Rozprawy doktorskie na temat „Malaria – Pathogenesis”

Severe anemia is a major life-threatening complication of malaria. Inappropriately low reticulocytosis in malaria patients with anemia suggests insufficient erythropoiesis, of which the mechanisms and implications are not clear. The principle growth factor that promotes erythropoiesis is erythropoietin (Epo). Studies determining the serum level of Epo in malaria infected patients have been inconclusive. Furthermore, the role of Epo and the erythropoietic response to Epo stimulation during malaria have never been examined. The purpose of the experiments performed in this thesis was, thus, to investigate the role of Epo and erythropoiesis in relation to anemia during blood-stage malaria using the murine model of Plasmodium chabaudi AS. A murine Epo specific ELISA, which was determined to be less biased by the presence of other cytokines in the samples as compared to the conventional Epo bioassay, was first developed to facilitate the research. The kinetics of Epo production in the kidney and the levels in the serum were characterized. It was demonstrated that Epo production during blood-stage malaria is mainly regulated by the degree of anemia and that renal cytokines may have only a minor effect on this response. Next, the roles of Epo and erythropoiesis during blood-stage malaria were investigated by neutralization of endogenous Epo or by administration of exogenous Epo. Timely onset of Epo-induced reticulocytosis was shown to be important for the alleviation of malarial anemia and survival. However, reticulocytosis in response to Epo stimulation is severely suppressed by infection with malaria. Dissection of the upstream events of erythropoiesis demonstrated that blood-stage malaria compromises the generation of reticulocytes by suppressing the proliferation, differentiation, and maturation of erythroid-lineage cells at various stages of erythroid development. Taken together, our data provide important insights for understanding the patho

In this thesis we will consider two mathematical models on malaria transmission and patho- genesis. The transmission model is a human-mosquito interaction model that describes the development of malaria in a human population. It accounts for the various phases of the disease in humans and mosquitoes, together with treatment of both sick and partially im- mune humans. The partially immune humans (termed asymptomatic) have recovered from the worst of the symptoms, but can still transmit the disease. We will present a mathematical model consisting of a system of ordinary differential equations that describes the evolution of humans and mosquitoes in a range of malarial states. A new feature, in what turns out to be a key class, is the consideration of reinfected asymptomatic humans. The analysis will include establishment of the basic reproduction number, R0, and asymptotic analysis to draw out the major timescale of events in the process of malaria becoming non-endemic to endemic in a region following introduction of a few infected mosquitoes. We will study the model to ascertain possible time scale in which intervention programmes may yield better results. We will also show through our analysis of the model some evidence of disease control and possible eradication. The model on malaria pathogenesis describes the evolution of the disease in the human host. We model the effect of immune response on the interaction between malaria parasites and erythrocytes with a system of delay differential equations in which there is time lag between the advent of malaria merozoites in the blood and the training of adaptive immune cells. We will study the model to ascertain whether or not a single successful bite of an infected mosquito would result in death in the absence of innate and adaptive immune response. Stability analysis will be carried out on the parasite free state in both the immune and non immune cases. We will also do numerical simulations on the model to track the development of adaptive immunity and use asymptotic methods, assuming a small delay to study the evolution of the disease in a naive individual following the injection of small amount of merozoites into the blood stream. The effect of different levels of innate immune response to the pathogenesis of the disease will be considered in the simulations to elicit a possible immune level that can serve as a guide to producing a vaccine with high efficacy level.

Malaria is caused by infection with the protozoan Plasmodium. About 40% of the world’s population is at risk, nearly all of them living in the least developed countries. While 300-500 million cases are reported per year, about 2 million people die annually. Most of the fatal cases develop severe malaria, which is a complication of Plasmodium fulciparum infection. Symptoms can include multi-organ dysfunction, anaemia, lung complications and cerebral malaria (CM). Although the pathogenesis is controversial, there are two main theories to explain the aetiology of CM: sequestration of parasitised red blood cells (pRBC) and leukocytes in the vasculature, and an over-reaction of the immune system. In the latter case, enhanced production of deleterious immune effectors such as cytokines is involved. Experimental models of CM (in vitro and in vivo) have been developed because clinical studies are hampered by ethical constraints and post-mortem human tissue is hard to obtain. CBA or C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) succumb to murine CM and show progressive behavioural, histopathological and immunological changes. In contrast, Plasmodium berghei K173 (PbK)-infected C57BL/6 mice die due to anaemia and hyperparasitaemia, but without neurological symptoms. Although it is important to study the disease in the whole animal, in vitro studies are helpful to elucidate mechanisms at the cellular level. It is known that various cell types contribute to the pathogenesis of CM. In particular, endothelial cells lining the cerebral microvasculature are involved in the development of CM. They form part of the blood-brain barrier (BBB) which tightly regulates the traffic of substances and cells into the brain parenchyma to protect cerebral cells and to maintain the intemeuronal milieu. The permeability of the BBB is increased early after infection (day 3-4 p.i.) in the murine experimental model of CM. This might lead to an influx of deleterious substances into the cerebral parenchyma, causing the neurological symptoms observed. Changes at the microvascular endothelium are thought to contribute to the leakage of the BBB. In response to various stimulants ECs increase the expression of cellular adhesion molecules (CAM) on their surface. Cells such as leucocytes (in murine CM) and pRBCs (in human CM) adhere to the vessels, recruiting an immune response to the area.

Malaria ist die Erkrankung, die durch Infektion eines Säugetiers mit dem eukaryotischen Parasiten Plasmodium entsteht. Die Symptome dieser Erkrankungen reichen von Fieber und Gelenkschmerzen bis zu schweren Organschäden in Hirn, Lunge, Niere und Leber bei einem geringen Teil der Erkrankten. Diese klinischen Symptome treten nur auf, während der Parasit sich asexuell in roten Blutzellen vermehrt. Die Zerstörung von Erythrozyten und die daraus resultierende Freisetzung von Zytokinen sind die Verursacher der malariatypischen wiederkehrenden Fieberzyklen. Der Mechanismus, der zur Entstehung von Gewebsschäden führt, ist hingegen nur unzureichend bekannt. Eine notwendige Bedingung für das Auftreten von Gewebeschäden ist, dass infizierte rote Blutzellen an das Endothel der Mikrovaskulatur bin. Die Schwere der Erkrankung ist direkt mit dem extrazellulären Auftreten von Stoffen verbunden, die normalerweise von neutrophilen Granulozyten im Zellinnern gespeichert werden. Neutrophile sind dafür bekannt sind, ein ganzes Arsenal an Waffen bereitwillig durch Degranulierung oder programmierten Zelltod einzusetzen. In dieser Studie berichten wir von einem direkten kausalen Zusammenhang zwischen dem aktiven inflammatorischen Zelltod (NETose) von Neutrophilen und der Entstehung von Organschäden bei einer Plasmodium-Infektion. Wir zeigen, dass NETs in Zirkulation freigesetzt und von extrazellulären DNase verdaut werden. Dadurch werden systemisch Aktivierungssignale für eine weitere Immunantwort zur Verfügung gestellt und es kommt zur Freisetzung von Zytokinen, Notfallgranulopoese und der Hochregulierung von zellulären Adhäsionsmarkern auf Endothelzellen. Dies erlaubt das Binden von infizierten Erythrozyten und Immunzellen an die Mikrovaskulatur bestimmter Organe. Wir zeigen außerdem, dass eine Intervention mit der Entstehung von NETs oder der Freisetzung neuer Neutrophiler diesen Prozess unterbindet und einen vielversprechenden therapeutischen Ansatz darstellt.
Malaria is the disease caused by an infection of a mammalian host by the mosquito borne eukaryotic parasite Plasmodium. The symptoms of the disease are diverse, ranging from fever and rigor in most patients to severe damage in solid organs such as brain, lung, kidney and liver in a small fraction of the afflicted. Clinical symptoms of the disease only occur when the parasite undergoes asexual replication within the red blood cells of the host. Destruction of these cells and subsequent release of cytokines are responsible for the recurring fever cycles of mild malaria. The mechanism underlying the occurrence of tissue damage however, remain mostly elusive. The adhesion of infected red blood cells to the endothelial wall of the microvasculature in the affected organs is a necessary requirement and pathology is associated with the activation of specific immune cells residing within the blood stream. Severity of disease is linked to extracellular accumulation of neutrophil proteins. Neutrophils are abundant white blood cells, known to readily deploy an arsenal of weaponry either by degranulation or by externalization of chromatin. In this study we report a direct causal relationship between the active inflammatory neutrophil cell death (NETosis) and the development of organ damage during a Plasmodium infection. We show that NETs are released in circulation, digested by extracellular DNase and thereby supply immune activation signals that drive inflammation. The systemic dissemination of these factors leads to the release of cytokines, emergency granulopoiesis and upregulation of cellular adhesion markers on endothelial cells thereby allowing for the binding of both infected red blood cells and immune cells to the microvasculature of specific organs. Furthermore we supply evidence, that repression of NETosis or inhibition of granulopoiesis abrogate these processes and present promising therapeutic strategies.

Cerebral malaria (CM) is a severe complication of malaria, and involves the central nervous system (CNS). Despite the significant negative impact of CM, its pathogenesis is not fully understood. Two theories, namely cerebral hypoxia and cytokine expression, are considered to be involved in the process. The present study investigated the potential interaction of these two theories in driving the development of CM. Astrocytes can be a major determinant of the outcome of CNS diseases, and we hypothesised that astrocytes, by responding to the pathways involved in the two theories, would drive the development of CM. The cytokines interferon-gamma (IFN-γ) and lymphotoxin-alpha (LT-α) are essential for the development of experimental CM in a murine model. The chemokine C-X-C motif ligand 10 (CXCL10) also is implicated in this process. Both Malawian paediatric and mouse CM brain samples showed increased cytokine expression and astrocyte activation. Furthermore, by the use of Ifnγ-/- mice, it was shown that IFN-γ was involved in this CM-associated astrocyte activation. Cultured human primary astrocytes were directly activated by IFN-γ and LT-α to produce synergistic levels of CXCL10. This finding provides a potential mechanism by which astrocytes could be involved in the pathogenesis of CM, and sheds light on the possible role of LT-α in CM. Hypoxia had an effect on astrocytes, but their response to cytokines was not altered by hypoxia. However, oxygen-glucose deprivation resulted in a decline in cytokine-induced CXCL10 release by cultured astrocytes. Decreased production of CXCL10 has the potential to translate into less blood brain barrier damage. Thus, the mechanisms underlying these two theories do interact at the astrocyte level, but astrocytes show both protective and pathological features as a result of this interaction. The present thesis shows that it is unlikely that the two mechanisms act together to reinforce the astrocytes’ pathological effects to cause CM.

Malaria is one of the most serious human infectious diseases. To date, the collection of studies suggest that the disease is determined by transmission dynamics and host age altogether with host genetics and immunological responses. The precise and direct contribution of parasite components to the activation of such immunological responses has not been fully unravelled. In addition to a role proposed for plasmodial GPI, different lines of evidence suggest that hemozoin (HZ) could also be a potential inflammatory agent. The role of HZ in the modulation of immune responses has remained a polemic subject, making it difficult to describe the contribution of this molecule in pathogenesis of malaria. However, our previous laboratory studies, suggest that HZ has a pro-inflammatory role. For this reason, our study was designed to further define the contribution of HZ to the pro-inflammatory events related to malaria immunopathology, and to identify the intracellular signals underlying the up-regulatory effects of HZ in the macrophage, one of the major sources of inflammatory mediators in malaria. In order to do that, we used a chemically characterized synthetic version of the native PfHZ, rcHZ; and evaluated its effects on macrophage nitric oxide (NO) production. Our first approach was to compare the effects of rcHZ with other morphologically different versions of this molecule (aHZ and scHZ) alone or in combination with IFN-gamma on macrophage NO production. In a second approach, we evaluated if the presence of serum proteins plays a role in the increased IFN-gamma induced-NO production by rcHZ. In the third part of our study, we explored if rcHZ is able to increase NO production by macrophages when incubated in combination with a molecule from another pathogen, such as gram-negative bacteria lipopolysaccaride (LPS). The present study is a functional study that uses a synthetic and morphologically identical version of the native PfHZ. Our results suggests that intrinsic physical characteristics, such as shape and size; presence of host serum proteins, and presence of other pathogenic molecules, are important determinants for the macrophage response to HZ in the context of NO production. Besides, it describes part of the signaling pathways that are involved, which may contribute in the future, not only to understand mechanisms of regulation; but also, to find new therapeutic targets against malaria.

A complex network of elements is responsible for cerebral malaria (CM) development, but interactions between these elements are still being explored. Annually, there are 212 million cases of malaria with 1-2% progressing to CM. Plasma microvesicles (MV) are increased in patients and mice with CM and blocking their release protects against CM. The miRNA content of circulating plasma MV and brain tissue during murine CM and non-CM was assessed using microarray and RT-qPCR techniques. Following infection, MV and brain miRNA were altered in CM mice, coinciding with neurological syndrome onset. Particularly, miR-146a and miR-193b were dysregulated in plasma MV from CM mice and play roles in apoptosis, cytokine regulation and inflammatory cell recruitment. Several other miRNA were dysregulated in the brains of CM mice and play roles in TGF- signalling, endocytosis, FoxO signalling and adherens junctions. Using confocal microscopy, we investigated monocyte, platelet, T cell, parasite and ICAM-1-positive cell accumulation in CM brains. These cells accumulate at vessel branch points, associated with haemorrhaging and the development of tissue hypoxia, consistent with previous findings. As monocytes were the most numerous cells in the brains of CM mice, we targeted them therapeutically. Treatment in vivo with immuno-modulatory particles and artesunate resulted in 88% protection when administered at CM onset, while monotherapies resulted in greatly reduced protection. All successfully treated mice displayed reduced clinical signs of CM and monocyte and MV numbers, and were immune to reinfection. This thesis reinforces existing findings regarding CM pathogenesis and highlights several novel candidates as players in CM development. We explore the potential of miRNA as biomarkers of the neurological syndrome. We show that monocytes displaying a Ly6Clo phenotype aggravate CM development and that therapy targeting this cell subset is a novel strategy to abrogate late-stage CM.

L'importante virulence de P. Falciparum a été reliée à la capacité du parasite à adhérer aux cellules endothéliales de la microvasculature ou aux syncytiotrophoblastes placentaires. La séquestration de globules rouges infectés dans différents organes conduit à l'apparition des symptômes cliniques de la maladie. Les protéines variables PfEMP1, exprimées à la surface des hématies parasitées, sont les ligands parasitaires majeurs responsables de la cytoadhérence. PfEMP1-VAR2CSA est le candidat vaccinal le plus prometteur pour lutter contre le paludisme gestationnel. Afin de mieux caractériser les interactions entre la protéine VAR2CSA et son ligand naturel, la CSA, nous avons généré des VEIH contre VAR2CSA. Nous avons obtenu 19 VHHs, en grand partie dirigés contre le domaine DBLIX. Quatre VHHs contre DBL1X étaient capable d'inhiber les interactions entre les hématies parasitées et la CSA, montrant que le domaine DBL1X fait partie ou est localisé très proche du site de liaison au récepteur. Le paludisme sévère a été associé la séquestration des hématies parasitées, exprimant les cassettes de domaines DC8 ou DC13, au récepteur endothélial de la protéine C (EPCR) exprimé à l'endothélium vasculaire. Dans cette étude nous avons montré que la cassette DC8 de IT4VAR19 adhère avec une plus grande affinité à l'EPCR et HBEC5i que le domaine CIDRŒ I. 1 seul. Nous avons observé que, même si IT4VAR19 est le variant préférentiellement exprimé par la souche IT4 après sélection à l'EPCR, l'immunité humorale contre IT4VAR19-DC8 cassette et IT4VAR19-CIDR n'est pas stimulé pendant un épisode de paludisme pédiatrique sévère au Bénin. En conclusion, ce projet de thèse permet de mieux comprendre les interactions moléculaires impliquées dans l'adhérence des globules rouges parasités aux cellules endothéliales de l'hôte et contribuera à l'élaboration d'approches vaccinales ou thérapeutiques visant à protéger les patients des symptômes cliniques du paludisme sévère
Sequestration of infected erythrocytes (IEs) is the prime mediator of disease and is mediated by members of the highly diverse PfEMP1. The aim of this thesis was to characterize the molecular mechanisms associated to pregnancy associated malaria (PAM) and severe malaria, in order to design new intervention strategies to protect patients against severe malaria clinical symptoms. PfEMP 1 -VAR2CSA stands today as the leading vaccine candidate aiming to protect future pregnant women against the severe clinical outcomes of PAM. In order to better characterize the interactions between PfEMPI-VAR2CSA and its receptor CSA, we generated VAR2CSA specific nanobodies. Following immunization of a Ilama with the full-length VAR2CSA recombinant protein, we obtained 19 nanobodies, mainly targeting the DBL1X. Four nanobodies targeting DBL1X reproducibly inhibited CSA adhesion of erythrocytes infected with the homologous NF54-CSA parasite strain, providing evidences that DBL1X domain is part or close to the CSA binding site. Severe malaria was recently associated with binding of IEs, expressing domain cassettes DC8 and DC13, to Endothelial Protein C Receptor (EPCR) pr'sent in the host endothelium. In this study we demonstrated that the IT4VAR19-DC8 binds to EPCR with a greater affinity than the CIDRa1. L domain alone and also the binding to EPCR-expressing endothélial cell line (HBEC5i) is more pronounced. We observed that although IT4VAR19 is the preferentially selected EPCR-binding variant from IT4 strain, the humoral immunity against the EPCR binding IT4VAR19-DC8 cassette or the CIDRa1. 1 domain is not boosted during a severe pediatric malaria episode in Benin. In conclusion, this thesis provide new insights on the molecular mechanisms underlying the binding of P. Falciparum infected erythrocytes to the host endothelium that will help in the development of anti-adhesive strategies to protect patients against severe malaria clinical outcomes

Endothelium plays a crucial role in the regulation of vascular homeostasis and permeability. Its adhesion proprieties are highly regulated to allow physiologic functions, such as the migration of leucocytes into tissue, and to avoid pathologic degenerations. In particular, the adhesion of red blood cells (RBC) to endothelial cells (ECs) is involved in pathologies such as sickle cell anaemia, diabetes and malaria. In severe malaria RBC infected with Plasmodium falciparum (Pf) undergo membrane alterations which cause a receptor-mediated adhesion to endothelium. This phenomenon is called cytoadherence and is responsible for the microvascular occlusions and consequent tissue hypoxia, which are associated with severe malaria. The mechanisms and the molecular pathways activated by cytoadherence are extensively studied due to the high necessity of new adjuvant therapies for severe malaria. The apoptosis of ECs by PRBC is one of the processes described in vitro and in vivo. It is considered critical for the development of the disease but its mechanisms and molecular consequences have not been clarified yet. Among the drugs used to treat malaria, including the severe forms, the derivatives of artemisinin, an active principle extracted from the plant Artemisia annua, are very important due to their high efficacy and tolerability. Besides their antimalarial activity, this class of molecules is studied for the antiangiogenic and antitumor effect. In a previous work from our laboratory we investigated the antiangiogenic effect of artemisone, one of the newest artemisinin derivatives. In particular, the inhibition of ECs growth and the production of pro-angiogenic cytokines have been demonstrated in different in vitro and ex vivo models. The aim of this work was to study the role of ECs in malaria considering two aspects: the effect of parasitized RBC (PRBC) and the effect of dihydroartemisinin (DHA), the active metabolite of most artemisinins. The first part of the project was performed at INSERM/UPMC UMR S511 Faculté de Médecine Pierre et Marie Curie, Paris, under the supervision of Dominique Mazier. We characterised two genes of Pf with putative pro-apoptotic function on pulmonary ECs. Using sera from immunised mice, we demonstrated the nuclear localisation of the proteins encoded by these genes. Therefore, it was not possible to proceed with the functional studies, which consisted in using the antibodies to inhibit the pro-apoptotic function. However, the information obtained is important to further investigate the function of these proteins. The second part of the thesis reports the studies on the effect of DHA on ECs in hypoxia, which is a condition common to the microvessels of severe malaria patients and to the tumour microenvironment. In the first case hypoxia may contribute to inflammation and endothelial damage, whereas in tumours it is a potent stimulus for the production of new vessels, fundamental to bring nutrients to the tumour mass. We demonstrated that the inhibition of ECs growth by DHA is strongly influenced by the oxygen tension: at low doses (similar to those found in the plasma of malaria patients) DHA is more active in hypoxia, whereas at high doses (more similar to the doses proposed as antiangiogenic and antitumor) it is more effective in normoxia. We verified that DHA induces apoptosis only at high doses and affects the cell cycle via induction of oxidative stress. Further studies are needed to understand the mechanisms of growth inhibition in hypoxia, evidenced at the low doses of DHA, and to envisage the possible therapeutic application of this phenomenon.

Introduction Malaria retinopathy is a set of visible changes in the retina which are specific to falciparum malaria. Studies to date have been mostly limited to comatose African children. Retinal changes in adults with severe malaria and severely unwell patients without malaria have been less well studied and the specificity, pathogenesis, diagnostic and prognostic value of malarial retinopathy in adults are not known. Methods A series of observational studies of retinopathy in Bangladesh, India and Malaysia were done from 2008-2012. The aims were to describe the spectrum of retinal changes in falciparum and knowlesi malaria in adults, determine their specificity for severe falciparum malaria, quantify the impact of malaria retinopathy on visual function, understand its pathogenesis and assess the potential contribution of retinopathy to confirming diagnosis of malarial coma, predicting prognosis and understanding pathogenesis of cerebral malaria. Results 495 patients were enrolled and underwent retinal photography (305 with P. falciparum malaria (112 cerebral, 68 noncerebral severe, 125 uncomplicated), 44 P. knowlesi, 43 sepsis, 41 encephalopathy and 62 healthy). Retinal whitening and white-centred haemorrhages were common and specific to severe falciparum malaria. Retinopathy was most common and severe in cerebral (88%) and fatal (91%) falciparum malaria. Moderate-severe retinopathy was 95% specific for cerebral malaria in comatose patients, and its severity correlated with depth of coma. Vessel whitening was not seen and papilloedema was rare. In noncerebral severe falciparum malaria, retinopathy predicted increased likelihood of later development of coma and death. Retinal findings in Bangladeshi children were similar to those in adults. Optic nerve sheath diameter was mildly increased and brain swelling minimal on MRI. Severity of retinopathy correlated with plasma lactate, serum bicarbonate, sequestered parasite load and red cell stiffness suggesting a central role for microvascular obstruction in the pathogenesis. Severity of retinal whitening correlated with decreased visual acuity. Conclusions Retinal changes seen in severe P. falciparum malaria in Asian adults is similar, but not identical, to that seen in African children. They have potential to help with diagnosis and prognosis of Asian adults with severe falciparum malaria. Microvascular obstruction is prominent in the pathogenesis of retinopathy and coma in adults whereas raised intracranial pressure is not.

Infectious diseases that spread from person-to-person and continent-to-continent are a cause for concern for any health entity. One such disease is malaria, a mosquito-borne infection instigated by the protozoan parasite, Plasmodium falciparum. Hundreds of millions of people are affected annually and it is responsible for nearly 1 million deaths. It is the most fatal species causing malaria and proliferates in human red blood cells with a life cycle occurring every 48 hours. At this time, the parasite’s late stage form or schizont bursts from the erythrocyte releasing immune-inducing particles and infective forms (merozoites) into the bloodstream. The merozoites go on to infect other red blood cells as human immunity leads to fever. Fever is a hallmark symptom of malaria and effectively inhibits the growth of late stage parasites. Plasmodium still manages to complete its life cycle as early stages or rings are not affected by febrile temperatures. It is this facet of parasite biology that prompts our research into identifying genetic factors associated with fever. The parasite’s response under elevated body temperature may offer further insight into its adaptive mechanism. A heat shock assay was developed in order to simulate fever in vitro. Mutant parasite cultures were subjected to 41°C for 8 hours and returned to normal body temperature or 37°C for the remainder of the life cycle. The piggyBac mutagenesis system allows for the evaluation of phenotypes associated with a particular genotype as the transposon inserts randomly into the gene. This often leads to changes in function that may cause delays in invasion or attenuation of growth. Determining the genes responsible for these phenotypes would be a great advantage to the field of drug discovery. Collaborative efforts to develop vaccines and new antimalarial drugs are underway as resistance to current methods of treatment is on the rise. Such circumstances require new technologies for detecting novel drug targets or pathways in the parasite that can be significantly affected by these therapeutics. QISeq is a next generation sequencing tool that identifies genes with a particular phenotype that may alter intraerythrocytic development of P. falciparum. This technique was utilized in our study to confirm the heat shock phenotype with a high-throughput approach. The genomic DNA of pooled parasite cultures was sequenced to reveal those mutants sensitive and/or resistant to febrile temperature exposure. Through bioinformatics analyses, functional associations between genes can be made that lead to biological pathways of interest for therapeutic research.

[Truncated abstract] Iron is essential to most biological systems. Under pathological conditions affecting the iron metabolic pathway, iron can be deposited in the tissue in various forms. The work presented in this thesis has exploited the relationship between the magnetic and the chemical properties of tissue iron deposits to further understanding of two major pathologies, the haemoglobinopathies termed thalassaemias and the malaria parasite Plasmodium falciparum, both amongst the most common health concerns in tropical countries. The iron-specific magnetic susceptibilities ¿Fe for spleen tissue samples from 7 transfusion dependent ß-thalassaemia (ß-thal) patients and 11 non-transfusion dependent ß-thalassaemia/Haemoglobin E (ß/E) patients were measured at 37°C. Both groups of patients were iron loaded with no significant difference in the distribution of spleen iron concentrations between the two groups. There was a significant difference between the mean ¿Fe of the spleen tissue from each group. The ß/E patients had a higher mean (± standard deviation) spleen ¿Fe (1.55 ± 0.23 × 10-6 m3.kgFe -1) than the ß-thal patients (1.16 ± 0.25 × 10-6 m3.kgFe -1). Correlations were observed between ¿Fe of the spleen tissue and the fraction of magnetic hyperfine split sextet in the 57Fe Mössbauer spectra of the tissues at 78 K (Spearman rank order correlation ¿ = -0.54, p = 0.03) and between ¿Fe of the spleen tissue and the fraction of doublet in the spectra at 5 K (¿ = 0.58, p = 0.02) indicating that ¿Fe of the spleen tissue is related to the chemical speciation of the iron 2 deposits in the tissue. The biological variability of the iron-specific magnetic susceptibility of the tissue iron examined would contribute a random uncertainty of 19% to magnetic susceptibility based non-invasive measurements of tissue iron concentration. ... Magnetic susceptibility measurements were also performed on malaria parasitised red blood cells. In vitro cultures of P. falciparum were magnetically enriched up to 61-fold using high field gradient magnetic separation columns, and the magnetic susceptibility of cell contents was directly measured. Forms of haem iron were quantified spectroscopically. Further fractionations were performed such that, by controlling the fluid velocity through the column, cells with more than a critical amount of paramagnetic 3 iron were preferentially extracted. A chloroquine-sensitive (CQS) laboratory strain of parasites converted approximately 60% of host cell haem iron to haemozoin and this product was the primary source of the increase in cell magnetic susceptibility. The volumetric magnetic susceptibility of the magnetically enriched cells was found to be 0.15 ± 0.03 × 10-7 relative to the suspension medium, accounting for the enrichment of mature parasites. Comparisons of fractionation samples of two pairs of CQS and chloroquine resistant (CQR) strains showed enrichment of mature parasites was significantly greater in the CQS than the CQR strains. The results suggest the possibility of using magnetic separation columns in identifying CQR strains of P. falciparum, potentially in a diagnostic or research setting. The study also underlines the need to identify and quantify the forms of iron in CQR and CQS parasite strains as the fate of haem iron will have implications in understanding the mechanisms of chloroquine resistance.

Inhibition of nitric oxide (NO) signalling may contribute to the pathogenesis of severe malaria. This thesis examines the impact of Plasmodium infection on three key determinants of nitric oxide synthase (NOS) biochemistry: substrate availability, substrate/inhibitor homeostasis and cofactor availability. Arginine, the NOS substrate, is depleted in human patients with severe Plasmodium falciparum malaria and mice infected with P. berghei ANKA. Using heavy isotope tracer infusions to quantify arginine metabolism in infected mice, we found no evidence of increased catabolism by the enzyme arginase, widely assumed to be responsible for arginine depletion. Genetic knock-out of parasite arginase had no effect on arginine depletion in mice. Instead, our findings link arginine depletion to decreased rates of arginine and citrulline appearance in the plasma of infected mice. Asymmetric dimethylarginine (ADMA) competes with arginine for binding to the NOS catalytic site. We observed elevation of the ADMA/arginine ratio in Gambian children with severe malaria, favouring NOS inhibition. In mice infected with P. berghei ANKA, we found evidence of degradation of dimethylarginine dimethylaminohydrolase 1 (DDAH1), the enzyme primarily responsible for ADMA metabolism. We also observed reduced DDAH activity and accumulation of intracellular ADMA in hepatic tissue of infected mice, suggesting that DDAH dysfunction could contribute to disruption of ADMA/arginine homeostasis. Tetrahydrobiopterin (BH4) is an essential NOS cofactor. In P. berghei ANKA-infected mice, BH4 concentrations were decreased in plasma, erythrocytes and brain tissue, which could inhibit NO synthesis and promote NOS-dependent superoxide production. To reverse deficiencies of NOS substrate and cofactor availability, we infused P. berghei ANKA-infected mice with citrulline, an arginine precursor, and sepiapterin, a BH4 precursor. Restoration of systemic arginine and BH4 availability in infected mice improved whole blood nitrite concentrations, a biomarker of NO synthesis, but did not prevent onset of disease symptoms. These studies have identified biochemical disturbances that may contribute to severe malaria pathogenesis by inhibiting NO synthesis.

INTRODUCTION Malaria is a major health problem, with more than 650.000 deaths and 200 million clinical cases each year. Respiratory distress as malaria associated acute respiratory distress syndrome (MA-ARDS) is a common complication. The pathogenesis of MA-ARDS is mainly inflammatory and one of the main observation is the presence of abundant monocytes and macrophages inside the blood capillaries, in the interstitium and also in alveolar spaces. Malaria pigment or haemozoin (Hz) is often seen in these cells reflecting active phagocytosis and leads to the production of cytokines and other inflammatory mediators. Multiple organ dysfunctions are described in MA-ARDS, including liver damages. ARDS in non malarious patients is often associated with disorders of the lung surfactant, which can lead to the increase in surface tension, alveolar collapse and loss of the liquid balance in the lungs. Surfactant is known to reduce the surface tension at the air–liquid interface of lung epithelia and to regulate the local host immune response. It can be separated into a surface active Large Aggregate fractions (LA), representing a reservoir for the surface film located at the air-liquid interface of the alveoli and a less surface active, Small Aggregate fraction (SA). It is not known at present if alterations of the surfactant also exist in MA-ARDS and how they may contribute to the pathology and the development of the inflammatory response. AIM The aim of our studies was to perform a comprehensive analysis of the local and systemic inflammatory response present in MA-ARDS and to analyse the lipid profile of the pulmonary surfactant, the lung and liver tissues and plasma using two different models of murine malaria of similar gravity, but different involvement of lungs or liver. In particular, we studied C57BL/6J mice infected with two different species of Plasmodium: Plasmodium berghei NK65 strain which induces MA-ARDS and Plasmodium chabaudi (PcAS), which does not. The two models allowed us to directly compare the different pathological manifestation of the same infection in order to identify peculiarities which could be exploited for novel therapeutic interventions. RESULTS AND DISCUSSION Macroscopic and functional analysis of lung tissues in the two strains. The lungs of PbNK65 infected mice were swollen, increased in weight and with a dark brown aspect due to micro haemorrhages and to the deposition of Hz clusters in concentrations significantly higher compared to the lungs of mice infected with PcAS. The expression of TNF-α and IFN- was increased only in PbNK65 mice, indicating that these cytokines are induced specifically during MA-ARDS and are not a consequence of malaria infection. This hypothesis was confirmed by the decrease of lung weight and of the CD8+ cells infiltrate, and the reduction /delay in mortality rates seen in PbNK65 mice treated with DEX without a concomitant reduction in parasitaemia. Therefore, DEX seems to ameliorate MA-ARDS, not by inhibiting parasite growth but rather by modulating the immunopathology and the inflammatory response. A significant increase of the total phospholipid (PL) content and cholesterol esters (ChoE) was observed in PbNK65 lungs and was reverted by DEX. Moreover, compared to the control mice (CTR), the fatty acid distribution of lung ChoE was characterized by higher levels of the polyunsaturated fatty acid and an high linoleic/oleic ratio typical of plasma ChoE. All these features confirm a strict correlation between the interstitial oedema and the infiltration of plasma lipoproteins during MA-ARDS. Protein and lipid composition of surfactant and plasma of infected mice. The total bronchoalveolar lavage (BAL) of PbNK65 mice showed a significant increase in the protein levels compared to CTR or PcAS mice, probably due to plasma derived proteins being incorporated into or associated with microstructures in the alveolar hypophase. This event is known to decrease the intrinsic surface activity of surfactant. The total content of PL was not different from CTR, whereas the PL profile of the LA and SA fractions in PbNK65 infected mice showed a significant increase in the amounts of sphingomyelin and a decrease in phosphatidylglycerol. These changes were absent in PcAS mice and may be related to the altered re-uptake and synthesis of PL by injured cells or to PL contamination due to inflammatory cells. The plasma levels of PL and triacylglycerol (TG) were significantly higher in PbNK65 mice than in CTR or PcAS mice. Compared to PcAS or CTR mice in PbNK65 group all the PL classes were significantly increased with the exception of lisophosphatidilcholine (LisoPC) that was decreased. These plasma alterations may be related to an impaired activity of the enzymes involved in the lipoprotein metabolism during infections or inflammatory diseases. The most important observation, both in PbNK65 and PcAS mice, was the significant increase of docosahexahenoic acid (C22:6 n-3, DHA) compared to CTR, which was only partially reverted by DEX treatment, suggesting that the increase of DHA is not related to lung pathology but rather to the malaria infection. Analysis of the liver tissue of infected mice. The hypothesis that the higher PL and TG content of PbNK65 plasma might be due to an enhanced hepatic lipogenesis was confirmed by the higher TG and ChoE content of the liver of PbNK65 mice compared to PcAS and CTR. An increased ratio linoleic (LA)/arachidonic acid (AA) was also present possibly due to the impairment of the elongation/desaturation pathway from LA to AA acid. Higher levels of Hz, compared to PcAS, were present in PbNK65 mice and, in agreement with the Hz capability of stimulating Kupffer cells, we found higher levels of TNF-α. Both Hz and TNF-α can induce lipoperoxidation as confirmed by the elevated levels of malondialdehyde (MDA) in the liver of PbNK65 mice. This finding was paralleled by the lower content of glutathione and of antioxidant enzymes particularly in the late stage of the pathology. CONCLUSIONS This is the first time that a comprehensive analysis of the lipid content and inflammatory response of different organs in a model of murine MA-ARDS has been performed. All together the data suggest that in MA-ARDS as in other severe non infectious pathologies, a pulmonary-liver metabolic interplay exist which may contribute to the pathology. In PbNK65 mice, Hz and the derived inflammation play an important role in the lung pathology inducing changes in the lipid composition of lung and surfactant, cellular infiltration and cytokine production. Lung pathology is associated with liver disorders and alterations in the lipoprotein profile. Hz accumulation may induce macrophages to produce TNF-α and ROS that can interfere with liver functions by inducing lipogenesis and affecting the lipid profile of liver and plasma, which in turn contribute to the altered lipid composition of the lung tissue. These results confirm that severe malaria is a multi-organ dysfunction in which inflammation has an important role in different organs and thus, in addition to antimalarial treatment, adjunct therapies with anti-inflammatory drugs can be envisaged.

La maladie d’Alzheimer (MA) est caractérisée par deux lésions neuropathologiques: les plaques séniles (composées essentiellement du peptide amyloïde Ab) et les dégénérescences neurofibrillaires (DNF, composées de formes hyperphosphorylées de protéines tau). Les mécanismes de formation des DNF sont encore mal compris et notre travail expérimental a eu pour objectifs d’étudier certaines hypothèses de formation des DNF.

1° Une hypothèse étiopathogénique de la MA est la “cascade amyloïde”, selon laquelle le peptide amyloïde Ab exercerait un effet toxique entraînant la phosphorylation de tau et la formation de DNF. Certaines formes familiales de MA sont dues à des mutations du gène du précurseur du peptide amyloïde (APP) ou des présénilines et nous avons voulu déterminer si la surexpression de ces protéines pouvait entraîner la formation de DNF. Nous avons d’abord étudié une lignée murine double transgénique surexprimant l’isoforme 0N3R de protéine tau humaine “sauvage” et une forme mutée de préseniline 1 (M146L). Nous y avons démontré une co-expression neuronale des deux protéines et une augmentation de la phosphorylation de tau mais nous n’y avons pas observé de formation de DNF, chez des animaux examinés jusqu’à 17 mois. Nous avons ensuite étudié une lignée murine triple transgénique surexprimant l’isoforme 0N3R de protéine tau “sauvage”, une forme mutée de préseniline 1 (M146L) et une forme mutée de l’APP 751 (mutations Swedish K670N, M671L et London V717I). Ces animaux ont développé précocement (2.5 mois) des dépôts extracellulaires de peptide Ab. Nous y avons observé une augmentation de la phosphorylation de tau dans les prolongements neuronaux en contact avec les dépôts amyloïdes et des anomalies de l’organisation du cytosquelette, mais pas de DNF, chez des animaux examinés jusqu’à 18 mois.

2° Certaines mutations du gène de tau sont responsables de formes familiales de démence frontotemporales dans lesquelles se développent des DNF. Ces mutations favoriseraient l’agrégation de tau où entraîneraient un déséquilibre de l’expression relative des isoformes de tau. Un tel déséquilibre pourrait également être induit dans les formes sporadiques de MA, en l’absence de mutations de tau. Afin d’investiguer cette hypothèse, nous avons étudié le profil d’expression des ARNm de tau et des isoformes de protéines tau dans plusieurs régions cérébrales de sujets contrôles ou atteints de MA. Un même profil d’expression a été observé dans les deux groupes. Une augmentation relative de l’expression de l’isoforme 0N3R de tau dans le cortex temporal pourrait être liée à la sensibilité de cette région au développement de DNF. Nous avons également étudié des lignées stables de cellules CHO exprimant des formes mutées (P301L, R406W) et non-mutées de protéines tau. Nous n’avons cependant pas observé d’augmentation de l’agrégation de tau dans les lignées exprimant les formes mutées de tau.

Nos résultats indiquent que la simple surexpression de formes mutées de l’APP et des présénilines, même en présence d’une protéine tau humaine, ne suffit pas à entraîner la formation de DNF. En outre, l’absence de différence dans le profil d’expression cérébrale des isoformes de tau entre sujets contrôles et atteints de MA suggère que les modifications post-traductionnelles de cette protéine jouent un rôle plus important dans la genèse des DNF.

Doctorat en sciences biomédicales
info:eu-repo/semantics/nonPublished

Depuis les annees 60, peu de travaux sur la pathogenese de la poliomyelite ont ete publies. Afin de donner un eclairage nouveau aux donnees moleculaires sur la neurovirulence du poliovirus, nous avons tente de definir le mode de propagation de ce virus in vivo. Dans le but d'identifier les cellules siege de la multiplication extraneurale a l'origine de la viremie persistante, nous avons teste l'hypothese des cellules endotheliales source de cette viremie. L'etude de la permissivite des cellules endotheliales in vitro au poliovirus a montre qu'un faible pourcentage de cellules sont sensibles a l'infection par le poliovirus immediatement avant la mise en culture, suggerant que ces cellules puissent etre un site de multiplication extraneurale du poliovirus in vivo. Concernant la multiplication intraneurale du poliovirus, nous avons pu montrer, par hybridation in situ, que la replication virale dans les neurones moteurs est la cause directe de la destruction de ces cellules. L'analyse comparative de la replication intraneuronale d'une souche attenuee et d'une souche neurovirulente nous a permis d'emettre une hypothese: l'attenuation resulterait d'une vitesse de multiplication virale moindre qui permettrait au systeme immunitaire de localiser l'infection. Parallelement a ces travaux, nous nous sommes interesses aux determinants moleculaires de la neurovirulence du poliovirus chez la souris. La sequence de 9 acides amines du site antigenique 1 de la souche lansing est suffisante pour conferer le phenotype neurovirulent chez la souris a la souche mahoney. Nous avons tente de definir plus precisement le ou les acides amines de ce site impliques dans le phenotype neurovirulent. Les resultats obtenus suggerent que la conformation du site antigenique, plutot que sa sequence primaire, joue un role determinant dans ce phenotype. Des elements fondamentaux sur la viremie et sur la multiplication intraneur

La malaltia de Parkinson (PD) s'associa amb la degeneració de les neurones dopaminèrgiques del mesencèfal ventral (vmDA) i l'acumulació d'inclusions citoplasmàtiques, els cossos de Lewy, compostos principalment per la α sinucleina agregada en les neurones vmDA supervivents. Aquest procés, juntament amb els mecanismes patogènics autònoms cel·lulars subjacents, s'ha modelat fins ara amb èxit utilitzant tecnologia de cèl·lules mare pluripotents induïdes de cada pacient (iPSC). En el nostre laboratori, vam generar astròcits de línies d'iPSC derivades de pacients amb PD familiar amb la mutació G2019S en el gen Leucine rich repeat kinase 2 (LRRK2), astròcits de pacients idiopàtics, així com d’individus sans de la mateixa edat (wildtype; WT). Per avaluar el possible paper dels mecanismes cel·lulars no autònoms durant la patogènesi del PD, es va concebre un sistema de co-cultiu entre astròcits derivats d’iPSC i neurones vmDA per avaluar la interacció neurona-glia. En les neurones WT vmDA, es van trobar signes morfològics de neurodegeneració (poques neurites, curtes, i en forma granulada) i una acumulació anormal d’α-sinucleina després del co-cultiu amb astròcits LRRK2-PD. Per altra banda, els astròcits LRRK2-PD en mono-cultiu mostren fenotips que recorden als observats en neurones LRRK2-PD vmDA, incloent alteracions en l'autofàgia i dinàmica mitocondrial, així com una acumulació progressiva de la α- sinucleina, en comparació amb els astròcits WT. Un fàrmac activador de l’autofàgia mediada per chaperones (CMA), QX77.1, va rescatar amb èxit la disfunció i, com a conseqüència, va eliminar l'α sinucleina acumulada prèviament en astròcits LRRK2-PD. Per contra, el co-cultiu de les neurones LRRK2-PD vmDA amb astròcits WT va impedir parcialment l'aparició de neurodegeneració relacionada amb la malaltia. Aquest paper neuroprotector sembla ser portat a terme a través de l'activació de les cèl·lules glials a un estat reactiu i suggereix que els astròcits LRRK2-PD tenen una relació alterada entre la neuroprotecció i la reactivitat, el que resulta en efectes de neurodegeneració. Els nostres resultats revelen una contribució important dels astròcits durant la patogènesi del PD i obren el camí per explorar noves estratègies terapèutiques destinades a bloquejar la interacció patògena entre neurones i cèl·lules glials.
Parkinson’s disease (PD) is associated with the degeneration of ventral midbrain dopaminergic (vmDA) neurons and the accumulation of cytoplasmic inclusions, known as Lewy Bodies, composed mainly of aggregated α synuclein in the surviving vmDA neurons. This process, along with the underlying cell-autonomous pathogenic mechanisms, has been successfully modeled using patient-specific induced pluripotent stem cell (iPSC) technology. Non-cell autonomous neurodegeneration during PD has been suggested by past observational studies, but remains to be experimentally tested. Here, we generated astrocytes from iPSC lines derived from familial Parkinson’s disease patients with the G2019S mutation on the Leucine rich repeat kinase 2 (LRRK2) gene, and astrocytes from Sporadic PD patients, as well as healthy age-matched individuals (to whom we will refer as wild type (WT)). To assess the possible non-cell autonomous role during PD pathogenesis, a co-culture system was devised between iPSC-derived astrocytes and vmDAn to assess the potential pathogenic neuron-glia crosstalk. WT vmDAn displayed morphological signs of neurodegeneration (such as few and short neurites, as well as beaded-like necklace neurites) and abnormal, astrocyte-derived, α synuclein accumulation when co-cultured on top of LRRK2-PD astrocytes. Upon further investigation, PD astrocytes alone displayed phenotypes reminiscent of those observed in PD-iPSC-derived vmDAn, those including alterations in autophagy and mitochondrial dynamics, as well as a progressive accumulation of α synuclein, when compared with WT astrocytes. A CMA activator drug, QX77.1, successfully rescued CMA dysfunction and as a consequence cleared the previously accumulated α-synucein in PD astrocytes. Conversely, the co-culture of LRRK2-PD vmDA neurons with WT astrocytes partially prevented the appearance of disease- related neurodegeneration. This neuroprotective role appears to be managed via the activation of glia to a reactive state, and suggests LRRK2-PD astrocytes have an impaired relation between neuroprotection and reactivity, which results in neurodamaging effects. Our findings unveil a crucial non-cell autonomous contribution of astrocytes during PD pathogenesis, and open the path to exploring novel therapeutic strategies aimed at blocking the pathogenic cross-talk between neurons and glial cells.

La maladie de Parkinson (MP) est un trouble moteur neurodégénératif progressif, caractérisé par la perte des neurones dopaminergiques (DA) de la substance noire et la présence d'inclusions cytoplasmiques composées principalement de synucléine α (syn α), appelées corps de Lewy. Les objectifs de mon travail de thèse étaient de caractériser un modèle de la MP développé chez la drosophile afin de comprendre comment l'accumulation de syn α dans les neurones DA peut perturber progressivement la locomotion et de rechercher de nouvelles protéines neuroprotectrices. Nous avons d'abord identifié des réseaux cérébraux impliqués dans la modulation de la réactivité locomotrice chez la drosophile, qui incluent des sous-ensembles de neurones DA associés aux corps pédonculés. Nous avons ensuite obtenu des évidences que l'expression de la syn α dans les neurones DA perturbe la dynamique mitochondriale à la fois dans ces neurones eux-mêmes et, par un processus non-autonome cellulaire, dans les neurones-cibles cholinergiques des corps pédonculés. Enfin, nous montrons que la protéine Argonaute Piwi est induite par le stress oxydatif et a un effet neuroprotecteur dans un modèle de MP sporadique chez la drosophile. Dans l’ensemble, ces travaux mettent en lumière des mécanismes pathologiques et neuroprotecteurs qui pourraient constituer de nouvelles cibles pour le traitement thérapeutique de la MP
Parkinson's disease (PD) is a progressive neurodegenerative motor disorder, characterized by the loss of dopaminergic neurons from the substantia nigra and the presence of cytoplasmic inclusions composed mainly of α-synuclein (α-syn), called Lewy bodies. The objectives of my thesis work were to characterize a PD model developed in Drosophila in order to understand how the accumulation of α-syn in DA neurons can progressively disturb locomotion and to search for new neuroprotective proteins. We first identified brain networks involved in modulating locomotor reactivity in Drosophila, which include subsets of DA neurons associated with the mushroom bodies. We then obtained evidence that the expression of α-syn in DA neurons disrupts mitochondrial dynamics both in these neurons themselves and, through a non-cell-autonomous process, in their cholinergic target neurons of the mushroom bodies. Finally, we show that the Argonaute Piwi protein is induced by oxidative stress and has a neuroprotective effect in a sporadic PD model in Drosophila. Our evidence suggests that Piwi could delay neuronal aging and PD progression by reducing deleterious transcription of transposable elements. Overall, these studies highlight pathological and neuroprotective mechanisms that may constitute novel targets for the therapeutic treatment of PD

Diversi studi sperimentali hanno evidenziato un possibile ruolo del sistema immune intestinale nella patogenesi del diabete di tipo 1(T1D). Lo scopo del nostro studio è stato quello di evidenziare un possibile link tra il sistema immune intestinale e il T1D, in particolare un possibile ruolo del glutine come antigene T cellulare nel T1D. Le cellule mononucleate del sangue periferico (PBMC) sono state isolate da 25 bambini con T1D (età da 3.4 a 19.6 anni), da 22 controlli sani (HC) (età da 3.5 a 17 anni) entrambi negativi per gli anticorpi antiendomisio e anti-transglutaminasi e da 15 bambini con malattia celiaca (CD) (età da 2.2 a 13.3 anni). Nella prima parte dello studio, in 6 bambini affetti da T1D, in 5 con CD, e in 6 HC, i PBMC sono stati coltivati con o senza OKT3 e anti-CD28mAb. Dopo 18 ore è stata valutata l’espressione dell’integrina β7 mediante citometria a flusso multiparametrica. Nei pazienti con T1D abbiamo osservato una alta espressione dell’integrina Beta7 su linfociti T CD4+ con fenotipo di memoria. Dopo attivazione policlonale abbiamo osservato una significativa riduzione dell’espressione dell’integrina Beta7 su linfociti T CD4+ con fenotipo di memoria nei pazienti con T1D rispetto ai controlli. Nella seconda parte dello studio, i PBMC di 19 bambini con T1D , 10 con CD e 17 HC, sono stati coltivati, a diverse concentrazioni, con/senza un digesto peptico-triptico di gliadina (PTG) e/o con il digesto di gliadina transglutaminata (TG)-PTG. La proliferazione dei PBMC è stata valutata al quinto giorno di coltura tramite la determinazione dell’incorporazione di [3H]-timidina. E’ stata inoltre valutata, nei sopranatanti delle colture cellulari, la produzione di IFN-gamma e IL-4 mediante metodica ELISA, e l’espressione dell’integrina Beta7 mediante citometria a flusso. Nei pazienti con T1D abbiamo evidenziato una risposta proliferativa dose-dipendente dei PBMC dopo stimolazione con PTG e/o TG-PTG. In 7 degli 11 pazienti con T1D (64%) abbiamo osservato una risposta proliferativa dopo stimolazione con PTG, con una media di indice di stimolazione (SI) maggiore nei pazienti con T1D rispetto ai controlli (2,95±2 vs.1,3±0,6 rispettivamente, p=0,02). Quando i PBMC sono stati stimolati con il TG-PTG abbiamo osservato una risposta proliferativa in 6 dei 13 pazienti con T1D (54%) and 2 dei 3 pazienti con CD (67%) (T1D 2,4±2 vs. HC 1,5±0,7). In 3 dei 7 T1D che hanno risposta alla gliadina (43%), ma in nessun dei controlli sani, la stimolazione con 100 µg/ml TG-PTG ha indotto la produzione di IFN-gamma. In 1 paziente con T1D abbiamo osservato, dopo stimolazione con gliadina, l’induzione di una discreta popolazione di cellule CD4+ con alta espressione dell’integrina β7. I nostri dati mostrano che cellule T che esprimono alti livelli di integrina β7 sono evidenziabili nel sangue periferico di pazienti con CD and T1D e abbiamo inoltre osservato un aumento della risposta T cellulare glutine-specifica nei pazienti con T1D. Questi dati supportano l’ipotesi che l’immunità verso antigeni orali è alterata nei pazienti con T1D. E’ tuttavia necessario un lungo follow-up per stabilire se i pazienti con T1D studiati hanno un aumentato rischio di sviluppare la malattia celiaca.
Accumulating data indicate that a dysregulation of the gut immune system may play a role in the development of Beta cell autoimmunity and type 1 diabetes (T1D). The aim of this study was to determine a possible link between the gut immune system and T1D, in particular, a possible role of gliadin as a T cell antigen in human T1D. Peripheral blood mononuclear cells (PBMC) were isolated from 25 children with T1D (aged from 3.4 to 19.6 yrs), 22 healthy controls (HC) (aged from 3.5 to 17 yrs), both negative for both anti-endomysial and anti-human tissue transglutaminase antibodies and 15 children with celiac disease (CD) (aged from 2.2 to 13.3 yrs). In the first part of the study, in 6 children with T1D, 5 with CD and in 6 HC, PBMC were cultured with or without OKT3 plus anti-CD28mAb. After 18 hours the expression of β7 integrin was determined by flow cytometry. In patients with T1D we observed a higher expression of Beta7 integrin on memory CD4+ T cells. After polyclonal stimulation we found a significant reduction of Beta7 expression on memory CD4+ T cell in T1D patients compared with healthy controls. In the second part of the study, in 19 children with T1D, 10 with CD and in 17 HC, PBMC were cultured with the peptic-tryptic digest of gliadin (PTG) and/or transgluatminase-treated (TG)-PTG at increasing concentrations, or left un-stimulated. PBMC proliferation was assessed on day 5 by [3H]-thymidine incorporation assay. We also assessed IFN-gamma and IL-4 production in culture supernatants by ELISA and we studied the expression of Beta7 integrin by flow cytometry. In T1D patients we detected a dose-response PBMC proliferation to both PTG and TG-PTG with the maximal proliferation at the concentration of 100 µg/ml. PBMC from 7 out of 11 T1D patients (64%) responded to 100 µg /ml PTG. Mean stimulation index (SI) in T1D patients was higher than in HC (2,95±2 vs.1,3±0,6 respectively, p=0,02). When PBMC were stimulated with TG-PTG we found that 6 out of 13 T1D (54%) and 2 out of 3 CD (67%) showed a proliferative response (T1D 2,4±2 vs. HC 1,5±0,7). In 3 of 7 T1D responders (43%) but not in HC, TG-PTG induced IFN-gamma production. In 1 patient with T1D we identified, after the stimulation with TG-PTG, a discrete population of CD4+ β7hi+ cells. Our data show that T cells expressing high levels of β7 integrin are detectable in peripheral blood of CD and T1D patients and we found an enhanced T cell-mediated gluten-specific immunity in T1D patients. This supports the hypothesis that immunity to oral proteins is altered in T1D patients. Long term follow-up is necessary to establish whether these subjects are at increased risk for developing celiac disease.

Parkinson’s disease (PD) is an incurable, chronically progressive disorder of old age leading to premature invalidity and death. Clinically, PD is characterized by classical motor syndrome linked to a progressive loss of dopamine-containing neurons (DAn) in the substantia nigra pars compacta, and disabling non-motor symptoms related to extranigral lesions. The identification of several genes associated to familiar PD have brought considerable insight into underlying pathogenic mechanisms. However, the unknown etiology of the sporadic forms (90% of patients) and the emerging view that non-neuronal cells could be also implicated in the pathophysiology of the disease, greatly impact on the development of accurate models and on the discovery of a cure. Here, I investigate the role of astrocytes in disease pathogenesis using a human iPSC-based model of Parkinson’s disease. First, I introduce Parkinson’s disease, its pathological hallmarks and the progression of the symptoms, and discuss genetic and environmental influences. Then, I elaborate on the different mechanisms involved in PD including mitochondrial dysfunction, oxidative stress and autophagy as well as on the inflammatory phenotypes observed in the disease and recent work describing the role of inflammation in PD animal models and post-mortem brain tissue. Subsequently, I examine the association of astrocytic dysfunctions with neuronal morphological and functional abnormalities that contribute to the progression of several neurodegenerative including Parkinson’s disease and the recent data showing an astrocyte-autonomous process mediating PD-associated degeneration of dopaminergic neurons, mainly via intracellular accumulation of α-synuclein aggregates in astrocytes and subsequent propagation of such toxic aggregates to surrounding neurons. In the results section, I describe the generation and characterization of iPSC- derived astrocytes of LRRK2-PD patients (LRRK2G2019S PD), healthy individual (Ctrl) and CRISPR/Cas9 gene edited isogenic control. I show that LRRK2G2019S PD astrocytes exhibited extensive perinuclear accumulation of fragmented mitochondria and a significant increase in DRP1 phosphorylation compared to control astrocytes. Fragmented mitochondria accumulated in LRRK2G2019S PD astrocytes was due to a defective mitophagy leading to an increase in oxidative stress. I also show that oxygen consumption rate, ATP production and mitochondrial membrane potential were significantly decreased in LRRK2G2019S PD astrocytes indicating altered mitochondrial function in PD astrocytes and that LRRK2G2019S PD astrocytes exhibited lower expression levels of mitochondrial biogenesis-related genes compared to control astrocytes. Importantly, correction of G2019S mutation in the LRRK2 gene by CRISPR-Cas9 gene editing normalized mitochondria morphology, clearance and function to those of control astrocytes. Then, I describe the effects of Urolithin A, a mitophagy activator drug, that was able to rescue mitochondrial fragmentation and accumulation in LRRK2G2019S PD astrocytes by inducing mitophagy, promoting expression of mitochondrial biogenesis-related genes and reducing ROS production in those astrocytes. Finally, in the last chapter, I show that, in a co-culture system established between LRRK2G2019S PD astrocytes and healthy DA neurons, the treatment with Urolithin A, prevented neuronal cell death, suggesting a potential astrocyte- targeted therapeutic. In conclusion, our findings provide the advantage for using iPSC-based modeling for assessing the consequences of mitochondrial dysfunctions in astrocytes and dissecting the initial mechanisms that lead to neuronal cell loss in PD. The present modeling has uncovered mitophagy dysfunction as a relevant altered mechanism in PD astrocytes whose activation might represent an interesting therapeutic option for counteracting PD-related neurodegeneration.

Introducció: La malaltia pulmonar obstructiva crònica (MPOC) és un factor de risc independent per el desenvolupament de càncer de pulmó (CP) en els pacients. Els mecanismes encara s’han de dilucidar per a comprendre les relacions entre MPOC i CP. Hipòtesi: Els components del microambient tumoral poden diferir en els tumors de pacients amb CP amb i sense MPOC. La immunoteràpia també pot reduir la càrrega tumoral a través de diversos mecanismes biològics. Objectius: 1) Pacients: estudiar el paper del microambient tumoral, les cèl·lules immunitàries, les característiques de l’estroma i la sobreactivació de PARP en el procés de tumorigènesi en pacients amb CP amb i sense MPOC. 2) Ratolins: avaluar els efectes de la immunoteràpia en la grandària tumoral mitjançant l’anàlisi de diversos mecanismes biològics com l’estrès oxidatiu, l’apoptosi i l’autofàgia. Mètodes: 1) Pacients: es van reclutar 90 pacients amb MPOC-CP i 43 pacients només amb CP procedents de la cohort Càncer de Pulmó Mar (Barcelona) des de l’any 2008 fins al 2019. Es van obtenir mostres pulmonars tumorals i no tumorals en els pacients mitjançant toracotomia/cirurgia toracoscòpica assistida per vídeo (VATS), sempre previ a la quimioteràpia i/o radioteràpia. 2) Ratolins: dos grups de ratolins Balb/c amb CP induït mitjançant la inoculació subcutània de cèl·lules d’adenocarcinoma pulmonar LP07: ratolins tractats i no tractats, n = 9/grup. Al grup tractat se li va administrar un còctel d’anticossos monoclonals (anti-PD-L1, anti-CTLA-4, anti-CD19 i anti-CD137) i una solució tampó (PBS) als ratolins control. Es van obtenir els tumors en tots els ratolins per dur a terme l’estudi (30 dies). Anàlisi biològic: es van utilitzar Western-blot, immunohistoquímica, ELISA, cultius cel·lulars, i immunofluorescència per avaluar els marcadors biològics objecte d’estudi en cada model i tipus de mostres. Resultats: 1) Pacients: els tumors pulmonars de pacients amb MPOC van mostrar nivells més baixos d’estructures limfoides terciàries (ETLs) i centres germinals (CG) respecte dels pacients sense MPOC. Els nivells més baixos de ETLs i cèl·lules B en els tumors pulmonars es van associar amb una pitjor supervivència a 10 anys, especialment en aquells amb MPOC. En l’estroma tumoral, la presència de MPOC no es va associar a diferències en els components de l’estroma com ara la matriu extracel·lular, els fibroblasts associats al càncer o les cèl·lules endotelials. A més, els nivells de dany de l’ADN i la consegüent activació de PARP estaven més elevats només en els tumors pulmonars dels pacients amb MPOC, mentre que l’expressió dels enzims PARP-1 i PARP-2 estaven disminuïdes en els tumors pulmonars respecte de les no tumorals, independentment de la presència de MPOC. 2) Ratolins: la immunoteràpia va reduir la càrrega tumoral a través de l’augment dels nivells de l’estrès oxidatiu, apoptosi, autofàgia i de vies de senyalització com NF-kB i sirtuin-1 en tumors dels ratolins tractats comparant amb els ratolins amb CP sense immunoteràpia. Conclusions: El microambient immunològic tumoral, els components de l’estroma i l’activitat de PARP s’expressen de forma clarament diferenciada en els tumors pulmonars de pacients amb CP amb MPOC respecte dels pacients sense MPOC. La reducció en la formació de ELTs i CGs, l’augment en el dany de l’ADN i la sobreactivació de PARP probablement contribueixin a la major susceptibilitat per desenvolupar CP en pacients amb MPOC. En ratolins tractats amb la immunoteràpia, l’augment dels nivells d’estrès oxidatiu juntament amb l’activació d’apoptosi i autofàgia poden ser part dels mecanismes mitjançant els quals la immunoteràpia redueix la càrrega tumoral. En resum, la presència de MPOC s’ha de tenir en compte en el disseny de teràpies per al CP, incloses la immunoteràpia i la inhibició de l’activació de PARP.
Introducción: La enfermedad pulmonar obstructiva crónica (EPOC) es un factor de riesgo independiente para el desarrollo de cáncer de pulmón (CP) en los pacientes. Los mecanismos aún se quedan por dilucidar a comprender las relaciones entre EPOC y CP. Hipótesis: Los componentes del microambiente tumoral pueden diferir en los tumores de pacientes con CP con y sin EPOC. La inmunoterapia también puede reducir la carga tumoral a través de varios mecanismos biológicos. Objetivos: 1) Pacientes: estudiar el papel del microambiente tumoral, las células inmunes, las características del estroma y la sobreactivación de PARP en el proceso de tumorigénesis en pacientes con CP con y sin EPOC. 2) Ratones: evaluar los efectos de la inmunoterapia en el tamaño tumoral mediante el análisis de varios mecanismos biológicos como el estrés oxidativo, la apoptosis y la autofagia. Métodos: 1) Pacientes: se reclutaron 90 pacientes con EPOC-CP y 43 pacientes solo con CP procedentes de la Cohorte Cáncer de Pulmón Mar, Barcelona, desde el año 2008 hasta el 2019. Se obtuvieron muestras pulmonares tumorales y no tumorales en los pacientes mediante toracotomía/cirugía toracoscópica asistida por video (VATS), siempre previo a la quimioterapia y/o radioterapia. 2) Ratones: dos grupos de ratones BALB /c con CP inducido mediante la inoculación subcutánea de células de adenocarcinoma pulmonar LP07: ratones tratados y no tratados, n= 9/grupo. Al grupo tratado se le administró un cóctel de anticuerpos monoclonales (anti-PD-L1, anti-CTLA-4, anti-CD19 y anti-CD137) y una solución tampón (PBS) a los ratones control. Se obtuvieron los tumores en todos los ratones al final del estudio (30 días). Análisis biológico: se utilizaron Western-blot, inmunohistoquímica, ELISA, cultivos celulares, y inmunofluorescencia para evaluar los marcadores biológicos objeto de estudio en cada modelo y tipos de muestras. Resultados: 1) Pacientes: los tumores pulmonares de pacientes con EPOC mostraron niveles más bajos de estructuras linfoides terciarias (ETLs) y centros germinales (CG) respecto de los pacientes sin EPOC. Los niveles más bajos de ELTs y células B en los tumores pulmonares se asociaron con una peor supervivencia a 10 años, especialmente en aquéllos con EPOC. En el estroma tumoral, la presencia de EPOC no se asoció a diferencias en los componentes del estroma tales como la matriz extracelular, los fibroblastos asociados al cáncer o las células endoteliales. Además, los niveles de daño del ADN y la consiguiente activación de PARP estaban más elevados solamente en los tumores pulmonares de los pacientes con EPOC, mientras que la expresión de las enzimas PARP-1 y PARP-2 estaban disminuidas en los tumores pulmonares respecto de las no tumorales, independientemente de la presencia de EPOC. 2) Ratones: la inmunoterapia redujo la carga tumoral a través del aumento de los niveles del estrés oxidativo, apoptosis, autofagia y de vías de señalización como NF-kB y sirtuin-1 en tumores de los ratones tratados comparando con los ratones con CP sin inmunoterapia. Conclusiones: El microambiente inmunológico tumoral, los componentes del estroma y la actividad de PARP se expresan de forma claramente diferenciada en los tumores pulmonares de pacientes con CP con EPOC respecto de los pacientes sin EPOC. La reducción en la formación de ELTs y CGs, el aumento en el daño del ADN y la sobreactivación de PARP probablemente contribuyan a la mayor susceptibilidad para desarrollar CP en pacientes con EPOC. En ratones tratados con la inmunoterapia, el aumento de los niveles de estrés oxidativo junto con la activación de apoptosis y autofagia pueden ser parte de los mecanismos mediante los cuales la inmunoterapia reduce la carga tumoral. En resumen, la presencia de EPOC debe tenerse en cuenta en el diseño de terapias para el CP, incluidas la inmunoterapia y la inhibición de la activación de PARP.
Background: Lung cancer (LC) is a leading cause of death worldwide. Chronic obstructive pulmonary disease (COPD) is a highly prevalent lung disease. COPD has been well established as an independent risk factor for lung tumorigenesis in patients. However, the biological mechanisms that explain the possible associations between lung cancer and COPD remain to be fully elucidated. Hypothesis: The tumor microenvironment components (immune profile, stroma, cytokines, and PARP activation) may differ in tumors of lung cancer patients with and without COPD. Immunotherapy may also reduce tumor burden through several biological events. Objectives: 1) Studies in patients: to elucidate the role of the biological events: tumor microenvironment, immune cell composition, stroma characteristics, and PARP overactivation in the process of tumorigenesis in tumors of patients with and without underlying COPD; 2) Mouse study: to evaluate the effects of immunotherapy on tumor burden through the analyses of several biological mechanisms such as oxidative stress, apoptosis, and autophagy. Methods: Two models were used: 1) Studies in patients: 90 LC patients with underlying COPD and 43 LC-only patients were recruited from 2008 to 2019 from the Lung Cancer Mar Cohort, Barcelona. Lung tumor and the surrounding non-tumor lung specimens were obtained from all study patients through thoracotomy or video-assisted thoracoscopic surgery (VATS) prior to chemotherapy and/or radiotherapy; 2) Mouse study: Two groups of wild-type BALB/C mice with experimental lung cancer (subcutaneous inoculation of LP07 adenocarcinoma cells in the left flank of mice) were established: treated and non-treated mice, n=9/group. In the treatment group, lung cancer mice were treated with a cocktail of monoclonal antibodies (intraperitoneal injection, anti-PD-L1, anti-CTLA-4, anti-CD19, and anti-CD137). Lung tumors were obtained from all mice. Biological analysis: laboratory techniques such as western-blot, immunohistochemistry, ELISA, cell culture, and immunofluorescence were used to assess the target biological markers in each study. Results: 1) Studies in patients: lung tumors of patients with underlying COPD showed lower levels of tertiary lymphoid structures (TLSs) compared to lung cancer only patients. Moreover, lower levels of TLS and B cells in lung tumors were associated with poorer 10-year overall survival rates of patients, especially in those with underlying COPD. In tumor stroma, the presence of COPD did not elicit any significant difference in levels of extracellular matrix, cancer-associated fibroblasts or endothelial cells. In addition, DNA damage and PARP activation levels were higher only in lung tumors of patients with underlying COPD, while PARP-1 and PARP-2 enzyme expression levels were lower in lung tumors compared to non-tumor specimens irrespective of the presence of COPD. 2) Mouse study: treatment with immunotherapy reduced tumor burden through increased levels of oxidative stress, apoptosis, autophagy, and signaling pathways such as NF-kB and sirtuin-1 in tumors of the treated mice compared to tumors of non-treated animals. Conclusions: Tumor immune microenvironment, stroma components, and PARP are differentially expressed in lung tumors of lung cancer patients with underlying COPD. The reduction in TLS and GC formation, the rise in DNA damage, and PARP overactivation probably contribute to the greater susceptibility of COPD patients to develop lung tumors. In mice treated with the combination of monoclonal antibodies, increased levels of oxidative stress along with activated apoptosis and autophagy may be part of the mechanisms whereby immunotherapy may reduce tumor burden. In conclusion, the presence of COPD should be considered when designing therapeutic strategies of lung cancer including immunotherapy as well as PARP activity inhibition.
Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina

Ce mémoire est consacré à l'élucidation des propriétés structurales des analogues du décapeptide de la région immunogène majeure (MIR) du récepteur de l'acétylcholine (RACh) afin de détecter certaine corrélation entre leur structure et activité d'une part, et du complexe peptide MIR/anticorps d'autre part, par modélisation moléculaire. Nous avons effectué une étude comparative de la structure à l'état libre (en solution dans le DMSO et en milieu micellaire) du MIR et de plusieurs de ses [Ala]-analogues plus ou moins affines envers l'anticorps anti-RACh, et sur la structure de l'analogue [A76]MIR complexé par un anticorps monoclonal. Pour atteindre les informations structurales, nous avons mené les calculs de dynamique moléculaire sous plusieurs aspects, dans le vide et en présence explicite de molécules de solvant (dans les boites de DMSO et d'eau). Le repliement Beta I/III de la séquence NPAD est indispensable à la reconnaissance du peptide par l'anticorps anti-RACh. La partie C-terminale adopte une structure peu courante formée par un pseudocycle à 11 atomes. On observe une réorientation des chaines latérales hydrophobes qui interviennent probablement dans la reconnaissance antigène-anticorps. Nous avons appliqué la technique de modélisation par homologie de séquence à la construction de la structure 3D du fragment scFv d'un anticorps anti-RACh pour modéliser son site de liaison. Ensuite, à partir de la conformation du ligand au contact de l'anticorps, nous avons reconstitué la structure du complexe antigène-anticorps par docking

L'infection quasi mondiale de l'homme par le virus d'epstein-barr (ebv) contraste avec, la restriction geographique de certaines pathologies qui lui sont associees, l'incidence et la distribution en fonction de l'age variant pour une meme maladie a travers le monde. Cette situation complexe suggere l'existence de differentes souches d'ebv, specifiques de regions geographiques ou de certaines pathologies. Le carcinome du rhinopharynx (npc) est une des pathologies associees a l'ebv, qui se caracterise par une incidence intermediaire en afrique du nord en affectant deux tranches d'age de la population : la premiere de 10 a 24 ans et la deuxieme de 45 a 55 ans. La maladie de hodgkin (mdh) est une autre pathologie associee a l'ebv relativement frequente en afrique du nord, ou elle predomine chez l'enfant alors qu'elle affecte les adultes dans les pays industrialises. Nos resultats dans l'analyse genotypique du virus d'epstein-barr implique dans le npc, dans la mdh ou present dans la population saine ebv-positive, ont montre que la souche d'ebv associee au npc d'afrique du nord est de type a/f/wi/xho1kept/h1h2, donc completement differente de celle associee au npc asiatique, de type a/f/wi/xho1lost/h. La meme souche de npc algerien est retrouvee dans les deux pics d'age et associee aux differents stades cliniques de la maladie. Cependant un autre type d'infection est associe a la maladie de hodgkin, qui se caracterise : premierement, par une coinfection avec le type a et le type b d'ebv, et deuxiemement, par le genotype h. Nos resultats ont montre aussi qu'en algerie le genotype h est dominant dans la population generale, dans les lcls etablies avec le virus oropharynge de patients npc ou d'individus sains ebv-positifs, dans la salive de personnes atteintes de npc ou de mdh. L'ensemble de ces resultats soutient l'hypothese d'une specificite pathologique du virus d'epstein-barr en afrique du nord.

Le but de ce travail etait d'etablir un modele de toxoplasmose experimentale chez le rat qui serait bien adapte a l'etude des infections humaines. Dans un premier temps, nous avons obtenu l'attenuation d'une toxoplasmose aigue due a une souche toxoplasmique tres virulente, chez des femelles rats gestantes. Notre objectif etait de comparer l'attenuation chez l'hote adulte immunocompetent par rapport a celle observee chez des nouveau-nes immuno-immatures. La suite des travaux a concerne l'immunodepression des rats femelles gestantes prealablement infestees avec t. Gondii. Un traitement immunodepresseur associant la ciclosporine a aux anticorps monoclonaux anti-cd4 et anti-cd8 a provoque une reactivation toxoplasmique chez les femelles et leurs petits. Nous avons voulu verifier dans quelle mesure l'immuno-immaturite foetale serait responsable d'un plus grand nombre de reactivations et/ou des reactivations plus precoces. Nous avons enfin etudie l'installation de deux phenomenes (attenuation et reactivation toxoplasmiques) au cours du temps. Une serie de prelevements sanguins issus d'un meme animal nous a permis de suivre l'installation des deux phenomenes. La detection d'adn toxoplasmique dans les echantillons de rat a ete possible grace a la pcr. Une variante de la pcr classique, la ap-pcr ou amplification aleatoire, a ete employee afin d'obtenir le typage des souches et clones toxoplasmiques provenant des animaux a differents statuts immunitaires

La synovite rhumatoïde représente un modèle de processus inflammatoire chronique ou le remodelage pathologique de la matrice extracellulaire est responsable de lésions anatomiques. Il dépend principalement des métalloprotéases matricielles, de certaines protéases à sérine comme les activateurs du plasminogène ou l'élastase des polynucléaires neutrophiles. Ces protéases concourent à une protéolyse focalisée qui dépend de la compartimentation des enzymes protéolytiques, de l'action d'inhibiteurs, spécifiques ou non, de la liaison enzyme-substrat, de l'éventuelle localisation membranaire des enzymes. Les polynucléaires neutrophiles sont présents en grand nombre dans le liquide synovial rhumatoïde. Les lymphocytes T et B, localisés dans la membrane synoviale, participent à la réaction inflammatoire et immune. Nous avons montre que l'élastase des polynucléaires neutrophiles préalablement actives par le PMA se localise en partie a la membrane plasmique et est capable d'activer la gélatinase-B de 92 KDA (MMP-9) dans l'environnement péricellulaire immédiat. D'autre part, nous nous sommes intéressés à la caractérisation des lymphocytes B en métalloprotéases et en inhibiteurs spécifiques tissulaires (TIMP). Nous avons isolé, purifié le TIMP-1 à partir de milieux de culture de lymphocytes B immortalisés par le virus d'Epstein-Barr par quatre étapes de chromatographie. Il a été identifié par spectrométrie de masse, caracterisé sur le plan de son pouvoir inhibiteur sur la gélatinase-B de 92 KDA préalablement activée par l'APMA. Les mécanismes de régulation de son expression ainsi que de celle de la MMP-9 ont été abordés par l'étude de l'influence de certaines cytokines, facteurs de croissance et ligands comme le PMA, le LPS et la concanavaline A, du calcium libre intracellulaire. La sécrétion de TIMP-1 par rapport à celle de MMP-9 dans les milieux de culture est 1000 fois plus importante sans que l'explication en soit claire. Elle est induite par l'IL-10, le TGFf, le LPS, le PMA. Les résultats suggèrent que l'expression de TIMP-1 et celle de la MMP-9, sont étroitement contrôlées. La chélation du calcium libre intracellulaire entraîne une baisse de la synthèse mais également de l'excrétion du TIMP-1. Nous avons dans une dernière partie étudié les taux synoviaux des gélatinases A ou MMP-2 et B ou MMP-9, du TIMP-1 et de la stromélysine-1 (MMP-3) dans 50 prélèvements. L'activité MMP-9, les taux de MMP-3 et de TIMP-1 permettent de caractériser et d'opposer les liquides mécaniques et inflammatoires.

Malaria, a parasitic disease, was commonly associated with third world countries, with the highest mortality in nations in Sub-Saharan Africa and Asia. But, travel increases the risk of spread to more temperate regions, such as Western Europe and the United States where Malaria has been successfully eradicated. In the past 40 years, with a better understanding of the mosquito vector and the parasite itself, advancements in treatment and containment have been made. Understanding the parasite as well as its pathogenesis is vital in formulating effective treatments. Following the incidences of Plasmodium falciparum, knowlesi, vivax, malaria, ovale, and less commonly cynomolgi and simium over time as well as region helps to better illuminate the methods of Malarial transmission, interplay with environmental factors, and methods of treatment. While each species of parasite is similar in terms of mode of infection, they differ slightly when considering incubation periods and diagnostic and treatment techniques. Many drugs have been developed to treat Malaria and include Chloroquine, Primaquine, and derivatives of Artemisinin. While the discovery of these drugs was a significant breakthrough that dramatically reduced incidence and deaths caused by Malaria, improper administration of treatment has led to a recent increase in strains of the parasite which have developed drug resistance to Artemisinin Combination Therapies (ACT’s). Of these species, P. falciparum and P. vivax, the most common causes of malaria, are also so far the only species to have developed drug resistance. The goal of this thesis is to explore popular interventions, both drug and public health based, and how research focus has now shifted to better understanding the mechanism of parasitic drug resistance, specifically linked to mutations found in the Kelch protein in P. Falciparum. The recent findings of Kelch mutations pave the way towards addressing the growing problem of anti-Malarial resistance.

Severe manifestations of malaria, including cerebral malaria (CM) and respiratory distress, result in approximately three million deaths annually worldwide. Currently, relatively little is known about severe disease pathogenesis. The development and outcome of severe malaria is determined by host-pathogen interactions, a complex interface of genetics and immune responses. Hypothetically, a spectrum of genetic susceptibility and resistance to severe disease exists within the host population, and malaria infection results in diverse host and parasite responses that impact disease outcome. The aim of this study was to identify differential host and parasite responses in a murine model of severe malaria, Plasmodium berghei ANKA (PbA), in CM-susceptible and CM-resistant mice; and to analyze host genetics in patients with severe disease due to Plasmodium falciparum. In vivo, expression microarray analysis showed that, in malaria target organs, differential responses were related to immune response – primarily interferon and complement pathways – and apoptosis. Histopathological examination of the brain confirmed an increased prevalence of apoptosis in CM-susceptible mice. Further examination of the role of complement in CM-susceptibility determined that early complement 5 (C5) activation conferred susceptibility to CM, and that C5 deficiency conferred resistance, which could be recapitulated by antibody blockade of activated C5 or its receptor in susceptible mice. Additionally, single nucleotide polymorphism (SNP) studies identified that complement receptor 1 SNPs were associated with disease severity in patients with P. falciparum malaria. PbA parasites displayed a unique transcriptional signature in each tissue examined (brain, liver, spleen and lung), showed differential gene expression between CM-resistant and susceptible hosts, and were most prominent in lung tissue. Closer examination of lung involvement in PbA infection revealed that PbA-infected C57BL/6 mice develop acute lung injury (ALI), defined by disruption of the alveolar-capillary membrane barrier. ALI susceptibility did not correlate with CM susceptibility, but was influenced by peripheral parasite burden and CD36-mediated parasite sequestration in the lung. PbA provides a clinically relevant experimental model for CM and ALI, through which important disease mechanisms can be identified and modulated. Ideally, the use of such models aids in the discovery of disease biomarkers and novel therapeutic strategies, which may be applied to human severe and cerebral malaria.

Plasmodium falciparum is responsible for the most virulent form of human malaria. The biology of the intra-erythrocytic stage of P. falciparum is the most well studied as it is this stage that marks the clinical manifestation of malaria. To establish a successful infection, P. falciparum brings about extensive remodeling of erythrocytes, its host compartment. The infected erythrocytes harbor several parasite induced membranous structures. Most importantly, pathogenesis related structures termed knobs, which impart cytoadherence, appear on the cell surface of the infected erythrocytes. For bringing about such eccentric renovations in its host compartment, the parasite exports 8% of its genome (~400 proteins) to various destinations in the host cell. Studies from our lab have shown that proteins belonging to heat shock protein40 (Hsp40) and heat shock protein70 (Hsp70) group of chaperones are also exported to the host compartment. We and others have implicated these chaperones in important processes such as protein trafficking and chaperoning assembly of parasitic proteins into the cytoadherent knobs. As detailed above, malaria parasite invests a lot of energy in exporting a large number of proteins including chaperones in the red blood cell to meet its pathogenic demands. In order to do so, it heavily relies on its secretory pathway. However, it is known that the parasite experiences a significant amount of oxidative stress on account of heme detoxification, its own metabolism and the immune system of the host. The parasite also effluxes large quantities of reduced thiols such as glutathione and homocysteine into the extracellular milieu indicative of redox perturbation. Additionally, the parasite lacks Peroxiredoxin IV, which otherwise localizes in the ER and carries out detoxification of peroxide generated as a result of oxidative protein folding. Together, these factors indicate that maintaining redox homeostasis is a challenging task for the parasite. It also implies that the ER, where the redox balance is even more critical as it requires oxidising environment for protein folding, is predisposed to stress. In light of this fact and the importance of secretory pathway in malaria pathogenesis, we decided to address the ways and mechanisms used by the parasite to tackle perturbations in its secretory pathway. Examination of a canonical unfolded protein response pathway in P. falciparum ER-stress is a condition arising whenever the load of unfolded proteins increases the folding capacity of the ER. However, eukaryotes have evolved a fairly well conserved homeostatic response pathway known as unfolded protein response (UPR) to tackle ER-stress. This signal transduction pathway is composed of three arms involving three ER-transmembrane signal transducers namely; IRE1, ATF6 and PERK. IRE1 brings about splicing of a bZIP transcription factor, XBP1/Hac1 and ATF6 becomes activated upon getting proteolytically cleaved in the Golgi. These transcription factors then migrate to the nucleus where they bind onto the ER-stress elements thereby, leading to the transcriptional up-regulation of the UPR targets such as ER chaperones and components of ER associated degradation (ERAD) pathway which rescue the function of the ER. PERK on the other hand brings about translational attenuation by phosphorylating eIF2α, thereby providing parasite the benefit of time to recover. We started our examination on UPR in Plasmodium by carrying out in silico analysis of the major components of UPR in the parasite by using Homo sapiens protein sequences as the query. We found that the parasite lacks the homologues of all the transcriptional regulators of canonical UPR. Only PERK component of the UPR was found to be present in the parasite. To rule out the existence of the canonical UPR in P. falciparum, we examined the status of UPR targets by subjecting the parasites to treatment with DTT. DTT perturbs the disulfide oxidation in the ER and thereby inhibits protein folding leading to ER-stress. Owing to the missing components of a canonical UPR, we did not find up-regulation of known UPR targets such as ER-chaperones including PfBiP, PfGrp94, PfPDI and ERAD marker Derlin1 at transcript as well as protein level. Owing to the presence of a PERK homologue, phosphorylation of eIF2α followed by attenuation of protein synthesis was observed upon subjecting the parasites to DTT mediated ER-stress. In the absence of a canonical UPR, the parasites were found to be hypersensitive to ER-stress in comparison to the mammalian counterpart. In the presence of DTT, the parasites showed perturbation in the redox homeostasis as indicated by increase in the levels of ROS. Next, we sought to examine if the parasites resorted to any alternate means of increasing the availability of chaperones in the ER. For this, we analysed the involvement of another Hsp70 family member, Hsp70-x which is homologous to BiP and which is known to traverse the ER while getting exported to the erythrocyte compartment. Interestingly, we found that upon exposure to ER-stress, the export of this protein is partially blocked and around 30% of the protein is retained in the ER. On the other hand, there was no effect on the trafficking of another exported chaperone KAHsp40. This indicates that the parasite possibly recruits this pool of retained Hsp70-x for the chaperoning of unfolded proteins in the ER. Global response to ER-stress in P. falciparum To dig deeper into the parasite specific strategies employed for dealing with ER-stress at a global level, we carried out high throughput transcriptomic and proteomic analysis upon subjecting the parasites to DTT mediated ER-stress. Microarray based gene expression profiling was carried out upon subjecting the parasites to DTT mediated ER-stress. We found that the parasite mounts a transcriptional response as indicated by up-regulation of 155 transcripts. In congruence with our biochemical analysis, we did not find up-regulation of ER chaperones as well as ERAD proteins. Functional grouping of the up-regulated genes revealed large number of hypothetical proteins in our list of differentially expressed genes. The genes encoding exported proteins represent yet another abundant class. In the course of examining the involvement of Plasmodium specific transcriptional regulators mediating response to DTT induced ER-stress, we identified 4 genes belonging to the family of AP2 transcription factors. AP2 (Apetela-2) are specific transcription factors which are possessed by apicomplexa and bring about regulation of developmental processes and stress response in plants. On comparing our list of up-regulated genes with the previously known targets of AP2 factors, we found that an entire cascade of AP2 factors is up-regulated upon DTT-mediated ER stress. Thus, AP2 factors appear to be the major stress response mediators as they are together responsible for the up-regulation of 60% of genes identified in this study. In addition, another striking observation made, was the up-regulation of a few sexual stage specific transcripts. 2D Gel electrophoresis and 2D-DIGE based Proteomic analysis indicated an up-regulation of secretory proteins and some components of vesicular trafficking and secretory machinery possibly to overcome the block in the functions of the secretory pathway. ER-stress triggers stage transition in P. falciparum Intrigued by the up-regulation of a few sexual stage specific genes, we were curious to examine if there was a functional significance of this observation. To this end, we decided to investigate the effect of ER-stress on induction of gametocytes, the only sexual stage found in humans. Indeed, we found a two fold induction in the numbers of gametocytes formed upon challenging the parasite with DTT mediated ER-stress. The induction of gametocytogenesis was also observed by using a clinical isolate of P. falciparum for the assay. The DTT treated cultures progressed through the gametocytogenesis pathway normally forming all the five morphologically distinct stages. Then we sought to examine if this phenomenon could be simulated in the physiological scenario as well. For this, we made use of a rodent model of malaria, P. berghei. Two different treatment regimes involving 1) direct injection of increasing concentration of DTT into P. berghei infected mice and 2) injection of DTT pretreated P. berghei infected erythrocytes into healthy mice were followed. In both cases, a significant increase in the gametocyte induction was observed. Having seen that Plasmodium undergoes gametocytogenesis upon exposure to ER-stress not only in in vitro cultures but also in in vivo scenario, we wanted to identify the players involved in the commitment to sexual stage. Recently, a transcription factor belonging to AP2 class of transcription factors, referred to as AP2-G has been implicated in committing the asexual parasites for transition to gametocyte stage. To examine the role of this factor in the phenotype observed by us, we looked at the effect of DTT on AP2-G. Interestingly, we found around 6 folds up-regulation in the expression of AP2-G levels under ER-stress. The downstream targets of AP2-G, many of which are the markers of gametocyte were also found to be up-regulated upon being exposed to DTT mediated ER-stress indicating the launch of a transcriptional program which together works in the direction of transition to gametocytes. Having seen that P. falciparum undergoes ametocytogenesis in response to DTT treatment both under in vitro and in vivo conditions, we sought to look for probable physiological analogue of DTT. Since glutathione is the major cellular redox buffer, critical for redox homeostasis, we quantitated the levels of both oxidized and reduced forms of this non protein thiol using Mass Spectrometric approach. We found that the levels of reduced forms of glutathione significantly increased upon treating the parasites with DTT. This indicates that the levels of glutathione could be one of the physiological triggers of gametocytogenesis. Conclusion In conclusion, our study analyses the ways and mechanisms employed by malaria parasite to cope with perturbations to its secretory pathway. We have established the absence of a canonical UPR in this parasite and our results suggest that Plasmodium has developed a three stage response to cope with ER stress: 1) an early adaptation to increase the local concentration of chaperones in the ER by partially blocking the export of a Hsp70 family member, 2) activation of gene expression cascade involving AP2 transcription factors and 3) a consequent switch to the transmissible sexual stage. Hence, our study throws light on a novel physiological adaptation utilised by malaria parasite to tackle stress to its secretory pathway. Gametocytogenesis, which can be transmitted to the mosquito vector, could hence serve as an effective means to escape ER-stress altogether. Importantly, while it is widely known that stress brings about switch towards sexual stages in P. falciparum, the molecular triggers involved in this process remain obscure in the field of malaria biology. Therefore, our findings also address this long standing question by providing the evidence of ER-stress being one such trigger required for switching to the transmissible sexual stages.

Plasmodium falciparum is responsible for the most virulent form of human malaria. The biology of the intra-erythrocytic stage of P. falciparum is the most well studied as it is this stage that marks the clinical manifestation of malaria. To establish a successful infection, P. falciparum brings about extensive remodeling of erythrocytes, its host compartment. The infected erythrocytes harbor several parasite induced membranous structures. Most importantly, pathogenesis related structures termed knobs, which impart cytoadherence, appear on the cell surface of the infected erythrocytes. For bringing about such eccentric renovations in its host compartment, the parasite exports 8% of its genome (~400 proteins) to various destinations in the host cell. Studies from our lab have shown that proteins belonging to heat shock protein40 (Hsp40) and heat shock protein70 (Hsp70) group of chaperones are also exported to the host compartment. We and others have implicated these chaperones in important processes such as protein trafficking and chaperoning assembly of parasitic proteins into the cytoadherent knobs. As detailed above, malaria parasite invests a lot of energy in exporting a large number of proteins including chaperones in the red blood cell to meet its pathogenic demands. In order to do so, it heavily relies on its secretory pathway. However, it is known that the parasite experiences a significant amount of oxidative stress on account of heme detoxification, its own metabolism and the immune system of the host. The parasite also effluxes large quantities of reduced thiols such as glutathione and homocysteine into the extracellular milieu indicative of redox perturbation. Additionally, the parasite lacks Peroxiredoxin IV, which otherwise localizes in the ER and carries out detoxification of peroxide generated as a result of oxidative protein folding. Together, these factors indicate that maintaining redox homeostasis is a challenging task for the parasite. It also implies that the ER, where the redox balance is even more critical as it requires oxidising environment for protein folding, is predisposed to stress. In light of this fact and the importance of secretory pathway in malaria pathogenesis, we decided to address the ways and mechanisms used by the parasite to tackle perturbations in its secretory pathway. Examination of a canonical unfolded protein response pathway in P. falciparum ER-stress is a condition arising whenever the load of unfolded proteins increases the folding capacity of the ER. However, eukaryotes have evolved a fairly well conserved homeostatic response pathway known as unfolded protein response (UPR) to tackle ER-stress. This signal transduction pathway is composed of three arms involving three ER-transmembrane signal transducers namely; IRE1, ATF6 and PERK. IRE1 brings about splicing of a bZIP transcription factor, XBP1/Hac1 and ATF6 becomes activated upon getting proteolytically cleaved in the Golgi. These transcription factors then migrate to the nucleus where they bind onto the ER-stress elements thereby, leading to the transcriptional up-regulation of the UPR targets such as ER chaperones and components of ER associated degradation (ERAD) pathway which rescue the function of the ER. PERK on the other hand brings about translational attenuation by phosphorylating eIF2α, thereby providing parasite the benefit of time to recover. We started our examination on UPR in Plasmodium by carrying out in silico analysis of the major components of UPR in the parasite by using Homo sapiens protein sequences as the query. We found that the parasite lacks the homologues of all the transcriptional regulators of canonical UPR. Only PERK component of the UPR was found to be present in the parasite. To rule out the existence of the canonical UPR in P. falciparum, we examined the status of UPR targets by subjecting the parasites to treatment with DTT. DTT perturbs the disulfide oxidation in the ER and thereby inhibits protein folding leading to ER-stress. Owing to the missing components of a canonical UPR, we did not find up-regulation of known UPR targets such as ER-chaperones including PfBiP, PfGrp94, PfPDI and ERAD marker Derlin1 at transcript as well as protein level. Owing to the presence of a PERK homologue, phosphorylation of eIF2α followed by attenuation of protein synthesis was observed upon subjecting the parasites to DTT mediated ER-stress. In the absence of a canonical UPR, the parasites were found to be hypersensitive to ER-stress in comparison to the mammalian counterpart. In the presence of DTT, the parasites showed perturbation in the redox homeostasis as indicated by increase in the levels of ROS. Next, we sought to examine if the parasites resorted to any alternate means of increasing the availability of chaperones in the ER. For this, we analysed the involvement of another Hsp70 family member, Hsp70-x which is homologous to BiP and which is known to traverse the ER while getting exported to the erythrocyte compartment. Interestingly, we found that upon exposure to ER-stress, the export of this protein is partially blocked and around 30% of the protein is retained in the ER. On the other hand, there was no effect on the trafficking of another exported chaperone KAHsp40. This indicates that the parasite possibly recruits this pool of retained Hsp70-x for the chaperoning of unfolded proteins in the ER. Global response to ER-stress in P. falciparum To dig deeper into the parasite specific strategies employed for dealing with ER-stress at a global level, we carried out high throughput transcriptomic and proteomic analysis upon subjecting the parasites to DTT mediated ER-stress. Microarray based gene expression profiling was carried out upon subjecting the parasites to DTT mediated ER-stress. We found that the parasite mounts a transcriptional response as indicated by up-regulation of 155 transcripts. In congruence with our biochemical analysis, we did not find up-regulation of ER chaperones as well as ERAD proteins. Functional grouping of the up-regulated genes revealed large number of hypothetical proteins in our list of differentially expressed genes. The genes encoding exported proteins represent yet another abundant class. In the course of examining the involvement of Plasmodium specific transcriptional regulators mediating response to DTT induced ER-stress, we identified 4 genes belonging to the family of AP2 transcription factors. AP2 (Apetela-2) are specific transcription factors which are possessed by apicomplexa and bring about regulation of developmental processes and stress response in plants. On comparing our list of up-regulated genes with the previously known targets of AP2 factors, we found that an entire cascade of AP2 factors is up-regulated upon DTT-mediated ER stress. Thus, AP2 factors appear to be the major stress response mediators as they are together responsible for the up-regulation of 60% of genes identified in this study. In addition, another striking observation made, was the up-regulation of a few sexual stage specific transcripts. 2D Gel electrophoresis and 2D-DIGE based Proteomic analysis indicated an up-regulation of secretory proteins and some components of vesicular trafficking and secretory machinery possibly to overcome the block in the functions of the secretory pathway. ER-stress triggers stage transition in P. falciparum Intrigued by the up-regulation of a few sexual stage specific genes, we were curious to examine if there was a functional significance of this observation. To this end, we decided to investigate the effect of ER-stress on induction of gametocytes, the only sexual stage found in humans. Indeed, we found a two fold induction in the numbers of gametocytes formed upon challenging the parasite with DTT mediated ER-stress. The induction of gametocytogenesis was also observed by using a clinical isolate of P. falciparum for the assay. The DTT treated cultures progressed through the gametocytogenesis pathway normally forming all the five morphologically distinct stages. Then we sought to examine if this phenomenon could be simulated in the physiological scenario as well. For this, we made use of a rodent model of malaria, P. berghei. Two different treatment regimes involving 1) direct injection of increasing concentration of DTT into P. berghei infected mice and 2) injection of DTT pretreated P. berghei infected erythrocytes into healthy mice were followed. In both cases, a significant increase in the gametocyte induction was observed. Having seen that Plasmodium undergoes gametocytogenesis upon exposure to ER-stress not only in in vitro cultures but also in in vivo scenario, we wanted to identify the players involved in the commitment to sexual stage. Recently, a transcription factor belonging to AP2 class of transcription factors, referred to as AP2-G has been implicated in committing the asexual parasites for transition to gametocyte stage. To examine the role of this factor in the phenotype observed by us, we looked at the effect of DTT on AP2-G. Interestingly, we found around 6 folds up-regulation in the expression of AP2-G levels under ER-stress. The downstream targets of AP2-G, many of which are the markers of gametocyte were also found to be up-regulated upon being exposed to DTT mediated ER-stress indicating the launch of a transcriptional program which together works in the direction of transition to gametocytes. Having seen that P. falciparum undergoes ametocytogenesis in response to DTT treatment both under in vitro and in vivo conditions, we sought to look for probable physiological analogue of DTT. Since glutathione is the major cellular redox buffer, critical for redox homeostasis, we quantitated the levels of both oxidized and reduced forms of this non protein thiol using Mass Spectrometric approach. We found that the levels of reduced forms of glutathione significantly increased upon treating the parasites with DTT. This indicates that the levels of glutathione could be one of the physiological triggers of gametocytogenesis. Conclusion In conclusion, our study analyses the ways and mechanisms employed by malaria parasite to cope with perturbations to its secretory pathway. We have established the absence of a canonical UPR in this parasite and our results suggest that Plasmodium has developed a three stage response to cope with ER stress: 1) an early adaptation to increase the local concentration of chaperones in the ER by partially blocking the export of a Hsp70 family member, 2) activation of gene expression cascade involving AP2 transcription factors and 3) a consequent switch to the transmissible sexual stage. Hence, our study throws light on a novel physiological adaptation utilised by malaria parasite to tackle stress to its secretory pathway. Gametocytogenesis, which can be transmitted to the mosquito vector, could hence serve as an effective means to escape ER-stress altogether. Importantly, while it is widely known that stress brings about switch towards sexual stages in P. falciparum, the molecular triggers involved in this process remain obscure in the field of malaria biology. Therefore, our findings also address this long standing question by providing the evidence of ER-stress being one such trigger required for switching to the transmissible sexual stages.

Infections or diseases are not just stressful for the one who encounters it. The pathogens causing the same also have to deal with the hostile environment present in the host. The maintenance of physiological homeostatic balance is must for survival of all organisms. This becomes a challenging task for the protozoan parasites which often alternate between two different hosts during their life cycle and thereby encounter several environmental insults which they need to acclimatize against, in order to establish a productive infection. Since their discovery as proteins up-regulated upon heat shock, heat shock proteins have emerged as main mediators of cellular stress responses and are now also known to chaperone normal cellular functions. Parasites like Plasmodium falciparum have fully utilized the potential of these molecular chaperones. This is evident from the fact that parasite has dedicated about 2% of its genome for this purpose. During transmission from the insect vector to humans, the malaria parasite Plasmodium falciparum experiences a temperature rise of about 10oC, and the febrile episodes associated with asexual cycle further add to the heat shock which the parasite has to bear with. The exact mechanism by which the parasite responds to temperature stress remains unclear; however, the induction of chaperones such as PfHsp90 and PfHsp70 has been reported earlier. In other eukaryotes, there are three main factors which regulate heat shock response (HSR): heat shock factor (HSF), heat shock element (HSE) and HSF binding protein (HSBP). Bioinformatics analysis revealed presence of HSE and HSBP in P. falciparum genome; however, no obvious homolog of HSF could be identified. Either the HSF homologue in P. falciparum is highly divergent or the parasite has evolved alternate means to tackle temperature stress. Therefore, we decided to biochemically characterize HSBP and understand the heat shock response pathway in the parasite using transcriptomics and proteomics. The expression for PfHSBP was confirmed at both mRNA and protein level and it was found to translocate into the nucleus during heat shock. As previously reported for HSBP in other organisms, PfHSBP also exists predominantly in trimeric and hexameric form and it interacts with PfHsp70-1. Nearly 900 genes, which represent almost 17% of the parasite genome, were found to have HSE in their promoter region. HSE are represented by three repeating units of nGAAn pentamer and its inverted repeat nCTTn; however, the most abundant class of genes in P. falciparum possessed an atypical HSE which had only 2 continuous repeat units. Next, we were interested to find out if these HSE could actually bind to any parasite protein. Therefore, we performed EMSA analysis with the parasite nuclear extracts using HSE sequence as the oligonucleotide. We observed retarded mobility of the oligonucleotide suggesting that it was indeed able to recruit some protein from the nuclear extract. The importance of transcriptional regulation during heat shock was further confirmed when parasite culture subjected to heat shock in the presence of transcription inhibitor did not show induction in the levels of PfHsp70. These evidences suggest that parasite indeed possesses all the components of heat shock response pathway with either a divergent homologue of HSF or an alternate transcription factor which would have taken its role. Next, we performed global profiling of heat shock response using transcriptomic analysis and 2DDIGE based proteomic profiling. Overall, the parasite’s response to heat shock can be classified under 5 functional categories which aim at increasing the folding capacity of the cell, prevent protein aggregation, increase cytoadhesion, increase host cell remodelling and increase erythrocyte membrane rigidity. Out of the 201 genes found to be up-regulated upon heat shock, 36 were found to have HSE in their promoter region. This suggested that HSE-mediated protein up-regulation could be responsible for the induction of only 18% of total number of genes up-regulated upon heat shock. How would the parasite bring about up-regulation of rest of the heat shock responsive genes? It has been previously reported that genes for some of the heat shock proteins in P. falciparum possess G-box regulatory elements in their promoters and recently, it was shown that these elements served as the binding site for one of the transcription factors (PF13_0235) of AP2 family. Therefore, we looked for the status of this AP2 factor and its targets in our transcriptome data. Although, PF13_0235 was itself not up-regulated, we found up-regulation of its target genes which included another AP2 factor gene PF11_0404. The target genes of PF11_0404 were also up-regulated upon heat shock, thereby suggesting the functioning of an AP2 factor mediated response to heat shock. The next major challenge which the malaria parasite has to deal with is the remodelling of the erythrocyte as these cells do not have a cellular machinery which the parasite can take control of. The parasite remodels the erythrocyte with the help of its large repertoire of exported proteins and develops protrusions known as “knobs” on the erythrocyte surface. These protrusions are cytoadherent in nature and constitute the main virulence determinants of malaria. They also represent variable antigens that allow immune escape. Our lab has previously demonstrated an exported PfHsp40, termed as KAHsp40, to be involved in knob biogenesis. Apart from KAHsp40, there are 19 other PfHsp40s which possess the PEXEL motif required for protein export to erythrocytes. Although, Hsp40s work with an Hsp70 partner, none of the parasitic Hsp70s were known to be exported and was always a missing link in the field of malaria chaperone biology. A genomic re-annotation event could fill this gap by re-annotating the sequence for a pseudogene, PfHsp70-x and described it to contain a functional ORF. According to the re-annotated ORF sequence, PfHsp70-x possessed an ER signal peptide and thus could be targeted to the secretory pathway. Following validation of the re-annotation using a PCR-based approach, we confirmed the expression of this protein at the protein level by immunoblot analysis. Using various subcellular fractionation approaches and immunolocalization studies we established that PfHsp70-x indeed gets exported to the erythrocyte compartment; however, it did not contain the PEXEL motif required for protein export. It gets secreted into the vacuole around the parasite via the canonical ER-Golgi secretory pathway. Its trafficking from vacuole into the erythrocyte was mediated by a hexameric sequence which was present just after the signal peptide cleavage site and before the beginning of ATP-binding domain. In the erythrocyte compartment, it was found to interact with KAHsp40 and MAHRP1, proteins previously implicated in knob biogenesis. Most importantly, PfHsp70-x interacted with the major knob component PfEMP1; however, itself did not become part of knobs. Instead, it localized to the Maurer’s clefts in the erythrocyte compartment. Inside the parasite, PfHsp70-x was present in a complex with Plasmepsin V and PfHsp101. These proteins have been shown to be essential for host cell remodelling process. Plasmepsin V recognizes the PEXEL motif and brings about its cleavage and PfHsp101 specifically targets these PEXEL-cleaved exported proteins to the translocon in vacuolar membrane thereby facilitating their export into the erythrocyte. Thus, PfHsp70-x could also be involved in directing the export of knob constituents apart from just facilitating their assembly. Since, we found out that heat shock or the febrile episodes encountered during the asexual cycling of the parasite promote host cell remodelling; we wanted to find out if PfHsp70-x has any specific role under conditions of temperature stress. PfHsp70-x gene expression was not influenced upon heat shock, however, its export into the erythrocyte was inhibited and the protein got accumulated within the parasite compartment. Surprisingly, immunolocalization studies revealed that the accumulated pool of PfHsp70-x localized into the nucleus instead of ER thus suggesting an alternate role to be associated with PfHsp70-x under stress. Overall, our study addresses two major aspects of malaria pathogenesis. First, response to heat shock and second, remodelling of the host cell. We, for the first time describe global profiling of the parasite’s heat shock response and identify a novel P. falciparum specific heat shock protein member to be involved in malaria pathogenesis.

Infections or diseases are not just stressful for the one who encounters it. The pathogens causing the same also have to deal with the hostile environment present in the host. The maintenance of physiological homeostatic balance is must for survival of all organisms. This becomes a challenging task for the protozoan parasites which often alternate between two different hosts during their life cycle and thereby encounter several environmental insults which they need to acclimatize against, in order to establish a productive infection. Since their discovery as proteins up-regulated upon heat shock, heat shock proteins have emerged as main mediators of cellular stress responses and are now also known to chaperone normal cellular functions. Parasites like Plasmodium falciparum have fully utilized the potential of these molecular chaperones. This is evident from the fact that parasite has dedicated about 2% of its genome for this purpose. During transmission from the insect vector to humans, the malaria parasite Plasmodium falciparum experiences a temperature rise of about 10oC, and the febrile episodes associated with asexual cycle further add to the heat shock which the parasite has to bear with. The exact mechanism by which the parasite responds to temperature stress remains unclear; however, the induction of chaperones such as PfHsp90 and PfHsp70 has been reported earlier. In other eukaryotes, there are three main factors which regulate heat shock response (HSR): heat shock factor (HSF), heat shock element (HSE) and HSF binding protein (HSBP). Bioinformatics analysis revealed presence of HSE and HSBP in P. falciparum genome; however, no obvious homolog of HSF could be identified. Either the HSF homologue in P. falciparum is highly divergent or the parasite has evolved alternate means to tackle temperature stress. Therefore, we decided to biochemically characterize HSBP and understand the heat shock response pathway in the parasite using transcriptomics and proteomics. The expression for PfHSBP was confirmed at both mRNA and protein level and it was found to translocate into the nucleus during heat shock. As previously reported for HSBP in other organisms, PfHSBP also exists predominantly in trimeric and hexameric form and it interacts with PfHsp70-1. Nearly 900 genes, which represent almost 17% of the parasite genome, were found to have HSE in their promoter region. HSE are represented by three repeating units of nGAAn pentamer and its inverted repeat nCTTn; however, the most abundant class of genes in P. falciparum possessed an atypical HSE which had only 2 continuous repeat units. Next, we were interested to find out if these HSE could actually bind to any parasite protein. Therefore, we performed EMSA analysis with the parasite nuclear extracts using HSE sequence as the oligonucleotide. We observed retarded mobility of the oligonucleotide suggesting that it was indeed able to recruit some protein from the nuclear extract. The importance of transcriptional regulation during heat shock was further confirmed when parasite culture subjected to heat shock in the presence of transcription inhibitor did not show induction in the levels of PfHsp70. These evidences suggest that parasite indeed possesses all the components of heat shock response pathway with either a divergent homologue of HSF or an alternate transcription factor which would have taken its role. Next, we performed global profiling of heat shock response using transcriptomic analysis and 2DDIGE based proteomic profiling. Overall, the parasite’s response to heat shock can be classified under 5 functional categories which aim at increasing the folding capacity of the cell, prevent protein aggregation, increase cytoadhesion, increase host cell remodelling and increase erythrocyte membrane rigidity. Out of the 201 genes found to be up-regulated upon heat shock, 36 were found to have HSE in their promoter region. This suggested that HSE-mediated protein up-regulation could be responsible for the induction of only 18% of total number of genes up-regulated upon heat shock. How would the parasite bring about up-regulation of rest of the heat shock responsive genes? It has been previously reported that genes for some of the heat shock proteins in P. falciparum possess G-box regulatory elements in their promoters and recently, it was shown that these elements served as the binding site for one of the transcription factors (PF13_0235) of AP2 family. Therefore, we looked for the status of this AP2 factor and its targets in our transcriptome data. Although, PF13_0235 was itself not up-regulated, we found up-regulation of its target genes which included another AP2 factor gene PF11_0404. The target genes of PF11_0404 were also up-regulated upon heat shock, thereby suggesting the functioning of an AP2 factor mediated response to heat shock. The next major challenge which the malaria parasite has to deal with is the remodelling of the erythrocyte as these cells do not have a cellular machinery which the parasite can take control of. The parasite remodels the erythrocyte with the help of its large repertoire of exported proteins and develops protrusions known as “knobs” on the erythrocyte surface. These protrusions are cytoadherent in nature and constitute the main virulence determinants of malaria. They also represent variable antigens that allow immune escape. Our lab has previously demonstrated an exported PfHsp40, termed as KAHsp40, to be involved in knob biogenesis. Apart from KAHsp40, there are 19 other PfHsp40s which possess the PEXEL motif required for protein export to erythrocytes. Although, Hsp40s work with an Hsp70 partner, none of the parasitic Hsp70s were known to be exported and was always a missing link in the field of malaria chaperone biology. A genomic re-annotation event could fill this gap by re-annotating the sequence for a pseudogene, PfHsp70-x and described it to contain a functional ORF. According to the re-annotated ORF sequence, PfHsp70-x possessed an ER signal peptide and thus could be targeted to the secretory pathway. Following validation of the re-annotation using a PCR-based approach, we confirmed the expression of this protein at the protein level by immunoblot analysis. Using various subcellular fractionation approaches and immunolocalization studies we established that PfHsp70-x indeed gets exported to the erythrocyte compartment; however, it did not contain the PEXEL motif required for protein export. It gets secreted into the vacuole around the parasite via the canonical ER-Golgi secretory pathway. Its trafficking from vacuole into the erythrocyte was mediated by a hexameric sequence which was present just after the signal peptide cleavage site and before the beginning of ATP-binding domain. In the erythrocyte compartment, it was found to interact with KAHsp40 and MAHRP1, proteins previously implicated in knob biogenesis. Most importantly, PfHsp70-x interacted with the major knob component PfEMP1; however, itself did not become part of knobs. Instead, it localized to the Maurer’s clefts in the erythrocyte compartment. Inside the parasite, PfHsp70-x was present in a complex with Plasmepsin V and PfHsp101. These proteins have been shown to be essential for host cell remodelling process. Plasmepsin V recognizes the PEXEL motif and brings about its cleavage and PfHsp101 specifically targets these PEXEL-cleaved exported proteins to the translocon in vacuolar membrane thereby facilitating their export into the erythrocyte. Thus, PfHsp70-x could also be involved in directing the export of knob constituents apart from just facilitating their assembly. Since, we found out that heat shock or the febrile episodes encountered during the asexual cycling of the parasite promote host cell remodelling; we wanted to find out if PfHsp70-x has any specific role under conditions of temperature stress. PfHsp70-x gene expression was not influenced upon heat shock, however, its export into the erythrocyte was inhibited and the protein got accumulated within the parasite compartment. Surprisingly, immunolocalization studies revealed that the accumulated pool of PfHsp70-x localized into the nucleus instead of ER thus suggesting an alternate role to be associated with PfHsp70-x under stress. Overall, our study addresses two major aspects of malaria pathogenesis. First, response to heat shock and second, remodelling of the host cell. We, for the first time describe global profiling of the parasite’s heat shock response and identify a novel P. falciparum specific heat shock protein member to be involved in malaria pathogenesis.