These studies have characterized the peptide transport systems of Candida albicans, with a view to the rational design of peptide antifungal agents exploiting the 'smugglin' concept. In initial studies, a series of polyoxin complexes (peptide-nucleoside antibiotics) and individual components, were isolated from a batch of agricultural fungicide (Polyoxin Z). Isolated fractions were toxic to a particulate chitin synthetase preparation from Candida albicans. Different strains of Candida albicans exhibited varied sensitivities to a series of peptide analogues. From a sensitive strain, B2630, spontaneous mutants were selected for resistance to each analogue; certain mutants showed cross-resistance to other analogues and associated defects in peptide transport. A bacilysin-resistant mutant was cross-resistant to the other analogues and to m- fluorophenylalanylalanylalanine a but retained sensitivity to m- fluorophenylalanylalanylalanine. This mutant showed defective dipeptide transport but normal oligopeptide transport, and was unable to utilize Ala-Ala as a sole nitrogen source. Thus, Candida albicans has distinguishable mechanisms for dipeptide and oligopeptide transport which can be exploited for uptake of peptide-drug adducts. Peptide transport was shown to be stimulated by the presence of peptides (peptone) in the growth medium. On transferring cells from minimal to peptone medium, this stimulatory effect was shown to be rapid, independent of protein synthesis and to override ammonia regulation of peptide transport. The reduction of transport activity on transferring cells from peptone to minimal medium was also rapid. It was speculated that regulation of peptide transport is achieved by a rapid, reversible activation of preformed transport components, or a mechanism of exocytotic insertion and endocytic retrieval of preformed transporters. The effect of protein-modification reagents on transport activity was also examined. Dipeptide transport was specifically inhibited by N-ethyl-5-phenylisoxazolium-3'-sulphonate (Woodwards Reagent K), offerring potential for the specific labelling of the component(s) of this system. Peptide transport was shown not to be sensitive to osmotic shock though a series of uncharacterized polypeptides was released by the shock treatment.

Background: Respiratory syncytial virus (RSV) is one of the leading causes of acute lower respiratory tract infection and childhood hospitalization worldwide. However, there are currently no vaccines or antivirals available to prevent or treat RSV infections. Of the 11 proteins encoded by RSV’s negative-sense single-stranded RNA genome, the nucleoprotein, phosphoprotein, and large polymerase interact through well characterized domains to form the RNA-dependent RNA polymerase complex. This polymerase complex is essential for viral replication and virulence, which makes it an excellent antiviral target. Previous studies have shown that the nucleoprotein-phosphoprotein interaction of the polymerase complex can be disrupted by synthetic peptides of the last 21 C-terminal (P220-241) or the first 29 N-terminal (P1-29) amino acids of the phosphoprotein. Objective: The Mahony lab has also previously demonstrated that P220-241 conjugated to a maltose binding protein (MBP) and HIV-1 Tat cell penetrating peptide (CPP) could inhibit up to 90% of RSV A replication in vitro. However, the bacterial derived MBP is immunogenic. This study builds on these findings by developing and evaluating the efficacy of a P220-241 peptide mimetic conjugated to human thioredoxin (hTrx) carrier protein and a P1-29 peptide mimetic conjugated to MBP. Methods and Results: Inverse PCR and In-Fusion® cloning was used to clone a hTrx-P220-241 plasmid, which was then expressed as a recombinant protein and purified by affinity chromatography for functional analysis. HTrx-P220-241 was shown to specifically interact with RSV nucleoprotein in a glutathione S-transferase (GST) pull down assays and it could successfully enter into LLC-MK2 cells. However, upon challenge with RSV A, LLC-MK2 cells that were incubated with increasing concentrations of hTrx-P220-241 did not inhibit RSV A replication when assessed by indirect immunofluorescence microscopy. The MBP-P1-29 construct did not exhibit any significant cytotoxicity in LLC-MK2 cells nor BEAS-2B cells. Upon challenge with RSV A, LLC-MK2 cells and BEAS-2B cells pre-treated with MBP-P1-29 demonstrated a dose-dependent inhibition of RSV replication in vitro, with a percent inhibition of infection of 80% and 60% respectively. Furthermore, MBP-P1-29 also reduced the release of infectious progeny virion by up to 74% in LLC-MK2 cells and 34% in BEAS-2B cells. Conclusion: Phosphoprotein peptide mimetics targeting essential nucleoprotein-phosphoprotein interaction are a promising approach in the development of therapeutic treatments for RSV. In this study, a P220-241 peptide mimetic conjugated to a human thioredoxin scaffold protein was not able to inhibit RSV A replication while a P1-29 peptide attached to a maltose binding protein was effective in reducing RSV replication in vitro. Thus, further studies are required to evaluate a P1-29 peptide mimetic against different RSV A and B strains and to find an appropriate human carrier protein to attach it to. / Thesis / Master of Science (MSc) / Respiratory syncytial virus is a respiratory illness that is one of the leading causes of childhood hospitalization worldwide. RSV infects almost all infants at least once by the age of two. It can also repeatedly infect individuals throughout their lives, which puts the elderly and individuals with weak immune, cardiac or pulmonary systems at risk. There are also no approved vaccines or antiviral treatments available to prevent or combat a RSV infection, which highlights the pressing need for the development of new antiviral drugs. This thesis focuses on developing and evaluating the efficacy of two different antiviral peptides, which both target and disrupt the formation of the viral machinery required for the replication of the RSV genome.

No description available.

<p>Virulence in <em>Staphylococcus aureus </em>is mediated by the <em>accessory gene regulator </em>(agr) quorum sensing system. This regulatory system is activated by a secreted thiolactone peptide termed autoinducing peptide (AIP) and its receptor histidine kinase, AgrC. Interaction of extracellular AIP with a cognate AgrC receptor generates an intracellular signal that is transduced by conformational changes and phosphorylation events in a two-component sensor histidine kinase system. At the heart of the <em>agr</em> quorum-sensing cascade lies the two-component histidine kinase, AgrC, and the response regulator protein, AgrA. Interaction of AgrC and AgrA, and the resulting phosphotransfer event results in expression from the divergent promoters P2 and P3, inducing expression of the master quorum-sensing regulator RNAIII and upregulating the <em>agr</em> operon respectively. Signal transduction systems function as intracellular information-processing pathways that link sensation of external stimuli to specific adaptive processes. In <em>S. aureus</em> , these include the up-regulation of virulence factors and hemolysis production, biofilm formation, and colonization-based regulation of surface proteins and adhesion factors. As such, the interactions of these systems have become key targets in the design of small inhibitor compounds.</p> <p>Through the creation of a protein truncation series, we proposed the development of a small protein for the inhibition of key protein-protein interactions involved in <em>S. aureus</em> <em>agr</em> two-component signaling. Herein, we demonstrate the efficacy of these protein truncations as dominant negative inhibitors of AgrC:AgrA interactions, likely acting as a dominant phosphoacceptor in place of endogenous AgrA. We provide evidence of this function through <em>in vitro </em>hemolysis assays and phosphate-detection based gel electrophoresis.</p> / Master of Science (MSc)

The arsenal of drugs available for treating Leishmania infections is limited and presents high toxicity. Therefore, new, effective, and less toxic leishmaniasis treatments are still needed. In this work, two series of derivatives, containing a semicarbazone or hydrazide-N- acylhydrazone scaffolds was designed and synthetized as protease inhibitors. From these series, derivatives LASSBio 1483, LASSBio 1705, LASSBio 1707 and LASSBio 1736 highlighted, showing in vitro and in vivo leishmanicidal activities. Thus, these derivatives presented a potent leishmanicidal activity against amastigotes of L. major (IC50 of 1.5 µΜ to LASSBio 1483, 8.5 µΜ to LASSBio 1705 and 1.,9 µΜ to LASSBio 1707) , L. amazonensis (IC50 of 3.5 µΜ to LASSBio 1483 and 84.0 µΜ to LASSBio 1736), L. braziliensis (IC50 of 31.7 µΜ to LASSBio 1483, 8.0 µΜ to LASSBio 1705 and 5.3 µΜ to LASSBio 1736) and L. chagasi (IC50 of 53.3 µΜ to LASSBio 1707 and 57,6 µΜ to LASSBio 1736). Also, the leishmanicidal activity of derivatives LASSBio-1483, LASSBio 1705, LASSBio 1707 and LASSBio 1736 were mediated via induction of apoptosis as evidenced by externalization of phospholipids, despolarization of mitochondrial membrane and elevation of activation of caspases. The ultrastructural morphological effects against the parasite of LASSBio 1483 and LASSBio 1736 against L. chagasi promastigotes were also verified. The treatment of L. amazonensis -infected BALB/c mice with derivatives LASSBio 1483 (effect of 30.5% and 33.3% in infected ear, by p.o and i.p., respectively), LASSBio 1705 1483 (effect of 58.5% in infected ear and 61.1% in lymph node, i.p.), LASSBio 1707 (effect of 56.5% in lymph node, i.p.) and LASSBio 1736 (effect of 53.6% in infected ear, i.p.)or the treatment of L. chagasi - infected hamsters with LASSBio 1707 (effect of 53.6, i.p.)and LASSBio 1736 (effect of 46.0%, i.p.) led to a significant reduction of parasite burden when compared to controls that received PBS. The treatment with these derivatives did not result in hepatic or renal toxicity in these animals models of leishmaniasis. These data make LASSBio 1483, LASSBio 1705, LASSBio 1707 and LASSBio 1736 new lead-candidates against cutaneous and visceral leishmaniasis. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O arsenal de fármacos disponíveis para o tratamento das infecções por Leishmania spp. é limitada e de elevada toxicidade. Portanto, novos tratamentos, eficazes e menos tóxicos para leishmaniose ainda são necessários. Neste trabalho, duas séries de derivados contendo as subunidades semicarbazona ou hidrazida-N-acilhidrazona foram desenhados e sintetizados como inibidores de proteases. A partir destas séries, os derivados LASSBio 1483, LASSBio 1705, LASSBio 1707 e LASSBio 1736 se destacaram, mostrando atividade leishmanicida in vitro e in vivo . Assim, esses derivados apresentaram atividade leishmanicida potente contra amastigotas de L. major (CI 50 de 1,5 µΜ para LASSBio 1483, 8,5 µΜ para LASSBio 1705 e 15,9 µΜ para LASSBio 1707) , L. amazonensis (CI 50 de 3,5 µΜ para LASSBio 1483 e 84,0 µΜ para LASSBio 1736) , L. braziliensis (CI 50 de 31,7 µΜ para LASSBio 1483, 8,0 µΜ para LASSBio 1705 e 5,3 µΜ para LASSBio 1736) e L. chagasi (CI 50 de 53,3 µΜ para LASSBio 1707 e 57,6 µΜ para LASSBio 1736). Além disso, a atividade leishmanicida dos derivados LASSBio 1483, LASSBio 1705, LASSBio1707 e LASSBio 1736 foi mediada via indução de apoptose como evidenciado pela externalização de fosfolipídeos de membrana, despolarização da membrana mitocondrial e ativação de caspases. Os efeitos morfológicos ultra-estruturais de LASSBio 1483 e LASSBio 1736 em promastigotas de L. chagasi também foram verificados. O tratamento de camundongos BALB/c infectados por L. amazonensis com os derivados LASSBio 1483 (efeito de 30,5% e 33,3% na orelha infectada, por v.o. e i.p., respectivamente), LASSBio 1705 (efeito de 58,1% na orelha infectada e de 61,1% no linfonodo, i.p.) , LASSBio 1707 (efeito de 56,5% no linfonodo, i.p.) e LASSBio 1736 (efeito de 38,8% na orelha infectada , i.p.) ou o tratamento de hamsters infectados por L. chagasi com LASSBio 1707 (efeito de 53,6%, i.p.) e LASSBio 1736 (efeito de 46,0%, i.p.), na dose de 30 µmols/kg/dia, levaram a uma redução significativa da carga parasitária quando comparados aos controles que receberam PBS. O tratamento com estes derivados não resultaram em toxicidade hepática ou renal nestes modelos animais de leishmaniose. Estes dados fazem de LASSBio-1483, LASSBio-1705, LASSBio-1707 e LASSBio-1736 novos candidatos a protótipos a fármacos contra leishmaniose cutânea e visceral

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Na presente tese foram desenvolvidas três plataformas para a construção de biossensores visando o diagnóstico molecular da hepatite C, hepatite B e artrite reumatoide, por técnicas eletroquímicas, ópticas e microscópicas, usando amostras reais. Os genossensores para hepatites C e B foram desenvolvidos sobre a superfície de um eletrodo de ouro modificado com nanomateriais, sendo esses o óxido de grafeno e o óxido de grafeno reduzido, respectivamente. Em todos os biossensores propostos a interação da sonda com o alvo foi efetivamente verificada pelas diferentes técnicas. No caso do genossensor para hepatite C, o óxido de grafeno foi modificado quimicamente com etilenodiamina e apresentou limites de detecção e quantificação de 1:483 (v/v) e 1:145 (v/v), respectivamente, usando amostras de soro de pacientes positivos. A interação da sonda específica do HCV: gRNA causou uma redução na amplitude de resposta de corrente de cerca de 2,9 vezes quando comparada ao controle negativo, usando a VPD. O genossensor para a hepatite B a sonda foi imobilizada sobre eletrodo de ouro contendo óxido de grafeno reduzido, ouro descoberto e nanoparticulas de ouro. A análise usando VPD indica que a adição de DNA genômico de HBV provocou um aumento de cerca de 1,4 vezes na amplitude de corrente de pico quando comparado ao controle negativo. Em adição, análises de SPR mostraram que as amostras positivas de HBV resultaram em uma alteração de cerca de 15 vezes em comparação com as amostras negativas. No biossensor desenvolvido para o diagnóstico da artrite reumatoide foi utilizado um eletrodo de grafite modificado com um filme poli(3-hidroxibenzóico), no qual foi imobilizado um peptídeo mimético que reconhece o anticorpo anti-CAIII. O sensor mimético desenvolvido permitiu a distinção entre amostras positivas e negativas para a artrite reumatóide, uma vez que apresentou uma diminuição expressiva no sinal de corrente de cerca de 2,2 vezes, quando comparado ao soro negativo. Assim, foi possível desenvolver plataformas analíticas, seletivas e específicas fornecendo novas abordagens para o diagnóstico clínico e aplicações point-of-care para o monitoramento de doenças inflamatórias e infecciosas. / In the present thesis, three biosensing platforms aiming the molecular diagnosis of hepatitis C, hepatitis B and rheumatoid arthritis were developed by electrochemical, optical and microscopic techniques using real samples. The genosensors for the diagnosis of hepatitis C and B were developed on a gold electrode modified with nanomaterials, being these graphene oxide and reduced graphene oxide, respectively. In all proposed biosensors the interaction of the probe with the target was effectively verified by the different techniques. In the case of the genossensor for hepatitis C, graphene oxide was chemically modified with ethylenediamine and showed limits of detection and quantification of 1:483 (v/v) and 1:145 (v/v), respectively, using serum samples from positive patients. The interaction of the HCV probe and the gRNA caused a reduction in current response amplitude of about 2.9 fold as compared to the negative control, using the DPV. The genossensor for hepatitis B probe was immobilized on a gold electrode containing reduced graphene oxide, gold disks and gold nanoparticles. Analysis using DPV indicates that the addition of HBV gDNA caused an increase of about 1.4 times in peak current amplitude, when compared to the negative control. In addition, SPR analyzes showed that positive samples of HBV resulted in a change of about 15- fold compared to negative samples. In the biosensor developed for the diagnosis of rheumatoid arthritis, a graphite electrode modified with a poly (3-hydroxybenzoic) film was used, in which a mimetic peptide that recognizes the anti-CAIII antibody was immobilized. The developed mimetic sensor allowed the distinction between positive and negative samples for rheumatoid arthritis, since it presented an decrease in the current signal of about 2.2 times, when compared to the negative serum. Thus, it was possible to develop analytical, selective and specific platforms, providing new approaches for clinical diagnosis and point-of-care applications, for the monitoring of inflammatory and infectious diseases. / Tese (Doutorado)