The heat shock or stress response is a transient response to stressful stimuli that protects vital cellular proteins from damage and irreversible aggregation. Heat shock proteins (Hsps) are molecular chaperones that bind to unfolded protein and inhibit their aggregation, thereby maintaining their solubility until they can be refolded to their native conformation. Hsp47 is an endoplasmic reticulum (ER)-resident protein that serves as a molecular chaperone during collagen production. Collagen is the major class of insoluble fibrous protein found in the extracellular matrix and in connective tissues. It is the single most abundant protein of the animal kingdom; at least 14 different forms exist, each with distinct structures and binding properties. The various types of collagen all possess protein regions with the distinct triple helical conformation. This complex physical structure requires very organized assembly and HSP47 has been established as an integral component of this process for collagen types I-V. Most of the previous studies examining the expression and function of hsp47 have been conducted with mammalian cultured cells. The present study represented the first investigation of the expression of hsp47 in the poikilothermic vertebrate, <i>Xenopus laevis</i>. Full-length <i>Xenopus</i> hsp47 nucleotide and amino acid sequences were obtained from Genbank and compared with hsp47 from chicken, mouse, rat, human and zebrafish. <i>Xenopus</i> HSP47 protein had an identity of approximately 77% with chicken, 73% with mouse, 72% with rat and human, and 70% with zebrafish. Most of the sequence identity between HSP47 from all investigated organisms occurred centrally in the amino acid sequence and in several carboxyl terminal regions. Three key features were conserved between HSP47 proteins from most species investigated: a hydrophobic leader sequence, two potential glycosylation sites and the ER-retention signal, RDEL. A partial cDNA clone encoding <i>Xenopus</i> hsp47 was obtained from the American Type Culture Collection (ATCC) and used to generate hsp47 antisense riboprobe for the purpose of investigating hsp47 mRNA accumulation in <i>Xenopus</i> A6 kidney epithelial cells and embryos. Northern blot analysis detected hsp47 mRNA constitutively in A6 cells. The expression pattern for hsp47 mRNA was compared with two other <i>Xenopus</i> heat shock proteins that have been previously characterized in our laboratory: hsp70, a cystolic/nuclear hsp and BiP, an ER-resident hsp. The results of hsp47 mRNA accumulation in A6 cells suggested that the expression pattern for <i>Xenopus</i> hsp47 was unique but, with respect to some stressors, resembled that of a cytosolic hsp rather than an ER-resident hsp. HSP47 protein levels were also examined in A6 cells. Heat shock, sodium arsenite and b-aminopropionitrile fumerate treatments enhanced hsp47 accumulation. In some experiments, western blot analysis revealed the presence of two closely sized protein bands. It is possible that minor differences in HSP47 protein size may be due to post-translational modification, namely phosphorylation or glycosylation. The present study also examined the accumulation and spatial pattern of hsp47 mRNA accumulation during <i>Xenopus laevis</i> early development. Hsp47 was constitutively expressed throughout <i>Xenopus</i> early development. Constitutive levels of hsp47 mRNA in unfertilized eggs, fertilized eggs and cleavage stage embryos indicated that these transcripts were maternally inherited. Constitutive hsp47 mRNA accumulation was enhanced in neurula and tailbud embryos compared to earlier stages. This finding may be explained by the shift towards organogenesis during these stages. Whole mount <i>in situ</i> hybridization revealed hsp47 message along the dorsal region of the embryo, in the notochord and somites, as well as in the head region including the eye vesicle. Hsp47 mRNA induction in <i>Xenopus</i> embryos was also examined in response to heat shock. Hsp47 mRNA accumulated in response to heat shock immediately following the midblastula transition (MBT). In tailbud stages, hsp47 mRNA accumulated in the notochord, somites and head region. Northern blot analysis and whole mount <i>in situ</i> hybridization results revealed an expression pattern that coincided well with the development of collagen-rich tissues thereby substantiating the proposed role of HSP47 as a procollagen molecular chaperone.

TRAP1 is a Heat Shock Protein (HSP) chaperone to retinoblastoma but also associated to the tumor necrosis factor receptor. HSPs are primarily up regulated in cancer. Work in our lab noted a down regulation of TRAP1 in some non-small cell lung cancers compared to normal lung. The first aim of this project was to evaluate the effect of the loss of TRAP1 on cell proliferation using a spheroid model. The presence of TRAP1 in spheroids promoted cell proliferation and a faster onset of hypoxia. This suggests an oncogenic role for TRAP1 since rapid hypoxia development equates to poor prognosis. Micro array analysis showed that TRAP1’s loss was associated with increased transcrpition of the Junctional Mediating and Regulatory protein (JMY). JMY possesses an oncogenic property due to its ability to facilitate cell motility. Additionally it has tumor suppressor activity in promoting p53 activation. The second aim of this project was to produce an anti-JMY antibody and use it to characterize JMY and additionally verify the association between TRAP1 and JMY. JMY was found to be widely expressed in normal tissues and in many types of tumors. In neoplastic tissues, comparing primary versus metastatic tumors, JMY was found to have significantly higher expression in the metastatic compared with the primary tumors. A pilot study showed that nuclear co-expression of JMY and P53 was associated with shorter overall survival suggesting that a possible tumorigenesis mechanism could be via a deregulation/mutation of JMY/p53 or both. Finally, using 3 dimensional constructions, I demonstrated the distinct morphological difference between an angiogenic tumor and a non-angiogenic tumor. Additionally, I showed a characteristic cytoplasmic p53 sequestration in the non-angiogenic phenotype that is absent in the angiogenic phenotype. This could be the mechanism that the non-angiogenic tumor uses to adapt to hypoxia. This would imply that there is a potential for cancers to escape therapy by switching between these 2 phenotypes.

Increases in temperature damage plant cells, and plants react to heat stress by inducing of a number of protective mechanisms. In this study, it has been shown that heat damages cells both directly while the plants are being heated, and indirectly through heat induced oxidative stress during recovery from heating. Different stress response pathways are induced in each case. Evidence implicates salicylic acid, abscisic acid, ACC (a precursor of ethylene), calcium ions and active oxygen species in pathways resulting in thermotolerance (i.e. increased survival at high temperature). Addition of these potential second messengers results in increased survival and decreased oxidative damage after heating (as measured using the TBARS assay), while silencing the pathways through use of mutants, inhibitors or transgenes results in decreased thermotolerance. In vivo calcium measurements show cytosolic calcium transients only at initiation of cooling after heat stress. Calcium chloride also induces thermotolerance when added after heating, as does ACC. These substances can induce early increases in ascorbate peroxidase activity after heating, and induce expression of antioxidant genes. Thus they may play a role in heat induced oxidative stress response pathways in recovery. Two different pathways induced during heating appear to result in the expression of genes for heat shock proteins. The predominant pathway induced at 30oC involves ABA, while that at 35-40oC involves an oxidative burst generated through the NADPH oxidase, atrbohB. Addition of SA can induce HSP (heat shock protein) expression, but there is little evidence that this occurs endogenously in plant cells, although nahG plants unable to signal via SA are thermosensitive. Results in this study indicate that there are several signalling pathways associated with heat shock. The pathway induced during recovery from heating involves calcium ions and ethylene, and results in increased antioxidant capacity. During heating two pathways induce expression of HSPs: one involving abscisic acid and possibly salicylic acid and one involving an oxidative burst. At least one further pathway is believed to exist, which involves protein kinases and phosphatases as heat shock causes up-regulation of expression of certain genes for these signalling components.

Molecular chaperones, commonly known as heat shock proteins (HSPs), are essential for mammalian cells to maintain homeostasis, and HSPs function by inducing an ATPase-coupled structural change, followed by interactions with diverse co-chaperones and over 200 client proteins implicated in many critical signaling networks. These highly expressed HSPs participate in the onset and progression of several human diseases including cancer, and their connection with tumorigenesis has facilitated research and clinical trials related to targeting HSPs as a novel anti-tumor therapy. The predominant mechanism of chaperone inhibition is through either disruption of the HSP association with client protein or an altered binding state that ultimately leads to proteasome-mediated degradation. Importantly, chaperone inhibition results in the degradation of several client proteins that play critical roles in many of the pathways known as the Hallmarks of Cancer, such as proliferation, angiogenesis, invasion, metastasis, and drug resistance. Here, we discuss: (1) the current knowledge of HSPs, particularly studies related to Hsp90-targeted cancer therapy, (2) the targeting of Hsp90-mediated signaling interactions to prevent emergence of core Hallmarks of Cancer, (3) the recent progression of Hsp90 inhibitors in clinical trials. Finally, we propose combinatorial therapy, additional inhibitor discovery, and location-specific inhibition of HSPs as necessary next steps in chaperone-targeted research relevant to cancer therapy.

The cells are capable of very quick and specific reactions on stress conditions. Influence of translation, specifically initiation of translation by inhibition factors, is one of the main regulatory process. Two of eIF4E-binding proteins (4E-BP), Eap1p and Caf20p, are known as cap-dependent translation repressors in yeast Saccharomyces cerevisiae. We used in vivo fluorescent microscopy analysis to show different reaction of Caf20p and Eap1p to heat stress. Protein Caf20p does not react on heat shock and stays difused in cytoplasm. Contrary to Caf20p reaction, protein Eap1p accumulates in cytoplasm close to stress granules (SGs). This work shows that Eap1p is involved in stress granules assembly. In the absence of Eap1p, yeast cells react to the heat stress with small and less focused SGs. Dele- tion of CAF20 does not affect SG assembly. This points to specific function of SG in distribution of factors connected with stress reaction. Polysomal analysis shows that deletion of one of initiation translation repressors does not affect heat induced global repression of translation. In permisive condition deletion of EAP1 may cause defect in addition of 60S ribosomal subunits. Absence of protein Eap1p causes morphological defect. That point to a different reactions of Eap1p and Caf20p on heat stress and possible...

M.Sc. / Heat shock (HS) proteins and HS transcription factors (HSFs) have been coined as the ‘Achilles Heel’ for cancer therapy, since they have been found to be overexpressed in cancer cells and are required for cell survival during tumour progression and metastasis. Hsp70 and other members of the Hsp family have been shown to inhibit apoptosis at several different stages, contributing to resistance to chemotherapy. NSAIDs, like salicylates and aspirin, are used for the treatment and prevention of cancers such as breast cancer. SA has been shown to enhance HSF-DNA binding and results in the increased expression of heat-induced Hsp70 which is antiapoptotic. We hypothesise that SA treatment can result in the potentiation of Hsp70 in MCF-7 cells further increasing their resistance to apoptosis and thus the aim of this study was to investigate the dose-responsive effects of salicylic acid (SA) in the presence and absence of heat shock on components of the pro and antiapoptotic components of the apoptotic pathway. MCF-7 cells, which naturally overexpress Hsp70, were treated with several doses of SA in the presence and absence of a mild heat shock, followed by analysis of Hsp70 and several pro and antiapoptotic members of intrinsic and extrinsic apoptotic pathways, including Bcl-2, Bax, caspase 6 and 8, JNK, AIF and APAF-1. Induced Hsp70 accumulation by the SA treatments in the presence and absence of heat shock enhanced apoptosis in cells exposed to SA whereas higher concentrations of SA combination with heat shock induced necrosis and a decrease in Hsp70 accumulation in MCF-7 cells. Identification of the effects which specific concentrations of SA in the presence and absence of heat shock had on the apoptotic pathway constituents helped highlight potential pathways by which cell death could occur in MCF-7 cells through the downregulation of Hsp70. It is most likely that MCF-7 cell death is occurring due to the release of reactive oxygen species (ROS) which in turn lead to necrosis or death may be achieved via a cathepsin-B-mediated cell death pathway where both of these possibilities need to be further investigated.

Caulobacter crescentus é uma bactéria Gram-negativa de vida livre cujo ciclo celular depende de eventos de diferenciação celular. A célula pré-divisional assimétrica dá origem a duas células filhas morfológica e funcionalmente distintas: a célula-talo e a célula móvel. A expressão do operon groESL é regulada por choque térmico e durante o ciclo celular a temperaturas normais, sendo a transcrição máxima na célula pré-divisional, com níveis baixos na célula-talo. Numa linhagem superexpressando &#963;32, os níveis do mRNA e da proteína GroEL estão aumentados, indicando que a transcrição ocorre a partir de um promotor ativado por &#963;32. A região regulatória também apresenta uma sequência repetida invertida, CIRCE, em que mutações de ponto aumentam a transcrição apenas a temperaturas normais de crescimento, indicando o papel inibitório desse elemento. Fusões de transcrição groESL-/acZ com mutações em CIRCE deixam de apresentar regulação temporal, bem como a síntese de GroEL numa linhagem hrcA-, em que o gene codificando o provável repressor que se liga em CIRCE está interrompido. Estes resultados indicam que o sistem CIRCE/HrcA está envolvido com a regulação da expressão de groESL durante o ciclo celular. Tentativas de se construir uma linhagem com groEL interrompido não tiveram sucesso, indicando ser este um gene essencial em todas as temperaturas. Um mutante condicional de groESL foi construído por recombinação homóloga e, em condições restritivas, o crescimento é inibido, os níveis de DnaK aumentam e as células se tomam filamentosas, porém não foi observada lise celular. As proteínas essenciais que são dependentes de GroEL/GroES para atingirem sua conformação funcional ainda não foram determinadas. / Caulobacter crescentus is an aquatic free-living Gram-negative bacterium whose cell cycle depends on cell differentiation. The asymmetrical predivisional cell gives rise to two morphologically and functionally different daughter cells: the swarmer cell an the stalked cell. The expression of the groESL operon is induced by heat shock and is cell cycle controlled at normal temperatures, with maximal transcription in the predivisional cell and very low levels in the stalked cell. In this work, it was demonstrated that, in a strain overexpressing &#963;32, the levels of groESL transcripts and the synthesis of GroEL are increased, confirming that this factor is responsible for the transcriptional activation of the &#963;32 -like promoter of this operon, that also presents a inverted repeat called CIRCE in its regulatory region. groESL-lacZ transcription fusions with point mutations in CIRCE indicated a negative role of this cis-acting element only at normal growth temperatures, with a minor effect on heat shock induction. In addition, the expression of these fusions are no longer cell-cycle regulated, as well as GroEL synthesis in a strain which does not have the HrcA protein, the putative repressor that binds CIRCE, indicating that the CIRCE-HrcA system are involved in cell cycle regulation of groESL in C. crescentus. It was also shown that groEL is an essential gene at normal growth temperatures, since a strain with groEL disrupted is not viable. A conditional mutant was obtained by homologous recombination and in restrictive conditions growth is inhibited, DnaK levels are increased and the cells become filamentous, but no celllysis was observed. The proteins that require GroEL/GroES for proper folding have not been identified yet.

Nosso objetivo, neste trabalho, foi verificar se a interação das células dendríticas (DCs) com antígenos da Hsp60 induz um efeito sinérgico no direcionamento de uma resposta imune reguladora, no sistema humano. Células dendríticas humanas maduras (mDC) e imaturas (iDC e iDC IL-10) foram geradas, in vitro, a partir de monócitos de 15 de indivíduos saudáveis. Estas células foram caracterizadas quanto à (i) morfologia, (ii) imunofentotipagem, (iii) produção de citocinas e, (iv) capacidade de estimular aloproliferação. Analisamos a auto-reatividade de linfócitos T (LT) dirigida a diferentes DCs (mDC, iDC e iDC IL-10). Na interação de antígenos da Hsp60 com essas diferentes DCs, verificamos: (i) a capacidade de induzir a produção de citocinas pelas DCs e de inibir a sua produção espontânea, (ii) a auto-reatividade de linfócitos T dirigida a esses antígenos (proliferação e produção de citocinas), (iii) a expressão gênica de um painel de moléculas reguladoras (TGFb, receptor de TGF-b, IL-10 e GATA3) e inflamatórias (IFNg, TNF-a e T-bet) em linfócitos, T no contexto de células dendríticas imaturas. As mDC apresentaram expressão de CD83, maior expressão de CD80, e CD86, assim como induziram respostas alogenéicas mais intensas do que as DCs imaturas. Apesar de haver variabilidade na produção de citocinas, apenas as DC imaturas produziram espontaneamente IL-10, e as DCs maduras produziram mais freqüentemente IFN-g e TNF-a. Analisando o efeito dos antígenos da Hsp60 sobre a produção de citocinas, observamos tanto indução quanto inibição da produção de IFN-g, TNF-a, IL-4 e IL-10 nos três grupos de DC. Porém, a inibição predominou sobre a produção nos três grupos de DC. A auto-reatividade proliferativa de LT dirigida às diferentes DCs foi mais freqüente nas culturas com as DCs maduras (6/10) do que com as DCs imaturas (4/10). Também detectamos produção das citocinas IFN-g, TNF-a, e IL-2 para todos os grupos de células, porém, mais freqüentemente na auto-reatividade contra as DCs maduras. Diversos antígenos da Hsp60 foram capazes de inibir esta auto-reatividade. O peptídeo N7 teve um efeito dominante na inibição da auto-reatividade proliferativa de linfócitos T dirigida às mDCs. A auto-reatividade a antígenos da Hsp60, de um modo geral, foi maior com as DCs imaturas. Diversos antígenos foram capazes de induzir proliferação e produção de citocinas. Todavia, o peptídeo C3 foi imunodominante (6/10) na indução de resposta linfoproliferativa, no contexto das iDCs. A expressão gênica de moléculas reguladoras e inflamatórias foi verificada em linfócitos T, na auto-reatividade a antígenos da Hsp60. Observamos modificações importantes de praticamente todas as moléculas estudadas. Verificamos um predomínio de modificações reguladoras para os genes TGFb, TGF-bR, GATA3, TNF-a e T-bet. O peptídeo N7 induziu modificações dominantemente reguladoras em todas as condições em que ele foi testado. Em conclusão, verificamos que antígenos da Hsp60 têm efeito direto na produção de citocinas das diferentes DCs. Também têm a capacidade de ativar, simultaneamente, em linfócitos T, na interação com as células dendríticas, genes funcionalmente antagônicos. Isto reafirma a diversidade funcional da Hsp60. Ademais, identificamos o peptídeo N7 como potencialmente imunorregulador e o consideramos um candidato a ser testado em protocolos para indução de tolerância. / The aim of the present study was to determine whether the interaction of dendritic cells (DCs) with antigens derived from Hsp60 is capable of inducing a synergistic effect in directing a regulatory immune response, using a human system. Human DCs with mature (mDC) and immature (iDC and iDC IL-10) phenotype were generated in vitro from monocytes obtained from 15 healthy subjects. These cells were characterized according to (i) morphology, (ii) expression of surface markers, (iii) cytokine production, and (iv) ability to stimulate alloproliferation. We analyzed the autoreactivity of T lymphocytes (TL) directed against different DC types (mDC, iDC, and iDC IL-10). For the interaction of Hsp60 antigens with these different DCs, we determined: (i) the ability to induce cytokine production by DCs as well as to inhibit their spontaneous production, (ii) the autoreactivity of TL to these antigens (proliferation and cytokine production), and (iii) gene expression levels of a panel of regulatory (TGFb, TGF-b receptor, IL-10, and GATA3) and inflammatory (IFN-g, TNF-a, and T-bet) molecules by TL when stimulated by mDC. mDC expressed CD83 and showed higher levels of CD80 and CD86 and induced stronger allogeneic responses than immature DCs. Although cytokine production varied, only immature DCs spontaneously produced IL- 10, and mature DCs more frequently produced IFN- and TNF-. An analysis of the effects of Hsp60 antigens on cytokine production showed both induction and inhibition of production of IFN-g, TNF-a, IL-4, and IL-10 by the three sets of DCs; however, inhibition predominated over induction in all three DC groups. The proliferative autoreactivity of LT directed towards the different DCs was more frequent in cultures containing mDCs (6/10) than in those containing immature DCs (4/10). We also detected production of IFN-g, TNFa, and IL-2 by all groups of cells; however this was more frequent in the context of autoreactivity against mDCs. Several Hsp60 antigens were capable of inhibiting this autoreactivity. Peptide N7 had a dominant effect on the inhibition of the proliferative autoreactivity of LT directed towards mDCs. Autoreactivity to Hsp60 antigens was generally greater in cultures containing immature DCs. Several antigens were capable of inducing proliferation and cytokine secretion. However, peptide C3 was immunodominant (6/10) in the induction of a lymphoproliferative response in cultures containing iDCs. Gene expression of regulatory and inflammatory molecules was determined in LTs in the context of autoreactivity to Hsp60 antigens. There were important modifications in virtually all molecules studied. There was a predominance of regulatory-oriented changes in expression of TGFb, TGF-bR, GATA3, TNFa, and T-bet. Peptide N7 induced dominantly regulatory changes in gene expression in all conditions in which it was tested. In conclusion, we have shown that Hsp60 antigens have a direct effect on cytokine production by different DCs. These antigens are also able to activate, during the interaction of LT with DCs, genes that are functionally antagonistic. This finding reinforces the functional diversity of Hsp60. Furthermore, we have identified peptide N7 as potentially immunoregulatory, and consider it as a candidate to be tested in protocols for the induction of tolerance.

The human brain functions within a narrow range of temperatures and variations outside of this range incur cellular damage and death and, ultimately, death of the organism. Other organisms, like the poikilotherm Drosophila melanogaster, have adapted mechanisms to maintain brain function over wide ranges in temperature and, if exposed to high temperatures where brain function is no longer supported, these animals enter a protective coma to promote survival of the organism once the acute temperature stress is alleviated. This research characterized the role of different neuronal cell types, including glia, in the protection of brain function during acute hyperthermia, specifically looking at two protective pathways: the heat shock protein (HSP) pathway and the cGMP-dependent protein kinase G (PKG) pathway. Whole animal behavioral assays were used in combination with tissue-specific genetic manipulation of protective pathways to determine the specific cell types sufficient to confer protection of neuronal function during acute hyperthermia. Using the neuromuscular junction (NMJ) preparation, calcium imaging techniques were combined with pharmacological and genetic manipulations to test the hypothesis that alterations in ion channel conductance via endogenous mechanisms regulating the cellular response to high temperature stress alter neuronal function. Expression of foraging RNAi to inhibit PKG expression in neurons or glia demonstrated protection of function during acute hyperthermia measured behaviorally through the extension of locomotor function. This extension of function with the tissue-specific inhibition of PKG was also confirmed at the cellular level using the genetically encoded calcium indicator (GECI), GCaMP3, to image calcium dynamics at the NMJ, where preparations expressing foraging RNAi could continue to elicit changes in calcium dynamics in response to stimulation. Over the course of this study, the mechanism underlying a novel glial calcium wave in the peripheral nervous system was characterized in order to elucidate glia’s role in the protection of neuronal function during acute hyperthermia. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Durante o ciclo de vida da Leishmania, amastigotas vivem no interior de fagolisossomas de células fagocíticas de hospedeiros vertebrados, enquanto promastigotas vivem no interior do vetor invertebrado. Proteases intracelulares como as caspases são as principais efetoras no processo apoptótico. Metacaspases (MCAs) são formas evolutivas distantes das caspases de metazoários, presentes em protozoários, plantas e fungos, e vistas como potenciais alvos para combate dos parasitas sem prejuízo do hospedeiro. Ligantes e moduladores das metacaspases são até hoje desconhecidos. O Phage Display é uma técnica baseada na expressão de proteínas sintéticas nos capsidíos de fagos, usada com o propósito de selecionar ligantes de proteínas, células ou tecidos. Produzimos a metacaspase recombinante de Leishmania L. amazonensis e aplicamos Phage Display para buscar peptídeos ligantes dessa enzima. Esses peptídeos permitiram identificar potenciais proteínas ligantes da MCA, como quinases e cinesinas, que fornecem informações sobre a regulação e controle de sua atividade. Futuramente testaremos se peptídeos ativadores da MCA poderão induzir apoptose do parasita e serem usados como drogas para o tratamento da leishmaniose. / During its life cycle, Leishmania amastigotes live inside phagolysosomes of phagocytic cells of vertebrate hosts, while promastigotes live inside the invertebrate vector. Intracellular proteases such as caspases are key effectors in the apoptotic process. Metacaspases (MCAs) are distant evolutionary forms of metazoan caspases found in protozoa, plants and fungi, and seen as potential targets to destroy the parasites without damage to the host. Ligands and modulators of metacaspases are so far unknown. Phage Display is a technique based on the expression of synthetic proteins in the phage capsid, and is used for selecting ligands of proteins, cells or tissues. We have produced the recombinant metacaspase of Leishmania (L.) amazonensis and employed Phage Display to find peptide ligands of this enzyme. These peptides led to the identification of potential binding proteins of the MCA, such as kinases and kinesin, which provide information about the regulation and control of MCA´s activity. In the future we will test whether peptide activators of MCA nduce apoptosis of the parasite and can be used as drugs for the treatment of leishmaniasis.