Diss. (sammanfattning) Linköping : Univ., 2004. / Härtill 4 uppsatser.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2018-04-19T13:14:58Z No. of bitstreams: 1 texto completo.pdf: 2407879 bytes, checksum: 24a29868ff91347f5c8b65b662cb7870 (MD5) / Made available in DSpace on 2018-04-19T13:14:58Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2407879 bytes, checksum: 24a29868ff91347f5c8b65b662cb7870 (MD5) Previous issue date: 2018-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Serine/Arginine protein kinases (SRPKs) são componentes chave da maquinaria de splicing através da regulação por fosforilação das proteínas SR, as quais são cruciais para a seleção dos sítios de splicing alternativo. Entretanto, as SRPKs são frequentemente encontradas superexpressas ou com atividade alterada em diversos tipos de cânceres, inclusive em leucemias. Dessa forma, a busca por pequenas moléculas inibidores destas quinases são de potencial interesse para o delineamento de novas estratégias terapêuticas. Nesta tese, descreve­se a avaliação da inibição farmacológica de SRPKs pelo inibidor seletivo N­(2­ (piperidin­1­il)­5­(trifluorometil)fenil)isonicotinamida (SRPIN340) sobre a viabilidade de linhagens leucêmicas linfoides e mieloides. Além de mostrar atividade citotóxica significativa, o efeito dos tratamentos na regulação da fosforilação das proteínas SR e na alteração da expressão dos genes MAP2K1, MAP2K2, VEGF e FAS foram também identificados. Além disso, a inibição farmacológica de SRPKs foi capaz de desencadear eventos precoces e tardios de apoptose. Por último, estudos de fluorescência intrínseca de triptofano, docking molecular e dinâmica molecular foram analisados a fim de se obter informações estruturais acerca do complexo SRPK/SRPIN340. No segundo estudo, é descrita a síntese de uma série de vinte e duas trifluorometil arilamidas baseadas na estrutura molecular do SRPIN340, além da avaliação dos seus efeitos antileucêmicos. Alguns dos derivados apresentaram efeitos citotóxicos superiores contra linhagens de leucemia mieloide e linfoide em comparação com o SRPIN340. Em particular, os compostos N­(2­(4­bromofenilamino)­5­(trifluorometil)fenil)­2­cloronicotinamida (24), N­(2­(4­bromofenilamino)­5­(trifluorometil)fenil)nicotinamida (30), e N­(2­(4­ bromofenilamino)­5­(trifluorometil)fenil)benzamida (36) apresentaram valores de IC 50 na faixa de 6,0 – 35,7 μM (μmol L ­1 ). Estes três compostos também foram capazes de desencadear eventos de apoptose e autofagia, além de exibir efeito sinergístico em combinação com o agente quimioterápico vincristina. Além disso, o composto 30 se mostrou mais eficiente que o SRPIN340 na diminuição da fosforilação das proteínas SR bem como na diminuição da expressão de isoformas oncogênicas dos genes MAP2K1, MAP2K2, VEGF, e RON. Tomados conjuntamente, estes resultados sugerem que inibidores de SRPKs são capazes de suprimir o crescimento celular através da regulação dos eventos de splicing e podem ser considerados como ponto de partida importante para desenvolvimento de novas estratégias terapêuticas contra leucemias e outros tipos de cânceres. / Serine/Arginine protein kinase (SRPK) are key components of the splicing machinery trough the phospho­regulation of SR Proteins, which are crucial for exon selection in the alternative splicing. However, SRPK have frequently been found overexpressed or/and with altered activity in a number of cancers, including leukemias. Thus, the discovery of small molecule inhibitors against these kinases is of potential interest to identify novel therapeutic opportunities. Here, it is described the pharmacological inhibition of SRPK by N­(2­(piperidin­1­yl)­5­ (trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, it was found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. In a second study, it is described the synthesis of a series of twenty­two trifluoromethyl arylamides based on the SRPIN340 scaffold and the evaluation of their antileukemia effects. Some derivatives presented superior cytotoxic effects against myeloid and lymphoid leukemia cell lines compared to SRPIN340. In particular, compounds N­(2­(4­bromophenylamino)­5­(trifluoromethyl)phenyl)­2­chloronicotinamide (24), N­(2­(4­bromophenylamino)­5­(trifluoromethyl)phenyl)nicotinamide (30), and N­(2­(4­ bromophenylamino)­5­(trifluoromethyl)phenyl)benzamide (36) presented IC 50 values within the 6.0 – 35.7 μM (μmol L ­1 ) range. In addition, these three compounds were able to trigger apoptosis and autophagy, and they exhibited synergistic effects in combination with the chemotherapeutic agent vincristine. Moreover, compound 30 was more efficient than SRPIN340 in impairing the intracellular phosphorylation status of SR proteins as well as the expression of MAP2K1, MAP2K2, VEGF, and RON oncogenic isoforms in leukemia cells. Taken together, these results suggest that SRPK pharmacological inhibitors may be considered for the development of novel therapeutic strategies against leukemias and other types of cancers.

L’activation des cellules T est initiée suite à la rencontre avec un antigène spécifique. Les études réalisées pour mieux comprendre ce processus d’activation se sont principalement focalisées sur le rôle des cellules présentatrices d'antigènes et des cytokines. Toutefois, des données récentes soulignent également l'importance du microenvironnement métabolique pour soutenir l’augmentation des besoins énergétiques et biosynthétiques liés à la stimulation antigénique. Cette reprogrammation métabolique est conditionnée par la disponibilité en nutriments et la teneur en oxygène qui peuvent être altérés en conditions pathologiques, comme dans des tumeurs. En effet, plusieurs groupes dont le nôtre ont montré qu’en cas de faible disponibilité en nutriments, une compétition peut se créer entre les cellules tumorales et les cellules T, impactant de ce fait négativement leurs fonctions anti-tumorales. Cet effet est dû, du moins en partie, aux profils métaboliques distincts des sous-populations de cellules T; alors que les cellules T effectrices (dont les cellules Th1) sont fortement glycolytiques, les cellules T régulatrices suppressives (Treg) présentent un métabolisme plus mixte avec des niveaux accrus d'oxydation lipidique. Il est donc important de déterminer comment les changements métaboliques des cellules T anti-tumorales affectent leur persistance et leur fonctionnalité. Ainsi, j'ai entrepris des travaux afin d’évaluer si le niveau d’expression du transporteur de glucose Glut1 permettait d’identifier et de sélectionner des cellules T ayant des fonctions effectrices distinctes. Nous avons confirmé cette hypothèse et notamment montré que les cellules T exprimant un niveau élevé de Glut1 possèdent un potentiel de sécrétion d’IFNg accru.De plus, nos travaux montrent que la disponibilité en nutriments extracellulaires est un élément clé pour la différenciation terminale des cellules Th1. En effet, l'activation des cellules T CD4 naïves en conditions limitantes en glutamine induit leur différenciation en cellules Treg Foxp3+. Plus surprenant encore, cette carence induit un blocage de la différenciation Th1 même lors d’une polarisation vers ce lignage. De plus, en conditions de carence en glutamine, nous avons découvert que l'alpha-cétoglutarate (aKG), un métabolite dérivé de la glutamine, rétablit cette différenciation terminale Th1. J'ai ensuite évalué l'impact de l’aKG dans les processus de différenciation Th1/Treg en condition non limitante en glutamine. Mes données montrent que, dans des conditions de polarisation Th1, l’ajout d’aKG améliore la différenciation des cellules T CD4 naïfs en cellules Th1 et augmente la production d’IFNg. A l’inverse, l’ajout d’aKG s’accompagne d’une diminution des cellules Foxp3+ et d’une augmentation de la sécrétion de cytokines inflammatoires dans des conditions de polarisation Treg. L'altération de la différenciation des cellules T médiée par l'aKG est notamment associée à une phosphorylation oxydative (OXPHOS) accrue ; ainsi, l'ajout d’un inhibiteur du cycle de Krebs et du complexe mitochondrial II /succinate déshydrogénase, atténue le blocage de la différenciation Treg induit par l'aKG. De façon remarquable, ces modifications de l'équilibre Th1/Treg médiées par l'aKG sont maintenues in vivo et impactent le devenir de cellules T exprimant un récepteur chimérique anti-tumoral (CAR) injectées chez des souris porteuses de tumeurs. En résumé, nos données montrent qu'une faible teneur en aKG intracellulaire liée à une disponibilité limitée en glutamine, favorise un phénotype Treg, alors que des niveaux élevés d’aKG modifient l'équilibre vers un phénotype Th1.En conclusion, les données générées au cours de ma thèse devraient permettre le développement de stratégies permettant de sélectionner des cellules T ayant des propriétés effectrices anti-tumorales améliorées. / T cells are stimulated upon interaction with their cognate antigen. While much research has focused on the role of antigen presenting cells (APC) and cytokines as important components of the T cell microenvironment, recent data highlight the importance of the metabolic environment in sustaining the energetic and biosynthetic demands that are induced upon antigen stimulation. The subsequent metabolic reprogramming of the T cell is conditioned by the nutrient composition and oxygen levels. Notably, this environment can be altered by pathological conditions such as tumors and data from our group, as well as others, have shown that the competition of T cells and tumor cells for limiting amounts of nutrients has a negative impact on T cells, inhibiting their anti-tumor effector functions. This effect is due, at least in part, to the distinct metabolic profiles of T lymphocyte subsets; T effector cells (including Th1 cells) are highly glycolytic while suppressive Foxp3+ regulatory T cells (Tregs) display a mixed metabolism with increased levels of lipid oxidation. It is therefore important to determine how changes in the metabolic programming of anti-tumor T cells impacts on their persistence and function. Indeed, in the context of my PhD research, I found that high levels of the glucose transporter Glut1 was associated with a significantly increased level of IFNγ secretion by both CD4 and CD8 T cells. Furthermore, there was a bias of CD8 over CD4 lymphocytes in the Glut1-hi T cell subset. These data point to the importance of metabolic alterations in the fate and effector function of T lymphocytes and during my PhD, I focused on elucidating the metabolic parameters that regulate effector and regulatory T cells, with the goal of improving the efficacy of anti-tumor T cells. In this context, I contributed to initial studies from our group, revealing a critical role for extracellular nutrient availability in terminal CD4+ T cell differentiation. Activation of naïve CD4+ T cells under conditions of glutamine deprivation caused them to differentiate into induced Treg (iTreg). Moreover, the skewing of glutamine-deprived naive CD4+ T cells to a Foxp3+ fate occurred even under Th1-polarizing conditions, blocking terminal Th1 differentiation. Under glutamine-deprived conditions, we found that alpha-ketoglutarate (αKG), a glutamine-derived metabolite, rescued Th1 differentiation. I then evaluated the impact of aKG under glutamine-replete conditions in the Th1/iTreg differentiation processes. My studies showed that, under Th1-polarizing conditions, aKG markedly enhanced naïve CD4+ T cell differentiation into Th1 cells and increased IFNg secretion. Moreover, under Treg-polarizing conditions, αKG decreased Foxp3 expression and increased the secretion of inflammatory cytokines such as IFNg, GM-CSF and IL-17. Notably, the aKG-mediated alteration in T cell differentiation was associated with an augmented oxidative phosphorylation (OXPHOS), and inhibiting the citric acid cycle and the mitochondrial complex II with malonate, an inhibitor of succinate dehydrogenase (SDH), alleviated the αKG-mediated block in Treg differentiation. Impressively, these aKG-mediated changes in the Th1/Treg balance were maintained in vivo, promoting a Th1-like profile in T cells expressing an anti-tumor chimeric antigen receptor (CAR) in tumor-bearing mice. Thus, our data show that low intracellular aKG content, caused by limited external glutamine availability, imposes a Treg phenotype while high aKG levels shift the balance towards a Th1 phenotype.Altogether, the data generated during my PhD will promote the development of metabolic strategies aimed at modulating T cell function and foster the design of nutrient transporter-based approaches that can be used to select T lymphocytes with enhanced anti-tumor effector properties.

No description available.

No description available.

The aim of this study was to investigate in vitro transdermal diffusion of a small peptide namely arginine vasopressin (AVP) with the aid of the novel PheroidTM drug delivery system. Generally, peptides seem unfit for transdermal permeation, but it was thought prudent to explore the suitability of this lipid-based system after success was achieved with entrapment of tuberculostatics, bacteria and viruses. Bestatin (a selective aminopeptidase inhibitor) was employed to circumvent any skin-related degradation of the active. Therefore, the effect of bestatin on the preservation of AVP during diffusion was investigated. Vertical Franz cell diffusion studies were conducted with female abdominal skin, with AVP at a concentration of 150 pglml in the donor phase and Hepes buffer as the receptor phase over a twelve-hour period. To prove entrapment of AVP within the lipid structures of the PheroidsTM, fluorescentlylabelled samples were monitored by means of confocal laser scanning microscopy (CLSM), which revealed definite entrapment. In vitro permeation profiles for AVP exhibited a biphasic character, with the majority of permeation occurring during the first two hours. The PheroidTM delivery system proved to be advantageous when applied as delivery medium. The inclusion of bestatin has an enhancing effect on permeation probably due to its protection of AVP. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

<p>The pathogenesis of Alzheimer’s disease (AD) is a critical unsolved question, and while recent studies have demonstrated a strong association between altered brain immune responses and disease progression, the mechanistic cause of neuronal dysfunction and death is unknown. We have previously described the unique CVN-AD mouse model of AD, in which immune-mediated nitric oxide is lowered to mimic human levels, resulting in a mouse model that demonstrates the cardinal features of AD, including amyloid deposition, hyperphosphorylated and aggregated tau, behavioral changes and age-dependent hippocampal neuronal loss. Using this mouse model, we studied longitudinal changes in brain immunity in relation to neuronal loss and, contrary to the predominant view that AD pathology is driven by pro-inflammatory factors, we find that the pathology in CVN-AD mice is driven by local immune suppression. Areas of hippocampal neuronal death are associated with the presence of immunosuppressive CD11c+ microglia and extracellular arginase, resulting in arginine catabolism and reduced levels of total brain arginine. Pharmacologic disruption of the arginine utilization pathway by an inhibitor of arginase and ornithine decarboxylase protected the mice from AD-like pathology and significantly decreased CD11c expression. Our findings strongly implicate local immune-mediated amino acid catabolism as a novel and potentially critical mechanism mediating the age-dependent and regional loss of neurons in humans with AD.</p><p>There is a large interest in identifying, lineage tracing, and determining the physiologic roles of monophagocytes in Alzheimer’s disease. While Cx3cr1 knock-in fluorescent reporting and Cre expressing mice have been critical for studying neuroimmunology, mice that are homozygous null or hemizygous for CX3CR1 have perturbed neural development and immune responses. There is, therefore, a need for similar tools in which mice are CX3CR1+/+. Here, we describe a mouse where Cre is driven by the Cx3cr1 promoter on a bacterial artificial chromosome (BAC) transgene (Cx3cr1-CreBT) and the Cx3cr1 locus is unperturbed. Similarly to Cx3cr1-Cre knock-in mice, these mice express Cre in Ly6C-, but not Ly6C+, monocytes and tissue macrophages, including microglia. These mice represent a novel tool that maintains the Cx3cr1 locus while allowing for selective gene targeting in monocytes and tissue macrophages.</p><p>The study of immunity in Alzheimer’s requires the ability to identify and quantify specific immune cell subsets by flow cytometry. While it is possible to identify lymphocyte subsets based on cell lineage-specific markers, the lack of such markers in brain myeloid cell subsets has prevented the study of monocytes, macrophages and dendritic cells. By improving on tissue homogenization, we present a comprehensive protocol for flow cytometric analysis, that allows for the identification of several cell types that have not been previously identified by flow cytometry. These cell types include F4/80hi macrophages, which may be meningeal macrophages, IA/IE+ macrophages, which may represent perivascular macrophages, and dendritic cells. The identification of these cell types now allows for their study by flow cytometry in homeostasis and disease.</p> / Dissertation

The Twin arginine translocase (Tat) system catalyzes movement of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. This transport process requires energy in the form of the transmembrane proton motive force (PMF). The Tat system can be studied in vitro using inner membrane vesicles (IMVs) from E. coli overproducing the Tat components, TatA, TatB and TatC. However, the transport efficiencies of current in vitro Tat transport assays are low. In this work, current in vitro Tat transport assays were compared and parameters that affect transport efficiencies were identified. Mild French press treatment of IMVs resulted in larger IMVs with higher transport efficiencies. Chloride ions were shown to inhibit Tat transport in vitro. Generation of a PMF by the activity of ATP synthase gave higher transport efficiencies than generating a PMF by NADH respiration. This understanding was applied to develop an optimized in vitro Tat transport assay that showed a higher transport efficiency than currently published methods. Fluorescently labelled Tat substrates were developed to allow quantitative analysis of Tat transport. The transport of the purified native Tat substrate, CueO into IMVs was characterized using the optimized in vitro Tat transport assay. It was shown that the proton concentration (ΔpH) component of the PMF was sufficient to support Tat transport in vitro. It was observed that transport of CueO ceased in a time-dependent manner in the in vitro Tat transport assays. This loss of transport efficiency could be due, at least in part, to the presence of a PMF since transport efficiency was reduced when IMVs were pre-energized. Substrates for future in vitro single molecule fluorescence microscopy studies of the Tat transport were developed in this work. One of the substrates is fluorescently labelled CueO. The second substrate is the native Tat substrate alkaline phosphatase PhoX from Vibrio fischeri which was able to cleave the fluorogenic compound AttoPhos® and can thus be used as an enzymatic reporter of Tat transport. The structure of a native Tat substrate from Pseudomonas fluorescens, PhoX, was solved by X-ray crystallography at a resolution of 1.4Å. PhoX is a monomeric six blade β propeller with two α-helical bundle subdomains. PhoX was shown to have optimum activity at pH8.0. PhoX has a novel catalytic site which requires two Fe³⁺ (including a Cys-coordinated Fe³⁺) and three Ca²⁺ as cofactors. Mutagenesis studies showed that all the metal ions are required for the integrity of the active site. Co-crystallization of PhoX with vanadate, an inhibitor of PhoX which mimics the transition state, showed that hydrolysis of phosphomonoesters does not involve formation of a covalent phosphoenzyme intermediate. Instead, dephosphorylation of substrates is proposed to occur via a SN2 reaction with OH- as the attacking nucleophile.

The Twin arginine translocation pathway translocates fully folded proteins across cellular membranes and is only utilised by proteins that fold before translocation. It is a unique process that is found in many bacteria, archaea and also in plant chloroplasts. Investigation of the bacterial and thylakoidal systems has revealed much of the substrates and the components involved in their translocation. Unfortunately, there are still many unanswered questions such as how substrates are directed to the membrane and the actual mechanism of translocation. This thesis specifically investigates the Tat pathway of halophilic and thermophilic archaea. To date, there has been a lack of research into the archaeal Tat pathway and it is possible that there are unique adaptations because of the extreme environments that these organisms inhabit. Chapter 3 specifically investigates the thermophiles Sulfolobus solfataricus and Sulfolobus tokodaii and attempts to purify their Tat complexes. By doing so it was hoped to learn more about the Tat components and their interactions. Further experiments were also performed to determine if the two S. solfataricus Tat operons provide specificity to the Tat substrates that translocate. Four separate areas of the Tat pathway of halophilic archaea (haloarchaea) were investigated in Chapters 4-7. Firstly, site-directed mutagenesis was used to analyse the signal peptides of haloarchaeal Tat substrates in more detail. Consequently, the resulting data led to the use of bioinformatics to analyse the Haloarchaeal signal peptide. The bioenergetics of the Tat system was then determined by analysing the effect of a variety of ionophores on translocation of the Tat substrates AmyH and SptA. Finally, a series of folding and stability assays were used to increase our understanding of AmyH, which could provide further information on why this protein, like many other haloarchaeal proteins, requires the Tat pathway for translocation.

Monoclonal antibodies (mAbs) represent one of the fastest growing classes of therapeutic proteins. This success is due to a number of attractive properties such as high binding affinity, specificity, low immunogenicity and high aqueous solubility. Despite this, mAbs can suffer from undesirable physical instabilities, especially reversible self-association (RSA), which can lead to aggregation and phase separation. One aspect of formulation is therefore to find solution conditions which minimise mAb aggregation propensity during storage at high concentrations. Hence, the buffer, excipient and pH must be carefully considered to obtain the optimal formulation. Currently, if a platform formulation process is non-ideal for a particular candidate mAb, then an alternative strategy is to utilise high-throughput screening to measure various physical parameters indicative of physical stability. Arginine (in the form of hydrochloride salt Arg·HCl) is often used in formulations exhibiting high RSA and a propensity for aggregation. The interaction of Arg with the protein surface is complex and dependent on both the salt form and concentration. Here the focus was on the glutamate salt of arginine (Arg·Glu), to quantify its effect on mAb conformational and colloidal stability under different pH conditions. Arg·Glu was able to decrease the propensity of the mAbs to aggregate, particularly at pH values closer to their pI.The work also included the use of in vitro cell culture models to examine cell viability in the presence of the various arginine salts over a range of osmolalities. Whilst Arg·Glu is composed of two naturally occurring amino acids and both of which are considered non-toxic individually, the effect of the increased concentrations of their combination, on cells has not been explored previously. In vitro cell lines were chosen to represent the subcutaneous tissue, the effect of Arg·Glu on cell viability was compared against NaCl, Arg·HCl and sodium glutamate (NaGlu). The work concluded there was no additional toxicity associated with the presence of Arg·Glu in the cell culture models studied, therefore Arg·Glu has the potential as an excipient as it reduces aggregation and is nontoxic. Another aspect of the work was to assess the use of solution NMR spectroscopy as an orthogonal technique in mAb formulation characterisation. 1H NMR spectroscopy was used to measure a number of experimental parameters for high concentration mAb solution. The work proposed that 1H NMR spectroscopy can serve as a valuable orthogonal method for mAb characterization and formulation.