L'effet pathologique du monoxyde d'azote est diminué dans les myocytes cardiaques hypertrophiés

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1-mutant myelodysplastic syndromes

The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3' splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3' splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.Leukemia advance online publication, 17 June 2016; doi:10.1038/leu.2016.149.

Exploration génétique de l'hypothyroïdie congénitale par dysgénésie thyroïdienne

L'hypothyroïdie congénitale par dysgénésie thyroïdienne (HCDT, ectopie dans plus de 80 %) a une prévalence de 1 cas sur 4000 naissances vivantes. L’HCDT est la conséquence d'une défaillance de la thyroïde embryonnaire à se différencier, à se maintenir ou à migrer vers sa localisation anatomique (partie antérieure du cou), qui aboutit à une absence totale de la thyroïde (athyréose) ou à une ectopie thyroïdienne (linguale ou sublinguale). Les HCDT sont principalement non-syndromiques (soit 98% des cas sont non-familiale), ont un taux de discordance de 92% chez les jumeaux monozygotes, et ont une prédominance féminine et ethnique (i.e., Caucasienne). La majorité des cas d’HCDT n’a pas de cause connue, mais est associée à un déficit sévère en hormones thyroïdiennes (hypothyroïdie). Des mutations germinales dans les facteurs de transcription liés à la thyroïde (NKX2.1, FOXE1, PAX8, NKX2.5) ont été identifiées dans seulement 3% des patients atteints d’HCDT sporadiques et l’analyse de liaisons exclue ces gènes dans les rares familles multiplex avec HCDT. Nous supposons que le manque de transmission familiale claire d’HCDT peut résulter de la nécessité d’au moins deux « hits » génétiques différents dans des gènes importants pour le développement thyroïdien. Pour répondre au mieux nos questions de recherche, nous avons utilisé deux approches différentes: 1) une approche gène candidat, FOXE1, seul gène impliqué dans l’ectopie dans le modèle murin et 2) une approche en utilisant les techniques de séquençage de nouvelle génération (NGS) afin de trouver des variants génétiques pouvant expliquer cette pathologie au sein d’une cohorte de patients avec HCDT. Pour la première approche, une étude cas-contrôles a été réalisée sur le promoteur de FOXE1. Il a récemment été découvert qu’une région du promoteur de FOXE1 est différentiellement méthylée au niveau de deux dinucléotides CpG consécutifs, définissant une zone cruciale de contrôle de l’expression de FOXE1. L’analyse d’association basée sur les haplotypes a révélé qu’un haplotype (Hap1: ACCCCCCdel1C) est associé avec le HCDT chez les Caucasiens (p = 5x10-03). Une réduction significative de l’activité luciférase est observée pour Hap1 (réduction de 68%, p<0.001) comparé au promoteur WT de FOXE1. Une réduction de 50% de l’expression de FOXE1 dans une lignée de cellules thyroïdienne humaine est suffisante pour réduire significativement la migration cellulaire (réduction de 55%, p<0.05). Un autre haplotype (Hap2: ACCCCCCC) est observé moins fréquemment chez les Afro-Américain comparés aux Caucasiens (p = 1.7x10-03) et Hap2 diminue l’activité luciférase (réduction de 26%, p<0.001). Deux haplotypes distincts sont trouvés fréquemment dans les contrôles Africains (Black-African descents). Le premier haplotype (Hap3: GTCCCAAC) est fréquent (30.2%) chez les contrôles Afro-Américains comparés aux contrôles Caucasiens (6.3%; p = 2.59 x 10-9) tandis que le second haplotype (Hap4: GTCCGCAC) est trouvé exclusivement chez les contrôles Afro-Américains (9.4%) et est absent chez les contrôles Caucasiens (P = 2.59 x 10-6). Pour la deuxième approche, le séquençage de l’exome de l’ADN leucocytaire entre les jumeaux MZ discordants n’a révélé aucune différence. D'où l'intérêt du projet de séquençage de l’ADN et l’ARN de thyroïdes ectopiques et orthotopiques dans lesquelles de l'expression monoallélique aléatoire dans a été observée, ce qui pourrait expliquer comment une mutation monoallélique peut avoir des conséquences pathogéniques. Finalement, le séquençage de l’exome d’une cohorte de 36 cas atteints d’HCDT a permis d’identifier de nouveaux variants probablement pathogéniques dans les gènes récurrents RYR3, SSPO, IKBKE et TNXB. Ces quatre gènes sont impliqués dans l’adhésion focale (jouant un rôle dans la migration cellulaire), suggérant un rôle direct dans les défauts de migration de la thyroïde. Les essais de migration montrent une forte diminution (au moins 60% à 5h) de la migration des cellules thyroïdiennes infectées par shRNA comparés au shCtrl dans 2 de ces gènes. Des zebrafish KO (-/- et +/-) pour ces nouveaux gènes seront réalisés afin d’évaluer leur impact sur l’embryologie de la thyroïde.

Phylum-wide transcriptome analysis of oogenesis and early embryogenesis in selected nematode species

Oogenesis is a prerequisite for embryogenesis in Metazoa. During both biological processes important decisions must be made to form the embryo and hence ensure the next generation: (1) Maternal gene products (mRNAs, proteins and nutrients) must be supplied to the embryo. (2) Polarity must be established and axes must be specified. While incorporation of maternal gene products occurs during oogenesis, the time point of polarity establishment and axis specification varies among species, as it is accomplished either prior, during, or after fertilisation. But not only the time point when these events take place varies among species but also the underlying mechanisms by which they are triggered. For the nematode model Caenorhabditis elegans the underlying pathways and gene regulatory networks (GRNs) are well understood. It is known that there the sperm entry point initiates a primary polarity in the 1-celled egg and with it the establishment of the anteroposterior axis. However, studies of other nematodes demonstrated that polarity establishment can be independent of sperm entry (Goldstein et al., 1998; Lahl et al., 2006) and that cleavage patterns, symmetry formation and cell specification also differ from C. elegans. In contrast to the studied Chromadorea (more derived nematodes including C. elegans), embryos of some marine Enoplea (more basal representatives) even show no discernible early polarity and blastomeres can adopt variable cell fates (Voronov and Panchin 1998). The underlying pathways controlling the obviously variant embryonic processes in non-Caenorhabditis nematodes are essentially unknown. In this thesis I addressed this issue by performing a detailed unbiased comparative transcriptome analysis based on microarrays and RNA sequencing of selected developmental stages in a variety of nematodes from different phylogenetic branches with C. elegans as a reference system and a nematomorph as an outgroup representative. In addition, I made use of available genomic data to determine the presence or absence of genes for which no expression had been detected. In particular, I focussed on components of selected pathways or GRNs which are known to play essential roles during C. elegans development and/or other invertebrate or vertebrate model systems. Oogenesis must be regulated differently in non-Caenorhabditis nematodes, as crucial controlling components of Wnt and sex determination signaling are absent in these species. In this respect, I identified female-specific expression of potential polarity associated genes during gonad development and oogenesis in the Enoplean nematode Romanomermis culicivorax. I could show that known downstream components of the polarity complexes PAR-3/-6/PKC-3 and PAR-1/-2 are absent in non-Caenorhabditis species. Even PAR-2 as part of the polarity complex does not exist in these nematodes. Instead, transcriptomes of nematodes (including C. elegans), show expression of other polarity-associated complexes such as the Lgl (Lethal giant larvae) complex. This result could pose an alternative route for nematodes and nematomorphs to initiate polarity during early embryogenesis. I could show that crucial pathways of axis specification, such as Wnt and BMP are very different in C. elegans compared to other nematodes. In the former, Wnt signaling, for instance, is mediated by four paralogous beta-catenins, while other Chromadorea have fewer and Enoplea only one beta-catenin. The transcriptomes of R. culicivorax and the nematomorph show that regulators of BMP (e.g. Chordin), are specifically expressed during early embryogenesis only in Enoplea and the close outgroup of nematomorphs. In conclusion, my results demonstrate that the molecular machinery controlling oogenesis and embryogenesis in nematodes is unexpectedly variable and C. elegans cannot be taken as a general model for nematode development. Under this perspective, Enoplean nematodes show more similarities with outgroups than with C. elegans. It appears that certain pathway components were lost or gained during evolution and others adopted new functions. Based on my findings I can conjecture, which pathway components may be ancestral and which were newly acquired in the course of nematode evolution.

The synthetic progestin norgestrel modulates Nrf2 signaling and acts as an antioxidant in a model of retinal degeneration

Retinitis pigmentosa (RP) is one of the most common retinal degenerative conditions affecting people worldwide, and is currently incurable. It is characterized by the progressive loss of photoreceptors, in which the death of rod cells leads to the secondary death of cone cells; the cause of eventual blindness. As rod cells die, retinal-oxygen metabolism becomes perturbed, leading to increased levels of reactive oxygen species (ROS) and thus oxidative stress; a key factor in the secondary death of cones. In this study, norgestrel, an FDA-approved synthetic analog of progesterone, was found to be a powerful neuroprotective antioxidant, preventing light-induced ROS in photoreceptor cells, and subsequent cell death. Norgestrel also prevented light-induced photoreceptor morphological changes that were associated with ROS production, and that are characteristic of RP. Further investigation showed that norgestrel acts via post-translational modulation of the major antioxidant transcription factor Nrf2; bringing about its phosphorylation, subsequent nuclear translocation, and increased levels of its effector protein superoxide dismutase 2 (SOD2). In summary, these results demonstrate significant protection of photoreceptor cells from oxidative stress, and underscore the potential of norgestrel as a therapeutic option for RP.

Identification of Genes Modulating Mitochondrial Biogenesis in Caenorhabditis elegans

Since Altmann recognized ubiquitously distributed "bioblasts" in 1890, understanding of mitochondria has evolved from "elementary organisms" living inside cells and carrying out vital functions, over the Harman's "free radical theory" in 1956, to one of the driving forces of aging and cause of multiple associated diseases impacting society today. While a tremendous amount of work has contributed to the understanding of mitochondrial biology in different model organisms, the precise molecular mechanisms of basic mitochondrial function have yet to be deciphered. By employing an RNA interference mediated screen in Caenorhabditis elegans, we identified two transcription factors: SPTF-3, a member of Sp1 family, and an uncharacterized, nematode specific W04D2.4. We propose that both proteins modulate expression of many genes with regard to mitochondrial function including mitochondrial single-stranded binding protein encoded by mtss-1, whose promoter was used as transcriptional reporter in the screen. Further, RNA sequencing data indicate that W04D2.4 indirectly regulates expression of mitochondrial DNA via control of genes functionally related to mitochondrial replication and translation machineries. We also demonstrate that from all interventions targeting cytosolic translation, MTSS-1 levels are elevated only upon knockdown of genes encoding cytosolic ribosomal proteins. Reduction of ribosomes leads to increased sptf-3 translation, most likely in an internal ribosome entry side (IRES) mediated manner, eventually inducing mtss-1 expression. Moreover, we identify a novel role for SPTF-3 in the regulation of mitochondrial unfolded stress response (UPRmt) activation, but not endoplasmatic reticulum or oxidative stress responses. Taken together, this study identifies two transcription factors previously not associated with mitochondrial biogenesis and UPRmt in C. elegans, establishing a basis for further investigation of mito-nuclear interactions.

Carotenoids and antioxidant enzymes as biomarkers of the impact of heavy metals in food chain

Antioxidant enzymes (catalase and peroxidase) and carotenoids (lutein and â-carotene) are often used as biomarkers of metal contamination of water and agricultural soils. In this study, the effects of heavy metals present in irrigation water on the aforementioned carotenoids of potatoes (Solanum tuberosum L.) and carrots (Daucus carota L.), cultivated in a greenhouse and irrigated with a water solution including different levels of Cr(VI) and Ni(II) were investigated. These results were compared to the levels of the same metabolites that had been assessed in market-available potato and carrot samples. The findings indicated that the levels of the examined metabolites on the treated with Cr and Ni samples, resemble the levels of the same parameters in the market samples, originating from polluted areas. Therefore, the antioxidant enzymes, catalase and peroxidase, and the carotenoids, lutein and â-carotene, could be handled as indicators of heavy metal pollution.

Investigation of the role of long non-coding RNAs in oncogene induced cellular senescence

Cellular senescence is a stable arrest of cell proliferation induced by several factors such as activated oncogenes, oxidative stress and shortening of telomeres. Senescence acts as a tumour suppression mechanism to halt the progression of cancer. However, senescence may also impact negatively upon tissue regeneration, thus contributing to the effects of ageing. The eukaryotic genome is controlled by various modes of transcriptional and translational regulation. Focus has therefore centred on the role of long non- coding RNAs (lncRNAs) in regulating the genome. Accordingly, understanding how lncRNAs function to regulate the senescent genome is integral to improving our knowledge and understanding of tumour suppression and ageing. Within this study, I set out to investigate the expression of lncRNAs’ expression within models of senescence. Through a custom expression array, I have shown that expression of multiple different lncRNAs is up-regulated and down regulated in IMR90 replicative senescent fibroblasts and oncogene-induced senescent melanocytes. LncRNA expression was determined to be specific to stable senescence-associated cell arrest and predominantly within the nucleus of senescent cells. In order to examine the function of lncRNA expression in senescence, I selected lncRNA transcript ENST0000430998 (lncRNA_98) to focus my investigations upon. LncRNA_98 was robustly upregulated within multiple models of senescence and efficiently depleted using anti-sense oligonucleotide technology. Characterisation and unbiased RNA-sequencing of lncRNA_98 deficient senescent cells highlighted a list of genes that are regulated by lncRNA_98 expression in senescent cells and may regulate aspects of the senescence program. Specifically, the formation of SAHF was impeded upon depletion of lncRNA_98 expression and levels of total pRB protein expression severely decreased. Validation and recapitulation of consequences of pRB depletion was confirmed through lncRNA_98 knock-out cells generated using CRISPR technology. Surprisingly, inhibition of ATM kinase functions permitted the restoration of pRB protein levels within lncRNA_98 deficient cells. I propose that lncRNA_98 antagonizes the ability of ATM kinase to downregulate pRB expression at a post-transcriptional level, thereby potentiating senescence. Furthermore, lncRNA expression was detected within fibroblasts of old individuals and visualised within senescent melanocytes in human benign nevi, a barrier to melanoma progression. Conversely, mining of 337 TCGA primary melanoma data sets highlighted that the lncRNA_98 gene and its expression was lost from a significant proportion of melanoma samples, consistent with lncRNA_98 having a tumour suppressor functions. The data presented in this study illustrates that lncRNA_98 expression has a regulatory role over pRB expression in senescence and may regulate aspects of tumourigenesis and ageing.

The metabolic and epigenetic effects of the isocitrate dehydrogenase-1 mutation in glioma

Cancer cells have been noted to have an altered metabolic phenotype for over ninety years. In the presence of oxygen, differentiated cells predominately utilise the tricarboxylic acid (TCA) cycle and oxidative phosphorylation to efficiently produce energy and the metabolites necessary for protein and lipid synthesis. However, in hypoxia, this process is altered and cells switch to a higher rate of glycolysis and lactate production to maintain their energy and metabolic needs. In cancer cells, glycolysis is maintained at a high rate, even in the presence of oxygen; a term described as “aerobic glycolysis”. Tumour cells are rapidly dividing and have a much greater need for anabolism compared to normal differentiated cells. Rapid glucose metabolism enables faster ATP production as well as a greater redistribution of carbons to nucleotide, protein, and fatty acid synthesis, thus maximising cell growth. Recently, other metabolic changes, driven by mutations in genes related to the TCA cycle, indicate an alternative role for metabolism in cancer, the “oncometabolite”. This is where a particular metabolite builds up within the cell and contributes to the tumorigenic process. One of these genes is isocitrate dehydrogenase (IDH) IDH is an enzyme that forms part of the tricarboxylic acid (TCA) cycle and converts isocitrate to α-ketoglutarate (α-KG). It exists in three isoforms; IDH1, IDH2 and IDH3 with the former present in the cytoplasm and the latter two in the mitochondria. Point mutations have been identified in the IDH1 and IDH2 genes in glioma which result in a gain of function by converting α-KG to 2-hydroxyglutarate (2HG), an oncometabolite. 2HG acts as a competitive inhibitor of the α-KG dependent dioxygenases, a superfamily of enzymes that are involved in numerous cellular processes such as DNA and histone demethylation. It was hypothesised that the IDH1 mutation would result in other metabolic changes in the cell other than 2HG production, and could potentially identify pathways which could be targeted for therapeutic treatment. In addition, 2HG can act as a potential competitive inhibitor of α-KG dependent dioxygenases, so it was hypothesised that there would be an effect on histone methylation. This may alter gene expression and provide a mechanism for tumourogenesis and potentially identify further therapeutic targets. Metabolic analysis of clinical tumour samples identified changes associated with the IDH1 mutation, which included a reduction in α-KG and an increase in GABA, in addition to the increase in 2HG. This was replicated in several cell models, where 13C labelled metabolomics was also used to identify a possible increase in metabolic flux from glutamate to GABA, as well as from α-KG to 2HG. This may provide a mechanism whereby the cell can bypass the IDH1 mutation as GABA can be metabolised to succinate in the mitochondria by GABA transaminase via the GABA shunt. JMJ histone demethylases are a subset of the α-KG dependent dioxygenases, and are involved in removing methyl groups from histone tails. Changes in histone methylation are associated with changes in gene expression depending on the site and extent of chemical modification. To identify whether the increase in 2HG and fall in α-KG was associated with inhibition of histone demethylases a histone methylation screen was used. The IDH1 mutation was associated with an increase in methylation of H3K4, which is associated with gene activation. ChiP and RNA sequencing identified an increase in H3K4me3 at the transcription start site of the GABRB3 subunit, resulting in an increase in gene expression. The GABRB3 subunit forms part of the GABA-A receptor, a chloride channel, which on activation can reduce cell proliferation. The IDH1 mutation was associated with an increase in GABA and GABRB3 subunit of the GABA-A receptor. This raises the possibility of GABA transaminase as a potential therapeutic target. Inhibition of this enzyme could reduce GABA metabolism, potentially reducing any beneficial effect of the GABA shunt in IDH1 mutant tumours, and increasing activation of the GABA-A receptor by increasing the concentration of GABA in the brain. This in turn may reduce cell proliferation, and could be achieved by using Vigabatrin, a GABA transaminase inhibitor licensed for use in epilepsy.

Nitric oxide synthase and NAD(P)H oxidase modulate coronary endothelial cell growth

Reactive oxygen species (ROS) including nitric oxide (NO) and superoxide anion (O2-) are associated with cell migration, proliferation and many growth-related diseases. The objective of this study was to determine whether there was a reciprocal relationship between rat coronary microvascular endothelial cell (CMEC) growth and activity/expressions (mRNA and protein) of endothelial NO synthase (eNOS) and NAD(P)H oxidase enzymes. Proliferating namely, 50% confluent CMEC possessed approximately three-fold increased activity and expression of both enzymes compared to 100% confluent cells. Treatment of CMEC with an inhibitor of eNOS (L-NAME, 100M) increased cell proliferation as assessed via three independent methods i.e. cell counting, determination of total cellular protein levels and [3H]thymidine incorporation. Similarly, treatment of CMEC with pyrogallol (0.3-3 mM), a superoxide anion (O2-)- generator, also increased CMEC growth while spermine NONOate (SpNO), a NO donor, significantly reduced cell growth. Co-incubation of CMEC with a cell permeable superoxide dismutase mimetic (Mn-III-tetrakis-4-benzoic acid-porphyrin; MnTBAP) plus either pyrogallol or NO did not alter cell number and DNA synthesis thereby dismissing the involvement of peroxynitrite (OONO-) in CMEC proliferation. Specific inhibitors of NAD(P)H oxidase but not other ROS-generating enzymes including cyclooxygenase and xanthine oxidase, attenuated cell growth. Transfection of CMEC with antisense p22-phox cDNA, a membrane-bound component of NAD(P)H oxidase, resulted in substantial reduction in [3H]thymidine incorporation, total cellular protein levels and expression of p22-phox protein. These data demonstrate a cross-talk between CMEC growth and eNOS and NAD(P)H oxidase enzyme activity and expression, thus suggesting that the regulation of these enzymes may be critical in preventing the initiation and/or progression of coronary atherosclerosis.

Macrophages and CD4 T-cells in rheumatoid arthritis and their modulation by JAK inhibitors

Background: Rheumatoid arthritis (RA) is a chronic inflammatory arthritis that causes significant morbidity and mortality and has no cure. Although early treatment strategies and biologic therapies such as TNFα blocking antibodies have revolutionised treatment, there still remains considerable unmet need. JAK kinase inhibitors, which target multiple inflammatory cytokines, have shown efficacy in treating RA although their exact mechanism of action remains to be determined. Stratified medicine promises to deliver the right drug to the right patient at the right time by using predictive ‘omic biomarkers discovered using bioinformatic and “Big Data” techniques. Therefore, knowledge across the realms of clinical rheumatology, applied immunology, bioinformatics and data science is required to realise this goal. Aim: To use bioinformatic tools to analyse the transcriptome of CD14 macrophages derived from patients with inflammatory arthritis and define a JAK/STAT signature. Thereafter to investigate the role of JAK inhibition on inflammatory cytokine production in a macrophage cell contact activation assay. Finally, to investigate JAK inhibition, following RA synovial fluid stimulation of monocytes. Methods and Results: Using bioinformatic software such as limma from the Bioconductor repository, I determined that there was a JAK/STAT signature in synovial CD14 macrophages from patients with RA and this differed from psoriatic arthritis samples. JAK inhibition using a JAK1/3 inhibitor tofacitinib reduced TNFα production when macrophages were cell contact activated by cytokine stimulated CD4 T-cells. Other pro-inflammatory cytokines such as IL-6 and chemokines such as IP-10 were also reduced. RA synovial fluid failed to stimulate monocytes to phosphorylate STAT1, 3 or 6 but CD4 T-cells activated STAT3 with this stimulus. RNA sequencing of synovial fluid stimulated CD4 T-cells showed an upregulation of SOCS3, BCL6 and SBNO2, a gene associated with RA but with unknown function and tofacitinib reversed this. Conclusion: These studies demonstrate that tofacitinib is effective at reducing inflammatory mediator production in a macrophage cell contact assay and also affects soluble factor mediated stimulation of CD4 T-cells. This suggests that the effectiveness of JAK inhibition is due to inhibition of multiple cytokine pathways such as IL-6, IL-15 and interferon. RNA sequencing is a useful tool to identify non-coding RNA transcripts that are associated with synovial fluid stimulation and JAK inhibition but these require further validation. SBNO2, a gene that is associated with RA, may be biomarker of tofacitinib treatment but requires further investigation and validation in wider disease cohorts.

Exploration génétique de l'hypothyroïdie congénitale par dysgénésie thyroïdienne

L'hypothyroïdie congénitale par dysgénésie thyroïdienne (HCDT, ectopie dans plus de 80 %) a une prévalence de 1 cas sur 4000 naissances vivantes. L’HCDT est la conséquence d'une défaillance de la thyroïde embryonnaire à se différencier, à se maintenir ou à migrer vers sa localisation anatomique (partie antérieure du cou), qui aboutit à une absence totale de la thyroïde (athyréose) ou à une ectopie thyroïdienne (linguale ou sublinguale). Les HCDT sont principalement non-syndromiques (soit 98% des cas sont non-familiale), ont un taux de discordance de 92% chez les jumeaux monozygotes, et ont une prédominance féminine et ethnique (i.e., Caucasienne). La majorité des cas d’HCDT n’a pas de cause connue, mais est associée à un déficit sévère en hormones thyroïdiennes (hypothyroïdie). Des mutations germinales dans les facteurs de transcription liés à la thyroïde (NKX2.1, FOXE1, PAX8, NKX2.5) ont été identifiées dans seulement 3% des patients atteints d’HCDT sporadiques et l’analyse de liaisons exclue ces gènes dans les rares familles multiplex avec HCDT. Nous supposons que le manque de transmission familiale claire d’HCDT peut résulter de la nécessité d’au moins deux « hits » génétiques différents dans des gènes importants pour le développement thyroïdien. Pour répondre au mieux nos questions de recherche, nous avons utilisé deux approches différentes: 1) une approche gène candidat, FOXE1, seul gène impliqué dans l’ectopie dans le modèle murin et 2) une approche en utilisant les techniques de séquençage de nouvelle génération (NGS) afin de trouver des variants génétiques pouvant expliquer cette pathologie au sein d’une cohorte de patients avec HCDT. Pour la première approche, une étude cas-contrôles a été réalisée sur le promoteur de FOXE1. Il a récemment été découvert qu’une région du promoteur de FOXE1 est différentiellement méthylée au niveau de deux dinucléotides CpG consécutifs, définissant une zone cruciale de contrôle de l’expression de FOXE1. L’analyse d’association basée sur les haplotypes a révélé qu’un haplotype (Hap1: ACCCCCCdel1C) est associé avec le HCDT chez les Caucasiens (p = 5x10-03). Une réduction significative de l’activité luciférase est observée pour Hap1 (réduction de 68%, p<0.001) comparé au promoteur WT de FOXE1. Une réduction de 50% de l’expression de FOXE1 dans une lignée de cellules thyroïdienne humaine est suffisante pour réduire significativement la migration cellulaire (réduction de 55%, p<0.05). Un autre haplotype (Hap2: ACCCCCCC) est observé moins fréquemment chez les Afro-Américain comparés aux Caucasiens (p = 1.7x10-03) et Hap2 diminue l’activité luciférase (réduction de 26%, p<0.001). Deux haplotypes distincts sont trouvés fréquemment dans les contrôles Africains (Black-African descents). Le premier haplotype (Hap3: GTCCCAAC) est fréquent (30.2%) chez les contrôles Afro-Américains comparés aux contrôles Caucasiens (6.3%; p = 2.59 x 10-9) tandis que le second haplotype (Hap4: GTCCGCAC) est trouvé exclusivement chez les contrôles Afro-Américains (9.4%) et est absent chez les contrôles Caucasiens (P = 2.59 x 10-6). Pour la deuxième approche, le séquençage de l’exome de l’ADN leucocytaire entre les jumeaux MZ discordants n’a révélé aucune différence. D'où l'intérêt du projet de séquençage de l’ADN et l’ARN de thyroïdes ectopiques et orthotopiques dans lesquelles de l'expression monoallélique aléatoire dans a été observée, ce qui pourrait expliquer comment une mutation monoallélique peut avoir des conséquences pathogéniques. Finalement, le séquençage de l’exome d’une cohorte de 36 cas atteints d’HCDT a permis d’identifier de nouveaux variants probablement pathogéniques dans les gènes récurrents RYR3, SSPO, IKBKE et TNXB. Ces quatre gènes sont impliqués dans l’adhésion focale (jouant un rôle dans la migration cellulaire), suggérant un rôle direct dans les défauts de migration de la thyroïde. Les essais de migration montrent une forte diminution (au moins 60% à 5h) de la migration des cellules thyroïdiennes infectées par shRNA comparés au shCtrl dans 2 de ces gènes. Des zebrafish KO (-/- et +/-) pour ces nouveaux gènes seront réalisés afin d’évaluer leur impact sur l’embryologie de la thyroïde.

Proteômica diferencial da Chromobacterium violaceum em resposta ao estresse oxidativo induzido por peróxido de hidrogênio

The β-proteobacterium Chromobacterium violaceum is a Gram-negative, free-living, saprophytic and opportunistic pathogen that inhabits tropical and subtropical ecosystems among them, in soil and water of the Amazon. It has great biotechnological potential, and because of this potential, its genome was completely sequenced in 2003. Genome analysis showed that this bacterium has several genes with functions related to the ability to survive under different kinds of environmental stresses. In order to understand the physiological response of C. violaceum under oxidative stress, we applied the tool of shotgun proteomics. Thus, colonies of C. violaceum ATCC 12472 were grown in the presence and absence of 8 mM H2O2 for two hours, total proteins were extracted from bacteria, subjected to SDS-PAGE, stained and hydrolysed. The tryptic peptides generated were subjected to a linear-liquid chromatography (LC) followed by mass spectrometer (LTQ-XL-Orbitrap) to obtain quantitative and qualitative data. A shotgun proteomics allows to compare directly in complex samples, differential expression of proteins and found that in C. Violaceum, 131 proteins are expressed exclusively in the control condition, 177 proteins began to be expressed under oxidative stress and 1175 proteins have expression in both conditions. The results showed that, under the condition of oxidative stress, this bacterium changes its metabolism by increasing the expression of proteins capable of combating oxidative stress and decreasing the expression of proteins related processes bacterial growth and catabolism (transcription, translation, carbon metabolism and fatty acids). A tool with of proteomics as an approach of integrative biology provided an overview of the metabolic pathways involved in the response of C. violaceum to oxidative stress, as well as significantly amplified understanding physiological response to environmental stress. Biochemical and "in silico" assays with the hypothetical ORF CV_0868 found that this is part of an operon. Phylogenetic analysis of superoxide dismutase, protein belonging to the operon also showed that the gene is duplicated in genome of C. violaceum and the second copy was acquired through a horizontal transfer event. Possibly, not only the SOD gene but also all genes comprising this operon were obtained in the same manner. It was concluded that C. violaceum has complex, efficient and versatile mechanisms in oxidative stress response

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins

International audience

Shaping the Dicot Fruit: Molecular and Genomic Approaches to Fruit Development

The fruit is one of the most complex and important structures produced by flowering plants, and understanding the development and maturation process of fruits in different angiosperm species with diverse fruit structures is of immense interest. In the work presented here, molecular genetics and genomic analysis are used to explore the processes that form the fruit in two species: The model organism Arabidopsis and the diploid strawberry Fragaria vesca. One important basic question concerns the molecular genetic basis of fruit patterning. A long-standing model of Arabidopsis fruit (the gynoecium) patterning holds that auxin produced at the apex diffuses downward, forming a gradient that provides apical-basal positional information to specify different tissue types along the gynoecium’s length. The proposed gradient, however, has never been observed and the model appears inconsistent with a number of observations. I present a new, alternative model, wherein auxin acts to establish the adaxial-abaxial domains of the carpel primordia, which then ensures proper development of the final gynoecium. A second project utilizes genomics to identify genes that regulate fruit color by analyzing the genome sequences of Fragaria vesca, a species of wild strawberry. Shared and distinct SNPs among three F. vesca accessions were identified, providing a foundation for locating candidate mutations underlying phenotypic variations among different F. vesca accessions. Through systematic analysis of relevant SNP variants, a candidate SNP in FveMYB10 was identified that may underlie the fruit color in the yellow-fruited accessions, which was subsequently confirmed by functional assays. Our lab has previously generated extensive RNA-sequencing data that depict genome-scale gene expression profiles in F. vesca fruit and flower tissues at different developmental stages. To enhance the accessibility of this dataset, the web-based eFP software was adapted for this dataset, allowing visualization of gene expression in any tissues by user-initiated queries. Together, this thesis work proposes a well-supported new model of fruit patterning in Arabidopsis and provides further resources for F. vesca, including genome-wide variant lists and the ability to visualize gene expression. This work will facilitate future work linking traits of economic importance to specific genes and gaining novel insights into fruit patterning and development.