Functional and therapeutical implications of ligand recognition by the scavenger-like lymphocyte receptors CD5 and CD6

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Leader succession: The impact of leaders’ background characteristics on organizations’ performance

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Mitigating the impact of occupational noise exposure for elderly workers: Setting the functional requirements for an ANC system

Noise affects people in very different aspects and in almost every aspect of our daily life. The most prominent impact of noise exposure is hearing loss. However, it can also impair people at their work settings due to other effects rather than hearing loss. Older works tend to be more susceptible to noise exposure effects at work, firstly because most of them already have some ‘natural’ hearing loss, as a results of the ageing process, and secondly because they also tend to be more susceptible at an psychological level. The current study is an attempt to describe the potential problem and to make a survey to identify the available active noise cancelation systems, as well as to specific the main requirements of this type of systems to be applied in such contexts. Several aspects of characteristics of the ANC systems were identified and are presented in this study. From the obtained results it was possible to have a clearer idea about the potential of this technology, and to confirm that this type of solution can be extremely important as a component of an active ageing program, as the preservation of hearing will also impact on the social life of the exposed workers.

Evolution of the functional properties of titanium–silver thin films for biomedical applications: Influence of in-vacuum annealing

In this work, the thermal stability of TiAgx thin films, deposited by magnetron sputtering, was evaluated, envisaging their application in biomedical devices, namely as electrodes for biosignal acquisition. Based on the composition and microstructural characterization, a set of four representative TiAgx thin films was selected in order to infer whether they are thermally stable in terms of functional properties. In order to achieve this purpose, the structural and morphological evolution of the films with annealing temperature was correlated with their electrical, mechanical and thermal properties. Two distinct zones were identified and two samples from each zone were extensively analysed. In the first zone (zone I), Ti was the main component (Ti-rich zone) while in the second, zone II, the Ag content was more significant. The selected samples were annealed in vacuum at four different temperatures up to 500 oC. For the samples produced within zone I, small microstructural changes were observed due to the recrystallization of the Ti structure and grain size increment. Also, no significant changes were observed with annealing temperature regarding the f l ’ functional properties, being thermally stable up to 500 oC. For higher Ag contents (zone II) the energy supplied by thermal treatments was sufficient to activate the crystallization of Ti-Ag intermetallic phases. A strong increase of the grain size of these phases was also reported. The structural and morphological organization proved to be determinant for the physical responses of the TiAgx system. The hardness and Y g’s modulus were significantly improved with the formation of the intermetallic phases. The silver addition and annealing treatments also played an important role in the electrical conductivity of the films, which was once again improved by the formation of Ti-Ag phases. The thermal diffusivity of the films was practically unchanged with the heat-treatment. This set of results shows that this intermetallic-like thin film system has good thermal stability up to high temperatures (as high as 500 oC), which in case of the highest Ag content zone is particularly evident for electrical and mechanical properties, showing an important improvement. Hardness increases about three times, while resistivity values become half of those from the lowest Ag contents zone. These set of characteristics are consistent with the targeted applications, namely in terms of biomedical sensing devices.

Cells with neuronal characteristics differentiate and persist for one year in rat optic nerve explants cultures.

Evidence concerning the presence or absence of common neuronglia lineages in the postnatal mammalian central nervous system is still a matter of speculation. We address this problem using optic nerve explants, which show an extremely long survival in culture. Morphological, immunocytochemical and immunochemical methods were applied. The results obtained from in vitro tissue were compared with optic nerves (ONs) and whole-brain samples from animals of different ages. Newborn rat ONs represented the starting material of our tissue culture; they are composed of unmyelinated axons, astrocytes and progenitor cells but devoid of neuronal cell bodies. At this age, Western blots of ONs were positively stained by neurofilament and synapsin I specific antibodies. These bands increased in intensity during postnatal in situ development. In explant cultures, the glia cells reach a stage of functional differentiation and they maintain, together with undifferentiated cells, a complex histotypic organization. After 6 days in vitro, neurofilaments and synapsin I could not be detected on immunoblots, indicating that 1) axonal degeneration was completed, and 2) neuronal somata were absent at the time. Surprisingly, after about 4-5 weeks in culture, a new cell type appeared, which showed characteristics typical of neurons. After 406 days in vitro, neurofilaments and synapsin I were unequivocally detectable on Western blots. Furthermore, both immunocytochemical staining and light and electron microscopic examinations corroborated the presence of this earlier-observed cell type. These in vitro results clearly show the high developmental plasticity of ON progenitor cells, even late in development. The existence of a common neuron-glia precursor, which never gives rise to neurons in situ, is suggested.

Functional sphere profiling reveals the complexity of neuroblastoma tumor-initiating cell model.

Neuroblastoma (NB) is a neural crest-derived childhood tumor characterized by a remarkable phenotypic diversity, ranging from spontaneous regression to fatal metastatic disease. Although the cancer stem cell (CSC) model provides a trail to characterize the cells responsible for tumor onset, the NB tumor-initiating cell (TIC) has not been identified. In this study, the relevance of the CSC model in NB was investigated by taking advantage of typical functional stem cell characteristics. A predictive association was established between self-renewal, as assessed by serial sphere formation, and clinical aggressiveness in primary tumors. Moreover, cell subsets gradually selected during serial sphere culture harbored increased in vivo tumorigenicity, only highlighted in an orthotopic microenvironment. A microarray time course analysis of serial spheres passages from metastatic cells allowed us to specifically "profile" the NB stem cell-like phenotype and to identify CD133, ABC transporter, and WNT and NOTCH genes as spheres markers. On the basis of combined sphere markers expression, at least two distinct tumorigenic cell subpopulations were identified, also shown to preexist in primary NB. However, sphere markers-mediated cell sorting of parental tumor failed to recapitulate the TIC phenotype in the orthotopic model, highlighting the complexity of the CSC model. Our data support the NB stem-like cells as a dynamic and heterogeneous cell population strongly dependent on microenvironmental signals and add novel candidate genes as potential therapeutic targets in the control of high-risk NB.

Infections and functional impairment in nursing home residents: a reciprocal relationship.

OBJECTIVES: To determine the relationship between infections and functional impairment in nursing home residents. DESIGN: Prospective cohort study (follow-up period, 6 months). SETTING: Thirty-nine nursing homes in western Switzerland. PARTICIPANTS: A total of 1,324 residents aged 65 and older (mean age 85.7; 76.6% female) who agreed to participate, or their proxies, by oral informed consent. MEASUREMENTS: Functional status measured every 3 months. Two different outcomes were used: (a) functional decline defined as death or decreased function at follow-up and (b) functional status score using a standardized measure. RESULTS: At the end of follow-up, mortality was 14.6%, not different for those with and without infection (16.2% vs 13.1%, P=.11). During both 3-month periods, subjects with infection had higher odds of functional decline, even after adjustment for baseline characteristics and occurrence of a new illness (adjusted odds ratio (AOR)=1.6, 95% confidence interval (CI)=1.2-2.2, P=.002, and AOR=1.5, 95% CI=1.1-2.0, P=.008, respectively). The odds of decline increased in a stepwise fashion in patients with zero, one, and two or more infections. The analyses predicting functional status score (restricted to subjects who survived) gave similar results. A survival analysis predicting time to first infection confirmed a stepwise greater likelihood of infection in subjects with moderate and severe impairment at baseline than in subjects with no or mild functional impairment at baseline. CONCLUSION: Infections appear to be both a cause and a consequence of functional impairment in nursing home residents. Further studies should be undertaken to investigate whether effective infection control programs can also contribute to preventing functional decline, an important component of these residents' quality of life.

Placebo effect characteristics observed in a single, international, longitudinal study in Huntington's disease.

BACKGROUND: Classically, clinical trials are based on the placebo-control design. Our aim was to analyze the placebo effect in Huntington's disease. METHODS: Placebo data were obtained from an international, longitudinal, placebo-controlled trial for Huntington's disease (European Huntington's Disease Initiative Study Group). One-hundred and eighty patients were evaluated using the Unified Huntington Disease Rating Scale over 36 months. A placebo effect was defined as an improvement of at least 50% over baseline scores in the Unified Huntington Disease Rating Scale, and clinically relevant when at least 10% of the population met it. RESULTS: Only behavior showed a significant placebo effect, and the proportion of the patients with placebo effect ranged from 16% (first visit) to 41% (last visit). Nondepressed patients with better functional status were most likely to be placebo-responders over time. CONCLUSIONS: In Huntington's disease, behavior seems to be more vulnerable to placebo than overall motor function, cognition, and function

Ageing-related cardiomyocyte functional decline is sex and angiotensin II dependent.

Clinically, heart failure is an age-dependent pathological phenomenon and displays sex-specific characteristics. The renin-angiotensin system mediates cardiac pathology in heart failure. This study investigated the sexually dimorphic functional effects of ageing combined with angiotensin II (AngII) on cardiac muscle cell function, twitch and Ca(2+)-handling characteristics of isolated cardiomyocytes from young (~13 weeks) and aged (~87 weeks) adult wild type (WT) and AngII-transgenic (TG) mice. We hypothesised that AngII-induced contractile impairment would be exacerbated in aged female cardiomyocytes and linked to Ca(2+)-handling disturbances. AngII-induced cardiomyocyte hypertrophy was evident in young adult mice of both sexes and accentuated by age (aged adult ~21-23 % increases in cell length relative to WT). In female AngII-TG mice, ageing was associated with suppressed cardiomyocyte contractility (% shortening, maximum rate of shortening, maximum rate of relaxation). This was associated with delayed cytosolic Ca(2+) removal during twitch relaxation (Tau ~20 % increase relative to young adult female WT), and myofilament responsiveness to Ca(2+) was maintained. In contrast, aged AngII-TG male cardiomyocytes exhibited peak shortening equivalent to young TG; yet, myofilament Ca(2+) responsiveness was profoundly reduced with ageing. Increased pro-arrhythmogenic spontaneous activity was evident with age and cardiac AngII overexpression in male mice (42-55 % of myocytes) but relatively suppressed in female aged transgenic mice. Female myocytes with elevated AngII appear more susceptible to an age-related contractile deficit, whereas male AngII-TG myocytes preserve contractile function with age but exhibit desensitisation of myofilaments to Ca(2+) and a heightened vulnerability to arrhythmic activity. These findings support the contention that sex-specific therapies are required for the treatment of age-progressive heart failure.

Analgesic efficacy of zoledronic acid and its effect on functional status of prostate cancer patients with metastasis

Objectives: A multi-centered observational study evaluated the efficacy of zoledronic acid for improving pain and mobility, and preventing skeletal-related events (SRE) (fracture, spinal compression, pain-relieving radiotherapy), in patients with prostate cancer and bone metastasis. Materials and Methods: Males (n = 218) with prostate cancer and bone metastasis undergoing oncologic therapy received zoledronic acid (4 mg iv/month) for 6 months. Parameters evaluated were: 1) pain and movement after 2 consecutive doses; 2) quality of life; 3) SRE incidence and time-to-appearance. Medication tolerance and treatment satisfaction were assessed using a questionnaire. Results: A total of 170 that matched all the inclusion criteria (78%) out of 218 were evaluable for efficacy. There was a measurable statistically significant reduction in pain at rest and on movement as well as an improvement in the quality of life compared with baseline. Best results were obtained with early treatment. Overall incidence of bone events was 11.2%. Of the 212 patients (97.2%) evaluable for safety, 16% suffered adverse events and 66% expressed satisfaction with the treatment Discussion: Zoledronic acid is effective for reducing pain, improving mobility, and increasing the quality of life in patients with prostate cancer with bone metastasis. Its easy administration and good tolerability make zoledronic acid one of the principal therapeutic tools in the management of patients with pain associated with bone metastasis from prostate cancer.

Lack of association between the protein tyrosine phosphatase non-receptor type 22 R263Q and R620W functional genetic variants and endogenous non-anterior uveitis.

OBJECTIVE Endogenous uveitis is a major cause of visual loss mediated by the immune system. The protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene encodes a lymphoid-specific phosphatase that plays a key role in T-cell receptor (TCR) signaling. Two independent functional missense single nucleotide polymorphisms (SNPs) located within the PTPN22 gene (R263Q and R620W) have been associated with different autoimmune disorders. We aimed to analyze for the first time the influence of these PTPN22 genetic variants on endogenous non-anterior uveitis susceptibility. METHODS We performed a case-control study of 217 patients with endogenous non-anterior uveitis and 718 healthy controls from a Spanish population. The PTPN22 polymorphisms (rs33996649 and rs2476601) were genotyped using TaqMan allelic discrimination assays. The allele, genotype, carriers, and allelic combination frequencies were compared between cases and controls with χ(2) analysis or Fisher's exact test. RESULTS Our results showed no influence of the studied SNPs in the global susceptibility analysis (rs33996649: allelic P- value=0.92, odds ratio=0.97, 95% confidence interval=0.54-1.75; rs2476601: allelic P- value=0.86, odds ratio=1.04, 95% confidence interval=0.68-1.59). Similarly, the allelic combination analysis did not provide additional information. CONCLUSIONS Our results suggest that the studied polymorphisms of the PTPN22 gene do not play an important role in the pathophysiology of endogenous non-anterior uveitis.

Assessment of particulate exposure and surface characteristics in association with urinary levels of 8-hydroxy-2'-deoxyguanosine, considered as marker of oxidative stress

Epidemiological studies have demonstrated that exposure to fine particles is associated to adverse health effects, including cancer, respiratory and cardiovascular diseases. However, mechanisms by which particles induce health effects remain unclear. According to one of the most investigated hypotheses, particles cause adverse effects through the production of reactive oxygen species (ROS), which are very hazardous compounds able to attack directly biological structures, including the DNA strand or the lipid bilayer of the cells. If the defense mechanisms, constituted of antioxidants, are not able to counter ROS, then these compounds will cause in the body a range of oxidation reactions called "oxidative stress". The aim of the present research project was to better understand mechanisms by which exposure to fine particles induces oxidative stress. The first point of this project was to check whether exposure to high levels of fine particles is directly linked to oxidative stress, and whether this oxidative stress is accompanied by the activation of the defense mechanisms (antioxidants). The second point was to study the role played by the particle surface characteristics in the oxidative stress process. For that purpose, a study was conducted in bus depots with the participation of 40 mechanics. First, occupational exposure to particles (PM4) and to other pollutants (NOx, O3) was measured over a two-day period. Then, urine samples of mechanics were collected in order to measure levels of 8-hydroxy-2'-deoxyguanosine (8OHdG) and antioxidants. 8OHdG is a molecule formed by the oxidation of DNA and allowing to assess the oxidative stress status of the mechanics. Finally, particles were collected on filters, and functional groups located on the particle surface were analyzed in the laboratory using a Knudsen flow reactor. This technique allows not only to quantify functional groups on the particle surface, but also to measure the reaction kinetics. Results obtained during the field campaign in bus depots showed that mechanics were exposed to rather low levels of PM4 (20-85 μg/m3) and of pollutants (NOx: 100-1000 ppb; O3: <15 ppb). However, despite this low exposure, urinary levels of the oxidative stress biomarker (8OHdG) increased significantly for non-smoking workers over a two-day period of shift. This oxidative stress was accompanied by an increase of antioxidants, indicating the activation of defense mechanisms. On the other hand, the analysis of functional groups on the particle surface showed important differences, depending on the workplace, the date and the activities of workers. The particle surface contained simultaneously antagonistic functional groups which did not undergo internal reactions (such as acids and bases), and was usually characterized by a high density of carbonyl functions and a low density of acidic sites. Reaction kinetics measured using the Knudsen flow reactor pointed out fast reactions of oxidizable groups and slow reactions of acidic sites. Several exposure parameters were significantly correlated with the increase of the oxidative stress status: the presence of acidic sites, carbonyl functions and oxidizable groups on the particle surface; reaction kinetics of functional groups on the particle surface; particulate iron and copper concentrations; and NOx concentration.

Functional analysis: evaluation of response intensities--tailoring ANOVA for lists of expression subsets.

Background: Microarray data is frequently used to characterize the expression profile of a whole genome and to compare the characteristics of that genome under several conditions. Geneset analysis methods have been described previously to analyze the expression values of several genes related by known biological criteria (metabolic pathway, pathology signature, co-regulation by a common factor, etc.) at the same time and the cost of these methods allows for the use of more values to help discover the underlying biological mechanisms. Results: As several methods assume different null hypotheses, we propose to reformulate the main question that biologists seek to answer. To determine which genesets are associated with expression values that differ between two experiments, we focused on three ad hoc criteria: expression levels, the direction of individual gene expression changes (up or down regulation), and correlations between genes. We introduce the FAERI methodology, tailored from a two-way ANOVA to examine these criteria. The significance of the results was evaluated according to the self-contained null hypothesis, using label sampling or by inferring the null distribution from normally distributed random data. Evaluations performed on simulated data revealed that FAERI outperforms currently available methods for each type of set tested. We then applied the FAERI method to analyze three real-world datasets on hypoxia response. FAERI was able to detect more genesets than other methodologies, and the genesets selected were coherent with current knowledge of cellular response to hypoxia. Moreover, the genesets selected by FAERI were confirmed when the analysis was repeated on two additional related datasets. Conclusions: The expression values of genesets are associated with several biological effects. The underlying mathematical structure of the genesets allows for analysis of data from several genes at the same time. Focusing on expression levels, the direction of the expression changes, and correlations, we showed that two-step data reduction allowed us to significantly improve the performance of geneset analysis using a modified two-way ANOVA procedure, and to detect genesets that current methods fail to detect.

"Adolescent-onset Still's disease": characteristics and outcome in comparison with adult-onset Still's disease.

OBJECTIVES: To determine if adolescent onset systemic juvenile idiopathic arthritis (JIA) and adult onset Still's disease (AOSD) represent the same clinical continuum of disease. METHODS: Retrospective review of available clinical data on all pediatric and adult patients diagnosed with Still's disease within the last 10 years at a university hospital. Assessment of functional outcomes at last visit by clinical evaluation and HAQ or c-HAQ. RESULTS: Nine patients were identified as adolescent onset systemic JIA and were compared with 10 patients with AOSD (onset > 18 years old). No statistically significant differences were found between the two groups in terms of clinical presentation at onset and outcome at follow up. CONCLUSION: Adolescent patients presenting with systemic JIA have a disease onset and course undistinguishable from that of AOSD patients, suggesting that they represent a continuum of a single disease entity.

Temporal SNR characteristics in segmented 3D-EPI at 7T.

Three-dimensional segmented echo planar imaging (3D-EPI) is a promising approach for high-resolution functional magnetic resonance imaging, as it provides an increased signal-to-noise ratio (SNR) at similar temporal resolution to traditional multislice 2D-EPI readouts. Recently, the 3D-EPI technique has become more frequently used and it is important to better understand its implications for fMRI. In this study, the temporal SNR characteristics of 3D-EPI with varying numbers of segments are studied. It is shown that, in humans, the temporal variance increases with the number of segments used to form the EPI acquisition and that for segmented acquisitions, the maximum available temporal SNR is reduced compared to single shot acquisitions. This reduction with increased segmentation is not found in phantom data and thus likely due to physiological processes. When operating in the thermal noise dominated regime, fMRI experiments with a motor task revealed that the 3D variant outperforms the 2D-EPI in terms of temporal SNR and sensitivity to detect activated brain regions. Thus, the theoretical SNR advantage of a segmented 3D-EPI sequence for fMRI only exists in a low SNR situation. However, other advantages of 3D-EPI, such as the application of parallel imaging techniques in two dimensions and the low specific absorption rate requirements, may encourage the use of the 3D-EPI sequence for fMRI in situations with higher SNR.