Avian erythrocytes have functional mitochondria, opening novel perspectives for birds as animal models in the study of ageing.

BACKGROUND: In contrast to mammalian erythrocytes, which have lost their nucleus and mitochondria during maturation, the erythrocytes of almost all other vertebrate species are nucleated throughout their lifespan. Little research has been done however to test for the presence and functionality of mitochondria in these cells, especially for birds. Here, we investigated those two points in erythrocytes of one common avian model: the zebra finch (Taeniopygia guttata). RESULTS: Transmission electron microscopy showed the presence of mitochondria in erythrocytes of this small passerine bird, especially after removal of haemoglobin interferences. High-resolution respirometry revealed increased or decreased rates of oxygen consumption by erythrocytes in response to the addition of respiratory chain substrates or inhibitors, respectively. Fluorometric assays confirmed the production of mitochondrial superoxide by avian erythrocytes. Interestingly, measurements of plasmatic oxidative markers indicated lower oxidative stress in blood of the zebra finch compared to a size-matched mammalian model, the mouse. CONCLUSIONS: Altogether, those findings demonstrate that avian erythrocytes possess functional mitochondria in terms of respiratory activities and reactive oxygen species (ROS) production. Interestingly, since blood oxidative stress was lower for our avian model compared to a size-matched mammalian, our results also challenge the idea that mitochondrial ROS production could have been one actor leading to this loss during the course of evolution. Opportunities to assess mitochondrial functioning in avian erythrocytes open new perspectives in the use of birds as models for longitudinal studies of ageing via lifelong blood sampling of the same subjects.

Functional assessment of chest wall integrity after methylmethacrylate reconstruction.

BACKGROUND: All patients with extensive resection of the anterolateral chest wall and the sternum followed by reconstruction with methylmethacrylate substitutes were assessed prospectively 6 months after the operation to delineate chest wall integrity with pulmonary function and cine-magnetic resonance imaging. METHODS: Twenty-six patients underwent chest wall reconstruction by use of methylmethacrylate between 1994 and 1998 due to primary tumors in 35%, metastases in 27%, T3 lung cancer in 19%, and debridement for radionecrosis and osteomyelitis in 19% of patients. Three to eight ribs were resected and additional sternum resection was performed in 39% of patients. RESULTS: There was no 30-day mortality. All patients were extubated after the operation without need for reintubation. Prosthesis dislocation occurred in 1 patient and infection in 2 patients during follow-up. Nineteen patients (73%) suffered no restrictions of daily activities. Clinical examination revealed normal shoulder girdle function in 77% of patients. There was no significant difference between preoperative and postoperative FEV1 (forced expiratory volume in 1 second) measurements in patients with lobectomy or wedge resections. Cinemagnetic resonance imaging revealed concordant chest wall movements during respiration in 92% of patients without paradoxical movements or implant dislocations being observed. CONCLUSIONS: Large defects of the anterolateral chest wall and sternum can be reconstructed efficiently with methylmethacrylate substitutes with minimal morbidity and excellent cosmetic and functional outcome.

NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies.

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

Characterization of a plant-derived peptide displaying water clarifying and antimicrobial activities

SUMMARY Drinking water is currently a scarce world resource, the preparation of which requires complex treatments that include clarification of suspended particles and disinfection. Seed extracts of Moringa oleifera Lam., a tropical tree, have been proposed as an environment- friendly alternative, due to their traditional use for the clarification of drinking water. However, the precise nature of the active components was unknown. Here, we show that recombinant or synthetic forms of a cationic seed polypeptide mediate efficient sedimentation of suspended mineral particles and bacteria. Unexpectedly, the polypeptide was also found to possesses a bactericidal activity capable of disinfecting heavily contaminated water. Furthermore, the polypeptide has been shown to efficiently kill several pathogenic bacteria, including antibiotic-resistant isolates of Pseudomona, Streptococcus and Legionella species. Structural modeling of the peptide coupled to the functional analysis of synthetic peptide derivatives delineated distinct structural determinants for the flocculation and antibacterial activities. Our results suggest that a glutamine-rich portion of the polypeptide is involved in the sedimentation process; alternatively, the antibacterial activity depends on a amphiphilic loop. Assembly of multiple copies of this loop into a branched peptide derivative strongly enhances antibacterial activity without displaying hemolytic effect. In conclusion, this polypeptide displays the unprecedented feature of combining efficient water purification and disinfectant properties indicating different molecular mechanisms involved in each case. This work not only identified the features responsible for these activities but also provides useful information that has implications for the further development of this cationic polypeptide as a potent antibacterial agent. RESUME L'eau potable est actuellement une ressource limitée dans le monde. La production d'eau propre à la consommation exige des traitements complexes, incluant la clarification des particules en suspension ainsi que sa désinfection par des additifs chimiques. Les extraits de la graine d'un arbre tropical, Moringa oleifera, sont utilisés traditionnellement en Afrique afin de clarifier l'eau. Quoique la nature exacte des composants actifs était inconnue, on a pu mettre en évidence un polypeptide cationique contenu dans ces graines, capable de sédimenter de manière efficace des particules minérales en suspension ainsi que des bactéries. Ce travail a aussi mis en évidence que ce polypeptide a une activité bactéricide, permettant une désinfection d'eau fortement contaminée. De plus, nous avons démontré que ce polypeptide est efficace contre de nombreuses souches bactériennes pathogènes, également celles résistantes aux antibiotiques comme Pseudomonas, Streptococcus et Legionella. L'analyse de la structure moléculaire de ce polypeptide, couplée à son analyse fonctionnelle a mis en évidence deux domaines structuraux distinct, un pour l'activité de floculation et l'autre pour l'activité antibactérienne. Nos résultats suggèrent que le domaine riche en glutamine est impliqué dans le processus de sédimentation et que l'activité antimicrobienne dépend d'un domaine formant une boucle amphiphilique. En ramifiant plusieurs copies de cette boucle on a pu augmenter de manière significative l'activité antibactérienne. En conclusion, nous avons pu démontrer que ce polypeptide à la capacité unique de combiner des propriétés de purification et de désinfection de l'eau, ce qui implique des mécanismes moléculaires distincts pour ces deux activités. Ce travail a permis d'identifier les domaines du polypeptide qui sont responsables de ses activités et offre une perspective pour le développement d'un nouvel agent antimicrobien.

BDNF overexpression in mouse hippocampal astrocytes promotes local neurogenesis and elicits anxiolytic-like activities.

The therapeutic activity of selective serotonin (5-HT) reuptake inhibitors (SSRIs) relies on long-term adaptation at pre- and post-synaptic levels. The sustained administration of SSRIs increases the serotonergic neurotransmission in response to a functional desensitization of the inhibitory 5-HT1A autoreceptor in the dorsal raphe. At nerve terminal such as the hippocampus, the enhancement of 5-HT availability increases brain-derived neurotrophic factor (BDNF) synthesis and signaling, a major event in the stimulation of adult neurogenesis. In physiological conditions, BDNF would be expressed at functionally relevant levels in neurons. However, the recent observation that SSRIs upregulate BDNF mRNA in primary cultures of astrocytes strongly suggest that the therapeutic activity of antidepressant drugs might result from an increase in BDNF synthesis in this cell type. In this study, by overexpressing BDNF in astrocytes, we balanced the ratio between astrocytic and neuronal BDNF raising the possibility that such manipulation could positively reverberate on anxiolytic-/antidepressant-like activities in transfected mice. Our results indicate that BDNF overexpression in hippocampal astrocytes produced anxiolytic-/antidepressant-like activity in the novelty suppressed feeding in relation with the stimulation of hippocampal neurogenesis whereas it did not potentiate the effects of the SSRI fluoxetine on these parameters. Moreover, overexpressing BDNF revealed the anxiolytic-like activity of fluoxetine in the elevated plus maze while attenuating 5-HT neurotransmission in response to a blunted downregulation of the 5-HT1A autoreceptor. These results emphasize an original role of hippocampal astrocytes in the synthesis of BDNF, which can act through neurogenesis-dependent and -independent mechanisms to regulate different facets of anxiolytic-like responses.

Structure-function analyses point to a polynucleotide-accommodating groove essential for APOBEC3A restriction activities.

Members of the human APOBEC3 family of editing enzymes can inhibit various mobile genetic elements. APOBEC3A (A3A) can block the retrotransposon LINE-1 and the parvovirus adeno-associated virus type 2 (AAV-2) but does not inhibit retroviruses. In contrast, APOBEC3G (A3G) can block retroviruses but has only limited effects on AAV-2 or LINE-1. What dictates this differential target specificity remains largely undefined. Here, we modeled the structure of A3A based on its homology with the C-terminal domain of A3G and further compared the sequence of human A3A to those of 11 nonhuman primate orthologues. We then used these data to perform a mutational analysis of A3A, examining its ability to restrict LINE-1, AAV-2, and foreign plasmid DNA and to edit a single-stranded DNA substrate. The results revealed an essential functional role for the predicted single-stranded DNA-docking groove located around the A3A catalytic site. Within this region, amino acid differences between A3A and A3G are predicted to affect the shape of the polynucleotide-binding groove. Correspondingly, transferring some of these A3A residues to A3G endows the latter protein with the ability to block LINE-1 and AAV-2. These results suggest that the target specificity of APOBEC3 family members is partly defined by structural features influencing their interaction with polynucleotide substrates.

Functional significance of first metatarso-phalangeal joint arthrodesis : FM101

Introduction: Indications for arthrodesis of the first metatarsophalangeal joint (MTP1) are commonly arthrosis (hallux rigidus), rheumatoid arthritis, failed hallux valgus surgery, severe hallux valgus, infectious arthritis, fractures and neuroarthropathies. Many reports focus on technical and radiological issues but few studies emphasize the functional outcome considering daily activities, sports and expectation of the patient. Method: We retrospectively reviewed the patients who underwent MTP1-arthrodesis from 2002 to 2005 in our institution. Clinical and radiological results were assessed but we specially focussed on the functional outcome. Scoring systems used were the SF-12, EQ-5D, PASI, FFI and AOFAS (10 points given to MTP1 mobility) scales. Results: 61 of 64 consecutive patients were evaluated. Female to male ratio was 49:15, mean age at surgery was 67 years, the average follow up was 29 month. Even if radiological consolidation was incomplete in 18 patients, all patients had a clinically stable and rigid arthrodesis. Mean AOFAS score was 87 (24-100) points at follow up. The FFI was 5.91% (0-66%). Patient satisfaction was excellent in 37 patients (60%), good in 18 (30%), fair in 5(8%) and poor in1 (2%). EQ- 5D was 0.7 (0.4-1).40 patients (66%) estimated their cosmetic result as excellent, 15 (25%) as good, 4(6%) as fair and 2 (3%)as poor. 10 patients (16%) had no shoe wear limitation , 48 (79%) had to wear comfortable shoes and 3 (5%) needed orthopaedic wearing. Professionally 34 patients (56%) had better performances, 18 (26%) had no change and 9 (18%) had aggravation of their capacities but this was due to other health reasons. In sports, 16 patients (26%) had better performances, 35 patients (57%) no change and 10 (17%) were worse as consequence of other health problems for 7. Finally, 56 patients (92%) would recommend the operation and 5 (8%) would not. Conclusion: Experience of clinical practice suggests that the idea of fusing the first MTP joint is initially frequently disregarded by the patients because they fear to be limited by a rigid forefoot. Our results show, in fact, that this procedure can be proposed for numerous pathological situations with the perspective of good to excellent outcome in terms of function and quality of life in the majority of cases.

Contributory factors for the functional independence of oldest old

OBJECTIVE To investigate the socioeconomic and clinical factors that contribute to the functional independence of the oldest old of a community. METHOD Cross-sectional quantitative study whose sample consisted of 214 elderly people registered in Basic Health Units. Data were collected through structured interviews and application of the Functional Independence Measure. We used descriptive statistics, and for association of the variables we used the Student t-test, ANOVA and Tukey's test for multiple comparisons. RESULTS The significant variables that contributed to the functional independence were remaining economically active, practicing physical and leisure activities, having a social life, eating fruits, vegetables and meat. The orientation to conduct these practices reduces the demand for care and help needed in everyday activities. CONCLUSION Maintaining independence is primordial to delay disability and presents itself as an excellent field of work for nursing.

Functional independence measure in patients with intermittent claudication

AbstractOBJECTIVEOf this study were to evaluate the functional independence of patients with intermittent claudication and to verify its association with sociodemographic and clinical variables, walking ability and physical activity level.METHODThis was a descriptive, exploratory, cross-sectional study with a quantitative approach. Fifty participants (66.4 years; 68% male) were recruited from Claudication Unit of a tertiary hospital. Functional Independence Measure were used to evaluate functional incapacity; the Baltimore Activity Scale, to estimate the physical activity level and the Walking Impairement Questionnaire, the walking ability.RESULTSParticipants had complete functional independence (124.8 + 2.0), low levels of physical activity (4.2 + 2.0), and impairment of walking ability; the worst performance was found in walking velocity domain (21.2 + 16.4). The functional independence score was associated with physical activity (r=0,402) and walking ability scores (distance, r=0,485; speed, r=0,463; stairs, r=0,337).CONCLUSIONIn conclusion, the level of functionality is associated with functional capacity in these patients.

Heart failure as a predictor of functional dependence in hospitalized elderly

AbstractOBJECTIVEIdentify whether Heart Failure (HF) is a predictor of functional dependence for Basic Activities of Daily Living (BADL) in hospitalized elderly.METHODSWe investigated medical records and assessed dependence to BADL (by the Katz Index) of 100 elderly admitted to a geriatric ward of a university hospital. In order to verify if HF is a predictor of functional dependence, linear regression analyzes were performed.RESULTSThe prevalence of HF was 21%; 95% of them were dependent for BADLs. Bathing was the most committed ADL. HF is a predictor of dependence in hospitalized elderlies, increasing the chance of functional decline by 5 times (95% CI, 0.94-94.48), the chance of functional deterioration by 3.5 times (95% CI, 1.28-11.66; p <0.02) and reducing 0.79 points in the Katz Index score (p <0.05).CONCLUSIONHF is a dependency predictor of ADL in hospitalized elderly, who tend to be more dependent, especially for bathing.

Validity of the Katz Index to assess activities of daily living by informants in neuropathological studies

Abstract OBJECTIVE To analyze the evidences of construct validity of the Katz Index for the retrospective assessment of activities of daily living (ADL) by informants, to assist neuropathological studies in the elderly. METHOD A cross-sectional study analyzed the functional ability of ADL measure by the Katz Index, of 650 cases randomly selected from the Brazilian Brain Bank of the Ageing Brain Study Group (BBBABSG) database. Sample was divided in two subsamples for the analysis (N=325, each) and then stratified according to cognitive decline assessed by the Clinical Dementia Rating Scale (CDR). Factor analyses with calculations of internal consistency and invariance were performed. RESULTS Factor analysis evidenced a unidimensional instrument with optimal internal consistency, in all subgroups. Goodness of fit indices were obtained after two treatments of covariance, indicating adequacy of the scale for assessing ADL by informants. The scale is invariant to cognitive decline meaning that it can be used for subjects with or without cognitive impairment. CONCLUSION Katz Index is valid for the retrospective assessment of basic ADL by informants, with optimal reliability.

Functional analysis of human POT1 and RPA : insights into the role of single stranded DNA binding proteins at telomeres

Abstract Telomeres, the natural ends of chromosomes, need to be protected from chromosome end fusions, aberrant homologous recombination and degradation. In humans, chromosome ends are specified through arrays of tandemly repeated 5'-TTAGGG-3' hexamers, ending in a 3' overhang. A complex formed by the six proteins TRF1, TRF2, hRap1, TIN2, TPP1 and POT1 specifically assocìates with and protects telomeres. Telomeres are maintained by semiconservative DNA replication and by a specialized reverse transcriptase, telomerase, that carries an RNA subunit which templates new telomeric repeat synthesis. The telomeric single stranded (ss) DNA binding protein POT1 protects the telomeric 3' overhang and modulates telomerase-mediated telomere elongation. It is possible that POT1 also influences DNA synthesis during semiconservative DNA replication, which is initiated by the DNA polymerase alpha-primase complex. The heterotrimeric ss DNA-binding protein RPA plays essential roles during DNA replication. RPA binds to ss DNA with high affinity in order to stabilize ss DNA and facilitate nascent strand synthesis at the replication fork. Here we investigate how the two proteins RPA and POT1 contribute to telomere maintenance by regulating semi-conservative DNA replication and telomerase. Using chromatin immunoprecipitation experiments, we show that RPA associates with telomeres during S-phase. Analysis of telomere structure in cells shRNA-depleted for RPA and POT1 reveals that loss of RPA and POT1 causes exposure of single-stranded DNA at telomeres, suggestive of incomplete DNA replication. Biochemical experiments using purified recombinant POT1 and RPA show that saturating telomeric oligonucleotides with POT1 or RPA reduces the primase activity of the DNA polymerase alpha-primase complex and the overall activity of telomerase. POT1 and RPA also increase the primer extension by DNA polymerase alpha-primase complex and the processivity of telomerase under certain conditions, although POT1 increases the activities to a greater extent than RPA. We propose that POT1 is required for proper replication of the lagging strand of telomeres and that some phenotypes observed in POT1-depleted cells may stern from incomplete DNA replication rather than de-protection of the single-stranded overhang. Résumé Les télomères, les extrémités normales des chromosomes linéaires, doivent être protégés des fusions chromosomiques, d'événements de recombinaison homologue aberrants et de phénomènes de dégradation. Chez l'Homme, les extrémités des chromosomes sont constitués d'ADN double brin répétitif de séquence 5'-TTAGGG-3', d'une extension simple brin 3' sortante et d'un complexe protéique formé des six facteurs TRF1, TRF2, hRap1, TIN2, TPP1 et POT1 qui, s'associant à cette séquence, protègent l'ADN télomèrique. Les télomères sont maintenus par la télomérase, une transcriptase inverse capable d'allonger l'extension 3' sortante télomérique. POT1 lie l'ADN simple brin télomérique et module l'élongation des télomères par la télomérase. POT1 pourrait en théorie également influencer la réplication semi-conservative de l'ADN. L'ADN-polymérase Pal alpha-primase amorce et initie la synthèse d'ADN. Pendant la réplication, l'ADN simple brin est stabilisé par RPA, un complexe hétérotrimèrique qui lie l'ADN simple brin. RPA facilite la synthèse du brin naissant à la fourche de réplication. Ici nous avons étudié comment ces deux protéines qui lient l'ADN simple brin, RPA et POT1, régulent la réplication des télomères par la télomérase et la machinerie classique de réplication de l'ADN. Par immunoprécipitation de chromatine (ChIP), nous montrons que RPA est localisé aux télomères lors de la phase S du cycle cellulaire. De plus, l'analyse de la structure des télomeres indique que !a perte de RPA ou de POT1 conduit à l'apparition d'ADN simple brin télomérique, suggérant une réplication incomplète de l'ADN télomérique in vivo. Par une approche complémentaire biochimique utilisant les protéines POT1 et RPA recombinantes purifiées, nous montrons également que la liaison de POT1 ou de RPA à des oligonucléotides télomériques bloque l'activité primase du complexe polymérase alpha/primase et réduit l'activité télomérase sur ces substrats. En revanche, leur liaison augmente l'activité ADN-polymérase du complexe polymérase alpha/primase, ainsi que fa processivité de la télomérase dans certaines conditions, POT1 étant le plus efficace des deux facteurs. Nous proposons que POT1 est nécessaire à la réplication du brin retardé au niveau des télomères, ce qui suggère que certains phénotypes des cellules déplétés en POT1 puissent résulter d'une réplication incomplète de l'ADN télémétrique plutôt que d'une déprotection de l'extrémité sortante des télomères.

Correlates of frailty, prediction of functional decline and preventative approaches - selected results from the lausanne cohort 65+ (lc65+) study (Switzerland) (Lausanne) and the longitudinal urban cohort ageing study (Lucas) (Germany)

Overall introduction.- Longitudinal studies have been designed to investigate prospectively, from their beginning, the pathway leading from health to frailty and to disability. Knowledge about determinants of healthy ageing and health behaviour (resources) as well as risks of functional decline is required to propose appropriate preventative interventions. The functional status in older people is important considering clinical outcome in general, healthcare need and mortality. Part I.- Results and interventions from lucas (longitudinal urban cohort ageing study). Authors.- J. Anders, U. Dapp, L. Neumann, F. Pröfener, C. Minder, S. Golgert, A. Daubmann, K. Wegscheider,. W. von Renteln-Kruse Methods.- The LUCAS core project is a longitudinal cohort of urban community-dwelling people 60 years and older, recruited in 2000/2001. Further LUCAS projects are cross-sectional comparative and interventional studies (RCT). Results.- The emphasis will be on geriatric medical care in a population-based approach, discussing different forms of access, too. (Dapp et al. BMC Geriatrics 2012, 12:35; http://www.biomedcentral.com/1471-2318/12/35): - longitudinal data from the LUCAS urban cohort (n = 3.326) will be presented covering 10 years of observation, including the prediction of functional decline, need of nursing care, and mortality by using a self-filling screening tool; - interventions to prevent functional decline do focus on first (pre-clinical) signs of pre-frailty before entering the frailty-cascade ("Active Health Promotion in Old Age", "geriatric mobility centre") or disability ("home visits"). Conclusions.- The LUCAS research consortium was established to study particular aspects of functional competence, its changes with ageing, to detect pre-clinical signs of functional decline, and to address questions on how to maintain functional competence and to prevent adverse outcome in different settings. The multidimensional data base allows the exploration of several further questions. Gait performance was exmined by GAITRite®-System. Supported by the Federal Ministry for Education and Research (BMBF Funding No. 01ET1002A). Part II.- Selected results from the lausanne cohort 65+ (Lc65 + ) Study (Switzerland). Authors.- Prof Santos-Eggimann Brigitte, Dr Seematter-Bagnoud Laurence, Prof Büla Christophe, Dr Rochat Stéphane. Methods.- The Lc65+ cohort was launched in 2004 with the random selection of 3054 eligible individuals aged 65 to 70 (birth year 1934-1938) in the non-institutionalized population of Lausanne (Switzerland). Results.- Information is collected about life course social and health-related events, socio-economics, medical and psychosocial dimensions, lifestyle habits, limitations in activities of daily living, mobility impairments, and falls. Gait performance are objectively measured using body-fixed sensors. Frailty is assessed using Fried's frailty phenotype. Follow-up consists in annual self-completed questionnaires, as well as physical examination and physical and mental performance tests every three years. - Lausanne cohort 65+ (Lc65 + ): design and longitudinal outcomes. The baseline data collection was completed among 1422 participants in 2004-2005 through self-completed questionnaires, face-to-face interviews, physical examination and tests of mental and physical performances. Information about institutionalization, self-reported health services utilization, and death is also assessed. An additional random sample (n = 1525) of 65-70 years old subjects was recruited in 2009 (birth year 1939-1943). - lecture no 4: alcohol intake and gait parameters: prevalent and longitudinal association in the Lc65+ study. The association between alcohol intake and gait performance was investigated.

Integrated methodologies to study human transcriptional regulation from functional genomics data

Abstract : The human body is composed of a huge number of cells acting together in a concerted manner. The current understanding is that proteins perform most of the necessary activities in keeping a cell alive. The DNA, on the other hand, stores the information on how to produce the different proteins in the genome. Regulating gene transcription is the first important step that can thus affect the life of a cell, modify its functions and its responses to the environment. Regulation is a complex operation that involves specialized proteins, the transcription factors. Transcription factors (TFs) can bind to DNA and activate the processes leading to the expression of genes into new proteins. Errors in this process may lead to diseases. In particular, some transcription factors have been associated with a lethal pathological state, commonly known as cancer, associated with uncontrolled cellular proliferation, invasiveness of healthy tissues and abnormal responses to stimuli. Understanding cancer-related regulatory programs is a difficult task, often involving several TFs interacting together and influencing each other's activity. This Thesis presents new computational methodologies to study gene regulation. In addition we present applications of our methods to the understanding of cancer-related regulatory programs. The understanding of transcriptional regulation is a major challenge. We address this difficult question combining computational approaches with large collections of heterogeneous experimental data. In detail, we design signal processing tools to recover transcription factors binding sites on the DNA from genome-wide surveys like chromatin immunoprecipitation assays on tiling arrays (ChIP-chip). We then use the localization about the binding of TFs to explain expression levels of regulated genes. In this way we identify a regulatory synergy between two TFs, the oncogene C-MYC and SP1. C-MYC and SP1 bind preferentially at promoters and when SP1 binds next to C-NIYC on the DNA, the nearby gene is strongly expressed. The association between the two TFs at promoters is reflected by the binding sites conservation across mammals, by the permissive underlying chromatin states 'it represents an important control mechanism involved in cellular proliferation, thereby involved in cancer. Secondly, we identify the characteristics of TF estrogen receptor alpha (hERa) target genes and we study the influence of hERa in regulating transcription. hERa, upon hormone estrogen signaling, binds to DNA to regulate transcription of its targets in concert with its co-factors. To overcome the scarce experimental data about the binding sites of other TFs that may interact with hERa, we conduct in silico analysis of the sequences underlying the ChIP sites using the collection of position weight matrices (PWMs) of hERa partners, TFs FOXA1 and SP1. We combine ChIP-chip and ChIP-paired-end-diTags (ChIP-pet) data about hERa binding on DNA with the sequence information to explain gene expression levels in a large collection of cancer tissue samples and also on studies about the response of cells to estrogen. We confirm that hERa binding sites are distributed anywhere on the genome. However, we distinguish between binding sites near promoters and binding sites along the transcripts. The first group shows weak binding of hERa and high occurrence of SP1 motifs, in particular near estrogen responsive genes. The second group shows strong binding of hERa and significant correlation between the number of binding sites along a gene and the strength of gene induction in presence of estrogen. Some binding sites of the second group also show presence of FOXA1, but the role of this TF still needs to be investigated. Different mechanisms have been proposed to explain hERa-mediated induction of gene expression. Our work supports the model of hERa activating gene expression from distal binding sites by interacting with promoter bound TFs, like SP1. hERa has been associated with survival rates of breast cancer patients, though explanatory models are still incomplete: this result is important to better understand how hERa can control gene expression. Thirdly, we address the difficult question of regulatory network inference. We tackle this problem analyzing time-series of biological measurements such as quantification of mRNA levels or protein concentrations. Our approach uses the well-established penalized linear regression models where we impose sparseness on the connectivity of the regulatory network. We extend this method enforcing the coherence of the regulatory dependencies: a TF must coherently behave as an activator, or a repressor on all its targets. This requirement is implemented as constraints on the signs of the regressed coefficients in the penalized linear regression model. Our approach is better at reconstructing meaningful biological networks than previous methods based on penalized regression. The method is tested on the DREAM2 challenge of reconstructing a five-genes/TFs regulatory network obtaining the best performance in the "undirected signed excitatory" category. Thus, these bioinformatics methods, which are reliable, interpretable and fast enough to cover large biological dataset, have enabled us to better understand gene regulation in humans.

Escherichia coli RuvBL268S: a mutant RuvB protein that exhibits wild-type activities in vitro but confers a UV-sensitive ruv phenotype in vivo.

The RuvABC proteins of Escherichia coli process recombination intermediates during genetic recombination and DNA repair. RuvA and RuvB promote branch migration of Holliday junctions, a process that extends heteroduplex DNA. Together with RuvC, they form a RuvABC complex capable of Holliday junction resolution. Branch migration by RuvAB is mediated by RuvB, a hexameric ring protein that acts as an ATP-driven molecular pump. To gain insight into the mechanism of branch migration, random mutations were introduced into the ruvB gene by PCR and a collection of mutant alleles were obtained. Mutation of leucine 268 to serine resulted in a severe UV-sensitive phenotype, characteristic of a ruv defect. Here, we report a biochemical analysis of the mutant protein RuvBL268S. Unexpectedly, the purified protein is fully active in vitro with regard to its ATPase, DNA binding and DNA unwinding activities. It also promotes efficient branch migration in combination with RuvA, and forms functional RuvABC-Holliday junction resolvase complexes. These results indicate that RuvB may perform some additional, and as yet undefined, function that is necessary for cell survival after UV-irradiation.