Artificial ribonucleases (ARs) are recognised as a potential strategy to selectively target and cleave biologically significant RNA in cells. However, in order to work as true enzymes they must exhibit catalytic turnover. Many of the reported ARs incorporate metal-containing centres (e.g. dysprosium, copper) in order to induce substantial phosphodiester cleavage, which is not amenable to use in vivo. Therefore, new strategic directions employing metal-independent ARs, such as peptidyl-oligonucleotide conjugates (POCs), need to be investigated. Previous work has shown that poor or non-complementary POCs demonstrate catalytic turnover of a HIV-1 substrate; however, sequence specificity is an issue. For POCs to be useful from a therapeutic standpoint they must only cleave specific RNA molecules and do so in a catalytic fashion, therefore removing the requirement for stoichiometric drug delivery and binding. Consequently, novel POC design strategies are required that allow selective RNA targeting but promote dissociation of the POC following phosphodiester cleavage. In this research, three types of different peptidyl-oligonucleotide conjugate designs have been implemented with the attempt to find an appropriate balance between selectivity and catalytic turnover.(i) Selective targeting and quantitative cleavage (97-100%) of a tRNAPhe target was achieved through phosphoramidate attachment of a 17-mer TΨC-targeting oligonucleotide to catalytic amphiphilic peptide sequences containing leucine, arginine and glycine. Although the half-life of tRNAPhe was less than 1 h on exposure to some of these POCs, hybridisation studies reveal that the POCs bind too tightly to their target RNA sequences and thus an excess of POC is required for efficient cleavage activity. The effect of peptide and oligonucleotide sequence variations as well as the role of enhanced conformational freedom via incorporating an abasic deoxyribose linker between the oligonucleotide targeting motif and catalytic peptide is also investigated. (ii) Most of ‘Dual’ peptidyl-oligonucleotide conjugates containing an amphiphilic RNA-cleaving peptide placed between two RNA recognition motifs directed towards the TΨC loop and 3’ acceptor stem of tRNAPhe demonstrate marked RNA binding and cleavage activities. Interestingly, those dual conjugates which showed poor or negligible binding ability in electrophoresis assays, demonstrated sufficient RNA cleavage (70%) within the vicinity of the 65GACAC61 target region. Therefore, weak POC:RNA complexes may exist which could facilitate substrate turnover. (iii) Finally, POCs were designed which induce bulge-loops in their target RNA region upon hybridisation. By introducing regions of non-complementarity into the oligonucleotide sequences, 2- to 5- membered bulges sizes were formed. Via attachment of a catalytic peptide to an internally modified oligonucleotide residue, catalytic peptides were placed directly adjacent to single-stranded RNA regions to promote cleavage by nuclease mimics. Through probing the hybridised complexes with RNase H, the presence of bulges were confirmed for all bulge-loop sizes, which will be followed by cleavage experiments to assess the possibility for reaction catalytic turnover. In conclusion, a variety of POCs have been synthesised, characterised and partially tested for their RNA cleaving and turnover activity. Based on the encouraging results presented POCs could be further developed to target disease specific RNA sequences such as micro- or messenger RNAs.

Les oligonucléotides sont de courtes séquences d’acide nucléique qui ont la capacité d’inhiber ou de moduler l’expression d’un gène cible par différents mécanismes. Cependant, leur potentiel thérapeutique est limité par leur faible internalisation. Pour pallier ce problème de vectorisation, il a été envisagé de conjuguer les oligonucléotides à des molécules biocompatibles. Ce travail de thèse porte sur des bioconjugués composés d’un nucléolipide à l’extrémité 5’ ou 3’ des oligonucléotides. Tout d’abord, les propriétés physico-chimique et d’hybridation de ces nouveaux composés ont été évaluées. Des études biologiques ont ensuite été réalisées in vitro et in vivo. Les résultats obtenus ont permis de mettre en évidence le mécanisme d’internalisation cellulaire mais aussi de prouver l’efficacité de transfection et d’inhibition de ces conjugués. En outre, le caractère amphiphile de ce type de composé rend possible leur auto-assemblage pour la formulation de substances actives. Dans ce cadre, différentes formulations ont été investiguées. Ainsi, dans ce travail de thèse une nouvelle technologie d’oligonucléotides conjugués a été développée. La séquence de ces molécules peut être modulée et maitrisée de manière à l’adapter à la cible thérapeutique visée. Actuellement, ce système est appliqué sur treize projets différents à l’échelle nationale. L’effet thérapeutique est ainsi évalué dans différentes pathologies telles que des cancers hormono-dépendants, des leucémies, des maladies neurologiques chroniques ou encore la résistance aux antibiotiques. / Oligonucleotides are short nucleic acid molecules able to modulate or inhibit gene expression. However the main drawback of oligonucleotides lies in their poor cellular internalization, which limit their therapeutic applications. Herein, to overcome this limitation, oligonucleotides were conjugated to biocompatible molecules as a nucleolipid to either the 5'- or the 3'-end. First, physico-chemical properties and binding behaviour of this newly compound were investigated. Then in vitro and in vivo biological assays were performed to characterize but also understand the cellular internalization pathways and their biological activities. Finally, the amphiphilic nature of the oligonucleotide-nucleolipid confers spontaneously self-assembling properties for drugs loading and vectorization purposes. This Ph.D. thesis focuses on new oligonucleotide bioconjugates for various biological applications. Sequences of nucleotides can also be modulated to specifically bind to the therapeutic target. This tuneable technology is actually used in 13 different projects, including hormone-dependent cancers, leukemia, chronic neurological disorders and antibiotic resistance, for example.

Bacteria are coated with repeating units of oligosaccharides that exhibit remarkable diversity. Often, glycan units of three or even two sugars are sufficient to identify a species of bacteria. Such specificity makes bacterial surface glycans attractive vaccine targets. However, efforts to create effective vaccines against carbohydrates have been hampered by poor vaccine design as well as the human immune tendency to respond to glycan antigens with non-specific, T-cell independent mechanisms. As a result, carbohydrate vaccines have historically produced only adequate memory responses in healthy individuals and poor responses in the elderly or immunocompromised. To circumvent these issues, a novel conjugate vaccine was developed that utilizes theQβ virus-like particle carrier that displays both a carbohydrate antigen as well as a Natural Killer T cell adjuvant. This unique vaccine has been reported to stimulate the production of high affinity (nanomolar) antibodies against carbohydrate antigens. To further conjugate vaccine research, the present work synthesizes two bacterial surface antigens: a trisaccharide from Streptococcus pneumoniae serotype 23F (Sp23F), and a pentasaccharide from Ruminococcus gnavus (Rg). Sp23F has been characterized as one of the more virulent and disease-causing strains of S. pneumoniae. Rg secretes highly immunostimulatory proteins and is associated with irritable bowel syndrome. The Sp23F antigen is synthesized with an alkyne at the reducing end of the sugar to facilitate coupling to Qβ. A selection reagent for Sp23F is also synthesized to enable the extraction of antibodies and B cells that bind the antigen. In conjunction with providing a conjugate vaccine antigen, the Rg pentasaccharide will be examined as a TLR4 ligand and was therefore synthesized without an alkyne. The Rg conjugate vaccine shows promise in treating irritable bowel syndrome as well as facilitating research into the role Rg plays in the human microbiome.

This diploma thesis deals with the synthesis of conjugates of cyclodextrin with the anticancer drug 5-fluorouracil and folic acid, which works as a targeting group. 5-Fluorouracil is connected to cyclodextrin via an acid-labile linker, which is expected to be cleaved in decreased pH in the proximity of malignant tissue or in the endosome. Malignant tissue also overexpresses receptor for folic acid, and this phenomenon is used for targeted delivery of therapeutic agents. Cyclodextrins are cyclic oligosaccharides, which are known for their ability to complex various compounds into their hydrophobic cavity and increase solubility, stability and bioavailability of these compounds. A synthetic approach for the preparation of conjugate of cyclodextrin with 5-fluorouracil and folic acid was designed and the conjugate was subsequently synthesized. Key words: cyclodextrin, fluorouracil, targeting group, folic acid, drug delivery

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18814号 / 理博第4072号 / 新制||理||1586(附属図書館) / 31765 / 京都大学大学院理学研究科化学専攻 / (主査)教授 丸岡 啓二, 教授 大須賀 篤弘, 教授 時任 宣博 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20947号 / 理博第4399号 / 新制||理||1632(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 丸岡 啓二, 教授 依光 英樹, 教授 大須賀 篤弘 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Thesis advisor: Amir H. Hoveyda / Chapter 1. Catalytic SN2”-Selective and Enantioselective Substitution Reactions. The first broadly applicable strategy for SN2”-selective and enantioselective catalytic allylic substitution will be presented. It will be shown that transformations can be promoted by 5.0 mol% of a sulfonate-containing NHC–Cu complex (NHC = N-heterocyclic carbene), and may be carried out by the use of a commercially available allenyl–B(pin) (pin = pinacolato) or a readily accessible silyl protected propargyl–B(pin). Products bearing a 1,3 diene, a silyl allenyl or a propargyl moiety were obtained in high efficiency and selectivities. Also provided is insight regarding several of the unique mechanistic attributes of the catalytic process, obtained on the basis of kinetic isotope effect measurements and DFT studies. These investigations indicated that cationic π-allyl–Cu complexes are the likely intermediates, clarifying the role of the s-cis and s-trans conformers of the intermediate organocopper species and their impact on E:Z selectivity and enantioselectivity. It will also be shown we were able to highlight the utility of the approach by chemoselective functionalization of various product types, through which the propargyl, allenyl, or 1,3-dienyl sites within the products can be converted catalytically and chemoselectively to several synthetically useful derivatives. Chapter 2. NHC–Copper–Hydride-Catalyzed Enantioselective Processes with Allenyl Boronates and its Application in Natural Product Synthesis. Here, the development of a catalytic process that delivers otherwise difficult-to-access organoboron compound will be detailed. These processes involve the combination of a hydride, an allenyl–B(pin) and an allylic phosphate. As will be discussed, two unique selectivity problems were solved: avoiding rapid Cu–H reduction of an allylic phosphate, while promoting its addition to an allenylboronate as opposed to the commonly observed Cu–B exchange. We were able to underscore the considerable utility of the approach by applications to preparation of the linear fragment of pumiliotoxin B (myotonic, cardiotonic) and the first enantioselective synthesis of netamine C (anti-tumor), which also served to confirm its stereochemical identity. Chapter 3. Catalytic Enantioselective Prenyl Conjugate Addition Reactions. In this final section, studies leading to the development of the first class of catalytic enantioselective strategies for prenyl conjugate additions will be detailed. At the core of these investigations was finding ways to overcome two problems. One challenge originated from the fact that highly activated allylmetal species often deliver product with low enantioselectivity. The other was that regioselectivity was difficult to control owing to a strong preference for γ-selective additions. As will be described, we were able to address these difficulties by the use of a hydroxy NHC-copper complex and 3,3-dimethyl allyl–B(pin) as a reagent. In the end, we were able to use acyclic as well as cyclic enoates as substrates. The results of DFT studies that provide insight regarding varying selectivity profiles with different chiral ligands will be discussed as well. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The progress of high performance computing platforms is dramatic, and most of the simulations carried out on these platforms result in improvements on one level, yet expose shortcomings of current CFD packages. Therefore, hardware-aware design and optimizations are crucial towards exploiting modern computing resources. This thesis proposes optimizations aimed at accelerating numerical simulations, which are illus- trated in OpenFOAM solvers. A hybrid MPI and GPGPU parallel conjugate gradient linear solver has been designed and implemented to solve the sparse linear algebraic kernel that derives from two CFD solver: icoFoam, which is an incompressible flow solver, and laplacianFoam, which solves the Poisson equation, for e.g., thermal dif- fusion. A load-balancing step is applied using heterogeneous decomposition, which decomposes the computations taking into account the performance of each comput- ing device and seeking to minimize communication. In addition, we implemented the recently developed pipeline conjugate gradient as an algorithmic improvement, and parallelized it using MPI, GPGPU, and a hybrid technique. While many questions of ultimately attainable per node performance and multi-node scaling remain, the ex- perimental results show that the hybrid implementation of both solvers significantly outperforms state-of-the-art implementations of a widely used open source package.

A blackbody optical fiber thermometer consists of an optical fiber whose sensing tip is given a metallic coating. The sensing tip of the fiber forms an isothermal cavity, and the emission from this cavity is approximately equal to the emission from a blackbody. Standard two-color optical fiber thermometry involves measuring the spectral intensity at the end of the fiber at two wavelengths. The temperature at the sensing tip of the fiber can then be inferred using Planck's law and the ratio of the spectral intensities. If, however, the length of the optical fiber is exposed to elevated temperatures, erroneous temperature measurements will occur due to emission by the fiber. This thesis presents a method to account for emission by the fiber and accurately infer the temperature at the tip of the optical fiber. Additionally, an estimate of the temperature profile along the fiber may be obtained. A mathematical relation for radiation transfer down the optical fiber is developed. The radiation exiting the fiber and the temperature profile along the fiber are related to the detector signal by a signal measurement equation. Since the temperature profile cannot be solved for directly using the signal measurement equation, two inverse minimization techniques are developed to find the temperature profile. Simulated temperature profile reconstructions show the techniques produce valid and unique results. Tip temperatures are reconstructed to within 1.0%. Experimental results are also presented. Due to the limitations of the detection system and the optical fiber probe, the uncertainty in the signal measurement equation is high. Also, due to the limitations of the laboratory furnace and the optical detector, the measurement uncertainty is also high. This leads to reconstructions that are not always accurate. Even though the temperature profiles are not completely accurate, the tip-temperatures are reconstructed to within 1%—a significant improvement over the standard two-color technique under the same conditions. Improvements are recommended that will lead to decreased measurement and signal measurement equation uncertainty. This decreased uncertainty will lead to the development of a reliable and accurate temperature measurement device.

This study evaluates the ecology of Streptococcus pneumoniae (SP) nasopharynx (NP) colonization in response to the pneumococcal conjugate vaccines, specifically 7-Valent Pneumococcal Conjugate Vaccine (2000-2009), 13-Valent Pneumococcal Conjugate Vaccine (2010-2023) and 20-Valent Pneumococcal Conjugate Vaccine (2023-future date). It is anticipated that the replacement of PCV13 with PCV20, a pneumococcal conjugate vaccine with 7 additional polysaccharide conjugates to CRM197 will enhance the protection against non-vaccine serotypes which are in circulation in communities. The project will evaluate the dynamic changes in pneumococcal colonization over the 5-year time line from 2021-2026. Pneumococcal nasopharynx colonization is detected through nasopharyngeal culture and molecular techniques. The primary source of pneumococcal transmission occurs among the pediatric population and between children and adults. The impact of PCV7 and 13 on pneumococcal colonization over the prior 20 years created a herd effect that resulted in a reduction in pneumococcal disease in unimmunized children and adults. Studies of NP colonization has led to a deeper understanding of pneumococcal conjugate vaccine (PCV) effectiveness and the role of herd immunity in protecting the population, the emergence of replacement serotypes, the variation in invasive capability of each serotype and evolution of antimicrobial resistance resulting from the evolving ecology. In this 5-year-study, researchers at the Pelton Lab in Boston Medical Center set out to understand the prevalence of NP carriage of 13vPnC serotypes, the 7 unique 20vPnC serotypes and NVST (non-vaccine serotypes) within the pediatric population prior to and subsequent to the introduction of PCV 20 (Fall 2023).