Um método de baixo custo é proposto para determinação direta de chumbo em açúcar por espectrometria de absorção atômica com forno de grafite e correção de fundo com fonte de deutério, empregando modificação química permanente com 250µg W + 200µg Rh e co-injeção de 5µg Rh. Os experimentos foram realizados com plataforma integrada ou ao no tubo de grafite com aquecimento longitudinal. Para avaliação da modificação química permanente, foram feitos experimentos na ausência de modificação e na presença de modificação química convencional com Pd+Mg. No decorrer do trabalho observou-se ser imprescindível a co-injeção de Rh para a estabilização térmica de chumbo na presença de açúcar. A amostra (8 g) é dissolvida em 100 ml de solução aquosa 0,2% v/v HNO3, e uma alíquota de 10 l é injetada com 5µl de solução de Rh na plataforma do tubo de grafite modelo Universal da Varian tratada com W e Rh. A massa característica (mo) e o limite de detecção do método foram 11 pg Pb e 5 ng g-1 respectivamente, e a vida útil do tubo de grafite foi de 860 queimas. O método apresentou boa reprodutibilidade com coeficiente de variação inferior a 2,5 % (n=3). As características analíticas foram comparadas com os métodos recomendados na literatura. A exatidão do método proposto para determinação direta de chumbo em açúcar foi avaliada pela comparação com método convencional utilizando Pd+Mg e nenhuma diferença estatística foi observada aplicando-se o teste t de Student ao nível de 95% de probabilidade, em amostras que receberam adição de chumbo / A simple method for the direct determination of lead in sugar by graphite furnace atomic absorption spectrometry (GFAAS) with platform treated with 250µg W + 200µg Rh and deuterium background correction is proposed. Samples (8 g) were dissolved in 100 ml of an aqueous solution containing 0,2% v/v HNO3 and 10µl aliquots were co-injected with 5µg Rh into the integrated platform modified with W-Rh of the Universal Varian longitudinal heated graphite atomizer. The characteristic mass (mo) and the method detection limit were 11 pg Pb and 5,0 ng g-1 Pb, respectively, and the tube life time was 860 firings. The relative standard deviation of measurements (n=3) was lower than 2,5 %. For the evaluation of the permanent chemical modifier, parallel experiments were carried in the presence of diluted nitric acid and with the conventional Pd+Mg chemical modifier. Experiments were made either with manual inserted graphite plataforms or integrated plataforms into longitudinal graphite tubes. Comparison of the proposed method with a method based on Pd+Mg chemical modifier showed no statistical differences by applying a t-test at 95% confidence level, for sugar sample solution spiked with lead

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Treating lesions in living tissues has become a health issue due to the late natural healing process, limited acceptance and compatibility with grafts. The development of biomaterials emerges as a new strategy. Thus, this study aimed to obtain bioactive porous composite, of different compositions and the evaluation of the influence of the composition on the physical, chemical, structural, morphological and biological properties for possible application in tissue engineering. The project started with the preparation and characterization of hydroxyapatite nanoparticles (HAP), via a wet precipitation route, and the modification of their surface by stearic acid (SA) (reflux). The presence of SA in the surface of HAP promoted significant changes in their general characteristics, especially in their morphology, size and thermal stability (TG/DTG). For the formation of composites, chitosan with different degrees of purity were chosen. To the homogeneous dispersion of ceramic in the polymer dispersion has required the incorporation of a viscosity agent, hydroxyethyl cellulose (HEC), which prevented the settling of HAP particles. Thus, six groups were evaluated and characterized by techniques that allowed to infer basic information about the degree of swelling, porosity, surface morphology, thermal stability, crystallinity and chemical environment (chemical groups). The cellular viability evaluated under the composites allows suggesting the biocompability of these. The study of the structural organization during lyophilization, using the small angle X-ray scattering, and evaluation of internal morphological structure of the composites under the influence of the composition was performed using X-ray Tomography. These analyzes showed that the presence of SA in the surface composition of nanoparticles inserted in composites influenced the agglomeration and deposition of HAP nanometric in the polymer structure, determining the structural characteristics of these composites. / Tratar lesões em tecidos vivos tornou-se uma problemática em saúde devido ao tardio processo de cicatrização natural, a uma limitada aceitação e compatibilidade com enxertos. O desenvolvimento de biomateriais surge como nova estratégia. Deste modo, este trabalho teve como objetivo principal a obtenção de compósitos bioativos porosos e avaliar a influência da composição nas características físicas, químicas, estruturais, morfológicas e biológicas para uma possível aplicação em tecido ósseo. A proposta iniciou-se com a obtenção e caracterização de nanopartículas de hidroxiapatita (HAP) (precipitação por via úmida) e com a modificação de sua superfície pelo ácido esteárico (AE) (refluxo). A presença do AE sob a superfície da HAP promoveu alterações significativas em suas características gerais, principalmente em suas morfologias, tamanhos e estabilidade térmica (TG/DTG). Para os compósitos, quitosanas com diferentes graus de pureza foram escolhidas. Com o intuito de homogeneizar a cerâmica sob a dispersão polimérica, fez necessária a incorporação de um agente de viscosidade, o hidroxietilcelulose (HEC). Sendo assim, seis grupos de compósitos foram avaliados e caracterizados por técnicas que permitiram inferir informações básicas a respeito do grau de intumescimento, da porosidade, da morfologia de superfície, da estabilidade térmica, da cristalinidade e do ambiente químico. A viabilidade celular avaliada sob os compósitos permitiu sugerir a biocompatibilidade destes. O estudo da organização estrutural durante a liofilização, utilizando-se o espalhamento de raios X a baixos ângulos, e a avaliação da estrutura morfológica interna dos compósitos sob influência da composição foi realizada através da tomografia de raios X. Tais análises permitiram inferir que a presença do AE na composição de superfície das nanopartículas inseridas nos compósitos influenciou na aglomeração e deposição das nanocargas de HAP sob a estrutura polimérica, determinando as características estruturais destes compósitos.

Orientador: Carmen Silvia Bertuzzo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T02:22:32Z (GMT). No. of bitstreams: 1 Furgeri_DanielaTenorio_D.pdf: 7382335 bytes, checksum: edfcfca14ac645a69fd4e8e2d2777246 (MD5) Previous issue date: 2013 / Resumo: A fibrose cística (FC) é uma doença autossômica recessiva com características de doença complexa. Complicações clínicas parece ser fator decisivo para o prognóstico dos pacientes. Os polimorfismos nos genes ADRA2A e TCF7L2 são importantes para elucidar parte da variabilidade encontrada nas características clínicas de doenças inflamatórias, incluindo a FC, que tem a Diabetes Mellitus como uma importante co-morbidade. Os objetivos deste estudo foram determinar a frequência do polimorfismo rs12255372 no gene TCF7L2 e sua associação com Diabetes Mellitus em pacientes com fibrose cística, e investigar a associação de 27 variáveis clínicas da FC com os polimorfismos rs553668 e rs10885122 do gene ADRA2A. Em nosso estudo, 145 pacientes foram avaliados em relação ao genótipo do polimorfismo rs12255372 no gene TCF7L2 e 176 pacientes foram avaliados em relação à associação dos polimorfismos rs553668 e rs10885122 no gene ADRA2A com 27 variáveis clínicas da FC. Todos os pacientes em atendimento no Ambulatório de Pediatria da Faculdade de Ciências Médicas da UNICAMP foram confirmados como tendo fibrose cística por dois testes de suor alterados (valor de sódio e de cloro superior a 60 mmol / L) e por análise de diferencial do epitélio da membrana do intestino através da dosagem de CFTR pela câmara Ussing. A identificação das mutações do gene CFTR foi realizada no laboratório de Genética Molecular da FCM/Unicamp. O rastreio do polimorfismo rs12255372 foi feito através da técnica de PCR associada à digestão enzimática específica. O rastreio dos polimorfismos rs553668 e rs10885122 no gene ADRA2A foi feito por PCR ARMS. Uma comparação genotípica foi realizada com as 27 variáveis clínicas, da FC considerando as mutações do gene CFTR. Encontramos associações clínicas, sem considerar as mutações no gene CFTR, com as variáveis categóricas: raça [para o polimorfismo rs553668 (p = 0,002), grupo haplotípico (p = 0,014)], íleo Meconial [para o polimorfismo rs553668 (p = 0,030) Quando consideradas as duas mutações no gene CFTR, encontramos associações com as variáveis íleo meconial (p = 0,0012) e IMC [para o polimorfismo rs553668 (p = 0,014)]. A associação com dados numéricos, sem considerar as mutações no gene CFTR, foi positiva para a idade ao diagnóstico [para o polimorfismo rs553668 (p = 0,022)]. Considerando as duas mutações no gene CFTR, a associação com dados numéricos foi positiva para o Escore de Bhalla [para o polimorfismo rs553668 (p = 0,014)], Escore de Shwachman-Kulczycki [para o polimorfismo rs553668 (p = 0,008) e haplótipos (p = 0,050)]. Os polimorfismos rs553668 e rs10885122 no gene ADRA2A parecem ser moduladores da gravidade da FC em nossa amostra. Em nossa amostra, não houve associação entre o polimorfismo rs12255372 no gene TCF7L2 e a Diabetes Mellitus / Abstract: Cystic fibrosis (CF) is an autosomal recessive disease with characteristics of complex disease. Clinical complications appear to be a decisive factor in the prognosis of patients. The ADRA2A and TCF7L2 gene polymorphisms are important to elucidate part of the variability encountered in clinical characteristics in inflammatory diseases, including CF, which has diabetes-associated as an important comorbidity. The aims of this study ware to determine the frequency of polymorphism rs12255372 in the TCF7L2 gene and its association with Diabetes Mellitus in Cystic Fibrosis patients and to investigate the association of 27 CF clinical variables with ADRA2A polymorphisms. In our study, 145 patients were evaluated in relation to the genotype of the rs12255372 polymorphism in the TCF7L2 gene. 176 patients were evaluated in relation to associate rs553668 and rs10885122 polymorphisms in the ADRA2A gene with 27 CF clinical variables. All patients in attendance at the Pediatric Clinic at the Faculty of Medical Sciences, UNICAMP, were confirmed as having cystic fibrosis by two altered sweat tests (sodium and chlorine value greater than 60 mmol/L) and by analysis of differential membrane epithelium of the intestine by the dosage of active CFTR through the Ussing chamber. The identification of CFTR gene mutations was performed in the laboratory of Molecular Genetics, FCM/Unicamp. The rs12255372 polymorphism was screening by PCR method associated with specific enzymatic digestion. The rs553668 and rs10885122 polymorphisms in ADRA2A gene were screening by ARMS-PCR. A genotypic comparison was performed with 27 CF clinical variables, considering CFTR mutations. We found clinical associations, without considering the mutations in the CFTR gene, with categorical variables: race [for polymorphism rs553668 (p = 0.002), haplotype group (p = 0.014)], meconium ileus [for polymorphism rs553668 (p = 0.030). Considering the two mutations in the CFTR gene, we find associations with categorical variables meconium ileus (p = 0.0012) and BMI [for polymorphism rs553668 (p = 0.014)]. The association with numerical data, without considering the mutations in the CFTR gene, was positive for age at diagnosis [for polymorphism rs553668 (p = 0.022)]. Considering the two mutations in the CFTR gene, the association with numerical data was positive for Bhalla score [for polymorphism rs553668 (p = 0.014)], Shwachman-Kulczycki score [for polymorphism rs553668 (p = 0.008) and haplotypes (p = 0.050)]. Polymorphisms rs553668 and rs10885122 in ADRA2A gene appear to be modulators of CF severity in our sample. In our sample, there was no association between the polymorphism rs12255372 in the TCF7L2 gene and Diabetes Mellitus / Doutorado / Clinica Medica / Doutora em Clínica Médica

The glial cells of the brain and the peripheral nervous system retain the capacity to divide and proliferate throughout the lifespan of an individual and thereby have the propensity to give rise to the most adult neurological tumours. Among them, the tumours which arise from different kinds of glial cells are referred to as gliomas. Of the various types of gliomas, astrocytomas are the most common central nervous system neoplasms which make upto 60% of all the primary brain tumours. Being the most prevalent type, the WHO classifies them into grades ranging from I to IV based on their intensity of malignancy. Grade IV astrocytoma or Glioblastoma (GBM) is considered to be the most malignant form with a median survival of 14.6 months, in spite of all therapeutic modalities. GBM is further classified as primary and secondary GBM. Primary GBM manifests de novo without any early history of pre-malignant lesions, on the other hand secondary GBM arises progressively from lower grades over a period of 5-10 years. Like other malignancies, GBM also arises from various genetic and epigenetic variations. Epigenetic variations include all such mitotically and meiotically heritable traits that do not involve changes in DNA sequence. There are three major areas of epigenetics - DNA methylation, histone modifications and non-coding RNAs which are known to have profound effects on gene expression. A lot being known about the genetic derailments in GBM, in this study we looked into the epigenetic aspects of GBM. In our lab, we have carried out various high throughput studies, which unveiled the distorted landscape of DNA methylation and miRNA expression in GBM. This indicates that, in addition to the genetic mechanisms of gene alterations like mutations, copy number aberrations, protein coding genes are also affected by changes in methylation as well as by miRNA misregulation. The study has been divided into two parts. Part one of the study deals with the identification of chromobox homolog 7 (Cbx7), as a hypermethylated and downregulated gene in GBM. More importantly, Cbx7 is a member of the polycomb repressive complex and brings about its function through chromatin modifications. Here we have investigated the role of Cbx7 in gliomagenesis, and why it has to be silenced by methylation for tumorigenesis to ensue. In part two, we elucidated two unique ways of miRNA regulation in GBM. In the first section, we identified miR-326 as a PI3 kinase regulated miRNA and demonstrated its tumour suppressive role in GBM. In the other section, we analysed the copy number aberration data from TCGA and identified miR- 4484 as a miRNA subjected to deletion in GBM. We further went ahead to demonstrate its growth suppressive role in GBM. Part 1: Epigenetic regulation of the chromatin modifier Cbx7; chromobox homolog 7 DNA methylation is involved in the normal cellular control of expression and thereby plays a crucial role in maintaining the homeostasis of the cell. The phenomenon of DNA methylation keeps the various loci of the genome such as the germline specific genes and the repetitive transposable elements silenced, whereas the tumour suppressors and other growth modulator genes are spared from the methylation induced gene repression. One of the important steps that promote tumorigenesis is aberrant hypermethylation, which leads to the silencing of tumour-suppressor genes. Another important epigenetic phenomenon that affects the transcriptibility of the genome is histone modifications, which control the accessibility of the chromatin to the transcriptional machinery. In this section, we identified Cbx7, which happens to be an essential component of the chromatin modifying machinery, as an epigenetically regulated gene in GBM. We observed from the methylation array carried out in our lab, that Cbx7 was one of the highly methylated genes. We also validated that Cbx7 is downregulated in GBM and the same observation was further corroborated from other data sets. The hypermethylated state of Cbx7 was confirmed by DNA bisulphite sequencing and the expression levels of Cbx7 also got alleviated after 5-Aza-2′-deoxycytidine treatment, which is a DNA methylation inhibitor. This indicated that the down regulation of Cbx7 could be attributed to the methylation of its promoter region. In order to figure out the role of Cbx7 in GBM, we carried out transcriptome analysis of Cbx7 overexpressing cells compared to vector control condition by RNA sequencing. Gene ontology analysis revealed a significant enrichment of pathways involved in cell cycle, migration and invasion like processes. In fact, the exogenous overexpression of Cbx7 leads to cell death, reduced colony formation, retarded migration and invasion of cells. In order to explain the above phenotypes brought about by the exogenous expression of Cbx7, we further examined the RNA sequencing data and observed that many of the top most downregulated genes in Cbx7 overexpression state belonged to the Hippo signaling pathway. The effectors of the Hippo pathway, YAP and TAZ which essentially antagonize the pathway activity, are well known for their role in proliferation, migration and invasion in cancer. So we carried out a Gene Set Enrichment Analysis (GSEA) and found that there was a significant negative enrichment of YAP/TAZ targets in the Cbx7 regulated gene set. We validated some of these targets that were downregulated by Cbx7 overexpression. One of the most downregulated genes that we validated was Connective Tissue Growth Factor (CTGF), which also happens to be a bonafide target of YAP/TAZ. Independent downregulation of CTGF also resulted in reduced migration, thereby phenocopying the effects as were produced by Cbx7 overexpression. Moreover, we also observed that SAPK/JNK was the only kinase whose activity was abolished upon Cbx7 overexpression. Since CTGF is known to activate SAPK/JNK, we assessed the SAPK/JNK activity upon CTGF silencing. We found that levels of phospho-SAPK/JNK were significantly reduced in CTGF silenced condition. In addition to that, the inhibition of the SAPK/JNK by synthetic inhibitor also hampered the migration ability of the cells. We were also able to rescue the loss of migratory potential of glioma cells by the exogenous overexpression of CTGF in Cbx7 stable background. A similar rescue was also achieved by the overexpression of a constitutively active form of SAPK/JNK. This indicates that Cbx7 activates Hippo pathway to inhibit YAP/TAZ dependent transcription, resulting in the downregulation of CTGF, thereby inhibiting CTGF mediated activation of SAPK and thus resulting in the inhibition of glioma cell migration. PART 2: ROLE OF MIRNAS IN GLIOMA DEVELOPMENT AND PROGRESSION miRNAs are a class of small non-coding RNAs that are not translated into functional proteins but still contribute to numerous cellular processes, thereby adding yet another realm of regulation and control. miRNAs bring about gene regulation at the post-transcriptional level, either by degrading the mRNA or by translational repression and in this manner fine tune the expression of protein coding genes. miRNAs are often located in the most fragile sites of the genome which exposes them to grave genetic alterations, thus providing a circumstantial evidence of their etiological role in tumorigenesis. In a malignant state, miRNAs have been found to play pivotal roles in cellular transformation by altering various cellular phenotypes. Owing to their participation in diverse cellular functions, miRNAs have gained a strong foothold in gene regulation. Though a lot has been deciphered about the functional aspect of miRNAs, not much is known about the precise mechanisms which lead to their misregulation and therefore demands in-depth study. The expression of miRNAs can be modulated by a variety of genetic and epigenetic mechanisms. Section I: Role of miR-326 – a PI3 kinase regulated miRNA, in gliomagenesis The TCGA group in the year 2008 identified three major pathways which go disarray in GBM. These include the pro-tumorigenic receptor tyrosine kinase (RTK) pathway, and the p53 and the pRB tumour-suppressive pathways. The RTK signalling includes the PI3 kinase pathway, which is pivotal in gliomagenesis and many other cancers. This directed us to elucidate the set of miRNAs which are controlled by the aberrant functioning of the PI3 kinase pathway. We used synthetic inhibitor LY294002 to abrogate the PI3 kinase signalling and examined the miRNA profile in two glioma cell lines U87 and U251, which have an activated PI3 kinase pathway. Indeed the abrogation of the PI3 kinase pathway resulted in the modulation of a wide array of miRNAs. We validated miR-326 as one of the miRNAs that was upregulated upon PI3 kinase pathway abrogation. Furthermore, we observed that miR-326 was a down regulated miRNA in GBM and different glioma cell lines, as well as in many other publicly available data sets. We also observed that miR-326 is an intragenic miRNA and its host gene Arrestin β1 (ARRB1) also exhibited similar upregulation upon PI3K pathway inhibition. Over-expression of miR-326 resulted in various anti-tumorigenic affects like reduced proliferation, reduced migration and colony suppression. In order to find the targets of miR-326, we analysed the transcriptome by RNA sequencing upon pre-miR-326 transfection. We shortlisted and validated some of the genes which were getting regulated through miRNA over-expression and also explain the functional role of miR-326. Section II: Role of miR-4484 – a copy number deleted miRNA, in gliomagenesis In the TCGA study mentioned above, it was also unfurled that there are many genes in the RTK, p53 and pRB signalling pathways which are made dysfunctional through gene deletions and amplifications. We envisaged whether it is only the protein coding genes which are subjected to such regulations or the non-coding genes like miRNAs as well. In this pursuit, we identified miR-4484 as one of the miRNAs located in the deleted region of uroporphyrinogen III synthase (UROS) gene in the chromosome 11 of the GBM genome. As conceived, miR-4484 was observed to be a downregulated miRNA in association with its host gene UROS. We further elucidated that the downregulation was due to the co-deletion of a locus harbouring both the protein coding gene and the miRNA. In addition, upon over-expression of miR-4484, we observed reduced migration and colony formation, indicating its role as a tumour–suppressor. For seeking the targets of miR-4484, we extracted RNA from miR-4484 over-expression condition and subjected it to RNA sequencing. We shortlisted and validated some of the genes which were getting regulated through miRNA over-expression and possibly explain the functional role of miR-4484.

Lubrication is critical in order to achieve high efficiency and reliability of machine elements such as gears, bearings, and other moving mechanical assemblies (MMA). In space applications, tribological properties of lubricants are quickly growing more important. Traditional space systems such as satellites imply MMA such as gyroscopes, antenna pointing mechanisms, and solar array drives. These MMA operate in high vacuum (<10-5 Pa) under lightly loaded conditions. Modern space missions on the other hand, such as remotely operated vehicles used for in-situ Mars exploration relies on different types of MMA. In these robotic systems, electromechanical actuators are being used extensively to provide controlled motion. Gears and bearings in these actuators operate in an atmosphere mainly consisting of CO2 at ~10+3 Pa under heavily loaded contact conditions. In these conditions, the tribosystem is likely to operate in the boundary lubricated regime, with consequent risk of high friction and wear. High molecular weight fluids have significant heritage in space because of their low vapor pressure. They are currently employed as lubricants in a wide range of space applications, as they meet high demands on resistance to vacuum outgassing. Unfortunately, the large molecules are susceptible to degradation under heavy load. Ionic liquids (ILs) on the other hand, are synthetic fluids that consist entirely of ion pairs with opposing charge. The resulting ion bonds enable inherently low vapor pressure of the fluid without the need for a high molecular weight. For this reason ILs have been advocated as potential lubricants for space applications, but so far compatibility issues have hampered their use as lubricants. Countless IL variations are possible, and solutions are thus likely to exist. Constituent ions can be designed individually and combined in various configurations. However, the fundamental understanding of the lubricating mechanism of ionic liquids is still incomplete, and consequently the optimum molecular structure for IL lubricants remain unknown. In this thesis, a stepwise approach to molecular design of IL lubricants is described, and the resulting hydrocarbon-mimicking ionic liquids are evaluated in tribological experiments. In this thesis, the experiments focus on tribological performance, using steel-steel tribopairs in air environment under boundary lubrication (Paper I). Boundary film formation under a range of contact pressures and temperatures, is analyzed after tribotesting by optical profilometry, scanning electron microscopy (SEM), and energy dispersive X- iii ray spectroscopy (EDS) in Paper II. The analysis reveal formation of a highly effective boundary film based on silicate, that can be further enhanced by amine additives. This thesis demonstrates the feasibility of improving tribological performance of ionic liquids by molecular design. / Projekt: Rymdforskarskolan 2015

In Canada, about 60% of contaminated sites involve petroleum hydrocarbon (PHC) contamination and most of these sites have been abandoned due to contamination. Among current technologies used for soil remediation, supercritical fluid extraction (SFE) is a relatively recent and potentially viable method. The main aim of this research was to investigate the application of SFE for removal of PHCs from contaminated soils. In the first phase, the effects of SFE operational parameters including fluid pressure, fluid temperature, time duration and mode of extraction on the removal efficiency of PHCs from a spiked sandy soil (with diesel fuel with a ratio of 5 wt%) were investigated. SFE experiments were performed at different pressures (15, 33 and 50 MPa) and temperatures (30, 75 and 120 °C). The combination of 10 min static mode followed by 10 min dynamic mode, repeated for 3 cycles (60 min in total) led to the highest PHC removal percentage. According to response surface methodology (RSM), the optimum pressure and temperature were found to be 50 MPa and 69.3 °C, respectively. According to experimental results, the optimum combination of pressure and temperature determined to be 33 MPa and 75 °C; which resulted in the extraction percentages of 99.2%, 91.7% and 86.1% for PHC F2, F3 and F4 fractions, respectively. In the second phase, the influence of several parameters including soil water content, soil pH and addition of modifier on PHCs removals from a field-contaminated sandy soil using SFE were experimentally investigated. SFE experiments were performed at 33 MPa pressure and temperatures of 45 and 75 °C. Three water content levels of 8%, 14% and 20% at two levels of pH 6.5 and 7.5 were investigated. The extraction of total petroleum hydrocarbon fractions (TPHF), the sum of F2, F3, and F4 fractions, decreased due to the increase in the water content from 8% to 20% at both pH 6.5 and 7.5. The difference of extractions of all PHC fractions at pH values of 6.5 and 7.5 were not statistically significant (at p < 0.05 confidence level) at all three water content levels and pH did not have a significant influence on the PHC removal efficiency. Addition of acetone as a modifier (33.7% TPHF removal) was more effective than hexanes (24.3% TPHF removal) to decrease the concentrations of PHCs for the field contaminated soil. In the third phase, the influence of soil texture and grain size on the extraction of PHC fractions was investigated. SFE experiments were performed at 33 MPa pressure and 75 °C temperature. Three types of soils (soil A, B and C) were spiked with diesel fuel with a ratio of 5 wt%. Soil A, B and C had different particle sizes and were categorized as sand, silt loam and clay, respectively. Soil A (sand) which had the largest particle size resulted in the highest TPHF removal percentage while soil C (clay) with the smallest particle size led to the lowest TPHF removal percentage. A higher clay content in soil C resulted in a lower extraction of PHCs. In the fourth phase, the effects of pressure and temperature on the extraction of PHC fractions from a clay soil spiked with diesel fuel with a ratio of 5 wt% were investigated. SFE experiments were performed at three pressures (15, 33 and 50 MPa) and temperatures (30, 75 and 120 °C). According to the statistical analysis including factorial design and RSM, the optimized combination of pressure and temperature was selected at 42.8 MPa and 120 °C; which resulted in the removal percentages of 74.9% and 65.6% for PHC F2 and F3 fractions, respectively. The optimum combination of pressure and temperature based on the experimental results was selected at 33 MPa and 120 °C that led to 70.3%, 58.4% and 32.6% removal of PHC F2, F3 and F4 fractions, respectively.

The thesis provides a quantitative survey and a detailed description of noun postmodification by single uncomplemented adjectives, i.e. cases where a modifying adjective phrase represented only by an adjective follows a head noun. The theoretical background of this thesis is based mainly on Randolph Quirk et al.'s A Comprehensive Grammar of the English Language (1985). The data for empirical corpus based research have been drawn from the British National Corpus by the means of a corpus query extracting the sequence noun+adjective+verb. This query returned 6,413 concordance lines out of which, after manual assessment, the resultant sample of 4,627 examples was compiled. The data obtained were further examined and categorized, revealing that up to eleven categories are needed to account for the syntactic, semantic, pragmatic factors, and other communicative implications and lexicalized conventions that motivate the use the postnominal position.

La mucoviscidose (CF) est la maladie génétique récessive létale la plus fréquente dans la population caucasienne. Elle est caractérisée par une obstruction et des infections des voies respiratoires et une inflammation chronique. La morbidité et la mortalité sont principalement dues à l'atteinte pulmonaire, qui est variable chez les patients, même lorsqu’ils sont porteurs du même génotype. Les facteurs responsables sont multiples : les mutations dans CFTR (le gène responsable de la maladie), les gènes modificateurs, mais aussi les facteurs environnementaux et les modifications épigénétiques. L'objectif principal de ce projet était de déterminer s'il y avait une corrélation entre la méthylation de l'ADN et la sévérité de l'atteinte pulmonaire chez les patients CF. Nous avons obtenu la cohorte METHYLCF (49 patients CF p.Phe508del homozygotes et 24 témoins sains) ainsi qu’une biobanque d'ADN à partir de sang total et de cellules épithéliales nasales (NEC). Les patients CF ont été stratifiés en fonction de leur VEMS, ajusté à l’âge. D’une part, nous avons analysé la méthylation de l'ADN dans CFTR plus 13 gènes modificateurs en utilisant la méthode de conversion au bisulfite et séquençage de nouvelle génération (plateforme 454 Roche). D’autre part, nous avons réalisé une analyse pan-génomique de la méthylation de l'ADN avec la plateforme 450k BeadChip (Illumina). Les sites différentiellement méthylés (DMS) sélectionnés ont été validés par pyroséquençage (PyroMark Q24, Qiagen). Deux gènes modificateurs ont été identifiés comme différentiellement méthylés chez les patients CF par rapport aux témoins: EDNRA dans le sang et HMOX1 dans le sang et dans les NEC. De façon intéressante, dans les NEC, la méthylation de EDNRA, HMOX1 et GSTM3 a été corrélée avec la sévérité de l’atteinte pulmonaire. De plus, de faibles niveaux de méthylation d'ADN dans GSTM3 ont été associés à la présence de l'allèle GSTM3*B, un polymorphisme de séquence qui a un effet protecteur chez les patients CF. Grâce à l'analyse tout-génome, nous avons identifié 1267 DMS, associés à 638 gènes, chez les patients CF par rapport aux témoins, et 187 DMS, associés à 116 gènes, chez les patients CF sévères par rapport aux modérés. Parmi ces gènes, il y a de nombreux gènes importants pour l’adhésion cellulaire et les réponses immunitaire et inflammatoire. Les DMS identifiés sont enrichis dans des régions prédites comme enhancers, pouvant représenter des séquences régulatrices, mais également en régions intergéniques. De façon intéressante, 80 gènes différentiellement méthylés sur 638 étaient différentiellement exprimés (méta-analyse de données transcriptomiques disponibles). Six sur neuf DMS sélectionnés ont été validés et cinq DMS sur six ont été répliqués dans une population indépendante. De plus, 23 DMS, dont 10 intergéniques, étaient corrélés avec le VEMS. Notre étude a montré que la méthylation de l'ADN est profondément modifiée dans le sang et dans les NEC des patients CF. Des faibles changements de méthylation de l'ADN ont été observés dans des gènes modificateurs connus ; des changements de méthylation plus importants ont été observés dans d'autres gènes qui pourraient représenter de nouveaux modificateurs de la fonction pulmonaire. Ensemble, ces gènes pourraient moduler la sévérité de l’atteinte pulmonaire chez les patients CF. / Cystic fibrosis (CF) is the most common life-threatening recessive genetic disease in the Caucasian population. It is characterized by airway obstruction, respiratory infection and inflammation. Morbidity and mortality are mainly due to lung disease, which is variable among CF patients, even for those having the same genotype. Contributing factors are mutations in CFTR (the disease-causing gene), modifier genes, but also environmental factors and epigenetics. The main goal of this project was to determine whether there was a correlation between DNA methylation and the severity of CF lung disease. We built the METHYLCF cohort (49 p.Phe508del homozygous CF patients and 24 healthy controls) and a DNA biobank from whole blood and nasal epithelial cells (NEC). CF patients were stratified accordion to their FEV1% predicted, adjusted to age. We profiled DNA methylation at 14 modifier genes using bisulfite conversion and next-generation sequencing (454 Roche). Genome-wide DNA methylation was analyzed with the 450K Beadchip (Illumina). Selected differentially methylated sites (DMS) were validated by pyrosequencing. Using the candidate modifier gene approach, we showed that two CF modifier genes were differentially methylated in CF patients compared to controls: EDNRA in blood and HMOX1 in blood and NEC. Methylation of EDNRA, HMOX1 and GSTM3 was associated with lung disease severity in NEC. Interestingly, low DNA methylation levels at GSTM3 were associated with the GSTM3*B allele, a polymorphic 3-bp deletion that has a protective effect on CF patients. In addition, through the genome-wide analysis, we identified 1267 DMS, associated with 638 genes, between CF patients and controls and 187 DMS, associated with 116 genes, between severe CF and mild CF patients. DMS were enriched at predicted enhancers, which may represent regulatory sequences, and also at intergenic regions. Gene ontology analyses highlighted cellular processes relevant to CF, i.e. cell adhesion and inflammatory and immune response. Interestingly, 80 out of 638 differentially methylated genes were differentially expressed in publicly available NEC transcriptomic data. Six out of 9 selected DMS were validated and five out of six DMS were replicated in an independent set of patients. Additionally, 23 DMS, 10 of which were intergenic, correlated with FEV1% predicted. Our study has shown that DNA methylation is altered in blood and NEC of CF patients. Small DNA methylation changes were observed at known CF modifier genes; more dramatic DNA methylation changes were found at other genes that may impact lung function. Collectively, these epigenomic variations may lead to different degrees of lung disease severity in CF patients.

Le secteur du transport en Europe dépend à 98% du pétrole, participe pour plus de 30% à la consommation énergétique et représente plus de 20% des émissions totales de gaz à effet de serre. Dans ce contexte, le développement des biocarburants permettrait une diversification énergétique et une diminution des émissions de gaz à effet de serre, les biocarburants ayant un impact environnemental réduit. De plus, le frottement dans les moteurs diesel est responsable de la perte de la consommation totale d'énergie de près de 15%. La plupart de ces pertes sont liées au contact piston-chemise. Développer les biocarburants peut alors contribuer à réduire d’une part la consommation d’énergie et d’autre part les émissions polluantes. L'additivation de biocarburants avec des modificateurs de frottement organiques (MF) peut être l’une des solutions pour répondre à cette problématique. Dans cette thèse, nous avons étudié les propriétés de réduction de frottement de différents types de MF organiques dans les matrices carburant (B0) et biocarburant (B7), en contact acier / acier. Une diminution significative du coefficient de frottement, de plus de 50%, a été observée en présence d'un mélange d'acides gras en matrice B7 à 100 °C. De plus, peu d’usure est causée durant le test en frottement grâce à la formation d'un tribofilm organique. Cette tribofilm semble être amorphe et généré par une réticulation accélérée des acides gras insaturés (MF) en présence d'esters (B7) dans les conditions tribologiques utilisées. Appuyé par des techniques d’analyses de surface, un mécanisme d'action de réduction de frottement par les additifs organiques dans les biocarburants est proposé et de nouvelles pistes de développement sont présentées. / Improve friction efficiency and environmental sustainability of engines have been increasing in importance since the end of 20th century. Indeed, friction in diesel engines is responsible for almost 15% loss of the total energy consumption. Most of these losses are related to the pistonliner contact. The development of new biofuels, allowing an energy diversification and decreasing the emissions of greenhouse gases due to their reduced environmental impact, can contribute to environmental sustainability and reduction in energy costs. The additivation of biofuels with organic friction modifiers additives (FM) can be an essential way to address this problem. In this thesis, we investigated the friction reduction properties of different kinds of organic FM in fuel (B0) and biofuel (B7) matrixes for steel/steel contact. A significant decrease of friction coefficient, more than 50%, was observed in the presence of a mixture of fatty acids blended to the B7 matrix at 100°C. Moreover, almost no wear is caused on the track thanks to the formation of an organic tribofilm. This organic-based tribofilm appears to be amorphous and generated by an easiest crosslinking of the unsaturated fatty acids (FMs) in the presence of esters under tribological conditions. Supported by advances surface analyses, a potential friction reduction action mechanism of acid-based additives in biofuels is proposed and new avenues for development are provided.

La reproduction sexuée conduit à l'alternance d'une phase haploïde et d'une phase diploïde, dont la durée relative est très variable entre taxons. La proportion du cycle de vie passée en phase haploïde et en phase diploïde a d'importantes conséquences sur de nombreux processus adaptatifs. Cette thèse combine des approches théoriques qui explorent l'effet de facteurs génétiques et écologiques sur l'évolution des cycles de vie, et un travail expérimental sur l'effet de la ploidy sur l'évolution de l'isolement reproducteur entre populations. La partie théorique a consisté à intégrer des composantes écologiques dans des modèles génétiques pour l'évolution des cycles de vie. En particulier, j'ai exploré l'interaction entre la différenciation de niche entre haploïdes et diploïdes (qui favorise le maintien de cycles biphasiques, impliquant le développement des deux phases) et l'effet d'allèles délétères (qui favorisent soit l'haploïdie, soit la diploïdie). Tandis que la différentiation de niche (ou plus simplement, une différence de valeur sélective intrinsèque entre phases) stabilise les cycles intermédiaires, la présence d'allèles délétères conduit souvent à un branchement évolutif, avec la coexistence stable d'allèles codant pour l'haploïdie et la diploïdie. Cependant, des fluctuations temporelles de l'habitat permettent d'empêcher ce branchement et de stabiliser les cycles biphasiques. La partie expérimentale a consisté à comparer la dynamique de l'isolement reproducteur entre petites populations de levure haploïdes et de diploïdes avec de taux de mutations élevés. Les résultats montrent que tandis que les hybrides haploïdes ont une valeur sélective plus faible que leurs parents, les hybrides diploïdes bénéficient du phénomène d'hétérosis en génération F1, et ont encore une valeur sélective plus élevée que leurs parents en génération F2. La variance de la valeur sélective des hybrides était cependant beaucoup plus élevée chez les haploïdes, avec la production de certains génotypes très performants. / Sexual reproduction leads to an alternation between haploid and diploid phases, whose relative length varies widely across taxa. The proportion of the life cycle spent in the haploid and diploid phase has important consequences on a number of adaptive processes. This thesis combines theoretical approaches exploring the effect of genetic and ecological factors on the evolution of life cycles, and experimental work on the effects of ploidy on the evolution of reproductive isolation between populations. The theoretical part consisted in integrating ecological components into genetic models for the evolution of life cycles. In particular, I explored the interplay between niche differentiation between haploids and diploids (known to favour the maintenance of biphasic life cycles, involving development in both phases) and the effect of deleterious alleles (known to favour either haploid or diploid life cycles). While niche differentiation (or more simply intrinsic fitness differences between phases) stabilizes biphasic cycles, the presence of deleterious alleles often lead to evolutionary branching and to the stable coexistence of alleles coding for haploid and diploid cycles. Branching is prevented, however, when temporal environmental fluctuations are included into the model. The experimental part consisted in comparing the dynamics of reproductive isolation between small populations of haploid and diploid yeasts with elevated mutation rate. The results show that while haploid hybrids tend to have a lower fitness than their parents, diploid hybrids benefit from heterosis in the F1 generation, and still have a higher fitness than the diploid homozygous parents in the F2 generation. However, the variance of hybrid fitness was much higher in haploids, with the production of some highly fit genotypes.