Dissertations / Theses on the topic 'Alpha variation'

Copy number variation (CNV) has been acknowledged as an important contributor to variation in the human genome, and copy-variable regions harbouring genes are interesting subjects of research for their potential phenotypic effects. However, despite the extensive and continuing discovery of CNVs, multiallelic loci remain technically challenging to measure and study. In this thesis, a PCR-based measurement system for the tandemly repeated CNV that harbours the DEFA1A3 locus has been developed. This locus can either have the DEFA1 or the DEFA3 gene in any given copy of the repeat; the two genes differ by a single nucleotide difference in the coding sequence. This CNV has previously been shown to vary from 4 to 11 copies in a sample of the UK population. The use of this measuring system has allowed a usefully accurate measure and some characterization of this CNV in Europeans, Asians (Chinese and Japanese) and African (Ibadan, Nigeria) samples from the HapMap project, agreeing with the previously found copy number range in Europeans and showing more variability in non-Europeans. Typing of three-generation CEPH families has allowed the inference of haplotype copy numbers from segregation analysis. Combining haplotype data for some CEPH HapMap samples that were part of these families with their SNP genotypes in the same LD block has shown copy number lineages that are surprisingly well-tagged by SNPs. SNP rs4300027 allows the division of copy number haplotypes into low (2 and 3-copy) and high (4 and 5-copy) groups in European HapMap samples, a result that has been corroborated in an independent set of European samples. The Asian and African HapMap samples have failed to show this particular association. However, Japanese samples show copy number lineages tagged by other SNPs, an association not observed in other populations. In the second part of the study, an attempt has been made to explore the phenotypic effects of this variable locus. Copy number-tagging SNPs have been used to investigate published GWAS for indirect signals of association with DEFA1A3 copy number. Preliminary experiments for studying protein expression levels of DEFA1 and DEFA3 have been carried out and the possible role of this CNV as a modifier locus in Cystic Fibrosis disease severity has been investigated through direct typing of CF samples with clinical data, and indirectly through interrogating a CF GWAS.

Alpha 1 antitrypsin deficiency (AATD) is the only established genetic predisposition to chronic obstructive pulmonary disease (COPD). The development of COPD in AATD is highly variable, probably relating to complex interactions between multiple genetic and environmental factors. This thesis will describe the COPD phenotypes observed in AATD and their inter-relationships, forming the basis for examining phenotypic associations with specific candidate genes and HLA class II type. Finally it examines the potential role of ambient air pollution. Associations were seen for TNFA with chronic bronchitis, SFTPB with FEV1, TGFB with small airways disease and GC with bronchiectasis, consistent with the role of protein products in pathogenesis. Of four MMPs studied, association with gas transfer occurred in two. HLA-DQA1*0301 and HLA-DRB1*04 contributed significantly to gas transfer in regression models, and anti elastin antibodies were higher in HLA-DRB1*04 and HLA-DQA1*0301 homozygotes. Ozone levels contributed to the burden of disease in cross sectional and longitudinal models of pollution exposure, whilst PM10, NO2 and SO2 were associated only in the longitudinal model. In conclusion this thesis demonstrates the importance of genetic variation and environmental factors in determining respiratory phenotype in AATD. It also suggests a key role for adaptive immunity in pathogenesis of emphysema.

At present several groups are analysing quasar absorption spectra to search for variation of the fine structure constant, alpha, across space and time. These studies compare the wavelengths of several transitions observed in the absorption clouds with those seen in the laboratory, and interpret anomalies as variation in alpha. One group has already presented evidence that alpha may have been smaller at an early epoch. Other groups using different telescopes see no variation. These studies use the ???many-multiplet??? method, which relies on the utilisation of many transitions in many ions to enhance the size of the effects and remove sources of systematic error. While this method offers an order-of-magnitude improvement in sensitivity over the previously used alkali-doublet method, the alpha-dependence (relativistic shift) of every transition used in the analysis must be calculated ab initio. In this thesis we present a method for the precise calculation of relativistic shifts, based on an energy calculation involving combination of the configuration interaction method and many-body perturbation theory. The many-multiplet method also introduces a potential systematic error: if the relative isotope abundances of the absorbers differ from terrestrial abundances then there can be spurious shifts in the measured wavelengths, which may be incorrectly interpreted as variation of alpha. A ???conspiracy??? of several isotopic abundances may provide an alternative explanation for the observed spectral anomalies. To account for these systematic errors we need accurate values of the isotope shift. We calculate these shifts using the finite-field method to reduce the problem to that of an energy calculation, which in turn is done using the same method used for the relativistic shift. We present the results of our calculations for a variety of atoms and ions seen in quasar absorption spectra. The results of this research should allow astrophysicists to measure isotope abundances in the absorbers directly. This can provide a test for models of nuclear reactions in stars and supernovae, and of the chemical evolution of the Universe. Our calculations can also be used in conjunction with measurements to extract changes in nuclear charge radii between isotopes.

Despite very extensive studies related to molecular processes underlying memory formation, still little known about the potential differences in the brain biochemistry between &ldquo
good&rdquo
and &ldquo
poor&rdquo
learners belonging to a random population of young animals. In the present study, an attempt was taken to correlate the individual variation in short- and long-term spatial memory in three different lines of young, healthy rats: inbred Wistar (W), outcrossed Wistar/Spraque Dawley (W/S) and pigmented Long-Evans rats, with hippocampal levels of selected enzymes known as &ldquo
memory molecules&rdquo
including neuronal (n), endothelial (e) and inducible (i) NOS, CaMKII&alpha
, PKA and ChAT. Additionally, in order to indirectly estimate the activity of CaMKII&alpha
and PKA, hippocampal levels of their phosphorylated forms (pCaMKII&alpha
and pPKA) were assessed. Rats were classified as &ldquo
good&rdquo
and &ldquo
poor&rdquo
learners on the basis of their performance in a partially baited 12-arm radial maze. The hippocampal protein levels were measured using Western Blot technique. In addition to individual variation in animals&rsquo
learning capacity, strain-depended differences have also been observed. Deficient performance recorded in inbred W rats compared to outcrossed W/S rats, and &ldquo
poor&rdquo
learners from both rat groups had predominantly related to the higher frequency of reference memory errors. The results of biochemical assays showed strain-depended differences in the NOS expression. The overall NOS levels were significantly higher in outcrossed W/S rats compared to inbred W rats. In both rat lines, the rate of learning positively correlated with hippocampal levels of nNOS and negatively correlated with iNOS levels. Hippocampal eNOS levels correlated negatively with animals&rsquo
performance but only in the W rats. These results suggested that all 3 NOS isoforms are implemented in the learning process playing, however, different roles in neural signaling. Experiments carried out on Long-Evans rats did not reveal a significant difference in the basal hippocampal levels of the CaMKII&alpha
, however, the level of the pCaMKII&alpha
, was significantly higher in &ldquo
good&rdquo
learners. Also, hippocampal levels of both PKA and pPKA, as well as that of ChAT were significantly higher in &ldquo
good&rdquo
as compared to &ldquo
poor&rdquo
learners. Taken together, the latter findings indicate that low hippocampal expression of PKA and ChAT as well as low CaMKII&alpha
or PKA activation may cause learning deficits in random population of young rats, and thus, these enzymes can be considered target molecules when looking for cognitive enhancers to treat memory deficits in young subjects.

Elevated total plasma homocysteine (tHcy) is a risk factor for cardiovascular disease and neurocognitive disease such as dementia. The B vitamins folate and B12 are the main de terminants of tHcy. tHcy concentration can also be affected by mutations in genes coding for receptors, enzymes and transporters important in the metabolism of Hcy. This thesis focuses on mutations in the genes for folate receptor-alpha and methylenetetrahydrofolate reductase (MTHFR) and the effect they have on tHcy concentrations. Six novel mutations in the gene for folate receptor-alpha were described in Paper I. Taken together they exist in a population with a prevalence of approximately 1% and thus are not unusual. There may be an association of –69dupA and –18C>T to tHcy but for the 25-bp deletion, –856C>T, –921T>C and –1043G>A there is probably no association to tHcy. Mutation screening was continued and four additional mutations, 1314G>A, 1816delC, 1841G>A and 1928C>T, were described in Paper II. The prevalences for the heterozygotes were between 0.5% and 13% in an elderly population. There was no significant difference in prevalence between the elderly subjects and patients with dementia. The 1816(–)-allele and the 1841A-allele were in complete linkage and the haplotype 1816(–)-1841A may possibly have a tHcy raising effect. The 1314G>A and 1928C>T mutations had no association to tHcy. The genotype prevalences and haplotype frequencies of the MTHFR 677C>T, 1298A>C and 1793G>A polymorphisms were determined in a population sample of Swedish children and adolescents (Paper III). The MTHFR 677T-allele was associated with increased tHcy concentrations in both children and adolescents. A small elevating effect of the 1298C-allele and a small lowering effect of the 1793A-allele could be shown. In an epidemiological sample of adults from the Canary Islands, Spain, data for serum folate and vitamin B12 were used for a broader study of the nutrigenetic impact on tHcy (Paper IV). The 677T-allele had a significant tHcy increasing effect in men but not in women. The 1298C-allele had a minor elevating effect on tHcy in men with the 677CT genotype. It was not possible to document any effect of the 1793A-allele on tHcy due to its low prevalence. A slightly superior explanatory power for the genetic impact was obtained using the MTHFR haplotypes in the analysis compared to the MTHFR 677C>T genotype-based approach in both the Swedish children and adolescents and in the Spanish adults. Therefore MTHFR haplotypes should be considered when analysing the impact of the MTHFR 677C>T, 1298A>C and 1793G>A polymorphisms on tHcy. Notwithstanding the large geographical distance between our study populations the haplotype composition is quite similar. The MTHFR 677T-allele is slightly more prevalent in Spain compared to Sweden but it has only an effect on tHcy in the Spanish men. Age, gender and factors linked to the ethnicity of the studied subjects, seem to be able to override the nutrigenetic impact of tHcy-raising genotypes or haplotypes in particular settings, such as in the Spanish women in our study. Gene-nutrient interactions on plasma tHcy levels thus may or may not exist in a certain population. The transferability of nutrigenetic findings may therefore be limited, and must be re-evaluated for each particular setting of age-gender-ethnicity.

Darbe taikyta Norvaišos ir Salopk (2002) metodologija funkcijos šiurkštumui nagrinėti remiantis modifikuotu funkcijos grafiko dėžučių skaičiaus indeksu. Funkcijos šiurkštumas nusakomas p-variacijos indeksu, kuris prie tam tikrų sąlygų lygus fraktalo dimensijai. Darbe ištirtos tiesinės regresijos, kuri vertina p-variacijos indeksą, liekanos ir pasiūlytas būdas kaip išpildyti balto triukšmo prielaidas. Rezultatai apibendrinti Monte Carlo procedūra. Sukonstruoti p-variacijos indekso pasikliautinieji intevalai -stabiliam ir trupmeniniam Brauno judesio procesams. Ištirtas p-variacijos indekso kintamumas laike „Vallourec” akcijų kainos procesui.
To estimate the roughness of the sample function the methodology introdused in Norvaiša and Salopek (2002) was applied. The roughness is defined as p-variation index of the sample function graph. Methodology is based on linear regression of the oscilation index. This master thesis tests the assumptions of linear regression residuals and constructs estimator which fulfill these assumptions. The model was used for the generated α-stable process and fractional Brownian motion. Conclusions are generalized using Monte-Carlo procedure. The confidence intervals for the p-variation index was constructed making assumption that the process is the realisation of -stable or fractional Brownian motion. The p-variation index was estimated for the „Vallourec” stock price data, sampled at irregular time. In addidion the variability in time of p-variation index was studied for different segments of intervals.

A key prediction of the Capital Asset Pricing Model (CAPM) is that idiosyncratic risk is not priced by investors because in the absence of frictions it can be fully diversified away. In the presence of constraints on diversification, refinements of the CAPM conclude that the part of idiosyncratic risk that is not diversified should be priced. Recent empirical studies yielded mixed evidence with some studies finding positive correlation between idiosyncratic risk and stock returns, while other studies reported none or even negative correlation. In this thesis we revisit the problem whether idiosyncratic risk is priced by the stock market and what the probable causes for the mixed evidence produced by other studies, using monthly data for the US market covering the period from 1980 until 2013. We find that one-period volatility forecasts are not significantly correlated with stock returns. On the other hand, the mean-reverting unconditional volatility is a robust predictor of returns. Consistent with economic theory, the size of the premium depends on the degree of 'knowledge' of the security among market participants. In particular, the premium for Nasdaq-traded stocks is higher than that for NYSE and Amex stocks. We also find stronger correlation between idiosyncratic risk and returns during recessions, which may suggest interaction of risk premium with decreased risk tolerance or other investment considerations like flight to safety or liquidity requirements. The difference between the correlations between the idiosyncratic volatility estimators used by other studies and the true risk metric - the mean-reverting volatility - is the likely cause for the mixed evidence produced by other studies. Our results are robust with respect to liquidity, momentum, return reversals, unadjusted price, liquidity, credit quality, omitted factors, and hold at daily frequency.

Ventricular dysfunction and dilated cardiomyopathy (DCM) develop among untreated HIV-infected people at much higher rates than among HIV-negative individuals, resulting in significant contributions to morbidity and mortality. Mechanisms underlying development of HIV-associated cardiomyopathy (HIVCM) are as yet poorly understood. The well-characterized simian immunodeficiency virus (SIV) model of HIV infection provides a unique context for HIVCM pathogenesis studies in that SIV-infected rhesus monkeys develop myocardial lesions and contractile dysfunction similar to those described in HIV-infected people, suggesting a shared disease mechanism. Lymphocytic myocarditis is a commonly reported finding in AIDS patients at autopsy and constitutes one of several conditions known to predispose to development of DCM, irrespective of HIV-infection status. As lymphocytic myocarditis also occurs with high frequency among SIV-infected rhesus monkeys, a retrospective analysis of rhesus monkey cardiac tissue collected at necropsy was performed to examine viral and cellular correlates of lymphocytic inflammation within myocardial tissue. One subpopulation of macrophages, which has been reported by other groups to be associated with an anti-inflammatory phenotype, was found to correlate inversely with lymphocytic infiltration and positively with numbers of virus infected cells, suggesting effects of an anti-inflammatory cytokine production profile. In contrast, the detrimental effects of inflammatory cytokines on myocardial structure and function are well-recognized and HIV infection in general is characterized by chronic immune activation and inflammatory cytokine dysregulation. To further investigate a role for myocardial cytokine production in development of HIVCM, a prospective study was conducted in which SIV-infected rhesus monkeys and uninfected controls were treated with recurrent administration of inactivated Mycobacterium aviumcomplex bacteria (MAC). SIV-infected, MAC-treated animals rapidly developed significant ventricular systolic dysfunction and chamber dilatation not seen in control groups, suggesting an exaggerated myocardial sensitivity to exogenous antigenic stimulation. Concurrent treatment with the TNFα antagonist etanercept completely abrogated development of these changes, strongly implicating a causative role for TNFα in evolution of the contractile dysfunction and chamber remodeling. Findings reported from the current studies suggest that characteristics of local myocardial macrophage populations and the myocardial tissue cytokine milieu may play more important roles than lymphocytic infiltration, cardiomyocyte damage, or viral proteins in the pathogenesis of HIVCM.

Entender padrões de diversidade e composição de espécies ao longo de múltiplas escalas espaciais constitui um dos principais objetivos em ecologia e biogeografia. A relativa importância dos mecanismos responsáveis por estruturar as comunidades de plantas e como eles interagem para influenciar estes padrões têm sido foco de intensos debates. No presente estudo, foram utilizados dados do Inventário Florístico Florestal de Santa Catarina a fim de investigar os padrões de diversidade de espécies de árvores e suas relações com a heterogeneidade ambiental sob uma das perspectivas oriundas da teoria de metacomunidades, conhecida como “sorteio de espécies”. A predição chave deste ponto de vista é a de que a composição de espécies varia em resposta a diferenças nas condições ambientais entre manchas de hábitat. O presente estudo é focado nessa predição e objetivou entender como processos relacionados a filtros ambientais interagem direta e indiretamente sobre os padrões de diversidade em uma área de 95000 km 2 (dados de 432 unidades amostrais). Foi utilizada modelagem de equações estruturais (PLS Path Modeling), a fim de investigar os efeitos interativos da topografia, clima, balanço de água e energia e geometria das manchas de floresta sobre os padrões de alfa (α) e beta (β) diversidade de uma metacomunidade de floresta atlântica no sul do Brasil. Fatores relacionados a filtros ambientais mostraram substanciais efeitos sobre a diversidade alfa e beta. A quantidade total da variação na beta diversidade explicada pela filtragem de hábitat foi alta (64%), corroborando a predição testada no nível de metacomunidades. Os fatores mais importantes para explicar a diversidade beta foram: extremos climáticos, balanço de água e energia e alfa diversidade, enquanto tamanho da mancha e balanço de água e energia foram os fatores chaves para a alfa diversidade. O teste de Mantel parcial mostrou que os efeitos ambientais ocorrem amplamente independente de efeitos espaciais, reforçando a predição testada. O estudo provê forte suporte empírico para a predição de que a beta diversidade reflete primariamente processos determinísticos associados com o nicho das espécies e suas respostas às condições ambientais na escala espacial considerada.
Understanding patterns of species diversity and composition across multiple scales is one of the main purpose in ecology and biogeography. The relative importance of the mechanisms that structure plant communities and how they interact to influence these patterns remains a topic of hot debate. In the present study, we use data from the Forest Inventory of Santa Catarina to investigate the patterns of species diversity of subtropical Atlantic forests and its relationships with environmental heterogeneity on a metacommunity perspective (species-sorting). The key prediction of this viewpoint is that community composition varies in response to differences in environmental conditions among habitat patches. Our study focused on this perspective, aiming to understand how environmental filtering processes interact directly and indirectly on diversity patterns in an area of 95000 km 2 (data from 432 forest plots). We employed structural equation modeling (PLS Path Modeling) to disentangle the interactive effects of topography, climate, water-energy balance, and geometry of forest patches upon the alpha and beta diversity of a subtropical forest metacommunity in southern Brazil. Factors related to environmental filtering showed substantial effects upon tree alpha and beta diversity. The total amount of variation in beta diversity explained by environmental filtering was high (64%) and was even more when together with alpha diversity (73%), corroborating the prediction of species-sorting model at the metacommunity level. Climatic extremes, water-energy balance and alpha diversity were the key determinants of beta diversity and patch size and water- energy balance the key determinants of alpha diversity in the South Brazilian Atlantic forests. Partial mantel test showed that environmental effects occurred largely independent of spatial effects, reinforcing the tested prediction. Our study provides strong empirical support for the prediction that beta diversity primarily reflects deterministic factors associated with species niches and their responses to environmental conditions in the studied spatial scale.

Plus de 1% de la population mondiale est chroniquement infectée par le virus de l'hépatite C (VHC). La découverte et la caractérisation du VHC représente un succès majeur des applications techniques de clonage dans l'identification de nouveaux agents infectieux. L'infection par le VHC peut conduire à une hépatite aiguë, une hépatite chronique, voire une cirrhose qui est fortement associée au développement de carcinome hépatocellulaire. Le génotype 1b est le plus fréquemment rencontré dans le monde, et il est caractérisé par un faible taux de réponse à l'interféron alpha. [. . . ]

Nous avons mis au point une méthode de mesure du déplacement de fréquence dû aux collisions entre atomes froids, c'est l'effet systématique qui limite le plus l'exactitude des fontaines à 133Cs (~1E-15 en valeur relative), on peut le mesurer au niveau de 0.5%. Ceci ouvre des perspectives d'améliorations des performances des fontaines en terme d'exactitude jusqu'à 1E-16. La fontaine à aussi obtenu une stabilité de l'ordre de 1.6E-14 à 1s. Nous avons découvert, à champ magnétique très faible (5 +/- 1 mG), des résonances de Feshbach. Nous avons aussi effectué une nouvelle mesure absolue de la transition hyperfine du 87Rb, qui est la plus précise jamais réalisée et sert maintenant de définition pour l'étalon secondaire de fréquence 87Rb. En comparant cette valeur avec celles mesurées les années précédentes, nous avons pu tester la stabilité de la constante de structure fine au niveau de 1E-15/ an. Nous avons comparé localement notre fontaine avec les autres fontaines du laboratoire, avec dans le meilleur des cas une stabilité combinée de 5E-14 à 1s. La différence de fréquence des deux horloges se moyenne comme du bruit blanc de fréquence jusqu'à 3E-16. Le bilan d'exactitude de la fontaine double à été évalué à ~7E-16 pour la partie césium et ~8E-16 pour la partie rubidium. Nous avons contribué à la réalisation de l'échelle de Temps Atomique International, par des séries de calibrations de masers à hydrogène. Une comparaison de fontaines atomiques par liaisons satellitaires a été expérimenté entre notre laboratoire et nos homologues Allemands. Cette mesure a permis de déterminer le bon accord qu'il y a entre les deux horloges.

Class of 2012 Abstract
Specific Aims: The epithelial sodium channels (ENaC) found on the apical membranes of epithelial cells including those lining the respiratory tract are the rate limiting step of the absorption of excess fluid from the airspace of the alveoli. ENaC function is modulated by the effects of various physiologic signals such as the adrenergic and purinergic pathways, in addition to other local channels which control the flow of negatively charged ions such as the cystic fibrosis transmembrane conductance regulator (CFTR). We sought to determine the influence of genetic variation on the alpha subunit of ENaC at amino acid position 663 on baseline exhaled ions and pulmonary function in patients with CF. Methods: We assessed pulmonary function ( forced vital capacity[FVC], forced expiratory volume in one second [FEV1], forced expiratory flow maximum[FEFmax]) using a Medical Graphics cardiopulmonary testing device (Minneapolis, MN). Measures of exhaled sodium (Na+) and chloride (Cl-) were obtained using exhaled breathe condensate collected on a Jaeger Ecoscreen condenser unit (Cardinal Health, Yorba Linda, CA) with Na+ quantification using an atomic absorption spectrophotometer (Analyst 100; Perkin Elmer, Norwalk, CT) and Cl- anion quantification using a Dionex AS11 HC column. Healthy n=31 (n=18[58%], 9[29%], and 4[13%] subjects; Body mass index (BMI)=23±1, 25±2, and 25±2kg/ m2 for AA, AT and TT groups respectively). CF n= 42 (n=33[79%], 7[16%], and 2[5%] subjects; BMI equals 23±7, 19±0.4, and 20±2.2kg/m2 for AA, AT and TT groups respectively). Main Results: We found that the distribution of genotypes in CF differed from healthy subjects, with the AA genotype in 80% of CF and 59% in healthy. No significant difference were demonstrated in healthy subjects between genotype groups for pulmonary function and exhaled chloride while the genotypes did differ in exhaled Na (Na=2.9±0.4, 1.7±0.3, and 3.7±1.1mmol/L for AA, AT, and TT respectively, ANOVA p=0.07). CF subjects with the AA genotype had a higher baseline exhaled Cl-, FEV1, and FEFmax than those in the AA group (Cl=0.125±0.038,0.0 27±0.007, and 0.033±0.02 mmol/L ; FEV1=71±5, 68±11, and 40±22L; FEFmax=86±4, 72±7, and 44±24L/sec; for AA, AT, and TT respectively, ANOVA p<0.05, Tukey [AA vs. TT] p<0.05) while exhaled Na+ and FVC were similar between genotypes. Conclusions: Our results suggest that CF subjects with the AA genotype of the alpha subunit of the ENaC have a higher baseline exhaled Cl- and a resulting increase in pulmonary function when compared to the overactive TT groupCF patients with the TT αENaC genotype are likely candidates for early identification and treatment with inhaled ENaC inhibitors or other modulators of this pathway in order to improve survival.

Class of 2012 Abstract
Specific Aims: The epithelial sodium channels (ENaC) found on the apical membranes of epithelial cells including those lining the respiratory tract are the rate limiting step of the absorption of excess fluid from the airspace of the alveoli. ENaC function is modulated by the effects of various physiologic signals such as the adrenergic and purinergic pathways, in addition to other local channels which control the flow of negatively charged ions such as the cystic fibrosis transmembrane conductance regulator (CFTR). We sought to determine the influence of genetic variation on the alpha subunit of ENaC at amino acid position 663 on baseline exhaled ions and pulmonary function in patients with CF. Methods: We assessed pulmonary function ( forced vital capacity[FVC], forced expiratory volume in one second [FEV1], forced expiratory flow maximum[FEFmax]) using a Medical Graphics cardiopulmonary testing device (Minneapolis, MN). Measures of exhaled sodium (Na+) and chloride (Cl-) were obtained using exhaled breathe condensate collected on a Jaeger Ecoscreen condenser unit (Cardinal Health, Yorba Linda, CA) with Na+ quantification using an atomic absorption spectrophotometer (Analyst 100; Perkin Elmer, Norwalk, CT) and Cl- anion quantification using a Dionex AS11 HC column. Healthy n=31 (n=18[58%], 9[29%], and 4[13%] subjects; Body mass index (BMI)=23±1, 25±2, and 25±2kg/ m2 for AA, AT and TT groups respectively). CF n= 42 (n=33[79%], 7[16%], and 2[5%] subjects; BMI equals 23±7, 19±0.4, and 20±2.2kg/m2 for AA, AT and TT groups respectively). Main Results: We found that the distribution of genotypes in CF differed from healthy subjects, with the AA genotype in 80% of CF and 59% in healthy. No significant difference were demonstrated in healthy subjects between genotype groups for pulmonary function and exhaled chloride while the genotypes did differ in exhaled Na (Na=2.9±0.4, 1.7±0.3, and 3.7±1.1mmol/L for AA, AT, and TT respectively, ANOVA p=0.07). CF subjects with the AA genotype had a higher baseline exhaled Cl-, FEV1, and FEFmax than those in the AA group (Cl=0.125±0.038,0.0 27±0.007, and 0.033±0.02 mmol/L ; FEV1=71±5, 68±11, and 40±22L; FEFmax=86±4, 72±7, and 44±24L/sec; for AA, AT, and TT respectively, ANOVA p<0.05, Tukey [AA vs. TT] p<0.05) while exhaled Na+ and FVC were similar between genotypes. Conclusions: Our results suggest that CF subjects with the AA genotype of the alpha subunit of the ENaC have a higher baseline exhaled Cl- and a resulting increase in pulmonary function when compared to the overactive TT groupCF patients with the TT αENaC genotype are likely candidates for early identification and treatment with inhaled ENaC inhibitors or other modulators of this pathway in order to improve survival.

Questo lavoro di tesi ha riguardato l’acquisizione e l’elaborazione di segnali elettroencefalografici (EEG) registrati su 18 volontari mentre veniva loro chiesto di eseguire uno specifico task motorio, ovvero flesso-estensione dell’arto superiore destro a due diverse velocità, per indagare possibili differenze nei segnali EEG associate alla velocità di esecuzione del movimento. Il protocollo sperimentale è stato strutturato in tre blocchi intervallati da una breve pausa di riposo. Ogni blocco consta di 90 trial: si susseguono in maniera random 30 comandi ‘slow’ (il soggetto deve muoversi lentamente), 30 comandi ‘fast’ (il soggetto deve muoversi velocemente), 30 comandi ‘rest’ (il soggetto deve restare fermo). Ogni trial inizia con 1 s di rilassamento; segue il primo segnale (‘cue’ una scritta con l’indicazione del task da eseguire); in corrispondenza alla sua scomparsa si presenta il secondo segnale (‘go’ un beep di 10 ms che indica al soggetto di eseguire il task); infine sono concessi al soggetto 5 s durante i quali eseguire il task. Le elaborazioni sono state condotte sui 16 segnali EEG e su un segnale accelerometrico, acquisiti rispettivamente mediante elettrodi premontati su cuffia e sensore inerziale fissato sul dorso della mano destra. Sono stati analizzati i seguenti correlati neurali del movimento: contingent negative variation, un potenziale di anticipazione motoria identificato nel dominio del tempo tra ‘cue’ e ‘go’; event-related de/synchronization, un incremento/decremento di potenza, osservabile in specifiche bande di frequenza (alpha e beta) e in specifici intervalli nelle mappe tempo-frequenza. Per valutare infine la significatività dei risultati ottenuti si è condotta un’analisi statistica (t-test). Sebbene i risultati ottenuti non evidenzino differenze significative nelle caratteristiche del segnale EEG tra i movimenti ‘slow’ e i movimenti ‘fast’, lo studio si pone come importante punto di partenza per investigazioni future.

Background. Ray tracing can be used to achieve hyper-realistic 3D rendering but it is a computationally heavy task. Since hardware support for real-time ray tracing was released, the game industry has been introducing this feature into games. However, even modern hardware still experience performance issues when implementing common rendering techniques with ray tracing. One of these problematic techniques is alpha testing. Objectives. The thesis will investigate the following: 1) How the texture format of the alpha map and the number of alpha maps affect the rendering times. 2) How tessellation of the alpha tested geometry affects the performance and if tessellation has the potential to fully replace the alpha test from a performance perspective. Methods. A DXR 3D renderer will be implemented capable of rendering alpha tested geometry using an any-hit shader. The renderer was used to conduct a computational performance benchmark of the rendering times while varying texture and geometry data. Two alpha tested tree models were tessellated to various levels and their related textures were converted into multiple formats that could be used for the test scenes. Results & Conclusions. When the texture formats BC7, R(1xfloat32), and BC4 were used for the alpha map, the rendering times decreased in all cases, relative RGBA(4xfloat32). BC4 showed to give the best performance gain, decreasing the rendering times with up to 17% using one alpha map per model and up to 43% using eight alpha maps. When increasing the number of alpha maps used per model the rendering times increased with up to 52% when going from one alpha map to two. A large increase in rendering times was observed when going from three to four alpha maps in all cases. Using alpha testing on the tessellated model versions increased the rendering times in most cases, at most 135%. A decrease of up to 8% was however observed when the models were tessellated a certain amount. Turning off alpha testing gave a significant decrease in rendering allowing higher tessellated versions to be rendered for all models. In one case, while increasing the number of triangles with a factor of 78 the rendering times were still decreased by 30% relative to the original alpha test implementation. This suggests that pre-tessellated models could potentially be used to replace alpha tessellated geometry when performance is highly required.
Bakgrund. Strålspårning(Ray tracing) kan användas för att uppnå hyperrealistisk 3D-rendering, men det är en mycket tung beräkningsuppgift. Sedan hårdvarustöd för att utföra strålspårning i realtid lanserades har spelindustrin introducerat funktionen i spel. Trots modern hårdvara upplevers fortfarande prestandaproblem när vanliga renderingstekniker kombineras med strålspårning. En av dessa problematiska tekniker är alfa-testning(alpha testing). Syfte. Denna avhandling kommer att undersöka följande: 1) Hur texturformatet på alfamasken(alpha map) och hur antalet alfamaskar påverkar renderingstiderna. 2) På vilket sätt tesselering av den alfa-testade geometrin påverkar prestandan och om tesselering har potentialen att ersätta alfa-testet helt ur ett prestandaperspektiv. Metod. En DXR 3D-renderare kommer att implementeras som kan rendera alfatestad geometri med hjälp av en “Any-hit” shader. Renderaren användes för att mäta och jämföra renderingstider givet varierande textur- och geometri-data. Två alfaprövade trädmodeller tesselaterades till olika nivåer och deras relaterade texturer konverterades till fyra format som användes i testscenerna. Resultat & Slutsatser. När texturformaten BC7, R(1xfloat32) och BC4 användes för alfamasken visade alla en minskad renderingstid relativ RGBA (4xfloat32). BC4 gav bästa prestandaökningen och minskade renderingstiden med upp till 17% med en alfamask per modell och upp till 43% med åtta alfamasker. När antalet alfamasker som användes per modell ökade renderingstiderna med upp till 52% när alfamaskerna ökade från en till två. En stor ökning av renderingstiden observerades när alfamaskerna gick från tre till fyra i alla testfall. När alfatestning användes på de tesselerade modellversionerna ökade renderingstiderna i de flesta fall, som högst 135%. En minskning på upp till 8% observerades emellertid när modellerna tesselaterades till en viss grad. Att stänga av alfatestning gav en signifikant ökning av prestandan, vilket tillät högre tesselerade versioner att renderas för alla modeller. Samtidigt som antalet trianglar ökade med en faktor på 78, i ett av fallen, minskades renderingstiden med 30%. Detta antyder att förtesselerade modeller potentiellt kan användas för att ersätta alfatestad geometri när prestanda är ett högt krav.

The generation of memory T cells following primary exposure to a pathogen is a critical feature of the vertebrate immune system which has evolved as a protective mechanism in order to defend the host against repeated assaults by the patnogen. Memory T cells are long-lived, undergo rapid proliferation upon re-activation, mediate a robust secondary response and clear the pathogen much more efficiently. These aspects have made the generation of memory T cells an attractive goal for the production of both prophylactic and therapeutic vaccines. However, the degeneracy of the T cell receptor, whereby a given T cell recognizes more than one epitope, allows the T cell to be modulated by epitope variants which could be self-ligands, ligands related to the original epitope but altered in sequence, or completely unrelated epitopes. Experiments in both mice and humans show that such cross-reactive stimulation of memory T cells results in complete, partial, or no activation of T cells, and in some cases, even alters the functional identity of the T cell (for example, T helper 1 cells start secreting IL-4, IL-5 and become part of a T helper 2 response). In the context of secondary infection of immune organisms with pathogens containing mutated or related T cell epitopes, such alterations at the cellular level translate into drastic changes in the overall clinical outcome of the infection. Thus, the presence of cross-reactive T cells in the memory population implies that the protective or pathologic nature of the secondary immune response is a consequence of the host's infection history. Although several murine models of heterologous infection resulting in altered pathological outcome have been studied, the exact immune correlates of protection versus immunopathology are still unclear. This thesis addresses this issue in dengue virus infections in humans. Dengue fever (DF) and Dengue Hemorrhagic Fever (DHF) are two disease manifestations caused by infections of humans by the dengue viruses. These are a group of 4 serologically distinct flaviviruses (D1-4) which often co-circulate among endemic populations. While primary infection with any of the four serotypes can result in the more severe clinical disease characterized by DHF, epidemiological data from several outbreaks show that 80% - 90% of DHF cases occur among individuals with secondary infection. This implies that prior immunity to dengue is actually a risk factor for developing severe disease. In these DHF cases, there are increased numbers of CD69+ CD8+ T cells in circulation, with increases observed in the frequency of epitope-specific T cells, and the serum levels of several T cell produced cytokines, chemokines, and immune receptors are highly elevated. Since the four serotypes share 65% - 75% amino acid sequence homology, the possibility that unconserved T cell epitope sequences stimulated cross-reactive responses was borne out in in vitroexaminations. In these studies, peripheral blood mononuclear cells (PBMC) and cloned T cells from both vaccinated and infected donors contained large populations of memory T cells that were cross-reactive for heterologous viral serotypes in proliferation and CTL assays. These data suggest that the severity of disease seen in DHF patients can be attributed to an immunopathologic secondary response during heterologous infection, and highlight a role for serotype cross-reactive T cells in this process. This thesis addresses the hypothesis that the recognition of the natural variants of dengue virus T cell epitopes by serotype cross-reactive CD8+ T cells of a dengue-immune donor results in an altered secondary response profile, with the changes reflected in both the quantitative and qualitative nature of the response. In order to compare the functional profile of the secondary response of dengue-immune PBMC re-activated with heterologous serotypes, we focused on a panel of 4 donors who were vaccinated with live attenuated monovalent vaccines corresponding to D1, D2, or D4 serotypes. We screened a panel of peptides predicted to bind to HLA-A*0201 for cytokine responses and identified 4 novel epitopes that were highly immunogenic in all four donors. Direct ex vivo stimulation of donor PBMC with the heterologous sequences of these epitopes also showed sizeable serotype cross-reactive T cell populations. CFSE- and intracellular staining for cytokines and chemokines showed that these cross-reactive T cells not only expanded but also produced IFNγ, TNFα, and MIP-1β. Multi-parameter staining revealed functionally diverse populations comprised of single cytokine (IFNγ+, TNFα+, MIP-1β+, double cytokine (IFNγ+TNFα+, IFNγ+MIP-1β+, TNFα+MIP-1β+, and triple cytokine (IFNγ+TNFα+MIP-1β+ secreting sub-sets. Stimulation with the epitope variants altered the magnitude of the overall response as well as the relative sizes of these sub-sets. The patterns of responses revealed the effects of epitope immunogenicity, infection history and donor-specific variability. All 4 donors showed the highest cytokine response to a -single epitope (NS4b 2353). The same two peptide variants (D2 NS4a 2148 and D3 NS4b 2343) induced the highest response in all 4 donors regardless of the serotype of primary dengue infection. Interestingly, the epitope variants which showed the highest immunogenecity in our donors corresponded to the D2 and D3 serotypes which have been documented as being more virulent as well as a viral risk factor for DHF. In one donor, the response to all peptide variants was dominated by the same cytokine sub-sets. These data suggested that the dengue-immune memory T cell repertoire was functionally diverse and underwent alterations in size after secondary stimulation. Therefore, we also investigated the effect of epitope variants on dengue-specific CD8+T cell clones isolated from vaccinated and infected donors in order to determine if epitope variants induced altered functional outcomes at the clonal level. The epitope variants functioned either as strong agonists (particularly the D2 and D3 sequences), partial agonists, or null ligands. Some variants were able to induce cytolysis but not other effector functions at low concentrations. The variant ligands also influenced the hierarchy of cytokine responses within each clone. The third part of this thesis focused on the characterization of the frequency and phenotypic profile of epitope-specific CD8+ T cells in patients with DHF and DF at different times in the disease course in order to better understand the kinetics of the response and delineate any differences between the immune profile of severe vs. moderate disease. Tetramer staining for a previously identified HLA-B*07 restricted epitope was combined with staining for activation markers (CD69, CD38, HLA-DR), homing receptors (CCR7, CD62L), and programmed death receptor 1 (PD-1). The DHF subjects had early T cell activation with higher frequencies of tetramer+CD69+ cells as compared to DF subjects, in whom T cell frequencies peaked around the time of defervescence. While each subject had a unique phenotypic profile of tetramer+ cells, there was a difference between DF and DHF subjects in terms of CCR 7 expression; all subjects expressed low levels of CCR7 during acute illness but only the DHF subjects did not show upregulation of CCR7 on tetramer+ cells during convalescence. These data suggest that there is a sustained alteration in memory phenotype in those who recovered from severe dengue disease. A majority of the tetramer+cells also expressed PD-1 during acute illness but not during convalescence. Double-staining with variant tetramers allowed us to directly visualize serotype cross-reactivity of the epitope-specific population, and showed that secondary stimulation did induce the expansion of cells with low avidity for that secondary serotype and higher avidity to the variant. Furthermore, the ratios of these sub-sets changed during the course of the response. Taken together, these studies suggest that the immune response to heterologous secondary dengue infection is mediated by a heterogeneous population of serotype-cross reactive T cells that have different functional avidities to epitope variants and is influenced by the serotype of the secondary infection as well as the prior infection history of the individual. The preferential expansion of clones which secrete IFNγ but not inflammatory MIP-1β or TNFα or a repertoire characterized by a higher ratio of cytolytic to cytokine producing clones could limit immune mediated damage while efficiently clearing the virus. This information will be useful in the design of vaccine strategies aimed at inducing protective cross-reactive responses against all 4 dengue serotypes while preventing immunopathological outcomes following secondary infection.

Fragestellung: Wir gingen der Frage nach, inwieweit genetische Variationen im Gen für den Tumor Nekrose Faktor-alpha einen Einfluß auf die Krankheitsprogression oder die Entstehung bzw. Ausprägung HIV-assoziierter Erkrankungen haben. Methoden: Die Promotorregion sowie die kodierenden Sequenzen des TNF-alpha-Gens wurden mittels SSCP-Analyse auf genetische Variationen untersucht. Anschließend erfolgte die Charakterisierung der häufigsten bekannten Promotorpolymorphismen mittels Restriktionsfragment-Längenpolymorphismus-Analyse (RFLP). Die Bestätigung der Polymorphismen der RFLP-Analyse erfolgte an ausgewählten Proben durch DNA-Fluoreszenzsequenzierung. In sämtlichen Fällen handelte es sich um singuläre Basentransitionen von Guanin zu Adenin. Ergebnisse: Unter Einbeziehung verschiedener Progressionsparameter wie der CD4-Zellzahl, des Zeitraumes vom ARC-Stadium zum AIDS-Stadium und AIDS-assoziierter Krankheiten wie dem Wasting Syndrom und der HIV-Enzephalopathie, erfolgte anschließend die statistische Analyse in Korrelation mit den ermittelten Genotypen. Es zeigte sich bei keiner der statistischen Analysen eine signifikante Assoziation mit einem bestimmten TNF-alpha-Genotyp. Schlußfolgerung: Es ist kritisch anzumerken, daß für einige Subanalysen die Größe der untersuchten Patientengruppe zu gering war, um eine statistische Aussagekraft für seltene Allele zu erreichen. Anhand der hier vorgelegten Ergebnisse hat der TNF-alpha-Genotyp weder einen Einfluß auf die Progression der HIV-Erkrankung noch auf die Ausbildung HIV-assoziierter Erkrankungen wie dem Wasting Syndrom oder der HIV-Enzephalopathie.
Objective: We determined whether variation of the tumor necrosis factor-alpha gene had an impact on HIV disease progression or the prevalence of hiv-associated diseases. Methods: The promotor region of the TNF-alpha gene were examined with SSCP analysis for polymorphisms in the promotor region. The most common promotor polymorphisms were characterized with restriction fragment length polymorphism analysis (RFLP). To confirm RFLP results DNA fluorescence sequenzing analyses were performed with selected samples. In all cases with diagnosis of promotor polymorphisms single base transitions from guanine to adenine were confirmed. Results: Statistical analyses correlated the genotypes with different markers for disease progression e.g. CD4-count, the period from ARC to AIDS and the occurance of HIV associated diseases (wasting syndrome, hiv encephalopathy). In none of the statistical analyses significant association with a certain TNF-alpha genotyp could be demonstrated. Conclusion: For some subanalysis the sample sizes were too small in order to be able to make safe statistical statements concerning rare allels. Regarding our results, none of the examined tumor necrosis factor-alpha promotor polymorphisms had an impact on HIV disease progression or the prevalence of hiv-associated diseases.

La segmentation de régions d'intérêt dans le cadre de l'analyse d'images médicales et biomédicales reste encore à ce jour un challenge en raison notamment de la variété des modalités d'acquisition et des caractéristiques associées (bruit par exemple).Dans ce contexte particulier, cet exposé présente une méthode de segmentation de type contour actif dont l ‘énergie associée à l'obtention de l'équation d'évolution s'appuie sur une mesure de similarité entre les densités de probabilités (en niveau de gris) des régions intérieure et extérieure au contour au cours du processus itératif de segmentation. En particulier, nous nous intéressons à la famille particulière des alpha-divergences. L'intérêt principal de cette méthode réside (i) dans la flexibilité des alpha-divergences dont la métrique intrinsèque peut être paramétrisée via la valeur du paramètre alpha et donc adaptée aux distributions statistiques des régions de l'image à segmenter ; et (ii) dans la capacité unificatrice de cette mesure statistique vis-à-vis des distances classiquement utilisées dans ce contexte (Kullback- Leibler, Hellinger...). Nous abordons l'étude de cette mesure statistique tout d'abord d'un point de vue supervisé pour lequel le processus itératif de segmentation se déduit de la minimisation de l'alpha-divergence (au sens variationnel) entre la densité de probabilité courante et une référence définie a priori. Puis nous nous intéressons au point de vue non supervisé qui permet de s'affranchir de l'étape de définition des références par le biais d'une maximisation de distance entre les densités de probabilités intérieure et extérieure au contour. Par ailleurs, nous proposons une démarche d'optimisation de l'évolution du paramètre alpha conjointe au processus de minimisation ou de maximisation de la divergence permettant d'adapter itérativement la divergence à la statistique des données considérées. Au niveau expérimental, nous proposons une étude comparée des différentes approches de segmentation : en premier lieu, sur des images synthétiques bruitées et texturées, puis, sur des images naturelles. Enfin, nous focalisons notre étude sur différentes applications issues des domaines biomédicaux (microscopie confocale cellulaire) et médicaux (radiographie X, IRM) dans le contexte de l'aide au diagnotic. Dans chacun des cas, une discussion sur l'apport des alpha-divergences est proposée
In the particular field of Computer-Aided-Diagnosis, the segmentation of particular regions of interest corresponding usually to organs is still a challenging issue mainly because of the various existing for which the charateristics of acquisition are very different (corrupting noise for instance). In this context, this PhD work introduces an original histogram-based active contour segmentation using alpha-divergence family as similarity measure. The method keypoint are twofold: (i) the flexibility of alpha-divergences whose metric could be parametrized using alpha value can be adaptedto the statistical distribution of the different regions of the image and (ii) the ability of alpha-divergence ability to enbed standard distances like the Kullback-Leibler's divergence or the Hellinger's one makes these divergences an interesting unifying tool.In this document, first, we propose a supervised version of proposed approach:. In this particular case, the iterative process of segmentation comes from alpha-divergenceminimization between the current probability density function and a reference one which can be manually defined for instance. In a second part, we focus on the non-supervised version of the method inorder to be able.In that particular case, the alpha-divergence maximization between probabilitydensity functions of inner and outer regions defined by the active contour is maximized. In addition, we propose an optimization scheme of the alpha parameter jointly with the optimization of the divergence in order to adapt iteratively the divergence to the inner statistics of processed data. Furthermore, a comparative study is proposed between the different segmentation schemes : first, on synthetic images then, on natural images. Finally, we focus on different kinds of biomedical images (cellular confocal microscopy) and medical ones (X-ray) for computer-aided diagnosis

Dans la cascade des cytokines qui accompagne le rejet de greffe de coeur comme dans le développement de complications majeures de la greffe de moelle, le tumor necrosis alpha (TNF-alpha) et l'interleukine 6 (IL6) semblent jouer un rôle important. Chez 186 patients ayant subi une greffe de moelle (106 autogreffés), les taux plasmatiques de TNF-alpha, d'IL6 et du récepteur soluble de l'IL-2 (IL-2-RS) ont été analysés rétrospectivement. L'évolution des cytokines des greffés a été suivie sur trois périodes : prégreffe, premier trimestre post-greffe, deuxième trimestre post-greffe. Des taux plasmatiques élevés de l'IL-2-RS et surtout de l'IL-6 ont été observés chez les patients développant des complications majeures liées à la greffe ou à la maladie du greffon contre l'hôte, aigue (GVHA) ou chronique (GVHC). Le TNF-alpha ne montre pas d'association avec la GVHA ou le TRC, mais avec la GVHC étendue. Chez 142 greffés de coeur, les analyses de l'IL-bet, de l'IL-6 et du TNF-alpha sur un specimen sanguin contemporain du prélèvement biopsique ont permis de dégager des différences statistiquement significatives entre les deux échantillons de patients, sans rejet sévère. Une étude complémentaire portant sur 27 greffés suivis dès la transplantation a permis notamment de mettre en évidence une implication très forte de l'élévation du TNF-alpha dans le rejet sévère. Le rôle prédictif du taux plasmatique du TNF-alpha sur le rejet sévère n'a cependant pas pu être établi de manière significative. La variabilité individuelle du gène TNF (polymorphismes extragéniques, microsatellites, région du promoteur) a été abordée par analyse de restriction et séquençage direct : il a été montré certaines relations avec le niveau de production du TNF-alpha, dont une différence significative selon la gravité du rejet pour ce qui concerne certains microsatellites au niveau du locus TNFa
Among the elements of the cytokine cascade occuring with a heart-graft rejection or developing when there are major bone marrow transplantation related complications (TRC), tumor necrosis alpha (TNF) alpha and interleukin-6 (IL-6) are highly implicated. We studied 186 patients who undervent a bone marrow transplantation (106 autografted and 80 allografted). They were investigated for retrospective plasma TNF alpha, IL-6 and soluble IL-2 receptor (sIL-2 R)levels in order to appreciate their possible implication in TRC development. The cytokine evolution was followed in the three periods : pre-graft, post-graft first quater, post-graft second quater. High levels of sIL-2-R and especially of IL-6 were noted in patients developing TRC or suffering from graft-versus host disease acute (GVHA) or chronic (GVHC). . TNF alpha showed no link between GVHA or TRC, but was associated with extensive GVHD. In a cohort of 142 patients, we analysed blood specimens contemporary with the histological biopsy. Significant differences of plasma interleukin-1 beta, IL-6 and TNF alpha levels appeared between patients without severe rejection. By studying weekly blood samples of a group of 27 patients from the day of the transplantation, it was possible to emphasize a specific TNF alpha increase in the severe rejection risk while it was not possible to demontrate the significance due to the too small number of our patient sample. The individual variabuility of TNF gene (extragenic polymorphisms, microsatellites, promotor sequence) was studied by restriction analysis and direct sequencing which demonstrated some interesting relations with TNF alpha production, especially a significant difference appearing for some TNFa microsatellites according to rejection gravity

Les abeilles sont des pollinisateurs essentiels pour les cultures et les plantes sauvages, mais l'intensification des pratiques agricoles a engendré une baisse importante de leur abondance et diversité. Afin de protéger efficacement les abeilles dans les paysages agricoles, il est nécessaire d'avoir une meilleure connaissance de leurs patrons de diversité. L'objectif général de cette thèse était de déterminer les patrons spatio-temporels de la diversité des abeilles et l'utilisation des ressource fleuries dans un système agricole intensif. L'échantillonnage spatialement extensif de l'activité de butinage des abeilles sauvages et domestiques nous a permis de recenser 45040 individus (29314 abeilles domestiques et 15726 sauvages), appartenant à 192 espèces recensées à l'échelle territoriale. Cette diversité représente près de 20% de la richesse des espèces apiformes connues à l'échelle nationale. Cette communauté est caractérisée par une forte proportion d'espèces rares (28,8%) et de fortes variations temporelles et spatiales, en particulier de l'échelle locale jusqu'à 10-20 km2. L'importance des habitats semi-naturels pour soutenir les populations d'abeilles sauvages a été confirmée dans cette étude. Durant les périodes de floraison des cultures oléagineuses, les abeilles sauvages étaient étroitement associées aux habitats semi-naturels alors que les abeilles domestiques ont montré une nette préférence pour les cultures à floraison massive. La diversité des abeilles sauvages dans les habitats semi- naturels était 3-4 fois supérieure à celle observée dans le colza ou le tournesol. L'importance de certains facteurs écologiques clefs pour la diversité des abeilles, comme la richesse floristique locale et la quantité d'habitats semi-naturels dans le paysage, a été confirmée et quantifiée. Il a également été démontré que ces effets varient en fonction de la saison et de l'échelle spatiale. Ces résultats mettent en évidence les processus écologiques responsables des partons de diversité des abeilles à différentes échelles spatiales, et peuvent contribuer à optimiser la conception des mesures de conservation visant à promouvoir la diversité des abeilles dans les agrosystèmes intensifs.

Bibliography: leaves 161-213.
xviii, 219, [48] leaves of plates :
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Analyses of six rare alpha 1-antitrypsin alleles were undertaken to determine their molecular basis, assess the biosynthesis directed by these alleles and, examine their clinical correlation.
Thesis (M.D.)--University of Adelaide, Dept. of Medicine, 1992

碩士
國立屏東科技大學
獸醫學系所
105
Alpha 1 acid glycoprotein (AGP), which is also named orosomucoid (ORM), is a moderate acute phase protein in cats and regulated by interleukin-1, interleukin-6, and tumor necrosis factor α. The feline AGP that raises after any tissue injury and inflammatory procedure, has been proven a diagnostically useful item. However, it is still not been widely applied in clinical practices. The common reason may be associated with the high cost and time consuming of the laboratory procedures. Thus, the aim of the study is to evaluate its values on clinical application. There are three parts in this study. First, we established the basic blood profiles, included concentrations of AGP, total proteins, albumin, and white blood cells (WBC) in clinical healthy cats. Second, we performed surgery (castration or ovariohysterectomy) on normal healthy cats and evaluated the correlations between levels of AGP and other hematological and biochemistry values postoperatively. Third, clinical applications of AGP on parvovirus infected cats. The results indicated the regional clinical healthy cat’s AGP reference range was under 800 mg/L. Peak concentration of AGP (1.9-2.6 fold) were observed on day 2 postoperatively. The AGP of females was significant higher on day 2 postoperatively than presurgery and there were no significant differences noted between sex. There is high positive relationship between AGP and WBC. For the parvovirus infected cats, there was high relation (r=-0.721) between AGP values and the levels of qPCR at the admission. Besides, AGP values were significantly lower (P<0.05) in recovered cats than died cats at the 5th, 7th, 9th, and 11th day after admission. To summarized the study: (1) in a healthy population, feline AGP concentration is under 800mg/L. (2) According to the variation of AGP concentration, the impaction of standard localized and sterilized surgery is less than generalized infection (3) Low Cq value of qPCR and high plasma AGP levels indicate the severity of feline panleokopenia and may be used to indicate the yield of clinical signs (4) Panleukopenia infected cats that survived longer than 5 days usually recovered and the plasma AGP levels may serve as a predictor of recovery.

Thesis (M.Sc. (Med.))--University of the Witwatersrand, Faculty of Health Sciences, 1998.
The issue of Malagasy ancestry has been controversial, and has still not been completely resolved. The historical, linguistic, archaeological and some genetic evidence points to the fact that modern Malagasy are the descendants of immigrants who arrived on the island over the past 2000 years, from South and Southeast Asia, Africa and the Near East. In more recent centuries, mainly in the twentieth century, there have been significant numbers of Indian, Chinese and French immigrants. In addition, archaeological and historical studies of specific regional populations of Malagasy suggest a complex pattern of internal migration within the island, extending back in time to the first European contacts with the island in the sixteenth century. The 22 Malagasy ethnic groups may be classified as "highland" or "lowland" depending on their geographic distribution on the island. Within the ethnic groups, the founding populations have made different genetic contributions: the highland groups are said to have a greater Indonesian contribution to their ancestry, while the lowlanders have a greater African contribution to their ancestry. Genetic studies on the Malagasy have been limited by small sample sizes, deficiencies in sampling procedures and in the limited number of polymorphisms studied. In light of the paucity of written records, the Department of Human Genetics, SAIMR, has undertaken a large study in Madagascar to reconstruct the biological history of its people, using genetic variation. This thesis forms a part of this study. Variation in the a- and p-globin cluster has been extensively studied in many parts of the world, and has been shown to be population specific, with specific variants having distinct geographical distributions. Thus haemoglobin and its related disorders have been the subject of extensive studies for determining the origin(s) of particular populations. In this study, some of the a- and p-globin variation present in the Malagasy was characterised. Seven RFLPs/HVRs in the a-globin gene cluster and seven RFLPs in the p-globin gene cluster were analysed. The common a- and p-globin gene cluster haplotypes differ between African and Asian populations. Frequencies also vary between populations in a specific geographical regions. The aim of this study was to characterise the haplotypes present in the Malagasy, to provide information on the relative genetic contributions of different populations to the peoples of Madagascar. DNA samples from randomly selected, haematologically normal individuals were analysed. Individuals were chosen from six Malagasy ethnic groups: two “highland” populations (Merina and Betsileo), two “lowland" populations (Antasaka and Tsimiheti) and two populations from the south-west of the island (Mahafaly and Vezo). The groups chosen cover a broad range of Madagascar and thus provide some representation of the Malagasy population as a whole. The number of individuals studied in each ethnic group are as follows: Merina: 88; Betsileo: 78; Antasaka: 67; Tsimiheti: 67; Mahafaly: 26; Vezo: 25. The frequencies of the a- and (B-globin RFLP sites and a-globin HVRs in the Malagasy vere calculated. 5' and 3’ p-globin haplotypes were constructed on the basis of homozygosity. A maximum-likelihood algorithm was used to obtain frequencies of 5’ P-globin haplotypes that could not be assigned on the basis of homozygosity. These data were then subject to statistical analysis. The frequencies of the 5’ p-globin haplotypes (consisting of the five sites Hindi 5' to e, Hindi 11 within Gy and Ay, Hindi within \|/P and 3' to it) were the most informative data set for comparing the Malagasy ethnic groups to each other and to other world populations. Unfortunately, the maximum-likelihood estimates of 5‘ p-globin haplotypes could not be used for comparative analyses due to the lack of similar data in other populations. However the strong correlation between the maximum-likelihood frequencies and the observed frequencies illustrated the ability of the algorithm to determine hapiotype frequencies from otherwise uninformative individuals. 5’ p-globin haplotypes were assigned unambiguously for 248 Malagasy chromosomes. Ten haplotypes were found; of these, nine have been reported previously in other world populations and one has not been reported and hns thus been called “rare” in this study. The frequencies of unambiguous 5’ p-globin haplotypes in the Malagasy and the proposed parental populations were initially analysed with x2 tests. For a more accurate comparison between these populations, genetic distances were calculated and used for the construction of phylogenetic trees, principle component analysis was carried out, and a study of heterozygosity versus distance from the centroid was performed. Admixture estimates of two African populations and one Indonesian population to Malagasy ancestry were calculated. Certain general trends were noted in all the analyses. The results are in agreement with the historical data which provides evidence for both African and Asian contributions to Malagasy ancestry. The highlanders were more closely affiliated to the Indonesian/Polynesian populations, while the south-west groups showed the strongest associations with the African populations. The lowlanders were consistently intermediate in position between the highlanders and the south-west groups, with the Antasaka being slightly more closely related to the African populations than the Tsimiheti. The Malagasy were shown to have high heterozygosities, similar to those of African populations, and this high degree of diversity is probably a reflection of the many sources of ancestry of the Malagasy. The south-west groups were the furthest outliers in the model of heterozygosity versus distance from the centroid, suggesting that these groups are the most genetically admixed of all the Malagasy groups that were studied. Estimates of ancestral population admixture confirmed these trends, with the highlanders having the highest proportional contribution by Indonesians (53%), but the lowest total African contribution (47%), while the south-west groups have the highest Bantu contribution (65%). The Indonesian and African contributions to the lowlanders are intermediate between those to highlanders and south-west groups. Overall the Malagasy subjects included in this study showed a 61% African admixture contribution and a 39% Indonesian admixture contribution. It is hoped that the results obtained in this study will contribute to the larger project concerning the origins of the Malagasy, and that they may be used to shed further light on the much debated issue of Malagasy ancestry.

碩士
國立清華大學
物理研究所
84
alpha-Al2O3 是一應用相當廣泛的陶瓷材料。它是氧化鋁化合物中純度最 高的一個，俗稱剛玉(corundum)。因為它的結構相當複雜，所以有較多低 指數和低能量的面。這些面已有相當多的研究成果。本文則是對其中的一 個面在不同的退火條件下的結果作進一步的探討。 alpha-Al2O3 在工業 及科學上是一個不可多得的材料，如高溫絕緣材料、光學鏡片、切割工具 、鍍膜基板、固體雷射．．．等。它之所以有這麼廣泛的應用要歸功於其 高機械硬度、高熔點、散熱快、絕緣性佳還有穩定的化學性質。 (1 0 -1 0) 面是幾個低指數面中能量較高的一個，退火時較不穩定，會分裂為 能量較低的兩個小面。到目前為止鮮少有人對這個面在不同退火溫度及不 同退火時間做進一步的探討。本文則要對此做深入探討。在 1400 °C 退 火 24 小時後有鋸齒狀的形貌出現，方向是 (1 1 -2 0)。其中一個面較 為平坦，另一個則非常粗糙。週期約為 50 nm 。但將退火溫度降為 1000 °C 時，其週期約只剩下 10 nm 左右。在 1400 °C及1000 °C 長 時間退火的條件下，形貌幾乎沒有改變。本文共分為八部份：一簡介；二 表面分析的重要性；三晶體結構；四相關的研究；五反射式電子顯微鏡簡 介；六實驗過程；七實驗結果；八結論；九進一步的工作。

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2017
Objective: Analyse the evolution of biological tumour necrosis factor alpha (TNFalpha) inhibitors utilisation in the treatment of rheumatoid arthritis (RA) and identify the reasons for the observed variation. Methods: Two western European countries were selected for this analysis, Portugal and the Netherlands. Country characteristics, treatment guidelines and RA prevalence were obtained from the literature. Patient characteristics were obtained from the literature and from the data made available by the University Medical Center Utrecht, for Portugal and the Netherlands, respectively. Annual utilisation rates of TNFalpha inhibitors between 2008 and 2013 were expressed as defined daily doses (DDDs)/1000 inhabitants/day. Results: TNFalpha inhibitors utilisation varied from 0.18, in 2008, to 0.46 DDDs/1000 inhabitants/day, in 2013, in Portugal and from 0.98, in 2008, to 1.64 DDDs/1000 inhabitants/day, in 2013, in the Netherlands. Clinical guidelines, variations in GDP per capita, total health expenditure, medical goods expenditure and drug distribution channel, appear to have had limited impact on TNFalpha inhibitors utilisation. On average, Dutch RA patients under therapy with TNFalpha inhibitors appear to younger than their Portuguese counterparts. Conclusions: TNFalpha inhibitors utilisation continues to be increasing, despite the negative influences caused by the economic recession and posterior austerity measures. This increase in TNFalpha inhibitors utilisation was not equal in Portugal and in the Netherlands, which lead to a bigger difference in utilisation between both countries. The high percentage of undiagnosed Portuguese rheumatic patients might be one of the leading reasons for the anaemic utilization of TNFalpha inhibitors. The number of rheumatologists per 100,000 inhabitants, improved clinical efficiency and a reduction in drug pricing all seem to have positively influenced utilisation.
Objetivo: Analisar a utilização dos biológicos inibidores do fator de necrose tumoral alfa (TNF-alfa) no tratamento de artrite reumatoide (AR) e identificar as razões para a variação de utilização observada. Metodologia: Dois países europeus ocidentais foram selecionados para esta analise, Portugal e a Holanda. As características dos países, guidelines de tratamento e a prevalência foram obtidos da literatura. As características dos doentes com AR foram obtidas da literatura e a partir dos dados disponibilizados pelo University Medical Center Utrecht, para Portugal e Holanda, respetivamente. Ratios de utilização anuais dos inibidores do TNF-alfa entre 2008 e 2013 foram demonstrados como Dose Definida Diária (DDD)/1000 habitantes/dia. Resultados: A utilização dos inibidores do TNF-alfa variaram de 0.18, em 2008, para 0.46 DDD/habitantes/dia, em 2013, em Portugal e de 0.98, em 2008, para 1.64 DDD/habitantes/dia, em 2013, na Holanda. Guidelines clinicas, variações no GDP per capita, despesa total em saúde, despesa com produtos de saúde e canal de distribuição dos medicamentos, parecem ter tido um impacto limitado na utilização dos inibidores do TNF-alfa. Doentes holandeses com artrite reumatóide sob terapia dos inibidores do TNF-alfa são, em média, mais novos que os seus contrapartes portugueses. Conclusão: A utilização de inibidores do TNF-alfa continua a aumentar, mesmo com os efeitos negativos causados pela recessão económica e das medidas de austeridade implementas posteriormente. O aumento de utilização de inibidores do TNF-alfa não foi igual em Portugal e na Holanda, levando assim a um aumento na diferença de utilização destes medicamentos entre os dois países. A elevada percentagem de doentes portugueses não-diagnosticados pode ser uma das principais razões por detrás da utilização anémica de inibidores do TNF-alfa. O número de reumatologistas por 100.000 habitantes, aumento da eficiência clinica e reduções no preço dos fármacos parecem todos ser fatores que influenciaram positivamente a utilização destes fármacos.

La prédiction des paramètres pharmacocinétiques/toxicocinétiques (PK/TK), tels que la clairance intrinsèque (CLint) et la clairance hépatique (CLh) des médicaments, demeure un défi majeur en modélisation quantitative. Selon « l’hypothèse du médicament libre », seul le médicament libre peut traverser la membrane plasmique et la CLh de ce médicament est calculée en fonction de sa fraction libre (fup). Néanmoins, la captation hépatique facilitée par l’albumine (ALB) représente clairement une violation de « l’hypothèse du médicament libre ». Cette captation hépatique se base sur la possibilité que le complexe ALB-médicament puisse assurer un apport supplémentaire en médicament aux hépatocytes. Ainsi, cela pourrait expliquer en grande partie les sous-prédictions observées de CLh. Par ailleurs, certains médicaments peuvent se lier fortement à plusieurs protéines plasmatiques telles que l’ALB et l’alpha-1-glycoprotéine acide (AGP). Ainsi, la forte liaison d’un même médicament à l’ALB, à l’AGP, ou aux deux, pourrait avoir des répercussions bien distinctes sur la prédiction de ces paramètres PK/TK. Cependant, aucune étude n’a été faite pour simuler la différence entre leurs effets. L’objectif principal de cette thèse est donc d’évaluer (avec plus d’exactitude et de précision), pour une série de médicaments, en condition in vivo (ou in situ), ces répercussions en présence des deux protéines plasmatiques, conjointement ou séparément. En outre, il est indispensable de vérifier si une approche générique en modélisation peut être appliquée. Cette thèse est répartie en trois objectifs spécifiques. Le premier est de proposer un arbre décisionnel pour faciliter la sélection des approches prédictives appropriées de CLhin vivo pour des médicaments ayant des caractéristiques différentes. Le second est d’évaluer les répercussions de fortes liaisons aux deux protéines plasmatiques ALB et AGP sur la CLh de deux xénobiotiques choisis (perampanel (PER) et fluoxétine (FLU)) ; ces médicaments ont de fortes affinités pour les deux protéines et un métabolisme exclusif (ou prédominant) dans le foie. Et, le dernier est de développer et valider un nouveau modèle prédictif de CLh pour les xénobiotiques ayant le potentiel de se lier fortement dans le plasma, à l’ALB ainsi qu’à l’AGP. Dans un premier temps, des données in vitro rapportées chez l’humain ont été colligées pour 19 médicaments (substrats des transporteurs OAT2 et OATP1B1), et ont été ensuite utilisées dans six modèles d’extrapolation in vitro-in vivo (IVIVE) pour prédire lesdits paramètres. Après une comparaison statistique, les résultats ont montré que l’approche 2 (c’est-à-dire « fup-adjusted model ») qui se base sur la captation hépatique facilitée par l’ALB, avait la meilleure performance prédictive. Cependant, l’approche 5 (c’est-à-dire « Extended Clearance Model ») qui se base sur le transport facilité, en était une très pertinente à appliquer pour les substrats de transporteurs membranaires. Lesdits substrats seraient potentiellement moins affectés par l’ALB. Ainsi, un arbre décisionnel a été proposé pour choisir rapidement et judicieusement la meilleure approche IVIVE servant à prédire la CLhin vivo pour chaque xénobiotique en présence de l’ALB. Dans un deuxième temps, les médicaments PER et FLU ont été sélectionnés à partir d’une collecte de données (N= 1907 médicaments) en fonction de certains critères (avoir un métabolisme exclusif ou prédominant dans le foie, pas de transport facilité par les transporteurs membranaires, une haute affinité pour les deux protéines ALB et AGP, et un ratio de liaison à l’AGP sur celle à l’ALB proche de l’unité). Cette sélection a été réalisée pour faire des expériences sur des foies isolés et perfusés de rats (IPRL), en présence et en absence des protéines ALB et AGP (c’est-à-dire quatre scénarios IPRL). Les résultats IPRL ont démontré que PER est faiblement à moyennement métabolisé (extraction hépatique= 0,2-0,7), tandis que FLU est fortement métabolisé (extraction hépatique= 0,8-0,99). Le modèle Michaelis-Menten a été ajusté aux cinétiques métaboliques, et différents paramètres Vmax, Km et Km, u ont été obtenus de ce modèle. À de faibles concentrations libres pour les deux médicaments (c’est-à-dire à des concentrations thérapeutiques) et en présence des protéines plasmatiques, les valeurs de CLint non liée ont augmenté pour PER (avec l’ALB et le mélange des deux protéines (MIX)) et FLU (avec l’ALB, l’AGP et le MIX) par rapport à celles obtenues du scénario sans protéine (sauf pour PER avec AGP, lesdites valeurs ont diminué). Par ailleurs, les calculs des ratios CLint (SANS versus AVEC protéine) ont permis d’indiquer l’occurrence d’une facilitation de la captation hépatique de médicaments par l’ALB ou l’AGP. Ces ratios ont aussi permis de vérifier si la cinétique métabolique pour PER et FLU suivait soit « l’hypothèse du médicament libre » soit celle de « la captation hépatique facilitée par les protéines plasmatiques ». Dans un dernier temps, une nouvelle approche prédictive de CLh (approche WO-to-MIX) est développée en se basant sur une nouvelle notion de liaison fractionnelle et en intégrant dans le « fup-adjusted model » de nouveaux paramètres tels que la fraction liée à l’ALB (fB-ALB) et celle liée à l’AGP (fB-AGP) à partir du scénario MIX. Ce modèle est basé sur la captation facilitée par l’ALB. Contrairement à l’approche WO-to-MIX, le « well-stirred model » (ou modèle conventionnel) est basé sur l’hypothèse du médicament libre. Ensuite, les paramètres Vmax et Km obtenus in situ pour PER et FLU lors des expériences IPRL sans protéines, ont été utilisés en combinaison avec le paramètre intrant de la fraction libre ajustée (fup-adjusted) pour le « fup-adjusted model » ou avec la fraction libre (fup) pour le « well-stirred model ». Une comparaison des performances prédictives globales des deux modèles a été faite. Les performances prédictives du nouveau modèle étaient prometteuses, en particulier pour FLU qui montrait le plus haut degré de captation hépatique médiée par l’ALB, par rapport au modèle conventionnel. L’approche WO-to-MIX est une première validation d’un nouveau modèle d’extrapolation proposé pour les médicaments comme FLU qui se lient à l’ALB et à l’AGP. Néanmoins, le modèle conventionnel reste utile à utiliser pour les médicaments comme PER. L’exactitude de prédiction était inférieure pour ce dernier médicament probablement parce que la captation hépatique par l’ALB ne semble pas être maximale, et, par conséquent, l’utilisation de fup-adjusted a surestimé la CLhin vivo. Par conséquent, plus de travail est nécessaire en particulier pour PER. Cette thèse démontre qu’une seule approche générique pour prédire la CLh n’existe pas. Néanmoins, le choix d’une approche IVIVE ayant une performance prédictive satisfaisante est maintenant possible. Les résultats de cette thèse contribuent à : 1) mieux comprendre les répercussions sur les paramètres PK/TK de la forte liaison des médicaments à l’ALB et à l’AGP ; 2) choisir la meilleure approche prédictive de CLh sur la base de l’affinité du xénobiotique (médicament ou contaminant) pour chacune des protéines plasmatiques et des mécanismes impliqués dans le foie ; et 3) prédire la CLh avec précision et exactitude des xénobiotiques qui se lient aux deux protéines plasmatiques. Ces approches IVIVE pour la CLh pourront assurément être intégrées dans des modèles PK/TK à base physiologique pour les xénobiotiques afin d’améliorer la prédiction de leur pharmacocinétique et d’accélérer le processus de développement de médicaments.
The prediction of pharmacokinetic/toxicokinetic (PK/TK) parameters such as intrinsic clearance (CLint) and hepatic clearance (CLh) for highly bound drugs is a major challenge in quantitative modeling. According to the ‘free drug hypothesis’, only the free drug can pass through the plasma membrane and the CLh of this drug is calculated according to its free fraction (fup). Nevertheless, the hepatic uptake facilitated by albumin (ALB) is a violation of the ‘free drug hypothesis’. This facilitated hepatic uptake is based on the possibility that the ALB-drug complex may provide additional drug intake to the hepatocytes. Thus, this could largely explain the underpredictions of CLh. In addition, some drugs can bind extensively in plasma, and to several plasma proteins such as ALB and alpha-1-glycoprotein acid (AGP). Thus, the high binding of the same drug to either ALB or AGP, or to both, could have distinct impacts on the prediction of these PK/TK parameters. However, no study has yet explored how to simulate the difference between these impacts. The main objective of this thesis is therefore to evaluate (with accuracy and precision) for a series of drugs, in the in vivo (or in situ) condition, these impacts in the presence of the two plasma proteins, jointly or separately. Also, it is important to verify if a generic model can be applied. This thesis is divided into three specific objectives. The first is to propose a decision tree to facilitate the selection of appropriate predictive approaches of CLhin vivo for drugs with different characteristics. The second is to assess the impacts of extensive binding to the two plasma proteins ALB and AGP on the CLh of two selected xenobiotics (perampanel (PER) and fluoxetine (FLU)); these drugs have strong affinities to both proteins and an exclusive (or predominant) metabolism in the liver. And the last objective is to develop and validate a new predictive model of CLh for xenobiotics with the potential to bind extensively to ALB as well as to AGP. Firstly, in vitro data obtained in humans were collected for 19 drugs (i.e. substrates of OAT2 and OATP1B1 transporters) and were then used in six in vitro-to-in vivo (IVIVE) extrapolation models to predict these PK/TK parameters. After a statistical comparison, the results showed that the approach 2 (i.e. ‘fup-adjusted model’) that is based on the ALB-facilitated hepatic uptake, had the best predictive performance. However, the approach 5 (i.e. ‘Extended Clearance Model’) that is based on the membrane transporter-mediated uptake, was very relevant to apply for the substrates of membrane transporters. These substrates would potentially be less affected by ALB. Thus, a decision tree has been proposed to quickly and judiciously select the best IVIVE approach to predict CLhin vivo for each xenobiotic in the presence of ALB. Secondly, the PER and FLU drugs were selected from a data collection of 1907 drugs depending on certain criteria (exclusive or predominant metabolism in the liver, no transport facilitated by membrane transporters, high affinity for the two proteins ALB and AGP, and having a binding ratio between AGP and ALB close to the unity). This selection was made to conduct experiments using the isolated and perfused rat liver (IPRL) apparatus, in the presence, and in the absence of the ALB and AGP proteins (i.e. four IPRL scenarios). The IPRL results showed that PER is low to moderately metabolized (hepatic extraction= 0.2-0.7), while FLU is highly metabolized (hepatic extraction= 0.8-0.99). The Michaelis-Menten model was fitted to the obtained metabolic kinetics, and different parameters Vmax, Km and Km, u were obtained from the model. At low free concentrations for both drugs (i.e. therapeutic concentrations) and in the presence of plasma proteins, the values of unbound CLint increased for PER (with ALB and the mixture of the two proteins (MIX)) and FLU (with ALB, AGP and MIX); when compared to those obtained from the protein-free scenario (except for PER with AGP, the unbound CLint values decreased). In addition, the calculations of CLint ratios (WITHOUT versus WITH protein) indicated the occurrence of a hepatic uptake facilitated by ALB or AGP. These ratios also helped in verifying whether the metabolic kinetics for PER and FLU followed either ‘the free drug hypothesis’ or that of ‘plasma protein-facilitated hepatic uptake’. Finally, a new predictive approach of CLh (WO-to-MIX approach) was developed based on a new notion of fractional binding and incorporating new parameters such as the ALB bound fraction (fB-ALB) and the AGP bound fraction (fB-AGP) from the MIX scenario into the ‘fup-adjusted model’. This model is based on the ‘ALB-facilitated hepatic uptake’. Unlike the WO-to-MIX approach, the ‘well-stirred model’ is based on ‘the free drug hypothesis’. Then, the Vmax and Km parameters that were obtained in situ for PER and FLU from the protein-free IPRL experiments, were used in combination with the fup-adjusted input parameter for the ‘fup-adjusted model’ or with the free fraction (fup) for the ‘well-stirred model’. A comparison of the two models’ overall predictive performances was made. The predictive performances of the new model were promising for FLU, which showed the highest degree of ‘ALB-mediated hepatic uptake’, compared to the conventional model. This WO-to-MIX approach is a first validation of a novel extrapolation model suggested for drugs such as FLU that bind to both ALB and AGP. The well-stirred model remains however a useful tool to predict the clearance for drugs such as PER. The prediction accuracy was lower for the latter drug probably because the ALB-mediated hepatic uptake does not seem to be maximal, and, hence, the use of fup-adjusted has overestimated its CLhin vivo. Therefore, more work is needed particularly for PER. This thesis shows that a generic approach to predict the CLh in vivo does not exist. Nevertheless, the choice of an IVIVE approach with satisfactory predictive performances is now possible. The results of this thesis contribute to: 1) better understand the impacts on the PK/TK parameters of extensive drug binding to ALB and AGP; 2) choose the best predictive approach to CLh based on the affinity of xenobiotic (drug or contaminant) to each of the plasma proteins and the mechanisms involved in the liver; and 3) predict accurately and with precision the output CLh of xenobiotics that bind to the two plasma proteins. These IVIVE approaches for CLh can certainly be integrated into physiologically based PK/TK models for xenobiotics to improve the prediction of their pharmacokinetics and to accelerate the drug development process.

碩士
國立臺灣大學
化學研究所
90
CD1 molecules are a family of MHC-like cell surface glycoproteins that present glycolipid antigens to T lymphocytes. Human CD1d and its mouse homolog are shown to present a special glycolipid, a-galactosyl ceramide (alpha-GalCer), to a subset of T lymphocytes called natural killer T (NKT) cells. It is suggested that NKT cells are activated through the TCR interaction with CD1d/ alpha-GalCer complex. NKT cells are thought to be immuno-regulators because they secrete large amounts of cytokines, such as IL-4 and INFgamma, after stimulation. They are also found to show antitumor activity and to control autoimmune responses. Among the reported glycolipids presented by human CD1d (or its mouse homolog), alpha-GalCer and alpha-GlcCer show the highest immuno-stimulatory activities. Some structural features of alpha-GalCer have been demonstrated necessary for antigen recognition by CD1-restricted NKT cells, including: (1) the alpha- anomeric glycosidic linkage between sugar and lipid moieties; (2) the 2-hydroxyl group on the pyranose ring; and (3) the presence of 3’ and 4’-hydroxyl groups on the phytosphingosine chain. Change of the antigen structure may alter the level of stimulatory activity and/or switch the cytokine pattern released by NKT cells. However, the information is still limited about how antigen structures influence the activation event. In order to obtain the scenario about structure-activity relationship of alpha-GalCer, we synthesized alpha-GalCer and its analogues based on the above-mentioned features. The synthetic approach was designed not only to increase versatility of the alpha-GalCer synthesis but also to accommodate the flexibility for further modification. The phytosphingosine moiety was replaced by corresponding serine ester or amide as the simplified lipids to afford analogues ANGX1-3. In addition, the model study for functional group replacement at sugar C-2 position was demonstrated. Finally, an ELISA assay was established to evaluate the stimulatory activities of these analogues and compare the results with that of alpha-GalCer.