No description available.

A la suite d’un criblage in silico nous avons identifié un nouveau gène codant pour une protéine à domaine BTB-POZ, XBTBD6. Nous avons déterminé que la protéine XBTBD6 est une protéine cytoplasmique. Dans les cellules Hela, CHO, U2OS et COS7 la protéine XBTBD6 est localisée dans des corpuscules cytoplasmiques, localisation similaire à celle des protéines XBTBD3, HBTBD1 et HBTBD2. Nous avons observé que la partie N-terminale de la protéine, contenant le domaine BTB-POZ, est localisée dans la cellule comme la protéine entière ; par contre la partie C-terminale est exclusivement nucléaire. De plus, nous avons observé que XBTBD6 est localisée de façon diffuse dans le cytoplasme des cellules Neuro2A, 9L et 518A2e. Nous avons montré que la protéine XBTBD6 homodimérise et hétérodimérise avec XBTBD3 et XBTBD2 et qu’elle interagit avec l’ubiquitine ligase E3 XCullin 3. L’ensemble de ces interactions nécessite la présence du domaine BTB-POZ. Ces données montrent que les protéines BTBD6, BTBD3, BTBD1 et BTBD2 possèdent des propriétés communes indiquant qu’elles appartiennent à un sous groupe de la famille des protéines à domaine BTB-POZ. Le profil d’expression a été analysé par la technique de protection à la RNAse et par hybridation in situ. Les résultats montrent que ce gène est fortement exprimé dans le système nerveux adulte et embryonnaire. Des expériences de surexpression par micro-injection d’ARNm ont permis de placer le gène XBTBD6 dans la cascade d’activation des gènes proneuraux en aval de XNgnr-1, XNeuroD, Xath3 et Xebf3. Ces résultats montrent que XBTBD6 est un marqueur neuronal chez le xénope. Au cours de l’étude de la fonction du gène XBTBD6, nous avons montré que la surexpression et la perte de fonction de ce gène dans l’embryon de xénope n’induit pas de variation du nombre de neurones dans la plaque neurale. Par contre nous avons observé que la surexpression du gène XBTBD6 dans des cellules Neuro2A en différentiation régule négativement la croissance des neurites. Nous avons élaboré un modèle de fonctionnement biochimique hypothétique où la protéine XBTBD6 fonctionnerait comme protéine adaptatrice dans un complexe d’ubiquitination permettant l’ubiquitination d’une protéine cible. Nous avons recherché les partenaires potentiels de XBTBD6 en utilisant la technique du double hybride en levure mais sans y parvenir.

No description available.

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes bibliographical references (leaves 126-136). Also available online.

The glial cell-line derived neurotrophic factor (GDNF) and neurturin (NTN) belong to a structurally related family of neurotrophic factors. NTN exerts its effect through a multi-component receptor system consisting of the GDNF family receptor alpha 2 (GFRα2), proto-oncogene RET and/or NCAM. GFRα2 is spliced into at least three isoforms, GFRα2a, GFRα2b and GFRα2c. The present study investigated the expression and functional differences of GFRα2 isoforms. These receptor isoforms are differentially expressed in specific human brain regions. Using Neuro2A model, GDNF and NTN promote neurite outgrowth via GFRα2a and GFRα2c, but not GFRα2b. These GFRα2 isoforms regulate different early response genes when stimulated with GDNF and NTN. Interestingly, using co-expression models, GFRα2b inhibits ligand induced neurites outgrowth of GFRα2a and GFRα2c, and also the related receptor, GFRα1a. More intriguingly, ligands activated GFRα2b was also able to attenuate neurite extension induced by an unrelated stimulation using retinoic acid. MAPK activation induced by GDNF was not attenuated by GFRα2b in a co-expression model, while the early response genes expression profile (up-regulation of FosB) was similar to that induced by GFRα2b alone. This study suggest that GFRα2b is not merely a dominant negative isoform, but signals through a yet to be determined mechanism to antagonize and inhibit neuritogenesis. Together, these data suggest a new paradigm for the regulation of growth factor signaling and neurite outgrowth via an inhibitory splice variant of the receptor. / Singapore-MIT Alliance (SMA)

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Nos Ãltimos anos, a prevalÃncia da hansenÃase no Brasil vem sendo reduzida, entretanto, o nÃmero de pessoas com incapacidades resultantes de neurite crÃnica continua alto. O tratamento padrÃo da neurite hansÃnica se constitui de corticoterapia oral. Em casos resistentes à corticoterapia, pode ser realizada intervenÃÃo cirÃrgica descompressiva, denominada neurolise. Dados sobre a efetividade da neurolise sÃo escassos na literatura. Com o objetivo de avaliar o dÃficit sensitivo-motor antes e apÃs neurolise, realizou-se um estudo retrospectivo. Foram incluÃdos portadores de hansenÃase submetidos à neurolise na referÃncia estadual de RondÃnia, nos anos de 2000 a 2003. Dados sÃcio-demogrÃficos e clÃnicos foram coletados do livro de registro de cirurgias do hospital, dos prontuÃrios e da base de dados do Sistema Nacional de Agravos de NotificaÃÃo. Para avaliar o grau de comprometimento sensitivo e motor, foram criados escores ordinais baseadas nas avaliaÃÃes clÃnicas . Dos 118 indivÃduos incluÃdos, que foram submetidos a 297 neurolises, 74 (62,7%) eram do sexo masculino, noventa e seis (81,4%) foram classificados como portadores da forma clÃnica dimorfa. Somente (53,4%) estavam em tratamento com poliquimioterapia no momento da cirurgia. A mediana do tempo entre o primeiro episÃdio de neurite e a neurolise foi de um ano (mÃximo = 12,3 anos). A mediana do escore semi-quantitativo sensitivo diminuiu de 5 para 3 (nervo ulnar), de 3 para 2 (nervo mediano), e de 9,5 para 7,5 (nervo tibial posterior; todos p<0,001). Noventa por cento dos pacientes com dÃficit sensitivo grave do nervo ulnar antes da cirurgia apresentaram um escore melhor apÃs a cirurgia. Na anÃlise, um escore sensitivo alto do nervo ulnar (OR ajustada = 1,9; IC 95%: 1,37 â 2,65; p<0,001) e do nervo tibial posterior antes da cirurgia (OR ajustada = 1,2; IC 95%: 1,02 â 1,39; p = 0,02), aumentaram a chance da melhora. O tempo entre o primeiro perÃodo de neurite e a cirurgia nÃo modificou o resultado clÃnico de forma significativa. Similar à avaliaÃÃo sensitiva, o escore motor dos nervos ulnar e fibular melhorou significativamente (ulnar: p = 0,03; fibular: p<0,001). Quase 60% dos pacientes submetidos à neurolise no nervo fibular apresentaram maior forÃa muscular apÃs a cirurgia. Os dados do estudo indicam que a neurolise à benÃfica em casos de neurite hansÃnica, mesmo apÃs um perÃodo prolongado de neurite. Faz-se necessÃrio elaborar estratÃgias para aperfeiÃoar a terapia da neurite hansÃnica com Ãnfase no monitoramento da funÃÃo neural. / In Brazil, the prevalence of leprosy has been reduced in the last years. However, there are still many people living with disabilities, resulting from chronic leprosy neuritis. Oral corticosteroid therapy is the standard treatment of neuritis. In case of unsuccessful treatment, a surgery, the so-called neurolysis, or external decompression, may be indicated. Data about the effectiveness of neurolysis are scanty. To assess the degree of sensory and motor loss before and after neurolysis, we performed a retrospective study. Leprosy patients were included, that had received neurolysis of peripheral nerve trunks in the reference hospital of RondÃnia State (North Brazil), between 2000 and 2003. Socio-demographic and clinical data were collected from the hospitalâs registry of surgeries, from patientsâ charts and from the Notifiable Diseases Database (âSistema Nacional de InformaÃÃo de Agravos de NotificaÃÃoâ). To assess the degree of sensory and motor deficiencies, we created an ordinal score based on the clinical evaluations. Of the 118 individuals included (in total 297 neurolyses), 74 (62,7%) were males. Ninety-six (81,4%) patients were classified clinically with borderline leprosy. Only (53,4%) of the patients were under multi-drug therapy at the moment of neurolysis. The median time between the first episode of neuritis and the surgery was one year (maximum = 12,3 years). The median of the score reduced from 5 to 3 (ulnar nerve), from 3 to 2 (median nerve) and from 9,5 to 7,5 (posterior tibial nerve; all p<0,001). Ninety % of patients with severe sensory deficiency before surgery presented with an improved score after neurolysis. In the multivariate analysis, only the sensitive score of the ulnar nerve (adjusted OR = 1,9; 95% CI: 1,38 â 2,65; p<0,001) and the sensitive score of the posterior tibial nerve (adjusted OR = 1,2; 95% CI: 1,02 â 1,39; p = 0,02) before surgery were factors increasing the chance of improvement. The clinical classification and the period between the first episode of neuritis and surgery did not modify the clinical result significantly. Similar to the sensory evaluation, the motor score of the ulnar and common peroneal nerves improved significantly after surgical intervention (ulnar nerve: p = 0,03; comnor peroneal nerve: p<0,001). Almost 60% of the patients operated on the common peroneal nerve presented more muscle strength. The data of the present study indicate that neurolysis is of important benefit in leprosy neuritis, even after a prolonged period of neuritis. Strategies need to be elaborated to improve the therapeutic options in the treatment of leprosy neuritis.

This thesis is involved in image processing of medical data and its implementation using Java programming language. The main contribution of this thesis is creation of algorithms for feature extraction from 3D data and subsequent verification of the results for the issue of imagining 3D brain data, and creation of image filters and their implementation in the program RapidMiner. Consequently, the segmentation process is created at the 2D and 3D level, and output of 3D level segmentation are segmented brain structures. Furthermore, segmentation algorithms were compared on the basis of the final form of segmented structures and this approach was compared with other works.

This study examines the potential of Schwann cells (SCs) to be manipulated by electric fields (EFs) in order to improve recovery from spinal cord injury (SCI). It had long been believed that the central nervous system (CNS) is incapable of regeneration, but recent studies have proven otherwise. SC transplants are known to be useful in promoting axon regeneration after SCI, but is not sufficient for functional recovery. EFs are known to exist in vivo, and have been known to drastically affect the morphology and behavior of cells in various tissues. It was the hypothesis of this study that the conditioning and observed alignment of SCs was a reproducible phenomenon that could promote the growth of axons. It was found that SCs could be aligned at various field strengths and preliminary data suggest that aligned SCs increased the length and directionality of axons extending from DRG explants.

α-synuclein is a small presynaptic protein whose misfolding and aggregation are considered drivers of the neurological disorders Parkinson’s disease, multiple system atrophy, dementia with Lewy bodies and related synucleopathies. α-synuclein exists in a dynamic state that changes from an α-helical conformation when bound to liposomes to natively unfolded in solution, the majority being in the latter state. The disease process by which native healthy α-synuclein undergoes a change in conformation to form β-sheet oligomers and fibrils is still unresolved. The fibrillation process has been widely studied by several different techniques and the structure of the fibrils has been determined by NMR, scanning transmission electron microscopy and X-ray diffraction. The early stages of aggregation into β-sheet rich oligomers, despite having been widely studied, has proven difficult to follow due to the heterogeneity of the species formed and the unpredictability of the process. The goal of the work reported here was to design and develop a novel, reproducible and quantitative assay to study the early stages of α-synuclein aggregation and to establish a platform for discovery of novel compounds that inhibit this process. These compounds could then be taken as a starting point for the development of new drugs for the treatment of synucleopathies. The assay developed herein has been designed, established and demonstrated to be suitable for the screening of α-synuclein aggregation inhibitors. The assay quantitatively measures aggregation using α- synuclein site-specifically labelled with green and red fluorescent dyes. Proteins labelled with the green dye are bound to microbeads. α-synuclein labelled with the red dye aggregates on the bead-linked green α-synuclein. The first part of the thesis describes the development of the tools required for the assay. α-synuclein single cysteine mutants were produced to introduce a specific attachment point to the protein. Single isomer carboxytetramethylrhodamine was synthesised in large scale for the label. Two different trifunctional tags that allow both the fluorescent labelling of the protein and the addition of a group for bead attachment in a single step were synthesised. Optimisation of the attachment of the functionalised proteins to beads of differing materials was accomplished enabling further development of the bead-based aggregation assay. With all tools established, the second part of the work comprised the development of the bead-based α-synuclein aggregation assay. Solid supports made of two different materials, TentaGel and Agarose, with two different types of bead surface attachment chemistry for α-synuclein were investigated, Ni-NTA on bead with His6-tag on the target or dibenzylcyclooctyne on bead and azide conjugation for the target. Only the combination of Ni-NTA agarose beads linking to His6-tag functionalised α-synuclein was found to be suitable for quantitative measurement of the aggregation process. Using 20 % EtOH, α-synuclein on-bead aggregation was reproducible within a 5 h time-frame with a linear dependence of aggregation rate as function of protein concentration on-bead. The third part of the thesis describes the research into novel starting points for the discovery of inhibitors of α-synuclein aggregation. In the peptides field, the most active peptides in the literature were selected and synthesised for study under the same conditions to find the most active ones. The most active peptide could be modified with non-natural amino acids to increase affinity and stability. While peptides and peptidomimetics would be applied in mechanistic studies, small molecular inhibitors of aggregation might represent lead compounds. One known inhibitor of α-synuclein aggregation was selected, NPT200-5, and an on-bead synthesis was developed so a diversity library could be generated around its four different building blocks. Finally the peptides, the NPT200-5 amide derivative and some known small molecule inhibitors of α-synuclein aggregation, such as curcumin, baicalein and EGCG amongst others, were screened on the bead-based α-synuclein aggregation assay. Strong inhibitory effects of curcumin and baicalein demonstrated the efficacy of the newly developed assay. In summary, the tools for the development of a novel micro-bead-based α-synuclein aggregation assay have been successfully produced. A novel bead-based α-synuclein early stage aggregation assay has been developed and optimised. Validation of this new technique was achieved with known small molecules inhibitors of α-synuclein aggregation.