Mandal, Manoj Kumar2026-02-192026-02-192026-01-08http://hdl.handle.net/10400.5/117216Tese de doutoramento em Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2026.A tuberculose (TB), é uma doença infeciosa causada nos humanos principalmente pela espécie bacteriana Mycobacterium tuberculosis (Mtb). A infeção continua a causar elevada mortalidade mundialmente e constitui um grande problema de saúde pública, apesar da existência de antibióticos e de vacinas preventivas. A TB ressurgiu na década de 1980 como a principal causa mundial de morte por um único agente infecioso, afetando milhões de pessoas anualmente após décadas de declínio. A pandemia do HIV foi uma das principais causas, pois esta infeção induz imunodepressão por depleção de linfócitos CD4+ e, consequentemente, o aumento da suscetibilidade à TB. Outros fatores contribuintes incluem a maior facilidade de viajar de regiões onde a TB é endémica e o surgimento de estirpes resistentes aos antibióticos após décadas da implementação do seu emprego. É urgente o desenvolvimento de novas estratégias terapêuticas para contribuir para melhorar o tratamento e aliviar a morbilidade e a mortalidade associadas a ambas as infeções. O objetivo deste trabalho é o de identificar estratégias terapêuticas alternativas que possam auxiliar no controlo das infeções por Mtb e HIV, particularmente durante a coinfeção, contexto este onde as interações medicamentosas e uma condição sindémica representam desafios relevantes. Assim, este trabalho focou-se no desenvolvimento de terapias direcionadas ao hospedeiro (HDTs) tendo como alvo inibidores de protease (IPs), incluindo saquinavir (SQV) redirecionado aqui para controlo da TB, e cistatinas, que são inibidores naturais de catepsinas lisossomais. Os resultados deste estudo sugerem que o SQV, o primeiro inibidor de protease utilizado para controlar a infeção pelo HIV, possui a capacidade de aumentar a atividade proteolítica das proteases endolisossomais, superando a inibição induzida durante a infeção por Mtb. Como o tratamento com SQV apresenta alguma toxicidade sistémica, foi desenvolvido um sistema de administração de fármacos lipossomais para direcionar o SQV aos macrófagos. Essa estratégia teve um impacto significativo na concentração intracelular do SQV, contribuindo substancialmente para a eliminação de Mtb, ao mesmo tempo em que levou a uma citotoxicidade reduzida.Tuberculosis (TB), primarily caused by the bacterial species Mycobacterium tuberculosis (Mtb), remains a widespread fatal illness and a major public health problem despite the availability of antibiotics and preventive vaccines. TB re-emerged in the 1980s as the world’s leading cause of death from a single infectious agent, affecting millions of people annually after decades of decline. The HIV pandemic was a major cause, as the infection led to immunodepression and to increased susceptibility to TB. Other contributing factors include the increased accessibility of travel from regions where TB is endemic, and the emergence of drug-resistant strains following decades of antibiotic therapy usage. The development of novel strategies is urgently required to improve treatment outcomes and alleviate the morbidity and mortality associated with both infections. The objective of this work is to identify alternative therapeutic strategies that may assist in the control of Mtb and HIV infections, particularly during coinfection were drug-drug interactions and a syndemic condition pose relevant challenges. Therefore, we focused on the development of host-directed therapies (HDTs) by targeting protease inhibitors (PIs), including saquinavir (SQV) repurposed here for TB, and cystatins which are natural inhibitors of lysosomal cathepsins. The findings of this study suggest that SQV, the first protease inhibitor used to control HIV infection, possesses the capacity to enhance the proteolytic activity of endolysosomal proteases overcoming the inhibition induced during infection by Mtb. Since treatment with SQV has some systemic toxicity, a liposomal drug delivery system was developed to target SQV to macrophages. This strategy had a significant impact on the intracellular concentration of SQV, achieving a substantial increase in Mtb killing, while concomitantly exhibiting reduced cytotoxicity. The observation that Mtb infection leads to a decreased proteolytic activity of lysosomal cathepsins in macrophages, and simultaneously some cystatins were overexpressed during the early phase of infection, specifically between 24 h and 48 h post-infection, led to a subsequent investigation into the modulation of CstC and CstF, which were identified as the most differentially expressed. Likewise, their depletion by RNA silencing has demonstrated to enhance the intracellular killing of Mtb, including during infection with multidrug-resistant clinical strains. Furthermore, it was demonstrated that depletion of CstF in macrophages could also impact the level of control over HIV in infected lymphocytes infection during co-culture. The results indicate that a decrease in the CstF released by phagocytes leads to an increase in the major pro-granzyme convertase, cathepsin C, of cytotoxic immune cells derived from peripheral blood lymphocytes. Consequently, an observed augmentation of the granzyme B cytolytic activity leads to a significant reduction in viral replication in HIV-infected CD4+ T-lymphocytes. Moreover, the development of drug delivery nanoparticle formulation based on chitosan used for the targeting of siRNA to primary macrophages results in a significantly higher level of silencing when compared to the conventional in vitro transfection method. Altogether, the results of this thesis suggest that the development of new HDT based on protease inhibitors could improve the control of infections with Mtb or HIV, including during coinfection. The graphical abstract summarizes the mechanisms by which protease inhibitors modulate host responses during TB/HIV coinfection. All except for the targeting of metalloproteases were investigated in this thesis.application/pdfeng101746954Mycobacterium tuberculosishost-directed therapiessaquinavircystatinsHIV coinfectiondoctoral thesis