Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 143-165).

Orientadores: Pedro Duarte Novaes, Mamie Mizusaki Iyomasa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T15:22:08Z (GMT). No. of bitstreams: 1 Pereira_YambaCarlaLara_D.pdf: 21699774 bytes, checksum: 6a8918dd0a94fb092cc529b6cdc3cd34 (MD5) Previous issue date: 2014 / Resumo: O estresse e as perdas dentais têm impacto sobre os músculos da mastigação favorecendo a dor, no entanto, a patogênese envolvida ainda é pouco compreendida. O objetivo deste trabalho foi investigar o efeito do estresse crônico variado em músculo masseter esquerdo de animais submetidos à alteração oclusal por exodontia unilateral, isolados ou associados, por meio de avaliação das atividades gelatinolíticas totais e da mieloperoxidase, análises morfológicas, imunoistoquímicas, histoquímicas e ultraestruturais ao Microscópio Eletrônico de Transmissão. Foram usados 20 ratos machos, Wistar, adultos jovens, pesando ± 200g, divididos aleatoriamente em 04 (quatro) grupos: (n=5): GI controle, sem alteração oclusal e sem estresse crônico variado (EV), G II- alteração oclusal isolada, G III EV isolado e G IV- alteração oclusal associada ao EV. Nos grupos da exodontia (GII e G IV), animais previamente anestesiados, tiveram os molares superiores esquerdos extraídos. Os grupos dos estresses (G III e G IV) foram submetidos a diferentes estressores a partir do 14º dia após a exodontia. Todos os animais foram eutanasiados no 24º dia. No feixe superficial, a análise dos resultados revelou que o músculo masseter, a exodontia e o EV, isolados ou associados não promoveram a infiltração de polimorfonucleares analisados por meio da mieloperoxidase e não alteraram os níveis das metaloproteinases (MMP-2) avaliados por meio da atividade de Gelatinase Total. No feixe anterior do músculo masseter profundo, a exodontia e o EV, isolados ou associados alteraram, em determinadas regiões, as características histológicas das fibras reveladas por coloração em hematoxilina e eosina; alteraram a ultraestrutura das mitocôndrias ao microscópio eletrônico de transmissão; revelaram regiões com lúmen reduzido, e outras com lúmen dilatado, em capilares marcados com anticorpo anti-laminina; revelaram uma tendência ao aumento da densidade de capilares imunomarcados por anti-alpha CD31 do endotélio, e não alteraram as escassas fibras imunomarcadas pela miosina do tipo I Neste mesmo feixe, diminuíram os níveis de espécies reativas de oxigênio no grupo exposto à exodontia (GII). No feixe médio posterior do músculo masseter profundo, os protocolos experimentais confirmaram a prevalência de fibras claras, por meio da reação histoquímica de succinato desidrogenase. Podemos concluir que o músculo masseter esquerdo manifestou alteração regional decorrente da exodontia e EV isolados ou associados, revelados por alterações morfológicas, ultraestruturais, hemodinâmicas e estresse oxidativo, em especial no feixe profundo anterior. Esses dados contribuem para a compreensão do desenvolvimento do ponto de gatilho. No entanto, novos estudos serão necessários para melhor entendimento da etiopatogenia da dor miofascial e da disfunção temporomandibular e sua relação com estresse e alteração oclusal / Abstract: Stress and tooth loss have an impact on the masticatory muscles favoring the pain. However, the pathogenesis involved is still little understood. The aim of this study was to investigate the effect of varied chronic stress in left masseter muscle of rats subjected to unilateral alteration by occlusal tooth extraction, alone or associated, through evaluation of total gelatinolytic activities and myeloperoxidase, morphological analysis, histochemical, immunohistochemical labeling and transmission electron microscope. Twenty male Wistar rats, young adults, weighing ± 200 g, were divided randomly into four groups (n=5): GI control without occlusal alteration and without varied chronic stress (EV), G II-occlusal alteration isolated, G III EV isolated and G IV-occlusal alteration associated with the EV. The rats from groups of dental extraction (GII and G IV) were previously anesthetized and had the superior left molars extracted. The groups of stresses (G III and G IV) were submitted to different stressors from the 14th day after the tooth extraction. All the animals were euthanized on the 24th day. On the surface, the beam analysis of results showed that the masseter muscle, dental extraction and the EV, alone or associated with not promoted the infiltration of polymorphonuclear scanned through the myeloperoxidase and not altered levels of metalloproteinases (MMP-2) evaluated by means of Total Gelatinase activity. In the anterior bundle of masseter muscle located evaluated through the Total Gelatinase activity. In anterior bundle of the deep masseter muscle, the dental extraction and the EV, alone or associated with altered, in some regions, the histological characteristics of the fibers revealed by hematoxylin and eosin staining; the ultrastructure of mitochondria in the transmission electron microscope; revealed areas with reduced lumen, and lumen distended in other capillaries marked with anti-laminina antibody; showed a tendency to increase in the density of capillaries labelled by endothelium, CD31 anti-alpha and have not altered the scarce labelled fibers by myosin type I in the same bundle, decreased levels of reactive oxygen species in the group exposed to dental extraction (GII). The average bundle of deep masseter muscle later, experimental protocols confirmed the prevalence of clear fibers by means of Immunohistochemistry reaction of succinate dehydrogenase. We can conclude that the masseter muscle change resulting from regional expressed left dental extraction and EV alone or associated, revealed by morphological, ultrastructural, and hemodynamic oxidative stress, especially in deep beam. These data contribute to the understanding of the development of the trigger point. However, further studies are needed to better understanding of the pathogenesis of myofascial pain and temporomandibular joint dysfunction and its relation with occlusal stress and occlusial alteration / Doutorado / Histologia e Embriologia / Doutora em Biologia Buco-Dental

<p>Ugljenične nanocevi (UNC) imaju sve veću primenu u elektronici, kompjuterskoj i optičkoj industriji, kao i u biomedicini. Dok proizvodnja jednoslojnih UNC nanocevi beleži sve veći rast poslednjih godina, rizik koji nosi izlaganje ovom nanomaterijalu ostaje nerazja&scaron;njen. Oskudni i često kontradiktorni podaci o toksičnim efektima jednoslojnih UNC ukazuju na potrebu za daljim ispitivanjima. U na&scaron;im istraživanjima ispitivane su promene u ćelijskom odgovoru kao i morfolo&scaron;ke promene nakon delovanja jednoslojnih ugljeničnih nanocevi na ćelijskoj liniji humanih fetalnih fibroblasta pluća MRC-5 i ćelijskoj liniji humanog adenokarcinoma pluća A549. U ovoj studiji kori&scaron;ćene su jednoslojne ugljenične nanocevi koje su sadržale rezidualne nečistoće poput gvožđa. Citotoksičnost jednoslojnih UNC (engl. single-walled carbon nanotubes &ndash; SWCNT) je ispitivana kolorimetrijskim MTT testom. Tokom 24 i 48h niske koncentracije jednoslojnih ugljeničnih nanocevi (&lt;250 &mu;g/mL) pokazale su nisku toksičnost na proliferaciju i vijabilnost u obe ispitivane ćelijske linije. Ipak, pri visokim koncentracijama UNC (250-750 &mu;g/mL) antiproliferativni efekat je bio blizu IC50 vrednostima. Na osnovu rezultata dobijenih MTT testom može se zaključiti da su maligne A549 ćelije osetljivije na delovanje jednoslojnih UNC u odnosu na normalne MRC-5 ćelije. Kombinacija ugljeničnih nanocevi sa prirodnim polifenolima (resveratrolom i proantocijanidolima) nije značajno uticala na citotoksičnost u MRC-5 ćelijama, za razliku od A549 ćelija gde je tretman kombinacijama umanjio toksičnost ugljeničnih nanocevi. Transmisionom elektronskom mikroskopijom ispitivan je efekat jednoslojnih ugljeničnih nanocevi na ćelijsku morfologiju i preživljavanje. Intracelularni agregati ugljeničnih nanocevi primećeni su u obe ćelijske linije, čime je potvrđeno da ugljenične nanocevi ulaze u ćelije. Imajući u vidu da nanomaterijali poput ugljeničnih nanocevi indukuju oksidativni stres i njime posredovanu apoptozu, na protočnom citometru je određivano prisustvo ćelija u apoptozi i nekrozi. Tretman ćelija sa jednoslojnim ugljeničnim nanocevima nije doveo do značajnog porasta broja apoptotskih ili nekrotičnih ćelija, &scaron;to ide u prilog niskoj toksičnosti ovog nanomaterijala, odnosno ukazuje na alternativne mehanizme toksičnosti. Međutim kombinacija jednoslojnih ugljeničnih nanocevi sa antioksidantima, resveratrolom i proantocijanidolima indukuje veći procenat apoptoze i nekroze u odnosu na tretman samo sa nanocevima. Promene u ekspresiji gena praćene su lančanom reakcijom polimeraza (PCR). Komparativna analiza rezultata genske ekspresije MRC-5 i A549<br />ćelija nakon tretmana sa jednoslojnim ugljeničnim nanocevima pojedinačno i u kombinaciji sa antioksidantima ukazala je na kompleksnost i raznolikost biolo&scaron;kog odgovora ispitivanih ćelija. U na&scaron;em istraživanju ispitivana je i promena aktivnosti enzima antioksidativne za&scaron;tite i količine glutationa u ćeliji. Primena jednoslojnih ugljeničnih nanocevi u MRC-5 ćelijama dovodi po smanjenja specifične aktivnosti enzima SOD i GR, povećava specifičnu aktivnost GPx i ne utiče na promenu specifične aktivnosti GST i količine glutationa u ćeliji Primena jednoslojnih ugljeničnih nanocevi u A549 ćelijama dovodi po smanjenja specifične aktivnosti enzima SOD, ne utiče na promenu specifične aktivnost enzima GR, GST i GPx, i dovodi do povećanja količine glutationa u ćeliji. Ćelijska vijabilnost, morfolo&scaron;ke promene, redoks homeostaza i ekspresija ispitivanih gena bile su promenjene nakon tretmana sa jednoslojnim ugljeničnim nanocevima. Iako su dobijeni rezultati značajni za procenu toksičnosti ugljeničnih nanocevi, neophodna su dalja istraživanja koja treba da doprinesu boljem razumevanju toksičnih efekata ugljeničnih nanocevi.</p> / <p>Carbon nanotubes are being actively introduced in electronics, computer science, and optics as well as for various biomedical applications. While production of single-walled carbon nanotubes (SWCNT) has escalated in recent years, the knowledge on risk associated with exposure remains unclear. Contradictory data on the toxic effects of single-walled carbon nanotubes highlights the urgent need for further studies. In this study we investigated the alterations in cellular response along with morphological changes induced by single-walled carbon nanotubes in human lung fibroblast cell line MRC-5 and adenocarcinoma human alveolar basal epithelial cells A549. In this study we used SWCNT containing large amounts of residual metallic impurities such is iron, and the iron concentration increased in dose dependent manner in cells exposed to SWCNT. Cytotoxicity was evaluated by MTT assay and SWCNT showed little cytotoxic effect on the proliferation and viability of two cell lines tested at the concentrations used (&lt;250 &mu;g/mL) within 24 and 48h. However exposing both cell lines to high concentrations (250-750 &mu;g/mL) resulted in near IC50 values. Based on MTT test SWCNT were more cytotoxic to A549 cell line. Cytotoxicity of SWCNT in combination with natural polyphenols (resveratrol and proanthocyanidins) did not noticeably affect the cytotoxicity of SWCNT to MRC-5 cells. However introduction of polyphenols did reduce the cytotoxicity of SWCNT to A549 cells. Transmission electron microscopy was used to complement cytotoxicity assays and to investigate the pathological effect of internalized SWCNT on cell morphology and survival. Intracellular bundles of CNTs, possibly aggregated/agglomerated were observed in both cell lines, confirming internalization after 24h exposure. Since nanoparticles like carbon nanotubes are toxic mainly because they cause oxidative stress, often associated with an increased apoptosis we checked for apoptotic and necrotic cells using flow cytometry. Incubation with SWCNT did not result in pronounced apoptosis or necrosis supporting its low toxicity and possibly alternative mechanism of cell damage. However incubation with SWCNT in combination with resveratrol and proanthocyanidins induced higher levels of both apoptosis and necrosis than SWCNT alone. Changes in gene expression following exposure to SWCNT were evaluated by polymerase chain reaction PCR array which indicated complex and diverse change in expression of genes involved in apoptosis, cell proliferation and oxidative stress. Finally we investigated the modulation of the antioxidant enzyme system and the changes in the cytosolic levels of GSH. SWCNT reduced the specific activity of SOD and GR enzymes, increased GPx activity. No changes in intracellular levels of GSH were observed in MRC-5 cell line. Same treatment in A549 cell reduced the specific activity of SOD, had no effect on GR, GST and GPx activity, but increased intracellular levels of GSH. Cell viability, morphologic changes, redox homeostasis and gene expression were affected by the presence of SWCNT. Although our findings are useful in predicting human response against SWCNT exposure, further study is needed for better understanding of the effects of SWCNT.</p>