Occurrence and Function of Hoogsteen Base Pairs in Nucleic Acids

Nucleic acids (DNA and RNA) play essential roles in the central dogma of biology for the storage and transfer of genetic information. The unique chemical and conformational structures of nucleic acids – the double helix composed of complementary Watson-Crick base pairs, provide the structural basis to carry out their biological functions. DNA double helix can dynamically accommodate Watson-Crick and Hoogsteen base-pairing, in which the purine base is flipped by ~180° degrees to adopt syn rather than anti conformation as in Watson-Crick base pairs. There is growing evidence that Hoogsteen base pairs play important roles in DNA replication, recognition, damage or mispair accommodation and repair. Here, we constructed a database for existing Hoogsteen base pairs in DNA duplexes by a structure-based survey from the Protein Data Bank, and structural analyses based on the resulted Hoogsteen structures revealed that Hoogsteen base pairs occur in a wide variety of biological contexts and can induce DNA kinking towards the major groove. As there were documented difficulties in modeling Hoogsteen or Watson-Crick by crystallography, we collaborated with the Richardsons’ lab and identified potential Hoogsteen base pairs that were mis-modeled as Watson-Crick base pairs which suggested that Hoogsteen can be more prevalent than it was thought to be. We developed solution NMR method combined with the site-specific isotope labeling to characterize the formation of, or conformational exchange with Hoogsteen base pairs in large DNA-protein complexes under solution conditions, in the absence of the crystal packing force. We showed that there are enhanced chemical exchange, potentially between Watson-Crick and Hoogsteen, at a sharp kink site in the complex formed by DNA and the Integration Host Factor protein. In stark contrast to B-form DNA, we found that Hoogsteen base pairs are strongly disfavored in A-form RNA duplex. Chemical modifications N1-methyl adenosine and N1-methyl guanosine that block Watson-Crick base-pairing, can be absorbed as Hoogsteen base pairs in DNA, but rather potently destabilized A-form RNA and caused helix melting. The intrinsic instability of Hoogsteen base pairs in A-form RNA endows the N1-methylation as a functioning post-transcriptional modification that was known to facilitate RNA folding, translation and potentially play roles in the epitranscriptome. On the other hand, the dynamic property of DNA that can accommodate Hoogsteen base pairs could be critical to maintaining the genome stability.

Function of the PDLIM2 protein in oncogenic Wnt signalling pathways

Aberrant regulation of the Wnt signalling pathway is a recurrent theme in cancer biology. Hyper activation due to oncogenic mutations and paracrine activity has been found in both colon cancer and breast cancer, and continues to evolve as a central mechanism in oncogenesis. PDLIM2, a cytoskeletal PDZ protein, is an IGF-1 regulated gene that is highly expressed in cancer cell lines derived from metastatic tumours. Suppression of PDLIM2 inhibits polarized cell migration, reverses the Epithelial to Mesenchymal transition (EMT) phenotype, suppresses the transcription of β-catenin target genes, and regulates gene expression of key transcription factors in EMT. This thesis investigates the mechanism by which PDLIM2 contributes to the maintenance of Wnt signalling in cancer cells. Here we show that PDLIM2 is a critical regulator of the Wnt pathway by regulating β-catenin at the adherens juctions, as also its transcriptional activity by the interaction of PDLIM2 with TCF4 at the nucleus. Evaluation of PDLIM2 in macrophages and co-culture studies with cancer cells and fibroblasts showed the influence exerted on PDLIM2 by paracrine cues. Thus, PDLIM2 integrates cytoskeleton signalling with gene expression by modulating the Wnt signalling pathway and reconciling microenvironmental cues with signals in epithelial cells. Negative correlation of mRNA and protein levels in the triple negative breast cancer cell BT549 suggests that PDLIM2 is part of a more complex mechanism that involves transcription and posttranslational modifications. GST pulldown studies and subsequent mass spectrometry analysis showed that PDLIM2 interacts with 300 proteins, with a high biological function in protein biosynthesis and Ubiquitin/proteasome pathways, including 13 E3 ligases. Overall, these data suggest that PDLIM2 has two distinct functions depending of its location. Located at the cytoplasm mediates cytoskeletal re-arrangements, whereas at the nucleus PDLIM2 acts as a signal transduction adaptor protein mediating transcription and ubiquitination of key transcription factors in cancer development.

The function of the Saccharomyces Cerevisiae ribonucleotide reductase second [beta] subunit in DNA repair

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

Sea-surface temperature reconstruction and calibration-function of sediment cores from the Southern Ocean

We provide a new multivariate calibration-function based on South Atlantic modern assemblages of planktonic foraminifera and atlas water column parameters from the Antarctic Circumpolar Current to the Subtropical Gyre and tropical warm waters (i.e., 60°S to 0°S). Therefore, we used a dataset with the abundance pattern of 35 taxonomic groups of planktonic foraminifera in 141 surface sediment samples. Five factors were taken into consideration for the analysis, which account for 93% of the total variance of the original data representing the regional main oceanographic fronts. The new calibration-function F141-35-5 enables the reconstruction of Late Quaternary summer and winter sea-surface temperatures with a statistical error of ~0.5°C. Our function was verified by its application to a sediment core extracted from the western South Atlantic. The downcore reconstruction shows negative anomalies in sea-surface temperatures during the early-mid Holocene and temperatures within the range of modern values during the late Holocene. This pattern is consistent with available reconstructions.

Unraveling the function of bacterial-type phosphoenolpyruvate carboxylase in vascular plants

Two distinct phosphoenolpyruvate carboxylase (PEPC) isozymes occur in vascular plants and green algae: plant-type PEPC (PTPC) and bacterial-type PEPC (BTPC). PTPC polypeptides typically form a tightly regulated cytosolic Class-1 PEPC homotetramer. BTPCs, however, appear to be less widely expressed and to exist only as catalytic and regulatory subunits that physically interact with co-expressed PTPC subunits to form hetero-octameric Class-2 PEPC complexes that are highly desensitized to Class-1 PEPC allosteric effectors. Yeast two-hybrid studies indicated that castor plant BTPC (RcPPC4) interacts with all three Arabidopsis thaliana PTPC isozymes, and that it forms stronger interactions with AtPPC2 and AtPPC3, suggesting that specific PTPCs are preferred for Class-2 PEPC formation. In contrast, Arabidopsis BTPC (AtPPC4) appeared to interact very weakly with AtPPC2 and AtPPC3, suggesting that BTPCs from different species may have different physical properties, hypothesized to be due to sequence dissimilarities within their ~10 kDa intrinsically disordered region. Recent RNA-seq and microarray data were analyzed to obtain a better understanding of BTPC expression patterns in different tissues of various monocot and dicot species. High levels of BTPC transcripts, polypeptides and Class-2 PEPC complexes were originally discovered in developing castor seeds, but the analysis revealed a broad range of diverse tissues where abundant BTPC transcripts are also expressed, such as the developing fruits of cucumber, grape, and tomato. Marked BTPC expression correlated well with the presence of ~116 kDa immunoreactive BTPC polypeptides, as well as Class-2 PEPC complexes in the immature fruit of cucumbers and tomatoes. It is therefore hypothesized that in vascular plants BTPC and thus Class-2 PEPC complexes maintain anaplerotic PEP flux in tissues with elevated malate levels that would potently inhibit ‘housekeeping’ Class-1 PEPCs. Elevated levels of malate can be used by biosynthetically active sink tissues such as immature tomatoes and cucumbers for rapid cell expansion, drought or salt stressed roots for osmoregulation, and developing seeds and pollen as a precursor for storage lipid and protein biosynthesis.

Who is in charge? An L2 Discourse Intonation Study on Four Prosodic Parameters to Exert the Pragmatic Function of Dominance and Control in the Context of L2 Non-specialist Public Speaking

This paper reports the findings from a study of the learning of English intonation by Spanish speakers within the discourse mode of L2 oral presentation. The purpose of this experiment is, firstly, to compare four prosodic parameters before and after an L2 discourse intonation training programme and, secondly, to confirm whether subjects, after the aforementioned L2 discourse intonation training, are able to match the form of these four prosodic parameters to the discourse-pragmatic function of dominance and control. The study designed the instructions and tasks to create the oral and written corpora and Brazil’s Pronunciation for Advanced Learners of English was adapted for the pedagogical aims of the present study. The learners’ pre- and post-tasks were acoustically analysed and a pre / post- questionnaire design was applied to interpret the acoustic analysis. Results indicate most of the subjects acquired a wider choice of the four prosodic parameters partly due to the prosodically-annotated transcripts that were developed throughout the L2 discourse intonation course. Conversely, qualitative and quantitative data reveal most subjects failed to match the forms to their appropriate pragmatic functions to express dominance and control in an L2 oral presentation.

Newcomer Integration in Online Knowledge Communities: Exploring the Role of Dialogic Textual Complexity

Using online knowledge communities (OKCs) as informal learning environments poses the question how likely these will integrate newcomers as peripheral participants. Previous research has identified surface characteristics of the OKC dialog as integrativity predictors. Yet, little is known about the role of dialogic textual complexity. This contribution proposes a comprehensive approach based on previously validated textual complexity indexes and applies it to predict OKC integrativity. The dialog analysis of N = 14 blogger communities with a total of 1937 participants identified three main components of textual complexity: dialog participation, structure and cohesion. From these, dialog cohesion was higher in integrative OKCs, thus significantly predicting OKC integrativity. This result adds to previous OKC research by uncovering the depth of OKC discourse. For educational practice, the study suggests a way of empowering learners by automatically assessing the integrativity of OKCs in which they may attempt to participate and access community knowledge.

Investigating the Function of a Small Secreted Protein Family in Physcomitrella Patens

The correct development of multicellular organisms depends upon the perception of signals secreted by cells in order to co-ordinate cell differentiation. The Physcomitrella patens genome encodes many components of potential signaling systems, including putative receptor proteins and putative secreted protein ligands, yet at present little characterization of these proteins has been carried out. We are currently attempting to characterize the expression pattern and function of a family of 6 secreted proteins exhibiting homology to PrsS, the ligand that controls self-incompatibility (SI) in Papaver rhoeas (field poppy). In poppy, PrsS interacts a receptor on the surface of pollen tubes, PrpS causing SI by programmed cell death. Homologues of this protein (SPH – S-Protein Homologues) exist in dicotyledonous plants and bryophytes but not in other plant taxa. We aim to determine spatiotemporal expression differences between these proteins via reporter gene analysis and qPCR of cDNA. In addition we are in the process of creating targeted gene knockouts for all 6 of the genes in P. patens. We are also searching for receptors of PrpS in Physcomitrella using a bioinformatic strategy alongside phage display. In accomplishing this we hope to determine the function of a small novel secreted protein family in Physcomitrella but in addition we also hope to elucidate the function of SPH proteins in Arabidopsis.

Magnetoresistance in a High Mobility Two-Dimensional Electron System as a Function of Sample Geometry

In a high mobility two-dimensional electron gas (2DEG) realized in a GaAs/Al0.3Ga0.7As quantum well we observe changes in the Shubnikov-de Haas oscillations (SdHO) and in the Hall resistance for different sample geometries. We observe for each sample geometry a strong negative magnetoresistance around zero magnetic field which consists of a peak around zero magnetic field and of a huge magnetoresistance at larger fields. The peak around zero magnetic field is left unchanged for different geometries.

THE DISTRIBUTION AND FUNCTION OF DENITRIFICATION GENES: EXPLORING AGRICULTURAL MANAGEMENT AND SOIL CHEMICAL IMPLICATIONS

Denitrification is a microbially-mediated process that converts nitrate (NO3-) to dinitrogen (N2) gas and has implications for soil fertility, climate change, and water quality. Using PCR, qPCR, and T-RFLP, the effects of environmental drivers and land management on the abundance and composition of functional genes were investigated. Environmental variables affecting gene abundance were soil type, soil depth, nitrogen concentrations, soil moisture, and pH, although each gene was unique in its spatial distribution and controlling factors. The inclusion of microbial variables, specifically genotype and gene abundance, improved denitrification models and highlights the benefit of including microbial data in modeling denitrification. Along with some evidence of niche selection, I show that nirS is a good predictor of denitrification enzyme activity (DEA) and N2O:N2 ratio, especially in alkaline and wetland soils. nirK was correlated to N2O production and became a stronger predictor of DEA in acidic soils, indicating that nirK and nirS are not ecologically redundant.

The organization, form and function of intermediate care services and systems in England: results from a national survey

This paper reports the results of a postal survey of intermediate care co-ordinators (ICCs) on the organization and delivery of intermediate care services for older people in England, conducted between November 2003 and May 2004. Questionnaires, which covered a range of issues with a variety of quantitative, ‘tick-box’ and open-ended questions, were returned by 106 respondents, representing just over 35% of primary care trusts (PCTs). We discuss the role of ICCs, the integration of local systems of intermediate care provision, and the form, function and model of delivery of services described by respondents. Using descriptive and statistical analysis of the responses, we highlight in particular the relationship between provision of admission avoidance and supported discharge, the availability of 24-hour care, and the locations in which care is provided, and relate our findings to the emerging evidence base for intermediate care, guidance on implementation from central government, and debate in the literature. Whilst the expansion and integration of intermediate care appear to be continuing apace, much provision seems concentrated in supported discharge services rather than acute admission avoidance, and particularly in residential forms of post-acute intermediate care. Supported discharge services tend to be found in residential settings, while admission avoidance provision tends to be non-residential in nature. Twenty-four hour care in non-residential settings is not available in several responding PCTs. These findings raise questions about the relationship between the implementation of intermediate care and the evidence for and aims of the policy as part of NHS modernization, and the extent to which intermediate care represents a genuinely novel approach to the care and rehabilitation of older people.

RESPONSE OF LOCALLY ADAPTED PEARL MILLET POPULATIONS TO S1 PROGENY RECURRENT SELECTION FOR GRAIN YIELD AND RESISTANCE TO RUST

In the semi-arid zones of Uganda, pearl millet ( Pennisetum glaucum (L.) R. Br.) is mainly grown for food and income; but rust (Puccinia substriata var indica (L.) R. Br.) is the main foliar constraint lowering yield. The objective of the study was to genetically improve grain yield and rust resistance of two locally adapted populations (Lam and Omoda), through two cycles of modified phenotypic S1 progeny recurrent selection. Treatments included three cycles of two locally adapted pearl millet populations, evaluated at three locations. Significant net genetic gain for grain yield (72 and 36%) were achieved in Lam and Omoda populations, respectively. This led to grain yield of 1,047 from 611 kg ha-1 in Lam population and 943 from 693 kg ha-1 in Omoda population. Significant improvement in rust resistance was achieved in the two populations, with a net genetic gain of -55 and -71% in Lam and Omoda populations, respectively. Rust severity reduced from 30 to 14% in Lam population and from 57 to 17% in Omoda population. Net positive genetic gains of 68 and 8% were also achieved for 1000-grain weight in Lam and Omoda, respectively. Traits with a net negative genetic gain in both populations were days to 50% flowering, days to 50% anthesis, days to 50% physiological maturity, flower-anthesis interval, plant height and leaf area.

Pattern and function of woody features on the grazing itineraries of Terra Fria, Trás–os–Montes – Portugal

The trees, hedgerows and woods are current configuration of the tree network in several ecological regions of the world. In Trás–os–Montes region, Northeast of Portugal, they are a traditional component of Terra fria landscape and they could be seen in several forms: scatter trees, fencerows, small woodlots, riparian buffer strips, among others. The extensive livestock systems in this region are based on a set of circuits across the landscape. In this practice, flocks interacts with these structures using them for different functions inducing an influence on the itineraries. Our purpose will be focused on the woody features of landscape regarding their configurations, abundance and spacial distribution; in order to examine how the grazing systems depends on the currency of these formations; particularly how species flocks behaviors are related on. Depending on spatial data, The investigation attain to compare the tree network within the agriculture matrix, to the grazed territory crossed by flocks. From the other side, the importance of spatial data on interpreting the issue by suggesting different parameter that may influence the circuits. The recognition of the pressure exerciced by the occurence of the woody structures on the grazed circuits is possible. We believe that the role of these woody structures features in supporting the tradicional silvopastoral systems has been sufficiently strong for change their distribution pattern.