Dissertations / Theses on the topic 'Gametocyt'

Gametocytes, the transmission stage of malaria parasites, are generally rare in infections. I explore whether this may be a parasite adaptation that paradoxically maximises fitness. I present an optimality analysis of gametocyte investment based on a discretely formulated within-host model. I show that low gametocyte investment is predicted as a result of the trade-off that exists between producing gametocytes and increasing parasite numbers. I predict that gametocyte investment should decrease as maximum asexual density occurs later in infections and also as parasite fecundity rises. I address statistical problems with estimating gametocyte investment from blood smears. I also consider the simpler case of estimating parasitaemias and gametocytaemias. Traditional methods of counting parasites in smears can produce biased estimates of parasitaemia, gametocytaemia and gametocyte conversion. I introduce an alternative method of counting based on inverse sampling. This method is unbiased, is consistently precise and the most time-efficient method of counting. I used the inverse sampling method to estimate gametocyte conversion (the observed outcome of investment) in P. chabaudi infections in mice, which had been manipulated to alter the time of maximum asexual density. Gametocyte conversion showed two peaks. The timing of the peaks depended on the time of maximum asexual density. Maximum conversion decreased as the time to maximum asexual density rose as predicted by my optimality analysis. An interesting finding was that gametocyte conversion decreased after reaching a maximum. This result is counter to most life history theory. I suggest this indicates that survival of infections into a chronic phase may be an important component of fitness. Maximum conversion occurred after maximum asexual density. This leads me to propose that contrary to the common view, gametocyte investment may be suppressed at times when there is a high risk of parasite clearance. Understanding the reasons for gametocyte rarity may help to predict how P. falciparum will respond to intervention and suggest new methods of malaria control.

Malaria is the parasitic disease which affects the most humans, with Plasmodium falciparum malaria being responsible for the majority of severe malaria and malaria related deaths. The asexual form of the parasite causes the signs and symptoms associated with malaria infection. The sexual form of the parasite, also known as a gametocyte, is the stage responsible for infectivity of the human host (patient) to the mosquito vector, and thus ongoing transmission of malaria and the spread of antimalarial drug resistance. Historically malaria therapeutic efficacy studies have focused mainly on the clearance of asexual parasites. However, malaria in a community can only be truly combated if a treatment program is implemented which is able to clear both asexual and sexual parasites effectively. In this thesis focus will be on the modeling of the key features of gametocytemia. Particular emphasis will be on the modeling of the time to gametocyte emergence, the density of gametocytes and the duration of gametocytemia. It is also of interest to investigate the impact of the administered treatment on the aforementioned features. Gametocyte data has several interesting features. Firstly, the distribution of gametocyte data is zero-inflated with a long tail to the right. The observed longitudinal gametocyte profile also has a nonlinear relationship with time. In addition, since most malaria intervention studies are not designed to optimally measure the evolution of the longitudinal gametocyte profile, there are very few observation points in the time period where the gametocyte profile is expected to peak. Gametocyte data collected from malaria intervention studies are also affected by informative censoring, which leads to incomplete gametocyte profiles. An example of informative censoring is when a patient who experiences treatment failure is “rescued", and withdrawn, from the study in order to receive alternative treatment. This patient can be considered to be in worse health as compared to the patients who remain in this study. There are also competing risks of exit from the study, as a patient can either experience treatment failure or be lost to follow-up. The above mentioned features of gametocyte data make it a statistically appealing dataset to analyze. In literature there are several modeling techniques which can be used to analyze individual features of the data. These techniques include standard survival models for modeling the time to gametocyte emergence and the duration of gametocytemia. The longitudinal nonlinear gametocyte profile would typically be modeled using nonlinear mixed effect models. These nonlinear models could then subsequently be extended to accommodate the zero-inflation in the data, by changing the underlying assumption around the distribution of the response variable. However, it is important to note that these standard techniques do not account for informative censoring. Failure to account for informative censoring leads to bias in parameter estimates. Joint modeling techniques can be used to account for informative censoring. The joint models applied in this thesis combined the longitudinal nonlinear gametocyte densities and the time to censoring due to either lost to follow up or treatment failure. The data analyzed in this thesis were collected from a series of clinical trials conducted be- tween 2002 and 2004 in Mozambique and the Mpumulanga province of South Africa. These trials were a part of the South East African Combination Antimalarial Therapy (SEACAT) evaluation of the phased introduction of combination anti-malarial therapy, nested in the Lubombo Spatial Development Initiative. The aim of these studies was primarily to measure the efficacy of sulfadoxine-pyrimethamine (SP) and a combination of artesunate and sulfadoxine-pyrimethamine (ACT), in eliminating asexual parasites in patients. The patients enrolled in the study had uncomplicated malaria, at a time of increasing resistance to sulfadoxine-pyrimethamine (SP) treatment. Blood samples were taken from patients during the course of 6 weeks on days 0, 1, 2, 3, 7, 14, 21, 28 and 42. Analysis of these blood samples provided longitudinal measurements for asexual 1 parasite densities, gametocyte densities, sulfadoxine drug concentrations and pyrimethamine drug concentrations. The gametocyte data collected in this study was initially analyzed using standard survival modeling techniques. Non-parametric Cox regression models and parametric survival models were applied to the data as part of this initial investigation. These models were used to investigate the factors which affected the time to gametocyte emergence. Subsequently, using the subset of the population which experienced gametocytemia, accelerated failure time models were applied to investigate the factors which affected the duration of gametocytemia. It is evident that the findings from the aforementioned duration investigation would only be able to provide valid duration estimates for patients who were detected to have gametocytemia. This work was extended to allow for population level duration estimates by incorporating the prevalence of gametocytemia into the estimation of duration, for generic patients with specific covariate patterns. The prevalence of gametocytemia was modeled using an underlying binomial distribution. The delta method was subsequently used to derive confidence intervals for the population level duration estimates which were associated with specific covariate patterns. An investigation into the factors affecting the early withdrawal of patients from the study was also conducted. Early exit from the study arose either through loss to follow-up (LTFU) or through treatment failure. The longitudinal gametocyte profile was modeled using joint modeling techniques. The resulting joint model used shared random effects to combine a Weibull survival model, describing the cause- specific hazards of patient exit from the study, with a nonlinear zero-adjusted gamma mixed effect model for the longitudinal gametocyte profile. This model was used to impute the incomplete gametocyte profiles, after adjusting for informative censoring. These imputed profiles were then used to estimate the duration of gametocytemia. It was found, in this thesis, that treatment had a very strong effect on the hazard of gametocyte emergence, density of gametocytes and the duration of gametocytemia. Patients who received a combination of sulfadoxine-pyrimethamine and artesunate were found to have significantly lower hazards of gametocyte emergence, lower predicted durations of gametocytemia and lower predicted longitudinal gametocyte densities as compared to patients who received sulfadoxine-pyrimethamine treatment only.

Malaria continues to be a global health burden that affects millions of people worldwide each year. Increasing demand for malaria control and eradication has led research to focus on sexual development of the malaria parasite. Sexual development is initiated when pre-destined intraerythrocytic ring stage parasites leave asexual reproduction and develop into gametocytes. A mosquito vector will ingest mature gametocytes during a blood meal. Sexual reproduction will occur in the midgut, leading to the production of sporozoites that will migrate to the salivary gland. The sporozoites will be injected to another human host during the next blood meal consequently, transmitting malaria. Due to decreased drug susceptibility of mature gametocytes, more investigation of the biology and metabolic requirements of malaria parasites during gametocytogenesis, as well as during the mosquito stages, are urgently needed to reveal novel targets for development of transmission-blocking agents. Furthermore, increasing drug resistance of the parasites to current antimalarials, including slowed clearance rates to artemisinin, requires the discovery of innovative drugs against asexual intraerythrocytic stages with novel mechanisms of action. Here, we have investigated the role of the apicoplast during Plasmodium falciparum gametocytogenesis. In addition, we describe drug-screening studies that have elucidated a novel mode of action of one compound from the Malaria Box, as well as identified new natural product compounds that may be serve as starting molecules for antimalarial development.
Ph. D.

In order to reduce malaria prevalence worldwide, a better understanding of parasite transmission and the effect of drug resistance is needed. The effect of drug resistance on malaria transmission has been examined for some drugs, but not for mitochondrial inhibitors such as atovaquone and the current basis of malaria therapy, artemisinin. Therefore, the goal of this study was to produce gametocytes, the life cycle stage that transmits from mosquito to human, in several different drug resistant patient isolates as well as to determine the effect of drug resistance on gametocyte development and transmission. Previous studies have shown that the mutation that confers resistance to atovaquone, a common antimalarial, occurs de novo after treatment and transmission of this resistance is not seen in the field. Therefore, to determine whether or not the resistance mutation can be transmitted, mosquito-feeding experiments were conducted using atovaquone resistant parasites and resulting oocyst DNA was analyzed. In addition to these atovaquone studies, artemisinin resistant gametocytes were also grown in vitro and drug pressure was added to determine if resistance mechanisms affect gametocyte development. This study is the first examine gametocyte development in these resistant strains and the first to report that transmission of the atovaquone resistant mutation may be possible. However, data is currently inconclusive on the effect of artemisinin resistance on gametocyte development.

Malaria is one of the deadliest parasitic diseases worldwide, causing 219 million infections and 435 thousand deaths per year. As such, this mosquito-borne illness is a major target for global eradication efforts. One critical arm of the eradication strategy is chemotherapy. For a therapeutic to advance the eradication agenda, it must cure the patient of infection and eliminate transmission stage parasites (called gametocytes) from the blood, thereby breaking the cycle of transmission. Currently, first-line treatments against malaria infection consist of an artemisinin derivative in combination with another antimalarial drug from a different drug class. Although artemisinin and its derivatives are highly efficacious at curing malaria, these drugs are ineffective at preventing disease transmission. However, recent in vivo studies have suggested that whole plant Artemisia annua (the botanical source of artemisinin) delivered as tea can cure patients of infection and eliminate transmission stage parasites from the bloodstream. To validate these in vivo results in vitro, experiments were performed to measure the killing efficacy of A. annua and A. afra tea infusions against three different stages of the parasite life cycle— one stage of the asexual cycle, immature gametocytes, and mature gametocytes. Killing effects were observed using light microscopy and gametocyte gene-specific RT-qPCR analyses. Results suggested that A. annua tea was nearly as effective as artemisinin at killing all three tested stages of the parasite. A. afra tea, which contains low levels of artemisinin, showed comparable killing efficacy against late stage gametocytes, but not against the other two tested stages. These results supported the notion that A. annua tea is an effective antimalarial and also provides evidence that both A. annua and A. afra teas may be a viable therapeutic option for eliminating gametocytes during human infection.

The aim of this study was to investigate the genetic basis of variation in gametocyte sex ratio in the human malaria parasite, Plasmodium falciparum. The gametocyte sex ratio was measured in progeny clones from the 3D7 x HB3 experimental genetic cross and found to be remarkably stable across replicates of different parasite clones. Significant differences in the sex ratio were observed between the parents of the cross. Progeny clones fell into two classes of sex ratio, one similar to that seen in parent 3D7 and the other like parent HB3, suggesting a single gene of major effect controlling sex ratio. Using a genetic map of the progeny and parental clones, QTL analysis revealed two highly significant loci, the first on chromosome 10 (LOD score = 8.8), and the second on chromosome 14 (LOD = 4.0), linked to gametocyte sex ratio. The locus on chromosome 10, spanning approximately 35kb, contained ten genes. This locus, named PfROS1 (Plasmodium falciparum Ratio of Sex 1), explained 95% of the variation in sex ratio. The second locus on chromosome 14, PfROS2 (Plasmodium falciparum Ratio of Sex 2), explained a small proportion of gametocyte sex ratio variation when combined with PfROS1, the two loci explained 99% of the variation in gametocyte sex ratio observed. As PfROS1 explains such a high percentage of the variation observed in the gametocyte sex ratio it represents a single controlling locus to define the sex ratio of gametocytes produced. This is the first report of a genomic locus influencing gametocyte sex ratio in any Plasmodium species. In addition, changes in the sex ratio of clones 3D7 and HB3, over the course of 16 days of gametocyte culture were investigated. The number of gametocytes, and especially male gametocytes, was observed to fall markedly in the last few days of culture, when the majority of gametocytes were stage V (mature). Fluctuations in temperature during the culture process were found to influence sex ratio, suggesting the loss of males was due to exflagellation of mature gametocytes. Parasite clone and day of culture were also significant explanatory variables in influencing sex ratio.

The in vitro production of gametocytes and oocysts of the apicomplexan parasite genus Eimeria is still a challenge in coccidiosis research. Until today, an in vitro development of gametocytes or oocysts had only been shown in some Eimeria species. For several mammalian Eimeria species, partial developments could be achieved in different cell types, but a development up to gametocytes or oocysts is still lacking. This study compares several permanent cell lines with primary fetal cells of the black rat (Rattus norvegicus) concerning the qualitative in vitro development of the rat parasite Eimeria nieschulzi. With the help of transgenic parasites, the developmental progress was documented. The selected Eimeria nieschulzi strain constitutively expresses the yellow fluorescent protein and a macrogamont specific upregulated red tandem dimer tomato. In the majority of all investigated host cells the development stopped at the second merozoite stage. In a mixed culture of cells derived from inner fetal organs the development of schizont generations I-IV, macrogamonts, and oocysts were observed in crypt-like organoid structures. Microgamonts and microgametes could not be observed and oocysts did not sporulate under air supply. By immunohistology, we could confirm that wild-type E. nieschulzi stages can be found in the crypts of the small intestine. The results of this study may be helpful for characterization of native host cells and for development of an in vitro cultivation system for Eimeria species.

Thematic Paper (M.C.T.M.(Clinical Tropical Medicine)--Mahidol University, 2008.
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Malaria is a vector-borne disease that is still responsible for high human morbidity and mortality. Of the five Plasmodium species that can cause malaria in humans, Plasmodium falciparum is regarded the most virulent species. The most fundamental component of sustained control and eradication efforts is the development of effective drugs for malaria treatment and prophylaxis. Plasmodium falciparum’s sexual stages (gametocytes) are not associated with malarial pathogenesis or the clinical symptoms, but they are responsible for the transmission of the disease from human hosts to mosquitos. As such, the development of gametocytocidal interventions that targets the transmission stage to break the disease’s lifecycle forms the basis of efforts towards malaria elimination and eradication. However, despite the importance of this developmental stage, the biology and pharmacology of gametocytes are still very poorly understood. This thesis has set out to gain a better understanding of the identity of gametocyte-active antimalarials and a deeper understanding of the mechanisms underpinning the activity. Using a newly generated luciferase-reporting transgenic line, pharmacodynamic gametocyte studies could be performed to help characterise the activity of selected known reference antimalarials, new potential gametocyte inhibitors in pre-clinical development as well as newly developed fully synthetic compounds designed against the sexual stages. This novel assay revealed that the efficacy of active tested compounds is highly stage-specific. Of all the tested reference antimalarial drugs, MB and DHA were the most potent antimalarial across all gametocyte stages and importantly they were active at clinically relevant levels. These observations were progressed further, developing a time- dependent killing assay that was performed with different concentrations of targeted drug over discrete time intervals to determine the drug’s kill rate. These parameters were then used to simulate the PK/PD relationship of the drug in order to estimate gametocyte clearance profiles during the human treatment period (Chapter 3 and 4). A main focus of the thesis was conducted to better understand the mechanism of drug activity of the 8-aminoquinolines against gametocytes. The ability of a series of 8-aminoquinolines (primaquine as the parent drug, synthesised metabolites in chapter 5, three novel analogues and tafenoquine in (chapter 6) to interact with CYP2D6 was tested by measuring their ability to specifically inhibit the metabolism of fluorescently-tagged tracer substrate by recombinant human CYP 2D6. Reaction products from the CYP metabolites and HLM were then used to test firstly their ability to kill gametocytes, and then to establish their ability to generate hydrogen peroxides and finally measure their haemolytic toxicity. At 10 µM, primaquine CYP metabolites showed activity against the gametocytes that was higher than that of the parent drugs, with the exception of tafenoquine which, interestingly, demonstrated good activity and haemolytic toxicity as a parent drug. These analyses are presented and discussed in the context of strategies that aim at the discovery and development of new transmission-reducing antimalarial drugs.

A detailed understanding of the genes and mechanisms that regulate oocyte growth and maturation underpins the development of improved methods of assisted conception. Gametocyte specific factor 1 (GTSF1)-a putative marker of gamete developmental competence, is highly conserved across species but in mammals demonstrates a sexual dimorphism in its function. In mice, male mutants for Gtsf1 have an infertile phenotype, whereas female mutants appear to have normal ovarian function. It is hypothesised that GTSF1 regulates oocyte development in monovular species such as the sheep. Initial studies characterised the expression and cellular distribution of GTSF1 across cDNA libraries spanning ovine oogenesis and embryogenesis and by using in situ hybridisation of fixed tissue. GTSF1 expression was confined to gonadal and embryonic tissues with highest expression in the ooplasm of germinal vesicle (GV)-staged secondary oocytes. The gene sequence of GTSF1 was obtained and the gene and predicted protein sequences revealed close homology across many species with two conserved CHHC zinc finger domains known to bind RNA. Functional analysis of the role of GTSF1 during sheep oocyte maturation was conducted using short interference RNA (siRNA injection) in conjunction with oocyte in vitro maturation (IVM) and oocytectomised cumulus shell co-culture. This system was validated using siRNA knockdown (kd) for a known oocyte-specific gene, Growth differentiation factor 9 (GDF9). The effect on GTSF1 kd was evaluated following the microinjection of 770 GV oocytes with siRNA target against the sixth exon of ovine GTSF1. The effects of GTSF1 kd were evaluated in 57 MII oocytes and cumulus shells. Targeted kd of GTSF1 in GV oocytes followed by IVM and cumulus shell co-culture did not affect oocyte meiotic progression or cumulus expansion. Microarray analysis using the bovine GeneChip Affymetrix array revealed that 6 down-regulated genes (TCOF1, RPS8, CACNA1D, SREK1IP1, TIMP1, MYL9) following the GTSF1 kd were associated with developmental competence, RNA storage, post-transcriptional modifications and translation. Immunofluorescent studies localized GTSF1 protein to the P-body in GV ovine oocytes. Collectively these results suggest a possible role of GTSF1 in post-transcriptional control of RNA processing, translational regulation and RNA storage which may impact on oocyte developmental competence.

The in vitro production of gametocytes and oocysts of the apicomplexan parasite genus Eimeria is still a challenge in coccidiosis research. Until today, an in vitro development of gametocytes or oocysts had only been shown in some Eimeria species. For several mammalian Eimeria species, partial developments could be achieved in different cell types, but a development up to gametocytes or oocysts is still lacking. This study compares several permanent cell lines with primary fetal cells of the black rat (Rattus norvegicus) concerning the qualitative in vitro development of the rat parasite Eimeria nieschulzi. With the help of transgenic parasites, the developmental progress was documented. The selected Eimeria nieschulzi strain constitutively expresses the yellow fluorescent protein and a macrogamont specific upregulated red tandem dimer tomato. In the majority of all investigated host cells the development stopped at the second merozoite stage. In a mixed culture of cells derived from inner fetal organs the development of schizont generations I-IV, macrogamonts, and oocysts were observed in crypt-like organoid structures. Microgamonts and microgametes could not be observed and oocysts did not sporulate under air supply. By immunohistology, we could confirm that wild-type E. nieschulzi stages can be found in the crypts of the small intestine. The results of this study may be helpful for characterization of native host cells and for development of an in vitro cultivation system for Eimeria species.

Malaria, babesiosis, trypanosomosis, and leishmaniasis are some of the most life-threatening parasites, but the range of drugs to treat them is limited. An effective, safe, and low-cost drug with a large activity spectrum is urgently needed. For this purpose, an aryl amino alcohol derivative called Alsinol was resynthesized, screened in silico, and tested against Plasmodium, Babesia, Trypanosoma, and Leishmania. In silico Alsinol follows the Lipinski and Ghose rules. In vitro it had schizontocidal activity against Plasmodium falciparum and was able to inhibit gametocytogenesis; it was particularly active against late gametocytes. In malaria-infected mice, it showed a dose-dependent activity similar to chloroquine. It demonstrated a similar level of activity to reference compounds against Babesia divergens, and against promastigotes, and amastigotes stages of Leishmania in vitro. It inhibited the in vitro growth of two African animal strains of Trypanosoma but was ineffective in vivo in our experimental conditions. It showed moderate toxicity in J774A1 and Vero cell models. The study demonstrated that Alsinol has a large spectrum of activity and is potentially affordable to produce. Nevertheless, challenges remain in the process of scaling up synthesis, creating a suitable clinical formulation, and determining the safety margin in preclinical models.
Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS)
Revisión por pares

For vector-borne parasites such as malaria, how within- and between-host processes interact to shape transmission is poorly understood. In the host, malaria parasites replicate asexually but for transmission via mosquitoes to occur, specialized sexual stages (gametocytes) must be produced. Once inside the mosquito vector, gametocytes immediately differentiate into male and female gametes, and motile male gametes must swim through the hostile environment of the bloodmeal to find and fertilise female gametes. Despite the central role that gametocytes play in disease transmission, explanations of why parasites adjust gametocyte production in response to in-host factors remain controversial. Furthermore, surprisingly little is known about the mating behaviour of malaria parasites once inside the mosquito. Developing drugs and/or vaccines that prevent transmission by disrupting sexual stages are major goals of biomedicine, but understanding variation in gametocyte investment and male gamete behaviour is key to the success of any intervention. First, I propose that the evolutionary theory developed to explain variation in reproductive effort in multicellular organisms provides a framework to understand gametocyte investment strategies in malaria parasites. I then demonstrate that parasites appear to change their reproductive strategies in response to environmental cues and in a manner consistent with our predictions. Next, I show how digital holographic microscopy can be used to characterise the morphology and motility of male gametes. I then provide evidence for non-random movement of male gametes and that gamete interactions with red blood cells appear to hinder mating success in a bloodmeal. Finally, I discuss the variation in gametocyte differentiation and fertilisation success when exposed to a number of factors implicated in gametocyte activation. The data presented here provides important information on the basic biology of malaria parasite reproductive stages and demonstrates considerable variation in parasite traits and behaviours in response environmental changes; both in the host and in the mosquito vector.

L’un des obstacles majeurs au développement de nouveaux antipaludiques est notre connaissance limitée de la biologie parasitaire et la rareté des cibles thérapeutiques potentielles identifiées. Les interactions protéines-protéines sont impliquées et essentielles dans divers processus biologiques incluant les modifications post-traductionnelles. Les interactions substrats-kinases ou phosphatases sont considérées comme une liaison transitoire et jouent un rôle central et essentiel dans le cycle cellulaire de Plasmodium. La Ser/Thr Protéine Phosphatase de Type 1 (PP1), l’une des phosphatases majeurs des eucaryotes, est essentielle à la survie du parasite Plasmodium falciparum, responsable du paludisme. Elle est régulée par diverses sous-unités régulatrices dont plus de 200 ont été identifiées chez l’Homme, mais seulement 4 chez Plasmodium.Afin d’explorer le réseau de régulation de la P. falciparum PP1 (PfPP1), nous avons utilisé trois approches complémentaires pour caractériser l’interactome de la PfPP1. La purification par co-affinité suivie d'une analyse par spectrométrie de masse a identifié 6 protéines interagissant avec la PfPP1 dont 3 contenaient le motif consensus d’interaction RVxF, 2 autres le motif Fxx[RK]x[RK], également connu pour interagir avec la phosphatase et une protéine avec les deux motifs de liaison. Le criblage par double hybride chez la levure a identifié 134 protéines dont 30 présentent le motif RVxF et 20 ont le motif de liaison Fxx[RK]x[RK]. Le criblage in silico du génome de P. falciparum en utilisant une séquence consensus du motif RVxF a révélé 55 partenaires potentiels de la PfPP1. Afin de confirmer l’interaction de certaines protéines, 35 partenaires candidats ont été validés par un test d’interaction de type ELISA. Les résultats ont permis de détecter aussi bien des partenaires conservés de la PP1 qu'un nombre élevé d'interacteurs spécifiques à la PP1 du parasite et montrent une grande diversité dans les fonctions biologiques impliquant la PP1 chez Plasmodium. Parmi ces candidats, un partenaire appelé Gametocyte EXported Protein 15 (GEXP15) a été confirmé comme un réel partenaire de la PfPP1 par différentes approches. De plus, GEXP15 est surexprimé chez les gamétocytes, stade responsable de la transmission du parasite chez le moustique. Ces résultats ainsi que des études fonctionnelles seront présentés
A major obstacle in the development of novel anti-malarials is our limited knowledge of basic parasite biology and the paucity of identified potential intervention targets. Protein-protein interactions are involved and essential in broad range of biological processes including the post-translational modifications. Substrate-kinase or phosphatase interactions are considered as transient binding and play a central and essential role in Plasmodium cell cycle. The Ser/Thr Protein Phosphatase Type 1 (PP1), one of the main contributors of eukaryotic phosphatase activity, is essential to malaria parasite Plasmodium falciparum and is highly regulated by many regulatory subunits. In humans, there are about 200 distinct regulators, however, only 4 have been so far reported in Plasmodium.To explore the P. falciparum PP1 (PfPP1) regulatory network as complete as possible, we carried out three complementary approaches to characterize the PfPP1 interactome. Co-affinity purification followed by Mass Spectrometry-based proteomics identified 6 PfPP1 interacting proteins (PIPs) of which 3 contained the RVxF consensus binding motif, 2 PIPs with a Fxx[RK]x[RK] binding motif, one with both binding motifs. The Yeast Two-Hybrid screening identified 134 proteins of which 30 have the RVxF binding motif and 20 contain the Fxx[RK]x[RK] binding motif. The in silico screen of the P. falciparum genome using a consensus RVxF motif as template revealed the presence of 55 potential PfPP1 interacting proteins. As further demonstration, 35 candidate partners were validated in an independent ELISA-based assay using recombinant proteins. The data reports several conserved PP1 interacting proteins as well as a high number of specific interactors to PfPP1, indicating a high diversity of biological functions for PP1 in Plasmodium. Among these candidates, one partner assigned as Gametocyte EXported Protein 15 (GEXP15) has been confirmed as a direct interactor of PfPP1 by different approaches. In addition, GEXP15 is over-expressed during gametocyte stage, responsible for the transmission of the parasite in the mosquito. These results as well as functional studies will be presented and discussed

Adaptive phenotypic plasticity, the ability of a genotype to give rise to different phenotypes in different environments, evolves to allow organisms to fine-tune their life-history traits according to the varying conditions they encounter during their lives. Reproductive investment - the manner in which organisms divide their resources between survival and reproduction - is well studied in evolutionary ecology because it is a key determinant of fitness. However, whilst plasticity in reproductive effort is well understood for free-living multicellular taxa (such as insects, birds, and mammals), the application of evolutionary theory for plasticity and life history strategies to unicellular parasites and pathogens is lacking. In this thesis, I use empirical and theoretical approaches to uncover how differential resource allocation to non-replicating, sexual stages (gametocytes) versus asexually replicating stages can be harnessed by the rodent malaria parasite Plasmodium chabaudi to maximise its fitness across the often very variable conditions it encounters during infections. Differential allocation between those stages is equivalent to the fundamental life-history trade-off between survival and reproduction because gametocytes are responsible for between-host transmission (i.e. reproduction of the infection) whereas asexual parasites mediate host exploitation and within-host survival. A suite of within-host models reveal that malaria parasites could gain considerable fitness benefits in the face of low levels of drug treatment if they reduce their investment into gametocyte production ("reproductive restraint"), thereby assuring the continuity of the infection and capitalising on opportunities for future transmission. In contrast, high levels of drug treatment typically select parasites to commit all of their resources to gametocyte production ("terminal investment"), to escape a host that does not offer much opportunity for future transmission. My experiments reveal that P. chabaudi increases both its reproductive investment and its asexual replication rate in anaemic hosts (i.e. host that have a low density of red blood cells), suggesting that parasites profit from host anaemia and can afford high investment in gametocytes ("affluent investment"). I also uncover plasticity in a number of traits that underpin asexual replication rate, including invasion preference for different ages of red blood cells, but it is plasticity in the number of progeny (merozoites) per infected cell that is the main contributor to asexual replication rate. My experiments also reveal genetic variance in plasticity of the life-history traits investigated, which has profound implications for their evolution. Furthermore, plastic modification of these traits is associated with minimal costs or constraints, so that parasites can rapidly match life-history traits appropriately to the within-host environment. Severe anaemia is one of the deadliest symptoms of malaria, so observing that virulence and infectiousness increases in anaemic hosts has also fundamental clinical implications. Finally, the empirical and theoretical observations of affluent investment, reproductive restraint and terminal investment match theoretical predictions of how organisms should behave in varying environments, confirming P. chabaudi as a useful model system to test life-history theory.

The genus Eimeria (Apicomplexa, Coccidia) provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain.

The genus Eimeria (Apicomplexa, Coccidia) provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain.

La recrudescence du paludisme a plasmodium falciparum, et l'apparition de formes chloroquino-resistantes ont montre la necessite d'une connaissance plus approfondie des mecanismes de controle de la multiplication et de la differenciation du parasite. Les proteines g et les proteines kinases ont un role essentiel dans la modulation de l'activite de nombreuses enzymes en reponse a des signaux developpementaux, environnementaux ou metaboliques. La mise en evidence de proteines g au cours de notre etude initiale a montre que des voies de signalisation intracellulaires semblables a celles des eucaryotes superieurs existaient chez plasmodium. Ceci nous a amene a etudier egalement les proteines kinases. Les travaux rapportes dans ce memoire concernent l'identification, par pcr a l'aide d'oligonucleotides degeneres, et la caracterisation moleculaire d'une nouvelle proteine serine/threonine kinase plasmodiale de type snf1 que nous avons denommee pfkin. La sequence complete du gene a ete obtenue a partir de clones genomiques et d'adnc. Pour cloner l'extremite 5' du gene la pcr inverse a ete utilisee. Le gene, present a raison d'une copie par genome localisee sur le chromosome xiii, comprend 2298 pb et code pour une proteine de poids moleculaire estime de 90 845 da dont le domaine catalytique contient 40% de residus identiques avec celui de la proteine kinase snf1 de saccharomyces cerevisiae. L'expression preferentielle de pfkin au stade gametocyte, stade terminal du developpement chez l'homme, a ete determinee par rt-pcr. Les proteines kinases apparentees a snf1 constituent un groupe d'enzymes de regulation dont une fonction est de proteger la cellule des stress environnementaux ou metaboliques. Nous proposons un role de pfkin dans la reponse adaptative du gametocyte lors de sa transmission depuis l'hote vertebre homeotherme jusqu'a l'intestin du vecteur poikilotherme

Ce travail avait pour objectif : 1) évaluer le portage de gamétocytes et leur génotype avant et après le traitement d’une part, et d’étudier leur infectivité ; 2) exprimer le domaine catalytique (PfMCA1-cd-Sc) de la métacaspase de Plasmodium falciparum (PfMCA1) chez la levure et 3) tester in vitro l’activité antiplasmodiale de nouvelles molécules synthétiques dérivées des pyrano et ferro-quinoléines sur des clones de laboratoire 3D7 et Dd2. Pour cela, le test in vivo de 28 jours de l’OMS, les marqueurs moléculaires de résistance et le « direct feeding » ont été utilisés pour le premier objectif. La culture des levures, l’expression des protéines de la métacaspase 1 de Plasmodium falciparum, le western blot, le test de prolifération et de survie, et les marqueurs de mort cellulaire ont servi pour le second objectif et enfin, la culture parasitaire et tests in vitro par la méthode de fluorimétrie au Sybr Green I ont permis l’évaluation de l’activité antiplasmodiale de nouvelles molécules. Nous avons démontré que les gamétocytes post-traitement étaient porteurs de mutations ponctuelles et plus infectants dans le groupe chloroquine ; que l’expression hétérologue du domaine catalytique de la métacaspase de Plasmodium falciparum (PfMCA1) dans la levure Saccharomyces cerevisiae entraînait une mort clonale de type apoptotique et un retard de croissance dépendant de l’activité VAD-Protéase et enfin, que les substitues aromatiques à base de pyrimidine ou de benzylméthylamine ferrocène révèlent une activité satisfaisante par rapport à la méthoxyéthylidene sur les clones 3D7 et Dd2
Plasmodium species use programmed cell death for the survival of their offspring as some prokaryotic parasites. This study was designed to - assess the gametocytes carrier and their genotypes before/ after treatment and studying their infectivity; - express the catalytic domain (PfMCA1-cd-Sc) of Plasmodium falciparum metacaspase (PfMCA1) in yeast; - Test the “in vitro” anti-plasmodial activity of pyrano and ferro- quinolines derived new synthetic molecules on 3D7 and Dd2 Chloroquine laboratory clones. The 28-day “in vivo” WHO test, molecular markers of resistance and direct feeding; yeast culture, protein expression of P. falciparum metacaspase 1, Western blot, proliferation and survival test, and cell death markers were used to achieve the first two objectives while parasite culture and in vitro tests by the method of fluorimetry in SYBR Green I was used to evaluate the anti-plasmodial activity of new molecules. Results show that post-treatment gametocytes were carriers of point mutations and the most infective in the Chloroquine group. The heterologous expression of PfMCA1 catalytic domain in Saccharomyces cerevisiae resulted in apoptotic clonal death and growth retardation activity-dependent Protease-VAD, showing the involvement of PfMCA1 in the process of cell death. The aromatic substitutes with pyrimidine or benzyldimethylamine ferrocene residues showed satisfactory activity against the methoxyethylidene on 3D7 and Dd2. The data suggest that the structural optimization of these compounds based on pyrimidine and ferrocene is more interesting from the standpoint anti-plasmodial activity for candidate molecules in the near future

Transmission of malaria requires that some parasites abandon asexual replication and develop into sexual stages termed gametocytes. The discovery of the transcription factor PfAP2-G as the master regulator of sexual conversion has boosted our understanding of sexual development. The pfap2-g locus is controlled by heterochromatin-based silencing, with only a few parasites activating the locus and committing to sexual development at each asexual cycle. How heterochromatin forms in pfap2-g is currently unknown. Moreover, the initial steps of sexual conversion after pfap2-g activation, together with the biology of sexually committed stages, remain poorly characterized. In this thesis, we identified a new route of sexual conversion involving direct conversion within the same cycle of initial PfAP2-G expression. We also developed a conditional activation system for PfAP2-G, achieving synchronous sexual conversion of the majority of parasites, which enables the characterization of sexually committed parasites and early sexual stages. Finally, in an attempt to elucidate the mechanisms behind heterochromatin formation in pfap2-g, we gained insight into the role of different elements in heterochromatin nucleation.
Per a la transmissió de la malària, alguns paràsits han de deixar de replicar-se de forma asexual i desenvolupar-se com a estadis sexuals anomenats gametòcits. El descobriment del factor de transcripció PfAP2-G com a regulador clau de la conversió sexual ha permès entendre millor el procés. El locus pfap2-g està silenciat per la presència d’heterocromatina i només alguns paràsits activen el locus i inicien el desenvolupament sexual a cada cicle asexual. Actualment es desconeix com es forma heterocromatina a pfap2-g. A més a més, els primers passos de la conversió sexual després de l’activació de pfap2-g, juntament amb la biologia dels estadis que acabaran desenvolupant-se sexualment, estan molt poc caracteritzats. En aquesta tesis, hem identificat una nova ruta de conversió que consisteix en la conversió sexual directa, sense un nou cicle de replicació, just després de l’activació de PfAP2-G. També hem desenvolupat un sistema d’activació condicional de PfAP2-G que indueix la conversió sexual sincrònica de la majoria dels paràsits i que permet la caracterització dels estadis sexuals primerencs. Finalment, en un intent per entendre els mecanismes darrera la formació d’heterocromatina a pfap2-g, hem obtingut informació rellevant sobre el paper de diferents elements en la nucleació d’heterocromatina.

Transmission of the malaria parasite from man to the mosquito requires the formation of sexual parasite stages, the gametocytes. The gametocytes are the only parasite stage that is able to survive in the mosquito midgut and to undergo further development – gamete formation and fertilization. Numerous sexual stage-specific proteins have been discovered, some of which play crucial roles for parasite transmission. However, the functions of many sexual stage proteins remain elusive. Amongst the sexual stage-specific proteins are the proteins of the PfCCp proteins family, which exhibit numerous adhesion domains in their protein structures. For four members of the protein family, PfCCp1 to PfCCp4 gene-disruptant parasite lines had been already studied. Amongst these, PfCCp2 and PfCCp3 showed an important role for development of the parasites in the mosquito. In the present work the study of gene-disrupted parasites of the PfCCp Protein family was completed. PfCCp5-KO and PfFNPA-KO parasite lines were characterized to a great extent and many properties were similar to those of other PfCCp proteins. The co-dependent expression previously reported to be a phenomenon of PfCCp proteins was also observed in these two mutants, although to lesser extent. When either PfCCp5 or PfFNPA were absent, all other proteins were detected in reduced abundance only. Co-dependent expression manifests exclusively on the protein level. Transcript levels were not altered as RT-PCR showed. Amongst PfCCp proteins numerous proteinproteins interactions are taking place. The previously described multimeric protein complexes also include further sexual stage-specific proteins like Pfs230, Pfs48/45 and Pfs25. Recently, a new component of PfCCp-based multimeric protein complexes had been identified. The protein was named PfWLP1 (WD repeat protein-like protein 1) due to its possession of several WD40 repeats. In the present study expression of this uncharacterized protein was investigated via indirect IFA. It was expressed in asexual blood stages and gametocytes. Upon gamete formation and fertilization its expression ceased. Another sexual stage protein studied in this work was PfactinII. It was shown to be exclusively expressed in sexual stages. In gametocytes it co-localizes with Pfs230 and correct localization of PfactinII depends on presence of Pfs230. Transcript analysis by means of RT-PCR revealed the expression of several components of the IMC in gametocytes. Furthermore, five or six myosin genes encoded in the P. falciparum genome were detected in gametocytes. Gametocyte egress was studied on the ultrastructural level via transmission electron microscopy and an inside-out type of egress was observed. Firstly, the membrane of the parasitophorous vacuole (PVM) was lysed and only thereafter the membrane of the red blood cell (RBCM) ruptured. Furthermore, a new inductor of gametogenesis was identified: The K+/H+ ionophore nigericin induced gametocytes activation in the absence of xanthurenic acid (XA), which is responsible for gamtetocyte activation in the mosquito midgut. Selective permeabilization of RBCM and PVM by the mild detergent saponin, showed that in the absence of these membranes male gametocytes were still able to perceive both XA and the drop in temperature. Thus, the receptors for both factors signaling the parasite transmission to the mosquito, seem to be of parasitic origin. LC/MS/MS analysis confirmed the ability of RBCs to take up XA. With malaria eradication on the agenda of malaria research targeting the sexual stages becomes a crucial part of intervention strategies. The sexual stages are especially attractive target as they represent a population bottleneck. The here reported findings on P. falciparum gametocytes provide several potential candidate proteins for developing tools to interrupt transmission from man to mosquito. Such tools might include Transmission blocking vaccines and drugs
Die Übertragung der Malaria vom menschlichen Wirt auf die Überträgermücke erfordert die Bildung von Sexualstadien, der Gametozyten. Dieses Parasitenstadium ist in der Lage im Mitteldarm der Mücke zu überleben und sich zu Gameten zu entwickeln, gefolgt von Befruchtung und ygotenbildung. Eine Vielzahl von in den Sexualstadien exprimierter Proteine wurde bereits entdeckt. Einige von diesen haben essentielle Funktionen für die Transmission der Parasiten auf die Mücke. Die Rolle der meisten dieser spezifisch exprimierter Protein ist jedoch ungeklärt. Zu den sexualstadienspezifischen Proteinen gehören die Proteine der PfCCp-Proteinfamilie. Für vier Proteine diese Proteinfamilie wurden bereits KO-Mutanten untersucht. Zwei Mutanten, PfCCp2-KO und PfCCp3-KO besitzen eine wichtige Funktion während der Entwicklung der Parasiten in der Mücke. In der vorliegenden Arbeit wurde die Studie der PfCCp-Proteine komplettiert. PfCCp5- und PfFNPA-defiziente Parasitenlinien wurden zu einem Großteil charakterisiert. Viele Eigenschaften dieser beiden Parasitenlinien wiesen Ähnlichkeiten zu den bisher untersuchten PfCCp-KO-Mutanten auf. Die ko-abhängige Expression welche in der PfCCp-Proteinfamilie vorkommt, wurde auch in diesen beiden Mutanten beobachtet, wenngleich in geringerem Ausmaß. In den Mutanten, in welchem entweder PfCCp5 oder PfFNPA fehlten, waren alle übrigen PfCCp-Proteine nur in reduzierter Menge nachzuweisen. Diese ko-abhängige Expression ist ausschließlich auf dem Proteinlevel zu beobachten. Die Transkription der jeweiligen Gene hingegen ist unbeeinflusst. Zahlreiche Protein-Protein-Interaktionen finden zwischen den Proteinen der Proteinfamilie statt. The zuvor beschriebenen multimeren Proteinkomplexe schließen auch weitere sexualstadienspezifische Proteine ein, wie Pfs230, Pfs48/45 und Pfs25. Kürzlich wurde eine neue Komponente der PfCCp-basierten Multiproteinkomplexe identifiziert. Dieses Protein wurde PfWLP1 (WD repeat protein-like protein 1) genannt, da es mehrere WD40 repeat Domänen besitzt. In der vorliegenden Arbeit wurde das bisher unbeschriebene Protein mittels indirekter immunfluoreszenzstudien charakterisiert. PfWLP1 ist sowohl in asexuellen Blutstadien als auch in Gametozyten exprimiert. Nach der Gametenbildung und Fertilisation nimmt die Expression des Proteins ab. Ein weiteres Protein der Sexualstadien, welches in dieser Arbeit untersucht wurde, ist PfactinII. Es wurde gezeigt, dass dieses Protein ausschließlich in den Sexualstadien vorliegt. In Gametozyten ko-lokalisiert es mit Pfs230 und die korrekte Lokalisierung ist abhängig von der Anwesenheit von Pfs230. Mittels RT-PCR wurden mehrere Komponenten des inneren Membrankomplexes in Gametozyten nachgewiesen. Weiterhin wurden Transkripte für fünf der sechs Myosin-Gene, welche im Genom von P. falciparum exprimiert sind, nachgewiesen. Der Austritt der Gametozyten aus der Wirtszelle wurde auf ultrastruktureller Ebene mittels Transmissionselektronenmikroskopie untersucht. Hierbei wurde gezeigt, dass die Lyse der den Parasiten umgebenden Membranen von innen nach außen geschieht. Das heißt, dass zunächst die Membran der parasitophoren Vakuole (PVM) lysiert wird, und erst anschließend die Erythrozyten-Plasmamembran (RBCM). Als neuer Induktor der Gametozytenaktivierung wurde Nigericin identifiziert. Nigericin ist ein K+/H+-Ionophor, welcher in Abwesenheit von XA in der Lage ist, die Gametenbildung zu identifizieren. Die selektive Permeabilisierung der beiden den Gametozyten umgebenden Membranen, PVM und RBCM, zeigte, dass männliche Gametozyten nach Entfernung der beiden Membranen, in der Lage sind, den Temperaturabfall und XA zu perzipieren. Somit kann geschlussfolgert werden, dass die Rezeptoren beider Stimuli parasitischen Ursprungs sind. LC/MS/MS-Analysen bestätigten, dass Erythrozyten in der Lage sind, XA aufzunehmen. Die Sexualstadien des Malariaparasiten nehmen mehr und mehr an Bedeutung zu, da langfristig nicht nur eine Eindämmung der Malaria in endemischen Gebieten sondern die Auslöschung der Malaria angestrebt wird. Die Sexualstadien sind ein attraktiver Angriffspunkt aufgrund ihrer Bedeutung für die Transmission der Krankheit. Zum anderen ist die auf den Vektor übertragene Parasitenanzahl vergleichsweise gering. Die Ergebnisse der vorliegenden Arbeit zeigen mehrere potentielle Kandidatenproteine auf, welche für die Entwicklung von Interventionsstrategien von Bedeutung sein könnten. Als Interventionsstrategien wären sowohl transmissionsblockierenden Vakzine als auch transmissionsblockierende Wirkstoffe denkbar

PhD. thesis: The role of bZIP transcription factors in the male gametophyte of Arabidopsis thaliana Abstract Sexual plant reproduction depends on the production and differentiation of functional gametes by the haploid gametophyte generation. Currently, we have a limited understanding of the regulatory mechanisms that have evolved to specify the gametophytic developmental programs. To unravel such mechanisms, it is necessary to identify transcription factors (TF) and their complexes that are part of such haploid regulatory networks. For that reason we selected candidate Arabidopsis bZIP TFs, which have been shown to have critical roles in plants, animals and other kingdoms. Here we describe the putative bZIP TFs regulatory network active in Arabidopsis thaliana pollen and highlight to the greater impact two members of this network - AtbZIP18 and AtbZIP34. We report the complex functional characterization of AtbZIP34 and AtbZIP18, which are widely expressed in both gametophytic and sporophytic tissues, however they possess significantly enhanced expression during late stages of pollen development. We have studied both genes using several genetic and molecular approaches. Several lines of evidence, including the AtbZIP34 expression pattern and the phenotypic defects observed, suggest a complex role of AtbZIP34...

v angličtině (English abstract) Tobacco male gametophyte has a strongly dehydrated cytoplasm and represents a metabolically inactive stage. Upon cytoplasm rehydration, pollen grain becomes metabolically active and after the activation is finished, the pollen tube growth through a selected pollen aperture starts. The rehydration together with metabolic activation are accompanied by the regulation of translation and post-translational modifications (mainly phosphorylation) of the existing proteins. In this Ph.D. thesis, there were identified phosphopeptides from tobacco (Nicotiana tabacum) mature pollen, pollen activated in vitro 5 min and pollen activated in vitro 30 min. The total proteins from the above male gametophyte stages were extracted. The protein extract was trypsinized and the acquired peptide mixture was enriched by MOAC (metal oxide/hydroxide affinity chromatography) with titanium dioxide matrix. The enriched fraction was subjected to liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Totally, there were identified 471 phosphopeptides, carrying 432 exactly localized phosphorylation sites. The acquired peptide identifications were mapped to 301 phosphoproteins that were placed into 13 functional categories, dominant of which were transcription, protein synthesis,...

Alba family proteins are highly conserved in all domains of life. They are involved in RNA metabolism in Archae and Eucarya, while they are involved in the chromatin organisation in Crenarchaea. In animals, ALBA proteins were identified to associate with RNase P/MRP subunits. The objective of my thesis was the characterization of ALBA family proteins in a model plant Arabidopsis thaliana. The Arabidopsis genome contains six genes with close homology, three from Rpp20-like subfamily and three of Rpp25-like subfamily. Here I present the localization of GFP-fused proteins in Arabidopsis stable lines harbouring constructs cloned by Gateway® Technology. ALBA proteins were localized in the cytoplasm and undefined particles in root differentiation zone and in mature pollen. The characterization of the respective T-DNA insertion lines did not reveal significant phenotype defects in growth and development of sporophyte and gametophyte in comparison to Columbia-0 plants, probably because of likely functional redundancy od the paralogs. Expression profiles and localization of ALBA proteins suggest their possible role in differentiation and dehydration stress response in Oryza. They were also observed to associate with repressed mRNA transcripts in storage EPP particles. Collectively, I propose the likely role...

Tese de mestrado. Biologia (Microbiologia Aplicada). Universidade de Lisboa, Faculdade de Ciências, 2013
Malaria is caused by a parasite of the genus Plasmodium and remains the most important parasitic disease. The emergence of Plasmodium falciparum parasites resistant to all known antimalarial drugs is of major concern. New antimalarial drugs are needed, not only a drug that overcome the undesirable side effects of current antimalarial drugs but new highly active ones against the asexual stages, as well as, drugs that could also eliminate the transmissible sexual form of the parasite to the mosquito. To test the susceptibility of the parasite to drugs a variety of sensitivity assays can be used to screen new compounds, such as: hypoxanthine incorporation, ELISA based assays like pLDH and HRP2, fluorometric and flow cytometric assays. Recently, a novel flow cytometric sensitivity assay based on hemozoin detection was described. Using this novel sensitivity assay new antimalarial compounds were screened at 1 and 3 μM. The SYBR green DNA staining assay and the HRP2 were also perfomed as mean of comparison. Results showed that none of the tested compounds presented inhibitory activity against P. falciparum strains 3D7 and Dd2 at 1 μM, independently of the method used. Only one of the compounds showed more than 50% inhibition at 3μM. The flow cytometric hemozoin detection method was also assessed for its potential to detect gametocytes. Gametocyte may have a different depolarizing profile, based on the underlying hemozoin distribution. Thus, we further investigated if they could be distinguished from other parasitie forms based on their higher degree of depolarization. A culture enriched in gametocytes was FACS sorted by selecting the higher depolarizing population. Results showed that gametocytes were selectively present only in the high depolarizing population and not in the middle and non-depolarizing events. Therefore, this recently described sensitivity assay based on hemozoin detection can be used as a novel approach to screen for new antimalarial drugs. This approach has as major advantages the fact that results can be obtained in only 24 h of incubation and no additional reagents or additional incubation times are required. Another important characteristic of this method is that it might be able to detect gametocytes based on the particular hemozoin distribution in these forms, which can lead to the use of this method to test antimalarial transmission blocking drugs.
A malária é uma doença que segundo os mais recentes dados da Organização Mundial de Saúde (OMS) foi responsável por cerca de 660 000 mortes e 219 milhões de casos em 2010. É a doença parasitária que mais mortes causa sendo a região mais afetada por esta doença a África subsariana, e com uma maior incidência em crianças até aos 5 anos de idade. A malária é causada pelo parasita do género Plasmodium. As cinco espécies que podem causar doença no humano são: P. falciparum, P. vivax, P. ovale, P. malariae e P. knowlesi. No entanto P. falciparum é a espécie responsável pelo maior número de casos e mortes por malária sendo também o que pode levar a uma maior severidade da doença. A malária manifesta-se por períodos de febres altas e calafrios, mal-estar generalizado, torpor e dor de cabeça, naúsea e dor abdominal, por vezes até vómitos e diarreia. No caso de doença severa esta implica anemia severa, malária cerebral, síndrome de dificuldade respiratória aguda, insuficiência renal e nos casos mais graves a morte. Segundo estes factos, a maária é uma doença que levanta preocupação e tem impacto a nível mundial. Isto porque, por várias circunstâncias se pensou que estaríamos no caminho da erradicação da doença e no entanto, apesar de o número de casos e mortes ter diminuído na última década, esta continua a afetar milhões de indivíduos. Hoje em dia, um dos maiores problemas face a esta doença prende-se com o facto de P. falciparum já ter apresentado resistência a todos os fármacos utilizados no tratamento da doença. Entre os antimaláricos já usados encontram-se a quinina, a cloroquina, a primaquina, a pirimetamina conjugada com a sulfadoxina, a mefloquina entre outros. No entanto, as atuais directrizes para o tratamento da malária correspondem a terapias de combinação com recurso a derivados da artemisinina (“Artemisinin-based combination therapies” – ACTs). Alguns exemplos destas combinações são: artesunato combinado com amodiaquina, artesunato com mefloquina, arteméter e lumefantrina, entre outros. O principal objectivo detas terapias é eliminar as formas do parasita que circulam no sangue, pois é este estadio que conduz à manifestação da doença. Plasmodium é um parasita que apresenta um ciclo de vida complexo. É transmitido ao humano através da picada de um mosquito infectado femea do género Anopheles. Durante a picada, o parasita que existe nas glândulas salivares do mosquito é injetado na corrente sanguínea do humano, e dirige-se até ao fígado. No fígado vários parasitas vão invadir os hepatócitos, dentro dos quais maturam e replicam antes de serem libertados novamente na corrente sanguínea. Esta fase hepática é assintomática. Uma segunda vez na corrente sanguínea, os parasitas vão invadir eritrócitos dentro dos quais vão maturar e replicar antes de lisarem o eritrócito, libertando novos parasitas para invadir novos eritrócitos. Uma vez dentro de um eritrócito, um parasita segue um ciclo de maturação que compreende as seguintas formas: forma de anel, trofozoíto, e esquizonte. No final, irá libertar vários merozoítos. No entanto, ocasionalmente alguns destes merozoitos, quanto invadem um eritrócito vão originar formas sexuadas do parasita, os gametócitos. Os gametócitos apresentamse sob a forma de percursores masculinos e femininos, e são estas formas que quando em circulação podem ser ingeridas por um mosquito durante uma nova picada. Após ingeridas pelo mosquito, estas formas vão recombinar genéticamente, dando origem a uma nova descendência que será novamente transmitida ao humano, recomeçando este ciclo. Colocam-se assim dois grandes objectivos à erradicação da malária: (i) um passa pelo desenvolvimento de novos agentes antimaláricos, devido à evidênca de resistência de P. falciparum a todos os actuais antimaláricos, incluindo os derivados da artemisinina que compreendem as atuais diretrizes de tratemento da doença; (ii) o outro prende-se com a eliminação das formas sexuadas do parasita em circulação no hospedeiro, pois eliminando estas formas, quebra-se o ciclo de transmissão hospedeiro-vector. Para a contínua pesquisa de novos compostos antimaláricos é necessário ensaios que detectem a sensibilidade dos parasitas aos diferentes fármacos. Os ensaios de sensibilidade actualmente existentes são: (i) o teste de microscópia da OMS; (ii) o teste por incorporação de hipoxantina; os testes baseados na detecção de anticorpos (Enzyme-Linked Immunosorbent Assay – ELISA) para quantificação de proteinas do parasita como a (iii) lactase desidrogenase (Parasite lactate dehydrogenase – pLDH e a (iv) proteina rica em histidina 2 (Histidine Rich Protein - HRP2); testes baseados na deteção do DNA do parasita por (v) ensaios fluorométricos e por (vi) citometria de fluxo. No entanto, como até agora não existe o teste de sensibilidade ideal, e devido a várias limitações de cada ensaio, novos ensaios são desenvolvidos. Um dos mais recentes baseia-se na deteção de hemozoina por citometria de fluxo. A hemozoína, também denominada pigmento malárico, é um bioproduto que resulta da destoxificação de heme livre produzido após a metabolização da hemoglobolina pelo parasita, e acumula-se no interior do eritrócito durante a maturação do mesmo. A hemozoína é um cristal com propriedades birefringentes que levam à despolarização da luz. Devido a esta propriedade, foi desenvolvido um ensaio de citometria de fluxo que detecta a hemozoína presente no interior de eritrócitos infectados. Assim, com o acumular de hemozoina ao longo do tempo, a deteção dos parasitas vai aumentando, havendo um maior número de eventos a despolarizar entre as 24h-30h de um primeiro ciclo de maturação, altura em que a maioria dos parasitas se apresenta como esquizonte, o estadio que também tem maior quantidade de hemozoína. Este ensaio permitiu detectar a sensibilidade de P.falciparum a vários antimaláricos e foi agora utilizado para testar a sensibilidade do parasita a novos antimaláricos cedidos pelo grupo do Professor Doutor Rui Moreira da Faculdade de Farmácia. A pesquisa da actividade dos novos compostos (“screening”) a 1 μM e a 3 μM foi realizada em duas estirpes de laboratório de P. flaciparum, a 3D7 (sensível à cloroquina) e a Dd2 (resintente à cloroquina). Após incubar a estirpe Dd2 em presença dos vários compostos, a sua inibição foi avaliada por citometria de fluxo, utilizando o ensaio da deteção de hemozoína e pelo ensasio da deteção de parasitas cujo DNA foi corado com SYBR green. Os resultados de ambos os ensaios demonstraram que nenhum dos 18 compostos testados a uma concentração de 1 μM levou à inibição do parasita. Com a estirpe 3D7 foram testados 24 novos compostos, não só utilizando os dois médotos de citometria de fluxo já referidos como também através de HRP2-ELISA. Os resultados demonstaram que independentemente do ensaio utilizado nenhum dos novos compostos apresentou actividade inibitória a 1 μM. Porém os compostos foram testados a uma concentração mais elevada, e a 3 μM um dos compostos demonstrou actividade inibitória (o composto 321). Determinou-se a concentração à qual este composto inibe 50 % do crescimento do parasita (CI50) de acordo com os três ensaios para detectar a sensibilidade do parasita obtendo-se um valor à cerca de 2 μM. No entanto, esta concentração é demasiado alta para o composto poder ser posteriormente testado como agente antimalárico na fase sanguínea do parasita, pois os actuais antimaláricos actuam na ordem nos nanomolar (nM). Para erradiacar a malária, a eliminação de gametócitos é um dos principais objectivos, de modo a quebrar o ciclo de transmissão da doença. Porém, existem muito poucos tratamentos que inibam estas formas sexuadas, e por isso é necessário continuar a investigar novos fármacos que actuem com mais impacto neste estadio do parasita. Para tal, uma vez mais são necessários ensaios que possam averiguar a sensibilidade dos gametócitos a diversos fármacos. No entanto, não exitem tantos ensaios para tal, como os que existem para testar a sensibilidade dos estadios assexuados. Uma vez que os gametócitos também apresentam hemozoína no seu interior, pressupôs-se que o ensaio de sensibilidade baseado na deteção de hemozoína permitisse também detectar gametócitos, retirando partido do facto de a hemozoína acumulada nos gametócitos apresentar-se de forma diferente nos esquizontes, pois ocorre sob a forma de vários e pequenos fragmentos ao contrário de um único e grande aglumerado. Assim, colocou-se a hipótese de se detectar os gametócitos por citometria de fluxo, e que estes corresponderiam a uma diferente população de acordo com a despolarização da sua hemozoína. Para corroborar esta ideia, estabeleceu-se a diferenciação de gametócitos a partir de culturas contínuas e posteriormente, através de análise por citometria de fluxo, os eventos da cultura de gametócitos foram separados (“sorting”) com base na sua depolarização. Partindo do princípio que os gametócitos têm mais cristais, estes podem levar a uma maior quantidade de luz a depolarizar, logo iriam localizar-se num nível de despolarização mais elevado do que os esquizontes. Assim, três populações foram separadas do seguinte modo: (i) população que não despolariza; (ii) população que despolariza a um nível médio (semelhante ao nivel de despolarização de esquizontes) e (iii) população a um nível mais elevado de despolarização. Após observação de cada uma das populações por microscopia, constatou-se que dos eventos adquiridos, os gametócitos só se encontravam na população cuja despolarização era a mais elevada. Com este recente ensaio baseado na deteção de hemozoína foi então possivel testar a actividade inibitória de novos compostos antimaláricos, obtendo as mesmas conclusões que outros dois métodos utilizados em paralelo. Este ensaio poderá assim ser utilizado no rastreio da actividade de novos compostos, como alternativa aos existentes actualmente, aos quais, há resistências desenvolvidas pelo parasita assim como o facto de conduzirem a efeitos secundários indesejáveis. Em relação aos outros ensaios de sensibilidade, este método apresenta-se como um método rápido de obter resultados (24h) e em tempo real, sem a necessidade de adição de reagentes ou tempos adicionais de incubação com reagentes. Este método permitiu ainda detetar uma população específica do parasita, os gametócitos, os quais representam um dos principais alvos para a eliminação da malária. Assim, este método poderá também ser investigado como ensaio de sensibilidade dirigido a gametócitos.

From RNA-to-protein, translation initiation and protein synthesis is mediated by trans-acting factors that recognize mRNA features common to almost all eukaryotes. Eukaryotic translation initiation factor 3 complex (eIF3) is a highly conserved protein complex that recognizes 5'-CAP elements of the mRNA to initiate translation. eIF3 consists of nine subunits, three of them having two isoforms: eIF3A, eIF2B1, eIF3B2, eIF3C1, eIF3C2, eIF3D, eIF3E, eIF3F, eIF3G1, eIF3G2, eIF3H and eIF3K. This work deals with functional characterization, expression and subcellular localization of eIF3B1, eIF3B2 and eIF3E in Arabidopsis thaliana male gametophyte and interaction of eIF3E with the Constitutive photomorphogenesis 9 (COP9) complex as a regulatory complex of eIF3E post-translational control. Here we show that depletion of eif3b1 or eif3b2 is not gametophytic lethal and that the two protein might function redundantly, whereas, knockout of eIF3E causes male gametophyte lethality. Interestingly, eif3b1 show post-fertilization defects during embryogenesis, suggesting that its redundancy with eIF3B2 is restricted to the gametophyte. Gene expression studies revealed high expression of eIF3 subunits in actively dividing zones of leaf primordia, root meristem and root elongation zones as well as in the vegetative...

abstract: The complex life cycle and widespread range of infection of Plasmodium parasites, the causal agent of malaria in humans, makes them the perfect organism for the study of various evolutionary mechanisms. In particular, multigene families are considered one of the main sources for genome adaptability and innovation. Within Plasmodium, numerous species- and clade-specific multigene families have major functions in the development and maintenance of infection. Nonetheless, while the evolutionary mechanisms predominant on many species- and clade-specific multigene families have been previously studied, there are far less studies dedicated to analyzing genus common multigene families (GCMFs). I studied the patterns of natural selection and recombination in 90 GCMFs with diverse numbers of gene gain/loss events. I found that the majority of GCMFs are formed by duplications events that predate speciation of mammal Plasmodium species, with many paralogs being neutrally maintained thereafter. In general, multigene families involved in immune evasion and host cell invasion commonly showed signs of positive selection and species-specific gain/loss events; particularly, on Plasmodium species is the simian and rodent clades. A particular multigene family: the merozoite surface protein-7 (msp7) family, is found in all Plasmodium species and has functions related to the erythrocyte invasion. Within Plasmodium vivax, differences in the number of paralogs in this multigene family has been previously explained, at least in part, as potential adaptations to the human host. To investigate this I studied msp7 orthologs in closely related non-human primate parasites where homology was evident. I also estimated paralogs’ evolutionary history and genetic polymorphism. The emerging patterns where compared with those of Plasmodium falciparum. I found that the evolution of the msp7 multigene family is consistent with a Birth-and-Death model where duplications, pseudogenization and gene lost events are common. In order to study additional aspects in the evolution of Plasmodium, I evaluated the trends of long term and short term evolution and the putative effects of vertebrate- host’s immune pressure of gametocytes across various Plasmodium species. Gametocytes, represent the only sexual stage within the Plasmodium life cycle, and are also the transition stages from the vertebrate to the mosquito vector. I found that, while male and female gametocytes showed different levels of immunogenicity, signs of positive selection were not entirely related to the location and presence of immune epitope regions. Overall, these studies further highlight the complex evolutionary patterns observed in Plasmodium.
Dissertation/Thesis
Doctoral Dissertation Biology 2016

Conclusions My thesis was devoted to the investigation of a complex regulatory network of transcription factors in the Arabidopsis thaliana male gametophyte. In addition to the abovementioned objective, the second aim of the thesis was the creation of a web-based transcriptome database and data mining tool characterized by an innovative concept of presentation and evaluation of the data from DNA chips. The results of my thesis can be summarized as follows: 1. We created a database called Arabidopsis Gene Family Profiler (arabidopsisGFP) (http://agfp.ueb.cas.cz/) which was made available on-line. This database was developed in order to provide a new tool for presentation of gene expression data (transcriptome), allowing the user to display the data in a quick and intuitive way using the innovative and when launched also unique concept of a virtual plant. The database employs a progressive idea "from simple to complex", but also provides the user with more traditional data presenting options. The aGFP database can serve as a reference manual for the expression of individual genes and gene families in Arabidopsis thaliana under physiological conditions at different phases of the life cycle and in individual organs and tissues or cell types. 2. A chapter reviewing available servers for deposition of the...

In plants, translation regulation plays an important role during progamic phase, fertilization and seed development. The process of translation is mostly regulated in its initiation phase, where Eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex initiation factor, consisting of 12 different subunits. In plants, single eIF3 subunit mutants caused various growth and development defects, depending on the particular subunit that was mutated. However, not all the plant eIF3 subunits were characterized to this date. The objective of this work was to functionally characterize the eIF3 subunit A using Arabidopsis thaliana as the main model plant. We described in this work that plant eIF3A proteins share high levels of homology and domain organization with eIF3A subunits from non-plant eukaryotic species but contain regions specific only to plants. Next we described that Arabidopsis thaliana AteIF3A gene is transcribed in highly proliferating tissues, its protein product localizes to cytoplasm and around pollen vegetative cell nucleus and observed an increased frequency of defective pollen grains and defects in seed formation in plants with T-DNA insertion localized to the AteIF3A gene. We also produced stable transgenic Nicotiana tabacum lines expressing heterologous AteIF3A...

(anglicky) Alba-family proteins were identified in Archaea and Eucarya and are classified among the oldest and the most conserved nucleic acid-binding proteins. The binding preferences and roles differ among certain evolution clades. In Crenarchaea they represent chromatin-binding proteins, while their role in RNA metabolism is suggested in Euryarchaea and Eukaryotes. ALBA proteins are well characterized in human, where they play a role in the RNAse P/MRP complex and in unicellular parasites, such as Plasmodium and Trypanosoma, where an involvement in the life cycle regulation is confirmed. In plants, their role is not yet well understood. The aim of this thesis is to increase a knowledge about the Alba-family proteins in the model plant Arabidopsis thaliana. Based on a minimal changes to development and reproduction in single mutants and high sequence similarity, a functional redundancy of the proteins was assumed. For better understanding of the ALBA proteins function, three smaller members of the family were edited by the same metod. The obtained triple mutant showed delay in flowering. ALBA dimer formation was confirmed in many organisms. BiFC method was used to determine Arabidopsis ALBA homodimerization. The data analysis showed potential homodimerization in most of them.

In the presented work the relationship between transcription factors and male gametophyte development was studied. The Ph.D. Thesis covers selection of candidate genes, wide-scale screening of T-DNA mutant lines and detailed analysis of a selected transcription factor on pollen development.

The development of plant flowers represents a complex process controlled by numerous mechanisms. The creation of double homozygous mutant of both β subunits (sometimes also referred to as basic transcription factor 3) of nascent polypeptide associated complex in Arabidopsis thaliana (further referred to as nacβ1 nacβ2) caused quite a strong defective phenotype including abnormal number of flower organs, shorter siliques with a reduced seed set, and inferior pollen germination rate together with a lower ovule targeting efficiency. Previously, NAC complex was described to be formed as a heterodimer composed of an α- and β-subunit, which binds ribosome and acts as a chaperone in Saccharomyces cerevisiae. In plants, NACβ is connected to stress tolerance and to plant development as a transcription regulator. However, little is known of NAC heterodimer function in plants. In this thesis, yeast two hybrid system (Y2H) and bimolecular fluorescence complementation (BiFC) assays were used to verify the NAC heterodimer formation in A. thaliana and to establish any potential interaction preferences between both NACβ paralogues and five NACα paralogues. To deepen the understanding about molecular mechanisms behind the nacβ1 nacβ2 phenotype, flower bud transcriptome of the nacβ1 nacβ2 double homozygous mutants...

5 Abstract Tobacco mature pollen rehydrates in vivo on a stigma tissue, and develops into the rapidly-growing pollen tube. This rehydration process is accompanied by the de-repression of stored mRNA transcripts, resulting in the synthesis of novel proteins. Furthermore, such metabolic switch is also likely to be regulated on the level of post-translational modifications of the already-present proteins, namely via phosphorylation, since it was shown to play a significant regulatory role in numerous cellular processes. Since only a minor part of proteins is phosphorylated in a cell at a time, the employment of various enrichment techniques is usually of key importance. In this diploma project, metal oxide/hydroxide affinity chromatography (MOAC) with aluminium hydroxide matrix was applied in order to enrich phosphoproteins from the mature pollen and the 30-minute in vitro activated pollen crude protein extracts. The enriched fraction was separated by both 2D-GE and gel-free liquid chromatography (LC) approaches with subsequent mass spectrometric analyses. Collectively, 139 phosphoprotein candidates were identified. Additionally, to broaden the number of phosphorylation sites identified, titanium dioxide phosphopeptide enrichment of trypsin-digested mature pollen crude extract was performed. Thanks to the...