Implementation of an hybrid machine learning methodology for pharmacological modeling

Tese de mestrado, Bioinformática e Biologia Computacional (Bioinformática) Universidade de Lisboa, Faculdade de Ciências, 2017Hoje em dia, especialmente na area biomedica, os dados contem milhares de variaveis de fontes diferentes e com apenas algumas instancias ao mesmo tempo. Devido a este facto, as abordagens da aprendizagem automatica enfrentam dois problemas, nomeadamente a questao da integracao de dados heterogeneos e a selecao das caracteristicas. Este trabalho propoe uma solucao eficiente para esta questao e proporciona uma implementacao funcional da metodologia hibrida. A inspiracao para este trabalho veio do desafio proposto no ambito da competicao AstraZeneca-Sanger Drug Combination Prediction DREAM Challenge em 2016, e da solucao vencedora desenvolvida por Yuanfang Guan. Relativamente a motivacao do concurso, e observado que os tratamentos combinatorios para o cancro sao mais eficientes do que as terapias habituais de agente unico, desde que tem potencial para superar as desvantagens dos outros (limitado espetro de acao e desenvolvimento de resistencia). No entanto, o efeito combinatorio de drogas nao e obvio, produzindo possivelmente o resultado aditivo, sinergico ou antagonico. Assim, o objetivo da competicao era prever in vitro a sinergia dos compostos, sem ter acesso aos dados experimentais da terapia combinatoria. No ambito da competicao foram fornecidos ficheiros de varias fontes, contendo o conhecimento farmacologico tanto experimental como obtido de ajustamento das equacoes, a informacao sobre propriedades quimicas e estruturais de drogas, e por fim, os perfis moleculares de celulas, incluindo expressao de RNA, copy variants, sequencia e metilacao de DNA. O trabalho referido envolveu uma abordagem muito bem sucedida de integração dos dados heterogeneos, estendendo o modelo com conhecimento disponivel dentro do projeto The Cancer Cell Line Encyclopedia, e tambem introduzindo o passo decisivo de simulacao que permite imitar o efeito de terapia combinatoria no cancro. Apesar das descricoes pouco claras e da documentacao da solucao vencedora ineficiente, a reproducao da abordagem de Guan foi concluida, tentando ser o mais fiel possivel. A implementacao funcional foi escrita nas linguagens R e Python, e o seu desempenho foi verificado usando como referencia a matriz submetida no concurso. Para melhorar a metodologia, o workflow de selecao dos caracteristicas foi estabelecido e executado usando o algoritmo Lasso. Alem disso, o desempenho de dois metodos alternativos de modelacao foi experimentado, incluindo Support Vector Machine and Multivariate Adaptive Regression Splines (MARS). Varias versoes da equacao de integracao foram consideradas permitindo a determinacao de coeficientes aparentemente otimos. Como resultado, a compreensao da melhor solucao de competição foi desenvolvida e a implementacao funcional foi construida com sucesso. As melhorias foram propostas e no efeito o algoritmo SVM foi verificado como capaz de superar os outros na resolução deste problema, a equacao de integracao com melhor desempenho foi estabelecida e finalmente a lista de 75 variaveis moleculares mais informativas foi fornecida. Entre estes genes, poderiam ser encontrados possiveis candidatos de biomarcadores de cancro.Nowadays, especially in the biomedical field, the data sets usually contain thousands of multi-source variables and with only few instances in the same time. Due to this fact, Machine Learning approaches face two problems, namely the issue of heterogenous data integration and the feature selection. This work proposes an efficient solution for this question and provides a functional implementation of the hybrid methodology. The inspiration originated from the AstraZeneca-Sanger Drug Combination Prediction DREAM Challenge from 2016 and the winning solution by Yuanfang Guan. Regarding to the motivation of competition, the combinatory cancer treatments are believed to be more effective than standard single-agent therapies since they have a potential to overcome others weaknesses (narrow spectrum of action and development of the resistance). However, the combinatorial drug effect is not obvious bringing possibly additive, synergistic or antagonistic treatment result. Thus, the goal of the competition was to predict in vitro compound synergy, without the access to the experimental combinatory therapy data. Within the competition, the multi-source files were supplied, encompassing the pharmacological knowledge from experiments and equation-fitting, the information on chemical properties and structure of drugs, finally the molecular cell profiles including RNA expression, copy variants, DNA sequence and methylation. The referred work included very successful approach of heterogenous data integration, extending additionally the model with prior knowledge outsourced from The Cancer Cell Line Encyclopedia, as well as introduced a key step of simulation that allows to imitate effect of a combinatory therapy on cancer. Despite unexplicit descriptions and poor documentation of the winning solution, as accurate as possible, reproduction of Guan’s approach was accomplished. The functional implementation was written in R and Python languages, and its performance was verified using as a reference the submitted in challenge prediction matrix. In order to improve the methodology feature selection workflow was established and run using a Lasso algorithm. Moreover, the performance of two alternative modeling methods was experimented including Support Vector Machine and Multivariate Adaptive Regression Splines (MARS). Several versions of merging equation were considered allowing determination of apparently optimal coefficients. As the result, the understanding of the best challenge solution was developed and the functional implementation was successfully constructed. The improvements were proposed and in the effect the SVM algorithm was verified to surpass others in solving this problem, the best-performing merging equation was established, and finally the list of 75 most informative molecular variables was provided. Among those genes, potential cancer biomarker candidates could be found

Avaliação de um novo método de modificação genética de microrganismos

Mestrado em BiotecnologiaNowadays recombinant DNA technology is broadly present in our lives, bringing both hopes and fears. Transformation, even if achievable by several techniques, still faces with unsatisfying efficiencies and lack of universality. In this work, an attempt to evaluate the potential of a new transformation methodology was made. The idea for this work came from food preservation where high hydrostatic pressure is utilized to inactivate microbial flora, mainly by destructive effects on membranes, as for instance cavities. This way, it is rational to think that sub-lethally affected cells would create pores allowing the uptake of DNA. Thus, in order to compel the method to work, a compromise between enough stress and maintenance of some cells still viable and capable to recover must be looked for. In this work, the assessment was performed on bacterial host - Escherichia coli TOP10 and small circular plasmid – pUC19 that provides resistance to ampicillin as a selection marker. Electroporation, the reference technique, resulted in transformation efficiency rates of and transformants/μg of DNA when 0,01 μg/mL plasmid was applied and of and transformants/μg of DNA with 100 μg/mL plasmid (double values are derived from assays carried at distinct days). Concerning pressure, treatments were limited to 50-400 MPa and the time ranged from 10 seconds to 5 minutes. Pressurizations included also examination of two values of compression rate: 5 and 10 MPa/sec and two cycle variants: single and triple. Treatments under 50 and 100 MPa during 2,5 and 5 min, as well as pressurization under 200, 300 and 400 MPa during 1 and 5 min, caused reduction of viability ≥99,99% of initial bacterial population. As a result, values of the potential stress factors were narrowed to 50–200 MPa lasting up to 1 minute at 5 MPa/sec compression rate, with the reduction of viability approximately 90%. Longer treatments were performed with multiple-cycles resulting in number of survivors of approximately 3% higher values (for cells pressurized without plasmid). It was concluded that E. coli may require some time to recover and so rich nutritionally broth should not be added immediately after pressurization, although fluctuations were not of great significance. In majority of the cases, addition of plasmid reduced the number of detected survivors but the variations were almost unnoticeable being in the range of few percent. Some of the experiments resulted in surprising but evident increase of cell number, reaching even up to 7,5-fold increase for the treatment under 100 MPa during 30 sec at normal compression rate with addition of pUC19. Such observations were supposed to origin from disaggregating or germination-inducing activity of pressure. With the results obtained, no successful transformation was observed. Since there exist several potential improvements of methodology (i.e. various host-plasmid sets, induction of cell competences, forms and sizes of DNA) future evaluation could hopefully bring positive results.Actualmente, a tecnologia de DNA recombinante está presente de modo substancial nas nossas vidas, levando as suas possíveis aplicações simultaneamente a esperança e o medo. A transformação genética, mesmo que realizável com várias técnicas, enfrenta ainda eficiências insatisfatórias em muitos casos e impossibilidade noutros. Neste trabalho, foi realizada uma tentativa para estudar o potencial de um novo método de transformação genética, concretamente de introdução do material genético na célula a transformar. A ideia para este trabalho foi originada da área de conservação dos alimentos, onde a alta pressão hidrostática é utilizada para inactivar microrganismos, principalmente pelos seus efeitos destrutivos sobre as membranas, por exemplo produzindo poros. Deste modo, é razoável supor que nas células stressadas sub-letalmente se criem poros permitindo a introdução de DNA. Assim, para que esta metodologia funcione, um compromisso entre stress suficiente e a manutenção da viabilidade das células deve ser utilizado. Neste trabalho, a avaliação efectuou-se com o hospedeiro bacteriano - Escherichia coli TOP10 e com um pequeno plasmídeo circular – pUC19, que confere uma resistência à ampicilina como marcador. A electroporação, usada como técnica da referência, resultou em taxas de eficiência de (9,47±2,00)×107 e (6,30±0,83)×107 transformantes/μg de DNA quando foi aplicada concentração de plasmídeo 0,01 μg/mL e de (1,18±0,37)×107 e (3,44±0,56)×107 transformantes/μg com 100 μg/mL de plasmídeo. Relativamente à pressão, os tratamentos situaram-se entre 50–400 MPa e o tempo variou dos 10 segundos até aos 5 minutos. Além disso, as pressurizações incluíram um estudo de dois valores da taxa de compressão: 5 e 10 MPa/seg e dois tipos de ciclos: singulares e triplos. Os tratamentos sob 50 e 100 MPa durante 2,5 e 5 min, assim como as pressurizações sob 200, 300 e 400 MPa durante 1 e 5 min, causaram uma redução da viabilidade ≥99,99% da população bacteriana inicial. Portanto, os valores dos potenciais factores de stress foram limitados a 50–200 MPa, até 1 minuto com uma taxa da compressão 5 MPa/seg, com a redução da viabilidade de aproximadamente 90%. Os tratamentos mais longos realizaram-se com os ciclos múltiplos resultando num número de sobreviventes aproximadamente 3% mais alto, para células pressurizadas sem o plasmídeo. Concluiu-se que E. coli pode precisar de algum tempo para recuperar, pelo que é preferível adicionar o meio enriquecido não imediatamente depois uma pressurização, embora as variações não sejam muito significativas. Na maioria dos casos, a adição de plasmídeo reduziu o número dos sobreviventes detectados. Algumas das experiências resultaram num inesperado mas evidente aumento do número das células, até valores 7,5 vezes mais altos para o tratamento sob 100 MPa durante 30 seg com uma taxa da compressão normal e com adição de pUC19. Estas observações podem dever-se à possibilidade da pressão poder causar desagregação de células ou a indução de germinação. Com os resultados obtidos não se observou transformação genética com sucesso. Como potencialmente existem vários parâmetros da metodologia (p. ex. vários conjuntos hospedeiro - plasmídeo, indução das competências celulares, as formas e tamanhos de DNA), uma nova futura avaliação da alta pressão para modificação genética deve ser tentada

Effects of genetic modifications to flax (Linum usitatissimum) on arbuscular mycorrhiza and plant performance

Although arbuscular mycorrhizal fungi (AMF) are known for their positive effect on flax growth, the impact of genetic manipulation in this crop on arbuscular mycorrhiza and plant performance was assessed for the first time. Five types of transgenic flax that were generated to improve fiber quality and resistance to pathogens, through increased levels of either phenylpropanoids (W92.40), glycosyltransferase (GT4, GT5), or PR2 beta-1,3-glucanase (B14) or produce polyhydroxybutyrate (M50), were used. Introduced genetic modifications did not change the degree of mycorrhizal colonization as compared to parent cultivars Linola and Nike. Arbuscules were well developed in each tested transgenic type (except M50). In two lines (W92.40 and B14), a higher abundance of arbuscules was observed when compared to control, untransformed flax plants. However, in some cases (W92.40, GT4, GT5, and B14 Md), the mycorrhizal dependency for biomass production of transgenic plants was slightly lower when compared to the original cultivars. No significant influence of mycorrhiza on the photosynthetic activity of transformed lines was found, but in most cases P concentration in mycorrhizal plants remained higher than in nonmycorrhizal ones. The transformed flax lines meet the demands for better quality of fiber and higher resistance to pathogens, without significantly influencing the interaction with AMF

Microtubules with different diameter, protofilament number and protofilament spacing in Ornithogalum umbellatum ovary epidermis cells.

Microtubules present in the epidermis of Ornithogalum umbellatum ovary in the area of lipotubuloids (i.e. aggregates of lipid bodies surrounded by microtubules) are 25-51 nm in diameter. They consist mainly of 10 and 11, sometimes 9 and 12 protofilaments. An average diameter of microtubule consisting of 9 subunits is about 32 nm, of 10-35 nm, of 11-38 nm and of 12-43 nm, however, individual microtubules in each category significantly vary in size. These differences result from varying distance between protofilaments in microtubule walls and diameters of protofilaments: in thin microtubules they are densely packed and smaller while in thicker ones they are loosely arranged and bigger. A hypothesis has been put forward that changes in microtubule diameter depend on structural changes associated with their functional status and are executed by modifications of protofilament arrangement density and their diameters in microtubule wall. The above hypothesis seems to be in agreement with the opinion formed on the basis of in vitro image of microtubules, that lateral contact between tubulin subunits in neighboring protofilaments indicates some flexibility and changeability during microtubule function

Cytochemical and immunocytochemical studies of the localization of histones and protamine-type proteins in spermatids of Chara vulgaris and Chara tomentosa.

Spermiogenesis in Chara algae, which has been divided into 10 phases (sp I-X), is similar to spermiogenesis in animals. The most important process during spermiogenesis in animals is remodeling of chromatin leading to "sleeping genome", being the result the exchange of histone proteins into protamine-like proteins. Cytochemical studies showed in both Chara species (C. vulgaris, C. tomentosa) that at spI-IV phases only histones were present, at spV-VIII phases--the amount of nuclear protamine-type proteins progressively increased and that of histones decreased while at spIX-X only pro-tamine-type proteins were present. This was also confirmed with capillar electrophoresis. In order to localize more precisely both histones and protamines the immunocytochemical studies with the use of anti-protamine antibodies (protamine-type proteins were obtained from C. tomentosa antheridia) and anti-histone H3 antibodies, have been carried out. More specific immunocytochemical studies confirmed cytochemical results including the exchange of histones into protamine-type during spermiogenesis (spV-VIII) in both Chara species. At phase V spermiogenesis these strong strand-like anti-protamine signals were observed in cytoplasm which might suggest that protamine synthesis took place in ER

Epigenetic Landscape of Pain in Diabetic Neuropathy

The available treatments for neuropathic pain are still unsatisfactory – they cope with low efficiency and serious side effects. To our knowledge, this is a pioneering study evaluating whole-genome DNA methylation in unique populations of type 2 diabetes mellitus patients that were histopalogically diagnosed with diabetic neuropathy, with or without neuropathic pain. We provided an evidence on the significant differences in methylation patterns between painful and painless phenotypes. Epigenomes of patients from two independent cohorts were assessed in whole blood samples with Infinium Methylation EPIC BeadChip. We performed differential analysis and identified epigenetic signals that highlight the dissimilarities between painful and painless subjects. We estimated epigenetic age of the patients and evaluated eventual acceleration of biological age in painful and painless phenotypes using set of epigenetic predictors. With the differential analysis we identified 27 CpG sites that reached the level of statistical significance in both studied cohorts, presented the methylation change between painful and painless diabetic neuropathy > 1% in one of the populations and had the direction of methylation change concordant between the two cohorts. 19 of selected probes were genic and resulted in a list of 19 unique genes. Multidimensional scaling analysis confirmed the potential of generated set of CpG sites to separate painful and painless subjects and to highlight the dissimilarities between two phenotypes. Evaluation of biological age showed that there was no association between painful phenotype and acceleration of biological age expressed by any of the assessed epigenetic clocks. DNA methylation based prediction of telomere length was found to vary between painful and painless groups in both studied cohorts. Obtained results confirmed the presence of epigenetic differences between painful and painless diabetic neuropathy patients. Promising genes were identified that may be linked to neuropathic pain through DNA methylation mechanisms

DGAT2 revealed by the immunogold technique in Arabidopsis thaliana lipid bodies associated with microtubules

The immunogold technique with anti-diacylglycerol acyltransferase 2 (DGAT2) antibody revealed inA. thaliana embryo and root meristematic cells gold particles manifesting the presence of DGAT2 in ER as wellas in lipid bodies. This being so, lipid synthesis could take place both in ER and in the lipid bodies. The presenceof microtubules around the lipid bodies was evidenced under transmission EM. Detection of tubulin around thelipid bodies using the immunogold technique with anti-a-tubulin is in agreement with the above observations.Connection of lipid bodies with microtubules was also detected by us in other plants where they probably participatedin lipid synthesis. A similar phenomenon may take place in A. thaliana.The immunogold technique with anti-diacylglycerol acyltransferase 2 (DGAT2) antibody revealed inA. thaliana embryo and root meristematic cells gold particles manifesting the presence of DGAT2 in ER as wellas in lipid bodies. This being so, lipid synthesis could take place both in ER and in the lipid bodies. The presenceof microtubules around the lipid bodies was evidenced under transmission EM. Detection of tubulin around thelipid bodies using the immunogold technique with anti-a-tubulin is in agreement with the above observations.Connection of lipid bodies with microtubules was also detected by us in other plants where they probably participatedin lipid synthesis. A similar phenomenon may take place in A. thaliana

Insight into the Structural Dynamics of the Lysenin During Prepore-to-Pore Transition Using Hydrogen–Deuterium Exchange Mass Spectrometry

Lysenin is a pore-forming toxin of the aerolysin family, which is derived from coelomic fluid of the earthworm Eisenia fetida. Upon binding to sphingomyelin (SM)-containing membranes, lysenin undergoes a series of structural changes promoting the conversion of water-soluble monomers into oligomers, leading to its insertion into the membrane and the formation of a lytic β-barrel pore. The soluble monomer and transmembrane pore structures were recently described, but the underlying structural details of oligomerization remain unclear. To investigate the molecular mechanisms controlling the conformational rearrangements accompanying pore formation, we compared the hydrogen–deuterium exchange pattern between lyseninWT and its mutant lyseninV88C/Y131C. This mutation arrests lysenin oligomers in the prepore state at the membrane surface and does not affect the structural dynamics of the water-soluble form of lysenin. In contrast, membrane-bound lyseninV88C/Y131C exhibited increased structural stabilization, especially within the twisted β-sheet of the N-terminal domain. We demonstrated that the structural stabilization of the lysenin prepore started at the site of lysenin’s initial interaction with the lipid membrane and was transmitted to the twisted β-sheet of the N-terminal domain, and that lyseninV88C/Y131C was arrested in this conformation. In lyseninWT, stabilization of these regions drove the conformational changes necessary for pore formation

Secondary structure and orientation of the pore-forming toxin lysenin in a sphingomyelin-containing membrane

AbstractLysenin is a sphingomyelin-recognizing toxin which forms stable oligomers upon membrane binding and causes cell lysis. To get insight into the mechanism of the transition of lysenin from a soluble to a membrane-bound form, surface activity of the protein and its binding to lipid membranes were studied using tensiometric measurements, Fourier-transform infrared spectroscopy (FTIR) and FTIR-linear dichroism. The results showed cooperative adsorption of recombinant lysenin-His at the argon–water interface from the water subphase which suggested self-association of lysenin-His in solution. An assembly of premature oligomers by lysenin-His in solution was confirmed by blue native gel electrophoresis. When a monolayer composed of sphingomyelin and cholesterol was present at the interface, the rate of insertion of lysenin-His into the monolayer was considerably enhanced. Analysis of FTIR spectra of soluble lysenin-His demonstrated that the protein contained 27% β-sheet, 28% aggregated β-strands, 10% α-helix, 23% turns and loops and 12% different kinds of aggregated forms. In membrane-bound lysenin-His the total content of α-helices, turns and loops, and β-structures did not change, however, the 1636cm−1 β-sheet band increased from 18% to 31% at the expense of the 1680cm−1 β-sheet structure. Spectral analysis of the amide I band showed that the α-helical component was oriented with at 41° to the normal to the membrane, indicating that this protein segment could be anchored in the hydrophobic core of the membrane