Bone defects and fracture non-unions remain a substantial challenge for clinicians due to a high occurrence of delayed union or non-union requiring surgical intervention. The current grafting procedures used to treat these injuries have many limitations and further long-term complications associated with them. This has resulted in research efforts to identify graft substitution therapies that are able to repair and replace tissue function. Many of these tissue engineered products include the use of growth factors to induce cell differentiation, migration, proliferation, and/or matrix production. However, current growth factor delivery methods are limited by poor retention of growth factors upon implantation resulting in low bioactivity. These limiting factors lead to the use of high doses and frequent injections, putting the patients at risk for adverse effects. The goal of this work was to develop and evaluate the efficacy of BMP-2 delivery systems to improve bone regeneration. We examined two approaches for delivery of BMP-2 in this work. First, we evaluated the use of a self-assembling lipid microtube system for the sustained delivery of BMP-2. We determined that sustained delivery of BMP-2 from the lipid microtube system was able to enhance osteogenic differentiation compared to empty microtubes, however did not demonstrate a significant advantage compared to a bolus BMP-2 dose in vitro. Second, we developed and assessed the functionality of an affinity-based system to sequester BMP-2 at the implant site and retain bioactivity by incorporating heparin within a collagen matrix. Incorporation of heparin in the collagen matrix improved BMP-2 retention and bioactivity, thus enhancing cell-mediated mineralized matrix deposition in vitro. Lastly, the affinity-based BMP-2 delivery system was evaluated in a challenging in vivo bone repair model. Delivery of pre-bound BMP-2 and heparin in a collagen matrix resulted in new bone formation with mechanical properties not significantly different to those of intact bone. Whereas delivery of BMP-2 in collagen or collagen/heparin matrices had similar volumes of regenerated mineralized tissue but resulted in mechanical properties significantly less than intact bone properties. The work presented in this thesis aimed to address parameters currently preventing optimal performance of protein therapies including stability, duration of exposure, and localization at the treatment site. We were able to demonstrate that sustained delivery of BMP-2 from lipid microtubes was able to induce osteogenic differentiation, although this sustained delivery approach was not significantly advantageous over a bolus dose. Additionally, we demonstrated that the affinity-based system was able to improve BMP-2 retention within the scaffold and in vitro activity. However, in vivo implantation of this system demonstrated that only delivery of pre-complexed BMP-2 and heparin resulted in regeneration of bone with mechanical properties not significantly different from intact bone. These results indicate that delivery of BMP-2 and heparin may be an advantageous strategy for clinically challenging bone defects.

Tissue repair is a complex process that is difficult to emulate. The addition of the glycosaminoglycan heparan sulfate (HS), a multi-potential regulator of numerous growth factors and cytokines endogenously expressed during the repair process, may represent a valuable tool for tissue engineering. The addition of exogenous HS into wound site has previously been shown to promote tissue repair in a number of models, however, the incorporation of HS into controlled release systems or biomaterials for tissue engineering had not been explored prior to the work presented here. Thus, this thesis explores the incorporation of HS and its analogue heparin into synthetic biodegradable polymer biomaterials with different potential applications, either as a slow releasing drug reservoir, or as a drug releasing cell scaffold. Polycaprolactone was used to make microcapsules and electrospun fibers for HS or heparin entrapment. These materials were characterized for their drug release profiles, biocompatibility and bioactivity. Microcapsules encapsulating heparin or HS were made by the oil - in - water solvent evaporation method which allowed fabrication of slow releasing drug reservoirs. Either pure water or a poly(vinyl alcohol) solution was used in the drug phase which resulted in capsules with similar size and drug loading. However the internal morphology and drug release profiles showed differences depending on the drug phase, in either case release was sustained for over 30 days. These capsules elicited no pro-inflammatory response from macrophages in vitro, and the released HS retained its bioactivity to induce the proliferation of human mesenchymal stem cells, an important cell type for bone tissue engineering. Heparin and HS were incorporated into electrospun fibers as a drug releasing scaffold for two different tissue engineering applications. Heparin fibers were studied as a drug releasing membrane that could be used in vascular repair to prevent the unwanted proliferation of vascular smooth muscle cells. Heparin release was sustained from the fibers for at least 2 weeks. The fibers did not induce a pro-inflammatory response from macrophages in vitro and the released heparin retained the ability to inhibit the proliferation in vascular smooth muscle cells. HS fibers were studied as a tissue engineering scaffold for bone repair using human mesenchymal stem cells. HS release was maintained for over 30 days which is thought to be an appropriate time for bone repair applications. The release profiles depended on the HS concentration in the spinning solution which affected the morphology of the fibers. The fibers did not elicit a pro-inflammatory response in cultured macrophages and supported the proliferation and mineralization of human mesechymal stem cells. The HS fibers were then taken through to an in vivo model to study ectopic bone formation of pre-osteoblast cells on HS releasing scaffolds. The fibers produced a chronic inflammatory response in vivo, which lead to the clearance of implanted cells and no mineralization of the scaffold. The HS and heparin materials made in this work showed sustained release over appropriate time frames for different tissue repair applications. The released HS and heparin maintained bioactivity and showed good biocompatibility in vitro, however, further in vivo studies are required to fully test their efficacy for tissue engineering.

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, İç Hastalıkları Anabilim Dalı, 2006. / Bibliyografya var.

Η HARP (Heparin Affin Regulatory Peptide), γνωστή και ως πλειοτροπίνη, είναι ένας αυξητικός παράγοντας που η έκφρασή του στα ενήλικα άτομα είναι περιορισμένη σε συγκεκριμένους ιστούς. Ωστόσο, έκφραση ή υπερέκφρασή της έχει παρατηρηθεί in vivo σε διάφορους όγκους και στον ορό του αίματος ασθενών με διάφορες μορφές καρκίνου, καθώς και in vitro σε διάφορες καρκινικές κυτταρικές σειρές. Παρόλο που οι in vivo βιολογικές δράσεις της HARP είναι αδιαμφισβήτητες, δεν έχει διασαφηνιστεί ο μηχανισμός με τον οποίο ασκεί τις δράσεις αυτές. Επίσης, υπάρχουν πολλά αντικρουόμενα αποτελέσματα αναφορικά με τις in vitro βιολογικές της δράσεις. Στη συγκεκριμένη εργασία διερευνήθηκε το εάν η διαφορετική έκφραση των υποδοχέων της HARP, RPTPβ/ζ και ALK, είναι ένας άλλος λόγος για τα αντικρουόμενα αυτά αποτελέσματα. Χρησιμοποιώντας την RNAi τεχνολογία, δημιουργήσαμε DU145 και PC3 κύτταρα (κυτταρικές σειρές από καρκίνο ανθρώπινου προστάτη), τα οποία σταθερά έχουν μειωμένα επίπεδα έκφρασης του RPTPβ/ζ και του ALK. Τα DU145 κύτταρα εκφράζουν μόνο τον RPTPβ/ζ, σε αντίθεση με τα PC3 κύτταρα που εκφράζουν και τους δύο υποδοχείς. Τα αποτελέσματα έδειξαν ότι ο RPTPβ/ζ καταστέλλει την επαγόμενη από HARP κυτταρική προσκόλληση και μετανάστευση, ενώ ο ALK επάγει την επαγόμενη από HARP κυτταρική μετανάστευση. Επιπλέον, η μελέτη της μεταγωγής σήματος αυτών των υποδοχέων έδειξε ότι ο RPTPβ/ζ καταστέλλει τα επίπεδα φωσφορυλίωσης της κινάσης Src, της Fak, της Pten/Akt και των Erk1/2, ενώ ο ALK επάγει την ενεργότητα της Akt και των Erk1/2. Επιπρόσθετα, η μείωση της έκφρασης του RPTPβ/ζ σχετίζεται με την επαγωγή EMT φαινοτύπου, αφού καταστέλλει την έκφραση της E-καντερίνης και επάγει την έκφραση της Ν-καντερίνης, των ιντεγκρινών-α5, -αv και β3, καθώς και της MMP9. Επιπλέον, είναι γνωστό ότι οι αυξητικοί παράγοντες αποτελούν υπόστρωμα για διάφορα πρωτεολυτικά ένζυμα του κυτταρικού μικροπεριβάλλοντος, με αποτέλεσμα την παραγωγή βιολογικά ενεργών πεπτιδίων που μπορούν να έχουν παρόμοιες ή και αντίθετες δράσεις με το ολικό μόριο. Η πλασμίνη, η τρυψίνη και η MMP2, πέπτουν την HARP και παράγουν πεπτίδια που αναστέλλουν την επαγωγική της δράση. Σύμφωνα με αυτές τις μελέτες, το ενδιαφέρον για την ανακάλυψη πεπτιδίων με αντικαρκινική δράση εντοπίζεται στο καρβοξυτελικό τμήμα της HARP, καθώς και στις δύο κεντρικές περιοχές που παρουσιάζουν ομολογία με τις επαναλαμβανόμενες αλληλουχίες της θρομβοσπονδίνης-1. Στην παρούσα εργασία μελετήθηκε ο μηχανισμός δράσης του P(122-131) και οι βιολογικές δράσεις των P(13-39) και P(65-97). Τα αποτελέσματα έδειξαν ότι το P(122- 131) μετά την πρόσδεσή του στον RPTPβ/ζ, μειώνει τα επίπεδα φωσφορυλίωσης της κινάσης Src, της Fak, της Pten και των Erk1/2 και καταστέλλει την in vitro προσκόλληση και μετανάστευση των DU145 και LNCaP κυττάρων. Επιπλέον, τα αποτελέσματα υποστηρίζουν την υπόθεση ότι το P(122-131) καταστέλλει αυτές τις διαδικασίες και μετά από τον ανταγωνισμό του με τη HARP για την πρόσδεση όχι μόνο στον ALK, αλλά και σε άλλους υποδοχείς. Τέλος, χρησιμοποιώντας το σύστημα της χοριοαλλαντοϊδικής μεμβράνης εμβρύου όρνιθας, παρατηρήσαμε ότι το P(122-131) καταστέλλει και την in vivo αγγειογένεση. Παρόμοια με το P(122-131), τα P(13-39) και P(65-97) καταστέλλουν την in vitro προσκόλληση και μετανάστευση των DU145 και PC3 κυττάρων μετά την πρόσδεσή τους στον RPTPβ/ζ. Συμπερασματικά, στην παρούσα εργασία καταδεικνύεται ο ρόλος των υποδοχέων RPTPβ/ζ και ALK στον μηχανισμό δράσης του αυξητικού παράγοντα HARP και των πεπτιδίων του. Για πρώτη φορά αποδεικνύεται ότι η ανασυνδυασμένη HARP προκαρυωτικής προέλευσης είναι βιολογικά ενεργή και ότι η δράση της εξαρτάται από τη συνισταμένη των δράσεων που έχει κάθε υποδοχέας της, αντικατοπτρίζοντας τον περίπλοκο μηχανισμό δράσης της HARP και των πεπτιδίων της. / HARP (Heparin Affin Regulatory Peptide), also known as Pleiotrophin, is a growth factor that is thought to be involved in carcinogenesis. Elevated concentrations of this growth factor are found in many types of tumors, as well as in the plasma of patients with different types of cancer. However, contradictory results have been published concerning the in vitro activities of HARP. Here, we investigated whether the differential expression of HARP receptors, namely RPTPβ/ζ and ALK, is another reason for these controversies. Using the RNAi technology, we stably transformed prostate cancer cell lines DU145 and PC3 to knockdown RPTPβ/ζ or ALK expression. DU145 cells express only RPTPβ/ζ, while PC3 cells express both RPTPβ/ζ and ALK. Our results showed that RPTPβ/ζ inhibits HARP-mediated cellular adhesion and migration, while ALK induces HARP-mediated cellular migration. Investigation of the transduction mechanism revealed that RPTPβ/ζ inactivates Src, Fak, Pten/Akt, and Erk1/2, while ALK activates Akt and Erk1/2. In addition, RPTPβ/ζ knockdown promotes a shift in expression form E- to N-cadherin, and induces the expression of integrin-α5, -αv, -β3, and MMP9. Growth factors can be hydrolyzed by proteases, leading to the production of biological active peptides. Previous studies indicate that HARP is cleaved by enzymes in the extracellular environment, such as plasmin, trypsin, chymotrypsin, and MMP2. Moreover, the resulting peptides exert altered biological functions compared to the whole molecule. Here, we investigated the effect of (P122-131), corresponding to the basic cluster of the C-terminal region of HARP, as well as the effect of P(13-39) and P(65-97) derived from the TSR domains of HARP. Our results demonstrated that P(122-131) interacts with RPTPβ/ζ, inactivates its catalytic activity, and triggers a signal transduction pathway that inhibits DU145 and LNCaP adhesion and migration, while in parallel interferes with ALK or other pleiotrophin receptors inhibiting pleiotrophin-induced cellular adhesion and migration. In addition, P(122-131) inhibits angiogenesis in vivo, as determined by the chicken embryo CAM assay. Furthermore, P(13-39) and P(65-97) interacts with RPTPβ/ζ and inhibits DU145 and PC3 adhesion and migration. Taken together, the results of this study demonstrate the effect of RPTPβ/ζ and ALK on HARP and its peptides-mediated biological actions. Our results support the hypothesis that the overall effect of pleiotrophin depends on the expression profile of its receptors. Concluding, we show that bacterial pleiotrophin is biological active and part of the diversity of pleiotrophin biological actions is due to RPTPβ/ζ and /or ALK and the complex way of their interactions and signaling.

O objetivo da presente tese foi avaliar os efeitos da heparina de baixo peso molecular (HBPM) na perda óssea alveolar em ratos Wistar. Para a melhor compreensão e entendimento dos efeitos da HBPM se elaborou um único artigo com 40 ratos machos da linhagem Wistar de 60 dias de nascidos, os quais foram dívidos em 4 grupos experimentais previamente randomizados: Grupo Controle (C), Grupos Doença Periodontal (DP), Grupo Heparina (Hp) e Grupo Heparina+Doença Periodontal (Hp+DP) com um período experimental de 60 dias. Um animal foi perdido no período de aclimação, dois animais foram perdidos na primeira de três coletas sanguíneas pré-programadas e um rato foi perdido na colocação da ligadura. Os resultados observados foram analisados são perda óssea alveolar induzida onde houve diferença significava entre os grupos (C) e (DP), entre o grupo (C) e (Hp+DP), entre o grupo (DP) e (Hp) e o grupo (Hp) e (Hp+DP). Foi avaliado perda óssea alveolar não induzida onde não existiu diferença entre os grupos. Foi avaliado o peso do início ao final do período experimental. Foram avaliados o consumo de ração e agua onde não houve diferença significativa entre os grupos. Foram avaliados o número de megacariócitos nos fémures, onde também não existiram diferenças estatísticas. Foram avaliados números de adipócitos no timo, não havendo diferença significativa entre os grupos. Foram avaliados as plaquetas e desvio padrão onde não existiu diferença significativa entre os grupos. Foram avaliados os leucócitos e desvio padrão onde não houve diferença significativa entre os grupos. Posteriormente foi avaliado a porcentagem de linfócitos onde se achou diferença estatisticamente significativa na segunda coleta sanguínea entre o grupo (C) e grupo (Hp+DP) e grupo (Hp) e grupo (Hp+DP). Foi assim que as conclusões deste trabalho foram que o presente estudo mostrou que a HBPM não foi capaz de produzir perda óssea alveolar nos ratos Wistar, mas foi capaz de aumentar a quantidade de leucócitos e linfócitos, indicando a presença de um processo inflamatório. / In order to better understand and understand the effects of (LMWH), a single article was elaborated with 40 male rats of the 60 day old Wistar line, which were divided into four previously randomized experimental groups: Control Group (C), Groups Periodontal Disease (PD), Heparin Group (Hp) and Heparin Group + Periodontal Disease (Hp + PD) with an experimental period of 60 days. One animal was lost in the acclimation period, two animals were lost in the first of three preprogrammed blood collections and one mouse was lost in the ligation placement. The observed results were analyzed for induced alveolar bone loss where there was significant difference between groups (C) and (PD), between group (C) and (Hp + PD), between (PD) and (Hp) group and Group (Hp) and (Hp + PD). Uninduced alveolar bone loss was assessed where there was no difference between the groups. The weight of the onset at the end of the experimental period was evaluated. The ration and water consumption were evaluated where there was no significant difference between the groups. The number of megakaryocytes in the femurs was evaluated, in which there were also no statistical differences. Adipocyte numbers were evaluated in the thymus, with no significant difference between the groups. Platelets and standard deviation were evaluated where there was no significant difference between the groups. Leukocytes and standard deviation were evaluated where there was no significant difference between the groups. Later, the percentage of lymphocytes where a statistically significant difference was found in the second blood collection between group (C) and group (Hp + PD) and group (Hp) and group (Hp + PD) was evaluated. Thus the conclusions of this study were that the present study showed that LMWH was not able to produce alveolar bone loss in Wistar rats, but was able to increase the amount of leukocytes and lymphocytes, indicating the presence of a process inflammatory.

O objetivo da presente tese foi avaliar os efeitos da heparina de baixo peso molecular (HBPM) na perda óssea alveolar em ratos Wistar. Para a melhor compreensão e entendimento dos efeitos da HBPM se elaborou um único artigo com 40 ratos machos da linhagem Wistar de 60 dias de nascidos, os quais foram dívidos em 4 grupos experimentais previamente randomizados: Grupo Controle (C), Grupos Doença Periodontal (DP), Grupo Heparina (Hp) e Grupo Heparina+Doença Periodontal (Hp+DP) com um período experimental de 60 dias. Um animal foi perdido no período de aclimação, dois animais foram perdidos na primeira de três coletas sanguíneas pré-programadas e um rato foi perdido na colocação da ligadura. Os resultados observados foram analisados são perda óssea alveolar induzida onde houve diferença significava entre os grupos (C) e (DP), entre o grupo (C) e (Hp+DP), entre o grupo (DP) e (Hp) e o grupo (Hp) e (Hp+DP). Foi avaliado perda óssea alveolar não induzida onde não existiu diferença entre os grupos. Foi avaliado o peso do início ao final do período experimental. Foram avaliados o consumo de ração e agua onde não houve diferença significativa entre os grupos. Foram avaliados o número de megacariócitos nos fémures, onde também não existiram diferenças estatísticas. Foram avaliados números de adipócitos no timo, não havendo diferença significativa entre os grupos. Foram avaliados as plaquetas e desvio padrão onde não existiu diferença significativa entre os grupos. Foram avaliados os leucócitos e desvio padrão onde não houve diferença significativa entre os grupos. Posteriormente foi avaliado a porcentagem de linfócitos onde se achou diferença estatisticamente significativa na segunda coleta sanguínea entre o grupo (C) e grupo (Hp+DP) e grupo (Hp) e grupo (Hp+DP). Foi assim que as conclusões deste trabalho foram que o presente estudo mostrou que a HBPM não foi capaz de produzir perda óssea alveolar nos ratos Wistar, mas foi capaz de aumentar a quantidade de leucócitos e linfócitos, indicando a presença de um processo inflamatório. / In order to better understand and understand the effects of (LMWH), a single article was elaborated with 40 male rats of the 60 day old Wistar line, which were divided into four previously randomized experimental groups: Control Group (C), Groups Periodontal Disease (PD), Heparin Group (Hp) and Heparin Group + Periodontal Disease (Hp + PD) with an experimental period of 60 days. One animal was lost in the acclimation period, two animals were lost in the first of three preprogrammed blood collections and one mouse was lost in the ligation placement. The observed results were analyzed for induced alveolar bone loss where there was significant difference between groups (C) and (PD), between group (C) and (Hp + PD), between (PD) and (Hp) group and Group (Hp) and (Hp + PD). Uninduced alveolar bone loss was assessed where there was no difference between the groups. The weight of the onset at the end of the experimental period was evaluated. The ration and water consumption were evaluated where there was no significant difference between the groups. The number of megakaryocytes in the femurs was evaluated, in which there were also no statistical differences. Adipocyte numbers were evaluated in the thymus, with no significant difference between the groups. Platelets and standard deviation were evaluated where there was no significant difference between the groups. Leukocytes and standard deviation were evaluated where there was no significant difference between the groups. Later, the percentage of lymphocytes where a statistically significant difference was found in the second blood collection between group (C) and group (Hp + PD) and group (Hp) and group (Hp + PD) was evaluated. Thus the conclusions of this study were that the present study showed that LMWH was not able to produce alveolar bone loss in Wistar rats, but was able to increase the amount of leukocytes and lymphocytes, indicating the presence of a process inflammatory.

Made available in DSpace on 2014-12-17T14:03:31Z (GMT). No. of bitstreams: 1 CeltonPM.pdf: 997721 bytes, checksum: 8ad0354bfbff2f87d7c01316f84484e6 (MD5) Previous issue date: 2004-11-05 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Heparan sulfate (HS) and Heparin (Hep) glycosaminoglycans (GAGs) are heterogeneous and highly charged polysaccharides. HS is structurally related to Hep but is much less substituted with sulfo groups than heparin and has a more varied structure (or sequence). Because of structural similiarities between these two polymers, they have been described together as heparinoids . Both chains bind a variety of proteins and mediate various physiologically important processes including, blood coagulation, cell adhesion and growth factor regulation. Heparinoids with structural characteristics similar to these described from HS and/or Hep from mammalian tissues have been isolated from different species of invertebrates, although only a few heparinoids from unusual sources have been characterized. The present study describes the presence of unusual heparinoids population from Artemia franciscana, isolated after proteolysis and fractionation by ion exchange resin and named, F-3.0M. The study model in vivo were hemostasis (rat tail scarification) and inflamatoty activity. The tests in vitro were used for coagulations assays (PT and APTT). The analyse of the heparinoids eluted with 3,0M NaCl showed electrophoretic migration in different buffer systems a single band with a behaviour intermediate between those of mammalian HEP and HS. The main products obtained from Artemia heparinoids after enzymatic degradation with heparitinases I and II from F. heparinum were N-sulphated disaccharides (?U-GlcNS,6S/ ?U,2S-GlcNS and ?U-GlcNS) and N-acetylated disaccharides (?U, GlcNAc). This heparinoid had a lower hemorrhagic effect (400?g/ml) when compared to unfractiionated heparins(25?g/ml).The results also suggest a negligible APTT activity of this heparinoid (62.2s). No action was observed on PT indicating that F-3.0M haven t action on the extrinsic pathway. The results showed that the fraction F- 3.0M have inhibitory effect on migration of leukocytes, 64.5% in the concentration of 10 ?g/ml (P<0.001). The search for new heparin and/or heparan sulphates analogs devoid of anticoagulant activity is an atractive alternative and may open up a wide variety of new therapeutic applications / Heparam sulfato (HS) e Heparina (Hep) s?o glicosaminoglicanos (GAGs), heterog?neos e altamente poliani?nicos. HS ? estruturalmente relacionado a heparina, mas cont?m uma quantidade menor de grupos sulfatados que a Hep. Devido ?s similaridades estruturais entre estes dois pol?meros, eles t?m sido denominados conjuntamente de heparin?ides. Ambas as cadeias ligam-se a uma variedade de prote?nas mediadas por v?rios processos fisiol?gicos importantes, incluindo a coagula??o sangu?nea, ades?o celular e regula??o dos fatores de crescimento. O presente estudo descreve a presen?a de uma popula??o de heparin?ides (F-3.0M) diferenciados presentes em Artemia franciscana os quais foram isolados por prote?lise e fracionados por cromatografia de troca i?nica com variadas concentra??es de NaCl. Neste trabalho, utilizamos modelos in vivo para avaliar a homeostasia e a atividade anti-inflamat?ria destes heparinoides. A hemostasia foi avaliada por modelo de escarifica??o em caudas de ratos Wistar enquanto que o processo inflamat?rio foi observado atrav?s de peritonite induzida por tioglicolato de s?dio a 3%. Os heparin?ides eluidos com 3,0M de NaCl mostraram por eletroforese em diversos tamp?es uma ?nica banda com comportamento intermedi?rio entre a Hep de mam?fero e HS. O principal produto obtido da fra??o F-3.0M de Artemia franciscana ap?s a degrada??o enzim?tica com heparitinases I e II de Flavo bacterium Heparinum foram dissacar?deos N-sulfatados (?U-GlcNS,6S/ ?U,2S-GlcNS e ?U-GlcNS) e dissacar?deos N-acetilados (?U, GlcNAc). Estes heparin?ides apresentaram uma baixa atividade hemorr?gica somente na concentra??o de 400?g/ml, ? similar a heparina na concentra??o de 25?g/ml. Os resultados sugerem ainda, que esta fra??o tem baixa a??o anticoagulante APTT (62,2s); n?o apresentando a??o sobre PT, indicando que n?o tem a??o sobre a via extr?nseca da cascata. Os testes de atividade anti-inflamat?ria, desta fra??o tem efeito inibit?rio sobre a migra??o dos leuc?citos da ordem de 64,5% na concentra??o de 10 ?g/ml (P<0.001)Heparam sulfato (HS) e Heparina (Hep) s?o glicosaminoglicanos (GAGs), heterog?neos e altamente poliani?nicos. HS ? estruturalmente relacionado a heparina, mas cont?m uma quantidade menor de grupos sulfatados que a Hep. Devido ?s similaridades estruturais entre estes dois pol?meros, eles t?m sido denominados conjuntamente de heparin?ides. Ambas as cadeias ligam-se a uma variedade de prote?nas mediadas por v?rios processos fisiol?gicos importantes, incluindo a coagula??o sangu?nea, ades?o celular e regula??o dos fatores de crescimento. O presente estudo descreve a presen?a de uma popula??o de heparin?ides (F-3.0M) diferenciados presentes em Artemia franciscana os quais foram isolados por prote?lise e fracionados por cromatografia de troca i?nica com variadas concentra??es de NaCl. Neste trabalho, utilizamos modelos in vivo para avaliar a homeostasia e a atividade anti-inflamat?ria destes heparinoides. A hemostasia foi avaliada por modelo de escarifica??o em caudas de ratos Wistar enquanto que o processo inflamat?rio foi observado atrav?s de peritonite induzida por tioglicolato de s?dio a 3%. Os heparin?ides eluidos com 3,0M de NaCl mostraram por eletroforese em diversos tamp?es uma ?nica banda com comportamento intermedi?rio entre a Hep de mam?fero e HS. O principal produto obtido da fra??o F-3.0M de Artemia franciscana ap?s a degrada??o enzim?tica com heparitinases I e II de Flavo bacterium Heparinum foram dissacar?deos N-sulfatados (?U-GlcNS,6S/ ?U,2S-GlcNS e ?U-GlcNS) e dissacar?deos N-acetilados (?U, GlcNAc). Estes heparin?ides apresentaram uma baixa atividade hemorr?gica somente na concentra??o de 400?g/ml, ? similar a heparina na concentra??o de 25?g/ml. Os resultados sugerem ainda, que esta fra??o tem baixa a??o anticoagulante APTT (62,2s); n?o apresentando a??o sobre PT, indicando que n?o tem a??o sobre a via extr?nseca da cascata. Os testes de atividade anti-inflamat?ria, desta fra??o tem efeito inibit?rio sobre a migra??o dos leuc?citos da ordem de 64,5% na concentra??o de 10 ?g/ml (P<0.001)

Made available in DSpace on 2014-12-17T14:03:31Z (GMT). No. of bitstreams: 1 JaniceSC.pdf: 2257338 bytes, checksum: 87ae25d4af8c910f8c675e38f8bfe103 (MD5) Previous issue date: 2005-04-08 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Berberine is an alkaloid used as a fluorochrome in the identification of heparin and DNA. Enerback, 1974, described the technique used until today to study granules rich in heparin of vertebrate mast cells. Santos et al., 2003, studied mast cells of the mollusk Anomalocardia brasiliana using biochemical and histological analysis. This work used the fluorescent dye berberine technique to improve characterization of these cells. Mollusk organs (ctenidium and mantle) were processed with routine histological techniques. Tissue sections were treated with berberine 0,02% in redistilled water acidified to pH 4, by the addition of citric acid for 20 minutes. The visualization was made through fluorescence microscopy with ultraviolet region emission. The mast cell fluorescence had a strong yellow color, where cell nuclei appeared more greenish. This result was very similar to the ones reported before. Mast cells are location at the epithelium surface is the same in both organs, mantle and ctenidium. The fluorescence was easily observed in the granules. Therefore, this technique showed to be good and sensitive to study mast cell of invertebrates / A berberina ? um alcal?ide usado como fluorcromo na identifica??o de heparina e DNA. Enerb?ck, em 1974, descreveu a t?cnica at? hoje empregada deste fluorcromo no estudo dos gr?nulos ricos em heparina dos mast?citos de vertebrados. Santos et al., em trabalhos anteriores, utilizando abordagens bioqu?mica e histol?gica, descreveram a presen?a de mast?citos no marisco Anomalocardia brasiliana. Baseado nestes estudos, testamos a t?cnica de fluoresc?ncia com berberina para a caracteriza??o adicional dessas c?lulas. Destarte, mantos e cten?deos dos esp?cimes foram submetidos a t?cnicas rotineiras de fixa??o e inclus?o em parafina. Os cortes histol?gicos foram desparafinados, hidratados e incubados com cloridrato de berberina (0,02%) em ?cido c?trico (1%) por 20 minutos, sendo o excesso retirado com ?cido c?trico (pH 4) por 5 minutos. A visualiza??o das se??es foi feita por microscopia de fluoresc?ncia com emiss?o no espectro ultravioleta. Pudemos evidenciar c?lulas com conte?do citoplasm?tico fluorescente amarelo-brilhante, correspondentes ?quelas descritas anteriormente como mast?citos, localizadas nos epit?lios dos ?rg?os. Algumas esp?cimes, logo ap?s a disseca??o foram submetidas a tratamento com desgranulantes e depois processadas como citado acima. O uso da fluoresc?ncia tornou mais f?cil a delimita??o do conte?do granular destas c?lulas em rela??o ao n?cleo (que emite fluoresc?ncia esverdeada). Sendo assim, a t?cnica apresentada mostrou ser bastante sens?vel e promissora para o estudo dos mast?citos de invertebrados

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / The Binder of Sperm Protein 1 (BSP1) is a protein produced by seminal vesicles of animals. It helps into the fertilization process of oocytes, promoting sperm capacitation, mediating the ligation from itself with the oviduct epithelium, and increasing the motility in the oviduct. When spermatozoa with purified BSP1 were incubated, acrosomal reaction rates were determined with and without the presence of heparin, as controls. The percentage of acrosomal reaction was higher (p <0.05) in control 1 (44.5%) compared with the other treatments (BSP1:10 Âg/mL: 24.5%; 20 Âg/mL: 25.5%; 40 Âg/mL: 26.0 %), and lower the rate (p> 0.05) when sperm were incubated only in Fert-TALP medium without heparin (14.5%). Cleavage and blastocyst rates were evaluated on days two and seven respectively, after fertilize oocytes in a medium containing BSP1. 1274 cumulus- oocyte complexes were recovered and incubated for 18 hours with frozen-thawed ejaculated semen in a Fert-TALP medium, containing: only heparin, 10, 20 or 40 &#956;g/mL of BSP1. Cleavage and blastocyst rates (34.1  4.4 vs 40.8  5.0%) were similar (p> 0.05) when incubation was made with 10 Âg/mL of BSP1 and heparin, respectively. However, the concentrations of 20 and 40 Âg/mL decreased the formation of blastocysts (22.4  2.9 and 19.3  4.1%) compared with heparin (40.8  5.1%, p <0.02). Subsequently, cumulus- oocyte complexes (n = 2239) were incubated with frozen-thawed ejaculated semen. Spermatozoa were selected by a gradient of 45% -90% Percoll containing: only heparin, 10, 20, 40 &#956;g/mL of BSP1 and there was a control 2 of Percoll without heparin or BSP1. Cleavage rate was similar (p> 0.05) for the incubation with concentrations of 10, 20 and 40 &#956;g/mL of BPS1 (67.7  3.0, 68.7  3.5, 75.2  3.8, respectively). However, these rates were similar when it was used just heparin and when oocytes were incubated with 40Âg/mL of BSP1 (78.9  1.7 vs 75.2  3.8%). For blastocyst rates was found that they were similar (p> 0.05) when the selection was made with heparin (44.1  4.3%) and 40 Âg/mL of BSP1 (30.0  3.3%). Finally, 1213 cumulus-oocyte complexes were recovered and incubated with freeze-thawed epididymal semen in Fert-TALP medium, which contained: only heparin, with heparin, and 10, 20 or 40 Âg/mL of BSP1. Cleavage and blastocyst rates were similar after incubation with and without heparin, but the concentration of 40Âg/mL of BSP1 produced higher cleavages rates (79.0  1.1%) than control 1 (68.5  1.3%, p <0.05). About blastocyst rates, there were rates significantly higher when concentrations of 20 and 40 Âg/mL of BSP1 were used (35.6  2.5 and 41.1  2.0%) than with (24.7  3.2; p <0.05) and without heparin (27.3  1.6%; p <0.0003). In conclusion, BSP1 allowed proper cleavage and development, when the protein was added to the fertilization medium and the Percoll gradient using ejaculated semen, and to the fertilization medium when used epididymal sperm, without requiring the use of heparin. / A âBinder of Sperm Protein 1â (BSP1) à uma proteÃna produzida pelas vesÃculas seminais dos animais, que ajuda no processo de fertilizaÃÃo do oÃcito promovendo a capacitaÃÃo espermÃtica, mediando a sua ligaÃÃo com o epitÃlio do oviduto e aumentando sua motilidade no oviduto. As taxas de reaÃÃo de acrossoma dos espermatozoides foram determinadas ao serem incubados com BSP1 purificado, e com e sem a presenÃa de heparina, como controles. A porcentagem de reaÃÃo de acrossoma foi maior (p<0.05) no controle 1 (44.5%) comparado com os tratamentos restantes (BSP1: 10 Âg/mL: 24.5%; 20 Âg/mL: 25.5%; 40 Âg/mL: 26.0%), sendo menor esta taxa (p>0.05) quando os espermatozoides sà foram incubados no meio Fert-TALP sem heparina (14.5%). As taxas de clivagem e de blastocistos foram avaliadas nos dias dois e sete respectivamente, depois de fertilizar oÃcitos em um meio que continha BSP1. Foram recuperados 1274 COCs e incubados durante 18 horas com sÃmen ejaculado congelado- descongelado em meio Fert-TALP, que continha: sà heparina, 10, 20 ou 40 Âg/mL de BSP1. As taxas de clivagem e de blastocistos (34.1  4.4 vs 40.8  5.0%) foram similares (p>0.05) quando se fez a incubaÃÃo com 10 Âg/mL de BSP1 e heparina, respectivamente. PorÃm, as concentraÃÃes de 20 e 40 Âg/mL diminuÃram a formaÃÃo de blastocistos (22.4  2.9 e 19.3  4.1%) em relaÃÃo com a heparina (40.8  5.1%; p < 0.02). Posteriormente foram incubados COCs (n= 2239), com sÃmen ejaculado congelado-descongelado que foi selecionado por um gradiente de Percoll 45%-90% que continha: sà heparina, 10, 20 ou 40 Âg/mL de BPS1 e foi realizado um controle 2 de Percoll sem heparina nem BSP1. A taxa de clivagem foi similar (p > 0.05) para a incubaÃÃo com as concentraÃÃes de 10, 20 e 40 Âg/mL de BSP1 (67.7Â3.0, 68.7Â3.5, 75.2Â3.8). PorÃm, estas taxas foram similares quando sà foi usada heparina e quando se incubou com 40 Âg/mL de BSP1 (78.9Â1.7 vs 75.2Â3.8%). Para as taxas de blastocistos encontrou-se que foram similares (p > 0.05) quando se fez a seleÃÃo com heparina (44.1Â4.3%) e com 40 Âg/mL de BSP1 (30.0Â3.3%). Finalmente, foram recuperados 1213 complexos cumulus-oÃcitos e incubados com espermatozoides epididimÃrios congelados-descongelados em meio Fert-TALP, que continham: sà heparina, sem heparina, e 10, 20 ou 40 Âg/mL de BSP1. As taxas de clivagem e de blastocistos foram similares apÃs as incubaÃÃes com e sem heparina, mas a concentraÃÃo de 40 Âg/mL de BSP1 produziu maiores taxas de clivagem (79.0  1.1%) que o controle 1 (68.5  1.3%; p < 0.05). Quanto Ãs taxas de blastocistos, encontraram-se maiores taxas quando foram usadas as concentraÃÃes de 20 e 40 Âg/mL de BSP1 (35.6  2.5 e 41.1  2.0%) que com (24.7  3.2; p < 0.05) e sem heparina (27.3  1.6%; p < 0.0003). Em conclusÃo, a BSP1 adicionada ao meio de fertilizaÃÃo e ao gradiente de Percoll quando usado o sÃmen ejaculado, e ao meio de fertilizaÃÃo quando usados espermatozoides epididimÃrios, permitiu clivagem e desenvolvimento prÃprio, sem a necessidade do uso de heparina.

The pre-analytical phase is known for being the most important step in the laboratory process to reach reliable test results. If handling, transport or preparation of the sample is performed incorrectly the results can deviate from the true value. Today, sampling tubes contains various additives to stabilize concentration levels. The aim of this study was to test a new sampling tube containing fluoride/citrate for glucose, lactate and homocysteine. It was also of interest to evaluate the stability of those three analytes in lithium-heparin, sodium-fluoride/potassium oxalate and fluoride/citrate tubes. To perform the sustainability study, a method verification was done for homocysteine in plasma. The study was performed in a hospital laboratory on the routine instrument Roche Cobas 6000 analyzer. Blood was drawn from 20 patients and was analyzed at the hospital laboratory in Gävle. The blood samples were transported frozen to the laboratory in Hudiksvall and were used in the method verification. For the sustainability study, blood was drawn from 10 healthy volunteers in lithium-heparin, sodium-fluoride/potassium oxalate and fluoride/citrate tubes. The method verification was approved. The results showed that glucose was stable for up to 72 hours in Vacuette Glycaemia tube with fluoride/citrate and this tube also gave more accurate results. Lactate and homocysteine were also stable in fluoride/citrate, but needs further studies. All three analytes were more stable if the sample tubes were centrifuged as soon as possible after blood collection. Fluoride/citrate tubes were stable without centrifugation directly.