of SH3GL2, DNA methyltransferase inhibition represents an effective strategy to re-express SH3GL2 in BC and normalize tumor cell behavior.
2020-07-03T00:00:00Z

Introduction: The etiology of sporadic peripheral neuropathy (PN) remains unclear in a significant proportion of patients. In European and North American surveys, genetic causes have been increasingly recognized in these patients. Little is known about Latin American populations. Objective: We assessed the diagnostic yield of a comprehensive next generation sequencing (NGS) panel in a cohort of Brazilian patients with PN without family history. Potential predictors of genetic etiology in this group were further explored. Methods: We evaluated 41 consecutive adult patients regularly followed with PN and 1. Negative family history of PN or other neurological disorders and 2. Unclear etiology despite extensive laboratorial/neurophysiological (Nerve Conduction Study+Electromyography [NCS+EMG]) workup. All patients then underwent genetic testing using a comprehensive (NGS) panel that included 72 genes known to cause PN. Only pathogenic or likely pathogenic variants according to American College of Medical Genetics and Genomics criteria were retrieved. For each subject, we recorded demographic, clinical and NCS+EMG data. Such variables were then compared between positive vs negative NGS subgroups using Fisher exact test (P < 0.05). Results: Fifteen patients had diagnostic NGS results (8 men, median age = 43 years old), whereas 26 patients (15 men, median age = 47 years old) had negative or unconclusive results. Diagnostic yield of the PN panel was 15/41 = 36.5%. Seventeen distinct variants were found in 5 different genes; PMP22 and SH3TC2 were the most frequently identified. Earlier age of PN onset (P = 0.013), demyelinating pattern on NCS+EMG (P = 0.021) and presence of distal atrophy on clinical examination (P= 0.043) were associated with positive NGS results. Conclusion: A significant proportion of Brazilian patients with sporadic PN has genetic etiology. NGS emerges as a diagnostically useful tool for idiopathic PN, particularly when there is earlier age at onset and demyelinating features.