Τα έντομα και άλλοι ποικιλόθερμοι οργανισμοί έχουν αναπτύξει διάφορους μοριακούς μηχανισμούς για την προστασία τους από ακραίες θερμοκρασίες και άλλες στρεσογόνες καταστάσεις που συμβαίνουν κατά τη διάρκεια της ζωής τους. Ένας από τους σημαντικότερους μηχανισμούς είναι η παραγωγή των θερμοεπαγόμενων πρωτεϊνών οι οποίες, ως μοριακοί συνοδοί, προστατεύουν τα κύτταρα από τη μετουσίωση και συσσωμάτωση των πρωτεϊνών τους. Πολλές από τις θερμοεπαγόμενες πρωτεΐνες συνθέτονται και σε φυσιολογικές συνθήκες και παίζουν σημαντικούς ρόλους στην ορθή αναδίπλωση, μεταφορά και αποικοδόμηση των πρωτεϊνών του κυττάρου. Το εργαστήριο μας ενδιαφέρεται για το ρόλο των θερμοεπαγόμενων πρωτεϊνών και τη ρύθμιση των γονιδίων τους στη Μεσογειακή μύγα. Τέσσερα θερμοεπαγόμενα γονίδια, hsp90, hsp70, hsp27 και hsp26, έχουν κλωνοποιηθεί και χαρακτηριστεί στο εργαστήριό μας. Στόχοι αυτής της μεταπτυχιακής εργασίας είναι α) η λειτουργική ανάλυση της 5’ ανοδικής περιοχής του γονιδίου hsp90 της Μεσογειακής μύγας και β) η έκφραση του γονιδίου σε συνθήκες ψυχρού στρες. Για να μελετήσουμε την ρύθμιση του γονιδίου hsp90, δημιουργήσαμε διαγονιδιακά στελέχη που έφεραν το γονίδιο της λουσιφεράσης (ως γονίδιο αναφοράς) υπό τον έλεγχο διαφορετικών 5’ περιοχών του γονιδίου hsp90. Η ανάλυση των στελεχών αυτών έδειξε ότι η ευρύτερη περιοχή του υποκινητή που εκτίνεται έως και το -3536 περιλαμβάνει όλα τα απαραίτητα στοιχεία για τη θερμοεπαγόμενη έκφραση του γονιδίου. Για να μελετήσουμε εάν η χαμηλή θερμοκρασία (0 oC) έχει κάποια επίδραση στη βιωσιμότητα του εντόμου και στην έκφραση του hsp90, ομάδες αρσενικών ενήλικων εντόμων επωάστηκαν για διαφόρους χρόνους στους 0 oC και ακολούθως μελετήθηκε η επιβίωσή τους και η έκφραση του θερμοεπαγόμενου γονιδίου με RT-PCR και qRT-PCR. Τα αποτελέσματά μας υποδηλώνουν ότι οι χαμηλές θερμοκρασίες αυξάνουν την έκφραση του hsp90 σχεδόν στα ίδια επίπεδα με εκείνα του θερμικού στρες. Επίσης η βιωσιμότητα των εντόμων βρέθηκε πολύ υψηλή στους 0 oC. / Insects and other poikilotherms have multiple molecular mechanisms for their protection from extreme environmental and other stressful conditions which happens during their life. One of these mechanisms is the production of heat shock proteins. These proteins, being molecular chaperones, protect the cells from protein denaturation and aggregation. Some of these proteins are produced under normal conditions, and they play important role in proper protein folding, transport and degradation. We are interested in the role of heat shock proteins and the regulation of their genes in Mediterranean fruit fly (medfly). Four heat shock genes have been cloned and characterized in our laboratory. These are hsp90, hsp70, hsp27 and hsp26. The aim of this master thesis is: a) to study the normal and heat-induced regulation of the medfly’s hsp90 gene and b) to study the expression of this gene under cold shock conditions. For the first aim we created transgenic flies which they had the lusiferase gene (as a reference gene) under the control of different 5’ upstream regions of hsp90 gene. The analysis of these transgenic strains showed that the region between the first transcription base pair and -3536 base pair is crucial for the expression of the gene under normal and heat shock conditions. In order to examine whether the low temperature (0 oC) has any effect in the medfly’s viability and the expression of hsp90 gene, male groups were exposed at different intervals at 0 oC and then it was examined the viability and the expression of the gene. Our results suggest that the low temperature increases the expression of hsp90 nearly to the same levels of the thermal stress and this temperature does not affect the viability of the insect.

Η απόκριση των κυττάρων στο στρες συνδέεται με την επαγωγή μιας ομάδας πρωτεϊνών που ονομάζονται θερμοεπαγόμενες πρωτεΐνες (Hsps). Οι πρωτεΐνες αυτές ανήκουν στην κατηγορία των κυτταρικών συνοδών μορίων (chaperons) και προστατεύουν τα κύτταρα από τη μετουσίωση και συσσωμάτωση των πρωτεϊνών τους. Οι περισσότερες Hsps συντίθενται και σε φυσιολογικές συνθήκες και παίζουν σημαντικούς ρόλους στην ορθή αναδίπλωση, μεταφορά και αποικοδόμηση των πρωτεϊνών του κυττάρου. Οι Hsps κατηγοριοποιούνται σε διακριτές οικογένειες σύμφωνα με τα μοριακά τους βάρη. Από τις οικογένειες αυτές η οικογένεια των μικρών Hsps (sHsps), με μοριακά βάρη12-43 kDa, χαρακτηρίζονται από την παρουσία μιας πολύ συντηρημένης επικράτειας της α-κρυσταλλίνης στο καρβoξυτελικό τους άκρο. Μία από τις πιο καλά μελετημένες sHsps είναι η Hsp27. Η Hsp27, εκτός από το κύριο ρόλο της ως μοριακού συνοδού, εμπλέκεται και σε άλλες κυτταρικές λειτουργίες όπως η διαφοροποίηση, η απόπτωση, ο σχηματισμός του κυτταροσκελετού και η ρύθμιση της οξειδωτικής ισορροπίας των κυττάρων. Από τα αποτελέσματα της παρούσας εργασίας φάνηκε ότι το γονίδιο Cchsp27 εκφράζεται κάτω από φυσιολογικές συνθήκες κατά την ανάπτυξη της μεσογειακής μύγας και ότι η έκφραση του ρυθμίζεται στα διάφορα αναπτυξιακά στάδια του εντόμου. Πειράματα στον εγκέφαλο, στις ωοθήκες και στους όρχεις ενηλίκων ατόμων έδειξαν ότι στον εγκέφαλο των αρσενικών ατόμων το Cchsp27 mRNA παρουσιάζει σημαντικά υψηλότερα επίπεδα έκφρασης συγκριτικά με τον εγκέφαλο των θηλυκών ατόμων. Ακόμη παρατηρήθηκαν υψηλότερα επίπεδα έκφρασης του Cchsp27 mRNA στους όρχεις από ότι στις ωοθήκες. Τα αποτελέσματα από την έκφραση του γονιδίου στους σιελογόνους αδένες προνυμφών έδειξαν υψηλά επίπεδα έκφρασης στους σιελογόνους αδένες 24 ώρες πριν την εκτίναξη των προνυμφών, ενώ σταδιακά η έκφραση μειώνεται μέχρι το στάδιο της εκτινασσόμενης προνύμφης. Η έκφραση του Cchsp27 mRNA αυξάνεται στο στάδιο του λευκού προβομβυκίου ενώ στη συνέχεια σταδιακά μειώνεται. Ύστερα από την καλλιέργεια σιελογόνων αδένων παρουσία εκδυσόνης φάνηκε ότι συμβαίνει καταστολή της έκφρασης γονιδίου σε υψηλές συγκέντρωσης της ορμόνης, ενώ παρατηρείται επαγωγή σε συγκέντρωση 10-6 M. Σε ότι αφορά στην καλλιέργεια ωοθηκών και όρχεων παρουσία εκδυσόνης, παρατηρήθηκε μέγιστη επαγωγή του Cchsp27 γονιδίου στις ωοθήκες στα νεοεκκολαπτόμενα θηλυκά, ενώ μέγιστη επαγωγή στους όρχεις παρατηρήθηκε στα αρσενικά άτομα δύο ημερών. Ακολούθως, δημιουργήθηκε ένα διαγονιδιακό στέλεχος που φέρει το υβριδικό γονίδιο hsp27-GFP υπό τον έλεγχο της ευρύτερης 5΄ περιοχής του hsp27 γονιδίου, με σκοπό την μελλοντική μελέτη της κυτταρικής και ενδοκυτταρικής κατανομής της πρωτεΐνης Hsp27 κατά την ανάπτυξη της μεσογειακής μύγας. Τέλος, μελετήθηκε η έκφραση των γονιδίων Cchsp27 και Cchsp23 σε συνθήκες ψυχρού στρες. Και τα δύο γονίδια έδειξαν σημαντική έκφραση αυξανόμενου χρόνου επώασης στους 0 οC, με το Cchsp27 να παρουσιάζει υψηλότερα επίπεδα έκφρασης σε σχέση με το Cchsp23. / The stress response in cells is connected with the induction of heat shock proteins (Hsps). The Hsps are part of the molecular chaperons system and their role is to protect the cells from the protein denaturation and aggregation. Most Hsps are produced under non-stress conditions and they play a significant role in the correct folding, transmission and degradation of the cell’s proteins. Hsps are being divided into families according to their molecular mass. One of these families is the small heat shock proteins (sHsps) with molecular mass 12-43 kDa. This family is being characterized with a highly conservative domain called α-crystallin, which is located in the C-terminal domain. One of the most well studied sHsp is Hsp27. Hsp27 has an important role as molecular chaperone but also it is implicated in other events such differentiation, apoptosis, cytoskeleton’s formation and the regulation of the oxidized balance of the cell. The results that had arisen from this project, have shown that the Cchsp27 gene is expressed under non-stress conditions during the medfly’s development and that its expression is being regulated during the insect’s developmental stages. Experiments that were performed in brain, ovaries and testes which were removed from individual adults, have shown that the Cchsp27 gene is expressed highly in the male brain and in testes, comparing with female brain and ovaries respectively. Experiments that were performed in larvae salivary glands have shown significant expression levels of the Cchsp27 gene, 24 hours before jumping stage. The expression levels of the Cchsp27 gene are being reduced until the jumping stage and then they increased in the white pupa stage. The expression levels of the Cchsp27 gene are being reduced for the next three stages. The salivary glands were incubated with the hormone ecdysone and the results from these cultures have shown that in high ecdysone concentrations there is a repression in the gene’s expression whereas in concentration of 10-6 M there has been an induction. Ovaries and testes were incubated also with ecdysone and the results indicate that the maximum Cchsp27 gene induction happens in ovaries that have been removed from newborn females, and in testes that have been removed from males of the age of two days. Also, part of this project was the construction of a transgenic strain that it carries the hybrid gene hsp27-GFP under the regulation of the 5΄upstream region of the hsp27 gene. This strain will be used in the future for the study of the cellular distribution of the Hsp27 protein during the medfly’s development. For the last part of this project we study the expression levels of Cchsp27 and Cchsp23 genes under cold shock conditions. Both genes are expressed and their expression levels are being raised as the time of incubation increases in 0 οC. The expression levels of Cchsp27 gene were higher in comparison with the expression levels of Cchsp23 gene.

A variety of genes that influence aging have been identified in a broad selection of organisms including Saccharomyces cerevisiae (yeast), Caenorhabditis elegans (worms), Drosophila (fruit flies), Macaca Mulatta (rhesus monkeys), and even Homo sapiens. Many of these genes, such the TOR’s, FOXO’s, AKT’s, and S6K’s are conserved across different organisms. All of these genes participate in nutrient sensing networks. Other conserved genetic networks may similarly affect lifespan. In this thesis, I explored genes from an iron metabolism family and a heat shock protein (HSP) gene family that have been identified, but not confirmed, to influence lifespan. Yeast is a reliable model for mitotic (replicative) aging. Using yeast, I tested whether the FET-genes, encoding a family of iron importer-related genes, are required for mitotic lifespan. I also tested whether another family of genes, the yeast SSA HSP70- encoding genes, related to mammalian HSP70s, influence mitotic aging. I primarily used the replicative lifespan (RLS) assay, in which I measured the mitotic capacity of multiple FET and SSA yeast mutants. I hypothesize that aging occurs when iron transport is misregulated, which may lead to an over-reliance on HSPs for lifespan maintenance. The results presented in this thesis support the hypothesis. First, FET3 was primarily involved in lifespan maintenance under normal conditions (2% glucose), while FET5 was primarily involved in the cellular lifespan extension characteristic of caloric restriction (0.01% glucose), a known anti-aging intervention. In addition, SSA2 appeared to facilitate lifespan maintenance in the absence of FET4, while the presence of SSA1 limited lifespan length. That the aging genes identified in this study are involved in iron metabolism or heat stress suggests that protein aggregation or reactive oxidative species production are common processes through which these genes interact.

Thermal scavenging is a unique behavior restricted to a few desert ant genera. Workers are among the most thermotolerant land animals known to this day, being able to survive body temperatures of sometimes more than 50°C for several minutes. Making use of their remarkable heat-hardiness, they search for food in plain day, a feat that other desert creatures cannot accomplish. They mostly feed on the corpses of heat-stricken, less tolerant arthropods that were unable to survive the blazing sun of the midday desert. Thermal scavenging has evolved independently at least three times in distantly related genera, geographical well segregated inside the different deserts of the world. First, the Cataglyphis genus ranges from the Sahara Desert and extends its distribution to reach minor Asia through the Mediterranean Basin. Second the Ocymyrmex genus can be found in the Namib and Karoo deserts of southern Africa, extending its range to eastern Africa savanna plains. Finally, the Melophorus genus can be found in Australia, with thermal scavenging species distributed in the central desert of the outback region.While this impressive behavior was already well-described by the start of this PhD project, little was known about the mechanisms supporting the remarkable heat-tolerance of workers. Using biophysical and physiological approaches in Cataglyphis and Ocymyrmex, we’ve been able to pinpoint key aspects underlying stress tolerance in those genera. First, from a biophysical standpoint, the Sahara silver ant Cataglyphis bombycina is covered with a unique and dense array of prismatic hairs reflecting visible wavelengths by total internal reflection. This allows reflection of up to 50% of the incident sunlight energy, thus shifting down the ant’s thermal equilibrium and sparing its body a few critical degrees. Second, in a comparative framework, we found numerous genes involved with critical cellular processes to be constitutively expressed or strongly up-regulated to heat in thermal scavenging ants, while their orthologs were not in mesophilic species. Those processes, such as molecular chaperoning, cell-cycle regulation, energy metabolism and muscular functions are keys that allow those ants to meet the higher requirement needed to scavenge for food at both stunning speed and under extreme heat-pressure. Overall, this work investigates the physiological and biophysical basis enabling thermal scavenging ants to survive extreme heat conditions. It provides a deeper understanding of cellular heat-tolerance pathways in non-model animals and contribute to our knowledge of life’s adaptation to extreme conditions. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.

Lysosomal storage disorders (LSDs) comprise a broad disease group of inherited metabolic disorders, the majority of which are associated with CNS pathology, significant disability and reductions in life expectancy. LSDs are caused by mutations in genes encoding proteins critical for the correct functioning of lysosomal homeostasis. The disruption of lysosomal homeostasis results in the abnormal accumulation of lysosomal content, initiating intracellular pathological events, including calcium dysregulation and lysosomal membrane permeablisation (LMP) affecting cell function and inducing cellular death mechanisms. These pathological events are particularly damaging within the CNS, due to its limited capacity for regeneration. Despite intensive scientific research into these disorders, and an increased understanding of the pathological events underlying these diseases, effective treatments are still lacking for most LSDs. Several therapeutic approaches have been investigated in the last 30 years, including enzyme replacement therapy, bone marrow transplantation, substrate reduction therapy, chemical chaperones and gene therapy. However, the CNS pathology in many of the LSDs remains unaddressed due to the restricted ability of many therapeutic agents to cross the blood-brain barrier. The heat-shock response (HSR) is an emerging element involved in the pathogenesis of a variety of disorders. The HSR is a physiological response to a wide range of cellular stresses. It functions to protect the cell from the aggregation of misfolded proteins and LMP. Of the HSR, several key players are integral to mounting a heat shock response, these include the heat-shock factor 1 (HSF-1) and HSP70. In this thesis, we provide proof-of-principle for the use of recombinant HSP70, and the small molecule up-regulator of the HSR, arimoclomol in treatment of a range of LSDs. We show that HSP70 is able to access the CNS, and increase the degradative capacity of lysosomal hydrolases. This provides differential behavioural, biochemical and survival effects in LSD models of Niemann-Pick type C, Sandhoff and Fabry disease. Additional studies using the HSF-1 upregulator arimoclomol, show a complex dose-response between the different models, possibly reflecting essential differences in the calcium dysregulation between these disease states.

Nosso objetivo, neste trabalho, foi verificar se a interação das células dendríticas (DCs) com antígenos da Hsp60 induz um efeito sinérgico no direcionamento de uma resposta imune reguladora, no sistema humano. Células dendríticas humanas maduras (mDC) e imaturas (iDC e iDC IL-10) foram geradas, in vitro, a partir de monócitos de 15 de indivíduos saudáveis. Estas células foram caracterizadas quanto à (i) morfologia, (ii) imunofentotipagem, (iii) produção de citocinas e, (iv) capacidade de estimular aloproliferação. Analisamos a auto-reatividade de linfócitos T (LT) dirigida a diferentes DCs (mDC, iDC e iDC IL-10). Na interação de antígenos da Hsp60 com essas diferentes DCs, verificamos: (i) a capacidade de induzir a produção de citocinas pelas DCs e de inibir a sua produção espontânea, (ii) a auto-reatividade de linfócitos T dirigida a esses antígenos (proliferação e produção de citocinas), (iii) a expressão gênica de um painel de moléculas reguladoras (TGFb, receptor de TGF-b, IL-10 e GATA3) e inflamatórias (IFNg, TNF-a e T-bet) em linfócitos, T no contexto de células dendríticas imaturas. As mDC apresentaram expressão de CD83, maior expressão de CD80, e CD86, assim como induziram respostas alogenéicas mais intensas do que as DCs imaturas. Apesar de haver variabilidade na produção de citocinas, apenas as DC imaturas produziram espontaneamente IL-10, e as DCs maduras produziram mais freqüentemente IFN-g e TNF-a. Analisando o efeito dos antígenos da Hsp60 sobre a produção de citocinas, observamos tanto indução quanto inibição da produção de IFN-g, TNF-a, IL-4 e IL-10 nos três grupos de DC. Porém, a inibição predominou sobre a produção nos três grupos de DC. A auto-reatividade proliferativa de LT dirigida às diferentes DCs foi mais freqüente nas culturas com as DCs maduras (6/10) do que com as DCs imaturas (4/10). Também detectamos produção das citocinas IFN-g, TNF-a, e IL-2 para todos os grupos de células, porém, mais freqüentemente na auto-reatividade contra as DCs maduras. Diversos antígenos da Hsp60 foram capazes de inibir esta auto-reatividade. O peptídeo N7 teve um efeito dominante na inibição da auto-reatividade proliferativa de linfócitos T dirigida às mDCs. A auto-reatividade a antígenos da Hsp60, de um modo geral, foi maior com as DCs imaturas. Diversos antígenos foram capazes de induzir proliferação e produção de citocinas. Todavia, o peptídeo C3 foi imunodominante (6/10) na indução de resposta linfoproliferativa, no contexto das iDCs. A expressão gênica de moléculas reguladoras e inflamatórias foi verificada em linfócitos T, na auto-reatividade a antígenos da Hsp60. Observamos modificações importantes de praticamente todas as moléculas estudadas. Verificamos um predomínio de modificações reguladoras para os genes TGFb, TGF-bR, GATA3, TNF-a e T-bet. O peptídeo N7 induziu modificações dominantemente reguladoras em todas as condições em que ele foi testado. Em conclusão, verificamos que antígenos da Hsp60 têm efeito direto na produção de citocinas das diferentes DCs. Também têm a capacidade de ativar, simultaneamente, em linfócitos T, na interação com as células dendríticas, genes funcionalmente antagônicos. Isto reafirma a diversidade funcional da Hsp60. Ademais, identificamos o peptídeo N7 como potencialmente imunorregulador e o consideramos um candidato a ser testado em protocolos para indução de tolerância. / The aim of the present study was to determine whether the interaction of dendritic cells (DCs) with antigens derived from Hsp60 is capable of inducing a synergistic effect in directing a regulatory immune response, using a human system. Human DCs with mature (mDC) and immature (iDC and iDC IL-10) phenotype were generated in vitro from monocytes obtained from 15 healthy subjects. These cells were characterized according to (i) morphology, (ii) expression of surface markers, (iii) cytokine production, and (iv) ability to stimulate alloproliferation. We analyzed the autoreactivity of T lymphocytes (TL) directed against different DC types (mDC, iDC, and iDC IL-10). For the interaction of Hsp60 antigens with these different DCs, we determined: (i) the ability to induce cytokine production by DCs as well as to inhibit their spontaneous production, (ii) the autoreactivity of TL to these antigens (proliferation and cytokine production), and (iii) gene expression levels of a panel of regulatory (TGFb, TGF-b receptor, IL-10, and GATA3) and inflammatory (IFN-g, TNF-a, and T-bet) molecules by TL when stimulated by mDC. mDC expressed CD83 and showed higher levels of CD80 and CD86 and induced stronger allogeneic responses than immature DCs. Although cytokine production varied, only immature DCs spontaneously produced IL- 10, and mature DCs more frequently produced IFN- and TNF-. An analysis of the effects of Hsp60 antigens on cytokine production showed both induction and inhibition of production of IFN-g, TNF-a, IL-4, and IL-10 by the three sets of DCs; however, inhibition predominated over induction in all three DC groups. The proliferative autoreactivity of LT directed towards the different DCs was more frequent in cultures containing mDCs (6/10) than in those containing immature DCs (4/10). We also detected production of IFN-g, TNFa, and IL-2 by all groups of cells; however this was more frequent in the context of autoreactivity against mDCs. Several Hsp60 antigens were capable of inhibiting this autoreactivity. Peptide N7 had a dominant effect on the inhibition of the proliferative autoreactivity of LT directed towards mDCs. Autoreactivity to Hsp60 antigens was generally greater in cultures containing immature DCs. Several antigens were capable of inducing proliferation and cytokine secretion. However, peptide C3 was immunodominant (6/10) in the induction of a lymphoproliferative response in cultures containing iDCs. Gene expression of regulatory and inflammatory molecules was determined in LTs in the context of autoreactivity to Hsp60 antigens. There were important modifications in virtually all molecules studied. There was a predominance of regulatory-oriented changes in expression of TGFb, TGF-bR, GATA3, TNFa, and T-bet. Peptide N7 induced dominantly regulatory changes in gene expression in all conditions in which it was tested. In conclusion, we have shown that Hsp60 antigens have a direct effect on cytokine production by different DCs. These antigens are also able to activate, during the interaction of LT with DCs, genes that are functionally antagonistic. This finding reinforces the functional diversity of Hsp60. Furthermore, we have identified peptide N7 as potentially immunoregulatory, and consider it as a candidate to be tested in protocols for the induction of tolerance.

Orientadores: Selma Giorgio, Fernanda Ramos Gadelha / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T14:07:59Z (GMT). No. of bitstreams: 1 Codonho_BarbaraSantoni_M.pdf: 2501497 bytes, checksum: d7ceeef1653b2df4a7d52b28ebd16543 (MD5) Previous issue date: 2014 / Resumo: As leishmanioses são um conjunto de doenças causadas pelo protozoário do genêro Leishmania, que atingem milhões de pessoas por ano. O tratamento é realizado primeiramente com antimoniais pentavalentes e, em casos de resistência, são indicadas a pentamidina ou anfotericina B. Todos estes fármacos são tóxicos e induzem efeitos colaterais nos pacientes. Devido a dificuldades no tratamento, o estudo de moléculas presentes no parasita se torna importante. Dentre essas, as heat shock proteins 70 (HSP70) são proteínas essenciais para o ciclo de vida da Leishmania. Durante a passagem do vetor para o hospedeiro vertebrado, o parasita encontra vários tipos de estresses que induzem a uma maior expressão da HSP70. Nesse projeto avaliou-se os efeitos da superexpressão da HSP70 em Leishmania (Leishmania) amazonensis, comparando-se parasitas que superexpressam a proteína HSP70 (pTEX-HSP70) com parasitas contendo somente o vetor (pTEX). Os resultados mostraram que os promastigotas transfectados pTEX e pTEX-HSP70 apresentaram vários aspectos ultraestruturais semelhantes aos não transfectados (WT), porém mostraram ser maiores e com o tamanho da área nuclear maior. A superexpressão da proteína HSP70 conferiu aos parasitas uma fase estacionária de proliferação mais estendida do que a observada em parasitas pTEX. Uma maior resistência e capacidade proliferativa foram observadas nos parasitas pTEX-HSP70 quando submetidos a diferentes condições de estresses (tratamentos com H2O2, choque térmico e ambiente hiperbárico), em relação a parasitas pTEX. Os resultados também mostraram que parasitas pTEX e pTEX-HSP70 infectam culturas de macrófagos peritoneais e macrófagos humanos derivados de sangue periférico, em taxas (% de infecção e número de amastigotas/macrófago) semelhantes a de parasitas WT. O processo de infecção em camundongos BALB/c mostrou que o tamanho da lesão induzida pelos parasitas pTEX e pTEX-HSP70 na pata foi diferente nas primeiras semanas, mas semelhante no curso final da infecção. Adicionalmente, as cargas parasitárias nas lesões dos camundongos BALB/c infectados com os parasitas pTEX e pTEX-HSP70 foram semelhantes, mas maiores que as cargas parasitárias nas lesões induzidas por WT. Além disso, os baços dos camundongos infectados com os parasitas pTEX e pTEX-HSP70 apresentaram visceralização. Ensaios da bioenergética destes promastigotas mostraram que parasitas pTEX-HSP70 apresentam maiores taxas de consumo de O2 do que parasitas pTEX, apesar de apresentarem produção de ATP semelhante. A produção de superóxido nos parasitas pTEX-HSP70 e pTEX foram similares, apesar da liberação de H2O2 ser bem inferior nos de parasitas pTEX-HSP70. Os resultados obtidos indicam que a superexpressão da proteína HSP70 protege a L.(L.) amazonensis de situações de estresse imediato, mas não interfere com a sua capacidade infectiva / Abstract: Leishmaniasis are a group of diseases caused by the protozoan genus Leishmania, which affect millions of people each year. The treatment is performed primarily with pentavalent antimony and resistance cases are indicated pentamidine or amphotericin B. All these drugs are toxic and induce side effects in patients. Due to difficulties in treatment, the study of molecules present in the parasite becomes important. Among these, the heat shock protein 70 (HSP70) proteins are essential for the life cycle of Leishmania. During the transition from vector to vertebrate host, the parasite finds various types of stresses that induce a higher expression of HSP70. In this project was evaluated the effects of overexpression of HSP70 in Leishmania (Leishmania) amazonensis, comparing parasites that overexpressing HSP70 (pTEX-HSP70) protein with parasites containing the empty vector (pTEX). The results showed that transfected promastigotes pTEX and pTEX-HSP70 showed several similar ultrastructural aspects similar to promastigotes of L.(L.) amazonensis untransfected (WT), but proved to be larger and the size of the largest nuclear area. Overexpression of HSP70 protein gave the parasites a stationary phase of proliferation more extended than that observed in parasites pTEX. Higher strength and better proliferative capacity were observed in parasites pTEX-HSP70 when submitted to different stress conditions (hydrogen peroxide, heat shock treatments and hyperbaric environment), in relation to parasites pTEX. The results also showed that pTEX and pTEX-HSP70 parasites infect cultures of peritoneal macrophages and human peripheral blood-derived macrophages in rate (% infection and the number of amastigotes / macrophage) similar to WT parasites. The process of infection in BALB/c mice showed that the size of the induced parasitic pTEX and pTEX-HSP70 foot injury was different in the first few weeks but similar in the final course of infection. Additionally, parasitic loads on the lesions of BALB/c mice infected with pTEX and pTEX-HSP70 parasites were similar, but larger than the parasitic loads in lesions induced by WT. Moreover, spleens from infected with pTEX and pTEX-HSP70 parasites mice showed visceralization. Assays of bioenergetics promastigotes showed that these pTEX-HSP70 parasites consume more O2 than pTEX parasites, despite showing similar ATP production. Superoxide production in parasites pTEX and pTEX-HSP70 were similar, despite the release of hydrogen peroxide is considerably lower than pTEX-HSP70 parasites. The results indicate that overexpression of HSP70 protein protect L.(L.) amazonensis in immediate situations of stress, but does not interfere with its infective capacity / Mestrado / Imunologia / Mestra em Genética e Biologia Molecular

M.Sc. / Field grown plants are constantly challenged with a variety of stressful factors, such as high temperatures, drought and pathogen infection that adversely affect crop production and quality. These stresses seldom occur as single entities in plants and in warm climates, heat stress is often a common dominator in combinatorial stress. The heat shock (HS) response in plants has priority over other stress responses, including the pathogen-induced stress response. Activation of the HS response prevents the normal plant defence strategy, leaving the plant vulnerable to pathogen attack. However, prior exposure to elevated temperatures confers protection from subsequent, otherwise lethal, temperatures (thermotolerance) and a variety of other stress conditions including heavy-metals, chilling injury and certain pathogens (cross tolerance). In general, litterature supports a central role for heat shock proteins (HSP), in particular the 70 kDa HSP (Hsp70), in thermotolerance. Incompatible host-pathogen interactions lead to the activation of an array of defence mechanisms, including the promotion of phenylpropanoid metabolism. Phenylalanine ammonia-lyase is a key regulator of this metabolic pathway, influencing the production of salicylic acid, lignin and phytoalexins among other essential defence products. In this study it was hypothesised that prior exposure to non-lethal HS confers protection from subsequent heat-related suppression of the phenylpropanoid pathway, induced as a defence mechanism during an incompatible plant-pathogen interaction. This hypothesis was verified by analysing the effect of thermotolerance on pathogen-related stimulation of PAL promoter activity, enzyme activity and lignin deposition. The tomato, Lycopersicon esculentum cultivar UC82B and Ralstonia solanacearum, the causative agent of bacterial wilt, were used as host-pathogen model. Specific objectives in the study were: (1) Development of PAL promoter-GUS reporter transformed Lycopersicon esculentum. (2) Establishment of a thermotolerance protocol that ensures optimal Hsp70 levels at subsequent HS. (3) Evaluation of the influence of prior heat treatment on phenylpropanoid metabolism after exposure to HS in combination with Ralstonia solanacearum. Results obtained support the hypothesis indicating that thermotolerance protects phenylpropanoid metabolism, in particular PAL promoter and enzyme activity, and to a certain extent lignin production, induced by avirulent Ralstonia solanacearum during a second severe HS. In contrast, HS without a prior heat treatment, suppressed phenylpropanoid metabolism. The protective potential of prior heat treatment during subsequent infection under hyperthermic conditions support the application of HSP in the development of novel plant protection strategies.

M.Sc. / Plants are in constant conflict with pathogens and have evolved intricate mechanisms to protect themselves against pathogens. The gene-for-gene response is regarded as the first line of defence when plant and pathogen meet. This interaction leads to the induction of defence proteins such as PR proteins that protect the plant from invading pathogens. A seemingly unrelated topic to plants and pathogens is heat shock proteins (HSP). HSP are a highly conserved group of defence proteins induced in all organisms in response to a variety of environmental stresses to provide protection from, and adaptation to cellular stress. HSP are in general not considered to be part of the defence response classically induced by avirulent pathogens and whether they are induced and play a role in plant-pathogen interactions is controversial. The protective chaperoning capacity of HSP makes them ideal proteins to exploit to target as endogenous defence proteins in the search for new strategies in the management of infectious diseases. In humans, HSP induction during infection is a complex phenomenon depending on the pathogen, whether the infection is acute or chronic, the host cell type and its differentiative state as well as environmental factors. In this investigation the expression of the inducible and constitutive isoforms of the 70kDa HSP (Hsp70/Hsc70) was investigated in tomato, Lycopersicon esculentum in response to virulent and avirulent strains of Ralstonia solanacearum, the causative agent of bacterial wilt. Expression of Hsp70 was studied in conjunction with the accumulation of PR-la and host cell viability. A quick, non-toxic, tetrazolium-based assay was developed from the Alamar Blue assay, commonly used in mammalian cells, and applied for the evaluation of host cell viability. The results shown suggest Hsp70/Hsc70 is significantly induced in tomato cell suspensions during an incompatible interaction 24h to 48 h following co-cultivation with the avirulent R. solanacearum strain compared to normal levels at this interval in cells exposed to the virulent strain. In both compatible and incompatible interactions Hsp70/Hsc70 levels eventually (72 h) accumulated correlating significantly with decreased viability. PR-la accumulation was significantly induced from 6 h to 18 h by the virulent as well as the avirulent R. solanacearum strains. In general, comparable results were obtained using leaf discs as an in vivo model. Based upon the differential induction of Hsp70/Hsc70 by virulent and avirulent pathogens it is proposed that HSP may play an important role in determining the outcome of the interaction between tomato and R. solanacearum. Successful defence may not only involve a limited number of defence genes but may result from a concerted action of a large number of defence genes.