Dissecting the role of lipocalin in Plasmodium falciparum

[Excerpt] Malaria, caused by Plasmodium parasites, is an infectious disease responsible for millions of cases and deaths yearly. Despite the efforts, the disease control has been continuedly threatened by the resilient capacity of the parasite to acquire resistance to antimalarial drugs treatment, which target the symptomatic intraerythrocytic developmental cycle.This work was supported by NORTE-01-0145- FEDER-028178 funded by NORTE 2020 Portugal Regional Operational Program under PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund and the by Fundação para a Ciência e Tecnologia (FCT). V Baptista thanks FCT for the SFRH/BD/145427/2019 grant. MI Veiga and S Catarino thank FCT for their contract funding provided through 2020.03113.CEECIND and 2020.00215.CEECIND, respectively

Optical spectrophotometry into hemozoin: towards sensitive malaria diagnosis

The lack of prompt and sensitive diagnosis hampers malaria control and elimination, highlighting the need for next generation technology alternative for the established optical microscopy and Rapid Diagnostic Tests (RDTs) as means of in situ point-of-care malaria parasite detection. Hemozoin (Hz), a byproduct of hemoglobin (Hb) degradation inside human infected red blood cells (RBCs), has been extensively explored as a malaria biomarker. Along with malaria parasite maturation inside the RBC, Hb and Hz proportion is inversely related, which originates specific optical spectra of healthy and infected samples. Herein, we characterized the optical spectra of Plasmodium falciparum-infected RBCs, aiming the development of an innovative diagnostic device, detecting malaria without finger prick blood sampling, measuring directly in patients’ skin. Absorbance and reflectance spectrophotometry demonstrate their potential by increasing the limit of detection (LoD: 12 parasites/μL of RBC) when compared with microscopy or RDT (LoD: 50-200 parasites/μL of RBC). This sensitivity, coupled with the possible integration into a low-cost, fast, and non-invasive diagnostic device meets the growing clinical demands for malaria control and elimination.This work was supported by Fundação para a Ciência e Tecnologia (FCT) national funds, under the national support to R&D units grant, through the reference project UIDB/04436/2020 and UIDP/04436/2020. It is also supported by NORTE-01-0145- FEDER-028178 funded by NORTE 2020 Portugal Regional Operational Program under PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund and the FCT. V. Baptista thanks FCT for the SFRH/BD/145427/2019 grant. Maria Isabel Veiga thanks FCT for her contract funding provided through 2020.03113.CEECIND. Susana Catarino thanks FCT for her contract funding provided through 2020.00215.CEECIND

Deeper into hemozoin formation pathway of Plasmodium falciparum

Plasmodium falciparum malaria remains a public health problem, accounting for half millions of lives yearly. Artemisinin-combination therapies are the front-line treatment against malaria, which efficacy is now threatened by parasite’s-acquired resistance. Most antimalarial drugs target the symptomatic intra-erythrocytic stage of infection. During parasite development inside the human erythrocytes, parasites digest hemoglobin as a source of nutrients. This causes the release of toxic free heme, which the parasite detoxifies into inert crystals, called hemozoin. It is known that parasites with abnormalities in this pathway are committed to death. However, despite its importance, the orchestration of hemoglobin digestion into hemozoin formation has not been entirely resolved, and its players have not been identified. Through reverse genetics, we explored the role of P. falciparum aspartic proteases and other genes thought to be involved in the formation of hemozoin and their capacity to modulate drug response. We believe that a deeper understanding of this pathway is of great interest and relevant for the identification of new targets, both for diagnosis and for the development of antimalarial drugs. Furthermore, it will also provide insights into drug resistance, overall contributing for better malaria control and management.This work was supported by Fundação para a Ciência e Tecnologia (FCT) national funds, under the national support to R&D units grant, through the reference project UIDB/04436/2020 and UIDP/04436/2020. It is also supported by NORTE-01-0145-FEDER-028178 funded by NORTE 2020 Portugal Regional Operational Program under PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund and the FCT. V. Baptista thanks FCT for the SFRH/BD/145427/2019 grant. Maria Isabel Veiga thanks FCT for her contract funding provided through 2020.03113.CEECIND. Susana Catarino thanks FCT for her contract funding provided through 2020.00215.CEECIND

Multilayer thin-film optical filters for reflectance-based malaria diagnostics

Malaria diagnosis relies on optical microscopy and/or rapid diagnostic tests based on detecting specific malaria antigens. The clinical sensitivity of these methods is highly dependent on parasite density, with low levels of detection at low parasite density, challenging the worldwide malaria elimination efforts. Therefore, there is a need for diagnostic methods with higher sensitivity, demanding innovative diagnostics devices able to detect malaria at low parasite density and at early stages of the disease. We propose an innovative optical device for malaria diagnosis, based on optical reflectance spectrophotometry, for the detection of parasites through the quantification of haemozoin. For this purpose, a set of eight thin-film optical filters, based on multilayer stacks of MgO/TiO2 and SiO2/TiO2 thin-films, with high transmittance and low full width at half maximum (FWHM) at specific wavelengths, was designed and fully characterized (both numerically and experimentally). A preliminary assessment of its potential to reconstruct the original spectra of red blood cells was performed, both in uninfected and Plasmodium falciparum-infected samples. The obtained results show that, although the experimental filters have a non-ideal performance characteristic, they allow us to distinguish, based on only 8 discrete points in the optical spectrum, between healthy and malaria infected samples, up to a detection limit of 12 parasites/μL of red blood cells. Those results enhance the potential of using such a device for malaria diagnostics, aiming for non-invasiveness.Project NORTE-01-0145-FEDER-028178 funded by NORTE 2020 Portugal Regional Operational Program under PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund and the Fundação para a Ciência e Tecnologia (FCT), IP. This work was also supported by national funds, through the Portuguese FCT, under the reference projects UIDB/04436/2020, UIDP/04436/2020, UIDB/50026/2020 and UIDP/50026/2020, and by the ICVS Scientific Microscopy Platform, member of the national infrastructure PPBIPortuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122). V. Baptista thanks FCT for the SFRH/BD/145427/2019 grant. Maria Isabel Veiga thanks FCT for her contract funding provided through 2020.03113.CEECIND. Susana Catarino thanks FCT for her contract funding provided through 2020.00215.CEECIN

Characterization of the malaria parasite optical features for development of a non-invasive diagnostic device

[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

Hemozoin: the future in malaria diagnosis

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)

Explorar a levedura como modelo para estudar a regulação da proteína pro-apoptótica humana Bax

Dissertação de mestrado em Bioquímica Aplicada (área de especialização em Biomedicina)Cell death has long been considered crucial for proper tissue shaping during embryonic development and for normal turnover of the cells in several tissues. Moreover, mis-regulation of apoptosis has been implicated in a diversity of abnormal functions and in the progression and development of diseases. Indeed, a decrease in apoptosis has been associated with ageing and tumour progression, whereas an increase in apoptosis is mainly associated with ageing-related neurodegenerative diseases including Alzheimer’s, Parkinson’s and Huntington’s diseases. The life and death switch is among others regulated by interactions between pro- and anti-apoptotic Bcl-2 family members, including Bax and Bcl-xL, respectively. Bax plays a central role in apoptosis, as it is involved in the formation of pores within the mitochondria outer membrane through which apoptogenic factors, including cytochrome c, are released and originate a cascade of events that culminate in cell death. Given the importance of this pro-apoptotic protein in cell death, its potential as a therapeutic target was quickly recognized, and its mode of action and regulation have been studied extensively. However, many questions still remain, and thus further understanding of Bax regulation was the general purpose of this thesis. To accomplish this, we took advantage of the genetically tractable yeast Saccharomyces cerevisiae, whose genome is devoid of genes coding apparent homologues of the human Bcl-2 family. Therefore, the heterologous expression of human Bax allows its study without interference of the apoptotic network. To achieve our goal, we first optimized the conditions for expression of human Bax (Bax α) in yeast and characterized the phenotype of expression of Bax α and of an active form, Bax c-myc. Then, conditions that led to Bax α activation were found, and its regulation through different proteins was explored. Herein, we describe for the first time that sub-lethal concentrations of acetic acid trigger Bax α-mediated cell death without disturbing plasma membrane integrity and mitochondrial mass but increasing superoxide anion accumulation and release of cytochrome c, which is partially reverted by the anti-apoptotic protein Bcl-xL. This finding allows us to mimic what happens in human cells, without the need to use non-natural mutants of Bax, such as phosphomimetic or non-phosphorylatable, or mitochondrial tagged versions. Thus, yeast continuously proves to be a valuable tool to express human Bax and perform studies regarding its function, regulation and interaction with other members of the Bcl-2 protein family and other partners.A morte celular é um evento fundamental para a correta formação dos tecidos durante o desenvolvimento embrionário e para o normal funcionamento das células. Devido ao seu papel fundamental a nível fisiológico, desregulações neste processo estão associadas a uma grande diversidade de funções anormais e, consequentemente, ao desenvolvimento e progressão de várias doenças. De facto, uma diminuição da morte celular relaciona-se com envelhecimento e a progressão de tumores, enquanto que um aumento está associado a doenças neurodegenerativas incluindo a doença de Alzheimer, Parkinson e Huntington. O destino de uma dada célula é regulado, entre outras formas, por interações entre membros pro-apoptóticos e anti-apoptóticos da família de proteínas Bcl-2, nomeadamente a Bax e a Bcl-xL, respetivamente. A proteína Bax está envolvida na formação de poros na membrana mitocondrial externa, através do qual são libertados fatores apoptóticos, incluindo o citocromo c, originando uma cascata de eventos que culmina na morte da célula. Considerando o papel importante desta proteína na morte celular, as suas capacidades como alvo terapêutico emergiram rapidamente. Por este motivo, o seu modo de ação e regulação têm sido extensivamente estudados. Contudo, várias questões permanecem eminentes e, por isso a melhor compreensão da sua regulação é o foco principal desta tese. Assim, escolhemos a levedura Saccharomyces cerevisiae, cujo genoma não possui genes codificantes de homólogos da família de proteínas humanas Bcl-2. Deste modo, a expressão heteróloga da proteína Bax humana na levedura permite o seu estudo sem interferência dos restantes membros da família. Para alcançarmos o nosso objetivo, primeiramente otimizamos as condições para expressão da proteína na levedura, e caracterizamos o seu fenótipo de expressão, bem como o de uma forma ativa, Bax c-myc. De seguida, determinamos condições que nos permitiram a ativação da proteína Bax humana e a sua regulação por outras proteínas. Nesta tese, descrevemos pela primeira vez que concentrações sub-letais de ácido acético desencadeiam um processo de morte celular mediado por Bax, sem provocar alterações na integridade da membrana plasmática e na massa mitocondrial, mas aumentando a acumulação de anião superóxido e causando a libertação de citocromo c, parcialmente revertida pela proteína anti-apoptótica Bcl-xL. Estes resultados permitiram-nos mimetizar o que acontece em células humanas, sem a necessidade de usar mutantes de Bax artificiais, tais como os mutantes fosfomiméticos e não fosforiláveis ou versões endereçadas para a mitocôndria. Deste modo, a levedura continua a revelar o seu valor como modelo celular para expressar a proteína Bax humana e realizar estudos no que toca à sua função, regulação e interação com outros membros da família Bcl-2 e outros parceiros.Fundação para a Ciência e Tecnologia (FCT) através do projeto UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) e dos financiamentos PD/BD/128032/2016 no âmbito do Programa Doutoral em Biologia Aplicada e Ambiental (DP_AEM) e SFRH/BPD/89980/2012

Design of a two-fluorophore pH biosensor targeted to the digestive vacuole of Plasmodium falciparum

The maintenance of pH homeostasis is critical for biological processes in all organisms, with the malaria parasite, Plasmodium falciparum, being no exception. The parasite food vacuole is a major acidic digestive organelle, where the pH is thought to be regulated by proton pumps in its membrane. The acidic environment promotes the action of enzymes involved in hemoglobin degradation, and polymerization of toxic heme into hemozoin. This biological process is vital for the parasite and a proven chemotherapeutic target. Thus, determining the pH dynamics along the parasite life cycle and in the context of antimalarial drugs might provide unique information about the parasite pathophysiology, cell metabolism and function, shedding light into drugs mechanism of action and possibly target(s) disclosure. To reliably measure the pH of P.falciparum digestive vacuole along its life cycle, we designed a genetically encoded fluorescent sensor strategy, consisting of fusing the ratiometric pHLuorin2 with emiRFP670 to the falcipain-2 protein, a cysteine protease located in this compartment.This work was supported by Fundação para a Ciência e Tecnologia (FCT) national funds, under the national support to R&D units grant, through the reference project UIDB/04436/2020 and UIDP/04436/2020. V. Baptista thanks FCT for the SFRH/BD/145427/2019 grant. Susana Catarino thanks FCT for her contract funding provided through 2020.00215.CEECIND. Maria Isabel Veiga thanks FCT for her contract funding provided through 2020.03113.CEECIND

Plasmodium falciparum heme species dynamics under antimalarial exposure

While the recently approved first malaria vaccine provides new hope with a reduction in deadly severe malaria, decreasing the disease burden towards elimination requires highly effective drug treatment. However, treatment efficacy is constantly challenged by the capacity of Plasmodium falciparum parasites to develop resistance. The present artemisinin-based combination therapy is no exception. As the portfolio of new drugs is limited, identifying new targets and developing new therapies to tackle malaria is urgent. Most antimalarial drugs somehow target the heme detoxification pathway, happening in the symptomatic intraerythrocytic developmental cycle (IDC) of parasite infection in the human host. During the IDC, parasites feed on hemoglobin, which is endocytosed to the digestive vacuole, originating free heme. Free heme is harmful to the parasite and, thus, it is converted into the inert hemozoin crystal. Herein, we explore a cell fractionation assay that allows to measure the parasite’s heme species in complex with pyridine and measured the levels of specific heme fractions along tightly synchronized parasites’ lifecycle in the presence of antimalarials. This approach led to characterize the dynamics of these heme species throughout the IDC, unveiling the stages where antimalarials most impact the heme species dynamic balance

Effect of the materials’ properties in the design of high transmittance and low FWHM SiO2/TiO2 thin film optical filters for integration in a malaria diagnostics device

Malaria is an infectious disease, highly prevalent in world regions with lacking healthcare conditions. Nowadays, malaria diagnostic methods in these endemic regions are mainly based on microscopy and rapid diagnostic tests by immunochromatographic assays. Here, it is presented an optical diagnostic method, based on reflectance spectrophotometry, through hemozoin (Hz) quantification, towards an innovative non-invasive malaria diagnostic device. Therefore, a set of optical filters, with high transmittance and low full width at half maximum (FWHM) at specific wavelengths, is designed for being integrated in the device. These allow the full reconstruction of the optical reflectance spectrum, able to distinguish between healthy and infected samples, with a detection limit up to 12.5 parasites/μl of red blood cells. This work presents the design, performance simulation, and optimization of 16 highly selective narrow band-pass optical filters, based on multilayer stacks of SiO2/TiO2 thin films. The optical properties of the thin films layer materials, in particular the refractive indexes, are the main focus in this study. Three different reflective indexes were evaluated and the results showed that, for all the simulated conditions, each filter is sensitive to a single wavelength with a FWHM 90%, but slight variations were observed for the different refractive indexes. The simulation results proved that these 16 optical filters designs are extremely sensitive to the material properties, although they are the best option regarding the required optical response, assuring feasibility and being adequate for the fabrication process.This work was supported by Project NORTE-01-0145-FEDER-028178 funded by NORTE 2020 Portugal Regional Operational Program under PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund and the Fundação para a Ciência e Tecnologia (FCT), IP. V. Baptista thanks FCT for the SFRH/BD/145427/2019 grant. Maria Isabel Veiga thanks FCT for her contract funding provided through DL 57/2016 (CRP)