Preclinical research using animals often precedes and informs clinical trials; however, most attempts to translate findings from “bench-to-bedside” fail. There is growing concern that an important cause of failed translations is that much of preclinical research is not reproducible, with poor experimental methodology believed to be a major contributor. Four studies were conducted: (1) an assessment of reported study designs of preclinical experiments published in leading cardiovascular journals; (2) an examination of sex bias in preclinical cardiovascular research; (3) a comparison of experimental practices between male and female preclinical cardiovascular researchers; and (4) an analysis of the influence of journal initiatives on preclinical research quality. These studies suggest that (1) methodological shortcomings are prevalent and persistent in preclinical cardiovascular research; (2) women’s involvement in preclinical cardiovascular research is positively associated with considering sex as a biological variable; and (3) journals can exert considerable influence on the quality of published data.

Objetivos: Comparar os efeitos dos tratamentos com montelucaste e dexametasona no recrutamento eosinofílico e na avaliação de células positivas para eotaxina, RANTES, fibronectina, IGF-I e NF-B tanto no parênquima pulmonar distal, quanto nas vias aéreas de cobaias com inflamação alérgica crônica. Métodos: As cobaias receberam inalação com ovoalbumina (grupo OVA- 2 vezes semanais, durante 4 semanas, totalizando 7 inalações). Após a quarta inalação, as cobaias foram tratadas com montelucaste (grupo OVA-M: 10mg/Kg/VO/dia) ou dexametasona ( grupo OVA-D: 5mg/Kg/IP/dia). Após 72 horas da sétima inalação, as cobaias foram anestesiadas e os pulmões foram removidos e submetidos a avaliação histopatológica. Resultados: Os tratamentos com montelucaste e dexametasona reduziram o número de eosinófilos tanto no parênquima pulmonar distal quanto nas vias aéreas, quando comparados ao grupo OVA (p<0.05). No parênquima pulmonary distal, ambos os tratamentos foram efetivos na redução de células positivas para RANTES, NF-B e fibronectina, quando comparados ao grupo OVA (p<0.001). O tratamento com montelucaste mostrou melhor eficácia na redução de células positivas para eotaxina, quando comparado ao tratamento com dexametasona (p<0.001), por outro lado, o tratamento com dexametasona mostrou-se mais significativo na redução de células positivas para IGF-I, quando comparado ao tratamento com montelucaste (p<0.001). Nas vias aéreas, ambos os tratamentos foram efetivos na redução de células positivas para IGF-I, RANTES e fibronectina, quando comparados ao grupo OVA (p<0.05). O tratamento com dexametasona foi mais efetivo na redução de células positivas para eotaxina e NF-B, quando comparado ao tratamento com montelucaste (p<0.05). Conclusões: Neste modelo animal, ambos os tratamentos foram efetivos no controle da resposta inflamatória, tanto no parênquima pulmonar distal, quanto nas vias aéreas / Aims: Compare the effects of montelukast or dexamethasone treatments on eosinophilic recruitment, eotaxin, RANTES, fibronectin, IGF-I and NF-B positive cells of distal lung parenchyma and also in airway walls of guinea pigs (GP) with chronic allergic inflammation. Methods: GP were inhaled with ovalbumin (OVA group-2x/week/4weeks). After 4th inhalation, GP were treated with montelukast (M group: 10mg/Kg/PO/day) or dexamethasone (D group: 5mg/Kg/IP/day). After 72 hrs of 7th inhalation, GP were anesthetised, lungs were removed and submitted to histopathological evaluation. Results: Montelukast and dexamethasone treatments reduced the number of eosinophils both in airway wall as well as in distal lung parenchyma compared to OVA group (p<0.05). On distal parenchyma both montelukast and dexamethasone were effective in reducing RANTES, NF-B and fibronectin positive cells compared to OVA group (p<0.001). Montelukast was more effective in reducing the eotaxin positive cells on distal parenchyma compared to dexamethasone treatment (p<0.001), while there was a more expressive reduction of IGF-I positive cells in OVA-D group (p<0.001). On airway walls, both montelukast and dexamethasone were effective in reducing IGF-I, RANTES and fibronectin positive cells compared to OVA group (p<0.05). Dexamethasone was more effective reducing the number of eotaxin and NF-kB positive cells than Montelukast (p<0.05). Conclusions: In this animal model, both treatments were effective in modulating the eosinophilic response in distal lung parenchyma and in airway wall, contributing to a better control of the inflammatory response in distal lung parenchyma as well as in airway walls. Dexamethasone treatment induced a greater reduction of NF-B expression in airway walls which suggests one of the mechanisms that explains the higher efficacy of this therapeutic approach

Made available in DSpace on 2016-04-29T13:17:52Z (GMT). No. of bitstreams: 1 Maria Isabel Clemencio Pires de Camargo.pdf: 787435 bytes, checksum: f6b7e6afb7d42cdc656677e7a742c35e (MD5) Previous issue date: 2014-03-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / While it is important to implement experimental findings into clinical practice, there is still a gap in the area. In order to clarify it, this research aimed to conduct a historical review of Brazilian theses and dissertations on experimental models of psychopathology in behavior analysis. Seven models were chosen for examination: two for anxiety (elevated plus-maze and conditioned suppression), three for depression (social isolation, learned helplessness and chronic mild stress) and two for schizophrenia (prepulse inhibition and latent inhibition). The study was conducted in two stages, focusing on the following aspects: (a) institution, (b) pathology, (c) experimental model, (d) experimental subject, (e) year of thesis defense, (f) author, (g) thesis advisor, (h) title, (i) examination board, (j) type of document, (k) purpose of the research, (l) processes and criteria, (m) results, (n) treatment, and (o) interface with the clinic. In total, 57 dissertations and 15 theses were identified concerning the subject. The University of São Paulo (USP) produced the majority of works, Maria Helena Hunziker being the main thesis advisor in this field. Most works revolved around depression (41), investigated specially by learned helplessness model, primarily using rats as experimental subjects from the year 2000. In addition, from 71 studies identified, only 13 mentioned the treatment of the investigated pathology, but none established an interface with the clinic. The results of this research indicate that the study of psychopathologies through experimental models is a growing area in Brazil, but there is still much to do in building a connection between laboratory and clinic / Embora seja importante transpor achados experimentais à prática clínica, ainda há uma lacuna na área. A fim de esclarecê-la, esta pesquisa teve por objetivo realizar uma revisão histórica de teses e dissertações brasileiras sobre modelos experimentais de psicopatologias na análise do comportamento. Foram escolhidos para exame sete modelos: dois para ansiedade (labirinto em cruz elevado e supressão condicionada), três para depressão (isolamento social, desamparo aprendido e chronic mild stress) e dois para esquizofrenia (inibição pré-pulso e inibição latente). O estudo foi conduzido em duas etapas, focando os seguintes aspectos: (a) instituição, (b) patologia, (c) modelo experimental, (d) sujeito experimental, (e) ano de defesa, (f) autor, (g) orientador, (h) título, (i) banca, (j) tipo de documento, (k) objetivo da pesquisa, (l) processos e critérios, (m) resultados, (n) tratamento e (o) interface com a clínica. No total, identificaram-se 57 dissertações e 15 teses sobre o tema. A USP produziu o maior número de trabalhos, sendo Maria Helena Hunziker a principal orientadora na área. A maioria dos trabalhos versou sobre depressão (41), investigada sobretudo pelo modelo de desamparo aprendido, principalmente utilizando ratos como sujeitos experimentais a partir do ano 2000. Além disso, das 71 pesquisas identificadas, apenas 13 fizeram menção ao tratamento da patologia investigada, mas nenhuma delas estabeleceu a interface com a clínica. Os resultados deste trabalho indicam que o estudo de psicopatologias por meio de modelos experimentais é uma área em expansão no Brasil, mas ainda há muito a se fazer na construção da ponte entre laboratório e clínica

Objetivos: Comparar os efeitos dos tratamentos com montelucaste e dexametasona no recrutamento eosinofílico e na avaliação de células positivas para eotaxina, RANTES, fibronectina, IGF-I e NF-B tanto no parênquima pulmonar distal, quanto nas vias aéreas de cobaias com inflamação alérgica crônica. Métodos: As cobaias receberam inalação com ovoalbumina (grupo OVA- 2 vezes semanais, durante 4 semanas, totalizando 7 inalações). Após a quarta inalação, as cobaias foram tratadas com montelucaste (grupo OVA-M: 10mg/Kg/VO/dia) ou dexametasona ( grupo OVA-D: 5mg/Kg/IP/dia). Após 72 horas da sétima inalação, as cobaias foram anestesiadas e os pulmões foram removidos e submetidos a avaliação histopatológica. Resultados: Os tratamentos com montelucaste e dexametasona reduziram o número de eosinófilos tanto no parênquima pulmonar distal quanto nas vias aéreas, quando comparados ao grupo OVA (p<0.05). No parênquima pulmonary distal, ambos os tratamentos foram efetivos na redução de células positivas para RANTES, NF-B e fibronectina, quando comparados ao grupo OVA (p<0.001). O tratamento com montelucaste mostrou melhor eficácia na redução de células positivas para eotaxina, quando comparado ao tratamento com dexametasona (p<0.001), por outro lado, o tratamento com dexametasona mostrou-se mais significativo na redução de células positivas para IGF-I, quando comparado ao tratamento com montelucaste (p<0.001). Nas vias aéreas, ambos os tratamentos foram efetivos na redução de células positivas para IGF-I, RANTES e fibronectina, quando comparados ao grupo OVA (p<0.05). O tratamento com dexametasona foi mais efetivo na redução de células positivas para eotaxina e NF-B, quando comparado ao tratamento com montelucaste (p<0.05). Conclusões: Neste modelo animal, ambos os tratamentos foram efetivos no controle da resposta inflamatória, tanto no parênquima pulmonar distal, quanto nas vias aéreas / Aims: Compare the effects of montelukast or dexamethasone treatments on eosinophilic recruitment, eotaxin, RANTES, fibronectin, IGF-I and NF-B positive cells of distal lung parenchyma and also in airway walls of guinea pigs (GP) with chronic allergic inflammation. Methods: GP were inhaled with ovalbumin (OVA group-2x/week/4weeks). After 4th inhalation, GP were treated with montelukast (M group: 10mg/Kg/PO/day) or dexamethasone (D group: 5mg/Kg/IP/day). After 72 hrs of 7th inhalation, GP were anesthetised, lungs were removed and submitted to histopathological evaluation. Results: Montelukast and dexamethasone treatments reduced the number of eosinophils both in airway wall as well as in distal lung parenchyma compared to OVA group (p<0.05). On distal parenchyma both montelukast and dexamethasone were effective in reducing RANTES, NF-B and fibronectin positive cells compared to OVA group (p<0.001). Montelukast was more effective in reducing the eotaxin positive cells on distal parenchyma compared to dexamethasone treatment (p<0.001), while there was a more expressive reduction of IGF-I positive cells in OVA-D group (p<0.001). On airway walls, both montelukast and dexamethasone were effective in reducing IGF-I, RANTES and fibronectin positive cells compared to OVA group (p<0.05). Dexamethasone was more effective reducing the number of eotaxin and NF-kB positive cells than Montelukast (p<0.05). Conclusions: In this animal model, both treatments were effective in modulating the eosinophilic response in distal lung parenchyma and in airway wall, contributing to a better control of the inflammatory response in distal lung parenchyma as well as in airway walls. Dexamethasone treatment induced a greater reduction of NF-B expression in airway walls which suggests one of the mechanisms that explains the higher efficacy of this therapeutic approach

New technologies to probe the global output of the normal and cancer genomes have recently reached widespread use. The resulting genomewide gene expression profiles, e.g, a gene expression measurement per gene and per tissue sample, remain challenging to analyze and interpret, but have already provided new insights into the pathophysiology of cancer and towards personalized care. In vitro cell culture-based experimental models are used to elucidate cancer onset and progression because experimentation in humans is difficult practically and ethically unacceptable, and because they provide simplified, reproducible and controlled systems to test hypotheses. The thyroid tumors and their in vitro experimental models are particularly suited to compare the molecular phenotypes of experimental models and tumors. From one type of cell, the thyrocyte, at least five distinct benign and malignant tumors can arise. In addition, many immortalized tumor-derived cell lines and primary cultures models of these cells exist. This thesis has focused on the bioinformatic comparison of these in vitro models to the in vivo tumors, from the point of view of their gene expression profiles, to gain insight into the pathogenesis of thyroid tumors, and of tumors in general. In a first study, we showed that primary cultures of freshly isolated normal thyroid cells where proliferation and differentiation through the TSHR/cAMP pathway was chronically activated experimentally resemble specifically the autonomous thyroid adenomas, a type of benign thyroid tumor, and provide insight into a general mechanism of tumor progression: the suppression of negative feedbacks that normally restrain excessive cell division. Subsequently, we found that immortalized thyroid tumor-derived cell lines have converged to a common phenotype regardless of their tumor subtype of origin. A TSHR/cAMP thyroid cell differentiation signature, derived from data obtained for the first study, was used to show that the cell lines were dedifferentiated. Accordingly, we showed that the cell lines resemble most the phenotype of the more dedifferentiated, clinically aggressive anaplastic thyroid cancers. Finally, using large databases of gene expression profiles publicly available, we extended the comparison of cell lines and tumors to cancers of five other organs: breast, colon, kidney, ovary and lung. We discuss the correct use of these models and advance an hypothesis regarding the nature of the state to which these cells have converged: they could represent a surviving subpopulation of tumors cells, cancer stem cells, capable of initiating and maintaining tumor growth. As other technologies designed to perturb the genome in experimental models are emerging, careful characterization and validation of the experimental models are needed to extrapolate the results in vivo. Although many differences exist between the experimental models and their in vivo disease counterparts, focusing on the similarities could provide a path to design successful therapeutic interventions more systematically.

New technologies to probe the global output of the normal and cancer genomes have recently reached widespread use. The resulting genomewide gene expression profiles, e.g, a gene expression measurement per gene and per tissue sample, remain challenging to analyze and interpret, but have already provided new insights into the pathophysiology of cancer and towards personalized care. In vitro cell culture-based experimental models are used to elucidate cancer onset and progression because experimentation in humans is difficult practically and ethically unacceptable, and because they provide simplified, reproducible and controlled systems to test hypotheses. The thyroid tumors and their in vitro experimental models are particularly suited to compare the molecular phenotypes of experimental models and tumors. From one type of cell, the thyrocyte, at least five distinct benign and malignant tumors can arise. In addition, many immortalized tumor-derived cell lines and primary cultures models of these cells exist. This thesis has focused on the bioinformatic comparison of these in vitro models to the in vivo tumors, from the point of view of their gene expression profiles, to gain insight into the pathogenesis of thyroid tumors, and of tumors in general. In a first study, we showed that primary cultures of freshly isolated normal thyroid cells where proliferation and differentiation through the TSHR/cAMP pathway was chronically activated experimentally resemble specifically the autonomous thyroid adenomas, a type of benign thyroid tumor, and provide insight into a general mechanism of tumor progression: the suppression of negative feedbacks that normally restrain excessive cell division. Subsequently, we found that immortalized thyroid tumor-derived cell lines have converged to a common phenotype regardless of their tumor subtype of origin. A TSHR/cAMP thyroid cell differentiation signature, derived from data obtained for the first study, was used to show that the cell lines were dedifferentiated. Accordingly, we showed that the cell lines resemble most the phenotype of the more dedifferentiated, clinically aggressive anaplastic thyroid cancers. Finally, using large databases of gene expression profiles publicly available, we extended the comparison of cell lines and tumors to cancers of five other organs: breast, colon, kidney, ovary and lung. We discuss the correct use of these models and advance an hypothesis regarding the nature of the state to which these cells have converged: they could represent a surviving subpopulation of tumors cells, cancer stem cells, capable of initiating and maintaining tumor growth. As other technologies designed to perturb the genome in experimental models are emerging, careful characterization and validation of the experimental models are needed to extrapolate the results in vivo.

No description available.

Η χειρουργική καταρράκτη διαμέσου μικρής τομής με τη χρήση αναδιπλούμενων ενδοφθάλμιων φακών είχε ως αποτέλεσμα την εμφάνιση μιας νέας επιπλοκής˙ της θόλωσης των φακών. Αρκετοί παράγοντες έχουν ενοχοποιηθεί για την αιτιολόγηση του φαινομένου. Μεταξύ αυτών αναφέρονται η μετεγχειρητική φλεγμονή, η χρήση διαλυμάτων πλύσης και ιξωδοελαστικών υλικών, η σιλικόνη, τα λιπαρά οξέα που εμπεριέχονται στο υδατοειδές υγρό. Προηγούμενες αναφορές έχουν αποδώσει την αιτία του φαινομένου της θόλωσης των ενδοφακών στην ασβεστοποίηση. Για τη μελέτη του φαινομένου της ασβεστοποίησης έχουν κατά καιρούς χρησιμοποιηθεί in vivo μοντέλα. Στα μοντέλα αυτά γίνονται απλοποιήσεις ώστε να προσομοιώνονται όσο το δυνατόν καλύτερα οι φυσικοχημικές συνθήκες των βιολογικών ρευστών που είναι σε επαφή με τους αντίστοιχους ιστούς. Ακριβείς θερμοδυναμικές μετρήσεις και μελέτες της κινητικής στις συνθήκες αυτές είναι δυνατόν να δώσουν χρήσιμες πληροφορίες όσον αφορά στην εμφάνιση και εξέλιξη με το χρόνο του φαινομένου της ασβεστοποίησης διάφορων ιστών ή οργάνων. Στην παρούσα εργασία, και με στόχο να προσομoιωθούν οι συνθήκες του προσθίου θαλάμου κατά την επαφή του φακού με το υδατοειδές υγρό, κατασκευάσθηκε από πολυαμίδιο διπλότοιχος θερμοστατούμενος αντιδραστήρας συνολικού όγκου 10 ml. Ο πυθμένας και το άνω μέρος του αντιδραστήρα ήταν από γυαλί, έτσι ώστε το σύνολο να είναι δυνατόν να διευθετηθεί στο έδρανο οπτικού μικροσκοπίου διερχομένου φωτός, προκειμένου να είναι δυνατή η συνεχής παρατήρηση των δοκιμίων. Το συνθετικό υδατοειδές υγρό παρασκευάστηκε με τριπλά απεσταγμένο νερό στο οποίο διαλύθηκαν συγκεκριμένες ποσότητες κρυσταλλικών αλάτων έτσι ώστε η τελική σύσταση να αντιστοιχεί στη σύσταση του υδατοειδούς υγρού, σύμφωνα με την βιβλιογραφία. Η ροή του συνθετικού υδατοειδούς υγρού προς τον αντιδραστήρα γινόταν με τη βοήθεια αντλίας σύριγγας ρυθμιζόμενης παροχής. Ο ρυθμός ροής ήταν 0.2ml/h όπως και στην περίπτωση της ροής του υδατοειδούς υγρού στον πρόσθιο θάλαμο του φακού. Εντός του αντιδραστήρα τοποθετήθηκαν σε ειδικό δειγματοφορέα τρεις υδρόφιλοι ακρυλικοί ενδοφθάλμιοι φακοί (Α, Β και Γ) με περιεκτικότητα σε νερό 26% κ.β. Η μελέτη του συστήματος έγινε σε συνθήκες αντιπροσωπευτικές του oργανισμού, δηλαδή pH=7.4 και θερμοκρασία=37˚C. Η παρακολούθηση των φακών με τη βοήθεια του οπτικού μικροσκοπίου, το οποίο ήταν εφοδιασμένο με βιντεοκάμερα, γινόταν καθημερινά και λαμβάνονταν φωτογραφίες για περαιτέρω ανάλυση. Οι φακοί Α, Β και Γ απομακρύνθηκαν από τον αντιδραστήρα στους πέντε, εννιά και δώδεκα μήνες αντίστοιχα, με σκοπό να μελετηθεί τόσο η επιφάνειά τους όσο και το εσωτερικό του πολυμερικού υλικού. Τα μορφολογικά χαρακτηριστικά των εναποθέσεων εξετάσθηκαν και μελετήθηκαν με τη βοήθεια ηλεκτρονικού μικροσκοπίου σάρωσης ενώ η χημική σύσταση των κρυσταλλιτών ταυτοποιήθηκε με μικροανάλυση με φασματοσκοπία ενεργειακής διασποράς ακτίνων Χ (EDX). Περαιτέρω ταυτοποίηση έγινε με φασματοσκοπικές μεθόδους (φασματοσκοπία Raman) και με περίθλαση ακτίνων Χ. Η τελευταία τεχνική, λόγω της μικρής αναλογίας κρυσταλλικό στερεό/πολυμερές, δεν έδωσε αποτελέσματα και ως εκ τούτου δεν είναι δυνατόν να χρησιμοποιηθεί ως τεχνική ταυτοποίησης. Η χρήση της προϋποθέτει διαφορετική γεωμετρία δείγματος-ανιχνευτή. Ο υπερκορεσμός είναι η κινητήρια δύναμη για την έναρξη της πυρηνογένεσης και της εν συνεχεία ανάπτυξης των σταθερών πυρήνων σε κρυσταλλίτες φωσφορικού ασβεστίου. Δεδομένου του ότι, η συγκέντρωση ασβεστίου του υδατοειδούς υγρού είναι χαμηλή, περίπου η μισή της αντίστοιχης του πλάσματος, υπετέθη, ότι κάθε αιτία τοπικής αύξησης του ασβεστίου και του φωσφόρου, εντός του υδατοειδούς υγρού, μπορεί ενδεχομένως να καταλήξει σε δυστροφική ασβεστοποίηση των φακών. Η μελέτη των φακών και η αξιολόγηση του φαινομένου στο πειραματικό μοντέλο που δημιουργήθηκε στην παρούσα εργασία έγιναν σε συνθήκες σταθερού υπερκορεσμού, δεδομένου του ότι υπήρξε συνεχής ανανέωση του υδατοειδούς υγρού εντός του αντιδραστήρα, κατά τρόπο κατάλληλο ώστε να επιτευχθεί ικανοποιητική προσομοίωση των in vivo συνθηκών. Τα περισσότερα βιολογικά ρευστά, περιλαμβανομένου του υδατοειδούς υγρού είναι υπέρκορα ως προς διάφορες φάσεις αλάτων φωσφορικού ασβεστίου, οι οποίες κατά σειρά μειωμένης διαλυτότητας είναι το διένυδρο φωσφορικό ασβέστιο (CaHPO42H2O, DCPD), το φωσφορικό τριασβέστιο (Ca3(PO4)2, TCP), το φωσφορικό οκτασβέστιο (Ca8H2(PO4)65 H2O, OCP) και ο υδροξυαπατίτης (Ca10(PO4)6(OH)2, HAP). Η διαλυτότητα των κρυσταλλικών αυτών φάσεων και ο υπερκορεσμός των διαλυμάτων καθορίζονται από παράγοντες όπως η θερμοκρασία, το pH κτλ. Η τάση για καταβύθιση και σχηματισμό συγκεκριμένης φάσης κρυσταλλικού ασβεστίου εντός του υδατοειδούς υγρού, μπορεί να καθοριστεί από το διάγραμμα διαλυτότητας και εξαρτάται από το pH του διαλύματος και από τη θερμοκρασία. Σε υψηλές τιμές υπερκορεσμού και σε διαλύματα με υψηλό pH, έχει ταυτοποιηθεί ο σχηματισμός και η σταθεροποίηση πρόδρομων φάσεων, ενώ σε συνθήκες χαμηλού υπερκορεσμού και μειωμένου pH σχηματίζεται απ’ ευθείας ΗΑΡ. Η διαδικασία της ασβεστοποίησης των ενδοφακών επηρεάζεται από παράγοντες όπως η δομή και η κατεργασία του πολυμερούς υλικού βάσης (μήτρα), η παρουσία πόρων και η περιεκτικότητά του σε νερό. Τα ακρυλικά πολυμερή διαθέτουν επιφανειακές ιονιζόμενες καρβοξυλικές ομάδες, η παρουσία των οποίων σε ιονισμένη μορφή (-COO-, pH>4) ευνοεί τη συμπλοκοποίηση με ιόντα ασβεστίου του υδατοειδούς υγρού. Τα επιφανειακά αυτά σύμπλοκα είναι πιθανόν να λειτουργούν ως ενεργά κέντρα για την πυρηνογένεση και περαιτέρω ανάπτυξη των κρυστάλλων του φωσφορικού ασβεστίου. Επιπλέον, η ασβεστοποίηση φαίνεται ότι είναι έντονη στους υδρόφιλους φακούς, λόγω της υψηλότερης ενυδάτωσης των υδρόφιλων πολυμερών, η οποία έχει ως αποτέλεσμα την υψηλότερη επιφανειακή συγκέντρωση των ιονισμένων ομάδων (ομάδες / m2) και συνεπώς και των επιφανειακών συμπλόκων- ενεργών κέντρων για την κρυσταλλική ανάπτυξη. Στην παρούσα εργασία και στο in vitro μοντέλο με τη χρησιμοποίηση ενός υδρόφιλου φακού, διαπιστώθηκε ότι η διαδικασία της ασβεστοποίησης λαμβάνει χώρα στο εσωτερικό των φακών, στην πολυμερική μήτρα. Οι σχηματισμοί στο πειραματικό μοντέλο εντοπίσθηκαν στο εσωτερικό των ενδοφακών και με την πάροδο του χρόνου, διαπιστώθηκε ότι μετατοπίζονταν προς τα επιφανειακά στρώματα των φακών. Επίσης, οι εναποθέσεις εμφανίσθηκαν ως γραμμικό μέτωπο παράλληλο προς την γραμμή της επιφάνειας των ενδοφακών. Τόσο η μορφολογική εξέταση των εναποθέσεων στο εσωτερικό όσο και η φασματοσκοπική τους ταυτοποίηση, έδειξε ότι η σύστασή τους ήταν εξ’ ολοκλήρου ΗΑΡ, χωρίς να αποκλείεται και ο σχηματισμός πρόδρομης φάσης OCP, δεδομένου του ότι βρέθηκαν κρυσταλλίτες οι οποίοι μορφολογικά παρουσιάζουν μεγάλη ομοιότητα στη φάση αυτή. Η ερμηνεία της διαπίστωσης του γεγονότος ότι η ασβεστοποίηση λαμβάνει χώρα στο εσωτερικό των ενδοφακών, συνίσταται στο ότι η διάχυση των ιόντων Ca2+ και PO43- εντός της πολυμερικής μήτρας, προχωρεί μέχρι τέτοιο βαθμό ώστε να δημιουργούνται στο εσωτερικό του πολυμερούς συνθήκες τοπικού υπερκορεσμού, κατάλληλες για τη δημιουργία των αντίστοιχων κρυστάλλων. Με την υπόθεση ότι οι τιμές των συντελεστών διάχυσής των ιόντων Ca2+ και PO43- εντός της πολυμερικής μήτρας, είναι της αυτής τάξεως μεγέθους είναι δυνατόν να εξηγηθεί και η εμφάνιση μετώπου συγκέντρωσης των. Η συσσώρευση των ιόντων τα οποία αποτελούν τα δομικά συστατικά του ΗΑΡ συνεχίζεται μέχρις ότου επιτευχθεί μία κρίσιμη τιμή υπερκορεσμού. Σε αυτό το σημείο σχηματίζονται οι πυρήνες και λαμβάνει χώρα η κρυσταλλική ανάπτυξη με τον ερχομό και συσσώρευση επιπρόσθετων ιόντων. Η ασβεστοποίηση των υδρόφιλων ενδοφακών είναι μία προοδευτική και συνεχώς εξελισσόμενη διαδικασία μετά την εμφύτευσή τους και όσο περισσότερο αφήνεται να εξελιχθεί τόσο αυξάνεται η πυκνότητα των εναποθέσεων στο εσωτερικό των φακών. Η επιφάνεια μπορεί να προσβληθεί μόνο σε όψιμες φάσεις και αρκετά χρόνια μετά την εμφύτευση των φακών. Στο σημείο αυτό κρύσταλλοι μπορεί να αναπτυχθούν και σε σημεία, στα οποία έχουν δημιουργηθεί στην επιφάνεια του πολυμερούς σχισμές και χαρακιές. Το θέμα της έναρξης της ασβεστοποίησης (εσωτερικό ή επιφάνεια) χρειάζεται περαιτέρω διερεύνηση. Σύμφωνα με κάποιες αναφορές στη βιβλιογραφία, προτείνεται ότι η έναρξη της ασβεστοποίησης γίνεται στην επιφάνεια των ενδοφακών. Στην παρούσα εργασία, σε συνθήκες χαμηλού υπερκορεσμού, ανάλογες με τις αντίστοιχες υγιούς υδατοειδούς υγρού η ασβεστοποίηση έλαβε χώρα στο εσωτερικό της πολυμερικής μήτρας του ενδοφακού και σε χρόνο και με τρόπο ο οποίος μπορεί να εξηγηθεί από τη (βραδεία) διάχυση των δομικών ιόντων του ΗΑΡ στο εσωτερικό της πολυμερικής μήτρας. Τα ευρήματα και τα συμπεράσματα από την ολοκλήρωση της πειραματικής διαδικασίας με τη χρήση υδρόφιλων ενδοφακών με υδρόφοβη επικάλυψη δείχνουν ότι το θέμα της έναρξης της ασβεστοποίησης˙ εσωτερικό ή επιφάνεια φαίνεται ότι εκτός από τον υπερκορεσμό, υψηλός ή χαμηλός, εξαρτάται και από τη φύση του πολυμερούς του φακού˙ υδρόφιλη ή υδρόφοβη. Τα ευρήματα δείχνουν ότι η υδρόφιλη φύση ευνοεί τη διάχυση, ενώ το υδρόφοβο υλικό την έναρξη της ασβεστοποίησης στην επιφάνεια του φακού. Τα ευρήματα δείχνουν ότι η υδρόφιλη φύση ευνοεί τη διάχυση, ενώ το υδρόφοβο υλικό την έναρξη της ασβεστοποίησης στην επιφάνεια του φακού. Συμπερασματικά, η όψιμη μετεγχειρητική ασβεστοποίηση των ενδοφακών συνιστά σοβαρή επιπλοκή και αιτία μείωσης της όρασης. Λόγω του ότι η διαδικασία και η εκδήλωση του φαινομένου έχει καθυστερημένη έναρξη, είναι σημαντική η μακροχρόνια και προσεκτική παρακολούθηση αυτών των ασθενών. Αρκετοί οφθαλμίατροι δεν είναι ενήμεροι ως προς αυτήν την κλινική οντότητα και αναγνωρίζοντάς την βοηθούν ώστε να μην υποβάλλουν τους ασθενείς σε ανώφελες επεμβάσεις. Η χειρουργική εξαίρεση του φακού συνιστά τη μοναδική θεραπευτική προσέγγιση, καθώς ασθενείς με ασβεστοποιημένους φακούς παρουσιάζουν σταδιακή μείωση της οπτικής τους οξύτητας και καμία περίπτωση αυτόματης υποστροφής δεν έχει παρατηρηθεί. / Small incision cataract surgery with foldable IOL implantation resulted in a new postoperative complication, IOL opacification. Various factors implicated in the phenomenon have been suggested, including inflammation, irrigation solutions and viscosurgical devices, silicone and fatty acids contamination. There is increasing evidence that IOL opacification is due to calcification. Modeling in vivo processes by reliable and reproducible in vitro methods is of key importance to understand the underlying mechanisms. Precise thermodynamic calculations of equilibrium speciation in combination with kinetics measurements at conditions simulating the eye environment are expected to yield mechanistic information concerning the formation of calcium phosphate deposits. In the present contribution we have developed an experimental approach for the investigation of the mechanism of calcification of hydrophilic acrylic IOLs. A double walled thermostated reactor was constructed, volume totaling 10 ml made of polyamide. The reactor had glass windows on top and bottom to allow for the direct observation of the IOL specimens in situ using an optical microscope combined with an image analysis system. In the external wall of the reactor, water supplied from a thermostat was circulated in order to maintain the temperature at 37.0  0.2˚C, while in the interior of the reactor constant flow of a simulated aqueous humor solution (SAH) was ensured with the help of a syringe pump. The SAH solution was introduced in the reactor in an once flow mode at a flow rate of 0.2ml/h, simulating the in vivo flow in the anterior chamber, where aqueous humor is fully renewed within 2 hours. Hydrophilic acrylic IOLs (A, B and C) in triplicate, made of Poly-HEMA with 26% water content were placed in a special holder. The observation of IOLs was done in situ daily by optical microscopy, for the assessment of the opacification progress. Five months after the initiation of the experiment, Lens A was removed in order to be inspected, both at the surface and in the interior. The morphology of the deposits was examined using Scanning Electron Microscopy (SEΜ). The composition of the deposits was identified by microanalysis with Energy Dispersive x-ray Spectroscopy (EDS). Lens B was removed on the ninth month, while Lens C was inspected one year after the onset of the experiment. Similar studies including SEM and EDS analysis were used for the investigation of both those lenses. Investigation showed deposits of calcium phosphate crystallites in the interior of opacified IOLs. These deposits however, were not observed on the IOL’s surface. The thermodynamic driving force for the formation of a salt from solution is the difference between the chemical potentials of the salt in solution from the equilibrium. Taking into account that the calcium concentration in normal aqueous humor is low, about half of the respective value in blood serum, it may be assumed that any cause of localized increase in calcium and phosphorus might result in dystrophic calcification. The experiments in the present work were done at conditions of practically constant supersaturation, since the solution in the experimental reactor was flown once through thus providing a reasonable simulation of the in vivo conditions. Heterogeneous nucleation, an almost ubiquitous phenomenon, is initiated at impurity sites and foreign surfaces in contact with supersaturated solutions. The induction time preceding the formation of nuclei of solids which grow to crystals depends on several factors, including temperature, pH, ionic strength, solution composition which determine the thermodynamic driving force for the formation of the solid phase. Biological fluids like blood serum or aqueous humor are supersaturated with respect to a number of different phases of calcium phosphate salts, in the order of decreasing solubility: Calcium phosphate dehydrate (CaHPO42H2O, DCPD), Tricalcium phosphate (Ca3(PO4)2, TCP), Octacalcium phosphate (Ca8H2(PO4)65 H2O, OCP) and Hydroxyapatite (Ca10(PO4)6(OH)2, HAP). HAP is the thermodynamically stable phase while the rest of the crystalline phases are precursors which may be formed and/or stabilized in supersaturated solutions depending on the conditions in the supersaturated solutions. The tendency for particular calcium phosphate phases to form in supersaturated solutions may be estimated from the solubility phase diagrams of calcium phosphates. Unstable precursor phases, if formed, they convert through hydrolytic processes to the thermodynamically most stable. The process of biomaterial calcification seems moreover to be influenced by factors such as polymer structure, polymer porosity and water content. The ionizable surface hydroxyl groups (present in the carboxyl functional groups) available on the surface of the acrylic polymers may act as sites for nucleation and growth of mineral phase, through surface complexation with calcium ions. The presence of larger numbers of –COOH groups on the polymers accelerate the process of Ca-P overgrowth. The higher extend of hydration in hydrophilic materials leads to higher ionization of the surface functional groups, thus promoting calcification through the formation of complexes with Ca2+ ions. The higher calcification incidents observed in IOLs with higher water content has been attributed to this fact. The polar functional groups (-COO) on the surface of the polymeric matrix of IOLs result in the significant increase in electron density and the subsequent reduction of the interfacial energy between the polymer and aqueous solution. The energy barrier to diffusion of calcium and phosphate species from the bulk solution to the substrate is also reduced, through the formation of surface complexes, which favor the accumulation of Ca2+ and PO43- ions, thus promoting calcification. Our in vitro model experiments have shown that IOL’s calcification is a process initiated at the interior of the IOLs tested. SEM investigation in combination with EDS microanalysis, confirmed the presence of HAP crystallites with sizes less than 100nm. Raman spectroscopy analyses of the opacified lenses corroborated the findings for HAP formation in the interior of the polymeric matrix. X-Ray diffraction measurements failed to identify the presence of minerals apparently because of the low content (by mass) of solid in the polymeric matrix. Supersaturation of the aqueous humor, with respect to calcium phosphate, is the driving force a necessary condition for nucleation of Ca-P salts. The formations observed in our experimental set-up which simulated closely in vivo conditions, were found exclusively in the interior of the IOLs. The solid deposits formed linear fronts parallel to the lens surfaces, advancing with time. It may be suggested therefore that the deposits fronts is the result of diffusion of Ca2+, PO43- and OH- ions through the polymer matrix in contact with the polymer. Assuming similar values for the diffusion coefficients of Ca2+, PO43- and OH- ions in the gel (bulk polymer) material the formation of linear deposits fronts may be explained from the formation of the supersaturated solutions at a depth in which a critical supersaturation was reached. At this point calcium phosphate nuclei form and grow with the arrival of additional ions. IOL’s calcification is an ongoing process after IOL implantation, and the longer the process proceeds, the density of the deposits in the interior of the IOL increases. In all of the cases surface was free of deposits and the distortions that were observed are thought to be due to changes in polymer structure in the IOL’s interior. Surface can be affected only in late phases of calcification and many years after IOL implantation. At this stage crystals may outgrow especially at places in which the polymer’s surface has developed fissures. The issue of calcification’s process initiation (interior or surface) needs further investigation. Reports in the literature, suggest that calcification is initiated on the surface of the IOL. In our experiment where low supersaturation conditions have been achieved, diffusion was favored resulting in calcification at the interior of the polymeric matrix. Investigation and analysis of IOLs with hydrophobic surface confirmed that the issue of calcification`s process initiation is more over influenced by factors others than supersaturation conditions, such as the hydrophilic or hydrophobic nature of the IOLs surface. The experimental analysis proved that hydrophilic IOLs favor ions diffusion while hydrophobic material limits calcification process on the surface. In conclusion, late postoperative IOL opacification causes a severe loss of visual acuity. Because calcification process appears to be of delayed onset it is important to be vigilant in the long-term follow-up of these patients. Many ophthalmologists are not aware of this clinical problem and recognizing this phenomenon will help prevent patients from undergoing useless procedures. IOL exchange is the only therapeutic approach, in such patients, as patients with calcified IOLs have gradual deterioration of their visual acuity and no case of spontaneous recovery has been observed.

A ventilação natural pode ser promovida por dois mecanismos: o denominado efeito chaminé e o efeito dos ventos, porém também podem ocorrer os dois mecanismos simultaneamente. A determinação das vazões de ventilação por efeito do vento, em pavilhões industriais, pode ser realizada a partir de ensaios de modelos reduzidos em túnel de vento, com medição das velocidades do escoamento nas aberturas, ou então, com medição dos coeficientes de pressão em modelos fechados, nas faces onde estão localizadas as aberturas. A metodologia com medição dos coeficientes de pressão, que exige o emprego de um modelo teórico para calcular as vazões, é a forma convencional de usar o túnel de vento como ferramenta no projeto de ventilação. O objetivo deste trabalho é avaliar os procedimentos citados, para calcular as vazões de ventilação por efeito do vento em pavilhões industriais. Para atingir esse objetivo foram ensaiados em túnel de vento dois modelos reduzidos (escala geométrica 1:200) de um pavilhão industrial: um sem aberturas, para a medição dos coeficientes de pressão, e outro, com aberturas de ventilação de área variável, para a medição das velocidades do vento com anemômetro de fio quente. Além da comparação das duas metodologias para determinação das vazões de ventilação, os resultados dos ensaios foram também utilizados para analisar as alterações nas vazões de ventilação e nas velocidades do escoamento do ar decorrentes do fechamento de algumas aberturas do modelo estudado. Conclui-se que as aberturas localizadas na cumeeira e o tipo de escoamento exercem grande influência nas vazões de ventilação. A utilização da metodologia de medição direta de velocidades através de anemômetro de fio quente permite a obtenção de vazões de ventilação sem iterações, com resultados compatíveis com os modelos teóricos que se baseiam na diferença de pressões externas e internas. / Natural ventilation in industrial buildings may be promoted by two mechanisms: one due to wind effects and other called chimney effect. However, the two mechanisms may also occur simultaneously. The determination of wind induced ventilation flows in industrial buildings may be performed through wind tunnel tests, with measurement of discharge velocity at openings, or else, with measurement of the pressure coefficients at the face of the buildings, where the openings are located. The methodology which employs the pressure coefficients requires the use of a theoretical model to calculate the flows, being the conventional way of using the wind tunnel as a tool in the ventilation project. The objective of this work is to evaluate the experimental procedures previously mentioned in order to be able to calculate the wind induced ventilation flows in industrial buildings. To reach this objective, two reduced models of an industrial building were tested at a geometric scale 1:200; one without openings, for the measurement of the pressure coefficients, and the other with ventilation openings with variable area, for the measurement of the wind velocities by the use of hot-wire anemometer. Different opening combinations were also tested and the flows analyzed. It is concluded that the opening types and the different flows exert a big influence in the ventilation flows and, therefore, in the natural ventilation itself. Also, the direct velocity measurement technique, through hot-wire anemometer, allows the ventilation flows to obtained directly, without interactions, being the results compatible with the theoretical models based on pressure differences.

A ventilação natural pode ser promovida por dois mecanismos: o denominado efeito chaminé e o efeito dos ventos, porém também podem ocorrer os dois mecanismos simultaneamente. A determinação das vazões de ventilação por efeito do vento, em pavilhões industriais, pode ser realizada a partir de ensaios de modelos reduzidos em túnel de vento, com medição das velocidades do escoamento nas aberturas, ou então, com medição dos coeficientes de pressão em modelos fechados, nas faces onde estão localizadas as aberturas. A metodologia com medição dos coeficientes de pressão, que exige o emprego de um modelo teórico para calcular as vazões, é a forma convencional de usar o túnel de vento como ferramenta no projeto de ventilação. O objetivo deste trabalho é avaliar os procedimentos citados, para calcular as vazões de ventilação por efeito do vento em pavilhões industriais. Para atingir esse objetivo foram ensaiados em túnel de vento dois modelos reduzidos (escala geométrica 1:200) de um pavilhão industrial: um sem aberturas, para a medição dos coeficientes de pressão, e outro, com aberturas de ventilação de área variável, para a medição das velocidades do vento com anemômetro de fio quente. Além da comparação das duas metodologias para determinação das vazões de ventilação, os resultados dos ensaios foram também utilizados para analisar as alterações nas vazões de ventilação e nas velocidades do escoamento do ar decorrentes do fechamento de algumas aberturas do modelo estudado. Conclui-se que as aberturas localizadas na cumeeira e o tipo de escoamento exercem grande influência nas vazões de ventilação. A utilização da metodologia de medição direta de velocidades através de anemômetro de fio quente permite a obtenção de vazões de ventilação sem iterações, com resultados compatíveis com os modelos teóricos que se baseiam na diferença de pressões externas e internas. / Natural ventilation in industrial buildings may be promoted by two mechanisms: one due to wind effects and other called chimney effect. However, the two mechanisms may also occur simultaneously. The determination of wind induced ventilation flows in industrial buildings may be performed through wind tunnel tests, with measurement of discharge velocity at openings, or else, with measurement of the pressure coefficients at the face of the buildings, where the openings are located. The methodology which employs the pressure coefficients requires the use of a theoretical model to calculate the flows, being the conventional way of using the wind tunnel as a tool in the ventilation project. The objective of this work is to evaluate the experimental procedures previously mentioned in order to be able to calculate the wind induced ventilation flows in industrial buildings. To reach this objective, two reduced models of an industrial building were tested at a geometric scale 1:200; one without openings, for the measurement of the pressure coefficients, and the other with ventilation openings with variable area, for the measurement of the wind velocities by the use of hot-wire anemometer. Different opening combinations were also tested and the flows analyzed. It is concluded that the opening types and the different flows exert a big influence in the ventilation flows and, therefore, in the natural ventilation itself. Also, the direct velocity measurement technique, through hot-wire anemometer, allows the ventilation flows to obtained directly, without interactions, being the results compatible with the theoretical models based on pressure differences.