B cells play crucial roles in the development of the two human autoimmune diseases, type 1 diabetes (T1D) and systemic lupus erythematosus (SLE). In the past decade, numerous studies showed positive responses of B cell depletion therapies in these two diseases. However, the beneficial effects are temporary and accompanied with adverse events. In this dissertation, we aimed to identify novel targets for a better modulation of disease development using mouse models. These diseases have circulating autoantibodies that are mostly mutated with an IgG isotype, indicating B cells that are producing them have been through the process of affinity maturation. Activation-induced cytidine deaminase (AID) is a core enzyme that regulates somatic hypermutation (SHM) and class switch recombination (CSR), the two key mechanisms in affinity maturation. We showed that genetic ablation of AID significantly inhibited the development of TID in NOD mice. Homologous recombination (HR) pathway is important for the repair of AID-induced DNA double strand breaks during CSR. 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid, also known as DIDS, is a small molecule that inhibits HR pathway and subsequently leads to apoptosis of class switching cells. DIDS treatment remarkably retarded the progression of TID, even when started at a relatively late stage, indicating the potential of this treatment for disease reversal. In both approaches, we observed a notable expansion of CD73+ B cells, which exerted an immunosuppressive role and could be responsible for T1D resistance. Next we examined the effect of targeting affinity maturation through these two approaches in lupus-prone mice. The genetic abrogation of AID in BXSB mice significantly ameliorated lupus nephritis and prolonged their lifespan. AID-deficient mice also exhibited improvement on disease hallmarks with increased marginal zone B cells and more normal splenic architecture. DIDS treatment notably reduced class switching when B cells were stimulated in vitro. However, the administration of DIDS did not strikingly alter the course of SLE in either BXSB mice or MRL/lpr mice. These findings demonstrated that affinity maturation could be a potential target for T1D and SLE, while further explorations into targeting other components in the repair pathway are warranted for SLE. Lastly, we assessed the effect of maternal AID modulation on the SLE development in the offspring using BXSB mouse model. Interestingly, the absence of maternal AID resulted in offspring that developed significantly more severe lupus nephritis compared to control. The offspring born to AID-deficient dams also exhibited elevated levels of pathogenic autoantibodies and exacerbated disease features. Therefore, the modulation of maternal AID could influence the SLE development in the offspring, and future investigations are needed to determine the underlying mechanisms responsible for the disease acceleration. / Doctor of Philosophy / The failure of the immune system to differentiate self from non-self leads to the development of autoimmune diseases. Type 1 diabetes (T1D) and systemic lupus erythematosus (SLE) are complex autoimmune diseases affecting millions of people in the world. Despite intensive research regarding these two diseases, no known cure is available indicating an imperative need for the development of novel therapies. With the importance of B cells in the pathogenesis of these two diseases, intensive research focused on whole B cell depletion therapies. However, these therapies exhibited high risks of infections as a result of depleting all the B cells. In this dissertation, we sought to selectively target specific B lymphocyte subsets that are crucial contributing factors in the development of T1D and SLE. While the effect of therapeutic treatment varied among different mouse models, the genetic manipulation of specific B cells successfully retarded the progression of both T1D and SLE and extended the lifespan of the mice. Further studies shed light on the possible mechanisms that are responsible for the disease inhibition. These data proved that targeting specific B cell compartment could be a potential disease management in T1D and SLE patients. In addition, using the established mouse model, we demonstrated the modulation of maternal factors significantly impact the SLE development in the offspring. Future experiments to identify the underlying mechanisms could provide more targets for the therapeutic development.

Ground state destabilization is a hypothesis to explain enzyme catalysis. The most popular interpretation of it is the entropic effect, which states that enzymes accelerate biochemical reactions by bringing the reactants to a favorable position and orientation and the entropy cost of this is compensated by enthalpy of binding. Once the enzyme-substrate complex is formed, the reaction could proceed with negligible entropy cost. Deamination of cytidine catalyzed by E.coli cytidine deaminase appears to agree with this hypothesis. In this reaction, the chemical transformation occurs with a negligible entropy cost and the initial binding occurs with a large entropy penalty that is comparable to the entropic cost of the uncatalyzed reaction. Our calculations revealed that this reaction occurs with different mechanisms in the cytidine deaminase and water. The uncatalyzed reaction involves a concerted mechanism and the entropy cost of this reaction appears to be dominated by the reacting fragments and first solvation shell. The catalyzed reaction occurs via a stepwise mechanism in which a hydroxide ion acts as the nucleophile. In the active site, the entropy cost of hydroxide ion formation is eliminated due to pre-organization of the active site. Hence, the entropic effect in this reaction is due to a pre-organized active site rather than ground state destabilization. In the second part of this thesis, we investigated peptide bond formation and peptidyl-tRNA hydrolysis at the peptidyl transferase center of the ribosome. Peptidyl-tRNA hydrolysis occurs by nucleophilic attack of a water molecule on the ester carbon of peptidyl-tRNA. Our calculations showed that this reaction proceeds via a base catalyzed mechanism where the A76 O2’ is the general base and activates the nucleophilic water. Peptide bond formation occurs by nucleophilic attack of the α-amino group of aminoacyl-tRNA on the ester carbon of peptidyl-tRNA. For this reaction we investigated two mechanisms: i) the previously proposed proton shuttle mechanism which involves a zwitterionic tetrahedral intermediate, and ii) a general base mechanism that proceeds via a negatively charged tetrahedral intermediate. Although both mechanisms resulted in reasonable activation energies, only the proton shuttle mechanism found to be consistent with the pH dependence of peptide bond formation.

Made available in DSpace on 2015-04-14T13:35:46Z (GMT). No. of bitstreams: 1 450558.pdf: 2693787 bytes, checksum: e4ffe9234a98b2ddfc9bc350c708c01c (MD5) Previous issue date: 2013-06-28 / Tuberculosis (TB), one of the oldest recorded human afflictions, is still one of the biggest killers among the infectious diseases. The HIV co-infection and the emergence of multidrug resistant TB have provided a very alarming challenge to global health and led us to focus on the research for new and more effective therapeutics against the disease. The modern approach to the development of new chemical compounds against complex diseases, especially the neglected endemic ones, such as TB, is based on the use of defined molecular targets. Enzymes from the pyrimidine biosynthesis pathway have been considered potential targets for identification or development of novel anti-mycobacterial agents since in bacteria, pyrimidine nucleotide interconvertion pathways are important in a number of essential processes, including DNA, RNA, and phospholipid biosynthesis. Cytidine 5 -monophosphate kinase from Mycobacterium tuberculosis (MtCMK) catalyzes the ATP-dependent phosphoryl group transfer preferentially to CMP and dCMP. Here, initial velocity studies and Isothermal Titration Calorimetry (ITC) measurements showed that MtCMK follows a random-order kinetic mechanism of substrate binding, and an ordered mechanism for product release. The thermodynamic signatures of CMP and CDP binding to MtCMK showed favorable enthalpy and unfavorable entropy, and ATP binding was characterized by favorable changes in enthalpy and entropy. The contribution of linked protonation events to the energetics of MtCMK:phosphoryl group acceptor binary complex formation suggested a net gain of protons. Values for the pKa of a likely chemical group involved in proton exchange and for the intrinsic binding enthalpy were calculated. The Asp187 side chain of MtCMK is suggested as the likely candidate for the protonation event. Data on thermodynamics of binary complex formation were collected to evaluate the contribution of 2 -OH group to intermolecular interactions. The data are discussed in light of functional and structural comparisons among CMP/dCMP kinases and UMP/CMP ones. / A tuberculose (TB), uma das doen?as mais antigas da humanidade, ainda ? uma das principais causas de morte entre as doen?as infecciosas. A coinfec??o com o HIV e a emerg?ncia de TB resistente a m?ltiplas drogas representam um desafio ? sa?de p?blica e tem estimulado a pesquisa por novos e mais efetivos agentes terap?uticos contra a doen?a. Novas abordagens para o desenvolvimento de compostos contra doen?as complexas, especialmente doen?as end?micas negligenciadas, s?o baseadas no uso de alvos moleculares definidos. Enzimas envolvidas no metabolismo de pirimidinas tornam-se alvos moleculares interessantes para compostos inibidores, uma vez que em bact?rias, as rotas de interconvers?o de nucleot?deos pirimid?nicos s?o importantes em in?meros processos essenciais, incluindo a bioss?ntese de DNA, RNA e fosfolip?dios. A citidina 5 -monofosfato quinase de Mycobacterium tuberculosis (MtCMK) em estudo, catalisa a transfer?ncia revers?vel de um grupamento fosforil a partir de ATP, preferencialmente para CMP e dCMP. Neste trabalho, os estudos de velocidade inicial e experimentos de Calorimetria de Titula??o Isot?rmica (ITC) demonstraram que a adi??o dos substratos (CMP e ATP) ? MtCMK segue um mecanismo cin?tico sequencial aleat?rio, e que a libera??o dos produtos ocorre de forma ordenada, onde o ADP ? o primeiro produto a ser liberado. As assinaturas termodin?micas da liga??o do CMP e do CDP ? MtCMK mostraram varia??es favor?veis da entalpia e desfavor?veis da entropia, e, a liga??o do ATP foi caracterizada por mudan?as favor?veis da entalpia e da entropia. As contribui??es energ?ticas oriundas dos eventos de protona??o, associados ? forma??o do complexo bin?rio MtCMK:receptor do grupamento fosforil, sugerem um ganho l?quido de pr?tons. Al?m disso, foram calculados os valores de pKa de um prov?vel grupo envolvido na troca de pr?tons, e da entalpia de liga??o intr?nseca. A cadeia lateral do Asp187 da MtCMK ? sugerido como prov?vel candidato para o evento de protona??o. As medidas termodin?micas da forma??o do complexo bin?rio foram coletados a fim de avaliar a contribui??o do grupo 2 -OH da pentose nas intera??es intermoleculares. Os dados obtidos foram discutidos comparando-se as caracter?sticas estrutural e funcional entre as CMKs j? estudadas e a UMP/CMP quinase humana.

Although spinal cord injury (SCI) is a debilitating condition that presents a large socioeconomic problem in the United States, there is currently no treatment that reliably reduces morbidity and mortality. Current research is aimed at identifying mechanisms involved in the pathophysiology of SCI and using this knowledge to develop rational treatments. We have observed plasma membrane compromise in the acute (within 10 minutes), sub-acute (3 days), and chronic phases (5 weeks) in a rat model of contusion SCI and postulate that it negatively affects neurological outcome. Holes/tears in the plasma membrane were assessed with a dye exclusion assay, in which a fluorescent cell-impermeant dye was injected into the cerebrospinal fluid prior to sacrifice; therefore, cellular uptake of the dye is indicative of plasma membrane compromise. As early as 10 minutes after SCI, widespread uptake of permeability markers was evident in neuronal cell bodies as well as axonal projections. The number of permeable cells and the size of the membrane breaches (measured by using permeability markers of various sizes) varied with distance from the injury site, with larger disruptions located closer to the epicenter. Greater cellular uptake was observed when the impact force was increased (200 > 150 > 100 kdyn > sham). At longer time points (3 days and 5 weeks), substantial permeability marker uptake was observed in axons but not in cell bodies. Cells with increased permeability displayed a variety of pathomorphological alterations, including swelling, blebbing, retraction bulb formation, neurofilament loss, and fragmentation, suggesting that increased plasma membrane permeability is detrimental to cell survival and function. We therefore investigated a clinically-relevant treatment strategy designed to restore plasma membrane integrity. Animals were treated with citicoline, a molecule utilized in the endogenous synthesis of phosphatidylcholine (the major membrane component in mammalian cells). Citicoline has been shown to be beneficial in numerous studies of neurological disease, improving overall outcome by increasing phospholipid synthesis and attenuating phospholipid destruction (by reducing phospholipase A2 activity). However, these mechanisms have not been explored in a model of SCI. When compared to injured animals receiving vehicle (saline) injections, citicoline treatment after SCI did not have a statistically significant effect on cytoplasmic PLA2 activity (at 24h post-injury), the density of permeable axons (at 3 days post-injury), or the lesion volume (at 3 days post-injury). Since citicoline may improve neurological outcome after SCI through mechanisms we did not directly assess, we then conducted a longer-term study to evaluate the overall efficacy of citicoline treatment in terms of longer-term functional and histological consequences. Citicoline did not have a biologically significant effect on behavioral recovery (evaluated during open field locomotion, grid walk and hyperalgesia testing weekly for up to 5 weeks post-injury) or lesion volume (at 5 weeks post-injury). The lack of citicoline-mediated effect may be attributed to experimental parameters (e.g., dosing or sensitivity of outcome measures) or biological inefficacy. Although we were not able to demonstrate that citicoline improves outcome after SCI, the finding that plasma membrane damage occurs in a persistent fashion and is associated with pathophysiological cellular alterations may provide fundamental knowledge necessary for developing treatments targeted at membrane repair. Future work examining the complex mechanisms causing prolonged membrane damage after SCI and evaluating strategies for manipulating these pathways (potentially using citicoline in combination with other pharmacological agents) may lead to a clinically effective therapy.

The binding of HIV-1 Vif to the cellular cytidine deaminase Apobec3G and subsequent prevention of Apobec3G virion incorporation have recently been identified as critical steps for the successful completion of the HIV-1 viral life cycle. This interaction occurs in the cytoplasm where Vif complexes with Apobec3G and directs its degradation via the proteasome pathway or sequesters it away from the assembling virion, thereby preventing viral packaging of Apobec3G. While many recent studies have focused on several aspects of Vif interaction with Apobec3G, the subcellular localization of Vif and Apobec3G during the viral life cycle have not been fully considered. Inhibition of Apobec3G requires direct interaction of Vif with Apobec3G, which can only be achieved when both proteins are present in the same subcellular compartment. In this thesis, a unique approach was utilized to study the impact of Vif subcellular localization on Vif function. The question of whether localization could influence function was brought about during the course of studying a severely attenuated viral isolate from a long-term non-progressor who displayed a remarkable disease course. Initial observations indicated that this highly attenuated virus contained a mutant Vif protein that inhibited growth and replication. Upon further investigation, it was found that the Vif defect was atypical in that the mutant was fully functional in in vitro assays, but that it was aberrantly localized to the nucleus in the cell. This provided the basis for the study of Vif localization and its contribution to Vif function. In addition to the unique Vif mutant that was employed, while determining the localization and replication phenotypes of the differentially localized Vif proteins, a novel pathway for Vif function was defined. Copious publications have recently defined the mechanism for Vif inhibition of Apobec3G. Vif is able to recruit Apobec3G into a complex that is targeted for degradation by the proteasome. However, this directed degradation model did not fully explain the complete neutralization of Apobec3G observed in cell culture. Other recent works have proposed the existence of a second, complementary pathway for Vif function. This pathway is defined here as formation of an aggresome that prevents Apobec3G packaging by binding and sequestering Apobec3G in a perinuclear aggregate. This second mechanism is believed to work in parallel with the already defined directed degradation pathway to promote complete exclusion of Apobec3G from the virion. The data presented here provide insight into two areas of HIV research. First, the work on the naturally occurring Vif mutant isolated from a long-term non-progress or confirms the importance of Vif in in vivo pathogenesis and points to Vif as a potentially useful gene for manipulation in vaccine or therapy design due to its critical contributions to in vivo virus replication. Additionally, the work done to address the subcellular localization of Vif led to the proposal of a second pathway for Vif function. This could have implications in the field of basic Vif research in terms of completely understanding and defining the functions of Vif. Again, a more complete knowledge about Vif can help in the development of novel therapies aimed at disrupting Vif function and abrogating HIV-1 replication.

Made available in DSpace on 2016-03-15T14:17:03Z (GMT). No. of bitstreams: 2 7.pdf: 1019934 bytes, checksum: df1b248bb9c258918248c73b73272de2 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Fármacos/Farmanguinhos. Rio de Janeiro, RJ, Brasil. / Uridina 5'-trifosfato trissódica (UTPt) e citidina 5'-monofosfato dissódica (CMPd) são nucleotídeos pirimidínicos do ácido nucleico. Eficácia e segurança de fármacos baseados na UTPt e CMPd, usados no tratamento para neuropatias periféricas já foram estudadas, no entanto informações sobre farmacocinética desses fármacos ainda não são conhecidas. O objetivo deste estudo foi propor metodologias para quantificar UTPt e CMPd em matrizes biológicas, baseando-se numa revisão sistemática da literatura. Levando em consideração que a biodisponibilidade das pirimidinas, durante as neuropatias periféricas é diferente da observada em voluntários sadios, os dados disponíveis acerca das concentrações plasmáticas do UTPt e CMPd não devem ser usados para estimar a dose de fármacos baseados nessas pirimidinas. Para diferenciar pirimidinas endógenas e exógenas em matrizes biológicas, estas últimas devem ser marcadas, antes da administração, com material radioativo tais como trício [3H] ou carbono 14 [14C]. Além disso, a cromatografia líquida de alta performance é a técnica mais aplicada para identificação e quantificação de pirimidinas radioativas. Nós concluímos que a radiomarcação de UTPt e CMPd, seguida de separação cromatográfica e detecção por UV e cintilografia líquida, seria uma metodologia factível para estudos de detecção e quantificação de derivados de UTPt e CMPd em matriz biológica / Pyrimidines uridine 5'-triphosphate trisodium (UTPt) and cytidine 5'-monophosphate disodium (CMPd) are standard nucleosides which make up nucleic acids. Efficacy and safety from UTPt and CMPd based drugs on peripheral neuropathies has already been studied. However, information regarding pharmacokinetics of UTPt and CMPd based drugs during pathological condition remains unknown. The aim of this study was to propose methodologies to quantify UTPt and CMPd in biological matrices, based on a systematic literature review. Concerning that the bioavailability of pyrimidines during peripheral neuropathies is different of observed in healthy volunteers, the available data regarding plasmatic levels of UTPt and CMPd should not be used to estimate the dose of UTPt and CMPd based drugs. Furthermore, to differentiate endogenous and exogenous pyrimidines in biological matrices the exogenous pyrimidines must be labeled with [3H] or [14C] before administration. Next, high-performance liquid chromatography (HPLC) has been the most applied technique for identification and quantitation of radiolabeled pyrimidines. We concluded that UTPt and CMPd radiolabelling, followed by chromatographic separation and detection by UV and liquid scintigraphy, is a feasible methodology for detection and quantitation of UTPt and CMPd derivatives in biological matrices.

Activation-induced cytidine deaminase (AID) is a key protein required for both class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes. AID is induced in B cells during an immune response. Lack of AID or mutant form of AID causes immunodeficiency; e.g., various mutations in the C terminus of AID causes hyper IgM (HIGM2) syndrome in humans. The C terminal 10 amino acids of AID are required for CSR but not for SHM. During both CSR and SHM, AID deaminates dCs within Ig genes, converting them to dUs, which are then either replicated over, creating mutations, or excised by uracil DNA glycosylase (UNG), leading to DNA breaks in Ig switch regions. Also, the mismatch repair (MMR) heterodimer Msh2-Msh6 recognizes U:G mismatches resulting from AID activity and initiates MMR, which leads to increased switch region double strand breaks (DSBs). DSBs are essential intermediates of CSR; lack of UNG or MMR results in a reduction of DSBs and CSR. The DSBs created in the Sμ and one of the downstream S-regions during CSR are recombined by non-homologous end joining (NHEJ) to complete CSR. Available data suggest that AID is required not only for the deamination step of CSR, but also for one or more of the steps of CSR that are downstream of deamination step. This study investigates the role of C terminus of AID in CSR steps downstream of deamination. Using retroviral transduction into mouse splenic B cells, I show that AID binds cooperatively with UNG and Msh2-Msh6 to the Ig Sμ region, and this depends on the AID C terminus. I also show that the function of MMR during CSR depends on the AID C terminus. Surprisingly, the C terminus of AID is not required for Sμ or Sγ3 DSBs, suggesting its role in CSR occurs during repair and/or recombination of DSBs.

HIV/AIDS is a disease of grave global importance with over 33 million people infected world-wide and nearly 2 million deaths each year. The rapid emergence of drug resistance, due to viral mutation, renders anti-retroviral drug candidates ineffective with alarming speed and regularity. Instead of targeting mutation prone viral proteins, an alternative approach is to target host proteins that interact with viral proteins and are critical for the HIV life-cycle. APOBEC3G is a host anti-HIV restriction factor that can exert tremendous negative pressure by hypermutating the viral genome and has the potential to be a promising candidate for anti-retroviral therapeutic research. The work presented in this thesis is focused on investigating the A3G catalytic domain structure and implications of various observed structural features for biological function. High-resolution crystal structures of the A3G catalytic domain were solved using data from macromolecular X-ray crystallographic experiments, revealing a novel intermolecular zinc coordinating motif unique to A3G. Major intermolecular interfaces observed in the crystal structure were investigated for relevance to biochemical activity and biological function. Co-crystallization with a small-molecule A3G inhibitor, discovered using high-throughput screening assays, revealed a cysteine residue near the active site that is critical for inhibition of catalytic activity by catechol moieties. The serendipitous discovery of covalent interactions between this inhibitor and a surface cysteine residue led to further biochemical experiments that revealed the other cysteine, near the active site, to be critical for inhibition. Computational modeling was used to propose a steric-hinderance based mechanism of action that was supported by mutational experiments. Structures of other human APOBEC3 homologs were modeled using in-silico methods examined for similarities and differences with A3G catalytic domain crystal structures. Comparisons based on these homology models suggest putative structural features that may endow substrate specificity and other characteristics to the APOBEC3 family members.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18891号 / 医博第4002号 / 新制||医||1009(附属図書館) / 31842 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 小川 誠司, 教授 野田 亮 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Human Immunodeficiency Virus Type 1 (HIV-1) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS), currently the leading cause of death from infectious diseases. Since HIV-1 co-opts the host cellular machinery, the study of cellular factors involved is a rational approach in discovering novel therapeutic targets for AIDS drug development. In this thesis, we present studies on two such proteins. APOBEC3G is from the family of cytidine deaminases known to keep endogenous retroviruses and retrotransposons at bay to maintain stability of the human genome. APOBEC3G targets Vif-deficient HIV-1 particles and renders them noninfectious, partially through deaminase-dependent hypermutation of the provirus during reverse transcription. APOBEC3G largely localizes in mRNA processing (P) bodies, cytoplasmic structures involved in RNA metabolism. Here we explore the significance of APOBEC3G localization in P bodies. We found that disrupting P bodies does not affect virion incorporation of endogenous APOBEC3G, implying that the APOBEC3G fraction in P bodies is not directly involved in the production of nascent, non-infectious particles. We also study UPF1, another host protein encapsidated by HIV-1. It is an essential protein mainly studied for its role in nonsense-mediated decay (NMD) pathway and belongs to the same helicase superfamily as MOV10, a recently identified antiviral factor. We found that UPF1 is incorporated in HIV-1 virions in a nucleocapsid-dependent manner and is required for single-cycle infectivity at an early, post-entry step of the viral life cycle. This novel function of UPF1 most likely does not involve NMD since depletion of UPF2 does not affect viral infectivity.