In vivo activation of PPAR target genes by RXR homodimers.

The ability of a retinoid X receptor (RXR) to heterodimerize with many nuclear receptors, including LXR, PPAR, NGF1B and RAR, underscores its pivotal role within the nuclear receptor superfamily. Among these heterodimers, PPAR:RXR is considered an important signalling mediator of both PPAR ligands, such as fatty acids, and 9-cis retinoic acid (9-cis RA), an RXR ligand. In contrast, the existence of an RXR/9-cis RA signalling pathway independent of PPAR or any other dimerization partner remains disputed. Using in vivo chromatin immunoprecipitation, we now show that RXR homodimers can selectively bind to functional PPREs and induce transactivation. At the molecular level, this pathway requires stabilization of the homodimer-DNA complexes through ligand-dependent interaction with the coactivator SRC1 or TIF2. This pathway operates both in the absence and in the presence of PPAR, as assessed in cells carrying inactivating mutations in PPAR genes and in wild-type cells. In addition, this signalling pathway via PPREs is fully functional and can rescue the severe hypothermia phenotype observed in fasted PPARalpha-/- mice. These observations have important pharmacological implications for the development of new rexinoid-based treatments.

Homeodomain binding motifs modulate the probability of odorant receptor gene choice in transgenic mice.

Odorant receptor (OR) genes constitute with 1200 members the largest gene family in the mouse genome. A mature olfactory sensory neuron (OSN) is thought to express just one OR gene, and from one allele. The cell bodies of OSNs that express a given OR gene display a mosaic pattern within a particular region of the main olfactory epithelium. The mechanisms and cis-acting DNA elements that regulate the expression of one OR gene per OSN - OR gene choice - remain poorly understood. Here, we describe a reporter assay to identify minimal promoters for OR genes in transgenic mice, which are produced by the conventional method of pronuclear injection of DNA. The promoter transgenes are devoid of an OR coding sequence, and instead drive expression of the axonal marker tau-β-galactosidase. For four mouse OR genes (M71, M72, MOR23, and P3) and one human OR gene (hM72), a mosaic, OSN-specific pattern of reporter expression can be obtained in transgenic mice with contiguous DNA segments of only ~300 bp that are centered around the transcription start site (TSS). The ~150bp region upstream of the TSS contains three conserved sequence motifs, including homeodomain (HD) binding sites. Such HD binding sites are also present in the H and P elements, DNA sequences that are known to strongly influence OR gene expression. When a 19mer encompassing a HD binding site from the P element is multimerized nine times and added upstream of a MOR23 minigene that contains the MOR23 coding region, we observe a dramatic increase in the number of transgene-expressing founders and lines and in the number of labeled OSNs. By contrast, a nine times multimerized 19mer with a mutant HD binding site does not have these effects. We hypothesize that HD binding sites in the H and P elements and in OR promoters modulate the probability of OR gene choice.

Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.

Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.

Circulating carotenoids and risk of breast cancer: pooled analysis of eight prospective studies.

Background Carotenoids, micronutrients in fruits and vegetables, may reduce breast cancer risk. Most, but not all, past studies of circulating carotenoids and breast cancer have found an inverse association with at least one carotenoid, although the specific carotenoid has varied across studies. Methods We conducted a pooled analysis of eight cohort studies comprising more than 80% of the world's published prospective data on plasma or serum carotenoids and breast cancer, including 3055 case subjects and 3956 matched control subjects. To account for laboratory differences and examine population differences across studies, we recalibrated participant carotenoid levels to a common standard by reassaying 20 plasma or serum samples from each cohort together at the same laboratory. Using conditional logistic regression, adjusting for several breast cancer risk factors, we calculated relative risks (RRs) and 95% confidence intervals (CIs) using quintiles defined among the control subjects from all studies. All P values are two-sided. Results Statistically significant inverse associations with breast cancer were observed for α-carotene (top vs bottom quintile RR = 0.87, 95% CI = 0.71 to 1.05, Ptrend = .04), β-carotene (RR = 0.83, 95% CI = 0.70 to 0.98, Ptrend = .02), lutein+zeaxanthin (RR = 0.84, 95% CI = 0.70 to 1.01, Ptrend = .05), lycopene (RR = 0.78, 95% CI = 0.62 to 0.99, Ptrend = .02), and total carotenoids (RR = 0.81, 95% CI = 0.68 to 0.96, Ptrend = .01). β-Cryptoxanthin was not statistically significantly associated with risk. Tests for heterogeneity across studies were not statistically significant. For several carotenoids, associations appeared stronger for estrogen receptor negative (ER(-)) than for ER(+) tumors (eg, β-carotene: ER(-): top vs bottom quintile RR = 0.52, 95% CI = 0.36 to 0.77, Ptrend = .001; ER(+): RR = 0.83, 95% CI = 0.66 to 1.04, Ptrend = .06; Pheterogeneity = .01). Conclusions This comprehensive prospective analysis suggests women with higher circulating levels of α-carotene, β-carotene, lutein+zeaxanthin, lycopene, and total carotenoids may be at reduced risk of breast cancer.

Fibrinogen and fibronectin binding cooperate for valve infection and invasion in Staphylococcus aureus experimental endocarditis.

The expression of Staphylococcus aureus adhesins in Lactococcus lactis identified clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) as critical for valve colonization in rats with experimental endocarditis. This study further analyzed their role in disease evolution. Infected animals were followed for 3 d. ClfA-positive lactococci successfully colonized damaged valves, but were spontaneously eradicated over 48 h. In contrast, FnBPA-positive lactococci progressively increased bacterial titers in vegetations and spleens. At imaging, ClfA-positive lactococci were restricted to the vegetations, whereas FnBPA-positive lactococci also invaded the adjacent endothelium. This reflected the capacity of FnBPA to trigger cell internalization in vitro. Because FnBPA carries both fibrinogen- and fibronectin-binding domains, we tested the role of these functionalities by deleting the fibrinogen-binding domain of FnBPA and supplementing it with the fibrinogen-binding domain of ClfA in cis or in trans. Deletion of the fibrinogen-binding domain of FnBPA did not alter fibronectin binding and cell internalization in vitro. However, it totally abrogated valve infectivity in vivo. This ability was restored in cis by inserting the fibrinogen-binding domain of ClfA into truncated FnBPA, and in trans by coexpressing full-length ClfA and truncated FnBPA on two separate plasmids. Thus, fibrinogen and fibronectin binding could cooperate for S. aureus valve colonization and endothelial invasion in vivo.

Strong EBV-specific CD8+ T-cell response in patients with early multiple sclerosis

Epstein-Barr virus (EBV) has been associated with multiple sclerosis (MS), however, most studies examining the relationship between the virus and the disease have been based on serologies, and if EBV is linked to MS, CD8+ T cells are likely to be involved as they are important both in MS pathogenesis and in controlling viruses. We hypothesized that valuable information on the link between MS and EBV would be ascertained from the study of frequency and activation levels of EBV-specific CD8+ T cells in different categories of MS patients and control subjects. We investigated EBV-specific cellular immune responses using proliferation and enzyme linked immunospot assays, and humoral immune responses by analysis of anti-EBV antibodies, in a cohort of 164 subjects, including 108 patients with different stages of MS, 35 with other neurological diseases and 21 healthy control subjects. Additionally, the cohort were all tested against cytomegalovirus (CMV), another neurotropic herpes virus not convincingly associated with MS, nor thought to be deleterious to the disease. We corrected all data for age using linear regression analysis over the total cohorts of EBV- and CMV-infected subjects. In the whole cohort, the rate of EBV and CMV infections were 99% and 51%, respectively. The frequency of IFN-gamma secreting EBV-specific CD8+ T cells in patients with clinically isolated syndrome (CIS) was significantly higher than that found in patients with relapsing-remitting MS (RR-MS), secondary-progressive MS, primary-progressive MS, patients with other neurological diseases and healthy controls. The shorter the interval between MS onset and our assays, the more intense was the EBV-specific CD8+ T-cell response. Confirming the above results, we found that EBV-specific CD8+ T-cell responses decreased in 12/13 patients with CIS followed prospectively for 1.0 +/- 0.2 years. In contrast, there was no difference between categories for EBV-specific CD4+ T cell, or for CMV-specific CD4+ and CD8+ T-cell responses. Anti-EBV-encoded nuclear antigen-1 (EBNA-1)-specific antibodies correlated with EBV-specific CD8+ T cells in patients with CIS and RR-MS. However, whereas EBV-specific CD8+ T cells were increased the most in early MS, EBNA-1-specific antibodies were increased in early as well as in progressive forms of MS. Our data show high levels of CD8+ T-cell activation against EBV--but not CMV--early in the course of MS, which support the hypothesis that EBV might be associated with the onset of this disease.

Severely presenting strokes : profile & outcome

Etat des connaissances: Les Accidents vasculaires cérébraux (AVC) sévères sont une cause importante de mortalité et de handicap en Suisse. Les buts de cette étude sont de déterminer les caractéristiques des patients avec un AVC à présentation initiale sévère "severely presenting", ainsi que d'identifier les facteurs prédicteurs en phase aigüe et subaigüe d'un devenir favorable chez ces patients. Methodologie: En utilisant le registre des AVC "Acute STroke Registry and Analysis of Lausanne (ASTRAL), nous avons comparé tous les patients avec un AVC "à présentation sévère", defini comme un score National Institute of Health Stroke Scale (NIHSS) > 20 à l'admission aux urgences, avec tous les autres patients du registre. Dans une analyse statistique multivariée, les associations avec les caractéristiques démographiques, cliniques, pathophysiologiques, métaboliques et radiologiques des patients on été déterminées. Dans un deuxième temps, nous avons analysé les facteurs prédicteurs d'un devenir favorable à 3 mois (modified Rankin scale (mRS) <3) dans ce groupe d'AVC à présentation sévère. Resultats: Parmi les 1 '915 patients consécutifs, 243 (12.7%) présentaient un AVC rejoignant la définition de sévère. Ceux-cis étaient associés de manière significative avec un méchanisme ischémique cardio-embolique (OR=1.74 / 95% CI 1.19 - 2.54), un début inconnu de la Symptomatologie (OR=2.35 / 95% CI 1.14 - 4.83), avaient plus de trouvailles d'origine ischémique aigüe lors de l'imagerie dJentrée (la majorité sont des CT-scan, OR=2.65 / 95% CI 1.79 - 3.92), plus d'occlusions dans l'imagerie vasculaire d'admission (OR=27.01 / 95% CI 11.5 - 62.9), moins d'anciens infarctus cérébraux sur l'imagerie (OR=0.43 / 95% 0.26¬0.72), un taux d'haemoglobine plus bas en g/1 (OR=0.97 / 95% CI 0.96 - 0.99), un taux de leucocytes plus élevé par 1000 cells/1 (OR=1.05 / 95%CI 1.00 - 1.11). parmi les 68 (28%) patients avec un devenir favorable malgré un AVC initialement sévère, leur évolution favorable à été associée avec un âge plus jeune (OR=0.94 / 95% CI 0.92 - 0.97), la présence d'évenements cérébrovasculaires antécédants (OR=3.00 / 95% CI 1.01 - 8.97), un traitement hypolipémiant déjà présent (OR= 3.82 / 95% CI 1.34 - 10.90), une température corporelle d'admission plus basse (0R=0.43 1 95% CI 0.23 - 0.78), une concentration subaigüe de glucose plus basse (OR=0.74 1 95% CI 0.56 - 0.97), et une recanalisation spontanée ou par thrombolyse à 24h (OR= 4.51 1 95%CI 1.96- 10.41). Conclusion: les AVC à présentation initiale severe sont associés à des facteurs prédicteurs cliniques, radiologiques, et métaboliques multiples, dont certains sont modifiables. Les facteurs prédicteurs des 28% de patients avec un devenir favorable en dépit d'un AVC intitialement sévère sont un pré-traitement par hypolipémiants, une temperature corporelle plus basse à l'admission, une glycémie plus basse à 24heures et la recanalisation artérielle.

Analysis of the alternative pathways for the beta-oxidation of unsaturated fatty acids using transgenic plants synthesizing polyhydroxyalkanoates in peroxisomes.

Degradation of fatty acids having cis-double bonds on even-numbered carbons requires the presence of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. Two alternative pathways have been described to degrade these fatty acids. One pathway involves the participation of the enzymes 2, 4-dienoyl-coenzyme A (CoA) reductase and Delta(3)-Delta(2)-enoyl-CoA isomerase, whereas the second involves the epimerization of R-3-hydroxyacyl-CoA via a 3-hydroxyacyl-CoA epimerase or the action of two stereo-specific enoyl-CoA hydratases. Although degradation of these fatty acids in bacteria and mammalian peroxisomes was shown to involve mainly the reductase-isomerase pathway, previous analysis of the relative activity of the enoyl-CoA hydratase II (also called R-3-hydroxyacyl-CoA hydro-lyase) and 2,4-dienoyl-CoA reductase in plants indicated that degradation occurred mainly through the epimerase pathway. We have examined the implication of both pathways in transgenic Arabidopsis expressing the polyhydroxyalkanoate synthase from Pseudomonas aeruginosa in peroxisomes and producing polyhydroxyalkanoate from the 3-hydroxyacyl-CoA intermediates of the beta-oxidation cycle. Analysis of the polyhydroxyalkanoate synthesized in plants grown in media containing cis-10-heptadecenoic or cis-10-pentadecenoic acids revealed a significant contribution of both the reductase-isomerase and epimerase pathways to the degradation of these fatty acids.

The half-size ABC transporters STR1 and STR2 are indispensable for mycorrhizal arbuscule formation in rice.

The central structure of the symbiotic association between plants and arbuscular mycorrhizal (AM) fungi is the fungal arbuscule that delivers minerals to the plant. Our earlier transcriptome analyses identified two half-size ABCG transporters that displayed enhanced mRNA levels in mycorrhizal roots. We now show specific transcript accumulation in arbusculated cells of both genes during symbiosis. Presently, arbuscule-relevant factors from monocotyledons have not been reported. Mutation of either of the Oryza sativa (rice) ABCG transporters blocked arbuscule growth of different AM fungi at a small and stunted stage, recapitulating the phenotype of Medicago truncatula stunted arbuscule 1 and 2 (str1 and str2) mutants that are deficient in homologous ABCG genes. This phenotypic resemblance and phylogenetic analysis suggest functional conservation of STR1 and STR2 across the angiosperms. Malnutrition of the fungus underlying limited arbuscular growth was excluded by the absence of complementation of the str1 phenotype by wild-type nurse plants. Furthermore, plant AM signaling was found to be intact, as arbuscule-induced marker transcript accumulation was not affected in str1 mutants. Strigolactones have previously been hypothesized to operate as intracellular hyphal branching signals and possible substrates of STR1 and STR2. However, full arbuscule development in the strigolactone biosynthesis mutants d10 and d17 suggested strigolactones to be unlikely substrates of STR1/STR2. Interestingly, rice STR1 is associated with a cis-natural antisense transcript (antiSTR1). Analogous to STR1 and STR2, at the root cortex level, the antiSTR1 transcript is specifically detected in arbusculated cells, suggesting unexpected modes of STR1 regulation in rice.

Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers.

The nuclear hormone receptors called PPARs (peroxisome proliferator-activated receptors alpha, beta, and gamma) regulate the peroxisomal beta-oxidation of fatty acids by induction of the acyl-CoA oxidase gene that encodes the rate-limiting enzyme of the pathway. Gel retardation and cotransfection assays revealed that PPAR alpha heterodimerizes with retinoid X receptor beta (RXR beta; RXR is the receptor for 9-cis-retinoic acid) and that the two receptors cooperate for the activation of the acyl-CoA oxidase gene promoter. The strongest stimulation of this promoter was obtained when both receptors were exposed simultaneously to their cognate activators. Furthermore, we show that natural fatty acids, and especially polyunsaturated fatty acids, activate PPARs as potently as does the hypolipidemic drug Wy 14,643, the most effective activator known so far. Moreover, we discovered that the synthetic arachidonic acid analogue 5,8,11,14-eicosatetraynoic acid is 100 times more effective than Wy 14,643 in the activation of PPAR alpha. In conclusion, our data demonstrate a convergence of the PPAR and RXR signaling pathways in the regulation of the peroxisomal beta-oxidation of fatty acids by fatty acids and retinoids.

The activity of S.pombe DSC-1-like factor is cell cycle regulated and dependent on the activity of p34cdc2.

In the eukaryotic cell cycle, there are major control points in late G2 to determine the timing of the initiation of mitosis, and in late G1, regulating entry into S phase. In yeasts, this latter control is called start. Traverse of the start control and progression to S phase is accompanied by an increase in the expression of some of the genes whose products are required for DNA synthesis. In Saccharomyces cerevisiae, the coordinate expression of these genes in late G1 is dependent on a cis-acting sequence element called the MluI cell cycle box (MCB). A transcription factor called DSC-1 binds these elements and mediates cell cycle regulated transcription, though it is unclear whether this is by cell cycle-dependent changes in its activity. A DSC-1-like factor has also been identified in the fission yeast S.pombe. This is composed of at least the products of the cdc10 and sct1/res1 genes, and binds to the promoters of genes whose expression increases prior to S phase. We demonstrate that p85cdc10 is a nuclear protein and that the activity of the S.pombe DSC-1 factor varies through the cell cycle; it is high in cells that have passed start, decreases at the time of anaphase, remains low during the pre-start phase of G1 and increases at the time of the next S phase. We also show that the reactivation in late G1 is dependent on the G1 form of p34cdc2.

Alterations of the 5'untranslated region of SLC16A12 lead to age-related cataract.

PURPOSE. Knowledge of genetic factors predisposing to age-related cataract is very limited. The aim of this study was to identify DNA sequences that either lead to or predispose for this disease. METHODS. The candidate gene SLC16A12, which encodes a solute carrier of the monocarboxylate transporter family, was sequenced in 484 patients with cataract (134 with juvenile cataract, 350 with age-related cataract) and 190 control subjects. Expression studies included luciferase reporter assay and RT-PCR experiments. RESULTS. One patient with age-related cataract showed a novel heterozygous mutation (c.-17A>G) in the 5'untranslated region (5'UTR). This mutation is in cis with the minor G-allele of the single nucleotide polymorphism (SNP) rs3740030 (c.-42T/G), also within the 5'UTR. Using a luciferase reporter assay system, a construct with the patient's haplotype caused a significant upregulation of luciferase activity. In comparison, the SNP G-allele alone promoted less activity, but that amount was still significantly higher than the amount of the common T-allele. Analysis of SLC16A12 transcripts in surrogate tissue demonstrated striking allele-specific differences causing 5'UTR heterogeneity with respect to sequence and quantity. These differences in gene expression were mirrored in an allele-specific predisposition to age-related cataract, as determined in a Swiss population (odds ratio approximately 2.2; confidence intervals, 1.23-4.3). CONCLUSIONS. The monocarboxylate transporter SLC16A12 may contribute to age-related cataract. Sequences within the 5'UTR modulate translational efficiency with pathogenic consequences.

Photodynamic Diagnosis of Non-muscle-invasive Bladder Cancer with Hexaminolevulinate Cystoscopy: A Meta-analysis of Detection and Recurrence Based on Raw Data.

BACKGROUND: Studies on hexaminolevulinate (HAL) cystoscopy report improved detection of bladder tumours. However, recent meta-analyses report conflicting effects on recurrence. OBJECTIVE: To assess available clinical data for blue light (BL) HAL cystoscopy on the detection of Ta/T1 and carcinoma in situ (CIS) tumours, and on tumour recurrence. DESIGN, SETTING, AND PARTICIPANTS: This meta-analysis reviewed raw data from prospective studies on 1345 patients with known or suspected non-muscle-invasive bladder cancer (NMIBC). INTERVENTION: A single application of HAL cystoscopy was used as an adjunct to white light (WL) cystoscopy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We studied the detection of NMIBC (intention to treat [ITT]: n=831; six studies) and recurrence (per protocol: n=634; three studies) up to 1 yr. DerSimonian and Laird's random-effects model was used to obtain pooled relative risks (RRs) and associated 95% confidence intervals (CIs) for outcomes for detection. RESULTS AND LIMITATIONS: BL cystoscopy detected significantly more Ta tumours (14.7%; p<0.001; odds ratio [OR]: 4.898; 95% CI, 1.937-12.390) and CIS lesions (40.8%; p<0.001; OR: 12.372; 95% CI, 6.343-24.133) than WL. There were 24.9% patients with at least one additional Ta/T1 tumour seen with BL (p<0.001), significant also in patients with primary (20.7%; p<0.001) and recurrent cancer (27.7%; p<0.001), and in patients at high risk (27.0%; p<0.001) and intermediate risk (35.7%; p=0.004). In 26.7% of patients, CIS was detected only by BL (p<0.001) and was also significant in patients with primary (28.0%; p<0.001) and recurrent cancer (25.0%; p<0.001). Recurrence rates up to 12 mo were significantly lower overall with BL, 34.5% versus 45.4% (p=0.006; RR: 0.761 [0.627-0.924]), and lower in patients with T1 or CIS (p=0.052; RR: 0.696 [0.482-1.003]), Ta (p=0.040; RR: 0.804 [0.653-0.991]), and in high-risk (p=0.050) and low-risk (p=0.029) subgroups. Some subgroups had too few patients to allow statistically meaningful analysis. Heterogeneity was minimised by the statistical analysis method used. CONCLUSIONS: This meta-analysis confirms that HAL BL cystoscopy significantly improves the detection of bladder tumours leading to a reduction of recurrence at 9-12 mo. The benefit is independent of the level of risk and is evident in patients with Ta, T1, CIS, primary, and recurrent cancer.

Identification and functional characterization of auxiliary enzymes of the β-oxidation in " Arabidopsis thaliana "

Abstract: The ß-oxidation is the universal pathway that allows living organisms to degrade fatty acids. leading to lipid homeostasis and carbon and energy recovery from the fatty acid molecules. This pathway is centred on four core enzymatic activities sufficient to degrade saturated fatty acids. Additional auxiliary enzymes of the ß-oxidation are necessary for the complete degradation of a larger array of molecules encompassing the unsaturated fatty acids. The main pathways of the ßoxidation of fatty acids have been investigated extensively and auxiliary enzymes are well-known in mammals and yeast. The comparison of the established ß-oxidation systems suggests that the activities that are required to proceed to the full degradation of unsaturated fatty acids are present regardless of the organism and rely on common active site templates. The precise identity of the plant enzymes was unknown. By homology searches in the genome of Arabidopsis thaliana, I identified genes. encoding for proteins that could be orthologous to the yeast or animal auxiliary enzymes Δ 3, Δ 2-enoyl-CoA isomerase, Δ 3,5, Δ 2,4 -dienoyl-CoA isomerase, and type 2 enoyl-CoA hydratase. I established that these genes are expressed in Arabidopsis and that their expression can be correlated to the expression of core ß-oxidation genes. Through the observation of chimeric fluorescent protein fusions, I demonstrated that the identified proteins are localized in the peroxisóme, the only organelle where the ß-oxidation occurs in plants. Enzymatic assays were performed with the partially purified enzymes to demonstrate that the identified enzymes can catalyze the same in vitro reactions as their non-plant orthologs. The activities in vivo of the plant enzymes were demonstrated by heterologous complementation of the corresponding yeast Saccharomyces cerevisiae mutants. The complementation was visualized using the artificial polyhydroxyalkanoate (PHA) production in yeast peroxisomes. The recombinant strains, expressing a Pseudomonas aeruginosa PHA synthase modified for a peroxisomal localization, produce this polymer that serves as a trap for the 3-hydroxyacyl-CoA intermediaries of the ßoxidation and that reflects qualitatively and quantitatively the array of molecules that are processed through the ß-oxidation. This complementation demonstrated the implication of the plant Δ 3, Δ 2-enoyl-CoA isomerases and Δ3,5, Δ2,4-dienoyl-CoA isomerase in the degradation of odd chain position unsaturated fatty acids. The presence of a monofunctional type 2 enoyl-CoA hydratase is a novel in eukaryotes. Downregulation of the corresponding gene expression in an Arabidopsis line, modified to produce PHA in the peroxisome, demonstrated thàt this enzyme participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2Eenoyl-CoA for further degradation through the core ß-oxidation cycle. Résumé: La ß-oxydation est une voie universelle de dégradation des acides gras qui permet aux organismes vivants d'assurer une homéostasie lipidique et de récupérer l'énergie et le carbone contenus dans les acides gras. Le coeur de cette voie est composé de quatre réactions enzymatiques suffisantes à la dégradation des acides gras saturés. La présence des enzymes auxiliaires de la ß-oxydation est nécessaire à la dégradation d'une gamme plus étendue de molécules comprenant les acides gras insaturés. Les voies principales de la ß-oxydation des acides gras ont été étudiées en détail et les enzymes auxiliaires sont déterminées chez les mammifères et la levure. La comparaison entre les systèmes de ß-oxydation connus suggère que les activités requises pour la dégradation complète des acides gras insaturés reposent sur la présence de site actifs similaires. L'identité précise des enzymes auxiliaires chez les plantes était inconnue. En cherchant par homologie dans le génome de la plante modèle Arabidopsis thaliana, j'ai identifié des gènes codant pour des protéines pouvant être orthologues aux enzymes auxiliaires Δ3 Δ2-enoyl-CoA isomérase, Δ 3,5 Δ 2,4-dienoyl-CoA isomérase et enoyl-CoA hydratase de type 2 d'origine fongique ou mammalienne. J'ai établi la corrélation de l'expression de ces gènes dans Arabidopsis avec celle de gènes des enzymes du coeur de la ß-oxydation. En observant des chimères de fusion avec des protéines fluorescentes, j'ai démontré que les protéines identifiées sont localisées dans le péroxysomes, le seul organelle où la ß-oxydation se déroule chez les plantes. Des essais enzymatiques ont été conduits avec ces enzymes partiellement purifiées pour démontrer que les enzymes identifiées sont capables de catalyser in vitro les mêmes réactions que leurs orthologues non végétaux. Les activités des enzymes végétales in vivo ont été .démontrées par complémentation hétérologue des mutants de délétion correspondants de levure Saccharomyces cerevisiae. La visualisation de la complémentation est rendue possible par la synthèse de polyhydroxyalcanoate (PHA) dans les péroxysomes de levure. Les souches recombinantes expriment la PHA synthase de Pseudomonas aeruginosa modifiée pour être localisée dans le péroxysome produisent ce polymère qui sert de piège pour les 3-hydroxyacylCoAs intermédiaires de la ß-oxydation et qui reflète qualitativement et quantitativement la gamme de molécules qui subit la ß-oxydation. Cette complémentation a permis de démontrer que les Δ3, Δ2-enoyl-CoA isomérases, et la Δ3.5, Δ2,4-dienoyl-CoA isomérase végétales sont impliquées dans la dégradation des acides gras insaturés en position impaire. L'enoyl-CoA hydratase de type 2 monofonctionelle est une enzyme nouvelle chez les eucaryotes. La sous-expression du gène correspondant dans une lignée d'Arabidopsis modifiée pour produite du PHA dans le péroxysome a permis de démontrer que cette enzyme participe in vivo à la dégradation des acides gras ayant une double liaison en conformation cis (Z) en position paire.

La pertinence en science forensique. Une (en)quête épistémologique et empirique

A criminal investigation requires to search and to interpret vestiges of a criminal act that happened in a past time. The forensic investigator arises in this context as a critical reader of the investigation scene, in search of physical traces that should enable her to tell a story of the offence/crime which allegedly occurred. The challenge of any investigator is to detect and recognise relevant physical traces in order to provide forensic clues for investigation and intelligence purposes. Inspired by this obser- vation, the current research focuses on the following questions : What is a relevant physical trace? And, how does the forensic investigator know she is facing one ? The interest of such questions is to provide a definition of a dimension often used in forensic science but never studied in its implications and operations. This doctoral research investigates scientific paths that are not often explored in forensic science, by using semiotic and sociological tools combined with statistical data analysis. The results are shown following a semiotic path, strongly influenced by Peir- ce's studies, and a second track, called empirical, where investigations data were analysed and forensic investigators interviewed about their work practices in the field. By the semiotic track, a macroscopic view is given of a signification process running from the discove- red physical trace at the scene to what is evaluated as being relevant for the investigator. The physical trace is perceived in the form of several signs, whose meaning is culturally codified. The reasoning should consist of three main steps : 1- What kind of source does the discovered physical trace refer to ? 2- What cause/activity is at the origin of this source in the specific context of the case ? 3- What story can be told from these observations ? The stage 3 requires to reason in creating hypotheses that should explain the presence of the discovered trace coming from an activity ; the specific activity that is related to the investigated case. To validate this assumption, it would depend on their ability to respond to a rule of relevancy. The last step is the symbolisation of the relevancy. The rule would consist of two points : the recognition of the factual/circumstantial relevancy (Is the link between the trace and the case recognised with the formulated hypothesis ? ) and appropriate relevancy (What investment is required to collect and analyse the discovered trace considering the expected outcome at the investigation/intelligence level?). This process of meaning is based on observations and a conjectural reasoning subject to many influences. In this study, relevancy in forensic science is presented as a conventional dimension that is symbolised and conditioned by the context, the forensic investigator's practice and her workplace environment (culture of the place). In short, the current research states relevancy results of the interactions between parameters from situational, structural (or organisational) and individual orders. The detection, collection and analysis of relevant physical traces at scenes depends on the knowledge and culture mastered by the forensic investigator. In the study of the relation relevant trace-forensic investigator, this research introduces the KEE model as a conceptual map that illustrates three major areas of forensic knowledge and culture acquisition, involved in the research and evaluation of the relevant physical trace. Through the analysis of the investigation data and interviews, the relationship between those three parameters and the relevancy was highlighted. K, for knowing, embodies a rela- tionship to the immediate knowledge allowing to give an overview of the reality at a specific moment ; an important point since relevancy is signified in a context. E, for education, is considered through its relationship with relevancy via a culture that tends to become institutionalised ; it represents the theoretical knowledge. As for the parameter E, for experience, it exists in its relation to relevancy in the adjustments of the strategies of intervention (i.e a practical knowledge) of each practitioner having modulated her work in the light of success and setbacks case after case. The two E parameters constitute the library resources for the semiotic recognition process and the K parameter ensures the contextualisation required to set up the reasoning and to formulate explanatory hypotheses for the discovered physical traces, questioned in their relevancy. This research demonstrates that the relevancy is not absolute. It is temporal and contextual; it is a conventional and relative dimension that must be discussed. This is where the whole issue of the meaning of what is relevant to each stakeholder of the investigation process rests. By proposing a step by step approach to the meaning process from the physical trace to the forensic clue, this study aims to provide a more advanced understanding of the reasoning and its operation, in order to streng- then forensic investigators' training. This doctoral research presents a set of tools critical to both pedagogical and practical aspects for crime scene management while identifying key-influences with individual, structural and situational dimensions. - Une enquête criminelle consiste à rechercher et à faire parler les vestiges d'un acte incriminé passé. L'investigateur forensique se pose dans ce cadre comme un lecteur critique des lieux à la recherche de traces devant lui permettre de former son récit, soit l'histoire du délit/crime censé s'être produit. Le challenge de tout investigateur est de pouvoir détecter et reconnaître les traces dites pertinentes pour fournir des indices forensiques à buts d'enquête et de renseignement. Inspirée par un tel constat, la présente recherche pose au coeur de ses réflexions les questions suivantes : Qu'est-ce qu'une trace pertinente ? Et, comment fait le forensicien pour déterminer qu'il y fait face ? L'intérêt de tels questionnements se comprend dans la volonté de définir une dimension souvent utili- sée en science forensique, mais encore jamais étudiée dans ses implications et fonctionnements. Cette recherche se lance dans des voies d'étude encore peu explorées en usant d'outils sémiotiques et des pratiques d'enquêtes sociologiques combinés à des traitements statistiques de données. Les résultats sont représentés en suivant une piste sémiotique fortement influencée par les écrits de Peirce et une seconde piste dite empirique où des données d'interventions ont été analysées et des investigateurs forensiques interviewés sur leurs pratiques de travail sur le terrain. Par la piste sémiotique, une vision macroscopique du processus de signification de la trace en élément pertinent est représentée. La trace est perçue sous la forme de plusieurs signes dont la signification est codifiée culturellement. Le raisonnement se formaliserait en trois principales étapes : 1- Quel type de source évoque la trace détectée? 2- Quelle cause/activité est à l'origine de cette source dans le contexte précis du cas ? 3- Quelle histoire peut être racontée à partir de ces observations ? Cette dernière étape consiste à raisonner en créant des hypothèses devant expliquer la présence de la trace détectée suite à une activité posée comme étant en lien avec le cas investigué. Pour valider ces hypothèses, cela dépendrait de leur capacité à répondre à une règle, celle de la pertinence. Cette dernière étape consiste en la symbolisation de la pertinence. La règle se composerait de deux points : la reconnaissance de la pertinence factuelle (le lien entre la trace et le cas est-il reconnu dans l'hypothèse fournie?) et la pertinence appropriée (quel est l'investissement à fournir dans la collecte et l'exploitation de la trace pour le bénéfice attendu au niveau de l'investigation/renseignement?). Tout ce processus de signification se base sur des observations et un raisonnement conjectural soumis à de nombreuses influences. Dans cette étude, la pertinence en science forensique se formalise sous les traits d'une dimension conventionnelle, symbolisée, conditionnée par le contexte, la pratique de l'investigateur forensique et la culture du milieu ; en somme cette recherche avance que la pertinence est le fruit d'une interaction entre des paramètres d'ordre situationnel, structurel (ou organisationnel) et individuel. Garantir la détection, la collecte et l'exploitation des traces pertinentes sur les lieux dépend de la connaissance et d'une culture maîtrisées par le forensicien. Dans l'étude du rapport trace pertinente-investigateur forensique, la présente recherche pose le modèle SFE comme une carte conceptuelle illustrant trois grands axes d'acquisition de la connaissance et de la culture forensiques intervenant dans la recherche et l'évaluation de la trace pertinente. Par l'analyse des données d'in- terventions et des entretiens, le rapport entre ces trois paramètres et la pertinence a été mis en évidence. S, pour savoir, incarne un rapport à la connaissance immédiate pour se faire une représentation d'une réalité à un instant donné, un point important pour une pertinence qui se comprend dans un contexte. F, pour formation, se conçoit dans son rapport à la pertinence via cette culture qui tend à s'institutionnaliser (soit une connaissance théorique). Quant au paramètre E, pour expérience, il se comprend dans son rapport à la pertinence dans cet ajustement des stratégies d'intervention (soit une connaissance pratique) de chaque praticien ayant modulé leur travail au regard des succès et échecs enregistrés cas après cas. F et E formeraient la bibliothèque de ressources permettant le processus de reconnaissance sémiotique et S assurerait la contextualisation nécessaire pour poser le raisonnement et formuler les hypothèses explicatives pour les traces détectées et questionnées dans leur pertinence. Ce travail démontre que la pertinence n'est pas absolue. Elle est temporelle et contextuelle, c'est une dimension conventionnelle relative et interprétée qui se doit d'être discutée. C'est là que repose toute la problématique de la signification de ce qui est pertinent pour chaque participant du processus d'investigation. En proposant une lecture par étapes du processus de signification depuis la trace à l'indice, l'étude vise à offrir une compréhension plus poussée du raisonnement et de son fonctionnement pour renforcer la formation des intervenants forensiques. Cette recherche présente ainsi un ensemble d'outils critiques à portée tant pédagogiques que pratiques pour la gestion des lieux tout en identifiant des influences-clé jouées par des dimensions individuelles, structurelles et situationnelles.