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Dissertations / Theses on the topic 'Amanitine'

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Published: 10 March 2023

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1

Le, Daré Brendan. "Xénobiotiques hépatotoxiques : études de métabolisme et mécanismes d’action." Thesis, Rennes 1, 2021. http://www.theses.fr/2021REN1B005.

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La toxicité hépatique des xénobiotiques constitue un sujet extrêmement riche, au vu des multiples mécanismes et acteurs impliqués. Ce travail de toxicologie translationnelle a pour but d’améliorer la compréhension des mécanismes hépatotoxiques de l’éthanol et des amanitines, puissantes toxines de champignons, afin d’apporter de nouveaux éléments permettant d’optimiser les prises en charge thérapeutiques des patients intoxiqués. Dans un premier temps, nous apportons des éléments supplémentaires de compréhension sur les mécanismes de réponse des macrophages à l’éthanol. Ces interactions xénobiotiques – cellules, montrées à travers l’exemple de l’induction des récepteurs P2X7, semblent participer à la sévérité des atteintes hépatiques alcooliques, et témoignent de la plasticité des macrophages en situation pathologique. Ces résultats suggèrent notamment l’intérêt du développement des antagonistes des récepteurs P2X7 dans le traitement des alcoolopathies. Dans un deuxième temps, nous appliquons l’outil de réseau moléculaire, permettant la visualisation de données complexes acquises par LC-MS/MS, à l’étude du métabolisme de xénobiotiques. L’exemple du métabolisme de l’acébutolol dans le cadre d’une intoxication médicamenteuse volontaire d’une part, et l’étude du métabolisme de la quétiapine de manière in vitro d’autre part, ont apporté des preuves consistantes sur l’intérêt du réseau moléculaire dans ce contexte. Dans un troisième et dernier temps, l’application du réseau moléculaire nous a permis d’écarter l’hypothèse d’un métabolisme des amanitines in vivo et in vitro. Par ailleurs, nos résultats montrent que le modèle cellulaire de cellules hépatiques HepaRG différenciés constitue un modèle pertinent dans l’étude des amanitines, et objectivent l’implication de la production des ROS mitochondriaux dans la toxicité de ces substances
Xenobiotic-induced hepatotoxicity is an extremely rich subject, given the multiple mechanisms and actors involved. This translational work aims to improve ethanol and amanitins (powerful fungal toxins) hepatotoxic mechanisms understanding, in order to provide new elements to optimize the therapeutic management of intoxicated patients. In a first step, we provide additional elements of understanding on macrophages response mechanisms to ethanol. These xenobiotic-cell interactions, shown through the P2X7 receptor induction example, seem to contribute in alcoholic liver damage severity, and testify to the macrophages plasticity in pathological situations. These results suggest in particular the interest of P2X7 receptor antagonist’s development in the treatment of alcoholism. In a second step, we are applied molecular networking, which allows the visualization of complex data acquired by LC-MS/MS, to xenobiotic metabolism study. The acebutolol metabolism example, in the context of voluntary drug intoxication on the one hand, and the in vitro quetiapine metabolism study on the other hand, have provided consistent evidence concerning molecular network interest in this context. In a third and final step, the molecular network application allowed us to rule out the hypothesis of an in vivo and in vitro amanitins metabolism. Moreover, our results show that the hepatocyte-like cellular model of differentiated HepaRG is a relevant model in amanitins study, and show the mitochondrial ROS production implication in these substances toxicity
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2

Nakaya, Helder Takashi Imoto. "Identificação e análise de expressão de RNAs intrônicos não codificadores humanos." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-08052007-144816/.

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Neste trabalho, nós mostramos estudos em larga-escala de RNAs não codificadores antisenso que são transcritos em regiões intrônicas de genes humanos. Alguns destes transcritos intrônicos possuem níveis de expressão correlacionados ao grau de diferenciação tumoral de câncer de próstata, apontando para uma relevância biológica destas mensagens em doenças complexas como o câncer. Nós também avaliamos a existência de um mecanismo comum de regulação de transcrição, compartilhado por mRNAs codificadores de proteína e RNAs intrônicos, através de análises de perfis de expressão de uma linhagem tumoral de próstata estimulada por andrógeno. A análise de ESTs e mRNAs depositados em bancos públicos de seqüências revelou mais de 55 mil RNAs Totalmente Intrônicos Não-codificadores (TIN), transcritos dos íntrons de 74% de todos os genes RefSeq únicos. Guiados por esta informação, nós desenhamos uma plataforma de oligonucleotídeos contendo sondas senso e antisenso para cada um de 7.520 transcritos TIN selecionados aleatoriamente, além de sondas para os genes codificadores de proteína correspondentes. Nós identificamos assinaturas intrônicas e exônicas de expressão tecido-específicas em fígado, próstata e rim. Os RNAs TIN antisenso mais altamente expressos eram transcritos de íntrons de genes codificadores de proteína enriquecidos na categoria ?Regulação da transcrição?. A inibição da RNA Polimerase II resultou num aumento de expressão de uma fração dos RNAs intrônicos em células em cultura, sugerindo que outras RNA Polimerases possam estar envolvidas em sua biossíntese. Um subconjunto das assinaturas intrônicas e exônicas localizadas nos mesmos loci genômicos possuíram padrões de expressão correlacionados, sugerindo que RNAs intrônicos regulem a abundância ou o padrão de uso de éxons de mensagens codificadoras de proteína. Nós identificamos diversos padrões de expressão de RNAs intrônicos, indicando que eles possam ter papéis regulatórios. Esta estratégia orientada pelo gene, que combina um microarray intrônico/exônico deve permitir análises comparativas futuras de transcrição intrônica sob várias condições fisiológicas e patológicas, avançando assim em nosso conhecimento sobre as funções biológicas destes RNAs não codificadores.
In this work, we show large-scale studies of antisense noncoding RNAs transcribed from intronic regions of human genes. The correlation of expression levels of some intronic transcripts to the degree of tumor differentiation in prostate cancer points to the biological relevance of these messages in complex diseases such as cancer. We also evaluated the existence of a common mechanism of regulation of transcription shared by protein-coding mRNAs and intronic RNAs by measuring the effect of androgen on the transcriptional profile of a prostate cancer cell line. Survey of mRNA and EST public databases revealed more than 55,000 Totally Intronic Noncoding (TIN) RNAs transcribed from the introns of 74% of all unique RefSeq genes. Guided by this information, we designed an oligoarray platform containing sense and antisense probes for each of 7,520 randomly-selected TIN transcripts plus probes for the corresponding protein-coding genes. We identified exonic and intronic tissue-specific expression signatures for human liver, prostate and kidney. The most highly expressed antisense TIN RNAs were transcribed from introns of proteincoding genes enriched in the \"Regulation of transcription\" class. RNA Polymerase II inhibition resulted in increased expression of a fraction of the intronic RNAs in cell cultures, suggesting that other RNA polymerases may be involved in their biosynthesis. A subset of intronic and protein-coding signatures transcribed from the same genomic loci has correlated expression patterns, suggesting that intronic RNAs regulate the abundance or the pattern of exon usage in protein-coding messages. We have identified diverse intronic RNA expression patterns, indicating that they may have regulatory roles. This gene-oriented approach, using a combined intronic/exonic microarray should permit further comparative analysis of intronic transcription under various physiological and pathological conditions, thus advancing current knowledge about the biological functions of these noncoding RNAs.
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3

Amaral, Paulo de Paiva Rosa. "Estudo da biossíntese e regulação de RNAs não-codificadores intrônicos em células humanas." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-03012007-000256/.

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Recentemente, tem sido demonstrado que a maioria dos RNAs transcritos em células humanas são RNAs não-codificadores de proteínas (ncRNAs) originados de íntrons ou regiões intergênicas. Em trabalhos anteriores realizados por nosso grupo, foram descritos longos ncRNAs transcritos de regiões intrônicas de genes codificadores e cuja expressão foi correlacionada ao grau de diferenciação de tumores de próstata, apontando para a relevância fisiológica desta classe de transcritos. Apesar de sua abundância, as propriedades, funções e regulação da grande maioria dos ncRNAs ainda não foram elucidadas. O objetivo do presente trabalho foi investigar a biossíntese de ncRNAs intrônicos em células humanas, primordialmente a contribuição da RNA Polimerase II (RNAP II), bem como aspectos de sua regulação. Primeiramente, o modelo de regulação da expressão gênica por hormônio andrógeno foi utilizado para avaliação da participação direta de um fator de transcrição de RNAP II, o Receptor de Andrógeno (AR), na modulação da transcrição de ncRNAs intrônicos. Utilizando-se a técnica de imunoprecipitação da cromatina, foi detectada a ligação do AR ao elemento de resposta a andrógeno (ARE) presente em um possível promotor de um transcrito intrônico antisenso (derivado do locus Myo5A), cuja expressão é aumentada em células da linhagem LNCaP tratadas com o hormônio. A ligação ao ARE foi induzida pelo tratamento, sugerindo que o efeito do andrógeno na expressão do ncRNA é mediado pelo AR. Em uma segunda abordagem, o efeito da inibição da transcrição por RNAP II com α-amanitina por 24 h em células LNCaP foi avaliado com o uso de microarranjos de oligonucleotídeos representando transcritos total ou parcialmente intrônicos, além de éxons de genes codificadores. A expressão de menos de 20 % dos transcritos intrônicos foi afetada, fração significativamente menor que a observada para os transcritos exônicos (40 %). Ainda que a maioria dos ncRNAs intrônicos diferencialmente expressos tenha sua abundância diminuída, interessantemente, 13 a 16 % foram aumentados, contrastando com aproximadamente 2 a 3 % de exônicos que aumentaram. Os resultados obtidos neste trabalho indicam que a RNAP II atua na transcrição de ncRNAs intrônicos, mas que uma fração considerável pode ser transcrita por outra RNA Polimerase.
It has been recently shown that the bulk of the transcription in human cells is comprised of non-protein-coding RNAs (or noncoding RNAs - ncRNAs) transcribed from introns and intergenic regions of the genome. Previous work from our group has demonstrated that expression of long intronic ncRNAs can be correlated to the degree of prostate tumor differentiation, underscoring the physiological relevance of these transcripts. However, the properties, functions, and regulation of this huge population of ncRNAs remain largely unknown. The present work aimed to investigate the biosynthesis of intronic ncRNAs and aspects of its regulation in human cells, focusing on the contribution of RNA Polymerase II (RNAP II). Initially, the model of regulation of gene expression by androgen hormone was used in order to evaluate the participation of the RNAP II transcription factor Androgen Receptor (AR) in the transcriptional regulation of intronic ncRNAs. Chromatin immunoprecipitation experiments revealed the binding of the AR in an androgen response element (ARE) present in a putative promoter driving the expression of an antisense intronic transcript in Myo5A locus in LNCaP cells. The interaction occurred in an androgen-inducible fashion, along with the up-regulation of the transcript, suggesting that hormone activation occurred in a direct manner mediated by the AR. In a different approach, the effect of RNAP II inhibition with α-amanitin for 24 h in LNCaP cells was analyzed using an oligoarray representing totally and partially intronic transcripts, as well as exons of proteincoding genes. The expression of less than 20 % of the intronic transcripts was affected by the treatment, contrasting to a significantly higher fraction observed for exonic messages (40 %). Moreover, most differentially expressed intronic transcripts were down-regulated, but strikingly 13 to 16 % were up-regulated in cells with blocked RNAP II, while this fraction for exonic transcripts was about 2 %. The results described here demonstrate that RNAP II in fact plays a role in intronic transcription in human cells, but also highlight that another transcriptional system may account for the biogenesis of a fraction of intronic ncRNAs.
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4

Brückner, Florian. "Molecular basis of translocation, alpha-amanitin inhibition, and CPD damage recognition by RNA polymerase II." Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-86461.

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5

Brückner, Florian. "Molecular basis of translocation, alpha-amanitin inhibition, and CPD damage recognition by RNA polymerase II." kostenfrei, 2008. http://edoc.ub.uni-muenchen.de/8646/.

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6

CHAUD, JEAN-PAUL. "L'intoxication phalloidienne : a propos de quatre observations recensees dans le departement des alpes-maritimes." Nice, 1988. http://www.theses.fr/1988NICE6539.

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7

BENVENUTO, VINCENT. "Intoxication phalloidienne : aspect botanique, toxicologique, clinique, therapeutique et preventif." Saint-Etienne, 1989. http://www.theses.fr/1989STET6215.

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8

Dietrich, David John. "Synthesis and evaluation of probes of RNA polymerase II : adaptation of the bicyclic scaffold of the octapeptide amanitin." Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/27427.

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This thesis covers the synthesis and evaluation of bicyclic octapeptides as intracellular probes of RNAP polymerase II. In Chapter 2, the synthetic methodology used to achieve the rigid bicyclic octapeptide and the introduction of modifications to gain spatio-temporal control and visualization is presented. This includes the synthesis of a nitrorveratryl protected derivative of hydroxyproline, and a diethylaminocoumarin labeled asparagine residue. These amino acids were incorporated into amatoxins through solid-phase peptide synthesis. The synthesis and properties of these amatoxins, including their photolability and fluorescent properties is discussed. Chapter 3 describes the initial evaluation of the amatoxin probes in various eukaryotic cell lines. The cytotoxicity of α-amanitin in a variety of cell lines was investigated, where Chinese hamster ovary cells proved to be most sensitive to the toxin. These cytotoxic effects were observed at ~1 μM, which is much higher than the reported binding constant (~ 3 nM). The cytotoxicity was slow, requiring 60-72 hours to achieve 100% cell death. This diminished activity was attributed to cell uptake. Cell permeabilizing agents digitonin and saponin were applied to improve α-amanitin uptake and toxicity. All synthetic amatoxins were shown to have minimal cytotoxic effects. Confocal microscopy demonstrated no cell uptake of a fluorescent amatoxin unless the cells were treated with detergent, pointing to critical issues of cell permeability. The optimization of amatoxin synthesis is presented in Chapter 4. It is shown that up to 20% water can inhibit epimerization during macrolactamization of amatoxins. A convergent synthetic approach to the bicyclic octapeptide was also developed. This allowed for the incorporation of a variety of amino acids at position three. This new synthetic approach was applied to the synthesis of tryptathionine-containing analogs of the opioid receptor agonist enkephalin. The attempted synthesis of the unnatural amino acid (2S),(3R),(4R)-dihydroxyisoleucine is described in Chapter 5. This was achieved through the use of a diastereoselective [3,3] sigmatropic rearrangement followed by Sharpless asymmetric dihydroxylation (AD). Surprising selectivity was noted during the AD reaction using phthalazine-based ligands, but this selectivity was reversed using pyrimidine-based ligands.
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9

Korsak, Barbara [Verfasser]. "DUPA α-Amanitin Conjugates for Targeted Prostate Cancer Therapy - Optimization and Evaluation of In Vivo Potency / Barbara Korsak." Bielefeld : Universitätsbibliothek Bielefeld, 2021. http://d-nb.info/1237815606/34.

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10

SEITZ, ISABELLE. "Pharmacologie et toxicologie de l'amanite tue-mouche : ses aspects historiques et modernes." Strasbourg 1, 1991. http://www.theses.fr/1991STR15023.

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11

HEURTEBIZE, PASCALE. "Intoxication phalloidienne : a propos de cinq cas traites par ceftazidime et silymarine." Besançon, 1990. http://www.theses.fr/1990BESA3095.

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12

Gallo, Francesca [Verfasser]. "α-Amanitin-based Small Format-Drug Conjugates for Prostate Cancer Therapy: Enabling Anti-Tumor Activity by Tuning Pharmacokinetic Properties / Francesca Gallo." Bielefeld : Universitätsbibliothek Bielefeld, 2021. http://d-nb.info/1237048532/34.

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13

Ranoux-Daniel, Muriel. "A propos de l'intoxication par Amanita muscaria et Amanita pantherina : cas personnels et observations recueillies en Aquitaine." Bordeaux 2, 2000. http://www.theses.fr/2000BOR2M104.

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14

Liu, Xiangyang [Verfasser]. "Structure of mammalian RNA polymerase II elongation complex bound by α-amanitin and study of mammalian transcription termination and 3’ end processing / Xiangyang Liu." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2019. http://d-nb.info/1219301183/34.

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15

Ribordy, François-Xavier, Guy Gaudreau, Annette Ribordy, and Micheline Tremblay. "Amanita Muscaria." Acfas-Sudbury, 2004. https://zone.biblio.laurentian.ca/dspace/handle/10219/60.

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16

Roth, Wilmar. "Entwicklung einer HPLC-Methode für die Bestimmung von Opiaten, synthetischen Opiaten, Alpha-Amanitin und anderen Substanzen im Urin und Blut mittels elektrochemischer Detektion und/oder Fluorometrie /." [S.l. : s.n.], 1993. http://www.gbv.de/dms/bs/toc/132415984.pdf.

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17

Bellard, Jean-Christophe. "Étude épidémiologique des intoxications par les champignons supérieurs auprès du centre antipoisons de Bordeaux entre 1993 et 1997." Bordeaux 2, 1998. http://www.theses.fr/1998BOR2P108.

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18

Namysl, Corinne. "Etude du métabolisme carboné des racines d'Epicea (Picea abies L. Karsten) soumises à une mycorhization par Amanita muscaria : effets de l'ozone et de la sécheresse sur la mycorhization de l'épicéa." Nancy 1, 1992. http://www.theses.fr/1992NAN10011.

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Une méthode pour l'obtention de mycorhizes en conditions axéniques a été utilisée. Plusieurs enzymes du catabolisme glucidique ont été examinées sur le champignon Amanita muscaria, sur les racines d'épicéa et sur l'association. Dans les mycorhizes, le cycle des pentoses phosphates joue un plus grand rôle que dans les racines alors que la voie de la glycolyse a tendance à diminuer. Les pools de nucléotides pyrimidiques et adényliques ont également été mesurés. La présence du partenaire fongique modifie profondément les concentrations trouvées dans les racines améliorant ainsi les transports de métabolites entre les deux partenaires. Les impacts d'un stress hydrique et de l'ozone ont été étudiés sur des racines et des mycorhizes de trois clones d'épicéa. Les capacités des enzymes du métabolisme carboné sont hautement modifiées. Ces changements induisent un déséquilibre entre les parties aériennes et souterraines provoquant sans doute une sénescence précoce de l'arbre.
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19

Mohd, Najib Mohd Idris. "Characterisation of THOC4 response to replicative stress." Thesis, Queensland University of Technology, 2018. https://eprints.qut.edu.au/122562/1/Mohd%20Idris_Mohd%20Najib_Thesis.pdf.

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Human cells are continuously subject to conditions that cause breaks to DNA. This project explored the interaction between molecules involved in repairing this damage, called hSSB1 and THOC4. Novel discoveries were made that expand our understanding of this important process and identified THOC4 as a potential novel therapeutic target in lung and breast cancer.
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20

Perrier, Anne-Lise. "Syndrome phalloïdien : à propos d'une intoxication chez une femme enceinte à treize semaines d'aménorrhée." Montpellier 1, 1998. http://www.theses.fr/1998MON11025.

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21

Cirronis, Marco. "Epatotossicità indotta da xenobiotici: studio su nuovi marcatori diagnostici con particolare focus sui microRNA." Doctoral thesis, 2021. http://hdl.handle.net/2158/1245695.

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22

Brückner, Florian [Verfasser]. "Molecular basis of translocation, α-amanitin [alpha-amanitin] inhibition, and CPD damage recognition by RNA polymerase II / Florian Brückner." 2008. http://d-nb.info/989450376/34.

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23

Musil, Karel. "Vývoj HPLC-DAD-MS/MS metody pro stanovení vybraných toxinů v Muchomůrce zelené." Master's thesis, 2015. http://www.nusl.cz/ntk/nusl-339497.

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(EN) This work has been focused on the development and optimization of analytical methodology, using high performance liquid chromatography with diode array detection (HPLC-DAD) and high performance liquid chromatography with diode array and tandem mass spectrometric detection (HPLC-DAD-MS/MS) for the determination of amatoxins (α- and β-amanitin) and phalotoxins (phallacidin and phalloidin) in the crude extract from cap of Amanita phalloides with the option to later use for clinical purposes. In order to guarantee the reliability of the analytical results the influence of various parameters on the quality of the separation and the determination of toxins was studied. The developed HPLC-DAD method achieves good linearity in the concentrations range 1 - 100 µg/ml, with correlation coefficients higher than 0,997. Limits of detection (LOD) and quantitation (LOQ) were calculated for all the studied toxins with following values: for α-amanitin 0,90 µg/ml (LOD); 2,99 µg/ml (LOQ), β-amanitin 1,07 µg/ml (LOD); 3,56 µg/ml (LOQ), phallacidin 2,17 µg/ml (LOD); 7,26 µg/ml (LOQ) and phalloidin 0,79 µg/ml (LOD); 2,64 µg/ml (LOQ). Key words: Amanita phalloides,-amanitin,-amanitin, phallacidin, phalloidin, solid-liquid extraction, HPLC-DAD, HPLC-DAD-MS/MS .
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24

Leite, Marta Sofia Carvalho Ferreira Malheiro. "Desenvolvimento e optimização de uma metodologia analítica para a determinação de a-e B-amanitina em urina humana por LC-MS/MS." Master's thesis, 2011. http://hdl.handle.net/10316/17606.

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A colheita de cogumelos silvestres é uma prática ainda muito presente nos dias de hoje, por todo o mundo. Apesar do consumo da maioria dos cogumelos silvestres existentes ser inofensiva para o ser humano, existem algumas espécies que possuem na sua constituição substâncias tóxicas, cuja penetração no organismo, via tracto digestivo, causa o desenvolvimento de efeitos nocivos em diferentes tecidos. O envenenamento por cogumelos Amanita é uma rara mas séria causa de intoxicação humana fatal. As amatoxinas, um grupo de octapeptídeos biciclícos produzidas pela espécie Amanita phalloides, são responsáveis pela elevada toxicidade destes fungos, sendo a α-amanitina e a β-amanitina as toxinas letais maioritárias. Estas causam necrose celular, especialmente no fígado e nos rins, conduzindo à morte por falência hepática e insuficiência renal aguda. O presente trabalho teve como objectivo o desenvolvimento, optimização e aplicação de uma metodologia analítica por cromatografia líquida acoplada a espectrometria de massa sequencial (LC-MS/MS), após um pré-tratamento para precipitação de proteínas por solvente orgânico e procedimento de extracção em fase sólida (SPE), para a determinação das amatoxinas α- e β-amanitina, em amostras de urina humana. Os parâmetros de validação definidos para este método englobaram linearidade, limites de detecção (LD) e de quantificação (LQ), selectividade, sensibilidade, repetibilidade e recuperação da extracção, de forma a garantir fiabilidade nos resultados analíticos obtidos. O método desenvolvido provou ser específico e selectivo, com valores de LD e LQ para a α-amanitina de 1.93 e 3.29 ng/mL e para a β-amanitina de 1.74 e 3.16 ng/mL, respectivamente. A lineariedade estudada em amostras fortificadas foi satisfatória (0.97 para a α-amanitina e 0.98 para a β-amanitina) num intervalo de 5 a 20 ng/mL. A repetibilidade foi verificada ao nível de 10 ng/mL (n=3) de α- amanitina e β-amanitina em amostras de urina fortificadas, com coeficientes de variação obtidos de 3.68 e 2.33%, respectivamente. A recuperação do método extractivo desenvolvido apresentou bons resultados para as baixas concentrações RESUMO xii analisadas, com percentagens entre os 64 e 70% para a α-amanitina, e entre os 64 e 68% para a β-amanitina. A metodologia descrita foi, posteriormente, aplicada a uma amostra real de urina de cadáver, disponibilizada pelo Serviço de Toxicologia Forense da Delegação do Centro do Instituto Nacional de Medicina Legal (INML, I.P.). A análise da amostra forense em questão visou demonstrar a capacidade da metodologia desenvolvida para aplicação e implementação em casos reais de intoxicação por amatoxinas. A metodologia analítica desenvolvida tanto a nível de extracção, como de sistema cromatográfico, é inovadora, apresentando um elevado potencial na identificação e detecção de α- e β-amanitinas em amostras de urina. O método descrito poderá ser aplicável à determinação de α- e β-amanitinas a outras amostras biológicas, como fígado e rins. Palavras-chave: α-Amanitina; β-Amanitina; Intoxicação; SPE; LC-MS/MS
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25

Liu, Xiangyang. "Structure of mammalian RNA polymerase II elongation complex bound by α-amanitin and study of mammalian transcription termination and 3’ end processing." Doctoral thesis, 2019. http://hdl.handle.net/21.11130/00-1735-0000-0005-12C1-C.

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