Ischemic A/D transition of mitochondrial complex I and its role in ROS generation

Mitochondrial complex I (NADH:ubiquinone oxidoreductase) is a key enzyme in cellular energy metabolism and provides approximately 40% of the proton-motive force that is utilized during mitochondrial ATP production. The dysregulation of complex I function – either genetically, pharmacologically, or metabolically induced – has severe pathophysiological consequences that often involve an imbalance in the production of reactive oxygen species (ROS). Slow transition of the active (A) enzyme to the deactive, dormant (D) form takes place during ischemia in metabolically active organs such as the heart and brain. The reactivation of complex I occurs upon reoxygenation of ischemic tissue, a process that is usually accompanied by an increase in cellular ROS production. Complex I in the D-form serves as a protective mechanism preventing the oxidative burst upon reperfusion. Conversely, however, the D-form is more vulnerable to oxidative/nitrosative damage. Understanding the so-called active/deactive (A/D) transition may contribute to the development of new therapeutic interventions for conditions like stroke, cardiac infarction, and other ischemia-associated pathologies. In this review, we summarize current knowledge on the mechanism of A/D transition of mitochondrial complex I considering recently available structural data and site-specific labeling experiments. In addition, this review discusses in detail the impact of the A/D transition on ROS production by complex I and the S-nitrosation of a critical cysteine residue of subunit ND3 as a strategy to prevent oxidative damage and tissue damage during ischemia–reperfusion injury.

Exogenous RNA interference exposes contrasting roles for sugar exudation in host-finding by plant pathogens

Plant parasitic nematodes (PPN) locate host plants by following concentration gradients of root exudate chemicals in the soil. We present a simple method for RNA interference (RNAi)-induced knockdown of genes in tomato seedling roots, facilitating the study of root exudate composition, and PPN responses. Knockdown of sugar transporter genes, STP1 and STP2, in tomato seedlings triggered corresponding reductions of glucose and fructose, but not xylose, in collected root exudate. This corresponded directly with reduced infectivity and stylet thrusting of the promiscuous PPN Meloidogyne incognita, however we observed no impact on the infectivity or stylet thrusting of the selective Solanaceae PPN Globodera pallida. This approach can underpin future efforts to understand the early stages of plant-pathogen interactions in tomato and potentially other crop plants.

Mitochondrial Transfer via Tunneling Nanotubes (TNT) is an Important Mechanism by which Mesenchymal Stem Cells Enhance Macrophage Phagocytosis in the in vitro and in vivo Models of ARDS

Mesenchymal stromal cells (MSC) have been reported to improve bacterial clearance in pre-clinical models of Acute Respiratory Distress Syndrome (ARDS) and sepsis. The mechanism of this effect is not fully elucidated yet. The primary objective of this study was to investigate the hypothesis that the anti-microbial effect of MSC in vivo depends on their modulation of macrophage phagocytic activity which occurs through mitochondrial transfer. We established that selective depletion of alveolar macrophages (AM) with intranasal (IN) administration of liposomal clodronate resulted in complete abrogation of MSC anti-microbial effect in the in vivo model of E.coli pneumonia. Furthermore, we showed that MSC administration was associated with enhanced AM phagocytosis in vivo. We showed that direct co-culture of MSC with monocyte-derived macrophages (MDMs) enhanced their phagocytic capacity. By fluorescent imaging and flow cytometry we demonstrated extensive mitochondrial transfer from MSC to macrophages which occurred at least partially through TNT-like structures. We also detected that lung macrophages readily acquire MSC mitochondria in vivo, and macrophages which are positive for MSC mitochondria display more pronounced phagocytic activity. Finally, partial inhibition of mitochondrial transfer through blockage of TNT formation by MSC resulted in failure to improve macrophage bioenergetics and complete abrogation of the MSC effect on macrophage phagocytosis in vitro and the anti-microbial effect of MSC in vivo.

Collectively, this work for the first time demonstrates that mitochondrial transfer from MSC to innate immune cells leads to enhancement in phagocytic activity and reveals an important novel mechanism for the anti-microbial effect of MSC in ARDS.

Mitochondrial plasticity in pathophysiological conditions

Both skeletal and cardiac muscles daily burn tremendous amounts of ATP to meet the energy requirements for contraction. So, it is not surprising that the maintenance of mitochondrial morphology, number, distribution and functionality in striated muscle are important for muscle homeostasis. In these tissues mitochondria present the added dimension of two populations, the intermyofibrillar (IMF) and the subsarcolemmal (SS) mitochondria, being IMF the most abundant one. In the present thesis, the molecular mechanisms harboured in mitochondria of striated muscles were studied using animal models, to better comprehend the role of mitochondrial plasticity in several pathophysiological conditions such as aging, diabetes mellitus and bladder cancer. The comparative analysis of IMF and SS populations isolated from heart evidenced a higher respiratory chain activity of mitochondria interspersed in the contractile apparatus. The higher susceptible of SS respiratory chain complexes subunits to carbonylation, but not to nitration, seems to justify the lower respiratory chain activity observed in this mitochondrial population. Our results showed that in heart from aged mice there is an accumulation of dysfunctional mitochondria. The age-related decrease of oxidative phosphorylation activity seems to be justified, at least partially, by the increased proneness of mitochondrial proteins as OXPHOS subunits and MnSOD to oxidative modifications. Moreover, a sedentary lifestyle seems to worsen the functional consequences of aging in heart by increasing mitochondrial proteins susceptibility to nitration. In skeletal muscle from rats with type 1 diabetes mellitus induced by streptozotocin administration, we verified the accumulation of dysfunctional mitochondria due, at least in part, to the impairment of PQC system. Indeed, the decreased activity of AAA proteases was accompanied by the accumulation of oxidatively modified mitochondrial proteins with impact in respiratory chain activity. The diminishing of mitochondria activity also underlies cancer-induced muscle wasting. Indeed, using a rat model of chemically induced urothelial carcinoma we verified that the loss of gastrocnemius mass was related to mitochondrial dysfunction due to, at least partially, the down-regulation of PQC system involving the mitochondrial proteases paraplegin and Lon. PQC impairment resulted in the accumulation of oxidatively modified mitochondrial proteins. In overall, regardless the pathophysiological stimuli that promote mitochondrial alterations, there are similarities in the pattern of disease-related mitochondrial plasticity. The diminished capacity for ATP production in striated muscle seems to be due to increased oxidative damage of mitochondrial proteins, namely subunits of respiratory chain complexes, metabolic proteins and MnSOD. Our data highlighted, for the first time, the impact of mitochondrial PQC system impairment in the accumulation of oxidized proteins, exacerbating the dysfunction of this organelle in striated muscle in several pathophysiological conditions.

Characterization of proteotoxic stress in zebrafish

A fidelidade da síntese proteica é fundamental para a estabilidade do proteoma e para a homeostasia celular. Em condições fisiológicas normais as células têm uma taxa de erro basal associada e esta muitas vezes aumenta com o envelhecimento e doença. Problemas na síntese das proteínas estão associados a várias doenças humanas e aos processos de envelhecimento. De facto, a incorporação de erros nas proteínas devido a tRNAs carregados pelas aminoacil-tRNA sintetases com o amino ácido errado causa doenças neurodegenerativas em humanos e ratos. Ainda não é claro como é que estas doenças se desenvolvem e se são uma consequência directa da disrupção do proteoma ou se são o resultado da toxicidade produzida pela acúmulação de proteínas mal traduzidas ao nível do ribossoma. Para elucidar como é que as células eucarióticas lidam com proteínas aberrantes e agregados proteicos (stress proteotóxico) desenvolvemos uma estratégia para destabilizar o proteoma. Para isso estabelecemos um sistema de erros de tradução em embriões de peixe zebra que assenta em tRNAs mutantes capazes de incorporar erradamente serina nas proteínas. As proteínas produzidas neste sistema despoletam as vias de resposta ao stress, nomeadamente a via da ubiquitina-proteassoma (UPP – “ubiquitin protesome pathway”) e a via do retículo endoplasmático (UPR – “unfolded protein response”). O stress proteotóxico gerado pelos erros de tradução altera a expressão génica e perfis de expressão de miRNAs, o desenvolvimento embrionário e viabilidade, aumenta a produção de espécies reactivas de oxigénio (ROS), leva ainda à acumulação de agregados proteicos e à disfunção mitocondrial. As malformações embrionárias e fenótipos de viabilidade que observámos foram revertidos por antioxidantes, o que sugere que os ROS desempenham papéis importantes nos fenótipos degenerativos celulares induzidos pela produção de proteínas aberrantes e agregação proteica. Estabelecemos ainda uma linha de peixe zebra transgénica para o estudo do stress proteotóxico. Este trabalho mostra que a destabilização do proteoma em embriões de peixe zebra com tRNAs mutantes é uma boa metodologia para estudar a biologia do stress proteotóxico visto que permite a agregação controlada do proteoma, mimetizando os processos de agregação de proteínas que ocorrem naturalmente durante o envelhecimento e em doenças conformacionais humanas.

Mitochondrial dysfunction in fatty acid β-oxidation disorders

Mitochondria are central organelles for cell survival with particular relevance in energy production and signalling, being mitochondrial fatty acid β–oxidation (FAO) one of the metabolic pathways harboured in this organelle. FAO disorders (FAOD) are among the most well studied inborn errors of metabolism, mainly due to their impact in health. Nevertheless, some questions remain unsolved, as their prevalence in certain European regions and how pathophysiological determinants combine towards the phenotype. Analysis of data from newborn screening programs from Portugal and Spain allowed the estimation of the birth prevalence of FAOD revealing that this group of disorders presents in Iberia (and particularly in Portugal) one of the highest European birth prevalence, mainly due to the high birth prevalence of medium chain acyl-CoA dehydrogenase deficiency. These results highlight the impact of this group of genetic disorders in this European region. The characterization of mitochondrial proteome, from patients fibroblasts with FAOD, namely multiple acyl-CoA dehydrogenase deficiency (MADD) and long chain acyl-CoA dehydrogenase deficiency (LCHADD), provided a global perspective of the mitochondrial proteome plasticity in these disorders and highlights the main molecular pathways involved in their pathogenesis. Severe MADD forms show an overexpression of chaperones, antioxidant enzymes (MnSOD), and apoptotic proteins. An overexpression of glycolytic enzymes, which reflects cellular adaptation to energy deficiency due to FAO blockage, was also observed. When LCHADD fibroblasts were analysed a metabolic switching to glycolysis was also observed with overexpression of apoptotic proteins and modulation of the antioxidant defence system. Severe LCHADD present increased ROS alongside with up regulation of MnSOD while moderate forms have lower ROS and down-regulation of MnSOD. This probably reflects the role of MnSOD in buffering cellular ROS, maintain them at levels that allow cells to avoid damage and start a cellular response towards survival. When ROS levels are very high cells have to overexpress MnSOD for detoxifying proposes. When severe forms of MADD were compared to moderate forms no major differences were noticed, most probably because ROS levels in moderate MADD are high enough to trigger a response similar to that observed in severe forms. Our data highlights, for the first time, the differences in the modulation of antioxidant defence among FAOD spectrum. Overall, the data reveals the main pathways modulated in FAOD and the importance of ROS levels and antioxidant defence system modulation for disease severity. These results highlight the complex interaction between phenotypic determinants in FAOD that include genetic, epigenetic and environmental factors. The development of future better treatment approaches is dependent on the knowledge on how all these determinants interact towards phenotype.!

Synthesis and computer-assisted design of mitochondrial electron transport-chain inhibitors as antimalarial agents

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2014

Analysis of RNA-seq data from the interaction of Coffea spp. - Colletotrichum kahawae

Tese de mestrado em Bioinformática e Biologia Computacional (Bioinformática), apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2014

Nuclear and mitochondrial control of the apoptosis machinery regulates neural stem cell fate

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2014

RT-Bst: an integrated approach for reverse transcription and enrichment of cDNA from viral RNA

The synthesis of cDNA from RNA is challenging due to the inefficiency of reverse transcription (RT). In order to address this, a method was developed known as RT-Bst for sequential RT of RNA and Bst DNA polymerase amplification for enrichment of cDNA in a single tube reaction. Using genomic RNA from bacteriophage MS2, the yield of cDNA produced by RT alone and RT-Bst were compared by analysis of PCR-amplified products. Using random primers a superior performance was observed when amplifying MS2 RNA following RT-Bst compared to RT alone, indicating that greater quantities of cDNA were present after RT-Bst. RT-Bst was also compared with RT alone for their relative ability to produce sufficient cDNA to amplify 8 target regions spanning the respiratory syncytial virus (RSV) genome. Six out of 8 targets were amplified consistently by PCR subsequent to RT-Bst amplification whereas only 3 out of 8 targets could be amplified after RT alone. RSV sequences were selectively amplified using RSV specific primers from a mixed template containing an excess of MS2 RNA in a RT-Bst reaction without amplifying MS2 sequences. This suggests that RT-Bst can be used to amplify RNA sequences non-specifically using random primers and specifically using sequence specific primers and enhances the yield of cDNA when compared to RT alone.

Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells

The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells.

Results of An International Study of Quantitative BCR/ABL Assays Using Defined RNA Samples

Molecular monitoring of BCR/ABL transcripts by real time quantitative reverse transcription PCR (qRT-PCR) is an essential technique for clinical management of patients with BCR/ABL-positive CML and ALL. Though quantitative BCR/ABL assays are performed in hundreds of laboratories worldwide, results among these laboratories cannot be reliably compared due to heterogeneity in test methods, data analysis, reporting, and lack of quantitative standards. Recent efforts towards standardization have been limited in scope. Aliquots of RNA were sent to clinical test centers worldwide in order to evaluate methods and reporting for e1a2, b2a2, and b3a2 transcript levels using their own qRT-PCR assays. Total RNA was isolated from tissue culture cells that expressed each of the different BCR/ABL transcripts. Serial log dilutions were prepared, ranging from 100 to 10-5, in RNA isolated from HL60 cells. Laboratories performed 5 independent qRT-PCR reactions for each sample type at each dilution. In addition, 15 qRT-PCR reactions of the 10-3 b3a2 RNA dilution were run to assess reproducibility within and between laboratories. Participants were asked to run the samples following their standard protocols and to report cycle threshold (Ct), quantitative values for BCR/ABL and housekeeping genes, and ratios of BCR/ABL to housekeeping genes for each sample RNA. Thirty-seven (n=37) participants have submitted qRT-PCR results for analysis (36, 37, and 34 labs generated data for b2a2, b3a2, and e1a2, respectively). The limit of detection for this study was defined as the lowest dilution that a Ct value could be detected for all 5 replicates. For b2a2, 15, 16, 4, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. For b3a2, 20, 13, and 4 labs showed a limit of detection at the 10-5, 10-4, and 10-3 dilutions, respectively. For e1a2, 10, 21, 2, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. Log %BCR/ABL ratio values provided a method for comparing results between the different laboratories for each BCR/ABL dilution series. Linear regression analysis revealed concordance among the majority of participant data over the 10-1 to 10-4 dilutions. The overall slope values showed comparable results among the majority of b2a2 (mean=0.939; median=0.9627; range (0.399 - 1.1872)), b3a2 (mean=0.925; median=0.922; range (0.625 - 1.140)), and e1a2 (mean=0.897; median=0.909; range (0.5174 - 1.138)) laboratory results (Fig. 1-3)). Thirty-four (n=34) out of the 37 laboratories reported Ct values for all 15 replicates and only those with a complete data set were included in the inter-lab calculations. Eleven laboratories either did not report their copy number data or used other reporting units such as nanograms or cell numbers; therefore, only 26 laboratories were included in the overall analysis of copy numbers. The median copy number was 348.4, with a range from 15.6 to 547,000 copies (approximately a 4.5 log difference); the median intra-lab %CV was 19.2% with a range from 4.2% to 82.6%. While our international performance evaluation using serially diluted RNA samples has reinforced the fact that heterogeneity exists among clinical laboratories, it has also demonstrated that performance within a laboratory is overall very consistent. Accordingly, the availability of defined BCR/ABL RNAs may facilitate the validation of all phases of quantitative BCR/ABL analysis and may be extremely useful as a tool for monitoring assay performance. Ongoing analyses of these materials, along with the development of additional control materials, may solidify consensus around their application in routine laboratory testing and possible integration in worldwide efforts to standardize quantitative BCR/ABL testing.

Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene.

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.

Regulatory RNA binding molecules : implication for diabetes pathogenesis

Le glucose est notre principale source d'énergie. Après un repas, le taux de glucose dans le sang (glycémie) augmente, ce qui entraine la sécrétion d'insuline. L'insuline est une hormone synthétisée au niveau du pancréas par des cellules dites bêta. Elle agit sur différents organes tels que les muscles, le foie ou le tissu adipeux, induisant ainsi le stockage du glucose en vue d'une utilisation future.¦Le diabète est une maladie caractérisée par un taux élevé de glucose dans le sang (hyperglycémie), résultant d'une incapacité de notre corps à utiliser ou à produire suffisamment d'insuline. A long terme, cette hyperglycémie entraîne une détérioration du système cardio-vasculaire ainsi que de nombreuses complications. On distingue principalement deux type de diabète : le diabète de type 1 et le diabète de type 2, le plus fréquent (environ 90% des cas). Bien que ces deux maladies diffèrent sur beaucoup de points, elles partagent quelques similitudes. D'une part, on décèle une diminution de la quantité de cellules bêta. Cette diminution est cependant partielle dans le cas d'un diabète de type 2, et totale dans celui d'un diabète de type 1. D'autre part, la présence dans la circulation de médiateurs de l'inflammation nommés cytokines est décelée aussi bien chez les patients de type 1 que de type 2. Les cytokines sont sécrétées lors d'une inflammation. Elles servent de moyen de communication entre les différents acteurs de l'inflammation et ont pour certaines un effet néfaste sur la survie des cellules bêta.¦L'objectif principal de ma thèse a été d'étudier en détail l'effet de petites molécules régulatrices de l'expression génique, appelées microARNs. Basé sur le fait que de nombreuses publications ont démontré que les microARNs étaient impliqués dans différentes maladies telles que le cancer, j'ai émis l'hypothèse qu'ils pouvaient également jouer un rôle important dans le développement du diabète.¦Nous avons commencé par mettre des cellules bêta en culture en présence de cytokines, imitant ainsi un environnement inflammatoire. Nous avons pu de ce fait identifier les microARNs dont les niveaux d'expression étaient modifiés. A l'aide de méthodes biochimiques, nous avons ensuite observé que la modulation de certains microARNs par les cytokines avaient des effets néfastes sur la cellule bêta : sur sa production et sa sécrétion d'insuline, ainsi que sur sa mort (apoptose). Nous avons en conséquence pu démontrer que ces petites molécules avaient un rôle important à jouer dans le dysfonctionnement des cellules bêta induit par les cytokines, aboutissant au développement du diabète.¦-¦La cellule bêta pancréatique est une cellule endocrine présente dans les îlots de Langerhans, dans le pancréas. L'insuline, une hormone sécrétée par ces cellules, joue un rôle essentiel dans la régulation de la glycémie. Le diabète se développe si le taux d'insuline relâché par les cellules bêta n'est pas suffisant pour couvrir les besoins métaboliques corporels. Le diabète de type 1, qui représente environ 5 à 10% des cas, est une maladie auto-immune qui se caractérise par une réaction inflammatoire déclenchée par notre système immunitaire envers les cellules bêta. La conséquence de cette attaque est une disparition progressive des cellules bêta. Le diabète de type 2 est, quant à lui, largement plus répandu puisqu'il représente environ 90% des cas. Des facteurs à la fois génétiques et environnementaux sont responsables d'une diminution de la sensibilité des tissus métabolisant l'insuline, ainsi que d'une réduction de la sécrétion de l'insuline par les cellules bêta, ce qui a pour conséquence le développement de la maladie. Malgré les différences entre ces deux types de diabète, ils ont pour points communs la présence d'infiltrat immunitaire et la diminution de l'état fonctionnel des cellules bêta.¦Une meilleure compréhension des mécanismes aboutissant à l'altération de la cellule bêta est primordiale, avant de pouvoir développer de nouvelles stratégies thérapeutiques capables de guérir cette maladie. Durant ma thèse, j'ai donc étudié l'implication de petites molécules d'ARN, régulatrices de l'expression génique, appelées microARNs, dans les conditions physiopathologiques qui aboutissent au développement du diabète. J'ai débuté mon étude par l'identification de microARNs dont le niveau d'expression était modifié lorsque les cellules bêta étaient exposées à des conditions favorisant à la fois le développement du diabète de type 1 (cytokines) et celui du diabète de type 2 (palmitate). Nous avons découvert qu'une modification de l'expression des miR-21, -34a et -146a était commune aux deux traitements. Ces changements d'expressions ont également été confirmés dans deux modèles animaux : les souris NOD qui développent un diabète s'apparentant au diabète de type 1 et les souris db/db qui développent plutôt un diabète de type 2. Puis, à l'aide de puces à ADN, nous avons comparé l'expression de microARNs chez des souris NOD pré-diabétiques. Nous avons alors retrouvé des changements au niveau de l'expression des mêmes microARNs mais également au niveau d'une famille de microARNs : les miR-29a, -29b et -29c. De manière artificielle, nous avons ensuite surexprimé ou inhibé en conditions physiopathologiques l'expression de tous ces microARNs et nous nous sommes intéressés à l'impact d'un tel changement sur différentes fonctions de la cellule bêta comme la synthèse et la sécrétion d'insulinè ainsi que leur survie. Nous avons ainsi pu démontrer que les miR-21, -34a, -29a, -29b, -29c avaient un effet délétère sur la sécrétion d'insuline et que la surexpression de tous ces microARNs (excepté le miR-21) favorisait la mort. Finalement, nous avons démontré que la plupart de ces microARNs étaient impliqués dans la régulation d'importantes voies de signalisation responsables de l'apoptose des cellules bêta telles que les voies de NFKB, BCL2 ou encore JNK.¦Par conséquent, nos résultats démontrent que les microARNs ont un rôle important à jouer dans le dysfonctionnement des cellules bêta lors de la mise en place du diabète.

Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts.

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the distribution of mitochondrial DNA (mtDNA) heteroplasmy amongst the hairs of an individual who displays point heteroplasmy in blood and buccal cells. A second aim of the exercise was to study reproducibility of mtDNA sequencing of hairs between laboratories using differing chemistries, further to the first mtDNA reproducibility study carried out by the EDNAP group. Laboratories were asked to type 2 sections from each of 10 hairs, such that each hair was typed by at least two laboratories. Ten laboratories participated in the study, and a total of 55 hairs were typed. The results showed that the C/T point heteroplasmy observed in blood and buccal cells at position 16234 segregated differentially between hairs, such that some hairs showed only C, others only T and the remainder, C/T heteroplasmy at varying ratios. Additionally, differential segregation of heteroplasmic variants was confirmed in independent extracts at positions 16093 and the poly(C) tract at 302-309, whilst a complete A-G transition was confirmed at position 16129 in one hair. Heteroplasmy was observed at position 16195 on both strands of a single extract from one hair segment, but was not observed in the extracts from any other segment of the same hair. Similarly, heteroplasmy at position 16304 was observed on both strands of a single extract from one hair. Additional variants at positions 73, 249 and the HVII poly(C) region were reported by one laboratory; as these were not confirmed in independent extracts, the possibility of contamination cannot be excluded. Additionally, the electrophoresis and detection equipment used by this laboratory was different to those of the other laboratories, and the discrepancies at position 249 and the HVII poly(C) region appear to be due to reading errors that may be associated with this technology. The results, and their implications for forensic mtDNA typing, are discussed in the light of the biology of hair formation.