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Neural injuries in cerebral malaria patients are a significant cause of morbidity and mortality. Nevertheless, a comprehensive research approach to study this issue is lacking, so herein we propose an in vitro system to study human cerebral malaria using cellular approaches. Our first goal was to establish a cellular system to identify the molecular alterations in human brain vasculature cells that resemble the blood–brain barrier (BBB) in cerebral malaria (CM). Through transcriptomic analysis, we characterized specific gene expression profiles in human brain microvascular endothelial cells (HBMEC) activated by the Plasmodium falciparum parasites. We also suggest potential new genes related to parasitic activation. Then, we studied its impact at brain level after Plasmodium falciparum endothelial activation to gain a deeper understanding of the physiological mechanisms underlying CM. For that, the impact of HBMEC-P. falciparum-activated secretomes was evaluated in human brain organoids. Our results support the reliability of in vitro cellular models developed to mimic CM in several aspects. These systems can be of extreme importance to investigate the factors (parasitological and host) influencing CM, contributing to a molecular understanding of pathogenesis, brain injury, and dysfunction., This research was funded by National funds through the Foundation for Science and Technology (FCT) SFRH/BD/131540/2017, SFRH/BD/5813/2020, COVID/BD/152416/2022 and UMINHO/BIM-CNCG/2022/143. This work has been funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBIPOCI-01-0145-FEDER-022122; by National funds, through the FCT—project UIDB/50026/2020 and UIDP/50026/2020. Moreover, this work was funded by IF/00143/2015/CP1294/CT0001, PTDC/SAU-PAR/2766/2021 and UIDB/04469/2020. O.M. is funded by the project NORTE-01- 0247-FEDER-045914, supported by POFC–COMPETE and FCT, under the programs PT2020 and NORTE2020. M.I.V. thanks FCT for her contract funding provided through 2020.03113.CEECIND., info:eu-repo/semantics/publishedVersion

Tese de doutoramento em Envelhecimento e Doenças Crónicas, In 2019, malaria caused half a million deaths worldwide, being elimination hampered by the ability of Plasmodium falciparum to evolve antimalarial resistance. The efficacy of artemisinin-based combination therapies (ACT) helped to reduce malaria mortality, however, resistance is a reality. Being ACT efficacy threatened, efforts to define the molecular basis of multidrug resistance and the search for new compound, ideally with new mechanisms of action, are urgently in need. Within the parasite, most of the available antimalarials act at the host’s intraerythrocytic stage. Here, many drugs are housed in the digestive vacuole of the parasite with flux promoted by transporter proteins, such as the Plasmodium falciparum multidrug resistance protein 1 (PfMDR1), a well-known ACT resistance player. We explored the interplay of known pfmdr1 resistance markers, namely, gene copy number variation with N86Y and Y184F single nucleotide polymorphisms to unravel the complex traits that might serve to maximize ACT resistance. Using genomic epidemiology, a global prevalence and temporal changes of pfmdr1 polymorphisms were assessed and, taking into account the information from this database, through a gene editing approach, we create in vitro edited parasite lines to evaluate the impact of these polymorphisms in the kinetics of the transporter. This data provided evidence of specific multicopy PfMDR1 with N86/184F haplotype, geographic selection and expansion in Southeast Asia. The genetic tools created could help on finding drugs with potential of reverting a multidrug resistance phenotype, as the herein explored synthetic compounds derived from steroids, a class of molecules with relevant biological activities. Structure–activity relationship led to the synthesis of steroid derivatives with promising antimalarial activity against the blood stage of the parasite’s life cycle with high selectivity and independent of PfMDR1. Exploring possible mechanisms of action of the best compound, revealed induction of oxidative stress inside the parasite and interference with the metabolic process that leads to hemozoin formation inside the digestive vacuole of the parasite. Overall, the findings presented could help tailor and optimize present antimalarial drug usage by taking into account the regional prevalence of pfmdr1 polymorphisms and highlights the high potential of the newly developed compounds, thereby underscoring the possibility to develop new antimalarial drugs based on steroids., Em 2019, a malária causou meio milhão de mortes mundialmente, sendo a eliminação dificultada pela capacidade do Plasmodium falciparum desenvolver resistência aos antimaláricos atuais. A eficácia da terapia de combinação baseadas em artemisinina (ACT) ajudou a reduzir a mortalidade provocada pela malária, no entanto, a resistência é uma realidade. A eficácia do ACT está comprometida, as tentativas para definir as bases moleculares da multirresistência e a procura por novos compostos, idealmente com novos mecanismos de ação, são urgentes. No parasita, a maioria dos antimaláricos atua no estadio intra-eritrocítico. Muitos são alojados no vacúolo digestivo do parasita através do fluxo promovido por proteínas transportadoras, como a “Plasmodium falciparum multidrug resistance protein 1” (PfMDR1), um fator envolvido na resistência aos ACTs. Neste trabalho foi explorada a interação de marcadores de resistência conhecidos no pfmdr1, ou seja, variação do número de cópias do gene com polimorfismos no nucleótido N86Y e Y184F para tentar descobrir as características que ajudam a maximizar a resistência aos ACTs. Usando epidemiologia genómica, a prevalência e alterações temporais dos polimorfismos no pfmdr1 foram avaliadas tendo em consideração a informação obtida desta base de dados, através de edição de genes, geramos in vitro parasitas editados para avaliar o impacto desses polimorfismos na cinética do transportador. Isto desvendou evidências acerca do haplótipo específico de multicópias com N86/184F, sobre a seleção geográfica e expansão no Sudeste Asiático. As ferramentas genéticas criadas podem auxiliar na descoberta de fármacos com potencial para reverter o fenótipo de resistência, como os compostos sintéticos derivados de esteróides explorados nesta tese, uma classe de moléculas com atividade biológica relevante. A relação estrutura-atividade levou à síntese de esteróides com uma atividade promissora contra o estadio intra-eritrocítico do ciclo de vida do parasita com grande seletividade e independência do PfMDR1. Os mecanismos de ação do melhor composto foi também explorados, revelando indução de stress oxidativo no parasita e uma interferência no processo metabólico que leva à formação de hemozoína. Concluindo, os resultados apresentados podem ajudar a adaptar e otimizar o uso dos antimaláricos atuais, tendo em consideração a prevalência regional dos polimorfismos no pfmdr1 e, destaca também o grande potencial dos compostos recentemente desenvolvidos, demonstrando a possibilidade de desenvolver novos antimaláricos baseados em esteróides., Financial support was provided by grants from the PD/BD/127826/2016, ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122; by National funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020; by the projects NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)

Work supported by project NORTE-01-0145-FEDER-028178, funded by NORTE 2020 Portugal Regional Operational Programme, under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund and by Fundação para a Ciência e Tecnologia (FCT).

[Excerpt] Early and accurate malaria diagnosis is critical for the disease control and elimination [1]. Microscopy and/or immuno-rapid tests remain the standard diagnosis [2, 3], nevertheless it requires a skin puncture for blood sampling and not sensitive enough for reliable detect lowdensity parasitemias, urging the need to develop more sensitive and non-invasive tools. Symptoms of the disease starts when parasites infect the red blood cell (RBC), suffering biochemical and morphological changes [4]. Parasite survival is dependent on hemozoin (Hz) formation as a by-product of heme detoxification process of the parasite upon haemoglobin (Hb) degradation and therefore a good unique feature to identify parasites presence in patients’. Taking advantage of the fact that the Hz and Hb molar extinction coefficients differ significantly, especially at certain wavelengths [4, 5], and their proportion is inversely related upon parasite maturation inside the RBC, each stage of malaria is characterized by specific absorbance and reflectance spectra, according to the Hb/Hz concentrations on the iRBC. [...], Work supported by project NORTE-01-0145-FEDER-028178, funded by NORTE 2020 Portugal Regional Operational Programme, under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund and by Fundação para a Ciência e Tecnologia (FCT), IP.

Dissertação de mestrado em Genética Molecular, Alkylating agents are one a major class of mutagens contributing to DNA damages and carcinogenesis. However, as alkylating agents are powerful inducers of DNA damage related cell death, and are commonly used in the chemotherapy. O6-methylguanine (O6meG) and O6- chloroethylguanine (O6ClethG) are the most cytotoxic lesions caused by Temozolomide (TMZ) and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), respectively. MGMT (O6-methylguanine-DNA methyltransferase) repair protein directly reverses O6alkylG lesions and, consequently, it is considered as a prognostic marker of resistance in cancer cells exposed to alkylating agents. Therefore, expression levels of this protein predict cancer cell susceptibility and the success of therapy. However, it has been observed that not all tumors with low MGMT activity respond to alkylating agents with increased cell death. Mismatch repair (MMR) system and functional status of p53 are also two most relevant factors in this response. The aims of this project were to study the genotoxic effects of alkylating drugs as detected by the CoMeth assay, and to determine the dependence of a functional MMR system and p53 in the sensitivity of cells to treatment. Thus, using a proliferating MMR-proficient cancer cell line Caco-2, we report that CoMeth assay allows the qualitative evaluation of O6meG lesions as well as O6ClethG, after their conversion to strand breaks. To determine whether the DNA damages (comet assay) and cell death (nuclear condensation assay) induced by (TMZ) and (BCNU) agents are mediated by MMR we used a MMR-deficient colon HCT116 cell line. We observed that even though MMR status seemed to be determinant for cells’ sensitivity to TMZ, this was not applied for the case of BCNU. Resistance to the O6-chloroethylating agent involves other mechanisms that are independent of functional MMR system. We found that induction of DNA damages by BCNU was independent of p53 status, however p53-wt cells showed more pronounced apoptosis compared with p53-null cells. Altogether, our data show that functional MMR system is required for genotoxicity (apoptosis) induced by TMZ. For BCNU, we show that the efficiency on cancer cells is not dependent on the MMR status but only the activity of MGMT. In p53-wt cells, cell death by BCNU was more pronounced. This project also demonstrated the value of the CoMeth assay as a tool for the study of anticancer chemotherapeutic drugs., Agentes alquilantes são uma das maiores classes de mutagénicos contribuindo para os danos no DNA e para a carcinogénese. Contudo, como os agentes alquilantes são indutores potentes de danos no DNA relacionados com a morte celular eles são frequentemente usados na quimioterapia. O6-metilguanina (O6meG) e O6-cloroetilguanina (O6-cletG) são as lesões mais citotóxicas causadas pela Temozolomida (TMZ) e 1,3-bis-(2-cloroetil)-1-nitrosureia (BCNU), respetivamente. MGMT (O6-metilguanina-DNA metiltransferase) repara a proteína e reverte diretamente as lesões O6-alquilG e, consequentemente, é considerado um marcador de prognóstico de resistência em células cancerígenas expostas a agentes alquilantes. Desta forma, os níveis de expressão desta proteína preveem a suscetibilidade das células cancerígenas e o sucesso da terapia baseada em agentes alquilantes. Contudo, tem sido observado que nem todos os tumores com baixa actividade da MGMT respondem a agentes alquilantes com morte celular aumentada. O sistema de reparação Mismatch repair (MMR – do inglês mismatch repair) e o estado funcional do p53 são também dois dos fatores mais relevantes nesta resposta. Os objetivos deste projeto foram estudar os efeitos genotóxicos de drogas alquilantes, sendo detetadas pelo ensaio do Cometa e determinar a dependência do estado funcional do sistema Mismatch repair e do p53 na sensibilidade às drogas alquilantes. Desta forma, usando uma linha celular cancerígena proliferativa Caco-2, com um sistema MMR eficiente, demonstramos que o ensaio do Cometa permite uma avaliação qualitativa das lesões O6meG bem como as O6-cletG, depois da sua conversão em quebras na cadeia de DNA. Para determinar se os danos no DNA (ensaio do cometa) e morte celular (ensaio de condensação nuclear) induzidos pelos agentes metilantes (TMZ) e cloroetilantes (BCNU) são mediados por reparação MMR usamos a linha celular HCT116 deficiente no sistema MMR. Observamos que apesar do estado MMR parecer ser determinante para a sensibilidade das células à TMZ, este facto não foi observado para o caso do BCNU. A resistência ao agente O6-cloroetilante envolve outros mecanismos que são independentes do sistema funcional MMR. Verificamos que a indução dos danos no DNA pelo BCNU foram independentes do estado do p53, contudo as células com p53 ativo apresentaram uma apoptose mais pronunciada comparado com as células com p53 inativo. De um modo geral os nossos resultados mostram que o sistema funcional MMR é necessário para a genotoxicidade (apoptose) induzidos pela TMZ. Para o BCNU, mostramos que a eficiência nas células cancerígenas não é dependente do estado do MMR mas apenas da actividade da MGMT. Nas células com p53 ativo a morte celular induzida pelo BCNU foi mais pronunciada. Este estudo também demonstrou o papel do ensaio do cometa como ferramenta no estudo de drogas anticancerígenas.