The aim of this licentiate thesis is to contribute to understanding of upper secondary teaching and learning of protein synthesis with a focus on domain-specific language. It is based on two studies, designated Studies I and II. Study I addressed upper secondary students’ understanding of protein synthesis through their usage of domain-specific concepts. Data collected through semi-structured group interviews show that students can better reason about core concepts than peripheral concepts, and they compartmentalise the concepts into five clusters. Study II focused on chemistry and biology textbooks’ presentation of protein synthesis through domain-specific concept usage and effects of context on these presentations. The textbooks were analysed using a content analysis approach involving data mining techniques implemented by a computer-generated algorithm. The results reveal that chemistry textbooks focus more on peripheral concepts and generally tend to identify fewer relationships among more concepts than biology textbooks, which emphasise core concepts and tend to highlight more relationships among fewer concepts. Jointly, Studies I and II reveal four facets of teaching and learning protein synthesis: ‘mechanistic or conceptual descriptions’, ‘compartmentalisation’, ‘mRNA as a core concept’ and ‘canonical representation’. By acknowledging the results reported herein, teaching can improve the facilitation and reduce the hindrance in learning protein synthesis through the awareness of the domain-specific language usage. / Proteins are crucial to life: no proteins – no life. Every picosecond, thousands of proteins are constructed in each cell in what is referred to as protein synthesis. Due to its importance for understanding the mechanism for life, protein synthesis is globally regarded as a cornerstone of molecular life sciences and education in the field. Like any area of expertise, the molecular life sciences share a domain-specific language. However, research on teaching and learning protein synthesis through this domain-specific language is scarce. The aim of this licentiate thesis is thereby to contribute to understanding of upper secondary teaching and learning of protein synthesis with a focus on domain-specific language. The thesis is based on two studies: Study I addressed students’ understanding where the results show that students can better reason about core concepts than peripheral concepts, and they compartmentalise the concepts into five clusters. Study II focused on chemistry and biology textbooks’ presentation and effects of context on these presentations. The results reveal that chemistry textbooks focus more on peripheral concepts and generally tend to identify fewer relationships among more concepts than biology textbooks, which emphasise core concepts and tend to highlight more relationships among fewer concepts. Jointly, Studies I and II reveal four facets of teaching and learning protein synthesis. By acknowledging the results reported herein, teaching can improve the facilitation and reduce the hindrance in learning protein synthesis through the awareness of the domain-specific language usage.

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>Magister Scientiae - MSc / Salt stress is a major abiotic stress that accounts for huge agricultural losses worldwide, which in turn threaten food security and sustainable agriculture. Salt triggers the excessive production of reactive oxygen species (ROS) which accumulate to levels that become toxic to plants, resulting in cell death and reduced plant growth. Part of the plant’s mechanisms to counteract ROS-induced cell death involves the scavenging ability of the antioxidant defense system to maintain redox homeostasis. Gallic acid (GA) is an antioxidant that has been shown to reduce salt-induced ROS in legume plants. However, its effects on wheat plants have not been elucidated. This study thus investigated the role of exogenous GA (250 μM) on the physiological responses and antioxidant system of wheat plants under salt stress (150 mM). In addition, this study also investigated how GA and salt stress influenced changes in the membrane proteome of wheat plants using LC-MS proteomic analysis. / 2022

A comparison was made of bacteriophage MS2 RNA translation in infected Escherichia coli cells and in a defined cell-free system. A number of temperature-sensitive mutants were used as hosts for viral RNA translation at permissive and restrictive temperatures. The amount of viral coat protein synthesis was determined after gel electrophoresis of proteins from the cell lysates. These results were compared to those obtained with cell-free translation assays conducted with ribosomes isolated from the same mutants. Compared with control cells, a reduced activity in vivo and in vitro was found for each mutant examined at elevated temperatures. A good correlation between the two types of translational assays was observed. These findings are discussed in terms of the translational defects known to be a characteristic of some of these mutant strains.

La traduction des ARNm est un processus très complexe qui en plus des nombreux facteurs impliqués, nécessite également des étapes de maturation des protéines et ARN pour la production fidèle des protéines. Parmi ces évènements, des modifications post-transcriptionnelles et post-traductionnelles, dont la méthylation est la plus fréquente, sont trouvées dans tous les composants et principalement chez les eucaryotes. Le rôle des méthylations dans la traduction est parfaitement illustré par la protéine Trm112, qui est un activateur essentiel pour la fonction de 4 méthyltransférases (MTase) (Trm9, Trm11, Bud23 et Mtq2) qui modifient des facteurs impliqués dans la synthèse des protéines. Chez la levure, les complexes Trm9-Trm112 et Trm11-Trm112 catalysent la formation de mcm5U34 et m2G10, respectivement sur certains ARNts. Le complexe Bud23-Trm112 modifie l’ARNr 18S pour former la m7G1575 tandis que le complexe Mtq2-Trm112 modifie le facteur de terminaison de classe I eRF1sur la chaine latérale de la glutamine du motif GGQ. Jusqu’à présent, des études structurales et fonctionnelles du réseau d’interaction de la protéine Trm112 se sont uniquement focalisées chez les eucaryotes alors que cette protéine est trouvée dans les 3 domaines du vivant. Dans cette étude, des expériences de co-immunoprécipitations couplées à de la LC-MS/MS ont permis d’étudier le réseau d’interaction de la protéine Trm112 chez l’archée H. volcanii. Celui-ci s’avère être composé de plus de MTase que chez les eucaryotes. Pour la première fois, la structure cristallographique d’un complexe Trm112-MTase d’archée a été déterminée, révélant un mode d’interaction conservé par rapport aux complexes eucaryotes malgré une très faible identité de séquence. De façon très intéressante, un des partenaires de Trm112 chez H. volcanii est orthologue d’une protéine humaine dont nous avons pu démontré qu’elle est une nouveau partenaire de la protéine TRMT112 humaine / Methylation is a widely distributed modification found in a variety of substrates involved in different steps of eukaryotic protein translation. Methylation reactions are catalyzed by enzymes called methyltransferases (MTases) generally using S-adenosyl-L- methionine (SAM or AdoMet) as the methyl donor. The effects of methylation on translation are perfectly illustrated by the Trm112 protein, which is an activating platform, essential for the function of four SAM-dependent MTases (Trm9, Trm11, Bud23 and Mtq2) modifying factors participated in protein synthesis. The Trm9-Trm112 and Trm11-Trm112 complexes methylate some tRNAs to form mcm5U34 and m2G10 respectively. The Bud23-Trm112 complex modifies 18S rRNA to form m7G1715 while the Mtq2-Trm112 complex methylates class I translation termination factor eRF1 at glutamine side chain of GGQ motif. Until now, the study of Trm112 network in eukaryotes has been quite clear structurally and functionally, however, little is known for corresponding proteins in Archaea.My PhD project aims to characterize the Trm112 network in archaea using Haloferax volcanii as a model organism and to decipher the mechanisms of substrate modification by Trm112-MTase complexes. This will help understanding the roles of these enzymes in protein synthesis from an evolutionary point of view.Towards this goal, I have generated several H. volcanii strains (Δtrm112, Δtrm112 Trm112-Flag, …). Co-immunoprecipitation of Trm112-Flag coupled to mass spectrometry allowed me identifying a significant number of methyltransferases (MTases), including putative orthologues of eukaryotic Trm112 partners, as potential interactors. I have next validated these new partners by biochemical approaches (co-purification, enzymatic assays, …) and determined the crystal structure for one Trm112-MTase complex. I have then convincing evidences that H. volcanii Trm12 has more MTase partners than the eukaryotic one. My work opens new routes towards the characterization of the role of Trm112 in archaea but has also led to the identification of a new MTase partner of the eukaryotic Trm112.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20312号 / 薬科博第81号 / 新制||薬科||9(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 大野 浩章, 教授 高須 清誠, 教授 竹本 佳司 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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H1 and core histone mRNA levels have been examined in the presence of portein synthesis inhibitors with different mechanisms of action. Total HeLa cell RNAs were analyzed by Northern Blot hybridization using cloned human histone genes as probes. Inhibition of DNA replication resulted in a rapid decline in histone mRNA levels. However, in the presence of cycloheximide or puromycin, H1 and core mRNAs did not decrease in parallel with DNA synthesis, but were stabilized and accumulated. Inhibition of DNA synthesis with hydroxyurea after the inhibition of protein synthesis did not lead to a decline in histone mRNA levels. These results suggest that synthesis of a protein(s) - perhaps a histone protein(s) - is required for the coordination of DNA synthesis and histone mRNA levels.

Bacteremia initiated by Staphylococcus aureus infections can be a serious medical problem. Although a number of different antibiotics are used to combat staphylococcal infections, resistance has continued to develop. Combination therapy for certain infections has been used to reduce the emergence of resistance when a single agent has become ineffective. We hypothesize that the use of rifampicin and ciprofloxacin in combination with azithromycin, known for its inhibitory effects on the bacterial ribosome, can create potential synergistic effects resulting from indirect effects on ribosomal subunit synthesis. To determine this we measured the effects of single and multiple antibiotics on cell growth rates, cell viability, and synthesis rates for DNA, RNA, and protein. We then measured synthesis rates of ribosomal subunits and the amounts of gyrase and RNAP. Effects of the antibiotic combinations on 70S ribosomes was assayed and the amounts of RNA and degradation was measured. We lastly studied the effects of these antibiotic combinations on mutation frequency in Staphylococcus aureus. Our data have shown support not only for the use of antibiotic combination therapy but have provided strong evidence of an increase in the inhibition of bacterial ribosome assembly in Staphylococcus aureus. The reduction of 50S ribosomal subunit synthesis and 23S ribosomal RNA in cells grown in the presence of azithromycin, already known for it’s inhibitory effects on the 50S subunit synthesis, in combination with rifampicin or in combination with rifampicin and ciprofloxacin was observed. This also resulted in a reduction or elimination in the frequency of resistant cells when grown in the presence of these combinations. These studies have shed light on the mechanism of action involved and synergistic effects occurring in combination antibiotic treatments and how ribosomal subunit assembly is affected. The insights gained through this research provide necessary information needed for the design of more potent antibiotic combinations. This will create a better understanding and new methods for eliminating the spread of harmful pathogens such as Staphylococcus aureus.

Cell-free protein synthesis (CFPS) using crude lysates has developed into a robust platform technology over the last 60 years to express numerous types of recombinant proteins. The open-nature, elimination of reliance on cell viability, and focus of all energy towards production of the protein of interest represent substantial advantages of CFPS over in vivo protein expression methods. CFPS has provided new opportunities across a series of research fields that include metabolic engineering, therapeutic and vaccine development, education, biosensors, and many more. In recent years, optimizations of CFPS have even allowed the platform to reach the industrial level of protein production. Although there have been many advancements toward CFPS development, the democratization of the platform to a wide variety of educational, research, and industrial institutions has lacked due to an absence of resources for new users as well as a limited number of developments toward redesigning the tedious and time-consuming protocols to generate robust cell extract. To address these challenges to CFPS implementation, a comprehensive review spanning numerous cell lines with their respective applications, methodologies, and reaction formats were provided in addition to detailed protocols outlining the process of going from E. coli cells to a completed CFPS reaction. Together, these resources provide the scientific community with easily accessible resources for CFPS implementation. Moreover, the aforementioned protocols were redesigned from a four-day process into one that may be completed in under 24-hour’s time with very little researcher oversight. The resulting workflow maintained the robustness of prior methods but generated 400% more extract compared to traditional methods via a set-it-and-forget-it approach. To date, the works presented herein have garnered tremendous viewership from the CFPS research community with a substantial following among all three of the articles. Moving forward, I anticipate that these works will continue to bring new users into the CFPS field through the ease of access to these resources and through the advance of the simplistic and reproducible new workflow for preparation of robust E. coli cell extract.