Yes / Functional genomics research can give us valuable insights into bacterial gene function. RNA Sequencing (RNA-seq) can generate information on transcript abundance in bacteria following abiotic stress treatments. In this study, we used the RNA-seq technique to study the transcriptomes of the opportunistic nosocomial pathogen Pseudomonas aeruginosa PAO1 following heat shock. Samples were grown at both the human body temperature (37 C) and an arbitrarily-selected temperature of 46 C. In this work using RNA-seq, we identified 133 genes that are differentially expressed at 46 C compared to the human body temperature. Our work identifies some key P. aeruginosa PAO1 genes whose products have importance in both environmental adaptation as well as in vivo infection in febrile hosts. More importantly, our transcriptomic results show that many genes are only expressed when subjected to heat shock. Because the RNA-seq can generate high throughput gene expression profiles, our work reveals many unanticipated genes with further work to be done exploring such genes products. / University of Malaya High Impact Research (HIR) UM-MOHE HIR Grants (UM.C/625/1/HIR/MOHE/CHAN/14/1, No. H-50001-A000027; UM.C/625/1/HIR/MOHE/CHAN/01, No. A000001-50001); PPP Grant (PG081-2015B)

Temperature is a fundamental environmental force shaping species abundance and distributions through its effects on biochemical reaction rates, metabolism, activity, and reproduction. In light of future climate shifts, mainly driven by temperature increases, how will organisms persist in warmer environments? One molecular mechanism that may play an important role in coping with heat stress is the heat shock response (HSR), which protects against molecular damage. To prevent and repair protein damage specifically, Hsps activate and become up-regulated. However, the functional diversity and relevance of heat shock proteins (Hsps) in extending upper thermal limits in taxonomic groups outside marine and model systems is poorly understood. Ants are a good system to understand the physiological mechanisms for coping with heat stress because they have successfully diversified into thermally stressful environments. To identify and characterize the functional diversity of Hsps in ants, I surveyed Hsp orthologues from published ant genomes to test for signatures of positive selection and to reconstruct their evolutionary history. Within Hymenoptera, ants utilize unique sets of Hsps for the HSR. Stabilizing selection was the prevailing force among Hsp orthologues, suggesting that protein activity is conserved. At the same time, regulatory regions (promoters) governing transcriptional up-regulation diversified: species differ in the number and location of heat shock elements (HSEs). Therefore, Hsp expression patterns may be a target for selection in warm environments. I tested whether Hsp expression corresponded with variation in upper thermal limits in forest ant species within the genus Aphaenogaster. Whole colonies were collected throughout the eastern United States and were lab acclimated. There was a positive relationship between upper thermal limits (Critical Thermal maxima, CTmax) and local temperature extremes. Upper thermal limits were also higher in ant species that lived in open habitats (shrub-oak and long-leaf pine savannah) than species occupying closed habitats (deciduous forest). Ant species with higher CTmax expressed Hsps more slowly, at higher temperatures, and at higher maximum levels than those with low CTmax. Because Hsps sense and repair molecular damage, these results suggest the proteomes of open relative to closed canopy forests are more stable. Although deciduous forest ant species may be buffered from temperature stress, it is likely that temperature interacts with other environmental stressors such as water and nutrient availability that may impact upper thermal limits. I measured the influence of dehydration and nutrition stress on upper thermal limits of forest ants from a single population. Ants that were initially starved were much less thermally tolerant than controls and ants that were initially desiccated. Because ants are likely to experience similar combination of stressors in the wild, upper thermal limits may be severely overestimated in single factor experiments. Therefore, realistic forecasting models need to consider multiple environmental stressors. Overall, adaptive tuning of Hsp expression that reflects better protection and tolerance of protein unfolding may have facilitated ant diversification into warm environments. However, additional stressors and mechanisms may constrain the evolution of upper thermal limits.

Ascaris suum were exposed to a number of stressors, including heavy metals and both high (40°C) and low (18°C) temperatures. The 70kD and 90kD heat shock proteins (HSPs) in the different A. suum tissues were analyzed by Western blot and quantitated by Macintosh Image Program.

In another study, hyperthermic treatment was applied to study whether HSP27 expression would be induced in un-injured RGCs, and whether this treatment performed after axotomy would have effects on HSP27 expression, RGC survival and/or regeneration into PN graft. Brief duration of heat shock that elevate the body temperature to 42°C did not up-regulate HSP27 in normal retina. About 8-10% increase in RGC survival in the hyperthermia group was observed compared to those received a 37°C treatment at one week post-axotomy and it depended on the number of post-injury heat treatments applied. At the same time, the number of HSP27-RGCs was also doubled, although the same increase occurred was irrespective of the number of hyperthermic treatments. Multiple heat shock application also significantly enhanced RGC regeneration into PN graft through increased the number of HSP27 regenerating RGCs. These results suggest that post-injury hyperthermic treatment enhance HSP27 induction in RGCs and lead to their successful regeneration into the PNG, whereas further studies are necessary to determine whether the protective effect on survival by heat shock is due to the increase in a subset of HSP27-RGCs. (Abstract shortened by UMI.) / In the second study, different neurotrophic factors were injected into the vitreous to enhance RGC survival and/or regeneration. Brain-derived neurotrophic factor (BDNF) significantly reduced RGC death transiently at 14 days after ON cut, but the expression of HSP27 was reduced compared to bovine serum albumin-injected controls. In peripheral nerve (PN)-grafted retina, BDNF suppressed RGC regeneration via reducing the number of HSP27-RGCs regenerating into the PN graft. In ciliary neurotrophic factor (CNTF)-injected group, although there was only a 10% increase in RGC survival, a 5-fold drastic increase in the number of RGCs which expressed HSP27 was observed, and some of these were found to undergo intra-retinal sprouting similar to VPN-transplanted retina. Combined treatment of intra-vitreal CNTF injection with PNG resulted in a 5 fold-increase in the number of regenerating RGCs as well as increasing the proportion of cells which expressed HSP27 from about 60% to about 80%. The data indicates that HSP27 participates in axonal regrowth especially under synergistic interaction of CNTF and PNG. Intra-vitreal injection of hepatocyte growth factor (HGF) significantly sustained RGC survival compared to BDNF at 28 days after axotomy, but the HSP27 expression in RGCs did not change correspondingly. In the PN-ON grafted retina, HGF promoted more RGCs regenerate without altering the number of HSP27-RGCs regrowing into the PNG. Such results indicate that some trophic factors can specifically enhance or suppress RGC regeneration by modulating HSP27 expression, while other trophic factors promote regeneration which is independent to HSP27. Therefore, it suggests that RGCs may regenerate through at least two different mechanisms. / In this study, the detailed in vivo expression of HSP27 in retinal ganglion cells (RGCs) of golden hamster following axotomy and regeneration stimulated by peripheral nerve grafting and neurotrophic factors have been examined. / In whole-mount normal retinas, HSP27 was constitutively expressed in astrocytes and blood vessels, but not in RGCs. Three days after optic nerve (ON) transection, a small subset of surviving RGC began to express HSP27, the number of which peaked at 7 days and dropped to a minimal level at two weeks post-axotomy. When axotomy was done more proximally to their cell bodies, RGCs survival was significantly decreased but HSP27 expression did not change. This suggests the HSP27 expression does not correlate with cell survival after axonal injury. When a viable peripheral nerve (VPN) was transplanted intravitreally into the eye after ON cut, it induced intra-retinal sprouting of RGCs. Although it did not promote RGC survival, VPN prolonged HSP27 expression up to 56 days after surgery and significantly increased the number of HSP27-RGCs. This protein was localized in the cell body, and especially, in dendritic sprouts and growth cones, indicating that it was transported to active growing sites where it may have a functional role associated with regenerative sprouting. / Wong, Wai Kai. / Adviser: Eric Cho. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3270. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 159-198). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

糖調節蛋白78 (Glucose-regulated protein 78),也稱之Bip,是70kDa熱休克蛋白家族成员之一。已有的研究表明，Bip 是一個具有多功能的蛋白，參與眾多的生物調控過程，包括蛋白折疊，調節鈣平衡，以及作為內質網緊張（ER stress) 的感應器。有研究表明，Bip可以在細胞膜上定位，作為Nodal信號通路的一個輔助受體發揮作用。大量的研究表明，Bip在疾病和代謝方面也發揮重要作用。它參與胰島素的生物合成，並可以提高長期高血糖下β細胞的功能。同時具有抗細胞凋亡的作用。然而Bip在胚胎髮育中的生物功能卻知之甚少。 / 高等脊椎動物腎臟發育中經歷形成3種腎臟形式：前腎，中腎和後腎。腎單位是這3種形式的基本結構和功能單位。在兩棲類，前腎在胚胎時期發揮作用，在胚胎的兩側各只有一個腎單位。這使得爪蟾成為前腎研究的一個非常好的模型。 / 在此項研究中，我們採用非洲爪蛙作為動物模型來研究Bip在胚胎髮育過程中，尤其是在前腎發育中的生物功能。Bip是一個母性因子，在尾芽期，Bip 表達在粘液腺，前腎，肝以及耳囊。 Bip在前腎清晰明確的表達，表明Bip可能在前腎的發育中發揮作用。我們利用BipMO來進行敲低功能實驗，免疫印記顯示BipMO能阻斷帶Flag標記Bip的翻譯。通過原位雜交技術檢測前腎的不同標記基因的表達發現，敲低Bip抑制前腎的形成，表明Bip的正常表達是前腎發育所必須的。 / 為了研究Bip調節前腎的發育的分子機制， 我們使用Affmetrix基因芯片分析在Bip敲低情況下的不同時期胚胎中基因的表達譜，發現在Bip敲低表達的胚胎中，視黃酸信號通路的一些重要的組分的表達受到抑制。爪蛙胚胎原腸胚的動物帽細胞具有多能性， 使用激活素和視黃酸一同處理動物帽細胞可以誘導其分化成為原腎組織。在此體外分化體系中敲低Bip表達，前腎標記基因表達降低，顯示在這一體外系統中前腎的分化受到抑制。該實驗結果與體內實驗結果一致。在體外培養的HEK293T細胞中敲低Bip，抑制視黃酸處理後視黃酸信號通路螢光素報告的活性。 lhx1是前腎發育早期表達標記之一，對於前腎原基的初始化具有重要的作用，同是它也是視黃酸信號通路的靶基因。共同註射BipMO和lhx1表明，前腎的異常可以明顯降低，顯示lhx1可以部份拯救由於Bip缺失所造成的腎臟發育缺陷。該實驗表明Bip通過調節視黃酸信號通路，來調控lhx1的表達前腎的形成。我們進一不發現，敲低Bip後，前腎異常形成的區域內，細胞凋亡增加，增殖減少。該結果在細胞水平上解釋了Bip敲低表達時前腎形成異常的一個原因。 / 综述所述，Bip正確表達对胚胎前肾的发育極為重要。它胚胎发育过程中通过視黃酸信号通路調控lhx1的表達，從而对前肾的形成发挥重要作用。 / Glucose-regulated protein 78 (Grp78), also known as Bip, belongs to heat shock protein 70kDa family. It has been implicated in various biological processes including protein folding, regulation of calcium homeostasis, and serving as a sensor of ER (Endoplasmic Reticulum) stress. Moreover, it can localize in cell membrane, acting as co-receptor of nodal signaling. It is essential for insulin biosynthesis. In addition, Bip plays important roles in a number of diseases. For example, BIP can improve β-cell function in the prolonged hyperglycemia. Knockdown of BIP in β-cell can induce apoptosis. However, little is known about its function during embryonic development. / In high vertebrate, three sets of nephric forms develop successively during embryonic kidney development. They are pronephros, mesonephros, and metanephros. Nephron is the basic structural and functional unit of all these three forms. In amphibian, the pronephros performs function at the embryonic stages, which has only one nephron on either side of the body. It makes Xenopus a very good model for pronephros study. / In this study, we took advantage of Xenopus leavis as an animal model to investigate Bip function during embryonic development, especially its role in pronephros development. We first examined the expression of Bip in developing embryos. Whole mount in situ hybridization showed that Bip was expressed in the cement gland, pronephros, liver and ear vesicle during tailbud stages. It was expressed in the pronephros strongly and clearly which suggested that Bip might play roles in pronephros development. We performed loss-of-function experiment by using morpholino oligonucleotide (MO) knock down translation of endogenous Bip expression. Depletion of Bip impaired formation of pronephros revealed by reduction expression of different pronephros maker genes. The pluripotent animal caps can differentiate into pronephros tissue when treated with activin and all-trans retinoic acid (atRA) in vitro kidney induction assay. In line with our in vivo observation, knockdown of Bip inhibited pronephros differentiation that can normally achieved by combined effects of activin and atRA in animal cap assay. / In order to investigate the molecular mechanisms as how Bip regulated pronephros development, we performed Affymetrix DNA microarray assay to generate gene expression profile in Bip morphants. We found that some components of RA signaling were inhibited when Bip was knockdown. Moreover, knockdown of Bip caused reduction of RA target genes expression after treatment with RA. Consistent with above observations, luciferase activities of RA signaling reporter was reduced in HEK293T cells when BIP expression was depleted by RNAi. lhx1 is one of RA target genes and has been implicated playing essential roles in pronephros development. The inhibition of pronephros formation induced by Bip depletion can be partially rescued by co-overpression, suggesting 1) lhx1 is downstream of Bip in the regulatory network of pronephros formation; and 2) Bip regulates pronephros formation through RA signaling via lhx1. We also found increased apoptosis and decreased cell proliferation at pronephros-forming region in Bip morphants. That could explain the reason of pronephros malformation when Bip is downregulated. / Taken together, Bip is essential for pronephros development. It functions through RA signaling during the complex developmental processes. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Shi, Weili. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 125-143). / Abstracts also in Chinese.

Une réponse rapide et adaptée est nécessaire à la survie des cellules soumises à un stress. La réponse cellulaire au stress (HSR pour Heat-Shock response) médié par le facteur de transcription HSF1 est induite par les contextes environnementaux (chaleur, hypoxie, …) et les processus biologiques normaux et pathologiques (vieillissement, inflammation, …) associés à une accumulation de protéines endommagées (Morimoto, 1998). Ces protéines endommagées forment des agrégats toxiques aux conséquences létales pour les cellules.Conservé chez tous les eucaryotes, HSF1 orchestre les actions nécessaires à la survie et à la croissance des cellules malgré le stress. Ses cibles les mieux connues sont les gènes codants pour les Heat Shock Protein (HSP) qui font office de chaperon moléculaire. Une caractéristique de la HSR chez l’Homme est l’accumulation massive du facteur HSF1 en foyers nucléaires nommés Nuclear Stress Bodies (nSBs). Curieusement, ces foyers ciblent l’hétérochromatine péricentrique composée de séquences répétées en tandem de type Satellite III (SATIII), particulièrement au niveau du locus 9q12. HSF1 induit une forte transcription en ARN SATIII Sens (Jolly et al., 2004). Le rôle des nSBs est une des problématiques majeures de notre équipe cependant jusqu’à présent aucune fonction n’a été confirmée pour ces structures.Les nSBs, spécifiques aux cellules humaines, n’ont été décrits que dans des cellules en culture. Mon projet de thèse a consisté dans un premier temps à montrer la présence des nSBs in vivo chez l’Homme. Cette étude, réalisée sur du tissu testiculaire nous a également permis d’identifier une nouvelle cible SATIII majeure pour HSF1, la région Yq12. Dans les testicules, les nSBs sont associés à des processus méiotiques et post-méiotiques, suggérant un rôle dans le remodelage de l’hétérochromatine. Dans un deuxième temps, nous avons cherché à mieux comprendre le rôle des nSBs lors de la HSR. Nous avons pu montrer que l’étape de transcription des SATIII induit une déstabilisation de l’hétérochromatine péricentrique caractérisée par une dissociation des facteurs HP1 (Heterochromatin Protein 1) alpha et beta et une perte de la marque répressive H3K9me3. Au cours de la période de récupération qui accompagne la reformation de l’hétérochromatine, une transcription séquentielle d’ARN SATIII Sens puis Anti-sens précède la restructuration des loci 9q12. Nous avons également pu montrer que la transcription des SATIII est associée à un blocage de la mitose. Nous montrons que dans les cellules stressées, une altération de ce point de contrôle par un Knock down des ARN sat III par des approches LNA conduisent à une l’instabilité génomique des cellules tumorales avec apparition de cellules polynucléées. / A rapid and well-adapted response is required for cell survival upon stress. The cellular stress response (HSR) is mediated by the transcription factor Heat Shock Factor 1 (HSF1) (Morimoto, 1998). It is activated by environmental stress (heat, hypoxia, ...) and by a series of patho-physiological contexts (aging, inflammation, ...) involving protein damages.The best-characterized targets of HSF1 are genes encoding for Heat Shock Protein (HSP) acting as molecular chaperone. A specific feature of the HSR in human cells is the presence of HSF1 nuclear foci named Nuclear Stress Bodies (NSBs). Surprisingly, nSBs target pericentric heterochromatin consisting in tandem repeats of type III Satellite (SATIII) sequences, primarily at the 9q12 locus. HSF1 triggers a strong transcriptional activation of this locus (Jolly et al., 2004). The role of nSBS is a major issue since no function related to these structures has been reported so far.So far, nSBs have been only identified in cells in culture. My thesis project has been to further explore whether these structures also existed in normal tissues. Indeed, we have been able to identify the presence of nSBs in testis where they were found to be associated to meiotic and post-meiotic stages, suggesting a role related to heterochromatin remodeling. Moreover, we have identified the Yq12 locus as a new target of nSBs in these tissues. Secondly, we have brought new evidence that sat III sequences triggers a transient dissociation of HP1 (heterochromatin Protein 1) α and β as well as a loss of the repressive epigenetic H3K9me3 histone mark at pericentric heterochromatin. Interestingly we have also found that, following stress, a sequential accumulation of SATIII RNA in a Sense and Antisense orientation occurs, suggesting that this specific pattern of expression plays an important role in heterochromatin reformation. Finally, we have found that the accumulation of SATIII RNA is associated with a slowdown of mitosis. Indeed we have found that in stressed cells, accumulation of sat III impcats the progression of mitosis and that a knock down of sat III RNA using LNA approaches releases this blockade, leading to genomic instability of tumor cells and to the appearance of poly nucleated cells.

Με στόχο την απομόνωση του γονιδίου hsp83 της μεσογειακής μύγας, πραγματοποιήθηκε διαλογή μιας cDNA βιβλιοθήκης από προνύμφες 3ου σταδίου, με ανιχνευτή το δεύτερο εξώνιο του hsp83 ομόλογου γονιδίου της Drosophila auraria. Από τη διαλογή αυτή προέκυψαν αρκετοί αλληλεπικαλυπτόμενοι κλώνοι, ο μεγαλύτερος των οποίων (CM-1) είχε μέγεθος 2.593 kb και περιελάμβανε ένα ανοιχτό αναγνωστικό πλαίσιο 715 αμινοξέων, από τη μετάφραση του οποίου προκύπτει ένα πολυπεπτίδιο προβλεπόμενου μοριακού βάρους 81,74 kDa. Η προβλεπόμενη αμινοξική αλληλουχία έδειξε πολύ μεγάλη ταυτότητα με όλα τα μέλη της οικογένειας HSP90 και ειδικότερα με τις HSP83 ομόλογες πρωτεΐνες της Drosophila. Επιπλέον, η προβλεπόμενη αμινοξική αλληλουχία περιείχε όλες τις συντηρημένες περιοχές των μελών της οικογένειας των HSP90 και στο καρβοξυτελικό της άκρο, έφερε το πενταπεπτίδιο MEEVD, το οποίο είναι χαρακτηριστικό όλων των κυτταροπλασματικών ισομορφών αυτής της οικογένειας. Με βάση αυτά τα αποτελέσματα ο κλώνος CM-1, ονομάστηκε Cchsp83. Ο κλώνος αυτός, εκτός από ολόκληρη την κωδική περιοχή του γονιδίου, περιείχε μέρος της 5’ μη μεταφραζόμενης περιοχής και ολόκληρη την 3’ μη μεταφραζόμενη περιοχή του hsp83 γονιδίου της μεσογειακής μύγας. Ανάλυση κατά Southern σε γονιδιωματικό DNA με διάφορα ένζυμα περιορισμού και κατάλληλους cDNA ανιχνευτές, έδειξε ότι το Cchsp83 γονίδιο υπάρχει σε ένα μόνο αντίγραφο στο γονιδίωμα της μεσογειακής μύγας. Ανάλυση “Northern” έδειξε την ύπαρξη ενός μεταγράφου με μέγεθος 2,7 kb περίπου. Το Cchsp83 γονίδιο, χαρτογραφήθηκε με in situ υβριδοποίηση σε μία θερμοεπαγόμενη χρωμοσωματική διόγκωση των πολυταινικών χρωμοσωμάτων των σιελογόνων αδένων του εντόμου (6R:94C). Η μελέτη του προτύπου έκφρασης του γονιδίου Cchsp83 έγινε σε επίπεδο RNA με RT-PCR σε ολικό RNA και σε επίπεδο πρωτεΐνης με ανάλυση Western σε ολικά πρωτεϊνικά εκχυλίσματα. Αντισώματα για την CcHSP83 αναπτύχθηκαν μετά από έκφραση ενός σημασμένου με 6 His τμήματος του Cchsp83 cDNA (490-690aa) σε βακτήρια E. coli. Η αντίδραση του Cchsp83 γονιδίου στην αύξηση της θερμοκρασίας είναι πολύ γρήγορη και ευαίσθητη. Μετάγραφα του γονιδίου ανιχνεύονται μετά από 5 λεπτά θερμικού στρες και σε θερμοκρασίες αρκετά χαμηλές (300C). Η επαγωγή του γονιδίου γίνεται μέγιστη μετά από 30-60 λεπτά θερμικού στρες στους 37-390C. Η επαναφορά της έκφρασης του γονιδίου στα φυσιολογικά επίπεδα μετά από θερμικό στρες είναι αργή, αφού για να γίνει αυτό απαιτούνται 4 ώρες ανάκαμψης στους 250C, μετά από μόλις 30 λεπτά θερμικού στρες στους 380C. Αναπτυξιακή μελέτη του προτύπου έκφρασης του Cchsp83 έδειξε ότι το γονίδιο εκφράζεται σε όλη τη διάρκεια της ανάπτυξης της μεσογειακής μύγας. Στους 250C τόσο τα επίπεδα του RNA, όσο και τα επίπεδα της πρωτεΐνης, είναι υψηλά στα εμβρυικά στάδια, χαμηλά στα προνυμφικά και μέτρια στα νυμφικά και ενήλικα στάδια. Μετά από θερμικό στρες στους 380C, τα επίπεδα των μεταγράφων αυξάνονται αρκετά, ιδιαίτερα στα στάδια που σε φυσιολογικές συνθήκες είναι χαμηλά. Όσον αφορά στα επίπεδα της πρωτεΐνης, τα αποτελέσματα ήταν παρόμοια μετά από θερμικό στρες στους 350C, αλλά όχι στους 37-390C, όπου δεν παρατηρήθηκε καθόλου επαγωγή. Το γεγονός αυτό υποδηλώνει ότι σε υψηλές θερμοκρασίες καταστέλλεται η ωρίμανση ή και η μετάφραση του Cchsp83 RNA. Με στόχο την απομόνωση των ρυθμιστικών περιοχών του γονιδίου Cchsp83 της μεσογειακής μύγας, πραγματοποιήθηκε διαλογή μιας χρωμοσωματικής λEMBL-4A βιβλιοθήκης, με ανιχνευτή το δεύτερο εξώνιο του hsp83 ομόλογου γονιδίου της Drosophila auraria. Από τη διαλογή αυτή απομονώθηκαν δύο κλώνοι, ένας από τους οποίους περιελάμβανε μέρος της κωδικής περιοχής, την 5’ μη μεταφραζόμενη περιοχή και 3,5 kb της 5’ ανοδικής περιοχής του γονιδίου Cchsp83. Σύγκριση της γονιδιωματικής αλληλουχίας με τη cDNA αλληλουχία, αποκάλυψε την ύπαρξη ενός μικρού εσωνίου 275 bp ανάμεσα στην 5’ μη μεταφραζόμενη περιοχή και στην αρχή της κωδικής περιοχής του γονιδίου. Βιοπληροφορική ανάλυση και πειράματα 5’ RACE, υποδηλώνουν ότι το σημείο έναρξης της μεταγραφής του γονιδίου βρίσκεται 144 bp ανοδικά του 5’ άκρου του εσωνίου και 23 bp καθοδικά ενός τυπικού στοιχείου TATA (ΤΑΤΑΑΑΤΑ). Δύο πιθανά στοιχεία απόκρισης στη θερμοκρασία (HSEs) εντοπίστηκαν στην εγγύς 5’ περιοχή του γονιδίου, 35 και 330 bp ανοδικά του στοιχείου TATA. Επιπλέον, βρέθηκαν 4 ακόμα πιο απομακρυσμένα HSEs, 1.595, 2.861, 2.880 και 2.890 bp, ανοδικά του σημείου έναρξης της μεταγραφής, καθώς και ένα HSE μέσα στο εσώνιο. Λειτουργική ανάλυση της εγγύς 5’ ανοδικής περιοχής του γονιδίου Cchsp83 πραγματοποιήθηκε με τη μέθοδο του γενετικού μετασχηματισμού. Τρία αλληλεπικαλυπτόμενα τμήματα του υποκινητή του γονιδίου Cchsp83, μήκους 519 bp (-380/+139, PL), 230 bp (-86/+144, PM) και 193 bp (-55/+139, PS) τοποθετήθηκαν μπροστά από το γονίδιο αναφοράς lacZ και εισήχθησαν στο γονιδίωμα της μεσογειακής μύγας με το σύστημα μετασχηματισμού Minos. Η έκφραση του γονιδίου αναφοράς, ελέγχθηκε σε όλες τις διαγονιδιακές σειρές που προέκυψαν με την ποσοτική ενζυματική μέθοδο της β-γαλακτοζιδάσης. Οι PM-lacZ και PS-lacZ σειρές δεν έδειξαν ανιχνεύσιμα επίπεδα έκφρασης του lacZ γονιδίου τόσο σε φυσιολογικές συνθήκες, όσο και σε συνθήκες θερμικού στρες. Αντιθέτως, οι περισσότερες από τις PL-lacZ σειρές έδειξαν σημαντικά επίπεδα συστατικής έκφρασης. Το αναπτυξιακό πρότυπο έκφρασης του γονιδίου αναφοράς μελετήθηκε σε μία PL-lacZ σειρά και βρέθηκε παρόμοιο με εκείνο του ενδογενούς γονιδίου, υποδηλώνοντας ότι η -380/+139 περιοχή του υποκινητή περιλαμβάνει όλα τα ρυθμιστικά στοιχεία που απαιτούνται για την ορθή χρονική έκφραση του Cchsp83 γονιδίου σε φυσιολογικές συνθήκες. Αν και η περιοχή αυτή του υποκινητή περιλαμβάνει δύο δυνητικά στοιχεία απόκρισης στη θερμοκρασία καθώς και την 5’ UTR, εντούτοις δεν είχε την ικανότητα να οδηγήσει σε θερμοεπαγόμενη έκφραση το γονίδιο αναφοράς. Τα αποτελέσματά μας υποδεικνύουν ότι η περιοχή -380/+139 του Cchsp83 γονιδίου μπορεί να χρησιμοποιηθεί για την ανάπτυξη διαγονιδιακών συστημάτων σήμανσης της μεσογειακής μύγας, τα οποία αναμένεται να συμβάλλουν στη διατήρηση και ανίχνευση κατάλληλων στελεχών που χρησιμοποιούνται σε προγράμματα βιολογικού ελέγχου του επιβλαβούς αυτού εντόμου. / By using the second exon of the D. auraria hsp83 gene as a probe, a number of overlapping cDNA clones were isolated from a Ceratitis capitata (medfly) cDNA library. The longest cDNA had a size of 2,593 bp and contained an open reading frame coding for a putative polypeptide of 715 amino acids with a predicted molecular weight of 81.74 kDa. In addition, it contained a part of the 5’-untranslated region and the complete 3’-untranslated region of a medfly hsp83 homolog, named Cchsp83. The predicted amino acid sequence showed a high degree of identity to all known sequences of the HSP90 family and was more closely related to the Drosophila HSP83 homologs. The putative medfly HSP83 contained all the conserved sequences of the members of the HSP90 family and was ended, at the C-terminal, with the pentapeptide MEEVD that characterizes all the cytosolic members of this family. Genomic Southern blot analysis, with several restriction enzymes and appropriate cDNA probes, indicated that the Cchsp83 gene exists as a single copy in the medfly genome. Northern blot hybridization revealed a single transcript of approximately 2.7 kb. In situ hybridization analysis showed that the Cchsp83 gene maps at the 94C region of the 6th chromosome (6R:94C), which corresponds to one of the major heat shock puffs of the medfly salivary gland polytene chromosomes. Evaluation of the Cchsp83 gene transcript and protein levels was performed by RT-PCR and western analysis, using total RNA and protein from synchronized animals. Anti-HSP83 antibodies were prepared against a histidine tag purified chimeric polypeptide from E. coli cells, transformed with a part of the coding region (aa 490-690) of the Cchsp83 cDNA. The response of the Cchsp83 gene to heat was fast and sensitive. Heat-induced transcript levels could be detected within 5 min at temperatures as low as 300C. Maximum transcript levels were obtained after 30-90 min treatments at 35-390C. Following recovery at 250C, after a 30 min heat shock, the accumulated transcripts remained at high levels for approximately 3h and declined to the non-induced levels 1h later. Developmental studies showed that the Cchsp83 gene is expressed constitutively throughout medfly development. At 250C, both transcript and protein levels were high in embryonic stages, low in larval stages and moderate in pupal and adult stages. Following heat shock at 380C, the transcript levels increased approximately 3- to 5-fold, depended on the developmental stage. Similar results were obtained for the protein levels after a heat shock at 350C, but not at 380C, suggesting that the Cchsp83 mRNAs are not translated efficiently at high temperatures. Screening of a genomic λEMBL-4A library from 24-h-old medfly embryos with the second exon of the D. auraria hsp83 gene, resulted in the isolation of a genomic clone containing part of the coding region, the untranslated leader region (5’ UTR) and 3.5 kb from the 5’ flanking region of the gene. Comparison of the genomic and cDNA nucleotide sequences revealed the presence of a small intron of 275 bp between the 5’ untranslated and coding regions of the gene. Thus the Cchsp83 gene is organized into two exons separated by a small intron. Computational analysis and 5’ RACE experiments, suggested that the putative transcription initiation site of the gene is located 144 bp upstream of the 5’ splicing site of the intron and 23 bp downstream of a typical TATA box (TATAΑΑΤΑ). Two putative heat shock elements (HSEs) were identified in the proximal 5’ flanking region of the gene, 35 and 330 bp, upstream of the TATA box. In addition to them, four distal HSEs, 1,595, 2,861, 2,880 and 2,890 bp, upstream of the putative transcription initiation site and one HSE inside the intron were identified. Functional analysis of the proximal 5’ flanking region of the Cchsp83 gene was performed by germline transformation. Three overlapping promoter fragments PL (-380/+139), PM (-86/+144) and PS (-55/+139), were fused to the lacZ reporter gene and the resulting constructs were introduced into the medfly genome via Minos-element mediated germline transformation. The expression of the reporter gene, in at least 8 homozygous transformed lines for each construct, was evaluated by quantitative β-galactosidase assays. The PM-lacZ and PS-lacZ lines did not show detectable levels of lacZ expression at neither normal or heat shock conditions. On the other hand, most of the PL-lacZ lines showed significant levels of constitutive lacZ expression. Developmental expression studies in one of these lines showed that the reporter gene exhibited similar developmental expression pattern to the endogenous one, suggesting that the PL promoter region includes all the necessary regulatory elements for driving correct temporal expression of the Cchsp83 at normal conditions. Although this promoter region contained the two proximal HSEs and the 5’ UTR, it was unable to drive heat-induced expression of the reporter gene suggesting that additional upstream and/or downstream sequences are necessary for the heat-induced expression of the Cchsp83 gene. Our data indicate that the PL promoter region of the Cchsp83 gene can be used as a driver for the development of robust transgenic marker systems in medfly. Such systems are important for detecting, maintaining and recognizing medfly strains that are used today in population control programs of this agricultural pest.

Orientador: Carlos Henrique Inacio Ramos / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T20:53:19Z (GMT). No. of bitstreams: 1 Cagliari_ThiagoCarlos_D.pdf: 4482929 bytes, checksum: a1439ac0cca9a21c77eb47d2e163c224 (MD5) Previous issue date: 2009 / Resumo: As proteinas sao macromoleculas que possuem importancia vital para o funcionamento celular, participando da maioria das reacoes biologicas e tambem como componentes estruturais. Para que uma proteina possa exercer sua funcao, precisa atingir sua estrutura nativa atraves de um processo denominado enovelamento proteico. Neste contexto, as chaperonas moleculares sao proteinas capazes de auxiliar no enovelamento de outras proteinas, atuando na prevencao de agregados, desagregacao, translocacao, ativacao, entre outros. Dentre os muitos tipos de chaperonas existentes, neste trabalho foram abordadas as chaperonas das familias Hsp100 e Hsp90, as quais estao relacionadas aos processos de desagregacao e auxilio do enovelamento de proteinas-substrato, respectivamente. O presente trabalho pretendeu produzir as proteinas recombinantes Hsp100 e Hsp82 de cana-de-acucar para a caracterizacao de suas respectivas relacoes estrutura-funcao. Para isto foram empregadas tecnicas como: dicroismo circular, fluorescencia, espalhamento dinamico de luz e ultracentrifugacao analitica. Assim, foi observado que a forca ionica do meio e capaz de influenciar a estrutura quaternaria da proteina Hsp100, a qual se apresenta hexamerica em menores concentracoes de sal. Alem disto, e capaz de reconhecer agregados proteicos formados pelas proteinas luciferase e citrato sintase em ensaios in vitro. Ja a proteina Hsp82 apresentou uma estrutura dimerica, a qual nao e influenciada pela presenca de nucleotideos e apresenta grande estabilidade termica. Finalmente, a proteina p23 humana, a qual e responsavel por auxiliar a proteina Hsp90 no enovelamento de muitas proteinas/complexos proteicos, tambem foi caracterizada. Foram observados indicios de que a regiao C-terminal, rica em residuos de aminoacidos carregados, pode possuir algum grau de estruturacao, apesar de alguns estudos na literatura indicarem o contrario. O estudo das chaperonas de cana-de-acucar foi direcionado por um trabalho previo de anotacao de sequencias relacionadas as chaperonas moleculares no banco de dados do projeto SUCEST (Sugarcane EST Genome Project), o qual foi realizado por nosso grupo de pesquisa. Alem disto, sao apresentados os resultados da anotacao das sequencias relacionadas as chaperonas de eucalipto no banco de dados FORESTs (Eucalyptus Genome Sequencing Project Consortium), possibilitando futuros estudos com estas proteinas. / Abstract: Proteins are macromolecules that are vital to the functioning cell, participating in most of the biological reactions as well as structural components. To perform its function, a protein need to achieve its native structure through a process called protein folding. In this context, the molecular chaperone proteins are able to assist in the folding of other proteins, acting in the prevention of aggregation, disaggregation, translocation, activation, among others. From all types of existing chaperones, here were highlight the Hsp100 and Hsp90 families, which are related to processes of disaggregation and assistance of substrateprotein folding, respectively. This study sought to produce the recombinant proteins Hsp100 and Hsp82 from sugar cane for the characterization of their structure-function relationships. In order to do this, some techniques were employed such as: circular dichroism, fluorescence, dynamic light scattering and analytical ultracentrifugation. As a result, it was observed that the ionic strength of the solvent is capable of influencing the quaternary structure of protein Hsp100, which presents as a hexamer in lower salt concentrations. Furthermore, it is capable of recognizing protein aggregates formed by luciferase protein and citrate synthase in in vitro essays. The Hsp82 protein showed a dimeric structure, which was not influenced by the presence of nucleotides and presented a great thermal stability. Finally, the human protein p23, which is responsible for assisting in the Hsp90 protein folding of many proteins/protein complexes, was also characterized. In spite of some studies indicating the contrary, we observed evidence that the C-terminal region, which is rich in charged amino acid residues, can possible have some structure. The sugarcane chaperones study was guided by a previous chaperone sequence annotation work in the SUCEST (Sugarcane EST Genome Project) databank performed by our research group. In addition, results regarding chaperone sequences annotation in the eucalyptus databank (FORESTs - Eucalyptus Genome Sequencing Project Consortium) were presented here as well, which can also lead to future chaperone proteins function and structure studies. / Doutorado

Indiana University-Purdue University Indianapolis (IUPUI) / The function of major histocompatability complex (MHC) class II molecules is to present antigenic peptides to CD4+ T cells. Typically, MHC class II molecules present peptides derived from exogenous sources. Yet, certain endogenous antigens (Ags) have been found to be presented by class II molecules. Studies suggest that specific heat shock protein family members may play a role in Ag processing and subsequent class II presentation. The studies presented here using B lymphoblasts demonstrate the importance of HSP90α, HSP90β, and possibly HSP70 in selectively regulating MHC class II presentation. Inactivation of HSP90 function using pharmacological inhibitors inhibited class II presentation of exogenous and endogenous GAD, but did not perturb the presentation of several other intra- and extracellular Ags. Individual knockdown of HSP90 isoforms using isoform specific siRNA selectively inhibited GAD Ag presentation. These results demonstrate a requirement for HSP90α and HSP90β in regulating MHC class II presentation of select Ags. Studies to explore mechanistically the roles of HSP90α and HSP90β in regulating GAD Ag presentation were pursued. The pathways of exogenous and endogenous MHC class II presentation of GAD Ag are distinct yet converge with shared terminal processing of GAD within endosomal/lysosomal vesicles. The effect of HSP90 manipulation on various shared components of the MHC class II pathway was examined. The studies presented here suggest that HSP90α and HSP90β regulate MHC class II presentation of GAD Ag at discrete steps most likely involving HSP90 binding to GAD Ag rather than perturbing overall MHC class II function. vi Studying the role of HSP90 in MHC class II presentation in B cells revealed the potential requirement for HSP70 in the presentation of select Ags. The studies presented here demonstrate a possible role for HSP70 in the presentation of Ags such as SMA or Ig kappa by MHC class II molecules. Also included in this work is a study of a rare case of diabetes caused by type B insulin resistance due to development of insulin receptor autoantibodies during the treatment of hepatitis C with interferon alpha and ribavirin. Clinical and laboratory findings in the case are presented.

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.