Caracterización de la contaminación atmosférica debida a aportes antropogénicos y naturales mediante la aplicación de modelos de mixturas finitas, de Markov homogéneos y otras técnicas de minería de datos

Son cuantiosos los recursos científicos que se dirigen al estudio de las fuentes de emisión de contaminantes atmosféricos en las áreas urbanas. Este estudio puede ser cuantitativo, determinando la contribución de cada fuente a la contaminación ambiente, o cualitativo, para conocer más sobre la composición de las emisiones que afectan a los residentes en las ciudades. En los países mediterráneos, además, la contaminación causada por fenómenos naturales, como el transporte de polvo desde las regiones áridas del Norte de África, también es de primordial importancia. Entre los instrumentos fundamentales de los que se dispone para medir la contaminación atmosférica, se encuentran las redes de vigilancia de la calidad del aire, integradas por estaciones de medida que se sitúan tanto en ambientes urbanos como en el medio rural, con el fin de determinar e informar sobre la calidad del aire que nos afecta. En las ciudades, algunas de estas estaciones de medida se sitúan en emplazamientos fuera del alcance directo de fuentes de emisión, para determinar la contaminación de fondo urbano, representativa de la exposición a la que la población se expone de forma general. Esta tesis ha tenido como objetivos los siguientes: 1. La caracterización exhaustiva de la contaminación atmosférica en entornos urbanos y rurales empleando la información obtenida de las redes de vigilancia de la calidad del aire, desarrollando para ello una metodología general para la gestión eficiente de las redes de monitorización. 2. Mejorar la metodología existente para la estimación del aporte de polvo transportado por las masas de aire cálido desde las regiones norteafricanas. 3. Comparar los niveles de contaminación atmosférica entre diferentes redes de monitorización urbanas, sin influencia industrial y localización geográfica distinta, proponiendo para ello una metodología con la que caracterizar la contaminación atmosférica ambiental y de fondo. Los resultados de esta tesis, apoyados en cada uno de estos objetivos, están avalados, respectivamente, por las siguientes publicaciones: 1. Gómez-Losada, Á., Lozano-García, A., Pino-Mejías, R., Contreras-González, J. 2014. Finite mixture models to characterize and refine air quality monitoring networks. Science of the Total Environment, 485-486: 292-9. 2. Gómez-Losada, Á.,Pires,J.C.M.,Pino-Mejías,R.2015.Time series clustering for estimating particulate matter contributions and its use in quantifying impacts from deserts. Atmospheric Environment, 117: 271-81. 3. Gómez-Losada, Á., Pires, J.C.M., Pino-Mejías, R. 2016. Characterization of background air pollution exposure in urban environments using a metric based on Hidden Markov Models. Atmospheric Environment, 127: 255-61.A wealth of scientific resources have been dedicated to the study of the sources of pollutant emissions to air in urban areas. Such studies may be quantitative, determining the contribution of each source of environmental pollution, or they may be qualitative, providing insight into the makeup of the emissions that afect a city's inhabitants. In Mediterranean countries, contamination may also be the result of natural phenomenon, such as the ow of dust from the arid regions of North Africa, and are therefore of primary importance as well. The ow of particulate matter transcends these geographic areas, passing over the Atlantic Ocean and reaching the American coasts. Among the fundamental tools available for measuring air pollution are the air-quality monitoring networks, made up of monitoring stations located both in urban areas and rural environments, with the aim of providing information on the air quality that afects us. In cities, some of these monitoring stations are located on sites that are outside of the direct range of emission sources and thus the determination of the urban background pollution, which is indicative of the generalised exposure of the population to air pollution, is possible. The objectives of this thesis were the following: To exhaustively characterise the air pollutants in urban and rural areas using the information obtained from the air-quality monitoring networks. To this end, a general methodology was developed to efciently manage the monitoring networks; To improve the existing methodology used to estimate the contribution of dust originating in the North African region that is carried by waves of warm air; To compare the air-pollution levels between the diferent urban-monitoring networks unafected by industrial pollution, and between diferent geographic locations, proposing a methodology that can be used to characterise environmental and background air pollution. In order to fulil the First objective, the primary and secondary air-pollution monitoring data were modelled using finite mixture models. Based on the calculation of the first and second moments of these mixtures, hierarchical cluster analysis, imputation using random forests, and principal component analysis were used. This methodological approximation enabled the detection of duplications within the parameters monitored by the monitoring stations, thus allowing these networks to be reconfigured and enabling the economic resources invested in them to be optimised. For the second objective, hidden Markov models (HMM) were introduced and the diferent regimes or PM10 concentration profiles were described in some of the time series (TS) studied, enabling an estimation of the contribution of each of the profiles to environmental pollution. The new method proposed for estimating the natural contribution of PM10 improves upon the reference methodology used in the European Union (monthly moving 40th percentile method) in three ways - it avoids the use of empirical approximation, it applies modelling that is especially designed for the treatment of time-series data, and it allows for obtaining a con_dence interval for the contribution estimations for PM10. For the third objective, hidden Markov models were also used, in this case to define and characterise the environmental and background pollution caused by primary air pollution in diferent urban areas of diferent cities. The attributable fraction for background air pollution was estimated using a new procedure based on the first concentration profile defined by the HMMs in the TS. The ratio and diference between environmental and background concentrations were also studied

The role of apoptosis in <i>in vitro</i> and <i>in vivo</i> models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease leading to motor neurons death. Although many studies were performed, the aetiology and the features of cellular death occurring in ALS are poorly understood, and studies carried out from autoptic samples do not allow to clarify early events occurring in ALS. To understand the basic mechanisms leading to motor neuronal death in ALS and to detect the different steps involved in motor neuron degeneration, alternative models are required. The wobbler mouse is one of the most reliable models of human motor neurodegenerative diseases. In the wobbler mouse neuromuscular deficits that are related to a selective vulnerability of cervical spinal cord motor neurons have an early onset and progress rapidly. In my project of thesis I first characterized apoptosis in primary cultures of motor neurons exposed to detrimental stimuli, and then I evaluated the possible involvement of apoptosis in the cervical spinal cord neurons of early symptomatic wobbler mice. In primary cultures of motor neurons I found that: I) BDNF deprivation produced apoptotic cell death, II) AMPA and kainate induced apoptotic or non apoptotic cell death depending on the experimental conditions utilized, III) EPO selectively protected motor neurons committed to dye apoptically. In wobbler mice I found that: I) mitochondrial activity is reduced but caspase9 -mediated apoptosis does not seem involved, II) glutamate induced excitotoxicity does not seem involved in motor neuron degeneration, III) markers of neuroinflammation, such as activated microglia and TNF-a, appeared highly increased in the cervical region of early symptomatic wobbler mice but this process does not seem to induce the activation of caspase 8-mediated apoptosis. Pharmacological treatments confirmed that an antiglutamatergic agent (RPR 119990) and an antiapoptotic agent (EPO) were ineffective, while riluzole showed protective effects. Although alternative cell death pathways should be investigated, my study excludes apoptosis as the mechanism of death in cervical motor neurons of wobbler mice

Modelling methodologies to assess glucose metabolism in type 2 diabetes

The aetiology and pathogenesis of type 2 diabetes (T20) are yet to be fully understood. However, there is a degree of agreement that the most important pathological factors of T20 are p-cell dysfunction and insulin resistance. Moreover T2D is characterised by varying degrees of impaired pancreatic p-cell responsiveness and/or insulin resistance. The ability to easily quantify insulin sensitivity is useful for investigating the role of impaired insulin secretion and action in the pathophysiology ofT2D. The current work provided novel knowledge in both clinical and methodological areas. On the clinical side, it provided new information about pathology of T2D. On the methodological side, it assessed validity, performance, and/or reproducibility of two powerful models to assess insulin responsiveness and sensitivity. The aim was to use modelling techniques employing data collected during tolerance tests to progress our understanding of pathology of type 2 diabetes. This research evaluated and/or validated two approaches namely, the insulin secretion model and the minimal model, respectively, to assess pancreatic p-cell responsiveness and insulin sensitivity. These methods were then applied to study the aetiology and pathology of T2D on its first clinical appearance (newly diagnosed SUbjects). The insulin secretion model was assessed with two reduced sampling schemes. The model was validated during the oral glucose tolerance test (OGTT). and its performance was compared during OGTT and the meal tolerance test (Mm. The reproducibility of the model indices was also assessed during MTT and OGTT. The one and two compartmental minimal model performance was evaluated and compared to the clamp in subjects with T20. The insulin secretion model and the one compartment minimal model were then applied to study newly presenting T2D in order to gain more understanding of the disease pathology. The output results showed the ability of these approaches to explain the inter-individual variability of important glucose clinical measures such as FPG and HbA1C• In conclusion the insulin secretion model and the one compartment minimal model demonstrated their validity and utility in assessing insulin sensitivity and p-cell responsiveness to provide better Wlderstanding of type 2 diabetes

Mechanical injury and inflammatory cytokines affect cartilage integrity and tissue homeostasis : a mass spectrometric analysis of proteins with relevance to arthritis

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.Includes bibliographical references.Osteoarthritis is characterized by synovial joint degeneration, and its cardinal pathological feature is degeneration and loss of the articular cartilage joint surface. While the aetiology of osteoarthritis is unknown, risk factors include gender, age, obesity, and prior joint injury. Joint injuries, including tears of the anterior cruciate ligament (ACL) and meniscus, increase the risk for the development of OA and involve both mechanical damage to cartilage, meniscus and synovial tissues, and tissue degradation associated with cytokine-induced inflammation. While the role of inflammatory cytokines in OA is still controversial, their role in rheumatoid arthritis is evidenced by the successful use of anti-TNF-ca and anti-IL-1 therapies to abrogate disease symptoms and progression. In vitro, both IL-13 and TNF-ca promote chondrocyte-mediated matrix degradation and inhibit cartilage matrix synthesis, while mechanical damage causes cell death, matrix damage, and decreased cell biosynthesis. Understanding the similarities and differences in cartilage responses to inflammatory cytokines and mechanical injury is important in understanding the catabolic-anabolic shifts that typify OA progression. Therefore, the objectives of this thesis were (1) to identify the role of TNF-a and IL-1 induced nitric oxide (NO) as a mediator of cartilage tissue damage;(cont.) (2) to characterize and compare the regulation by IL- 1 P, TNF-cc, and mechanical injury of secreted factors, matrix degradation, and mechanisms of chondrocyte cell death using an SDS-PAGE-LC/MS/MS protein profiling approach; and (3) to further quantify the effects of IL-1 3, TNF-ca and injury using an isobaric isotope labeling (iTRAQ) based 2D-LC/MS/MS approach. Together these studies were designed to provide better understanding of matrix degradation, cell death, immune response, and evidence of cell-mediated repair processes. NO is produced by chondrocytes in response to inflammatory cytokines TNF-a, IL-1 3 and IL-17, and can mediate cellular and extracellular events through cGMP signaling, protein modifications (e.g., S-nitrosation or tyrosine nitration), altered transcript stability, and altered sugar and lipid chemistry. Cartilage was treated with IL-13 or TNF-ca left untreated in the presence or absence of the NO synthase inhibitor, L-N-methylarginine (L-NMA), and changes in gene expression and matrix breakdown were measured. We found that L-NMA treatment partially inhibited TNF-a-induced, aggrecanase-mediated aggrecan degradation as indicated by a decrease in sGAG loss to the medium and by an increase in the generation of aggrecanase-specific aggrecan fragments.(cont.) No change was observed upon addition of L-NMA to IL- 1P treated explants, but addition of L-NMA to combined IL-1 3 and TNF-a treated explants increased sGAG loss, suggesting that the effects of NO may be contextual. We hypothesized that this might be due to differences in aggrecanase expression (ADAMTS4 vs. ADAMTS5) or post-translational modification, but no aggrecanase was consistently identified in the samples. No difference in MMP expression or activation was noted following addition of L-NMA, and no change in NO chemistry between IL-1 3 and TNF-a treatment was evident by nitrate and nitrite production. Gene expression analysis was conducted on a battery of 32 genes, including matrix proteins, inflammatory mediators, proteases, cytokines and growth factors, and housekeeping proteins. While IL-113 and TNF-a both increased the expression of proteases and inflammatory mediators, addition of L-NMA did not significantly affect expression of the genes tested. We concluded that the effects of TNF-a and IL-1 p-induced NO production may depend on differences in cellular responses to each of these cytokines and possibly to differences in signaling or aggrecanase expression.(cont.) In the second study, newborn bovine calf cartilage explants were treated with 10 ng/ml IL-1 p, 100 ng/ml TNF-a, radially-unconfined injurious compression (strain: 50%; strain rate 1000/o/sec), or no treatment, and cultured for five days. Pooled medium was subjected to SDS-PAGE-LC/MS/MS, and data were analyzed by Spectrum Mill proteomics software, focusing on protein identification, differences between treatments and matrix protein proteolysis. Over 250 proteins were identified among the four protein groups including CD 109, platelet derived growth factor like protein, and scrapie responsive protein, which have not been previously identified in cartilage. IL-13 and TNF-a caused an increase in YKL39, YKL40, complement factor B, MMP-3, ECM-1, haptoglobin, serum amyloid A3, and clusterin. Injurious compression caused the release of intracellular proteins including GRP58, GRP78, alpha 4 actinin, pyruvate kinase, and vimentin, suggesting a loss of membrane integrity in a population of chondrocytes. Data on actin release within the first 24 hours suggested that this loss of membrane integrity occurred by mechanical cell disruption. Injurious compression also caused proteolysis of collagen type VI subunits, collagen type II, and COMP. Thrombospondin 1 fragments were seen in all treatment groups, and aggrecan proteolysis was predominant with cytokine treatment.(cont.) Cartilage explants subjected to injurious compression released intracellular proteins and showed enhanced degradation of matrix proteins, while explants subjected to IL- 13 or TNF-ct released proteins involved in innate immunity and stress response. In the third study, cartilage explants were subjected to injurious compression, TNF-a (100 ng/ml) or IL-13 (10 ng/ml), or no treatment, cultured in equal volumes of medium, and the medium was collected, pooled and the proteins deglycosylated by treatment with chondroitinase ABC. The proteins were subjected to trypsinization, and the peptides were labeled with one of four iTRAQ labels each containing a unique signature ion. The labeled peptides were subjected to nano-2D-LC/MS/MS on a QStar, quadrupole time of flight instrument. The study was done in analytical replicate on a pooled sample of greater than 70 explants from a total of 6-12 different animals. Data were analyzed by ProQuant to obtain a ProGroup peptide report containing identified spectra, which were combined to achieve a peptide, and then a protein level output of mean ratios, standard deviations of those ratios, and significance based on either Wilcoxan sign rank or Student's t-test both corrected for multiple comparisons.(cont.) Because of our interest in catabolic and anabolic shifts, a targeted data analysis approach was taken in addition to a systems level PCA and K-means clustering approach. By focusing on particular protein domains, we identified a decrease in the synthesis of most fibrillar collagen subunits (p<0.05), and an increase in the release of the aggrecan G2 and G3 domains with IL-13 and TNF-ta treatment (p<0.05). We also noted a significant increase in MMP-1, MMP-3, MMP-9, and MMP-13 in at least one condition and, in most cases, all conditions compared to the untreated sample. Increases in proteins involved in innate immunity and immune cell recruitment were noted with IL-1 [3 and TNF-a treatment, while an increase in intracellular protein release was seen most dramatically with mechanical compression injury. Since anabolic effects are often driven by the insulin-like growth factor family and the TGF-[3 superfamily, we specifically identified members of these pathways to understand which factors may mediate early repair processes.(cont.) At the systems level, 2 principal components were sufficient to describe 97% of the covariance in the data. IL- 1 and TNF-a caused a similar response in proteins identified; in contrast, a 'Y'-shaped distribution was observed upon projection of proteins based on their response injury vs. cytokine treatment. K-means clustering revealed six main clusters to further characterize the biology of mechanical injury versus cytokine effects on cartilage.by Anna L. Stevens.Ph.D