An integrated genetic and functional analysis of the role of type II transmembrane serine proteases (TMPRSSs) in hearing loss.

Building on our discovery that mutations in the transmembrane serine protease, TMPRSS3, cause nonsyndromic deafness, we have investigated the contribution of other TMPRSS family members to the auditory function. To identify which of the 16 known TMPRSS genes had a strong likelihood of involvement in hearing function, three types of biological evidence were examined: 1) expression in inner ear tissues; 2) location in a genomic interval that contains a yet unidentified gene for deafness; and 3) evaluation of hearing status of any available Tmprss knockout mouse strains. This analysis demonstrated that, besides TMPRSS3, another TMPRSS gene was essential for hearing and, indeed, mice deficient for Hepsin (Hpn) also known as Tmprss1 exhibited profound hearing loss. In addition, TMPRSS2, TMPRSS5, and CORIN, also named TMPRSS10, showed strong likelihood of involvement based on their inner ear expression and mapping position within deafness loci PKSR7, DFNB24, and DFNB25, respectively. These four TMPRSS genes were then screened for mutations in affected members of the DFNB24 and DFNB25 deafness families, and in a cohort of 362 sporadic deaf cases. This large mutation screen revealed numerous novel sequence variations including three potential pathogenic mutations in the TMPRSS5 gene. The mutant forms of TMPRSS5 showed reduced or absent proteolytic activity. Subsequently, TMPRSS genes with evidence of involvement in deafness were further characterized, and their sites of expression were determined. Tmprss1, 3, and 5 proteins were detected in spiral ganglion neurons. Tmprss3 was also present in the organ of Corti. TMPRSS1 and 3 proteins appeared stably anchored to the endoplasmic reticulum membranes, whereas TMPRSS5 was also detected at the plasma membrane. Collectively, these results provide evidence that TMPRSS1 and TMPRSS3 play and TMPRSS5 may play important and specific roles in hearing.

Fine mapping and functional analysis of the multiple sclerosis risk gene CD6.

CD6 has recently been identified and validated as risk gene for multiple sclerosis (MS), based on the association of a single nucleotide polymorphism (SNP), rs17824933, located in intron 1. CD6 is a cell surface scavenger receptor involved in T-cell activation and proliferation, as well as in thymocyte differentiation. In this study, we performed a haptag SNP screen of the CD6 gene locus using a total of thirteen tagging SNPs, of which three were non-synonymous SNPs, and replicated the recently reported GWAS SNP rs650258 in a Spanish-Basque collection of 814 controls and 823 cases. Validation of the six most strongly associated SNPs was performed in an independent collection of 2265 MS patients and 2600 healthy controls. We identified association of haplotypes composed of two non-synonymous SNPs [rs11230563 (R225W) and rs2074225 (A257V)] in the 2(nd) SRCR domain with susceptibility to MS (P max(T) permutation = 1×10(-4)). The effect of these haplotypes on CD6 surface expression and cytokine secretion was also tested. The analysis showed significantly different CD6 expression patterns in the distinct cell subsets, i.e. - CD4(+) naïve cells, P = 0.0001; CD8(+) naïve cells, P<0.0001; CD4(+) and CD8(+) central memory cells, P = 0.01 and 0.05, respectively; and natural killer T (NKT) cells, P = 0.02; with the protective haplotype (RA) showing higher expression of CD6. However, no significant changes were observed in natural killer (NK) cells, effector memory and terminally differentiated effector memory T cells. Our findings reveal that this new MS-associated CD6 risk haplotype significantly modifies expression of CD6 on CD4(+) and CD8(+) T cells.

Analysis of Centrosome duplication in "Caenorhabditis elegans" : the role of "sas" genes

Abstract: The centrosome is the major microtubule organizing center (MTOC) of most animal cells. As such, it is essential for a number of processes, including polarized secretion or bipolar spindle assembly. Hence, centrosome number needs to be controlled precisely in coordination with DNA replication. Cells early in the cell cycle contain one centrosome that duplicates during S-phase to give rise to two centrosomes that organize a bipolar spindle during mitosis. A failure in this process is likely to engage the spindle assembly checkpoint and threaten genome stability. Despite its importance for normal and uncontrolled proliferation the mechanisms underlying centrosome duplication are still unclear. The Caenorhabditis elegans embryo is well suited to study the mechanisms of centrosome duplication. It allows for the analysis of cellular processes with high temporal and spatial resolution. Gene identification and inactivation techniques are very powerful and a wide set of mutant and transgenic strains facilitates analysis. My thesis project consisted of characterizing three sas-genes: sas-4, sas-5 and sas-¬6. Embryos lacking these genes fail to form a bipolar spindle, hence their name (spindle assembly). I established that sas-4(RNAi) and sas-6(RNAi) embryos do not form daughter centrioles and thus do not duplicate their centrosomes. Furthermore, I showed that both proteins localize to the cytoplasm and are strikingly enriched at centrioles throughout the cell cycle. By performing fluorescent recovery after photobleaching (FRAP) experiments and differentially labeling centrioles, I established that both proteins are recruited to centrioles once per cell cycle when daughter centrioles form. In contrast, SAS-5, PLK-1 and SPD-2 shuttle permanently between the cytoplasm and centrioles. By showing that SAS-5 and SAS-6 interact in vivo, I established a functional relationship between the proteins. Testing the putative human homologue of SAS-6 (HsSAS-6) and a distant relative of SAS-4 (CPAP), I was able to show that these proteins are required for centrosome duplication in human cells. In addition I found that overexpression of GFP¬HsSAS-6 leads to formation of extra centrosomes. In conclusion, we identified and gained important insights into proteins required for centrosome duplication in C. elegans and in human cells. Thus, our work contributes to further elucidate an important step of cell division in normal and malignant tissues. Eventually, this may allow for the development of novel diagnostic or therapeutic reagents to treat cancer patients. Résumé: Le centrosome est le principal centre organisateur des microtubules dans les cellules animales. De ce fait, il est essentiel pour un certain nombre de processus, comme l'adressage polarisé ou la mise en place d'un fuseau bipolaire. Le nombre de centrosome doit être contrôlé de façon précise et en coordination avec la réplication de l'ADN. Au début du cycle cellulaire, les cellules n'ont qu'un seul centrosome qui se duplique au cours de la phase S pour donner naissance à deux centrosomes qui forment le fuseau bipolaire pendant la mitose. Des défauts dans ce processus déclencheront probablement le "checkpoint" d'assemblage du fuseau et menaceront la stabilité du génome. Malgré leurs importances pour la prolifération normale ou incontrôlée des cellules, les mécanismes gouvernant la duplication des centrosomes restent obscures. L'embryon de Caenorhabditis elegans est bien adapté pour étudier les mécanismes de duplication des centrosomes. Il permet l'analyse des processus cellulaires avec une haute résolution spatiale et temporelle. L'identification des gènes et les techniques d'inactivation sont très puissantes et de larges collections de mutants et de lignées transgéniques facilitent les analyses. Mon projet de thèse a consisté à caractérisé trois gènes: sas-4, sas-5 et sas-6. Les embryons ne possédant pas ces gènes ne forment pas de fuseaux bipolaires, d'où leur nom (spindle assembly). J'ai établi que les embryons sas-4(RNAi) et sas-6(RNAi) ne forment pas de centrioles fils, et donc ne dupliquent pas leur centrosome. De plus, j'ai montré que les deux protéines sont localisées dans le cytoplasme et sont étonnamment enrichies aux centrioles tout le long du cycle cellulaire. En réalisant des expériences de FRAP (fluorscence recovery after photobleaching) et en marquant différentiellement les centrioles, j'ai établi que ces deux protéines sont recrutées une fois par cycle cellulaire aux centrioles, au moment de la duplication. Au contraire, SAS-5, PLK-1 et SPD-2 oscillent en permanence entre le cytoplasme et les centrioles. En montrant que SAS-5 et SAS-6 interagissent in vivo, j'ai établi une relation fonctionnelle entre les deux protéines. En testant les homologues humains putatifs de SAS-6 (HsSAS-6) et de SAS-4 (CPAP), j'ai été capable de montrer que ces protéines étaient aussi requises pour la duplication des centrosomes dans les cellules humaines. De plus, j'ai montré que la surexpression de GFP-HsSAS-6 entrainait la formation de centrosomes surnuméraires. En conclusion, nous avons identifié et progressé dans la compréhension de protéines requises pour la duplication des centrosomes chez C. elegans et dans les cellules humaines. Ainsi, notre travail contribue à mieux élucider une étape importante du la division cellulaire dans les cellules normales et malignes. A terme, ceci devrait aider au développement de nouveaux diagnostics ou de traitements thérapeuthiques pour soigner les malades du cancer.

Functional heterogeneity of memory CD4 T cell responses in different conditions of antigen exposure and persistence.

Memory CD4 T cell responses are functionally and phenotypically heterogeneous. In the present study, memory CD4 T cell responses were analyzed in different models of Ag-specific immune responses differing on Ag exposure and/or persistence. Ag-specific CD4 T cell responses for tetanus toxoid, HSV, EBV, CMV, and HIV-1 were compared. Three distinct patterns of T cell response were observed. A dominant single IL-2 CD4 T cell response was associated with the model in which the Ag can be cleared. Polyfunctional (single IL-2 plus IL-2/IFN-gamma plus single IFN-gamma) CD4 T cell responses were associated with Ag persistence and low Ag levels. A dominant single IFN-gamma CD4 T cell response was associated with the model of Ag persistence and high Ag levels. The results obtained supported the hypothesis that the different patterns observed were substantially influenced by different conditions of Ag exposure and persistence.

Effective diagnosis of Alzheimer's disease by means of large margin-based methodology.

BACKGROUND Functional brain images such as Single-Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) have been widely used to guide the clinicians in the Alzheimer's Disease (AD) diagnosis. However, the subjectivity involved in their evaluation has favoured the development of Computer Aided Diagnosis (CAD) Systems. METHODS It is proposed a novel combination of feature extraction techniques to improve the diagnosis of AD. Firstly, Regions of Interest (ROIs) are selected by means of a t-test carried out on 3D Normalised Mean Square Error (NMSE) features restricted to be located within a predefined brain activation mask. In order to address the small sample-size problem, the dimension of the feature space was further reduced by: Large Margin Nearest Neighbours using a rectangular matrix (LMNN-RECT), Principal Component Analysis (PCA) or Partial Least Squares (PLS) (the two latter also analysed with a LMNN transformation). Regarding the classifiers, kernel Support Vector Machines (SVMs) and LMNN using Euclidean, Mahalanobis and Energy-based metrics were compared. RESULTS Several experiments were conducted in order to evaluate the proposed LMNN-based feature extraction algorithms and its benefits as: i) linear transformation of the PLS or PCA reduced data, ii) feature reduction technique, and iii) classifier (with Euclidean, Mahalanobis or Energy-based methodology). The system was evaluated by means of k-fold cross-validation yielding accuracy, sensitivity and specificity values of 92.78%, 91.07% and 95.12% (for SPECT) and 90.67%, 88% and 93.33% (for PET), respectively, when a NMSE-PLS-LMNN feature extraction method was used in combination with a SVM classifier, thus outperforming recently reported baseline methods. CONCLUSIONS All the proposed methods turned out to be a valid solution for the presented problem. One of the advances is the robustness of the LMNN algorithm that not only provides higher separation rate between the classes but it also makes (in combination with NMSE and PLS) this rate variation more stable. In addition, their generalization ability is another advance since several experiments were performed on two image modalities (SPECT and PET).