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Doctoral Thesis
Full name
Fernanda Viana Cabral
Knowledge Area
Date of Defense
São Paulo, 2021
Ribeiro, Martha Simões (President)
Fontes, Adriana
Lindoso, José Angelo Lauletta
Smith, Terence Kenneth
Title in English
Towards new therapeutic strategies for cutaneous leishmaniasis
Keywords in English
chitosan nanoparticles
cutaneous leishmaniasis
nitric oxide
photodynamic therapy
Abstract in English
Cutaneous leishmaniasis (CL) is a zoonotic disease developed by protozoa parasites of genus Leishmania. It promotes destructive and ulcerated lesions with limited treatment options. There is an urgent need for the development of topical, cost-effective and efficacious treatments with minimized side effects to treat affected patients. The parasite-host interaction is of great importance since Leishmania parasites survive and replicate within host macrophages. As phagocytic cells, the activated macrophages produce reactive oxygen species (ROS) and nitric oxide (NO), which are toxic to pathogens, hence preventing parasites proliferation. However, Leishmania parasites can evade the host immune response and subvert antimicrobial macrophage defenses, thereby surviving within these cells even in harsh conditions. Indeed, the role played by ROS and NO in the control of CL has been under debate over the past years, emerging as potential alternatives to tackle this important neglected disease. In this regard, we aimed to evaluate the role of both NO and ROS towards antileishmanial activity using two different therapeutic strategies: (1) nitric oxide-releasing chitosan nanoparticles (NONPs) and (2) antimicrobial photodynamic therapy (PDT). For this, we focused on development and investigation of the potential of NONPs in vitro and in vivo against Leishmania amazonensis, one of the causative agents of CL. To assess the role of ROS, photodynamic therapy was investigated against different Leishmania species. Firstly, we evaluated the potential of organic light-emitting diodes (OLEDs) as a novel light source to inactivate in vitro promastigotes of L. major and L. amazonensis, using three phenothiazine dyes: Methylene blue, new methylene blue and 1,9-dimethyl methylene blue (DMMB). Then, we addressed the underlying mechanisms of DMMB-PDT upon promastigotes of L. amazonensis wild-type (WT) and miltefosineresistant (MFR) strains. DMMB-PDT effectiveness was also evaluated against intracellular amastigotes of WT and MFR together with cytotoxicity assay on mammalian cells. Our findings demonstrate that either NONPs or PDT are promising strategies to target CL and should be further explored for future preclinical and clinical trials.
Title in Portuguese
Buscando novas estratégias terapêuticas para o tratamento de leishmaniose cutânea
Keywords in Portuguese
leishmaniose cutânea
nanopartículas de quitosana
óxido nítrico
terapia fotodinâmica
Abstract in Portuguese
Leishmaniose é uma zoonose desenvolvida por protozoários do gênero Leishmania. A leishmaniose cutânea (LC) abrange lesões destrutivas e ulceradas que podem evoluir para condições mais graves culminando em óbito dos hospedeiros. Tratamentos alternativos têm sido implementados com a finalidade de promover maior eficácia e menores efeitos colaterais aos pacientes. A interação patógeno-hospedeiro é de fundamental importância já que os parasitos se multiplicam no interior de macrófagos dos hospedeiros. Essas células são capazes de produzir grandes quantidades de oxigênio (ROS) e óxido nítrico (NO), promovendo a morte de diversos patógenos. Entretanto, esses protozoários são capazes de evadir o sistema imune do hospedeiro sobrevivendo no interior dessas células mesmo em ambientes hostis. Dessa forma, o equilíbrio entre parasito e resposta imune do hospedeiro possui fundamental importância na susceptibilidade e resistência do indivíduo à infecção por Leishmania. De fato, ROS e NO possuem uma função muito relevante no controle da leishmaniose. Nesse contexto, nós nos propusemos a avaliar as funções de ambas as moléculas como potenciais agentes terapêuticos no combate à LC. No presente estudo foram sintetizadas nanopartículas contendo doadores de óxido nítrico (NONPs) e avaliadas na inativação in vitro e in vivo de L. amazonensis. Para avaliarmos o papel das ROS, os efeitos da terapia fotodinâmica (PDT) foram investigados contra diferentes espécies do protozoário. Inicialmente, avaliamos o potencial de LEDs orgânicos (OLEDs) como fonte de luz para PDT na inativação das espécies de L. amazonensis e L. major utilizando três fotossesibilizadores pertencentes à classe das fenotiazinas: Azul de metileno, novo azul de metileno e 1,9-dimetil azul de metileno (DMMB). Posteriormente investigamos os possíveis mecanismos de ação envolvidos na morte de promastigotas de L. amazonensis WT e resistentes à miltefosina (MFR). Finalmente, investigamos a eficácia do DMMB-PDT na morte de amastigotas intracelulares de L. amazonensis WT e MFR. Ensaios de citotoxicidade em fibroblastos e macrófagos também foram investigados. Nossos resultados demonstram que tanto as NONPs quanto a PDT são estratégias promissoras para o tratamento de LC, portanto, podem ser exploradas em futuros ensaios pré-clínicos e clínicos.
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