Tuberculosis (TB) remains the leading cause of death due to a single infectious agent with more than 1.5 million people killed each year. In 2018, the World Health Organization (WHO) estimated that one third of the world’s population was infected with Mycobacterium tuberculosis (Mtb), the pathogen responsible for the disease.In 2000, EthR, a mycobacterial transcriptional repressor, was identified as a key modulator of ethionamide (ETH) bioactivation. ETH is one of the main second-line drugs used to treat drug-resistant strains and it is a prodrug that is activated in Mtb by the mono-oxygenase EthA and then inhibits InhA, an enzyme involved in the mycolic acid biosynthesis. In 2009, it was demonstrated that co-administration of ETH with the drug-like inhibitors of EthR was able to boost ETH activity by a factor three in a mouse-model of TB-infection, thus validating EthR protein as a target for a new therapeutic strategy. The first part of this thesis deals with the validation and deep characterization of the solved EthR-ligand structures based on all analysis of how each ligand bind to the EthR. In this section, based on the study of both co-crystal structures and the physicochemical properties of the ligands, we have rationalized the information currently available and understood the interaction of all EthR inhibitors in order to lead to more effective inhibitor design.More recently, another mycobaterial repressor, denoted EthR2, was identified as a putative target that appears to be functionally comparable to EthR (then the locus has been termed EthA2/EthR2, due to its similarity to the EthA/EthR locus). Furthermore, a spiroisoxazoline family of small-molecules, generically denoted as SMARt, has been identified as effective ligand of EthR2. However, according to the data present in the literature, this spiroisoxazoline family can also bind to the former EthR. In order to investigate this proposition, I have solved these small molecules in complex with EthR and compared their binding interactions to the EthR2 protein as well. The opportunity for the design small-molecules is capable of targeting both repressors, thereby opening the way to a dual-target approach.Finally, the third part of this thesis is devoted to the mycobacterial transcriptional factor MabR (Rv2242). Several studies identified this protein as a regulatory transcription factor of the fatty acid synthase II operon, which is mainly responsible for the mycolic acid biosynthesis in Mtb. I therefore purified to homogeneity and characterized the MabR protein as well as I determined the crystal structure of its C-terminal part. Finally, the functional role of MabR is largely discussed, and the way on how to interfere with its DNA binding ability is commented with respect to our results. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / Accurate DNA replication is vital for maintaining genomic stability. Consequently, the machinery required to drive this process is designed to ensure the meticulous maintenance of information. However, random misincorporation of errors reduce the fidelity of the DNA and lead to pre-mature aging and age-related disorders such as cancer and neurodegenerative diseases. Some of the incorporated errors are the result of the error prone DNA polymerase alpha (Pol α), which initiates synthesis on both the leading and lagging strand. Lagging strand synthesis acquires an increased number of polymerase α tracks because of the number of Okazaki fragments synthesized per round of the cell cycle (~50 million in mammalian cells). The accumulation of these errors invariably reduces the fidelity of the genome. Previous work has shown that these pol α tracks can be removed by two redundant pathways referred to as the short and long flap pathway. The long flap pathway utilizes a complex network of proteins to remove more of the misincorporated nucleotides than the short flap pathway which mediates the removal of shorter flaps. Lysine acetylation has been reported to modulate the function of the nucleases implicated in flap processing. The cleavage activity of the long flap pathway nuclease, Dna2, is stimulated by lysine acetylation while conversely lysine acetylation of the short flap pathway nuclease, FEN1, inhibits its activity. The major protein players implicated during Okazaki fragment processing (OFP) are known, however, the choice of the processing pathway and its regulation by lysine acetylation of its main players is yet unknown. This dissertation identifies three main findings: 1) Saccharomyces cerevisiae helicase, petite integration frequency (Pif1) is lysine acetylated by Esa1 and deacetylated by Rpd3 regulating its viability and biochemical properties including helicase, binding and ATPase activity ii) the single stranded DNA binding protein, human replication protein A (RPA) is modified by p300 and this modification stimulates its primary binding function and iii) lysine acetylated human RPA directs OFP towards the long flap pathway even for a subset of short flaps.

京都大学 / 0048 / 新制・課程博士 / 博士(情報学) / 甲第19107号 / 情博第553号 / 新制||情||98(附属図書館) / 32058 / 京都大学大学院情報学研究科知能情報学専攻 / (主査)教授 黒橋 禎夫, 教授 石田 亨, 教授 河原 達也 / 学位規則第4条第1項該当 / Doctor of Informatics / Kyoto University / DFAM

This work investigates the feasibility to perform an experiment at CERN n_TOF to study the fission cross section and fission fragment angular distribution (FFAD) of the 230Th(n,f) reaction. An analysis of fission fragment energy losses in the experimental target resulted in a choice of target thickness of 0.1 µm (100 µg/cm2 ), which yields good transmission out of the target at up to 45° emission angles from the target normal. A detection setup using ten PPAC detectors with nine thorium targets interleaved in between them was investigated, where the detectors and targets were tilted 45° with respect to the neutron beam. This makes it possible to measure all emission angles needed with respect to the neutron beam in order to determine the FFAD. For the experimental area EAR2 at n_TOF, a prediction of the count rate in the experiment resulted in low statistical uncertainties after a few weeks of beam time, which indicates that an experiment like this is feasible. / Detta projekt undersöker genomförbarheten av ett experiment vid CERN n_TOF för att mäta tvärsnittet och fördelningen av emissionsvinklar av fissionsfragment (FFAD) från 230Th(n,f)-reaktionen. En analys av energiförlusterna av fissionsfragment inuti torium-provet resulterade i en optimal provtjocklek på 0.1 µm (100 µg/cm2 ), vilket medför att fissionsfragment som emitteras i vinklar upp till 45° från provets normal har hög sannolikhet att transmitteras ut ur provet. En detektionsuppställning med tio PPAC-detektorer med nio toriumprov mellan dem undersöktes, där detektorerna och proven antogs vara snedställda med 45° från neutronstrålens riktning. Detta möjliggör detektion av fissionsfragment i alla vinklar som är nödvändiga för att kunna mäta hela FFAD. För experimentanläggningen EAR2 vid n_TOF, resulterade en uppskattning av antalet detekterade fissionsevent per sekund i låga mätosäkerheter efter ett par veckor av mättid, vilket antyder att experimentet är görbart.

As part of this Special Section on critical rhetoric, this article examines the role of collaboration in the future of critical rhetoric. Building on McKerrow’s original eight principles of praxis, the authors advocate for a missing ninth principle that reflects the need for critical rhetoric to be a shared venture across both individual projects and larger discourses. As an example of this type of work, they provide ProfsDoPop.com, an academic, online blog designed to bring academic sensibilities and concepts to popular audiences through the critique of popular culture.

In severe cases of ulcerative colitis (UC) unresponsive to current treatment options, patients require a complete proctocolectomy, or surgical removal of the colon. Ileal pouch anal anastomosis (IPAA) has become the preferred surgical technique for patients who require surgery, as this method restores rectal function. This procedure is also used to treat colorectal cancers such as adenocarcinoma and familial adenomatous polyposis (FAP). The surgery involves an abdominal colectomy with the construction of an ileal pouch created from folded tissue recovered from the ileal portion of the small intestine. Up to 50% of patients who require IPAA surgery experience an episode of pouchitis, a non-specific inflammation of the constructed ileal pouch with unknown etiology. Several hypotheses have been proposed regarding the pathogenesis of pouchitis. Current theories include bacterial overgrowth due to fecal stasis, microbial imbalance (dysbiosis), immune alteration, genetic susceptibility, metaplasia, ischemic complications of surgery, a recurrence of UC, or even a novel form of inflammatory bowel disease. The efficacy of antibiotics and probiotics in treating pouchitis and maintaining remission underscores the importance of gut microbiota in the development of this condition. In the study, we aimed to characterize the intestinal bacterial communities that inhabit IPAA pouches of both UC and FAP patients, in an effort to investigate the hypothesis that bacterial dysbiosis is involved in the pathogenesis of pouchitis. Mucosal biopsy and stool samples were analyzed from patients with UC and pouchitis (UCP), healthy UC controls (HUC) and healthy pouches with a background of FAP (FAP). Samples were examined through analysis of terminal restriction fragment length polymorphisms (TRF) and DNA sequencing. The data presented here demonstrate that a microbial imbalance exists in pouchitis, as bacterial communities in pouchitis differ significantly from healthy UC pouches and pouches constructed for FAP. Both methods identified potential groups of organisms that may play a role in the development of pouchitis, including decreases in protective Lactobacillus and Bacteroides and increases in mucin-degrading Clostridium and Akkermansia. A better understanding of the factors driving the pathogenesis of pouchitis will not only benefit patients with this disease, but also lead to a better understanding of the complex relationship that exists between the human host and the diverse community of organisms that inhabit the gastrointestinal tract.

Biological motion constitutes a key and indispensable element of all biomolecules, as dynamics tightly link spatial architecture with function. Several computational and experimental techniques have been developed to study biomolecular dynamics. Nevertheless, few label-free and atomic or sub-atomic resolution techniques are able to capture biological motions at close to native conditions. Indeed, the only label-free technique giving atomic level access to dynamics from picoseconds down to seconds is nuclear magnetic resonance (NMR) spectroscopy. In this dissertation, I identify the imperfections and inaccuracies accompanying the routine and well-accepted methods of probing protein dynamics via 15N spin relaxation NMR measurements. Subsequently, I propose and develop solutions and experimental approaches to overcome the limitations and eliminate artefacts. The routine procedures applying heavy water as an internal locking standard lead to artifacts in every type of relaxation rate of 15N amides due to reaction with exchangeable deuterons. The deviations from correct values are most pronounced for highly dynamic and exposed protein fragments. I introduce a novel set of directly detected 15N spin relaxation experiments yielding an unprecedent resolution resolving the signal overlap, although of lower sensitivity. I propose a more accurate. Finally, I present how the 15N spin relaxation techniques and improved routines can be applied to understand biological processes that cannot be described without monitoring molecular motions. Using the example of human BTB domains, which are directly linked to human cancer, I demonstrate the ability to detect cryptic binding sites on the surfaces of proteins. The cryptic binding site was verified by a comprehensive NMR-monitored fragment-based screening that revealed a hit-rate only for MIZ1BTB, which was the only protein displaying slow segmental motions. I also managed to track subtle and biologically-relevant dynamic modulations of an exposed H3 histone tail affected by H1 histones or other histone variants. Enhancement of H3 tail dynamics led to increased H3K36 methylation, while restriction of motions resulted in the opposite effect. These observed correlations unequivocally support the essential role of molecular mobility in biological functions.

No abstract provided. / Thesis / Master of Science (MSc) / Scope and contents: The probability of fission as a function of primary fragment velocities has been obtained by removing the neutron emission and instrumental dispersions from the velocities determined by Stein with time-of-flight techniques for the thermal neutron fission of u235. Each velocity was increased by 0.69% to make the average kinetic energy per fission agree with the calorimetric value of 167.1 Mev. Excitation energy distributions were obtained by using the primary fragment masses given by Cameron and assuming that the most probable charge distribution for a given mass ratio i s that which leads to the greatest energy release. Evaporation theory was used to determine the number of prompt neutrons emitted. When the excitation energy is divided equally between the fragments and a nuclear temperature of 0.59 Mev is used, the average number of neutrons emitted is 2.95 per fission.

Selective molecular recognition between proteins is a fundamental event in biological processes that governs cellular growth, function, survival, and differentiation. The immune system, for example, is a complex network of cellular processes regulated by protein-protein interactions (PPIs) between cells, receptors, and secreted molecules. Generating and maintaining an appropriate immune response and regulation requires coordination across many cell types and components, while dysregulation of these interactions can lead to disease. A major obstacle in small molecule therapy development towards these PPIs is their restriction to small protein-protein interfaces and a well-defined hydrophobic pocket. Most PPIs have large contact surface areas and lack traditional binding pockets making them historically challenging for the development of potent small-molecule modulators. To address this limitation, we utilized two binding-based approaches, a unique peptidomimetic fragment library and high-throughput small-molecule microarrays to design and discover molecules that target three important immunoregulatory PPIs: the DQ8-insulin complex, the KEAP1-Nrf2 complex, and the IL-4/IL-4R receptor complex. Many autoimmune diseases involve the ternary PPI complex between immunogenic peptides presented to T cell receptors through the major histocompatibility complex (MHC). Inhibiting this interaction may provide a therapeutic approach for delaying or preventing disease. To target type 1 diabetes, we developed a unique library consisting of 125 fragment-sized molecules that mimic glutamic acid and tyrosine residues from the immunogenic insulin B:9-23 peptide responsible for CD4+ T cell activation. Screening of our library after generation of the MHC class II protein responsible for insulin B:9-23 presentation, DQ8, has resulted in identification of 15 lead fragment compounds to date. Application of our fragment library towards pharmaceutically validated target for inflammation and neurogenerative diseases, the Kelch like ECH-associated protein 1 (KEAP1) and nuclear factor erythroid 2 like 2 (Nrf2), resulted in a 30% hit rate. These are promising results for the further development of selective compounds to inhibit these interactions. For treating inflammatory diseases, such as asthma or cancer, we report the identification of a first-in-class small molecule inhibitor to the cytokine Interleukin-4 (IL-4). The PPI between IL-4 and its receptor complex (IL-4Rα) contains no conventional binding pockets and binding is driven through clusters of complementary residues. Through the combination of small-molecule microarrays and cell-based assays we identified the lead compound, Nico-52, with micromolar inhibitory potency and micromolar affinity. A library of 60 analogs of Nico-52 was synthesized and preliminary structure activity relationships suggest amenability of the p-fluorophenyl substituent and importance of the diol substituent to retain binding potency. These studies resulted in development of a more potent inhibitor to IL-4 with a p-aniline substituent, which could be developed into a targeting ligand to deliver additional therapeutic payloads to an IL-4 enriched microenvironment. In summary, we have developed a peptidomimetic small molecule fragment library as a toolkit for screening against challenging PPI targets with applications towards type 1 diabetes and developed a first-in-class small molecule inhibitor towards IL-4 with applications towards inflammatory diseases. / 2025-09-21T00:00:00Z

The aim of this thesis is to explore the concept of spolia and its analytical potential. The historical development of the concept from Roman antiquity to the present is examined, focusing on specific turning points, and two artefacts – a set of columns and a vase from the National Museum in Stockholm – are analysed. The different sections of the thesis are intertwined through methodological thematizations of power, violence and temporality. By activating older meanings of spolia, as well as introducing new ones, the concept is expanded through the study. It becomes a critical tool useful in understanding composite objects, which are analysed in terms of form, function and migratory paths. The study revolves around a contemporary museum setting but moves between several contexts and time periods. The expanded, critical concept that this thesis develops can therefore be used in other studies.