The exposure to heat can adversely affect animal performance and productivity, particularly when associated with pregnancy. The comprehension of the physiological and cellular responses during heat stress assists the decisions to improve the productivity of goats in a tropical environment. In this context, this study evaluated the physiological and cellular responses of Saanen goats in acute and chronic stress conditions. Healthy Saanen goats were randomly assigned to heat stress treatment, short-term, under solar radiation and in the long-term, in climatic chamber. Data were analyzed using the Statistical Analysis System (SAS). The normality was confirmed using the Shapiro-Wilk test. In acute heat stress, solar radiation caused the increase of cortisol release, respiratory rate and reduced T3 and T4 to restore homeothermia. The expression of p53 (pro-apoptotic protein), Bcl-2 (anti-apoptotic protein), HSP60, HSP70 and HSP90 was higher in leukocyte cells for heat stressed goats. In chronic heat stress at the end of gestation, goats significantly mobilized the increase of respiratory rate to lose heat, with cortisol elevation on the 15 days previous to parturition (P < 0.05), which was even greater at day 15 postparturition. At the cellular level, HSP70 was the most expressed during and after heat challenge, with more transcripts at day 15 postparturition for the heat stressed goats. In mammary epithelial cells, there were an increase in apoptosis-related transcripts, p53 and Bax, for the group undergoing heat stress before parturition. The expression of HSP27 was higher before and after parturition for the same group when compared to the animals in thermal neutrality. During lactation, the stressed goats presented lower milk yield. The heat challenge increased the percentage of fat and decreased lactose. The somatic cell count was higher for stressed goats during all lactation. During lactation, the gene expression of prolactin receptor (PRLr) was lower for heat stress goats, which may explain the reduction in milk production at subsequent lactation. In conclusion, our findings suggest that respiratory rate and HSP70 are reliable biomarkers for assessing the thermal comfort and thermotolerance of Saanen goats under acute and chronic heat stress. In addition, chronic thermal stress at final gestation impaired milk production and milk composition from subsequent lactation. / A exposição ao calor pode afetar negativamente o desempenho e a produtividade dos animais, particularmente quando associados à gestação. Compreender as relações entre as respostas fisiológicas e celulares, quando em estresse por calor, auxilia na tomada de decisões para melhorar a produtividade de caprinos em ambiente tropical. Neste contexto, este estudo avaliou as respostas fisiológicas e celulares de cabras Saanen em condição de estresse agudo e crônico. Cabras da raça Saanen saudáveis foram aleatoriamente expostas ao estresse por calor, de curto prazo sob radiação solar e a longo prazo em câmara climática. Os dados foram analizados utilizando o software estatístico \"Statistical Analysis System\" (SAS, 2008). A normalidade foi confirmada com o teste de Shapiro-Wilk. Em estresse agudo, a radiação solar promoveu o aumento da liberação de cortisol, elevou a frequência respiratória e reduziu T3 e T4, para reestabelecer a homeotermia. Observou-se nas células leucocitárias das cabras sob estresse, aumento da expressão da p53 (proteína pró-apoptótica), Bcl-2 (proteína anti-apoptótica), HSP60, HSP70 e HSP90. Sob estresse crônico no final da gestação, as cabras aumentaram a frequência respiratória para perder calor, com elevação do cortisol no dia 15 pré-parto (P <0,05), que foi ainda maior no dia 15 pós-parto. A nível celular, a HSP70 foi a mais expressa durante e após o desafio com calor, com mais transcritos no dia 15 pós-parto para as cabras sob estresse. Nas células epiteliais mamárias, houve aumento dos transcritos relacionados à apoptose, p53 e Bax, para o grupo sob estresse por calor antes do parto. A expressão da HSP27 foi maior antes e após o parto para o mesmo grupo quando comparado aos animais em termoneutralidade. Durante a lactação, as cabras sob estresse apresentaram menor produção de leite. O desafio de calor aumentou o percentual de gordura e diminuiu a lactose. A contagem de células somáticas foi maior para as cabras sob estresse durante toda a lactação. A expressão gênica do receptor de prolactina (PRLr) foi menor para as cabras sob estresse, 15 dias após o parto, o que pode explicar a redução na produção de leite na lactação subsequente. Em conclusão, nossos achados sugerem que a frequência respiratória e a HSP70 são biomarcadores confiáveis para avaliar o conforto térmico e a termotolerância de cabras Saanen sob estresse por calor agudo e crônico. Além disso, o estresse por calor crônico pré-parto prejudicou a produção e a composição do leite da lactação subsequente.

The purpose of this study was to establish whether curcumin protects renal proximal tubule cells against ischemic injury, determine whether this postulated cytoprotective effect is mediated through the upregulation of HSP70, and investigate whether the mechanism by which curcumin induces HSP70 expression and confers its protective effect is through activation of the Unfolded Protein Response. LLC-PK1 cells were cultured on collagen-coated filters to mimic conditions of in vivo renal proximal tubule cells and induce cell polarization. Injury with and without curcumin treatment was studied by using chemically-induced ATP-depletion which mimics renal ischemic injury. Cell injury was assessed using a TUNEL assay in order to evaluate DNA cleavage associated with ischemia-induced apoptosis and actin staining used to assess cytoskeletal disruption. Renal ischemic damage was further investigated by determining detachment of the Na-K ATPase from the basolateral membrane, which represents loss of cell polarity. Cells were incubated with curcumin in a dose- and time-response fashion and subsequent levels of HSP70 expression were assessed. Cells were then incubated with AEBSF, an inhibitor of the Unfolded Protein Response (UPR) and HSP70 and BiP/GRP78 (an ER resident chaperone that is upregulated by the UPR) expression levels were evaluated. Results demonstrated that treatment with curcumin during two hours of injury results in significantly less injury-related apoptosis and cytoskeletal disruption compared to control injured cells. It was demonstrated that curcumin induces HSP70 in both a dose- and time-response fashion. Moreover, curcumin treatment resulted in profound stabilization of Na-K ATPase on the basolateral membranes as there was significantly less Na-K ATPase detachment in cells treated with curcumin during two hours of injury compared to control injured cells. Finally, treatment with AEBSF inhibited HSP70 upregulation in curcumin-treated cells as well as inhibiting the GRP78 over-expression otherwise demonstrated in curcumin-treated cells. Protection of proximal tubule cells against renal ischemic injury by curcumin was therefore indicated to be mediated by the activation of the UPR through which HSP70 is upregulated. Curcumins activation of the UPR and induction of HSP70 explains the stabilization of Na-K ATPase on the cytoskeleton and also provides a potential mechanism explaining many of curcumins therapeutic and protective qualities.

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.

The type III secretion system (T3SS) of pathogenic Yersinia pseudotuberculosis is involved in virulence. The syringe-like secretion system spans both bacterial membranes and is responsible for the ability of Yersinia to transfer toxic proteins (Yop proteins) into the eukaryotic target cell. The T3SS is believed to have evolved from the flagellum and regulation of the T3SS is a complex event that involves a series of regulatory proteins, whereby two are YscP and YscU. In a regulatory model, called the substrate specificity switch, both proteins act together to ensure proper T3SS structure and function by regulating a stop in YscF needle protein export with a shift to Yop effector secretion. YscU undergoes autoproteolysis at a conserved motif consisting of amino acids Asparagine-Proline-Threonine-Histidine (NPTH). Processing generates a C-terminal 10 kDa peptide, YscUCC. Processing is crucial for proper T3SS regulation and function both in vitro and in vivo. Full-length YscU does not support Yop secretion and after cleavage, YscUCC remains attached to the rest of YscU and acts as a negative block on T3S. Relief of this negative block is suggested to occur through displacement of YscUCC from the rest of YscU. Thorough control of many different cellular processes is brought by the heat shock proteins (HSPs) DnaK and DnaJ. Due to their multiple regulatory functions, mutations in the hsp-genes lead to pleiotropic effects. DnaK and DnaJ are essential for proper flagellum driven motion of bacteria, but more so; they ensure proper Yersinia T3SS function in vivo. Furthermore, DnaJ interacts with YscU and may be directly involved in T3SS regulation. Virulence of Yersinia is regulated on many levels. A previously identified virulence associated protein, VagH, is now characterized as an S-adenosyl-methionine dependent methyltransferase. The targets of the methylation activity of VagH are release factors 1 and 2 (RF1 and RF2), that are important for translation termination. The enzymatic activity of VagH is important for Yop secretion and a vagH mutant up-regulates a T3SS negative regulatory protein, YopD. Furthermore, a vagH mutant is avirulent in a mouse infection model, but is not affected in macrophage intracellular survival. The importance of VagH in vivo makes it a possible target for novel antimicrobial therapy.

Oligodendrocyte progenitors are highly sensitive to oxidative insults. Among the factors postulated to contribute to this susceptibility are high levels of intracellular iron and low antioxidant content. During ischemia, the neurotransmitter dopamine (DA) is released and may contribute to oxidative stress and oligodendrocyte injury in the hypomyelinating disorder, periventricular leucomalacia (PVL). In this thesis, I investigated the role of iron in DA-induced toxicity in primary cultures of oligodendrocyte progenitors, and assessed the contribution of the antioxidant defenses (glutathione (GSH), glutathione peroxidase (GPx) and superoxide dismutase (SOD)) and other survival factors (heat shock proteins and the protein kinase Akt) in determining the response of the cells to DA. / Addition of iron to cultures increased DA-induced expression of the stress protein heme oxygenase-1 (HO-1), and toxicity as assessed by mitochondrial activity, cellular release of lactate dehydrogenase, nuclear condensation and caspase-3 activation. In contrast, an iron chelator reduced these events. Furthermore, DA induced accumulation of superoxide, which was also reduced by the iron chelator. Surprisingly, a mimetic of the superoxide detoxifying enzyme, SOD potentiated DA toxicity, suggesting that generation of hydrogen peroxide via superoxide dismutation may be contributing to toxicity. Both a mimetic of the peroxide-scavenging enzyme, GPx and a GSH analog blocked DA-induced superoxide accumulation, HO-1 expression and caspase-3 activation. In addition, the GPx mimetic blocked caspase-3 activation induced by the combination of DA with iron. In contrast, an inhibitor of glutathione synthesis potentiated DA-induced HO-1 expression and cell death. / Finally, in further examining the cellular defense mechanisms, I found that various heat shock proteins increased in expression levels during oligodendroglial differentiation, however only heat shock protein-90 (HSP-90) was detected in oligodendrocyte progenitors. An HSP-90 inhibitor decreased activated Akt levels, induced caspase-3 activation, increased nuclear condensation, reduced oligodendrocyte progenitor viability, and potentiated DA-induced apoptosis. In addition, an Akt inhibitor alone exacerbated DA toxicity and in combination with the HSP-90 inhibitor caused synergistic potentiation of DA toxicity by enhancing caspase-3 activation. / In conclusion, elevated levels of iron, superoxide, deficient detoxification of peroxides by glutathione peroxidase and inadequate defense by glutathione contribute to the susceptibility of oligodendrocyte progenitors to DA-induced toxicity. On the other hand, HSP-90 alone or in concert with Akt play important roles in oligodendrocyte progenitors survival following an insult that produces oxidative stress.

Įvairios širdies ligos, kurių metu vyksta kardiomiocitų žuvimas, yra viena iš pagrindinių mirtingumo priežasčių visame pasaulyje. Regeneracinė ląstelių terapija, naudojant pirmines raumeninės kilmės ląsteles širdies ligų gydyme, sulaukia vis didesnio dėmesio. Dėl įvairių patologinių procesų, vykstančių pažeistame audinyje, didelė dalis transplantuotų ląstelių žūva. Todėl viskas, kas gali padėti transplantuotiems mioblastams išgyventi ir geriau prigyti, verta nuodugnesnių tyrimų. Šiame darbe buvo tirtas streso baltymo Hsp70 vaidmuo suaugusio triušio raumens kamieninių ląstelių įvairių viduląstelinių sistemų, atsakingų už ląstelių žuvimą ar išgyvenimą, reguliacijoje. Suaugusio triušio raumens kamieninės ląstelės buvo transfekuotos plazmide, turinčia hsp70 geną. Transfekuotų ląstelių padidėjusio atsparumo toksiniams poveikiams veikimo mechanizmai tirti lyginant jas su paprastomis kamieninėmis miogeninėmis ląstelėmis. Gauti rezultatai parodė, kad padidėjusi Hsp70 baltymo raiška apsaugo triušio raumens kamienines ląsteles nuo apoptozinio žuvimo po poveikio cheminėmis medžiagomis (naftazarinu, vandenilio peroksidu ir natrio prusidu) generuojančiomis reaktyvius deguonies darinius (ROS) ir sukeliančiomis oksidacinį stresą. Padidintas Hsp70 baltymo kiekis po poveikio osidacinį stresą indukuojančiomis medžiagomis keičia mitogenais aktyvinamos kinazės ERK 1,2, streso kinazės JNK trumpalaikį/pro-apoptozinį veikimą paprastose pirminėse miogeninėse ląstelėse į ilgalaikį/ani-apoptozinį... [toliau žr. visą tekstą] / Various heart diseases concomited with the death of cardiomyocytes is one of the major causes of death-rate. Regenerative stem cell therapy of damaged heart applying muscle-derived stem cells attains more and more attention. Patological environment in the damaged heart is major cause of death of newly transplanted muscle-derived stem cells. Therefore, everything increasing survival of transplanted myogenic cells is of research interest. This work has been designed to investigate role of stress protein Hsp70 in the regulation of intracellular systems responsible for the death or survival of primary myogenic cells. Rabbit muscle-derived stem cells were transfected with the plasmid carrying hsp70 gene. Various intracellular mechanisms responsible for the resistance of transfected cells have been investigated and compared with the regular non transfected primary myogenic cells. Our results show that Hsp70 protein protects rabbit muscle-derived stem cells from the toxic effect of compounds (naftazarin, hydrogen peroxide and sodium pruside) generating reactive oxigen species (ROS) and causing oxidative stress. Increased amount of intracellular Hsp70 protein converts transient/proapototic mode of action of ERK1,2 and JNK in regular primary myogenic cells to sustained/antiapoptotic action in transfected cells and significantly decreased total amount of JNK after the treatment with naftazatin. Transfected cells also showed sustained/anti-apoptotic mode of activation of transcription... [to full text]

Os fogachos são a queixa mais comum das mulheres peri- e pós-menopausa e estão fortemente relacionados com a diminuição dos níveis de estrogênio. No entanto, a fisiopatologia deste sintoma vasomotor muito desagradável é ainda desconhecido. Por outro lado, o estradiol (E2) apresenta a capacidade de induzir a expressão de HSP72, um membro da família de 70 kDa das proteínas de choque térmico (HSP70), que são citoprotetoras e cardioprotetoras. Sabe-se que a expressão HSP70 é comprometida em doenças inflamatórias relacionadas com o envelhecimento. Por isso, questionamos se a capacidade do organismo de desencadear uma resposta ao choque térmico (Heat Shock Response) robusta, estaria ainda presente após a retirada, via castração, do E2. Para tanto, foram estudados os efeitos do choque térmico (Heat Shock - HS), através de uma sessão de banho de imersão, em ratas Wistar submetidas a ovariectomia bilateral (OVX), após um período de washout hormonal de 7 dias. Doze horas após o HS, os animais foram mortos e o arco aórtico foi excisado cirurgicamente para análises moleculares. Os resultados foram comparados com os marcadores de estresse oxidativo no plasma (superóxido dismutase, catalase e lipoperoxidação), pois é bem estabelecido que a expressão de HSP70 é sensível a regulação redox. A relação entre a iHSP70 (intracelular) e a eHSP70 (extracelular/plasma), proposto como um índice do estado inflamatório sistêmico, também foi investigada. Os resultados mostraram que a Heat Shock Response continua preservada em animais OVX, como inferido a partir da expressão de HSP70 (até 40% de aumento, p <0,01) nas aortas, o que não foi acompanhado por nenhuma outra alteração em marcadores de estresse oxidativo, parâmetros hematológicos e no controle glicêmico. Desta forma, sugerimos que a avaliação periódica do status de HSP70 (iHSP70 vs eHSP70) pode ser de extrema relevância clínica, pois a diminuição da capacidade de defesa do organismo via Heat Shock Response está no centro do aparecimento de disfunções relacionadas com a menopausa. / Hot flashes, the most common complaint of peri- and postmenopausal women, are tightly related to decrease in estrogen levels. However, the pathophysiology of this very unpleasant vasomotor symptom is greatly unknown. On the other hand, estradiol (E2) has been found to induce the expression of HSP72, a member of the 70 kDa family of heat shock proteins (HSP70), which are cytoprotective and cardioprotective. Since it has been noticed that HSP70 expression is compromised in age-related inflammatory diseases, we argued whether the capacity of triggering a robust heat shock (HS) response would be still present after E2 withdrawal. Hence, we studied the effects of HS treatment (hot tub) in female Wistar rats subjected to bilateral oophorectomy (OVX) after a 7 day washout period. Twelve hours after HS, the animals were killed and aortic arches were surgically excised for molecular analyses. The results were compared with oxidative stress markers in the plasma (superoxide dismutase, catalase and lipoperoxidation) because HSP70 expression is sensitive to redox regulation. Extracellular (plasma) to intracellular HSP70 ratio, an index of systemic inflammatory status, was also investigated. The results showed that HS response was preserved in OVX animals, as inferred from HSP70 expression (up to 40% rise, p<0.01) in the aortas, which was accompanied by no further alterations in oxidative stress, hematological parameters and glycemic control either. As a consequence, periodic evaluation of HSP70 status (iHSP70 vs eHSP70) may be of clinical relevance because decreased HS response capacity is at the center of the onset of menopause-related dysfunctions.

Made available in DSpace on 2015-03-26T13:46:38Z (GMT). No. of bitstreams: 1 texto completo.pdf: 4898999 bytes, checksum: a1929863574a39c391ec971fb2ed4731 (MD5) Previous issue date: 2007-03-20 / The expression of HSP70 and HSP90 was evaluated through immunoistochemistry studies in bovine inoculated with synthetic peptides SBbo23290 and SBm7462 applied simultaneously, in association or in individual presentation. The animals received three applications with 30 days of interval among them and after challenged with the Babesia bovis strain UFV1 - 9th passage. The histopathological studies showed reactivity of the lymphoid organ seven days after the first immunization with germinative centers increased. After the second immunization, it was evident the hyperplasia of the paracortical and medular areas, with subsequent decrease of the reactivity of the germinative centers after this period, suggesting formation of a memory response to the immunogens. The heat shock proteins presented basal expression. HSP70 expressed preferentially in the paracortical area, while HSP90 showed more visible immunomarker in the paracortical and medular areas. Immunoistochemical studies showed a coincidence in the expression of HSP90 and of the peptides SBbo23290 and SBm7462, when were considered the same periods of immunization, suggesting the formation of a complex HSP- peptide. The glucose, lactate, haptoglobin and C-reactive protein levels stayed inside of the values considered physiologic for the bovine species. The basal cellular immunoreaction of HSP70 and HSP90, as well as the maintenance of the normal levels of the biochemical parameters during the whole period of the experiment, suggest that the stress provoked by the synthetic peptides was insufficient to cause damage to the organism. In other hand, the maintainance of the health condition of the animals influenced the immunological response, and the basal levels of the heat shock proteins might have been enough to promote cytoprotection after the challenge. / A expressão de HSP70 e HSP90 foi avaliada por meio de estudos imunoistoquímicos, em bovinos inoculados com peptídeos sintéticos SBbo23290 e SBm7462, aplicados simultaneamente, em associação ou em apresentação individual. Os animais receberam 3 aplicações em intervalos de 30 dias entre si e posteriormente desafiados com cepa de Babesia bovis (UFV1- 9ª passagem). Os estudos histopatológicos mostraram reatividade do órgão linfóide sete dias após a primeira imunização, com centros germinativos aumentados. Após a segunda imunização, ficou evidente a hiperplasia da região paracortical e medular, com subseqüente diminuição da reatividade dos centros germinativos após este período, sugerindo formação de uma resposta de memória aos imunógenos. As proteínas de choque térmico apresentaram expressão basal. A HSP70 expressou, preferencialmente, na região paracortical, enquanto que a HSP90 mostrou imunomarcação mais visível nas regiões paracortical e medular. Estudos imunoistoquímicos mostraram uma coincidência na expressão de HSP90 com os peptídeos SBbo23290 e SBm7462, quando se consideraram os mesmos períodos de imunização, sugerindo a formação de um complexo HSP-peptídeo. Os níveis de glicose, lactato, haptoglobina e proteína C-reativa mantiveram-se dentro dos valores considerados fisiológicos para a espécie bovina. A imunomarcação celular basal de HSP70 e HSP90, bem como a manutenção dos níveis normais das variáveis bioquímicas durante todo o período do experimento, sugerem que o estresse provocado pelos peptídeos sintéticos foi insuficiente para causar dano ao organismo. Em contrapartida, a manutenção do estado de saúde dos animais influenciou a reposta imunológica, e os níveis basais das proteínas de choque térmico podem ter sido suficientes para promover citoproteção após o desafio.

Os fogachos são a queixa mais comum das mulheres peri- e pós-menopausa e estão fortemente relacionados com a diminuição dos níveis de estrogênio. No entanto, a fisiopatologia deste sintoma vasomotor muito desagradável é ainda desconhecido. Por outro lado, o estradiol (E2) apresenta a capacidade de induzir a expressão de HSP72, um membro da família de 70 kDa das proteínas de choque térmico (HSP70), que são citoprotetoras e cardioprotetoras. Sabe-se que a expressão HSP70 é comprometida em doenças inflamatórias relacionadas com o envelhecimento. Por isso, questionamos se a capacidade do organismo de desencadear uma resposta ao choque térmico (Heat Shock Response) robusta, estaria ainda presente após a retirada, via castração, do E2. Para tanto, foram estudados os efeitos do choque térmico (Heat Shock - HS), através de uma sessão de banho de imersão, em ratas Wistar submetidas a ovariectomia bilateral (OVX), após um período de washout hormonal de 7 dias. Doze horas após o HS, os animais foram mortos e o arco aórtico foi excisado cirurgicamente para análises moleculares. Os resultados foram comparados com os marcadores de estresse oxidativo no plasma (superóxido dismutase, catalase e lipoperoxidação), pois é bem estabelecido que a expressão de HSP70 é sensível a regulação redox. A relação entre a iHSP70 (intracelular) e a eHSP70 (extracelular/plasma), proposto como um índice do estado inflamatório sistêmico, também foi investigada. Os resultados mostraram que a Heat Shock Response continua preservada em animais OVX, como inferido a partir da expressão de HSP70 (até 40% de aumento, p <0,01) nas aortas, o que não foi acompanhado por nenhuma outra alteração em marcadores de estresse oxidativo, parâmetros hematológicos e no controle glicêmico. Desta forma, sugerimos que a avaliação periódica do status de HSP70 (iHSP70 vs eHSP70) pode ser de extrema relevância clínica, pois a diminuição da capacidade de defesa do organismo via Heat Shock Response está no centro do aparecimento de disfunções relacionadas com a menopausa. / Hot flashes, the most common complaint of peri- and postmenopausal women, are tightly related to decrease in estrogen levels. However, the pathophysiology of this very unpleasant vasomotor symptom is greatly unknown. On the other hand, estradiol (E2) has been found to induce the expression of HSP72, a member of the 70 kDa family of heat shock proteins (HSP70), which are cytoprotective and cardioprotective. Since it has been noticed that HSP70 expression is compromised in age-related inflammatory diseases, we argued whether the capacity of triggering a robust heat shock (HS) response would be still present after E2 withdrawal. Hence, we studied the effects of HS treatment (hot tub) in female Wistar rats subjected to bilateral oophorectomy (OVX) after a 7 day washout period. Twelve hours after HS, the animals were killed and aortic arches were surgically excised for molecular analyses. The results were compared with oxidative stress markers in the plasma (superoxide dismutase, catalase and lipoperoxidation) because HSP70 expression is sensitive to redox regulation. Extracellular (plasma) to intracellular HSP70 ratio, an index of systemic inflammatory status, was also investigated. The results showed that HS response was preserved in OVX animals, as inferred from HSP70 expression (up to 40% rise, p<0.01) in the aortas, which was accompanied by no further alterations in oxidative stress, hematological parameters and glycemic control either. As a consequence, periodic evaluation of HSP70 status (iHSP70 vs eHSP70) may be of clinical relevance because decreased HS response capacity is at the center of the onset of menopause-related dysfunctions.

Apesar do sucesso do transplante na clínica, os importantes efeitos adversos dos imunossupressores, usados para prevenir e tratar a rejeição, apontam para a necessidade de novas terapias imunorreguladoras. A via oral tem sido efetiva na indução de imunorregulação, em diversos modelos experimentais, principalmente de doenças autoimunes. A Hsp60/65 é uma molécula com grande potencial imunoterapêutico, por sua capacidade de induzir respostas imunes pró-inflamatória e imunorreguladora. Testamos se a terapia oral com o probiótico Lactococcus lactis que expressa a Hsp65, combinada à administração de aloantígenos do doador (AloAg-doador), atua sinergicamente na indução de tolerância ao enxerto de pele semialogeneico murino, ou no aumento de sua sobrevida. Testamos diferentes combinações de terapia oral, assim como a influência da utilização de um anti-inflamatório, inibidor seletivo de COX-2 (celecoxibe). O transplante de pele foi realizado 10 dias após a última administração oral dos probióticos e aloantígenos do doador. Não observamos efeitos benéficos na sobrevida do enxerto no grupo de animais que receberam L.lactis que produz Hsp65, sozinho ou em combinação com AloAg-doador e/ou o anti-inflamatório. Em contraste, a terapia oral combinada com o probiótico L.lactis selvagem e AloAg-doador aumentou significativamente a sobrevida do enxerto (p=0,01), em comparação com o grupo não tratado. Nesse grupo que teve maior sobrevida do aloenxerto (L,lactis selvagem e AloAg-doador), também observamos maior quantidade de epitélio preservado (p=0,02) e maior expressão de TGF-beta (p=0,04), no enxerto, em comparação com o grupo sem tratamento. Não observamos diferenças significativas na expressão, in situ, de FOXP3 e IL-17, que foi baixa em todos os grupos experimentais. Concluímos que a Hsp65 não induziu efeito imunorregulador capaz de prolongar a sobrevida do enxerto. No entanto, a manipulação da microbiota com a terapia combinada com o L.lactis selvagem e a exposição a antígenos do doador, previamente, ao transplante, induz mecanismos imunorreguladores capazes de controlar, mesmo que parcialmente, as respostas inflamatórias dirigidas ao aloenxerto de pele, provavelmente, com a participação de TGF-beta / Despite the success of clinical transplantation, the significant side effects induced by immunosupressants used to prevent and treat rejection, indicate the need for novel immunoregulatory therapies. The oral route has been effective in inducing immunoregulation in several experimental models, mostly in pathological autoimmunity. Heat Shock protein 60/65 (Hsp) displays great immunotherapeutic potential due to its capacity to induce both pro-inflammatory and immunregulatory responses. We tested whether oral therapy with the probiotic Lactococcus lactis that expresses Hsp65, in combination with donor alloantigens (Donor-Allo-Ag), acted synergically, inducing immunotolerance or increasing graft survival, in a murine model of semiallogeneic skin transplantation. We tested different oral therapy combinations, as well as the association with a COX-2 selective nonsteroidal anti-inflammatory drug (celecoxib). Skin transplantation was performed 10 days after the last oral administration of probiotics and Donor-Allo-Ag. We observed no beneficial effect on graft survival in the group that received L.lactis that produce Hsp65, alone or in combination with Donor-Allo-Ag/and/or the anti-inflammatory drug. In contrast, combined oral therapy with wild type L.lactis and Donor-Allo-Ag significantly prolonged graft survival (p=0.01), in comparison to non-treated animals. In this prolonged-survival group (L.lactis and Donor-Allo-Ag), we also found higher extension of preserved epithelium (p=0.02) and higher expression of TGF-beta (p=0.04), within the graft, in comparison to non-treated animals. We found no significant differences in the intragraft expression of FOXP3 and IL-17, which was essentially absent or very low. We conclude that Hsp65 did not induce immunoregulatory effects capable of prolonging graft survival. However, the microbiota manipulation with the combined oral therapy with wild type L.lactis and Donor-Allo-Ag, prior to transplantation, induce immunoregulatory mechanisms capable of partially controlling the inflammatory responses to the graft, most likely involving the participation of TGF-beta