Development of a retroviral strategy that efficiently creates nested chromosomal deletions in mouse embryonic stem cells and its exploitation for functional genomics

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

A compendium of myeloma-associated chromosomal copy number abnormalities and their prognostic value.

To obtain a comprehensive genomic profile of presenting multiple myeloma cases we performed high-resolution single nucleotide polymorphism mapping array analysis in 114 samples alongside 258 samples analyzed by U133 Plus 2.0 expression array (Affymetrix). We examined DNA copy number alterations and loss of heterozygosity (LOH) to define the spectrum of minimally deleted regions in which relevant genes of interest can be found. The most frequent deletions are located at 1p (30%), 6q (33%), 8p (25%), 12p (15%), 13q (59%), 14q (39%), 16q (35%), 17p (7%), 20 (12%), and 22 (18%). In addition, copy number-neutral LOH, or uniparental disomy, was also prevalent on 1q (8%), 16q (9%), and X (20%), and was associated with regions of gain and loss. Based on fluorescence in situ hybridization and expression quartile analysis, genes of prognostic importance were found to be located at 1p (FAF1, CDKN2C), 1q (ANP32E), and 17p (TP53). In addition, we identified common homozygously deleted genes that have functions relevant to myeloma biology. Taken together, these analyses indicate that the crucial pathways in myeloma pathogenesis include the nuclear factor-κB pathway, apoptosis, cell-cycle regulation, Wnt signaling, and histone modifications. This study was registered at http://isrctn.org as ISRCTN68454111.

Abnormalities on 1q and 7q are associated with poor outcome in sporadic Burkitt's lymphoma. A cytogenetic and comparative genomic hybridization study.

Comparative genomic hybridization (CGH) studies have demonstrated a high incidence of chromosomal imbalances in non-Hodgkin's lymphoma. However, the information on the genomic imbalances in Burkitt's Lymphoma (BL) is scanty. Conventional cytogenetics was performed in 34 cases, and long-distance PCR for t(8;14) was performed in 18 cases. A total of 170 changes were present with a median of four changes per case (range 1-22). Gains of chromosomal material (143) were more frequent than amplifications (5) or losses (22). The most frequent aberrations were gains on chromosomes 12q (26%), Xq (22%), 22q (20%), 20q (17%) and 9q (15%). Losses predominantly involved chromosomes 13q (17%) and 4q (9%). High-level amplifications were present in the regions 1q23-31 (three cases), 6p12-p25 and 8p22-p23. Upon comparing BL vs Burkitt's cell leukemia (BCL), the latter had more changes (mean 4.3 +/- 2.2) than BL (mean 2.7 +/- 3.2). In addition, BCL cases showed more frequently gains on 8q, 9q, 14q, 20q, and 20q, 9q, 8q and 14q, as well as losses on 13q and 4q. Concerning outcome, the presence of abnormalities on 1q (ascertained either by cytogenetics or by CGH), and imbalances on 7q (P=0.01) were associated with a short survival.

Primary Immunodeficiency Associated with Chromosomal Aberration - an ESID Survey

Patients with syndromic features frequently suffer from recurrent respiratory infections, but little is known about the spectrum of immunological abnormalities associated with their underlying chromosomal aberrations outside the well-known examples of Down and DiGeorge syndromes. Therefore, we performed this retrospective, observational survey study.

Utilisation du logiciel Copilote Insertion pour l'évaluation des gains en employabilité de sujets ayant participé à un Service d'Intégration Socioprofessionnelle.

La situation du marché de l'emploi a subi ces dernières années, des changements de structure en profondeur. Le résultat est qu'au Québec, le taux réel d'inactivité révèle que près d'un adulte sur quatre est sans emploi. Parmi les personnes les plus touchées par ces changements on retrouve celles qui sont sans formation spécifique, sans diplôme ou bien sans expérience de travail. Pour rendre apte au travail cette mam d'oeuvre inactive, le Ministère de l'éducation a mis sur pied au milieu des années 90, une mesure d'aide à l'insertion appelée Services d'Intégration Socioprofessionnelle ou S.I.S. Dans le présent essai, nous avons cherché à évaluer le développement de l'employabilité de participants aux S.I.S. Selon les objectifs visés par le Service, on suppose que chaque personne aura acquis certaines attitudes et aptitudes au travail, susceptible de l'aider à retrouver du travail et de le conserver. Nous avons utilisé la structure de recherche test-retest sur le même échantillon. En effet, chaque participant s'est vu administrer le même questionnaire, au début et à la fin des S.I.S. Nous avons ainsi pu suivre l'évolution des facteurs en causes dans une insertion professionnelle. À cela s'ajoutent des données recueillies dans un journal de bord par l'évaluateur/animateur. Celles-ci sont reprises pour donner un sens dans l'analyse des résultats. La mesure utilisée est le questionnaire informatisé Copilote Insertion version 2.20. Celui-ci fut administré une première fois, à seize personnes présentes en amorce des S.LS. Pour la mesure post, nous avions plus que 8 personnes toujours disponibles et respectant le devis de recherche. Pour ces sujets, nous avons comparé chacun des vingt facteurs de Copilote, ainsi que l'évolution des stratégies d'insertion. Pour chacun d'eux, nous avons pu déterminer lesquels des facteurs d'employabilité évalué par le logiciel, démontraient une progression. Il a été démontré que l'ensemble des participants s'accordait beaucoup de difficulté à surmonter l'image de dépendance et d'incapacité, intériorisé avec l'expérience du chômage. Comme quoi, cette clientèle a à faire face à un certain nombre de deuil non résolu. À ce niveau, la structure des S.I.S semble permettre ce nécessaire recadrage de l'expérience chômage. Nous avons aussi observé que la composition du groupe n'a pas été réalisée en respectant le développement de l'employabilité de chacun des participants. Par conséquent, les plus enthousiasmes ont eu à subir l'inconfort de devoir faire face aux discours démotivant des personnes non-prêtes à s'insérer en emploi. La difficulté de sélectionner des personnes dont les besoins en matière de développement de l'employabilité ne seraient pas homogènes pourrait être surmontée si des activités individuelles personnalisées, sont présentées en alternance avec le travail de groupe. Autre constat réalisé est que malgré le temps passé à l'intérieur des SIS, des participants s'accordent plus de difficultés à la fin qu'au début, pour des facteurs d'employabilités. C'est le cas des facteurs du thème Communication de Copilote. La difficulté de mettre en branle une campagne visant la mise en marché de son potentiel de travail, cela vient confirmer que le participant, bien qu'il ait réalisé un bout du trajet le séparant du monde du travail, qu'il n'est pas parvenu à destination.

The RGBarrier assay: the parallel study of gene regulatory element performance at defined chromosomal locations

Vertebrate genomes are organised into a variety of nuclear environments and chromatin states that have profound effects on the regulation of gene transcription. This variation presents a major challenge to the expression of transgenes for experimental research, genetic therapies and the production of biopharmaceuticals. The majority of transgenes succumb to transcriptional silencing by their chromosomal environment when they are randomly integrated into the genome, a phenomenon known as chromosomal position effect (CPE). It is not always feasible to target transgene integration to transcriptionally permissive “safe harbour” loci that favour transgene expression, so there remains an unmet need to identify gene regulatory elements that can be added to transgenes which protect them against CPE. Dominant regulatory elements (DREs) with chromatin barrier (or boundary) activity have been shown to protect transgenes from CPE. The HS4 element from the chicken beta-globin locus and the A2UCOE element from a human housekeeping gene locus have been shown to function as DRE barriers in a wide variety of cell types and species. Despite rapid advances in the profiling of transcription factor binding, chromatin states and chromosomal looping interactions, progress towards functionally validating the many candidate barrier elements in vertebrates has been very slow. This is largely due to the lack of a tractable and efficient assay for chromatin barrier activity. In this study, I have developed the RGBarrier assay system to test the chromatin barrier activity of candidate DREs at pre-defined isogenic loci in human cells. The RGBarrier assay consists in a Flp-based RMCE reaction for the integration of an expression construct, carrying candidate DREs, in a pre-characterised chromosomal location. The RGBarrier system involves the tracking of red, green and blue fluorescent proteins by flow cytometry to monitor on-target versus off-target integration and transgene expression. The analysis of the reporter (GFP) expression for several weeks gives a measure of the protective ability of each candidate elements from chromosomal silencing. This assay can be scaled up to test tens of new putative barrier elements in the same chromosomal context in parallel. The defined chromosomal contexts of the RGBarrier assays will allow for detailed mechanistic studies of chromosomal silencing and DRE barrier element action. Understanding these mechanisms will be of paramount importance for the design of specific solutions for overcoming chromosomal silencing in specific transgenic applications.

The Impact of a Classroom Performance System on Learning Gains in a Biology Course for Science Majors

This study was conducted to determine if the use of the technology known as Classroom Performance System (CPS), specifically referred to as “Clickers”, improves the learning gains of students enrolled in a biology course for science majors. CPS is one of a group of developing technologies adapted for providing feedback in the classroom using a learner-centered approach. It supports and facilitates discussion among students and between them and teachers, and provides for participation by passive students. Advocates, influenced by constructivist theories, claim increased academic achievement. In science teaching, the results have been mixed, but there is some evidence of improvements in conceptual understanding. The study employed a pretest-posttest, non-equivalent groups experimental design. The sample consisted of 226 participants in six sections of a college biology course at a large community college in South Florida with two instructors trained in the use of clickers. Each instructor randomly selected their sections into CPS (treatment) and non-CPS (control) groups. All participants filled out a survey that included demographic data at the beginning of the semester. The treatment group used clicker questions throughout, with discussions as necessary, whereas the control groups answered the same questions as quizzes, similarly engaging in discussion where necessary. The learning gains were assessed on a pre/post-test basis. The average learning gains, defined as the actual gain divided by the possible gain, were slightly better in the treatment group than in the control group, but the difference was statistically non-significant. An Analysis of Covariance (ANCOVA) statistic with pretest scores as the covariate was conducted to test for significant differences between the treatment and control groups on the posttest. A second ANCOVA was used to determine the significance of differences between the treatment and control groups on the posttest scores, after controlling for sex, GPA, academic status, experience with clickers, and instructional style. The results indicated a small increase in learning gains but these were not statistically significant. The data did not support an increase in learning based on the use of the CPS technology. This study adds to the body of research that questions whether CPS technology merits classroom adaptation.

Prevalence of SOS-mediated control of integron integrase expression as an adaptive trait of chromosomal and mobile integrons

BACKGROUND Integrons are found in hundreds of environmental bacterial species, but are mainly known as the agents responsible for the capture and spread of antibiotic-resistance determinants between Gram-negative pathogens. The SOS response is a regulatory network under control of the repressor protein LexA targeted at addressing DNA damage, thus promoting genetic variation in times of stress. We recently reported a direct link between the SOS response and the expression of integron integrases in Vibrio cholerae and a plasmid-borne class 1 mobile integron. SOS regulation enhances cassette swapping and capture in stressful conditions, while freezing the integron in steady environments. We conducted a systematic study of available integron integrase promoter sequences to analyze the extent of this relationship across the Bacteria domain. RESULTS Our results showed that LexA controls the expression of a large fraction of integron integrases by binding to Escherichia coli-like LexA binding sites. In addition, the results provide experimental validation of LexA control of the integrase gene for another Vibrio chromosomal integron and for a multiresistance plasmid harboring two integrons. There was a significant correlation between lack of LexA control and predicted inactivation of integrase genes, even though experimental evidence also indicates that LexA regulation may be lost to enhance expression of integron cassettes. CONCLUSIONS Ancestral-state reconstruction on an integron integrase phylogeny led us to conclude that the ancestral integron was already regulated by LexA. The data also indicated that SOS regulation has been actively preserved in mobile integrons and large chromosomal integrons, suggesting that unregulated integrase activity is selected against. Nonetheless, additional adaptations have probably arisen to cope with unregulated integrase activity. Identifying them may be fundamental in deciphering the uneven distribution of integrons in the Bacteria domain.

Lack of geographic variation in Y-chromosomal introns of red deer (Cervus elaphus)

Intra-specific Y-chromosomal sequence variation is useful for analysing the male contribution to a species’ spatial genetic structure. In red deer (Cervus elaphus) this is especially relevant, because geographic dispersal and game translocations occur mainly through the males. However, Y-chromosomal markers for wild organisms are scarce and frequently non-polymorphic within species. We assessed the intra-specific variation of two Y-chromosomal introns in red deer, one in the DBY (or DDX3Y) gene and the other in the UBE1Y gene. The introns were amplified using previously published exonic primers and directly sequenced in individuals of five red deer subspecies from across Eurasia. However, no nucleotide polymorphism was observed, which rebuts the usefulness of these introns for studies of red deer phylogeography and on illegal transport of red deer within this region. Male-based phylogeographic studies should thus be focused on other Y-chromosomal markers for this species.

Heritability, repeatability, and genetic gains in a improvement population of cajuízeiro.

Cajuí (Anacardium spp) is an endemic fruit trees species in the Northeastern region of Brazil. Thus, this trial aimed to estimate the repeatability coefficients of the agrotechnological variables and to predict the genetic gains of a cajuí improvement population located at the Embrapa Meio-Norte.

Molecular And Morphological Evidence Reveals A New Species In The Phyllomedusa Hypochondrialis Group (hylidae, Phyllomedusinae) From The Atlantic Forest Of The Highlands Of Southern Brazil.

The taxonomic status of a disjunctive population of Phyllomedusa from southern Brazil was diagnosed using molecular, chromosomal, and morphological approaches, which resulted in the recognition of a new species of the P. hypochondrialis group. Here, we describe P. rustica sp. n. from the Atlantic Forest biome, found in natural highland grassland formations on a plateau in the south of Brazil. Phylogenetic inferences placed P. rustica sp. n. in a subclade that includes P. rhodei + all the highland species of the clade. Chromosomal morphology is conservative, supporting the inference of homologies among the karyotypes of the species of this genus. Phyllomedusa rustica is apparently restricted to its type-locality, and we discuss the potential impact on the strategies applied to the conservation of the natural grassland formations found within the Brazilian Atlantic Forest biome in southern Brazil. We suggest that conservation strategies should be modified to guarantee the preservation of this species.

Comparative Cytogenetics Of Physalaemus albifrons And Physalaemus Cuvieri Species Groups (anura, leptodactylidae).

Recently, Physalaemus albifrons (Spix, 1824) was relocated from the Physalaemus cuvieri group to the same group as Physalaemus biligonigerus (Cope, 1861), Physalaemus marmoratus (Reinhardt & Lütken, 1862) and Physalaemus santafecinus Barrio, 1965. To contribute to the analysis of this proposition, we studied the karyotypes of Physalaemus albifrons, Physalaemus santafecinus and three species of the Physalaemus cuvieri group. The karyotype of Physalaemus santafecinus was found to be very similar to those of Physalaemus biligonigerus and Physalaemus marmoratus, which were previously described. A remarkable characteristic that these three species share is a conspicuous C-band that extends from the pericentromeric region almost to the telomere in the short arm of chromosome 3. This characteristic is not present in the Physalaemus albifrons karyotype and could be a synapomorphy of Physalaemus biligonigerus, Physalaemus marmoratus and Physalaemus santafecinus. The karyotype of Physalaemus santafecinus is also similar to those of Physalaemus marmoratus and Physalaemus biligonigerus owing to the presence of several terminal C-bands and the distal localization of the NOR in a small metacentric chromosome. In contrast, the Physalaemus albifrons karyotype has no terminal C-bands and its NOR is located interstitially in the long arm of submetacentric chromosome 8. The NOR-bearing chromosome of Physalaemus albifrons very closely resembles those found in Physalaemus albonotatus (Steindachner, 1864), Physalaemus cuqui Lobo, 1993 and some populations of Physalaemus cuvieri Fitzinger, 1826. Additionally, the Physalaemus albifrons karyotype has an interstitial C-band in chromosome 5 that has been exclusively observed in species of the Physalaemus cuvieri group. Therefore, we were not able to identify any chromosomal feature that supports the reallocation of Physalaemus albifrons.

Undulatory Physical Resistance Training Program Increases Maximal Strength In Elderly Type 2 Diabetics.

To investigate the effects of a specific protocol of undulatory physical resistance training on maximal strength gains in elderly type 2 diabetics. The study included 48 subjects, aged between 60 and 85 years, of both genders. They were divided into two groups: Untrained Diabetic Elderly (n=19) with those who were not subjected to physical training and Trained Diabetic Elderly (n=29), with those who were subjected to undulatory physical resistance training. The participants were evaluated with several types of resistance training's equipment before and after training protocol, by test of one maximal repetition. The subjects were trained on undulatory resistance three times per week for a period of 16 weeks. The overload used in undulatory resistance training was equivalent to 50% of one maximal repetition and 70% of one maximal repetition, alternating weekly. Statistical analysis revealed significant differences (p<0.05) between pre-test and post-test over a period of 16 weeks. The average gains in strength were 43.20% (knee extension), 65.00% (knee flexion), 27.80% (supine sitting machine), 31.00% (rowing sitting), 43.90% (biceps pulley), and 21.10% (triceps pulley). Undulatory resistance training used with weekly different overloads was effective to provide significant gains in maximum strength in elderly type 2 diabetic individuals.

In Vivo Evaluation Of The Genetic Toxicity Of Rubus Niveus Thunb. (rosaceae) Extract And Initial Screening Of Its Potential Chemoprevention Against Doxorubicin-induced Dna Damage.

Rubus niveus Thunb. plant belongs to Rosaceae family and have been used traditionally to treat wounds, burns, inflammation, dysentery, diarrhea and for curing excessive bleeding during menstrual cycle. The present study was undertaken to investigate the in vivo genotoxicity of Rubus niveus aerial parts extract and its possible chemoprotection on doxorubicin (DXR)-induced DNA damage. In parallel, the main phytochemicals constituents in the extract were determined. The animals were exposed to the extract for 24 and 48h, and the doses selected were 500, 1000 and 2000mg/kg b.w. administered by gavage alone or prior to DXR (30mg/kg b.w.) administered by intraperitoneal injection. The endpoints analyzed were DNA damage in bone marrow and peripheral blood cells assessed by the alkaline alkaline (pH>13) comet assay and bone marrow micronucleus test. The results of chemical analysis of the extract showed the presence of tormentic acid, stigmasterol, quercitinglucoronide (miquelianin) and niga-ichigoside F1 as main compounds. Both cytogenetic endpoints analyzed showed that there were no statistically significant differences (p>0.05) between the negative control and the treated groups with the two higher doses of Rubus niveus extract alone, demonstrating absence of genotoxic and mutagenic effects. Aneugenic/clastogenic effect was observed only at 2000mg/kg dose. On the other hand, in the both assays and all tested doses were observed a significant reduction of DNA damage and chromosomal aberrations in all groups co-treated with DXR and extract compared to those which received only DXR. These results indicate that Rubus niveus aerial parts extract did not revealed any genotoxic effect, but presented some aneugenic/clastogenic effect at higher dose; and suggest that it could be a potential adjuvant against development of second malignant neoplasms caused by the cancer chemotherapic DXR.