Development of computational tools for the integrated analysis of DNA microarray data with applications in cancer research

The MAP-i Doctoral Program of the Universities of Minho, Aveiro and Porto

Integrating data from heterogeneous DNA microarray platforms

DNA microarrays are one of the most used technologies for gene expression measurement. However, there are several distinct microarray platforms, from different manufacturers, each with its own measurement protocol, resulting in data that can hardly be compared or directly integrated. Data integration from multiple sources aims to improve the assertiveness of statistical tests, reducing the data dimensionality problem. The integration of heterogeneous DNA microarray platforms comprehends a set of tasks that range from the re-annotation of the features used on gene expression, to data normalization and batch effect elimination. In this work, a complete methodology for gene expression data integration and application is proposed, which comprehends a transcript-based re-annotation process and several methods for batch effect attenuation. The integrated data will be used to select the best feature set and learning algorithm for a brain tumor classification case study. The integration will consider data from heterogeneous Agilent and Affymetrix platforms, collected from public gene expression databases, such as The Cancer Genome Atlas and Gene Expression Omnibus.

Search for vector-like B quarks in events with one isolated lepton, missing transverse momentum and jets at s√= 8 TeV with the ATLAS detector

A search has been performed for pair production of heavy vector-like down-type (B) quarks. The analysis explores the lepton-plus-jets final state, characterized by events with one isolated charged lepton (electron or muon), significant missing transverse momentum and multiple jets. One or more jets are required to be tagged as arising from b-quarks, and at least one pair of jets must be tagged as arising from the hadronic decay of an electroweak boson. The analysis uses the full data sample of pp collisions recorded in 2012 by the ATLAS detector at the LHC, operating at a center-of-mass energy of 8 TeV, corresponding to an integrated luminosity of 20.3 fb−1. No significant excess of events is observed above the expected background. Limits are set on vector-like B production, as a function of the B branching ratios, assuming the allowable decay modes are B→Wt/Zb/Hb. In the chiral limit with a branching ratio of 100% for the decay B→Wt, the observed (expected) 95% CL lower limit on the vector-like B mass is 810 GeV (760 GeV). In the case where the vector-like B quark has branching ratio values corresponding to those of an SU(2) singlet state, the observed (expected) 95% CL lower limit on the vector-like B mass is 640 GeV (505 GeV). The same analysis, when used to investigate pair production of a colored, charge 5/3 exotic fermion T5/3, with subsequent decay T5/3→Wt, sets an observed (expected) 95% CL lower limit on the T5/3 mass of 840 GeV (780 GeV).

Search for a Charged Higgs Boson Produced in the Vector-boson Fusion Mode with Decay H±→W±Z using pp Collisions at s√=8 TeV with the ATLAS Experiment

A search for a charged Higgs boson, H±, decaying to a W± boson and a Z boson is presented. The search is based on 20.3 fb−1 of proton-proton collision data at a center-of-mass energy of 8 TeV recorded with the ATLAS detector at the LHC. The H± boson is assumed to be produced via vector-boson fusion and the decays W±→qq′¯ and Z→e+e−/μ+μ− are considered. The search is performed in a range of charged Higgs boson masses from 200 to 1000 GeV. No evidence for the production of an H± boson is observed. Upper limits of 31--1020 fb at 95% CL are placed on the cross section for vector-boson fusion production of an H± boson times its branching fraction to W±Z. The limits are compared with predictions from the Georgi-Machacek Higgs Triplet Model.

Search for production of vector-like quark pairs and of four top quarks in the lepton-plus-jets final state in pp collisions at root 8=8 TeV with the ATLAS detector

A search for pair production of vector-like quarks, both up-type (T) and down-type (B), as well as for four-top-quark production, is presented. The search is based on pp collisions at s√=8 TeV recorded in 2012 with the ATLAS detector at the CERN Large Hadron Collider and corresponding to an integrated luminosity of 20.3 fb−1. Data are analysed in the lepton-plus-jets final state, characterised by an isolated electron or muon with high transverse momentum, large missing transverse momentum and multiple jets. Dedicated analyses are performed targeting three cases: a T quark with significant branching ratio to a W boson and a b-quark (TT¯→Wb+X), and both a T quark and a B quark with significant branching ratio to a Higgs boson and a third-generation quark (TT¯→Ht+X and BB¯→Hb+X respectively). No significant excess of events above the Standard Model expectation is observed, and 95% CL lower limits are derived on the masses of the vector-like T and B quarks under several branching ratio hypotheses assuming contributions from T→Wb, Zt, Ht and B→Wt, Zb, Hb decays. The 95% CL observed lower limits on the T quark mass range between 715 GeV and 950 GeV for all possible values of the branching ratios into the three decay modes, and are the most stringent constraints to date. Additionally, the most restrictive upper bounds on four-top-quark production are set in a number of new physics scenarios.

MOEA/VAN: Multiobjective Evolutionary Algorithm based on Vector Angle Neighborhood

Natural selection favors the survival and reproduction of organisms that are best adapted to their environment. Selection mechanism in evolutionary algorithms mimics this process, aiming to create environmental conditions in which artificial organisms could evolve solving the problem at hand. This paper proposes a new selection scheme for evolutionary multiobjective optimization. The similarity measure that defines the concept of the neighborhood is a key feature of the proposed selection. Contrary to commonly used approaches, usually defined on the basis of distances between either individuals or weight vectors, it is suggested to consider the similarity and neighborhood based on the angle between individuals in the objective space. The smaller the angle, the more similar individuals. This notion is exploited during the mating and environmental selections. The convergence is ensured by minimizing distances from individuals to a reference point, whereas the diversity is preserved by maximizing angles between neighboring individuals. Experimental results reveal a highly competitive performance and useful characteristics of the proposed selection. Its strong diversity preserving ability allows to produce a significantly better performance on some problems when compared with stat-of-the-art algorithms.

Clinical characteristics of women diagnosed with carcinoma who tested positive for cervical and anal high-risk human papillomavirus DNA and E6 RNA

High-risk human papillomavirus (hrHPV) is an essential cause of cervical carcinoma and is also strongly related to anal cancer development. The hrHPV E6 oncoprotein plays a major role in carcinogenesis. We aimed to evaluate the frequency of hrHPV DNA and E6 oncoprotein in the anuses of women with cervical carcinoma. We analyzed 117 women with cervical cancer and 103 controls for hrHPV and the E6 oncogene. Positive test results for a cervical carcinoma included 66.7 % with hrHPV-16 and 7.7 % with hrHPV-18. One case tested positive for both HPV variants (0.9 %). The samples from the anal canal were positive for HPV-16 in 59.8 % of the cases. Simultaneous presence of HPV in the cervix and anal canal was found in 53.8 % of the cases. Regarding expression of E6 RNA, positivity for HPV-16 in the anal canal was found in 21.2 % of the cases, positivity for HPV-16 in the cervix was found in 75.0 %, and positivity for HPV-18 in the cervix was found in 1.9 %. E6 expression in both the cervix and anal canal was found in 19.2 % of the cases. In the controls, 1 % tested positive for HPV-16 and 0 % for HPV-18. Anal samples from the controls showed a hrHPV frequency of 4.9 % (only HPV16). The presence of hrHPV in the anal canal of women with cervical cancer was detected at a high frequency. We also detected E6 RNA expression in the anal canal of women with cervical cancer, suggesting that these women are at risk for anal hrHPV infection.

Microscopy- or DNA-based analyses: Which methodology gives a truer picture of stream-dwelling decomposer fungal diversity?

We assessed aquatic hyphomycete diversity in autumn and spring on oak leaves decomposing in five streams along a gradient of eutrophication in the Northwest of Portugal. Diversity was assessed through microscopy-based (identification by spore morphology) and DNA-based techniques (Denaturing Gradient Gel Electrophoresis and 454 pyrosequencing). Pyrosequencing revealed five times greater diversity than DGGE. About 21% of all aquatic hyphomycete species were exclusively detected by pyrosequencing and 26% exclusively by spore identification. In some streams, more than half of the recorded species would have remained undetected if we had relied only on spore identification. Nevertheless, in spring aquatic hyphomycete diversity was higher based on spore identification, probably because many species occurring in this season are not yet connected to ITS barcodes in genetic databases. Pyrosequencing was a powerful tool for revealing aquatic hyphomycete diversity on decomposing plant litter in streams and we strongly encourage researchers to continue the effort in barcoding fungal species.

Polymer electrolyte based on DNA and N,N,N-trimethyl-N-(2-hydroxyethyl)ammonium bis(trifluoromethylsulfonyl)imide

Combining ionic liquids (ILs) with polymers offers the prospect of new applications, where they surpass the performance of conventional media, such as organic solvents, giving advantages in terms of improved safety and a higher operating temperature range. In this work we have investigated the morphology, thermal and electrochemical properties of polymer electrolytes prepared through the addition of con- trolled quantities of the cholinium based IL N,N,N-trimethyl-N-(2-hydroxyethyl)ammonium bis(trifluo- romethylsulfonyl)imide ([N1 1 1 2(OH)] [NTf2]) to a deoxyribonucleic acid (DNA) host network. These novel IL-based electrolytes have been analyzed aiming at applications in electrochemical devices. An optimized sample showed good thermal stability up to 155 °C and a wide electrochemical window of ~3.5 V. The highest conductivity was registered for the DNA[N1 1 1 2(OH)][NTf2] (1:1) (2.82 × 10-5 and 1.09 × 10-3 S cm-1 at 30 and 100 °C, respectively).

HPV DNA testing with cytology triage in cervical cancer screening: Influence of revealing HPV infection status

BACKGROUND: Knowledge of cervical human papillomavirus (HPV) status might influence a cytotechnician's assessment of cellular abnormalities. The authors compared original cytotechnicians' Papanicolaou (Pap) readings for which HPV status was concealed with Pap rereads for which HPV status was revealed separately for 3 screening populations. METHODS: Previously collected cervical Pap smears and clinical data were obtained from the Canadian Cervical Cancer Screening Trial (study A), the Democratic Republic of Congo Community-Based Screening Study (study B), and the Brazilian Investigation into Nutrition and Cervical Cancer Prevention (study C). Smears were reread with knowledge of HPV status for all HPV-positive women as well as a sample of HPV-negative women. Diagnostic performance of Pap cytology was compared between original readings and rereads. RESULTS: A total of 1767 Pap tests were reread. Among 915 rereads for HPV-positive women, the contrast between "revealed" and "concealed" Pap readings demonstrated revisions from negative to positive results for 109 women (cutoff was atypical squamous cells of undetermined significance or worse) and 124 women (cutoff was low-grade squamous intraepithelial lesions [LSIL] or worse). For a disease threshold of cervical intraepithelial neoplasia of grade 2 or worse, specificity significantly declined at the atypical squamous cells of undetermined significance cutoff for studies A (86.6% to 75.3%) and C (42.5% to 15.5%), and at the LSIL cutoff for study C (61.9% to 37.6%). Sensitivity remained nearly unchanged between readings, except in study C, in which reread performance was superior (91.3% vs 71.9% for the LSIL cutoff). CONCLUSIONS: A reduction in the diagnostic accuracy of Pap cytology was observed when revealing patients' cervical HPV status, possibly due to a heightened awareness of potential abnormalities, which led to more false-positive results. Cancer (Cancer Cytopathol) 2015. (c) 2015 American Cancer Society.

Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-d pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3'-phosphatase in spinocerebellar ataxia Type 3 pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

DNA evidence collecting in criminal investigation: Portuguese and Thai legal approaches

Doctoral Thesis in Juridical Sciences (Specialty in Public Legal Sciences)

Vector of coordinates, velocities and accelerations

This chapter describes the how the vector of coordinates are defined in the formulation of spatial multibody systems. For this purpose, the translational motion is described in terms of Cartesian coordinates, while rotational motion is specified using the technique of Euler parameters. This approach avoids the computational difficulties associated with the singularities in the case of using Euler angles or Bryant angles. Moreover, the formulation of the velocities vector and accelerations vector is presented and analyzed here. These two sets of vectors are defined in terms of translational and rotational coordinates.

Microfluidic based on aqueous two-phase system for plasmidic DNA purification

Dissertação de mestrado em Bioquímica Aplicada (área de especialização em Biotecnologia)