Identification and characterization of novel methyltransferases

So far 172 different types of nucleic acid modifications had been identified. However, for many RNA and DNA modifications the function(s) and the modifying enzymes (”writers”) are still not known. In my Ph.D. project, I focused on the identification of novel RNA and DNA methyltransferases. To identify novel enzymes, I systematically screened a collection of potential methyltransferases on the range of substrates in in vitro methyltransferase assay. I found four enzymes that exhibit robust methyltransferases activity towards RNA. Size exclusion chromatography in combination with labelling approaches and mass spectrometry allowed me to narrow down potential substrates and to identify the modified nucleosides for two candidate enzymes. I characterized METTL6 as a tRNA methyltransferases that specifically catalyzes methylation of cytosines at position 3 (m3C). By performing methyltransferase assays as well as sequence-specific purification of individual tRNAs, I identified the specific tRNA isoacceptors that are METTL6 targets and the precise position of the methylated C. RNA-seq and Ribosome profiling of KO mES cell lines revealed global changes in the transcriptome and translatome. In line with these changes, Mettl6 KO cells showed slower proliferation rates. Interestingly, METTL6 depletion resulted in slower hepatocellular tumor growth in in vitro models. In agreement with this, patients with high METTL6 expression levels have a reduced survival rate. I identified, METTL5 as a novel m6A RNA methyltransferase. Mass spectrometry analysis of RNA from Mettl5 KO mES cells revealed a decrease in m6A levels in 18S rRNA. By performing m6A-immunopreciptaion followed by sequencing in wt and Mettl5 KO cells, I mapped the transcript wide distribution of m6A in non-ribosomal RNAs. In addition to this, Mettl5 KO mES cells are less pluripotent and compromised in their ability to differentiate into neuronal precursors cells. In a complementary approach, I wanted to identify the interactome of METTL proteins. For this, I generated stable cells lines expressing tagged METTL family members, purified the complexes, and analysed interaction partners by mass spectrometry. The systematic identification of the complexes in which these potential enzymes act provides a useful resource for the epitranscriptomics community as it helps to understand METTL proteins function(s) and substrate specificities. Overall, in my Ph.D. project, I profiled the enzymatic activities and the interactome of 13 proteins of the METTL family. For two novel RNA methyltransferases, I characterized the substrates and the type of methylation they catalyse, profiled changes in the transcriptome and translatome, and characterized the phenotypes of mES cells upon KO of these two enzymes.Bisher wurden 172 verschiedene Arten von Nukleinsäuremodifikationen identifiziert. Für viele dieser RNA- und DNA-Modifikationen sind die Funktion (en) und die modifizierenden Enzyme ("Writer") jedoch noch nicht bekannt. In meinem Promotionsprojekt beschäftigte ich mich mit der Identifizierung neuartiger RNA- und DNA- Methyltransferasen. Um neue Enzyme zu identifizieren, habe ich systematisch eine Sammlung potenzieller Methyltransferasen in in-vitro-Methyltransferase-Assays auf verschiedenen Substraten untersucht. Ich habe vier Enzyme identifiziert, die robuste Methyltransferaseaktivität gegenüber RNA aufweisen. Größenausschlusschromatographie in Kombination mit Markierungsansätzen und Massenspektrometrie ermöglichten es mir, potenzielle Substrate einzugrenzen und die modifizierten Nukleoside für zwei positive Kandidaten zu identifizieren. Ich charakterisierte METTL6 als tRNA-Methyltransferase, die spezifisch m3C katalysiert. Durch Methyltransferase-Assays sowie sequenzspezifische Aufreinigung einzelner tRNAs identifizierte ich spezifische tRNA-Isoakzeptoren als METTL6-Tagets und die genaue Position des methylierten Cytosins. RNA-seq- und Ribosomen-Profiling von KO-mES-Zelllinien ergab globale Veränderungen im Transkriptom und Translatom. In Übereinstimmung mit diesen Änderungen zeigten Mettl6-KO-Zellen geringere Proliferationsraten. Interessanterweise führte METTL6-Depletion in in vitro-Modellen zu einem langsameren Wachstum hepatozellulärer Tumore. In Übereinstimmung damit haben Patienten mit hoher METTL6 Exoression eine verringerte Überlebensrate. Ich identifizierte METTL5 als eine neue m6A-RNA-Methyltransferase. Die massenspektrometrische Analyse von RNA aus Mettl5 KO mES-Zellen ergab eine Abnahme der Menge an m6A in 18S-rRNA. Durch das Durchführen von m6A- Immunpräzipationen, gefolgt von Sequenzierung in wt- und Mettl5 KO-Zellen, kartierte ich die transkriptweite Verteilung von m6A in nicht-ribosomalen RNAs. Diese METTL5-KO mES-Zellen verlieren Pluripotency und ihre Fähigkeit sich korrekt in neuronale Vorläuferzellen zu differenzieren. In einem komplementären Ansatz wollte ich das „Interaktom“ von METTL-Proteinen identifizieren. Zu diesem Zweck erzeugte ich stabile Zelllinien, die getagte METTL Protein exprimieren, reinigte die Komplexe und analysierte die Interaktionspartner durch Massenspektrometrie. Die systematische Identifizierung von Komplexen, in denen diese potenziellen Enzyme wirken, ist eine nützliche Ressource für das Epitranscriptomics Field, da sie zum Verständnis der Funktionen und Substratspezifitäten von METTL-Proteinen beitragen kann. Insgesamt habe ich in meiner Doktorarbeit die enzymatische Aktivität und das Interaktom von 13 Proteinen der METTL-Familie analysiert. Für zwei neue RNA- Methyltransferasen charakterisierte ich die Substrate und die Art der Methylierung, die sie katalysieren, profilierte Veränderungen im Transkriptom und Translatom und identifizierte die Phänotypen von mES-Zellen auf KO dieser beiden Enzyme

Influence of Gut Microbiota on Behavior and Its Disturbances

Hippocrates statement that “All disease begins in the gut” continues to be up to date more than 2000 years later. Growing number of scientific reports focus on the important role of intestinal microorganisms for modulation of many systems and human behavior. As a key component of the gut brain, gut microbiota influences the development and maturation of the hypothalamic-pituitary-adrenal axis, affects the development and function of the immune system, regulates the blood-brain barrier, modulates the synthesis and recognition of neurotransmitters, regulates neurogenesis, formation of myelination and supports the development and function of the brain. Disruption of gut-brain axis function is associated with alterations in the stress response and might contribute to neuropsychiatric diseases as depression, autistic spectrum disorders, rapid eye movement sleep behavior disorder, Parkinson disease, Alzheimer disease and other mental conditions. Studies in animal models are crucial for guiding research on brain-gut-microbiome axis in humans, as the impact of microbiota on specific brain regions and aspects of animal behavior will help in the selection of tasks for cognitive assessment. Exploring the interaction of gut microbes and human brain will not only allow us to better understand the pathogenesis of neuropsychiatric disorders, but will also provide us new opportunities for the design of novel immuno- or microbe-based therapies

Biomarkers in Multiple Sclerosis

Clinical, biological, and radiological evidence are currently needed to diagnose MS, but lack of preclinical biomarkers hinders the earliest possible diagnosis and treatment. Conventional biomarkers target immunity, blood-brain barrier disruption, demyelination, and neuronal and axonal damage, as well as mitochondrial activity. An increase of specific brain metabolites with 30–40% is registered before detection of MRI lesions in MS. Potential lipid biomarkers are fatty acids, phospholipids, and oxysterols. The role of proteoforms in the pathogenesis of MS was confirmed. Serum neurofilament light chains (sNfL) are currently being studied as a readily available biomarker for prognosis and response to treatment in MS. The sNfL levels reflect ongoing neuroaxonal damage caused by inflammation, and the sNfL levels predict disease activity over the next few years. The retinal nerve fiber layer (RNFL) thinning is reliable as a biomarker of disability worsening. The neutrophil-to-lymphocyte ratio and CRP are also MS biomarkers. The development of rationally targeted therapeutic agents that allow preventive treatment to stop the disease is also delayed without definite biomarkers

Social Cognition Impairments in Patients with Multiple Sclerosis and Comparison with Imaging Studies, Disease Duration and Grade of Disability

Cognitive impairments in multiple sclerosis (MS) are heterogeneous and their rate varies between 43% and 70%. A less studied aspect of cognition is social cognition, which is not a uniform theoretical construct. It includes emotion perception, prosody, empathy, theory of mind (ToM) and assessment of mood. In addition to progressive physical disability, social cognitive impairments are a reason for job loss in 24–80% of patients with MS, increased divorce rate, dissolution of partnerships and social communication difficulties

PERFORMING CULTURE OF FUTURE BACHELOR: CRITERIA AND LEVEL OF ITS FORMEDNESS

В статье описан оценочно диагностический инструментарий. В качестве основных критериев сформированности исполнительской культуры выделены: нормативный, интерактивный, регулятивный. Охарактеризованы уровни проявления исполнительской культуры: репродуктивно-алгоритмический, конструктивно-аналоговый, продуктивно-созидательный. Представлены основные результаты изучения сформированности исполнительской культуры будущих бакалавров. Наблюдается, что у большинства будущих бакалавров исполнительская культура находится на репродуктивно-алгоритмическом уровне.The assessment diagnostic tools are described in the article. The main criteria of formedness of performing culture are: normative, interactive, regulative. The levels of manifestation of performing culture are defined: reproductively-algorithmic, constructive-analogue, productively-creative. The main results of the research of the formedness of future bachelors’ performance culture are presented. It has turned out that the performing culture of the majority of future bachelors is on the reproductively-algorithmic level

Включение будущих бакалавров в разработку информационно-познавательной модели приобщения к социально-педагогическим ценностям

This paper describes the features of the development of an information-cognitive model of introducing future bachelors of social majors to social-pedagogical values, as well as the methods, techniques and means of the implementation of this process in high school on the basis of different social subjects and humanities. The development of “the model” is associated with providing assistance to students in the systematization of knowledge about social-pedagogical values, understanding their essence. In this connection, the paper analyzes the stages of constructing “the model”; considers the heuristic methods used in the course of learning; characterizes the basic professionally significant socialpedagogical valuesВ данной статье описываются особенности разработки информационно-познавательной модели приобщения к социально-педагогическим ценностям будущих бакалавров социально- ориентированных направлений подготовки, методы, приемы и средства реализации данного процесса в вузе на базе различных социально-гуманитарных дисциплин. Разработка «модели» связывается с оказанием помощи студентам в систематизации знаний о социально- педагогических ценностях, понимании их сущности. В этой связи анализируются этапы конструирования «модели»; рассматриваются эвристические методы, используемые в ходе обучения; характеризуются базовые профессионально значимые социально-педагогические ценност

ПРОФЕССИОНАЛЬНО-КУЛЬТУРНЫЕ ПРАКТИКИ БУДУЩЕГО БАКАЛАВРА И ИХ СТРУКТУРНЫЕ ХАРАКТЕРИСТИКИ

Educational activities at the university are presented as a variety of practices many of which have professional and cultural character and a sufficient potential for the development of the students’ personal initiative and for their reconsideration and transformation of their own life and the formation of social responsibility.In this paper there has been explicated the idea of «professional and cultural practices of the future bachelor» and their basic types that have been singled out and characterized. The involvement of the future bachelors in these practices has been also considered. The results of the performed study show that the level of involvement of the future bachelors in these practices (2012–2014) is characterized as indifferent-positive which indicates the need to activate their involvement in the given form of professional and cultural activities to focus on the higher level – sustainable-positive.Образовательная деятельность в вузе представлена многообразием практик, многие из которых носят профессионально-культурный характер и обладают достаточным потенциалом для развития личной инициативыобучающихся, переосмысления и преобразования имисобственной жизнедеятельности и формирования социальной ответственности.В статье эксплицировано понятие «профессионально-культурные практики будущего бакалавра», выделеныи охарактеризованы их основные виды, обсуждается вопрос об участии будущих бакалавров в данных практиках. Результаты проведенного исследования показали, что уровень участия будущих бакалавров в данных практиках (2012–2014 гг.) характеризуется как индифферентно-положительный, что свидетельствует о необходимости активизировать их участие в данной форме профессионально-культурной работы с ориентацией на более высокий уровень – устойчиво-положительный

IKT grįsto mokymosi personalizavimo įgalinimas konstrukcionistinių mokymosi veiklų diegimo kontekste

How to enable students to create a personalized learning environment? What are the criteria of evaluation of the ICT-based learning process personalization affordance? These questions are answered by conducting multiple case study research of the innovative ICT-based learning process in iTEC (Innovative Technologies for Engaging Classrooms) project in Lithuania. Analysis of the research data led to discernment of evaluation criteria of personalization affordance and creation of a framework for the ICT-based learning personalization affordance, comprised of the following three groups of evaluation criteria: (I) "extremely important" criteria, (II) "essential" criteria, (III) "important" criteria. Students, who performed various constructionist ICT-based learning activities as part of innovative learning scenarios, evaluated these according to the "extremely important" criteria. A majority of students have agreed that the proposed learning activities enable them to feel more confident and engaged into the learning process. The framework for ICT-based learning personalization affordance should help other teachers enable personalized learning in daily practice

ОЦЕНКА КАЧЕСТВА ИСПОЛНЕНИЯ ИНФОРМАЦИОННО-ПОИСКОВОЙ ДЕЯТЕЛЬНОСТИ БУДУЩИМ БАКАЛАВРОМ В ОБРАЗОВАТЕЛЬНОМ ПРОЦЕССЕ

The article considers information-search activity of a future bachelor as a special kind of educational and professional activity, definition was explicated, structural components and results were specified. Levels of the execution information-search activity were distinguished, a grading scale and questionnaire were designed based on their. The results of the study of this activity type were distinguished, showing the spontaneity of its execution in the educational process that, according to the authors, is a prerequisite for the need to assist students in organi-zing these type of activity.В статье рассмотрена информационно-поисковая деятельность будущего бакалавра как особый вид учебно-профессиональной деятельности, эксплицировано определение, выявлены структурные компоненты, уточнены результаты. Выделены уровни оценки качества исполнения информационно-поисковой деятельности, на основе которых разработана оценочная шкала и опросник. Представлены результаты исследования данного вида деятельности, показавшие стихийность ее протекания в образовательном процессе, что, по мнению авторов, является предпосылкой для необходимости оказании помощи обучающимся в организации данной деятельности

Включение будущих бакалавров в разработку информационно-познавательной модели приобщения к социально-педагогическим ценностям

This paper describes the features of the development of an information-cognitive model of introducing future bachelors of social majors to social-pedagogical values, as well as the methods, techniques and means of the implementation of this process in high school on the basis of different social subjects and humanities. The development of “the model” is associated with providing assistance to students in the systematization of knowledge about social-pedagogical values, understanding their essence. In this connection, the paper analyzes the stages of constructing “the model”; considers the heuristic methods used in the course of learning; characterizes the basic professionally significant socialpedagogical valuesВ данной статье описываются особенности разработки информационно-познавательной модели приобщения к социально-педагогическим ценностям будущих бакалавров социально- ориентированных направлений подготовки, методы, приемы и средства реализации данного процесса в вузе на базе различных социально-гуманитарных дисциплин. Разработка «модели» связывается с оказанием помощи студентам в систематизации знаний о социально- педагогических ценностях, понимании их сущности. В этой связи анализируются этапы конструирования «модели»; рассматриваются эвристические методы, используемые в ходе обучения; характеризуются базовые профессионально значимые социально-педагогические ценност