Acute myeloid leukemia (AML) is a disease characterized by overproduction of abnormally differentiated, hyper-proliferative myeloid cells known as blasts in bone-marrow and blood. Our laboratory has previously demonstrated that loss of epigenetic repression by the polycomb repressive complex 2 (PRC2), which is mediated by complex member metal response element binding transcription factor 2 (MTF2), drives chemo-resistance resulting in refractory AML. In this study, to identify MTF2-specific synthetic lethal interactions, a genome-scale CRISPR Knock-out (GeCKO) synthetic lethal screen was performed in matched MTF2-deficient and rescued THP-1 cells both in the absence and presence of the induction chemotherapeutic cytarabine. Following careful analysis of screening data using specialized software, 104 highly significant MTF2-specific synthetic lethal interactions as well as 15 cytarabine-specific synthetic lethal interactions were identified. Reduced stringency upon analysis helped to identify an additional seven MTF2-specific synthetic lethal interactions that could be targeted with commercially available small-molecule inhibitors. Among eight small molecule inhibitors, two DNA Polymerase A/Ribonucleotide Reductase Catalytic Subunit M1 (POLA/RRM1) dual inhibitors (clofarabine and fludarabine) were shown to induce toxicity with specificity for MTF2-deficient THP-1 cells at low concentrations only in the absence of cytarabine. In the future, further testing of the therapeutic potential of clofarabine and fludarabine in treating MTF2-deficient AML will be conducted in patient derived bone-marrow aspirates which better represent the true clonal and hierarchical nature of this life-threatening malignancy. Furthermore, lentiviral delivery of short-hairpin RNAs (shRNAs) targeting highly significant, non-enzymatic MTF2 and cytarabine-specific synthetic lethal interactions will be performed in both THP-1 cells as well as in patient derived bone-marrow aspirates. Eventually, in vitro validated targets will be validated under in vivo conditions using a patient derived xenograft (PDX) preclinical animal model of AML using immunocompromised NOD scid gamma (NSG) mice.

Tripodal borate ligands, including Tp and Tm, are some of the most widely used in organometallic chemistry and were originally prepared, as anions, from the reaction of the relevant heterocycle with an alkali metal borohydride. However, an alternate route, allowing access to zwitterionic, charge-neutral, scorpionates was recently developed within the Bailey group using tris(dimethylamino)borane as the boron source. This thesis describes the expansion of the borane synthetic route to create new, charge-neutral, zwitterionic, tris(methimazolyl)borate (ZTm) ligands containing B-N, B-O and B-C coordinate bonds. Unusual reactivity with isonitrile donors is also presented which has allowed access to boron substituted anionic Tm ligands from the charge-neutral starting material, (HNMe2)ZTm. Attempts to control the helical chirality of ZTm complexes, by using chiral imidazoline donors on the central boron are also described. The borane synthetic route has allowed access to the novel ligand ZThp, the first example of a tripod based on 2-hydroxypyridine ligand arms. As with Tm, this ligand exhibits helical chirality upon complexation and demonstrates how individual atom hybridisation within the ligand arms affects the helicity and thus the chirality of flexible scorpionate ligands. Coordination studies of both zwitterionic and boron-substituted anionic Tm ligands have shown a tendency for the formation of ‘sandwich’ complexes of the form L2M with some metal precursors, whilst the formation of the corresponding ‘half-sandwich’ complexes of these ligands with ruthenium and rhodium was found to be disfavoured.

Orientador: Alexandrina Sartori / Resumo: A leishmaniose visceral (LV) é uma doença negligenciada causada por Leishmania donovani na Índia e África ou Leishmania infantum na Europa e América Latina. O desenvolvimento de resposta imune eficaz e subsequente eliminação destes patógenos, requer o reconhecimento inicial da Leishmania, o qual é intermediado por receptores de reconhecimento padrão expressos por células da imunidade inata, entre eles os receptores de ligação a nucleotídeo (NLRs). Alguns NLRs ativam uma plataforma de proteínas, os inflamassomas, responsáveis pela ativação da caspase-1 e maturação de IL-1β e IL-18 e outra classe de NLRs, chamada NODs, ativam vias que culminam na ativação de NF-κB e produção de mediadores inflamatórios. O envolvimento desses receptores na LV ainda é pouco elucidado. Mesmo diante dos mecanismos de defesa do hospedeiro, esses parasitas conseguem sobreviver dentro dos macrófagos utilizando várias estratégias para escapar da resposta imune. Para um melhor entendimento dos mecanismos imunes envolvidos na LV, caracterizamos o perfil transcricional e a formação de inflamassomas e NODsomas de células THP-1 infectadas com L. infantum. Os resultados mostram que a L. infantum não induziu produção de TNF-α, IL-6 e IL-1β e nem ativação de caspase-1 após 8, 24 e 48 horas de infecção. Além disso, a infecção resultou em padrão de expressão gênica similar às células sem estímulo e distinto de células estimuladas com LPS, indicando que os parasitas entram nas células de forma mais silenciosa. Ap... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Visceral leishmaniasis (VL) is a neglected infectious disease caused by Leishmania donovani in India and Africa or Leishmania infantum in Europe and Latin America. The development of an effective immune response and subsequent elimination of these pathogens requires the initial recognition of the Leishmania that is mediated by pattern recognition receptors expressed in innate immunity cells, such as nucleotide-binding receptors (NLRs). Some NLRs activate a multiprotein platform named inflammasomes, responsible for the activation of caspase-1 and consequent maturation of IL-1β and IL-18; and another class of NLRs, the NODs, activate pathways that trigger NF-κB activation and production of inflammatory mediators. The involvement of NLRs in LV is poorly elucidated. Even in the presence of host defense mechanisms, these parasites can survive within the macrophages by employing successful strategies to escape from immune response. For a better understanding of the immune mechanisms involved in LV, we characterized the transcriptomic profiling and assembly of inflammasomes and NODsomas during infection with L. infantum in THP-1 cells. The results show that L. infantum did not induce the production of TNF-α, IL-6 and IL-1β nor activation of caspase-1 after 8, 24 and 48 hours of infection. In addition, the infection resulted in a pattern of gene expression similar to the non-stimulated cells and distinct from LPS-stimulated cells, indicating that the parasites enter inside cells in a... (Complete abstract click electronic access below) / Doutor

Submitted by Mariana Gatto (marianagatto11@hotmail.com) on 2018-05-21T17:47:50Z No. of bitstreams: 1 Tese Mariana Gatto.pdf: 3235524 bytes, checksum: b7c9938cd744aff0ff8a8ee5f858831e (MD5) / Approved for entry into archive by Sulamita Selma C Colnago null (sulamita@btu.unesp.br) on 2018-05-22T14:28:44Z (GMT) No. of bitstreams: 1 gatto_m_dr_bot.pdf: 3235524 bytes, checksum: b7c9938cd744aff0ff8a8ee5f858831e (MD5) / Made available in DSpace on 2018-05-22T14:28:44Z (GMT). No. of bitstreams: 1 gatto_m_dr_bot.pdf: 3235524 bytes, checksum: b7c9938cd744aff0ff8a8ee5f858831e (MD5) Previous issue date: 2018-04-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A leishmaniose visceral (LV) é uma doença negligenciada causada por Leishmania donovani na Índia e África ou Leishmania infantum na Europa e América Latina. O desenvolvimento de resposta imune eficaz e subsequente eliminação destes patógenos, requer o reconhecimento inicial da Leishmania, o qual é intermediado por receptores de reconhecimento padrão expressos por células da imunidade inata, entre eles os receptores de ligação a nucleotídeo (NLRs). Alguns NLRs ativam uma plataforma de proteínas, os inflamassomas, responsáveis pela ativação da caspase-1 e maturação de IL-1β e IL-18 e outra classe de NLRs, chamada NODs, ativam vias que culminam na ativação de NF-κB e produção de mediadores inflamatórios. O envolvimento desses receptores na LV ainda é pouco elucidado. Mesmo diante dos mecanismos de defesa do hospedeiro, esses parasitas conseguem sobreviver dentro dos macrófagos utilizando várias estratégias para escapar da resposta imune. Para um melhor entendimento dos mecanismos imunes envolvidos na LV, caracterizamos o perfil transcricional e a formação de inflamassomas e NODsomas de células THP-1 infectadas com L. infantum. Os resultados mostram que a L. infantum não induziu produção de TNF-α, IL-6 e IL-1β e nem ativação de caspase-1 após 8, 24 e 48 horas de infecção. Além disso, a infecção resultou em padrão de expressão gênica similar às células sem estímulo e distinto de células estimuladas com LPS, indicando que os parasitas entram nas células de forma mais silenciosa. Após a infecção houve aumento da expressão de alguns genes como ACTG1, ACTB, CD36 e DUSPs relacionados com vias de motilidade celular e regulação de MAPKs. Os genes CSF1 e CDC20 foram dois dos 30 mais expressos após infecção e estão relacionados com ciclo celular e diferenciação de macrófagos para um perfil anti-inflamatório. O gene GBP1, associado com ativação de inflamassomas, foi sub expresso após a infecção. Além disso, infecção com L. infantum resultou na expressão de poucos genes relacionados com a via dos NLRs, destacando-se entre esses o TNFAIP3 e IL1RN, referentes à modulação negativa dessa via. Os resultados obtidos indicam que a L. infantum entra nas células THP-1 de forma mais silenciosa, desativa vias inflamatórias, entre essas a via de receptores NLRs e evita a montagem de uma resposta imunológica efetora. Provavelmente o parasita usa esses recursos como mecanismos adicionais de escape para garantir sua sobrevivência dentro das células. / Visceral leishmaniasis (VL) is a neglected infectious disease caused by Leishmania donovani in India and Africa or Leishmania infantum in Europe and Latin America. The development of an effective immune response and subsequent elimination of these pathogens requires the initial recognition of the Leishmania that is mediated by pattern recognition receptors expressed in innate immunity cells, such as nucleotide-binding receptors (NLRs). Some NLRs activate a multiprotein platform named inflammasomes, responsible for the activation of caspase-1 and consequent maturation of IL-1β and IL-18; and another class of NLRs, the NODs, activate pathways that trigger NF-κB activation and production of inflammatory mediators. The involvement of NLRs in LV is poorly elucidated. Even in the presence of host defense mechanisms, these parasites can survive within the macrophages by employing successful strategies to escape from immune response. For a better understanding of the immune mechanisms involved in LV, we characterized the transcriptomic profiling and assembly of inflammasomes and NODsomas during infection with L. infantum in THP-1 cells. The results show that L. infantum did not induce the production of TNF-α, IL-6 and IL-1β nor activation of caspase-1 after 8, 24 and 48 hours of infection. In addition, the infection resulted in a pattern of gene expression similar to the non-stimulated cells and distinct from LPS-stimulated cells, indicating that the parasites enter inside cells in a more silent way. After infection, there was increased expression of some genes, such as ACTG1, ACTB, CD36 and DUSPs related to cellular motility and regulation of MAPKs pathways. The CSF1 and CDC20 genes were two of the 30 most expressed after infection and were related to cell cycle pathway and macrophage differentiation to an anti-inflammatory profile. The GBP1 gene, associated with inflammasome activation, was downregulated after infection. In addition, infection with L. infantum resulted in the expression of few genes related to the NLRs pathway, such as TNFAIP3 and IL1RN that are related to down modulation of this pathway. The results indicate that L. infantum enters inside the THP-1 cells more quietly, deactivates inflammatory pathways, including the NLR receptor pathway, and avoids the assembly of an effector immune response. Probably the parasite uses these strategies as additional escape mechanism to ensure its survival within host cells.

The human monocyte-derived THP-1 cell line was incubated with 10mM AAPH in Earle’s Balanced Salt Solution at 37°C for up to 24 hours. Protein hydroperoxide formation occurred after an 8 hour lag phase which corresponded to glutathione loss observed in the cells. SDS-Page analysis confirmed protein degradation occurred after 6 hours. Cell viability measured by the MTT reduction assay also dropped after 8 hours. Reduction of intracellular glutathione levels using BSO caused reduction of the lag phase seen in protein hydroperoxide formation. Cell viability of BSO-treated cells was lower than control cells, indicating the initiation of apoptotic events. Flow cytometry analysis showed no difference between BSO-treated and control cells, indicating that GSH levels do not have an effect on the type of cell death observed in AAPH-induced oxidative damage on THP-1 cells. These results confirmed previous data in the lab suggesting THP-1 cells undergo AAPH-induced necrosis as a result of cellular damage, including the loss of GSH and the formation of protein hydroperoxides.

Angiotensin II (Ang II) is an important modulator for development of atherosclerosis from early stage foam cell formation to advanced stage plaque rupture. Recently, the importance of locally generated Ang II, especially in macrophages, has become more evident. Generation of Ang II by several enzymes other than ACE and renin has been shown mainly in vitro. Cathepsin G is one these enzymes which is expressed in neutrophils and macrophages. Macrophages are one of the primary and crucial cells in atherosclerotic lesions which become lipid-laden foam cells through lipoprotein uptake. We hypothesized that activation of nuclear factors in foam cells increases Ang II by modulation of the renin angiotensin system (RAS) genes and cathepsin G. We also hypothesized that cathepsin G, through its Ang II generating activity and its other catalytic functions, promotes atherosclerosis. The present study assessed the Ang I and II levels and expression of the RAS genes in THP-1 cells, a human acute monocytic leukemia cell line, and in peritoneal and bone marrow-derived macrophages after exposure to acetylated LDL (ac-LDL). I also evaluated how RAS blockade would affect foam cell formation in THP-1 cells. In parallel, I assessed the role of cathepsin G in Ang II generation and in the progression of atherosclerosis in cathepsin G heterozygous knockout mice on an Apoe-/- background (Ctsg+/-Apoe-/- mice). Ac-LDL treatment increased Ang I and Ang II levels in cell lysates and media from THP-1 cells but not in peritoneal or bone marrow-derived macrophages from wild type C57BL/6 mice. In ac-LDL-treated THP-1 cells, ACE and cathepsin G mRNA levels and activities were elevated. Angiotensinogen mRNA is increased but not the angiotensinogen protein concentration. Renin mRNA level and activity were not altered by ac-LDL treatment. Blocking RAS by an AT1 receptor blocker, ACE inhibitors or a renin inhibitor decreased cholesteryl ester content of THP-1 cells after exposure to ac-LDL. To confirm that the Ang II effect on foam cell formation was not unique to ac-LDL, we treated the THP-1 macrophages with a renin inhibitor or an AT1 receptor inhibitor after exposure to oxidized LDL (ox-LDL). RAS blockade in ox-LDL-treated cells also abolished cholesteryl ester formation. To see how Ang II plays a role in foam cell formation we assessed the effect of RAS inhibitors on SR-A, the principal receptor for mediating ac-LDL entry into the cells and on acyl-CoA:cholesterol acyl transferase (ACAT-1), the enzyme responsible for intracellular cholesterol esterification. RAS blockade in both ac-LDL- and ox-LDL-treated cells decreased SR-A and ACAT-1 protein levels. Cathepsin G partial deficiency on an Apoe-/- background did not change Ang II levels in peritoneal or bone marrow-derived macrophage cell lysates or media. This deficiency also did not affect immunoreactive angiotensin peptide levels in atherosclerotic lesions. After 8 weeks on a high fat diet Ctsg+/-Apoe-/- mice were similar to Ctsg+/+Apoe-/- mice in terms of lesion size and serum cholesterol levels but the Ctsg+/+Apoe-/- mice had more advanced lesions with more collagen and smooth muscle cells and fewer macrophages. Moreover, Ctsg+/+Apoe-/- mice had more apoptotic cells than their Ctsg+/-Apoe-/- littermates. Overall, our findings indicate that Ang II is increased in foam cells and this endogenous Ang II is involved in cholesteryl ester formation, possibly by regulating the levels of ACAT-1 and SR-A. We did not find any role for cathepsin G in generation of Ang II in mice but cathepsin G does, nevertheless, promote the progression of atherosclerotic lesions to a more advanced stage.

Angiotensin II (Ang II) is an important modulator for development of atherosclerosis from early stage foam cell formation to advanced stage plaque rupture. Recently, the importance of locally generated Ang II, especially in macrophages, has become more evident. Generation of Ang II by several enzymes other than ACE and renin has been shown mainly in vitro. Cathepsin G is one these enzymes which is expressed in neutrophils and macrophages. Macrophages are one of the primary and crucial cells in atherosclerotic lesions which become lipid-laden foam cells through lipoprotein uptake. We hypothesized that activation of nuclear factors in foam cells increases Ang II by modulation of the renin angiotensin system (RAS) genes and cathepsin G. We also hypothesized that cathepsin G, through its Ang II generating activity and its other catalytic functions, promotes atherosclerosis. The present study assessed the Ang I and II levels and expression of the RAS genes in THP-1 cells, a human acute monocytic leukemia cell line, and in peritoneal and bone marrow-derived macrophages after exposure to acetylated LDL (ac-LDL). I also evaluated how RAS blockade would affect foam cell formation in THP-1 cells. In parallel, I assessed the role of cathepsin G in Ang II generation and in the progression of atherosclerosis in cathepsin G heterozygous knockout mice on an Apoe-/- background (Ctsg+/-Apoe-/- mice). Ac-LDL treatment increased Ang I and Ang II levels in cell lysates and media from THP-1 cells but not in peritoneal or bone marrow-derived macrophages from wild type C57BL/6 mice. In ac-LDL-treated THP-1 cells, ACE and cathepsin G mRNA levels and activities were elevated. Angiotensinogen mRNA is increased but not the angiotensinogen protein concentration. Renin mRNA level and activity were not altered by ac-LDL treatment. Blocking RAS by an AT1 receptor blocker, ACE inhibitors or a renin inhibitor decreased cholesteryl ester content of THP-1 cells after exposure to ac-LDL. To confirm that the Ang II effect on foam cell formation was not unique to ac-LDL, we treated the THP-1 macrophages with a renin inhibitor or an AT1 receptor inhibitor after exposure to oxidized LDL (ox-LDL). RAS blockade in ox-LDL-treated cells also abolished cholesteryl ester formation. To see how Ang II plays a role in foam cell formation we assessed the effect of RAS inhibitors on SR-A, the principal receptor for mediating ac-LDL entry into the cells and on acyl-CoA:cholesterol acyl transferase (ACAT-1), the enzyme responsible for intracellular cholesterol esterification. RAS blockade in both ac-LDL- and ox-LDL-treated cells decreased SR-A and ACAT-1 protein levels. Cathepsin G partial deficiency on an Apoe-/- background did not change Ang II levels in peritoneal or bone marrow-derived macrophage cell lysates or media. This deficiency also did not affect immunoreactive angiotensin peptide levels in atherosclerotic lesions. After 8 weeks on a high fat diet Ctsg+/-Apoe-/- mice were similar to Ctsg+/+Apoe-/- mice in terms of lesion size and serum cholesterol levels but the Ctsg+/+Apoe-/- mice had more advanced lesions with more collagen and smooth muscle cells and fewer macrophages. Moreover, Ctsg+/+Apoe-/- mice had more apoptotic cells than their Ctsg+/-Apoe-/- littermates. Overall, our findings indicate that Ang II is increased in foam cells and this endogenous Ang II is involved in cholesteryl ester formation, possibly by regulating the levels of ACAT-1 and SR-A. We did not find any role for cathepsin G in generation of Ang II in mice but cathepsin G does, nevertheless, promote the progression of atherosclerotic lesions to a more advanced stage.

The human monocyte-derived THP-1 cell line was incubated with 10mM AAPH in Earle’s Balanced Salt Solution at 37°C for up to 24 hours. Protein hydroperoxide formation occurred after an 8 hour lag phase which corresponded to glutathione loss observed in the cells. SDS-Page analysis confirmed protein degradation occurred after 6 hours. Cell viability measured by the MTT reduction assay also dropped after 8 hours. Reduction of intracellular glutathione levels using BSO caused reduction of the lag phase seen in protein hydroperoxide formation. Cell viability of BSO-treated cells was lower than control cells, indicating the initiation of apoptotic events. Flow cytometry analysis showed no difference between BSO-treated and control cells, indicating that GSH levels do not have an effect on the type of cell death observed in AAPH-induced oxidative damage on THP-1 cells. These results confirmed previous data in the lab suggesting THP-1 cells undergo AAPH-induced necrosis as a result of cellular damage, including the loss of GSH and the formation of protein hydroperoxides.

abstract: Tobacco and alcohol are the most commonly abused drugs worldwide. Many people smoke and drink together, but the mechanisms of this nicotine (NIC) -ethanol (EtOH) dependence are not fully known. EtOH has been shown to affect some nicotinic acetylcholine receptors (nAChRs), which potentially underlies NIC-EtOH codependence. Ventral Tegmental Area (VTA) dopamine (DA) and γ-aminobutyric acid (GABA) neurons express different nAChR subtypes, whose net activation results in enhancement of DA release in the Prefrontal Cortex (PFC) and Nucleus Accumbens (NAc). Enhancement of DA transmission in this mesocorticolimbic system is thought to lead to rewarding properties of EtOH and NIC, clarification of which is relevant to public health and clinical diseases. The aim of this study was to elucidate pharmacological mechanisms of action employed by both NIC and EtOH through nAChRs in VTA neurons by evaluating behavioral, network, synaptic and receptor functions therein. It was hypothesized that VTA GABA neurons are controlled by α7 nAChRs on presynaptic GLUergic terminals and α6 nAChRs on presynaptic GABAergic terminals. NIC and EtOH, via these nAChRs, modulate VTA GABA neuronal function. This modulation may underlie NIC and EtOH reward and reinforcement, while pharmacological manipulation of these nAChRs may be a therapeutic strategy to treat NIC or EtOH dependence. This data demonstrates that in VTA GABA neurons, α7 nAChRs on GLUergic terminals play a key role in the mediation of local NIC-induced firing increase. α6*-nAChRs on GABA terminals enhances presynaptic GABA release, and leads to greater inhibition to VTA GABA neurons, which results in an increase VTA DA neuron firing via a disinhibition mechanism. Genetic knockout of these nAChRs significantly prevents EtOH-induced animal conditioned place preference (CPP). Furthermore, levo-tetrahydropalmadine (l-THP), a compound purified from natural Chinese herbs, blocks nAChRs, prevents NIC-induced DA neuronal firing, and eliminates NIC CPP, suggesting it as a promising candidate in a new generation of interventions for smoking cessation. Improved understanding of underlying mechanisms and development of new drugs will increase the number of successful quitters each year and dramatically improve the quality of life for millions suffering from addiction, as well as those around them. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2015

Anti-inflammatory cytokines have an important role in the development of inflammatory reactions. If an acute inflammation turns into chronical it is very often a pathological phenomenon. Chronicle inflammations accompany a whole number of serious diseases with an unclear prognosis, such as some of the autoimmune diseases. Usually, the cause of these diseases is not quite clear and the treatment is mainly symptomatic with an effort to suppress the immunity system. For this purpose we use various immunosuppressant drugs, and biological treatment is used, too. Another possibility is to use bioactive secondary metabolites produced by various microorganisms. In this group there are for example macrolides antibiotics, and a big potential is also seen in the recently discovered polyketides. The objective of this work is to test the newly acquired secondary metabolites that were isolated in the Laboratory of Molecular Biology of Actinomycetes at the Czech Academy of Sciences. Tested were manumycin A, manumycin B, colabomycin E, asukamycin A, asukamycin D, β-rubromycin, deoxynybomycin. As comparative substances were used the macrolides antibiotics clarithromycin and azithromycin dehydrate, all of them commercial pharmaceuticals. These substances were tested on the monocytic line THP-1. Cells were stimulated...