Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2016-11-11T17:07:10Z No. of bitstreams: 0 / Made available in DSpace on 2016-11-11T17:07:10Z (GMT). No. of bitstreams: 0 / Nas últimas décadas tem aumentado o interesse pelos enxertos de pele humana radioesterilizadas, para aplicação principalmente em queimaduras extensas e profundas. Isto se deve ao fato destes enxertos apresentarem rápida aderência e menor potencial antigênico, em comparação com os demais tratamentos utilizados. A proposta deste estudo foi avaliar a histoarquitetura do enxerto de pele humana irradiada com doses de 25 kGy, 50 kGy e não irradiada, durante o processo de reparação tecidual, em camundongos Nude submetidos a enxertia de pele na região dorsal. Três grupos de animais receberam enxertos de pele humana irradiada (25 kGy e 50 kGy) e não irradiada e foram eutanasiados no 3º, 7º e 21º dia após a realização da cirurgia. Após os procedimentos histológicos de rotina, as amostras de tecido foram coradas com hematoxilina e eosina (HE) para a quantificação de queratinócitos, fibroblastos, células de defesa e vasos sanguíneos e a reação de imunofluorescência (IF) foi realizada para a determinação da expressão de colágeno do tipo I humano e do colágeno dos tipos I e III de camundongo. A quantificação, tanto das células quanto dos tipos de colágeno foi realizada por análise de imagem, utilizando o programa Image-Pro PLus 6.0. Os resultados histológicos demostraram que a pele humana irradiação, quando enxertada, influencia o aumento do número de células no local de cicatrização ao longo do tempo, principalmente na dose de 25 kGy, além de proporcionar uma melhor dispersão destas células. No 21º dia, os três grupos de animais com enxertia de pele humana tiveram parte do enxerto incorporado no processo de cicatrização. O grupo não irradiado apresentou maior incorporação do enxerto (43%), porém menor produção de colágeno do tipo III de camundongo (22%). Já os grupos com enxertia de pele irradiada apresentaram menor incorporação do enxerto (6 e 15%), mas com maior produção de colágeno do tipo III de camundongo (35% e 28%, para 25 kGy e 50 kGy, respectivamente). Com este estudo pôde-se concluir que o grupo irradiado a 25 kGy, apresenta maior proliferação celular e formação de vasos,além de melhor remodelamento da região de cicatrização. / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

nÃo hà / Enxertos compostos com artÃria torÃcica interna esquerda (ATIE) tem aumentado sua aplicabilidade na cirurgia de RevascularizaÃÃo MiocÃrdica (RM). A confirmaÃÃo de patÃncia do enxerto de ATIE à pedra fundamental na RM. O melhor parÃmetro de patÃncia calculado pelo ecoDopplercardiograma à a fraÃÃo diastÃlica (FD) ≥ 0,5. O objetivo geral deste estudo foi estabelecer parÃmetros ecoDopplercardiogrÃficos de patÃncia do enxerto composto de ATIE, quando revasculariza a artÃria interventricular anterior (AIA) e outro ramo do sistema coronariano esquerdo. O especÃfico foi definir a sensibilidade e a especificidade de trÃs variÃveis: RelaÃÃo da velocidade-pico na diÃstole sobre a velocidade-pico na sÃstole (VPD/VPS), integral da velocidade-tempo na diÃstole (VTID) e FD quanto à patÃncia do enxerto composto utilizando FD ≥ 0,5 como padrÃo de referÃncia. O estudo foi realizado segundo um desenho em duas fases, fase controle e fase estudo. Na fase controle, todos os pacientes tinham a patÃncia dos enxertos confirmadas por cineangiocoronariografia (CINE). Estes pacientes tinham registro das variÃveis VPD/VPS, VTID e FD. Foram entÃo estabelecidos pontos de cortes para essas variÃveis baseando-se nos cÃlculos de sensibilidade e especificidade atravÃs da curva ROC (ReceiverOperationCharacteristic) com o objetivo de diferenciar enxertos compostos de enxertos simples quando a ATIE somente revasculariza a AIA. Esses pontos de corte foram aplicados nos pacientes com enxerto composto da fase estudo. Foi construÃda uma tabela de contingÃncia 2x2 para o cÃlculo de sensibilidade e especificidade, tendo como indicador de patÃncia uma FD≥0,5. Na fase controle, observou-se diferenÃas estatisticamente significativas na anÃlise das trÃs variÃveis em diferenciar o enxerto simples do composto. Os parÃmetros de patÃncia do enxerto composto estabelecidos pela fase estudo foram VPD/VPS ≥ 0,71, VTId ≥ 0,09 e FD ≥ 0,58. A especificidade para todas essas variÃveis foi de 100%. A sensibilidade foi de 40% para VPD/VPS ≥ 0,71, 36,4% para VTId ≥ 0,09 e 68% para FD ≥ 0,58. Conclui-se que valores maiores ou iguais aos estabelecidos para cada variÃvel representam um provÃvel indicador de patÃncia do enxerto composto. Valores abaixo do estabelecido apresentam grande proporÃÃo de falsos negativos, nÃo sendo conclusivo quanto à patÃncia. / Composite grafts with left internal thoraic artery (LITA) has incresed its applicability in Coronary Artery Bypass Surgery (CAGB). Confirmation of patency of the LITA graft is the cornerstone of Miocardial Revascularization. The best measure of patency calculated by Doppler echocardiogram (Doppler) is the diastolic fraction (DF) ≥ 0.5. The aim of this study was to establish Doppler echocardiographic parameters which could suggest the presence of a composite graft of LITA, when it revascularizes the anterior interventricular artery (AIA) and another branch of the left coronary artery system. The endpoint of this study was to define sensitivity and specificity considering three variables: ratio of peak velocity in diastole over the systolic peak velocity (DPV / SPV), the mean velocity-time integral in diastole (VTID) and FD of the composite graft patency using FD ≥ 0.5 as the reference standard. The study was conducted according to a design in two stages. It was defined a control group and study group. In the control group, all patients had graft patencies confirmed by coronary angiography (CINE) and the variables DPV / SPV,VTID and DF measured. So, we use this data to establish cutoff points for these variables , based on the calculation of sensitivity and specificity using the ROC curve (Receiverv Operation Characteristic) in order to differentiate composite from simple graft when ATIE only revascularizes the AIA. These cutoff points were applied in patients with composite graft group study. We built a 2x2 contingency table to calculate sensitivity and specificity, considering the indicator of patency a DF ≥ 0.5. In the control group, we observed statistically significant differences in the analysis of the three variables in differentiating simple from composite grafts. The parameters of composite graft patency established by the study group were DPV / SPV ≥ 0.71,VTID ≥ 0.09 and FD ≥ 0.58. The specificity for all these variables was 100%. The sensitivity was 40% for DPV / SPV ≥ 0.71, ≥ 36.4% for VTId≥ 0.09 and 68% for FD ≥ 0.58. We conclude that values greater than or equal to those established for each variable represents a likely indicator of graft patency compound. Values below the established are not conclusive to exclude composite grafts due to a large proportion off alse negatives.

Le développement de nouvelles stratégies vaccinales et le traitement de nombreuses pathologies dermatologiques font partis des enjeux majeurs des années à venir. L'étude et la compréhension des mécanismes de réponses immunitaires cutanées sont ainsi essentielles. Les cellules de Langerhans épidermiques et les cellules dendritiques dermiques jouent un rôle central dans l'immunité adaptative spécifique à un pathogène, et dans l'immunité innée, de par leur fonction de présentation des antigènes (APC), mais également d'activateur et de régulateur de l'action des autres cellules immunitaires. Durant ce travail de recherche, nous avons mis au point et testé plusieurs modèles d'étude des mécanismes de l'immunité cutanée. Un premier modèle de " souris humanisées " a été mis au point, à partir de souris immunodéprimées NSG HLA-A2 tg et de greffes de peau humaine. Une fois le modèle techniquement mis au point, nous avons vérifié de la conservation et de la fonctionnalité des APC. La faible conservation des APC au-delà de plusieurs semaines, nous a conduit à mettre au point un deuxième modèle de " souris humanisées " qui bénéficiaient d'injections intra-hépatiques de progéniteurs hématopoïétiques humains. La colonisation en APCs cutanées humaines n'étant pas suffisante, il a fallu injecter aux souris des APCs provenant du même sang que les progéniteurs hématopoïétiques afin d'obtenir une réponse immunitaire suffisante. La faible concentration en APC cutanées de ce modèle, la lourdeur et le coût de mise au point sont des limites non négligeables pour l'utilisation de ce support d'étude. Ces souris humanisées sont d’excellents modèles d’étude, mais ils présentent chacun leurs limites. Lorsque cela est possible il est ainsi plus simple d’utiliser des explants cutanés frais. Nous avons quantifié les APC cutanées en fonction du site d’origine (abdomen, seins…) et de l’âge du donneur de ces explants issus de chirurgie plastique. Sur les 21 explants testés, nous n’avons pas retrouvé de différence significative dans la concentration en APC quel que soit le site et l’âge du donneur. Ces 3 modèles offrent des possibilités d’étude des mécanismes de l’immunité cutanée, nous les utilisons actuellement au sein du laboratoire pour des tests vaccinaux, et pour l’étude du rôle de l’IL-32 produite par les kératinocytes impliqués dans l’activation des cellules de Langerhans. / The development of new vaccination strategies and the treatment of many dermatological pathologies are among the major challenges of the years to come. The study and understanding of the mechanisms of cutaneous immune responses are thus essential. Epidermal Langerhans cells and dermal dendritic cells play a central role in pathogen-specific adaptive immunity, and in innate immunity, by their antigen presenting function (APC), but also as an activator and regulator of the action of other immune cells.During this research, we developed and tested several models for the study of the mechanisms of cutaneous immunity. A first model of "humanized mice" was developed from immunosuppressed NSG HLA-A2 tg mice and human skin grafts. Once the model was technically developed, we verified the conservation and functionality of the APCs. The low PCA retention beyond several weeks, led us to develop a second model of "humanized mice" that benefited from intrahepatic injections of human hematopoietic progenitors. Since colonization in human skin APCs was not sufficient, it was necessary to inject the APCs from the same blood as the hematopoietic progenitors to obtain a sufficient immune response. The low concentration of skin APCs in this model, the cumbersomeness and the cost of development are not insignificant limits for the use of this study support.These humanized mice are excellent models of study, but they each have their limitations. Where possible, it is thus easier to use fresh skin explants. We quantified the cutaneous APCs according to the site of origin (abdomen, breasts ...) and the age of the donor of these explants resulting from plastic surgery. Of the 21 explants tested, there was no significant difference in APC concentration at any site and age of the donor.These three models offer possibilities for studying the mechanisms of skin immunity, we are currently using them in the laboratory for vaccine tests, and for studying the role of IL-32 produced by the keratinocytes involved in l activation of Langerhans cells.

Objectifs : caractériser (1) la morphométrie de la crosse afin d’examiner les critères favorisant les complications et la faisabilité d’endoprothèses (EP) standards. (2) les propriétés mécaniques de l’aorte ascendante (AA) chez le porc, homme sain et en cas de dissection aortique (DAo).Matériels et méthodes :Caractérisation morphométrique : A partir d’angioscanner aortiques les paramètres: (1) morphométriques élémentaires (2) géométriques ; (3) troncs supra-aortiques (TSAo), sont mesurésCaractérisation mécanique : In vivo : à partir de données échographiques et hémodynamiques, les paramètres élastiques sont calculés.In vitro : tests de traction bi-axiale sur l’AA plus une analyse histomorphométrique et microstructurale.Résultats :Caractérisation morphométrique : > de75% des patients ont une orientation des TSAo à ± 15° par rapport à la moyenne, et une variabilité de la distance entre les ostia TSAo de ± 4 mm.Les facteurs indépendants associés aux endofuites, défaut d’apposition et mal-positionnement sont respectivement:(1) collet proximal court; (2) angulation de la zone d’ancrage (valeur seuil 51°); et (3) indice tortuosité (valeur seuil 1.68) Caractérisation mécanique : In vivo : une grande compliance de l’aorte porcine comparé à l’homme et une rigidification en cas de DAo.In vitro : l’aorte porcine a un comportement linéaire comparé au caractère non linéaire chez l’homme. Conclusion : Ce travail montre :- le lien entre morphométrie et complications après EP, et la faisabilité d’EP standards pour la crosse.- le modèle porcin est inapproprié pour tester les EP de l’AA. - la rigidification de la paroi en cas de DAo influencera le choix des futurs EP. / Objectives: characterize (1) arch morphometry to examine criteria favoring complications after thoracic endovascular aortic repair (TEVAR) and feasibility of « off-the-shelf » fenestrated devices.(2) mechanical properties of ascending aorta (AA) in swine and humans, with and without aortic dissection.Materials and methods : Morphometric characterization : Computed tomographic angiography were analysed to calculate elementary morphometric, geometric and supra-aortic trunks data Mechanical characterization : In vivo: arterial pressure and diameters measured with echocardiophy are used to calculate elastic parameters.Ex vivo: biaxial tensile testing performed on AA plus histological and microstructural analysis. Results :Morphometric characterization : In > 75% of cases supra-aortic branches are positioned within 15° of each other and distances between them have a variability of ± 4 mm.Independant factors associated with endoleak, bird beak and mis-positioning are respectively : (1) short proximal neck (2) landing zone angulation (cut-off value: 51°); and (3) tortuosity index (cut-off value: 1.68). Mechanical characterization : in vivo: greater compliance of swine aorta compared to humans and a stiffer aorta in case of aortic dissectionBiaxial testing: linear stress-strain behavior of swine aorta, compared to a non linear one in human. Conclusion : our study reveals :- the impact of anatomy on complications occurrence after TEVAR, and suitable arguments for « off-the-shelf » fenestrated devices.- swine model is inappropriate to test AA dedicated stent-graft.- stiffer wall in aortic dissection has consequences on the choice of futur devices dedicated to AA.

Factors currently at the forefront of human in vitro fertilization (IVF) that collectively influence treatment success in the form of blastocysts development were investigated during early mammalian embryology with concentration on infertile patients presenting with diminished ovarian reserve or preliminary ovarian failure. A novel experimental technique, Graft Transplant-Embryonic Stem Cells (GT-ESC) was introduced in the mouse model, as the first inclusive approach for embryo selection in IVF treatments resulting in successful graft integration of sibling cells, stage-dependent (day 4) blastocysts. E-Cadherin-catenin bonds play an integral role in trophectoderm cell viability and calcium removal, inducing disruption of cell-to-cell bonds at the blastocyst stage was detrimental to continued blastocyst development. One of the leading methods for embryo selection for uterine transfer in human IVF is application of pre-implantation genetic screening (PGS) methods such as next generation sequencing (NGS). Female patients <35 y do not benefit from this treatment when outcome is measured by presence of fetal heart beats at 10 weeks of gestation. Patients 35-37 y benefit from PGS with no significant difference of outcome based on form of PGS method utilized. Therefore, small nucleotide polymorphism array (snp-array) or targeted-NGS should be selected for this age range to lessen the financial burden of the patient. Embryos from women >40 y have a higher rate of mosaic cell lines which can be detected by NGS. Therefore NGS is most beneficial for women >40 y. Additionally, ovarian stimulation of the patient during human IVF can notably influence outcome. Anti-Müllerian hormone (AMH) is a more conducive indicator of blastocysts development per treatment compared to basal follicle stimulating hormone (FSH). Actionable variables included in a decision tree analysis determined a negative influence (0% success, n=11) of high dose gonadotropin use (>3325 IUs) in good prognosis patients (>12 mature follicles at trigger, AMH >3.15 ng/mL). A positive relationship exists (80% success, n=11) between poor responders (AMH <1.78 ng/mL, <12 mature follicles at trigger) and high dose gonadotropin use (>3025 IUs). Utilizing the decision tree during IVF treatment can be beneficial to treatment success. Moreover, a parallel relationship of the fundamental principles of culture medium pH, pCO2 and pO2 was found with respect to blastocyst development. Human infertility patients' gametes predisposed to primary stressors (i.e., age, genetics and etiology) are negatively impacted (~30% success, n=7) for cleavage stage (day 3) embryo development when primary culture medium has pCO2 <30mmHg given age >31 y and <14 oocytes retrieved. When day 3 embryo development is measured at >65% good quality embryos per treatment (based on SART grading criteria), blastocysts development success is highest when secondary culture medium pO2 is 69-88 mmHg (~90% success, n=12). Thus, IVF treatment outcome can be optimized with utilization of predictive model analyses in the form of decision trees providing greater success for the IVF laboratories, ultimately decreasing the emotional and financial burden to infertility patients.

Possibilities of Portal Vein Reconstruction during Surgical Treatment of Pancreatic Cancer - Experiment on a Large Animal Introduction: Pancreatic cancer is a fatal malignancy that is known as one of the leading causes of cancer mortality worldwide. The only potentially curative treatment is radical surgical resection. Because of the lack of early symptoms, the diagnosis is usually made in advanced stages of the disease. In the majority of patients, the tumor is already locally advanced or it has distant metastases at the time of diagnosis. Pancreatic cancer tends to infiltrate the portal vein (PV) or the superior mesenteric vein (SMV). Nowadays, resection of infiltrated parts of PV/SMV is recommended in specialized centers. There are several established techniques of PV/SMV reconstruction. The use of allogeneic venous grafts seems to be a method with minimal risk of adverse effects but there is only limited experience with these grafts. The optimal anatomical origin of allogeneic venous grafts for PV/SMV reconstruction remains unknown. Aims: The aim of this experiment was to compare two types of allogeneic venous grafts used for PV reconstruction in a large animal model of pancreatico- duodenectomy. These grafts were harvested from the systemic venous system (inferior caval vein grafts - IVC grafts) and...

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Year 2019, football, rugby, netball and skiing had most occurring ACL injuries, listed by United Kingdom National Ligament Report (NLR). The standard procedure treatment of complete laceration of the ACL, is performed by tissue autograft implantation designed from a patellar tendon, for replacement of damaged tissue using orthopaedic surgery. The aim of this thesis is to design and fabricate an ACL graft, attempting to mimic the natural ACL, for the purpose of tissue reconstruction. The desired graft analogues exhibited properties imitating native connective tissue, reducing pain through drug delivery with great biocompatibility and enhance suture mechanical strength. Various biomaterials were implemented into this study, utilising strategies; polymer solution fabrication, electrospinning, hydrogel synthesis, mechanical braiding and graft assembly to fabricate an ACL graft. The polymeric material poly (E- caprolactone) (PCL) was researched, utilising its ability to fabricate scaffolds. Results showed, three analogue ACL grafts (Braided PCL-BP, Braided PCL + Hydrogel-BPH & Braided PCL + Sutures-BPS) created utilising the properties of braiding, hydrogels and sutures, ultimately improving the versatility of electrospinning for tissue engineering and reconstruction. Graft analogues were tested and compared against patellar tendons producing similar tensile properties. Poly vinyl alcohol (PVA) hydrogels successfully held ibuprofen, revealing drug delivery characteristics, polyester threads improved mechanical properties of electrospun grafts and dry degradation showed that PCL did not lose significant mass over two months. Conclusion, tensile strength of patella tendon was 395x, 790x & 56x of analogue grafts (BP, BPH & BPS) respectively, having potential for improvement of tensile parameters for ligament reconstruction.

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Objetivos: O objetivo desse estudo foi avaliar comparativamente o potencial osteogênico in vitro de células obtidas do ramo mandibular (RM, área doadora) e do seio maxilar enxertado com uma mistura de RM e osso bovino inorgânico (OBI), previamente à instalação de implantes de titânio (SM, sítio do seio maxilar enxertado). Material e Métodos: As células foram obtidas de três pacientes submetidos a procedimentos de aumento do soalho do seio maxilar com a proporção de 1:1 de RM e OBI (GenOx Inorg®). No momento da realização dos enxertos no seio maxilar e após 08 meses, antes da inserção dos implantes de titânio, fragmentos ósseos foram colhidos do RM e do SM, respectivamente, e submetidos à digestão enzimática com tripsina e colagenase para obtenção de células primárias. As células foram subcultivadas e crescidas sob condições osteogênicas por até 21 dias, tendo sido avaliados os seguintes parâmetros: proliferação/viabilidade celular, expressão gênica de marcadores osteoblásticos, atividade de fosfatase alcalina (ALP) e conteúdo de cálcio, por extração do vermelho de Alizarina. Culturas primárias derivadas do RM foram expostas ao GenOx Inorg® por 7 dias, quando se avaliou a atividade de ALP. Os resultados foram comparados por ANOVA two-way, seguido do teste de Tukey, ou pelo teste de Mann-Whitney. Resultados: Culturas do SM exibiram uma redução significante do potencial osteogênico se comparado ao de culturas do RM, com um aumento progressivo na proliferação celular associado a uma redução da expressão dos marcadores osteoblásticos, da atividade de ALP e do conteúdo de cálcio. A exposição do GenOx Inorg® às células primárias derivadas do RM inibiram a atividade de ALP. Conclusão: Esses resultados sugerem que o uso do GenOx Inorg® em associação a fragmentos do RM para aumento do soalho do seio maxilar inibe a diferenciação de células osteoblásticas no sítio de inserção de implantes de titânio após 8 meses de enxertia. / Objectives: This study aimed to comparatively evaluate the in vitro osteogenic potential of cells obtained from the mandibular ramus (MR, autogenous bone donor site) and from the maxillary sinus bone grafted with a mixture of anorganic bovine bone (ABB) and MR prior to titanium implant placement (MS, grafted implant site). Material and methods: Cells were obtained from three patients subjected to maxillary sinus floor augmentation with a 1:1 mixture of ABB (GenOx Inorg®) and MR. At the time of the sinus lift procedure and after 8 months, prior to implant placement, bone fragments were taken from MR and MS, respectively, and subjected to trypsin-collagenase digestion for primary cell culturing. Subcultured cells were grown under osteogenic condition for up 21 days and assayed for proliferation/viability, osteoblast marker mRNA levels, alkaline phosphatase (ALP) activity and calcium content/Alizarin red staining. ALP activity was also determined in primary explant cultures exposed to GenOx Inorg® (1:1 with MR) for 7 days. Data were compared using the two-way ANOVA followed by the Tukey test; otherwise, the Mann-Whitney test was used. Results: MS cultures exhibited a significantly lower osteogenic potential compared with MR cultures, with a progressive increase in cell proliferation together with a downregulation of osteoblast markers, reduced ALP activity and calcium content. Exposure of MR-derived primary cultures to GenOx Inorg® inhibited ALP activity. Conclusion: These results suggest that the use of GenOx Inorg® in combination with MR fragments for maxillary sinus floor augmentation inhibits the osteoblast cell differentiation at the implant site in the longterm.