Étude du transcriptome des cellules non tumorales de l’épithélium de surface de l’ovaire des femmes porteuses d’une mutation des gènes BRCA1 et BRCA2

Nous avons étudié le transcriptome de neuf échantillons d'ARN extraits de cultures primaires de cellules non tumorales de l’épithélium de surface de l’ovaire (NOSE) provenant de quatre donneuses non porteuses de mutation, deux mutées sur BRCA1 et trois sur BRCA2, ainsi que de quatre échantillons d’ARN extraits de cultures primaires de cellules tumorales de l’ovaire (TOV) provenant de trois donneuses porteuses de mutation sur BRCA1 et une sur BRCA2. Nous avons identifié, pour la première fois, les signatures moléculaires associées à la présence d’une mutation de BRCA1 et BRCA2 dans les cellules NOSEs ainsi que la signature associée à la transformation tumorale des cellules NOSEs en TOVs chez les porteuses de mutation de BRCA1. Nous avons également localisé les domaines chromosomiques comportant des gènes corégulés en association avec la présence d’une mutation de BRCA1 dans les cellules NOSEs. Les allèles sauvage et muté de BRCA2 étaient exprimés dans les cellules TOVs provenant des porteuses de la mutation 8765delAG sur BRCA2. Nous avons observé que le niveau d’expression des transcrits de BRCA2 était plus élevé dans les cellules provenant des tumeurs ovariennes les plus agressives chez les femmes porteuses de la mutation 8765delAG sur BRCA2, les transcrits correspondants à l’allèle muté contribuant avec un pourcentage élevé du niveau d’expression total du gène. Le phénotype tumoral observé chez les Canadiennes Françaises porteuses de cette mutation pourrait résulter d’un effet de dosage de l’allèle muté.

TaNF-YC11, one of the light-upregulated NF-YC members in Triticum aestivum, is co-regulated with photosynthesis-related genes

NF-Y is a heterotrimeric transcription factor complex. Each of the NF-Y subunits (NF-YA, NF-YB and NF-YC) in plants is encoded by multiple genes. Quantitative RT-PCR analysis revealed that five wheat NF-YC members (TaNF-YC5, 8, 9, 11 & 12) were upregulated by light in both the leaf and seedling shoot. Co-expression analysis of Affymetrix wheat genome array datasets revealed that transcript levels of a large number of genes were consistently correlated with those of the TaNF-YC11 and TaNF-YC8 genes in 3-4 separate Affymetrix array datasets. TaNF-YC11-correlated transcripts were significantly enriched with the Gene Ontology term photosynthesis. Sequence analysis in the promoters of TaNF-YC11-correlated genes revealed the presence of putative NF-Y complex binding sites (CCAAT motifs). Quantitative RT-PCR analysis of a subset of potential TaNF-YC11 target genes showed that ten out of the thirteen genes were also light-upregulated in both the leaf and seedling shoot and had significantly correlated expression profiles with TaNF-YC11. The potential target genes for TaNF-YC11 include subunit members from all four thylakoid membrane bound complexes required for the conversion of solar energy into chemical energy and rate limiting enzymes in the Calvin cycle. These data indicate that TaNF-YC11 is potentially involved in regulation of photosynthesis-related genes.

How Effective Is The Data On Co-Occurrence Of Domains In Multi-Domain Proteins In Prediction Of Protein-Protein Interactions?

We explore the fuse of information on co-occurrence of domains in multi-domain proteins in predicting protein-protein interactions. The basic premise of our work is the assumption that domains co-occurring in a polypeptide chain undergo either structural or functional interactions among themselves. In this study we use a template dataset of domains in multidomain proteins and predict protein-protein interactions in a target organism. We note that maximum number of correct predictions of interacting protein domain families (158) is made in S. cerevisiae when the dataset of closely related organisms is used as the template followed by the more diverse dataset of bacterial proteins (48) and a dataset of randomly chosen proteins (23). We conclude that use of multi-domain information from organisms closely-related to the target can aid prediction of interacting protein families.

Nuclear dispositions of subtelomeric and pericentromeric chromosomal domains during meiosis in asynaptic mutants of rye (Secale cereale L.)

EI Mikhailova, SP Sosnikhina, GA Kirillova, OA Tikholiz, VG Smirnov, RN Jones and G Jenkins (2001). Nuclear dispositions of subtelomeric and pericentromeric chromosomal domains during meiosis in asynaptic mutants of rye (Secale cereale L.). Journal of Cell Science, 114 (10), 1875-1882. Sponsorship: Russian Foundation for Basic Research (grants 00-04-48522/ 99-04-48182) RAE2008

miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer and control cell cycle progression in a synergistic and Rb-dependent manner

microRNAs (miRNAs) are small non-coding RNAs that are frequently involved in carcinogenesis. Although many miRNAs form part of integrated networks, little information is available how they interact with each other to control cellular processes. miR-34a and miR-15a/16 are functionally related; they share common targets and control similar processes including G1-S cell cycle progression and apoptosis. The aim of this study was to investigate the combined action of miR-34a and miR-15a/16 in non-small cell lung cancer (NSCLC) cells.

The Ethics of a Co-regulatory Model for Farm Animal Welfare Research

Standards for farm animal welfare are variously managed at a national level by government-led regulatory control, by consumer-led welfare economics and co-regulated control in a partnership between industry and government. In the latter case the control of research to support animal welfare standards by the relevant industry body may lead to a conflict of interest on the part of researchers, who are dependent on industry for continued research funding. We examine this dilemma by reviewing two case studies of research published under an Australian co-regulated control system. Evidence of unsupported conclusions that are favourable to industry is provided, suggesting that researchers do experience a conflict of interest that may influence the integrity of the research. Alternative models for the management of research are discussed, including the establishment of an independent research management body for animal welfare because of its public good status and the use of public money derived from taxation, with representation from government, industry, consumers, and advocacy groups.

Electro-oxidation of CO at the Ru(0001) single-crystal electrode surface

In situ FTIR spectroscopic and electrochemical data and ex situ (emersion) electron diffraction (LEED) and RHEED) and Auger spectroscopic data are presented on the structure and reactivity, with respect to the electro-oxidation of CO, of the Ru(0001) single-crystal surface in perchloric acid solution. In both the absence and the presence of adsorbed CO, the Ru(0001) electrode shows the potential-dependent formation of well-defined and ordered oxygen-containing adlayers. At low potentials (e.g., from -80 to +200 mV vs Ag/AgCl), a (2 × 2)-O phase, which is unreactive toward CO oxidation, is formed, in agreement with UHV studies. Increasing the potential results in the formation of (3 × 1) and (1 × 1) phases at 410 and 1100 mV, respectively, with a concomitant increase in the reactivity of the surface toward CO oxidation. Both linear (CO ) and three-fold-hollow (CO ) binding CO adsorbates (bands at 2000-2040 and 1770-1800 cm , respectively) were observed on the Ru(0001) electrode. The in situ FTIR data show that the adsorbed CO species remain in compact islands as CO oxidation proceeds, suggesting that the oxidation occurs at the boundaries between the CO and O domains. At low CO coverages, reversible relaxation (at lower potentials) and compression (at higher potentials) of the CO adlayer were observed and rationalized in terms of the reduction and formation of surface O adlayers. The data obtained from the Ru(0001) electrode are in marked contrast to those observed on polycrystalline Ru, where only linear CO is observed.

Identification de nouvelles cibles transcriptionnelles d’ETV6, un facteur de transcription fréquemment altéré dans la leucémie aiguë lymphoblastique

La leucémie aiguë lymphoblastique (LAL) est le cancer pédiatrique le plus fréquent. Plusieurs réarrangements chromosomiques ont été associés à cette maladie, dont la translocation t(12;21), qui est observée dans 25% des cas de LAL de type pré-B. Cette translocation engendre l’expression de la protéine de fusion ETV6-AML1. Toutefois, celle-ci n’est pas suffisante pour initier seule une leucémie, ce qui suggère que des mutations additionnelles sont nécessaires à la transformation oncogénique. Or, on observe que l’allèle non-réarrangé d’ETV6 est perdu dans 75% des cas de t(12;21). Cette délétion entraîne l’inactivation complète du facteur de transcription ETV6 et l’abolition de sa fonction biologique. Puisqu’ETV6 semble jouer un rôle de suppresseur de tumeurs, nous croyons que son inactivation favoriserait le développement de la leucémie via la dérégulation de ses gènes cibles. Ce projet visait donc à identifier de nouvelles cibles transcriptionnelles d’ETV6, afin d’élucider son implication dans la leucémie. Une expérience de RNA-Seq a permis d’identifier plus de 200 gènes dont l’expression est corrélée avec celle d’ETV6 dans des cellules souches hématopoïétiques CD34+. Parmi ceux-ci, plusieurs gènes sont impliqués dans la réponse immunitaire et inflammatoire, la migration cellulaire, l’homéostasie ionique et la signalisation intracellulaire. Nous avons également mis en place une approche d’immunoprécipitation de la chromatine afin d’identifier les régions auxquelles le facteur de transcription ETV6 peut se lier. À l’aide de cette méthode, nous avons démontré une interaction entre ETV6 et SLCO2B1, un gène dont l’expression est également co-régulée avec ETV6. Finalement, notre étude suggère qu’ETV6 contribuerait à la leucémogenèse en dérégulant l’expression de certains gènes ayant des propriétés oncogéniques.

Identifying co-regulating microrna groups

Background: Current miRNA target prediction tools have the common problem that their false positive rate is high. This renders identification of co-regulating groups of miRNAs and target genes unreliable. In this study, we describe a procedure to identify highly probable co-regulating miRNAs and the corresponding co-regulated gene groups. Our procedure involves a sequence of statistical tests: (1) identify genes that are highly probable miRNA targets; (2) determine for each such gene, the minimum number of miRNAs that co-regulate it with high probability; (3) find, for each such gene, the combination of the determined minimum size of miRNAs that co-regulate it with the lowest p-value; and (4) discover for each such combination of miRNAs, the group of genes that are co-regulated by these miRNAs with the lowest p-value computed based on GO term annotations of the genes.
Results: Our method identifies 4, 3 and 2-term miRNA groups that co-regulate gene groups of size at least 3 in human. Our result suggests some interesting hypothesis on the functional role of several miRNAs through a "guilt by association" reasoning. For example, miR-130, miR-19 and miR-101 are known neurodegenerative diseases associated miRNAs. Our 3-term miRNA table shows that miR-130/19/101 form a co-regulating group of rank 22 (p-value =1.16 × 10^-2). Since miR-144 is co-regulating with miR-130, miR-19 and miR-101 of rank 4 (p-value = 1.16 × 10^-2) in our 4-term miRNA table, this suggests hsa-miR-144 may be neurodegenerative diseases related miRNA. Conclusions: This work identifies highly probable co-regulating miRNAs, which are refined from the prediction by computational tools using (1) signal-to-noise ratio to get high accurate regulating miRNAs for every gene, and (2) Gene Ontology to obtain functional related co-regulating miRNA groups. Our result has partly been supported by biological experiments. Based on prediction by TargetScanS, we found highly probable target gene groups in the Supplementary Information. This result might help biologists to find small set of miRNAs for genes of interest rather than huge amount of miRNA set.

The yield of essential oils in Melaleuca alternifolia (Myrtaceae) is regulated through transcript abundance of genes in the MEP pathway

Medicinal tea tree (Melaleuca alternifolia) leaves contain large amounts of an essential oil, dominated by monoterpenes. Several enzymes of the chloroplastic methylerythritol phosphate (MEP) pathway are hypothesised to act as bottlenecks to the production of monoterpenes. We investigated, whether transcript abundance of genes encoding for enzymes of the MEP pathway were correlated with foliar terpenes in M. alternifolia using a population of 48 individuals that ranged in their oil concentration from 39 -122 mg x g DM(-1). Our study shows that most genes in the MEP pathway are co-regulated and that the expression of multiple genes within the MEP pathway is correlated with oil yield. Using multiple regression analysis, variation in expression of MEP pathway genes explained 87% of variation in foliar monoterpene concentrations. The data also suggest that sesquiterpenes in M. alternifolia are synthesised, at least in part, from isopentenyl pyrophosphate originating from the plastid via the MEP pathway.

A GFP-equipped bidirectional expression module well suited for monitoring tetracycline-regulated gene expression in mouse

Doxycycline (Dox)-sensitive co-regulation of two transcriptionally coupled transgenes was investigated in the mouse. For this, we generated four independent mouse lines carrying coding regions for green fluorescent protein (GFP) and β-galactosidase in a bicistronic, bidirectional module. In all four lines the expression module was silent but was activated when transcription factor tTA was provided by the α-CaMKII-tTA transgene. In vivo analysis of GFP fluorescence, β-galactosidase and immunochemical stainings revealed differences in GFP and β-galactosidase levels between the lines, but comparable patterns of expression. Strong signals were found in neurons of the olfactory system, neocortical, limbic lobe and basal ganglia structures. Weaker expression was limited to thalamic, pontine and medullary structures, the spinal cord, the eye and to some Purkinje cells in the cerebellum. Strong GFP signals were always accompanied by intense β-galactosidase activity, both of which could be co-regulated by Dox. We conclude that the tTA-sensitive bidirectional expression module is well suited to express genes of interest in a regulated manner and that GFP can be used to track transcriptional activity of the module in the living mouse.

The chromosomal distribution of mouse odorant receptor genes.

Odorant receptors (ORs) on nasal olfactory sensory neurons are encoded by a large multigene family. Each member of the family is expressed in a small percentage of neurons that are confined to one of several spatial zones in the nose but are randomly distributed throughout that zone. This pattern of expression suggests that when the sensory neuron selects which OR gene to express it may be confined to a particular zonal gene set of several hundred OR genes but select from among the members of that set via a stochastic mechanism. Both locus-dependent and locus-independent models of OR gene choice have been proposed. To investigate the feasibility of these models, we determined the chromosomal locations of 21 OR genes expressed in four different spatial zones. We found that OR genes are clustered within multiple loci that are broadly distributed in the genome. These loci lie within paralogous chromosomal regions that appear to have arisen by duplications of large chromosomal domains followed by extensive gene duplication and divergence. Our studies show that OR genes expressed in the same zone map to numerous loci; moreover, a single locus can contain genes expressed in different zones. These findings raise the possibility that OR gene choice may be locus-independent or involve consecutive stochastic choices.

Self-regulated learning in higher education : strategies adopted by computer programming students

Trabalho apresentado em PAEE/ALE’2016, 8th International Symposium on Project Approaches in Engineering Education (PAEE) and 14th Active Learning in Engineering Education Workshop (ALE)

Characterisation of the TaNF-Y family of transcription factors in wheat

Light plays a unique role for plants as it is both a source of energy for growth and a signal for development. Light captured by the pigments in the light harvesting complexes is used to drive the synthesis of the chemical energy required for carbon assimilation. The light perceived by photoreceptors activates effectors, such as transcription factors (TFs), which modulate the expression of light-responsive genes. Recently, it has been speculated that increasing the photosynthetic rate could further improve the yield potential of three carbon (C3) crops such as wheat. However, little is currently known about the transcriptional regulation of photosynthesis genes, particularly in crop species. Nuclear factor Y (NF-Y) TF is a functionally diverse regulator of growth and development in the model plant species, with demonstrated roles in embryo development, stress response, flowering time and chloroplast biogenesis. Furthermore, a light-responsive NF-Y binding site (CCAAT-box) is present in the promoter of a spinach photosynthesis gene. As photosynthesis genes are co-regulated by light and co-regulated genes typically have similar regulatory elements in their promoters, it seems likely that other photosynthesis genes would also have light-responsive CCAAT-boxes. This provided the impetus to investigate the NF-Y TF in bread wheat. This thesis is focussed on wheat NF-Y members that have roles in light-mediated gene regulation with an emphasis on their involvement in the regulation of photosynthesis genes. NF-Y is a heterotrimeric complex, comprised of the three subunits NF-YA, NF-YB and NF-YC. Unlike the mammalian and yeast counterparts, each of the three subunits is encoded by multiple genes in Arabidopsis. The initial step taken in this study was the identification of the wheat NF-Y family (Chapter 3). A search of the current wheat nucleotide sequence databases identified 37 NF-Y genes (10 NF-YA, 11 NF-YB, 14 NF-YC & 2 Dr1). Phylogenetic analysis revealed that each of the three wheat NF-Y (TaNF-Y) subunit families could be divided into 4-5 clades based on their conserved core regions. Outside of the core regions, eleven motifs were identified to be conserved between Arabidopsis, rice and wheat NF-Y subunit members. The expression profiles of TaNF-Y genes were constructed using quantitative real-time polymerase chain reaction (RT-PCR). Some TaNF-Y subunit members had little variation in their transcript levels among the organs, while others displayed organ-predominant expression profiles, including those expressed mainly in the photosynthetic organs. To investigate their potential role in light-mediated gene regulation, the light responsiveness of the TaNF-Y genes were examined (Chapters 4 and 5). Two TaNF-YB and five TaNF-YC members were markedly upregulated by light in both the wheat leaves and seedling shoots. To identify the potential target genes of the light-upregulated NF-Y subunit members, a gene expression correlation analysis was conducted using publically available Affymetrix Wheat Genome Array datasets. This analysis revealed that the transcript expression levels of TaNF-YB3 and TaNF-YC11 were significantly correlated with those of photosynthesis genes. These correlated express profiles were also observed in the quantitative RT-PCR dataset from wheat plants grown under light and dark conditions. Sequence analysis of the promoters of these wheat photosynthesis genes revealed that they were enriched with potential NF-Y binding sites (CCAAT-box). The potential role of TaNF-YB3 in the regulation of photosynthetic genes was further investigated using a transgenic approach (Chapter 5). Transgenic wheat lines constitutively expressing TaNF-YB3 were found to have significantly increased expression levels of photosynthesis genes, including those encoding light harvesting chlorophyll a/b-binding proteins, photosystem I reaction centre subunits, a chloroplast ATP synthase subunit and glutamyl-tRNA reductase (GluTR). GluTR is a rate-limiting enzyme in the chlorophyll biosynthesis pathway. In association with the increased expression of the photosynthesis genes, the transgenic lines had a higher leaf chlorophyll content, increased photosynthetic rate and had a more rapid early growth rate compared to the wild-type wheat. In addition to its role in the regulation of photosynthesis genes, TaNF-YB3 overexpression lines flower on average 2-days earlier than the wild-type (Chapter 6). Quantitative RT-PCR analysis showed that there was a 13-fold increase in the expression level of the floral integrator, TaFT. The transcript levels of other downstream genes (TaFT2 and TaVRN1) were also increased in the transgenic lines. Furthermore, the transcript levels of TaNF-YB3 were significantly correlated with those of constans (CO), constans-like (COL) and timing of chlorophyll a/b-binding (CAB) expression 1 [TOC1; (CCT)] domain-containing proteins known to be involved in the regulation of flowering time. To summarise the key findings of this study, 37 NF-Y genes were identified in the crop species wheat. An in depth analysis of TaNF-Y gene expression profiles revealed that the potential role of some light-upregulated members was in the regulation of photosynthetic genes. The involvement of TaNF-YB3 in the regulation of photosynthesis genes was supported by data obtained from transgenic wheat lines with increased constitutive expression of TaNF-YB3. The overexpression of TaNF-YB3 in the transgenic lines revealed this NF-YB member is also involved in the fine-tuning of flowering time. These data suggest that the NF-Y TF plays an important role in light-mediated gene regulation in wheat.

School-based physical activity programs for adolescent wellness improvement : an investigation of the association between wellness and physical activity

This thesis by publication included seven manuscripts that advanced contemporary understanding of the association between physical activity and wellness among adolescents. The findings suggested that due to potential interrelatedness between various aspects of wellness, changes in physical activity may also influence co-existing wellness domains; highlighting the potential for physical activity interventions to have a broad range of benefits among youth. These findings also added to the body of literature supporting the potential inclusion of physical activity as a component within multifaceted youth wellness programs. Findings reported in this thesis have implications for those seeking to initiate youth wellness interventions.