Novel aspects for methodology and utilization of PET/CT imaging in head and neck cancer

Avainsanat: PET, PET/TT, pään ja kaulan alueen syöpä, [18F]EF5, [18F]FDG, hypoksia, molekulaarinen kuvantaminen, sädehoidon suunnittelu Positron emission tomography (PET), combined with computed tomography (CT),plays a key role in the management of head and neck cancer (HNC). In this thesis, novel aspects for PET/CT imaging of HNC regarding low oxygen levels, or hypoxia, and detection of glucose metabolism were evaluated. Hypoxia is a frequently observed hallmark of cancer contributing to radiotherapy resistance and poor prognosis. Enhanced glucose metabolism is characteristic of a malignant tumor, which is exploited in an everyday clinical application of [18F]FDG PET imaging. This study aimed to further investigate the feasibility of a novel hypoxia PET tracer [18F]EF5 and the potential of dynamic [18F]FDG PET/CT imaging in HNC. The first study indicated a favorable human biodistribution and radiation dosimetric profile of the hypoxia tracer [18F]EF5. The second preclinical study showed that the growth rate of human HNC xenografts in nude mice during the exponential growth period correlated with [18F]EF5 uptake in PET/CT images. In the third study, paired [18F]EF5 PET/CT scans performed for untreated HNC patients with a median time interval of seven days presented predominantly highly repeatable results. In the fourth study, advanced mathematical methodology for tracer uptake analysis was evaluated using dynamic [18F]FDG PET/CT in patients who were referred to chemoradiotherapy for oropharyngeal cancer. However, the method showed only a modest performance in the distinction of malignant, inflammatory and healthy tissues. In conclusion, further evaluation of [18F]EF5 PET/CT imaging and dynamic [18F]FDG PET/CT imaging seems important in the development of more effective strategies for the management of HNC

Sustainable Functional Food Processing

Functional nutrition is deeply connected with healthy lifestyle and sustainable food production, due to its positive health benefits and the use of economically underexplored and natural raw materials. Expectedly, it appeals to large number of interested consumers while becoming lucrative segment of the food industry with a fast-growing market fueled by new sociodemographic trends. Accordingly, functional juices and beverages made of indigenous fruits are interesting niche for various food market stakeholders. Here, biologically active compounds (BACs) and probiotics that have positive health effects in functional foods (juices) are mostly thermolabile. This is especially important for industry that still employs classical heat treatments (e.g., pasteurization), while being concerned with degradation of food quality in the final products. To prevent this, focus is on designing economic and ecological technologies that are able to preserve nutritional and sensory quality while maintaining microbiological stability in products. Such approaches are based on low-energy consumption and low-impact processing, e.g. “hurdle technology” that combines advanced and conventional methods (e.g., high-power ultrasound, pulse electric field). Food design is another important focus point for consumers’ sensory appeal and economic success of foods. Hence, technologies as 3D food printing can be particularly useful for manufacturing. Based on the above, presented topics are relevant to sustainable functional food production, functional fruit juices, BACs, “hurdle technology,” advanced food processing, 3D food printing, and authentic fruits

La farmacogenética como herramienta de la medicina personalizada: desarrollo de estrategias para su implementación en la práctica clínica e identificación de nuevas asociaciones

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 28-03-2019La Farmacogenética (PGx) representa en la actualidad una de las herramientas más importantes del paradigma de la medicina personalizada permitiendo evitar Reacciones Adversas a Medicamentos (RAM) así como maximizar la eficacia de los tratamientos farmacológicos y, en definitiva, optimizar el proceso de selección de la mejor pauta terapéutica para cada paciente. El primer objetivo de esta tesis doctoral, fue el desarrollo de una estrategia para la implementación de la PGx en la rutina clínica de un hospital de primer nivel asistencial adscrito al Sistema Nacional de Salud (SNS). Esta estrategia está basada en una plataforma de microarray de SNPs que permite el genotipado simultáneo de 180 polimorfismos de respuesta a fármacos. A lo largo de este período (2013-2018) se han realizado un total de 3532 estudios PGx para distintos fármacos y en pacientes de distintas patologías dentro de la rutina clínica del hospital. Por otro lado, se evaluó la utilidad de un protocolo multidisciplinar para la implementación del genotipado anticipado de CYP2C19 en una cohorte de pacientes hematológicos previo al trasplante de progenitores hematopoyéticos, definida como población de riesgo para el tratamiento o profilaxis con Voriconazol. Esta estrategia permitió aumentar notablemente el porcentaje de pacientes que alcanzaban el rango terapéutico diana así como reducir el tiempo necesario para alcanzarlo, mejorando de este modo el éxito del tratamiento. El último objetivo de este trabajo fue la identificación de nuevas asociaciones PGx que permitiesen mejorar los algoritmos monogénicos existentes para la optimización del tratamiento con Tacrolimus y Voriconazol, mediante la incorporación de nuevos biomarcadores asociados a la variabilidad en su farmacocinética. A lo largo de esta tesis doctoral se proponen nuevos biomarcadores no incluidos hasta la fecha en los algoritmos clínicos que podrían resultar de interés para la individualización del tratamiento con estos fármacos. En definitiva, en este trabajo se ha desarrollado una estrategia que ha permitido la implementación de la PGx en la práctica clínica de nuestro centro. Además, se ha demostrado la utilidad en la práctica clínica de un protocolo de genotipado anticipado en una cohorte de pacientes hematológicos previo al trasplante de progenitores hematopoyéticos definida como población de riesgo para el tratamiento o profilaxis con Voriconazol. Finalmente, se proponen modelos poligénicos de predicción del perfil farmacocinético de dos fármacos de uso clínico frecuente, Voriconazol y Tacrolimus, que parecen mejorar los algortimos clínicos monogénicos desarrollados hasta la fecha.Pharmacogenetics (PGx) constitutes an essential tool for personalized medicine. It minimizes Adverse Drug Reactions (RAM) and maximizes treatment efficacy; and ultimately optimizes treatment selection for each patient. The first goal of this doctoral thesis was the development of a strategy for the implementation of PGx in the clinical routine of a third-level care hospital of the Spanish National Health Service. This strategy was based on a SNP-array platform allowing simultaneous genotyping of 180 SNPs related to drug response. During this period (2013-2018) we have performed 3532 PGx tests for different drugs and diseases in the clinical routine of our center. We have also evaluated the utility of a multidisciplinary protocol for the implementation of preemptive genotyping of CYP2C19 in a cohort of haematological patients, defined as risk population for treatment or prophylaxis with Voriconazole. This strategy resulted in a significant increase in the percentage of patients achieving Voriconazole goal therapeutic range and reduced the time window required to achieve it, and therefore improved treatment success. The last aim of this work was the identification of new PGx associations that improved the existing monogenic algorithms for Tacrolimus and Voriconazole treatment optimization, by the incorporation of new biomarkers related to their PGx interindividual variability. During this doctoral thesis we propose new biomarkers, not previously included in clinical prediction algorithms that may be of interest for treatment individualization with these drugs in the clinical practice. In summary, we have developed a strategy that allowed the implementation of PGx in the clinical routine of our center. In addition, we have demonstrated the clinical utility of a preemptive genotyping protocol in a cohort of haematological patients before allogenic hematopoietic cell transplantation defined as a risk population for treatment or prophylaxis with Voriconazole. Finally, we here propose two polygenic predictive models of the pharmacokinetic profile of Tacrolimus and Voriconazole that seem to improve the existing monogenic predictive algorithms

Contributions to information extraction for spanish written biomedical text

285 p.Healthcare practice and clinical research produce vast amounts of digitised, unstructured data in multiple languages that are currently underexploited, despite their potential applications in improving healthcare experiences, supporting trainee education, or enabling biomedical research, for example. To automatically transform those contents into relevant, structured information, advanced Natural Language Processing (NLP) mechanisms are required. In NLP, this task is known as Information Extraction. Our work takes place within this growing field of clinical NLP for the Spanish language, as we tackle three distinct problems. First, we compare several supervised machine learning approaches to the problem of sensitive data detection and classification. Specifically, we study the different approaches and their transferability in two corpora, one synthetic and the other authentic. Second, we present and evaluate UMLSmapper, a knowledge-intensive system for biomedical term identification based on the UMLS Metathesaurus. This system recognises and codifies terms without relying on annotated data nor external Named Entity Recognition tools. Although technically naive, it performs on par with more evolved systems, and does not exhibit a considerable deviation from other approaches that rely on oracle terms. Finally, we present and exploit a new corpus of real health records manually annotated with negation and uncertainty information: NUBes. This corpus is the basis for two sets of experiments, one on cue andscope detection, and the other on assertion classification. Throughout the thesis, we apply and compare techniques of varying levels of sophistication and novelty, which reflects the rapid advancement of the field

Health-Promoting Effects of Traditional Foods

Food cannot be only considered a combination of constituents with different nutritional values, but its relevance for humans can be fully understood by also taking into account other aspects such as history, culture, ecology, and the environment. Overall, assuming that access to food is secured for all people, traditional dietary patterns are considered safe in terms of longevity, healthy ageing, and morbidity. Indeed, healthy diets have been associated with a reduced risk and incidence of chronic degenerative diseases including cardiovascular disease, type 2 diabetes, metabolic syndrome, certain types of cancers, and neurodegenerative disorders. In general, healthy dietary habits include a low consumption of refined sugars, red meat, and saturated fats, as well as a high intake of fruit, vegetables, legumes, low-fat dairy products, and healthy lipids (from seafood). As an example, the Mediterranean diet can be considered the archetype of a health-promoting lifestyle by virtue of the phytochemical diversity of its food components

Early detection of Alzheimer’s disease - Twin study on episodic memory and imaging biomarkers of neuroinflammation and β-amyloid

The disease process of Alzheimer’s disease (AD) causes damage to the brain for several years leading to the development of mild cognitive impairment (MCI) and finally to dementia which interferes with independent living. The early detection of AD disease process is key for the prevention and treatment of disease. The aim of this thesis was to improve the assessment of episodic memory (EM) and cognitive performance with a telephone interview and neuroimaging of early AD. The study population belonged to the older Finnish Twin Cohort study. 2631 twins (856 pairs) participated in the telephone interview (TELE, TICS) during 1999–2007 and 1817 twins (559 pairs) participated in the interview (TELE, TICS, TICS-m) during 2013– 2017. Cognitively discordant twin pairs were asked to participate in more detailed examinations. 11 twin pairs participated in [11C]PBR28 positron emission tomography (PET) imaging measuring neuroinflammation during 2014–2017 and 45 twin pairs participated in [11C]PiB PET imaging measuring β-amyloid (Aβ) deposits during 2005–2017. Twins who had co-twins with dementia (n=101) performed poorer than average in a word list learning test. When using the telephone interview TICS-m, the education‐adjusted classification resulted in a higher proportion of apolipoprotein (APOE) ε4 allele carriers among those identified as having MCI. Twins with poorer EM performance (n=10) had higher cortical [11C]PBR28 uptake compared to their better-performing co-twins. In addition, higher cortical [11C]PiB uptake was associated with poorer EM performance. The results from the telephone interview studies indicate that poorer word list learning performance may be an early marker of dementia risk and that the use of education‐adjustment may increase the accuracy of MCI classification. The twin pair setting controlling for genetic and environmental effects indicated that brain Aβ load and neuroinflammation have a negative association with EM performance.Alzheimerin taudin varhainen havaitseminen – Kaksostutkimus episodisesta muistista ja neuroinflammaation ja β-amyloidin kuvantamisbiomarkkereista Alzheimerin taudin (AT) prosessi vaurioittaa aivoja vuosien ajan ja johtaa lievään kognitiiviseen heikentymiseen (MCI) ja lopulta itsenäistä selviytymistä häiritsevään dementiaan. AT:n dementiaan johtavan prosessin varhainen havaitseminen on avainasemassa ehkäisyn ja hoidon kannalta. Tämän väitöskirjatutkimuksen tavoitteena oli kehittää puhelinhaastattelun käyttöä episodisen muistin (EM) ja muiden tiedonkäsittely- eli kognitiivisten toimintojen arvioimisessa sekä AT:n varhaista kuvantamista. Tutkimusjoukko kuului vanhempaan suomalaisen kaksoskohorttitutkimukseen. 2631 kaksosta (856 paria) osallistui puhelinhaastatteluun (TELE, TICS) 1999–2007 aikana ja 1817 kaksosta (559 paria) osallistui haastatteluun (TELE, TICS, TICS-m) 2013–2017 aikana. Kognitiivisesti diskordantit kaksosparit kutsuttiin tarkempiin jatkotutkimuksiin. 11 kaksosparia osallistui neuroinflammaatiota mittaavaan [11C]PBR28-merkkiaineen positroniemissiotomografia (PET) - kuvaukseen 2014–2017 aikana ja 45 kaksosparia osallistui aivojen β-amyloidikertymää mittaavaan [11C]PiB-merkkiaineen PET-kuvaukseen 2005–2017 aikana. Sellaisten kognitiivisesti normaalien ikääntyneiden kaksosten (n=101), joiden sisaruksella oli dementia, havaittiin suoriutuvan keskimääräistä heikommin sanalistan oppimista mittaavassa testissä. Käytettäessä TICS-m-puhelinhaastattelua koulutuskorjauksen käyttäminen johti siihen, että MCI:tä sairastavien joukossa oli suurempi osuus apolipoproteiini E:n (APOE) ε4-alleelin kantajia. Kaksosilla (n=10), jotka suoriutuivat heikommin EM-testeissä, oli suurempi aivokuoren [11C]PBR28-kertymä verrattuna paremmin suoriutuviin sisaruksiinsa. Myös suurempi aivokuoren [11C]PiB-kertymä oli yhteydessä heikompaan EM-suoritukseen. Puhelinhaastattelujen tulokset viittaavat siihen, että sanalistan oppiminen voi olla dementiariskistä kertova varhainen merkki ja että koulutuskorjauksen käyttö voi lisätä MCI-luokittelun tarkkuutta. Kaksosasetelma, joka kontrolloi geneettisten ja ympäristötekijöiden vaikutusta, osoitti, että aivojen β-amyloidikertymä ja neuroinflammaatio ovat negatiivisessa yhteydessä EM:n toiminnan kanssa

Biomimetic Radical Chemistry and Applications

The enormous importance of free radical chemistry for a variety of biological events, including ageing and inflammation, has attracted a strong interest in understanding the related mechanistic steps at the molecular level. Modelling the free radical chemical reactivity of biological systems is an important research area. When studying free-radical-based chemical mechanisms, biomimetic chemistry and the design of established biomimetic models come into play to perform experiments in a controlled environment that is suitably designed to be in strict connection with cellular conditions. This Special Issue gives the reader a wide overview of biomimetic radical chemistry, where molecular mechanisms have been defined and molecular libraries of products are developed to also be used as traces for the discovery of some relevant biological processes. Several subjects are presented, with 12 articles and 6 reviews written by specialists in the fields of DNA, proteins, lipids, biotechnological applications, and bioinspired synthesis, having “free radicals” as a common denominator

Translation elongation factor P and its post-translational modification enzyme EpmA

Protein translation is a non-uniform process, whereby especially proline-containing motifs lead to ribosome stalling events. Elongation factor P (EF P) rescues the ribosome by stimulation of peptidyl transfer. The four known subgroups of bacterial EF-Ps either require post-translational modification (PTM) established by EpmABC/EarP/YmfI or are functional without PTM. In Escherichia coli, the aminomutase EpmB converts (S)-α-lysine into (R)-β-lysine, which is subject to a two-step reaction catalyzed by lysyl-tRNA synthetase paralog EpmA. First, (R)-β-lysyl-adenylate is formed, from which the β-lysyl moiety is subsequently transferred to the ε-amino group of lysine 34 of EF P. This thesis focuses on the interplay of EF-P and EpmA which ensures EF P functionality in vivo and was used to modify EF-P with seven unnatural substrates in vitro. To detect PTM status two universal peptide antibodies, nonselective for the bacterial origin of EF-P, were generated and three subtypes of one-dimensional isoelectric focusing were established (native/denaturating horizontal, vertical). EpmA and EF-P protein copy numbers indicated balanced coordination to ensure outright modification status of EF-P during all growth phases, but no mutual regulation. EpmA’s donor substrate promiscuity was pinpointed to permit C6 scaffolds with at least an amino group at α- ((R/S)-α-lysine, 5-hydroxy-(S)-α-lysine), β- ((R/S)-β-lysine, (R)-3-aminocapronic acid) or ε-position (6-aminocapronic acid). In addition, EpmA variant A298G enabled modification of EF-P with (S)-α-ornithine. For the first time, known natural PTMs of EF P were expanded by seven synthetic PTMs. In vitro transcription translation assay demonstrated superiority of (R)-β-lysylation in ribosome rescue, explaining its evolutionary selection. Modification of EF-P with (S)-α-lysine was successfully achieved in vivo, when (R)-β-lysine synthesis was impeded (E. coli ΔepmB) and epmA(_A298G) overexpressed. In Bacillus subtilis, the ratio of unmodified-to-modified EF P varied over time. Out of 13 tested aminotransferase genes dat, epsN, gsaB, ilvK and yhdR are potentially involved in the yet unsolved modification pathway. In summary, the present work not only provides new biochemical insights into the functionalization of EF-P, but also paves the way to modify proteins post-translationally using EpmA.Die Translation von Proteinen ist kein gleichförmiger Prozess. Besonders bei Polyprolinmotiven treten Verzögerungen des Ribosoms auf. Hier übernimmt Elongationsfaktor P (EF-P) eine helfende Funktion und stimuliert den Peptidyltransfer. Die vier bekannten Gruppen von bakteriellen EF-P benötigen entweder posttranslationale Modifikation (PTM) durch EpmABC/EarP/YmfI, oder sind ohne PTM funktional. In Escherichia coli wandelt die Aminomutase EpmB (S)-α-Lysin in (R)-β-Lysin um. Ein Paralog der Lysyl-tRNA-Synthetase, EpmA, katalysiert die folgende zweistufige Reaktion. Erst wird (R)-β-Lysyladenylat gebildet, dessen β-Lysylgruppe dann auf die ε-Aminogruppe von Lysin 34 von EF-P übertragen wird. Diese Dissertation widmet sich dem Zusammenspiel von EF-P und EpmA. Dieses stellt in vivo die Funktion von EF-P sicher, in vitro erlaubt es die Modifikation von EF P mit sieben unnatürlichen Substraten. Zur Detektion des PTM Status wurden zwei universelle Peptidantikörper etabliert, die EF-P unabhängig von dessen bakterieller Herkunft detektieren, sowie drei Formen der eindimensionalen Isoelektrischen Fokussierung (nativ/denaturierend horizontal, vertikal). Die Kopienzahlen von EpmA und EF-P sind aufeinander abgestimmt, um vollständige Modifikation von EF-P in allen Wachstumsphasen sicherzustellen. Sie regulieren sich aber nicht gegenseitig. Die Promiskuität von EpmA erlaubt Donorsubstrate mit C6-Ketten, die zumindest eine Aminogruppe in α- ((R/S)-α-Lysin, 5-Hydroxy-(S)-α-lysin), β- ((R/S)-β-Lysin, (R)-3-Aminohexansäure) oder ε-Position (6-Aminohexansäure) haben. Zusätzlich ermöglicht die Enzymvariante EpmA_A298G (S)-α-Ornithylierung von EF-P. Erstmals konnten so die natürlichen PTMs von EF-P um sieben synthetische PTMs erweitert werden. In vitro Transkriptions/Translations-Assays zeigten die wirkungsvollste Ribosomen-rettung bei (R)-β-lysyliertem EF-P, was dessen evolutionäre Auswahl erklärt. In vivo gelang die Modifikation von EF-P mit (S)-α-Lysin, wenn die Fähigkeit zur (R)-β-Lysinsynthese fehlte (E. coli ΔepmB) und epmA(_A298G) überexprimiert wurde. In Bacillus subtilis variiert das Verhältnis von unmodifiziertem zu modifiziertem EF-P. Von 13 untersuchten Genen, die Aminotransferasen kodieren, sind dat, epsN, gsaB, ilvK und yhdR möglicherweise am ungeklärten Modifikationsweg beteiligt. Zusammengefasst liefert die vorliegende Arbeit nicht nur neue biochemische Einsichten in die Funktionalisierung von EF-P, sondern eröffnet auch einen neuen Weg, Proteine mittels EpmA posttranslational zu modifizieren

Translation elongation factor P and its post-translational modification enzyme EpmA

Protein translation is a non-uniform process, whereby especially proline-containing motifs lead to ribosome stalling events. Elongation factor P (EF P) rescues the ribosome by stimulation of peptidyl transfer. The four known subgroups of bacterial EF-Ps either require post-translational modification (PTM) established by EpmABC/EarP/YmfI or are functional without PTM. In Escherichia coli, the aminomutase EpmB converts (S)-α-lysine into (R)-β-lysine, which is subject to a two-step reaction catalyzed by lysyl-tRNA synthetase paralog EpmA. First, (R)-β-lysyl-adenylate is formed, from which the β-lysyl moiety is subsequently transferred to the ε-amino group of lysine 34 of EF P. This thesis focuses on the interplay of EF-P and EpmA which ensures EF P functionality in vivo and was used to modify EF-P with seven unnatural substrates in vitro. To detect PTM status two universal peptide antibodies, nonselective for the bacterial origin of EF-P, were generated and three subtypes of one-dimensional isoelectric focusing were established (native/denaturating horizontal, vertical). EpmA and EF-P protein copy numbers indicated balanced coordination to ensure outright modification status of EF-P during all growth phases, but no mutual regulation. EpmA’s donor substrate promiscuity was pinpointed to permit C6 scaffolds with at least an amino group at α- ((R/S)-α-lysine, 5-hydroxy-(S)-α-lysine), β- ((R/S)-β-lysine, (R)-3-aminocapronic acid) or ε-position (6-aminocapronic acid). In addition, EpmA variant A298G enabled modification of EF-P with (S)-α-ornithine. For the first time, known natural PTMs of EF P were expanded by seven synthetic PTMs. In vitro transcription translation assay demonstrated superiority of (R)-β-lysylation in ribosome rescue, explaining its evolutionary selection. Modification of EF-P with (S)-α-lysine was successfully achieved in vivo, when (R)-β-lysine synthesis was impeded (E. coli ΔepmB) and epmA(_A298G) overexpressed. In Bacillus subtilis, the ratio of unmodified-to-modified EF P varied over time. Out of 13 tested aminotransferase genes dat, epsN, gsaB, ilvK and yhdR are potentially involved in the yet unsolved modification pathway. In summary, the present work not only provides new biochemical insights into the functionalization of EF-P, but also paves the way to modify proteins post-translationally using EpmA.Die Translation von Proteinen ist kein gleichförmiger Prozess. Besonders bei Polyprolinmotiven treten Verzögerungen des Ribosoms auf. Hier übernimmt Elongationsfaktor P (EF-P) eine helfende Funktion und stimuliert den Peptidyltransfer. Die vier bekannten Gruppen von bakteriellen EF-P benötigen entweder posttranslationale Modifikation (PTM) durch EpmABC/EarP/YmfI, oder sind ohne PTM funktional. In Escherichia coli wandelt die Aminomutase EpmB (S)-α-Lysin in (R)-β-Lysin um. Ein Paralog der Lysyl-tRNA-Synthetase, EpmA, katalysiert die folgende zweistufige Reaktion. Erst wird (R)-β-Lysyladenylat gebildet, dessen β-Lysylgruppe dann auf die ε-Aminogruppe von Lysin 34 von EF-P übertragen wird. Diese Dissertation widmet sich dem Zusammenspiel von EF-P und EpmA. Dieses stellt in vivo die Funktion von EF-P sicher, in vitro erlaubt es die Modifikation von EF P mit sieben unnatürlichen Substraten. Zur Detektion des PTM Status wurden zwei universelle Peptidantikörper etabliert, die EF-P unabhängig von dessen bakterieller Herkunft detektieren, sowie drei Formen der eindimensionalen Isoelektrischen Fokussierung (nativ/denaturierend horizontal, vertikal). Die Kopienzahlen von EpmA und EF-P sind aufeinander abgestimmt, um vollständige Modifikation von EF-P in allen Wachstumsphasen sicherzustellen. Sie regulieren sich aber nicht gegenseitig. Die Promiskuität von EpmA erlaubt Donorsubstrate mit C6-Ketten, die zumindest eine Aminogruppe in α- ((R/S)-α-Lysin, 5-Hydroxy-(S)-α-lysin), β- ((R/S)-β-Lysin, (R)-3-Aminohexansäure) oder ε-Position (6-Aminohexansäure) haben. Zusätzlich ermöglicht die Enzymvariante EpmA_A298G (S)-α-Ornithylierung von EF-P. Erstmals konnten so die natürlichen PTMs von EF-P um sieben synthetische PTMs erweitert werden. In vitro Transkriptions/Translations-Assays zeigten die wirkungsvollste Ribosomen-rettung bei (R)-β-lysyliertem EF-P, was dessen evolutionäre Auswahl erklärt. In vivo gelang die Modifikation von EF-P mit (S)-α-Lysin, wenn die Fähigkeit zur (R)-β-Lysinsynthese fehlte (E. coli ΔepmB) und epmA(_A298G) überexprimiert wurde. In Bacillus subtilis variiert das Verhältnis von unmodifiziertem zu modifiziertem EF-P. Von 13 untersuchten Genen, die Aminotransferasen kodieren, sind dat, epsN, gsaB, ilvK und yhdR möglicherweise am ungeklärten Modifikationsweg beteiligt. Zusammengefasst liefert die vorliegende Arbeit nicht nur neue biochemische Einsichten in die Funktionalisierung von EF-P, sondern eröffnet auch einen neuen Weg, Proteine mittels EpmA posttranslational zu modifizieren

Deep learning methods for knowledge base population

Knowledge bases store structured information about entities or concepts of the world and can be used in various applications, such as information retrieval or question answering. A major drawback of existing knowledge bases is their incompleteness. In this thesis, we explore deep learning methods for automatically populating them from text, addressing the following tasks: slot filling, uncertainty detection and type-aware relation extraction. Slot filling aims at extracting information about entities from a large text corpus. The Text Analysis Conference yearly provides new evaluation data in the context of an international shared task. We develop a modular system to address this challenge. It was one of the top-ranked systems in the shared task evaluations in 2015. For its slot filler classification module, we propose contextCNN, a convolutional neural network based on context splitting. It improves the performance of the slot filling system by 5.0% micro and 2.9% macro F1. To train our binary and multiclass classification models, we create a dataset using distant supervision and reduce the number of noisy labels with a self-training strategy. For model optimization and evaluation, we automatically extract a labeled benchmark for slot filler classification from the manual shared task assessments from 2012-2014. We show that results on this benchmark are correlated with slot filling pipeline results with a Pearson's correlation coefficient of 0.89 (0.82) on data from 2013 (2014). The combination of patterns, support vector machines and contextCNN achieves the best results on the benchmark with a micro (macro) F1 of 51% (53%) on test. Finally, we analyze the results of the slot filling pipeline and the impact of its components. For knowledge base population, it is essential to assess the factuality of the statements extracted from text. From the sentence "Obama was rumored to be born in Kenya", a system should not conclude that Kenya is the place of birth of Obama. Therefore, we address uncertainty detection in the second part of this thesis. We investigate attention-based models and make a first attempt to systematize the attention design space. Moreover, we propose novel attention variants: External attention, which incorporates an external knowledge source, k-max average attention, which only considers the vectors with the k maximum attention weights, and sequence-preserving attention, which allows to maintain order information. Our convolutional neural network with external k-max average attention sets the new state of the art on a Wikipedia benchmark dataset with an F1 score of 68%. To the best of our knowledge, we are the first to integrate an uncertainty detection component into a slot filling pipeline. It improves precision by 1.4% and micro F1 by 0.4%. In the last part of the thesis, we investigate type-aware relation extraction with neural networks. We compare different models for joint entity and relation classification: pipeline models, jointly trained models and globally normalized models based on structured prediction. First, we show that using entity class prediction scores instead of binary decisions helps relation classification. Second, joint training clearly outperforms pipeline models on a large-scale distantly supervised dataset with fine-grained entity classes. It improves the area under the precision-recall curve from 0.53 to 0.66. Third, we propose a model with a structured prediction output layer, which globally normalizes the score of a triple consisting of the classes of two entities and the relation between them. It improves relation extraction results by 4.4% F1 on a manually labeled benchmark dataset. Our analysis shows that the model learns correct correlations between entity and relation classes. Finally, we are the first to use neural networks for joint entity and relation classification in a slot filling pipeline. The jointly trained model achieves the best micro F1 score with a score of 22% while the neural structured prediction model performs best in terms of macro F1 with a score of 25%