Orientador: Solange Maria de Almeida / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-05T09:03:13Z (GMT). No. of bitstreams: 1 Domingos_AndreadeCastro_D.pdf: 10036948 bytes, checksum: 7cd917fe62aa2831d762e0ac4943e2b4 (MD5) Previous issue date: 2005 / Resumo: O objetivo do presente trabalho foi avaliar morfologicamente e imunohistoquimicamente os efeitos agudos da radiação gama em glândulas parótidas de ratos. Vinte e um animais foram divididos em 2 grupos experimentais, controle e irradiado, sendo o primeiro grupo composto por animais não expostos à radiação e o segundo formado for animais que tiveram a região de cabeça e pescoço irradiada com uma dose única de 15 Gy. As glândulas parótidas dos animais irradiados foram removidas nos tempos 4, 8, 12, 24, 48 e 72 horas após sua exposição e, posteriormente, submetidas ao processamento tecidual por meio de métodos imunohistoquímicos, para a avaliação da proteína laminina, e pela coloração por hematoxilina-eosina, para avaliação morfológica. Não foram observadas diferenças estatisticamente significantes entre a intensidade demarcação da laminina do grupo controle e do grupo irradiado. Por outro lado, verificou-se o espessamento e a presença de conglomerados de laminina nas membranas basais acinares dos subgrupos irradiados, tendo sido detectadas diferenças significativas entre o grupo controle e o subgrupo 72 horas (p = 0,0315). A análise morfológica revelou a presença de figuras de necrose nuclear, vacuolizações e severa atrofia dos ácinos, especialmente nos grupos 8 e 12 horas. O início do processo de reparo se deu no tempo 24 horas, mas não foi observada recuperação completa nos últimos tempos avaliados. Os resultados sugeriram que os efeitos deletérios da radiação ionizante podem ser um dos fatores responsáveis pelo remodelamento da matriz extracelular / Abstract: The aim of this study was to evaluate the morphological and immunohistochemical characteristics of gamma radiation acute effects on rat parotid glands. Twenty-one animals were divided into two groups: control and irradiated ones. The former was composed by non-exposed animals, while the latter was formed by rats which had their necks and heads irradiated with 15 Gy. The parotid glands were excised at 4, 8, 12, 24, 48 and 72 hours after irradiation and, then, were submitted to immunohistochemical methods, for the evaluation of the laminin, and to hematoxylin-eosin staining, for the analysis of the morphology. Laminin staining intensities between irradiated and non-irradiated salivary glands showed no statistically significant differences. Nevertheless, it was observed that laminin thickening and laminin-positive conglomerations demonstrated significant differences between the control and the 72-hour-subgroup (p= 0,0315). The morphological analysis revealed the presence of vacuolation, acinar atrophy and necrosis, especially by 8 and 12 hours after irradiation. Regeneration of the tissue started by 24 hours, but it was not complete at 48 and 72 hours after exposure. The results showed that radiation may be one of the factors that induce extracellular matrix remodeling / Doutorado / Radiologia Odontologica / Doutor em Radiologia Odontológica

During chronic and severe acute liver injury, regeneration is thought to occur through hepatic progenitor cells (HPCs). Understanding the regulation of HPCs may offer therapeutic opportunities to enhance liver regeneration. HPCs are associated with an increase in laminins in the extracellular matrix. Laminins are heterotrimeric proteins, composed of an alpha, beta and gamma chain. There are 5 alpha chains with different distributions and functions, but the relative contributions of these in HPC-mediated liver regeneration are not known. My aims were to describe the laminin alpha chains associated with the HPC response and to define the functional effects of specific laminin chains on HPCs. I examined the laminin alpha chains in two mouse models of HPC activation: a transgenic model using conditional deletion of Mdm2 in hepatocytes, and a dietary model using 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). The laminin alpha 5 (Lama5) chain is significantly upregulated in both models and forms a basement membrane which surrounds the progenitor cells. I have also demonstrated Lama5 expression in the ductular reaction seen in human liver disease. Using primary mouse cell cultures, I have shown that Lama5 is produced predominantly by the HPCs themselves, rather than by stellate cells. The HPCs express the cell surface receptor alpha-6 beta-1 integrin, a binding partner of Lama5. I then studied the functional effects of matrix on cell behaviour in vitro using recombinant laminins and a line of spontaneously immortalised mouse HPCs. Compared to other laminin chains, Lama5 selectively promotes HPC adhesion and spreading. These effects are partially blocked by antibodies against beta-1 integrin. Lama5 also significantly enhances HPC migration, resulting in an increase in cell migration. Furthermore, only Lama5 enhances HPC survival in serum-free medium, with an increase in cell viability. Culturing HPCs on HPCs maintained in culture on plastic synthesise Lama5 chain. Knock-down of endogenous Lama5 production using siRNA results in reduced proliferation and increased hepatocytic differentiation, with increased albumin production. I then studied the effects in vivo using transgenic Cre-lox mouse strains that allow conditional knock-out of either laminin alpha 5 or beta-1 integrin in HPCs. The effects of gene deletion were examined in healthy mice and two dietary models of HPC activation: the DDC diet and a choline-deficient, ethionine-supplemented (CDE) diet. Although these experiments were limited by a low number of experimental animals and low recombination rates, there was a suggestion of impaired HPC expansion associated with loss of laminin alpha 5. There was also a significant increase in hepatocellular injury and fibrosis in response to the DDC diet seen with loss of laminin alpha 5 expression. Laminin alpha 5-containing matrix is deposited around HPCs during liver regeneration and supports progenitor cell attachment, migration and maintenance of an undifferentiated phenotype. This work identifies a novel target for enhancing liver regeneration.

The skeletal muscle is a dynamic tissue that has the ability to change and modify itself to fit the level of required activity; a phenomenon called muscle plasticity. Most studies of muscle plasticity are carried out in situ, a condition for which it is difficult to study and discern between the intrinsic properties of skeletal muscle, the myokines released by muscle fibers and the neurotrophic factors released by neurons innervating skeletal muscles that play various roles in the mechanisms of muscle plasticity. Another approach is to study the morphological and contractile properties of single adult muscle fibers under culture conditions for which one can fully control the level of activity and exogenous factors affecting muscle plasticity. However, the survival of single muscle fiber in culture is very low as most fibers degenerated or supercontracted within 5-7 days. The first objective of this study was to optimize fiber survival in culture. The application of chronic stimulation and beta-adrenergic agonists are two major factors that prevent muscle atrophy and loss of force in denervated muscles in situ. So, objective two was to determine if chronically stimulated single fibers in culture also improve fiber survival and contractile characteristic under culture conditions. The third objective was the same for salbutamol, a beta 2-adrenergic agonist. In regard to the optimization of fiber survival, the Minimum Essential Medium (MEM) was a better medium than Dulbecco’s Modified Eagle Medium (DMEM), changing 50% of the culture medium every two days also improved fiber survival compared to changing the medium every day. Interestingly, inhibiting the proliferation of satellite cells with AraC largely improved fiber survival when fibers were kept under resting conditions, but not when they were chronically stimulated. Finally, under conditions in which proliferation of satellite cells was inhibited, the use of a collagen/laminin mixture as adhering substrate to improve fiber adhesion to glass coverslip gave rise to a better fiber survival than Matrigel that contains not only collagen and laminin but several growth factors. The results suggest i) that when satellite cells (or fibroblasts) are allowed to proliferate they appear to contribute to the degeneration of fibers under resting conditions and ii) that the release of myokines by skeletal muscle fibers (or cytokines by other cells) likely play a role in fiber survival. Contrary to the situation in situ, neither the chronic stimulation nor salbutamol improved fiber survival and contractile characteristics of muscle fibers in culture suggesting that some important factors in culture are missing to allow chronic stimulation and salbutamol to reduce muscle atrophy and loss of force.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20562号 / 医博第4247号 / 新制||医||1022(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 江藤 浩之, 教授 開 祐司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Obstructive sleep apnea syndrome (OSAS) is a complex disorder characterized by repetitive bouts of upper airway collapse during sleep, causing subsequent intermittent hypoxia, hypercapnia, and fragmented sleep and is also associated with significant morbidity including daytime sleepiness, hypertension, and elevated cardiovascular risk. OSAS affects at least 4% of men and 2% of women; unfortunately, it is estimated that 80% to 90% of adults with OSAS remain undiagnosed. Both clinical characteristics and complex genetic and environmental interactions have made it difficult to understand OSAS disease etiology and identifying patients at risk is still elusive. A pattern of gene expression in cells or tissues related to a disease state for OSAS would provide beneficial information to be most effective in screening or diagnosing this disease. Objectives: The objectives of this study were to: 1) map out the study design and bench assay strategies by which to investigate this issue; 2) find out if there are specific differences in the global gene expression profiles of adult females with OSAS compared to those without OSAS, under conditions in which subjects were clinically similar (BMI, diabetes, cardiovascular disease, etc.); and 3) assess the protein expression differences that could potentially be linked via well-established molecular pathways associated with any differences found in global gene expression profiles in the presence and absence of OSAS. Methods: Subjects were overweight premenopausal Caucasian women with untreated OSAS (n=6; age = 40.7 ± 3.4; BMI = 49.04 ± 6.97; apnea-hypopnea index = 27.3 ± 16.02), and control subjects (n=10) (age = 38.2 ± 7.6; BMI = 47.94 ± 6.15; apnea-hypopnea index < 5), and matched for other clinical characteristics (diabetes, cardiovascular disease status, medications, etc.) recruited from either Carilion Clinic Pulmonary/Sleep Medicine or Carilion Clinic Bariatric Surgery practices. Subjects provided a fasting blood sample in which the monocytes were isolated from whole blood. The RNA was extracted from the monocytes, assessed for purity and quantity, frozen and shipped to collaborators at Dana-Farber Cancer Institute and hybridized to Affymetrix whole human genome chips on a gene chip. The initial computational evaluation and interpretation generated the hypothesis. Two-step quantitative real time polymerase chain reaction (qPCR) was performed to verify the results from the microarray analysis. The laminin enzyme immunoassay (EIA), and cellular adhesion assays were performed to determine if genomic changes resulted in proteomic and phenotypic assessments. Results: OSAS subjects had nine aberrantly regulated genes, of which three genes (LAMC-1, CDC42, and TACSTD2) showed a pattern in segregation between OSAS and controls subjects based on expression patterns. In addition, qPCR indicated a 2.1 fold increase in LAMC-1 and a 1.1 fold increase CDC42 expression unique to the tissue samples of patients with OSAS. Though the serum laminin EIA did not differ between groups, a statistically significant increase in peripheral blood mononuclear cells (PBMC) cellular adhesion in OSAS patients versus control subjects was found. The OSAS subjects had a well cell count of 9.27 ± 1.54 cells vs. controls 5.75 ± 0.78 cells (p Ë‚ 0.05), which is relative to the 103 cells/field that were plated. Conclusions: Cells isolated from women with moderate-severe OSAS show an abnormality in cellular adhesion, a process driven in part by the gene LAMC-1, which was also aberrantly expressed in these subjects. This suggests that inflammation may be linked to the pathogenesis of OSAS. This pilot study has provided the framework and preliminary data needed to propose a larger study with extramural research funding. / Ph. D.

In Anlehnung an die Definition beim Pferd (SCHOON et al. 1992, 1997) wird die bovine Endometrose als endometriale periglanduläre und/oder stromale Fibrose mit Alteration der betroffenen Drüsen definiert (RODENBUSCH 2011). Eine eingehende histomorphologische und immunhistologische Charakterisierung der Endometrose existiert bisher bei der Stute (KENNEY u. DOIG 1986, SCHOON et al. 1992, HOFFMANN et al. 2009), jedoch nicht beim Rind. Das Ziel dieser Arbeit ist daher die histomorphologische und immunhistologische Charakterisierung der verschiedenen Endometroseformen beim Rind. Die Auswertung der Proben erfolgt am Institut für Veterinär-Pathologie der Universität Leipzig. In Abhängigkeit von den klinisch-gynäkologischen Befunden werden diese Proben in drei Gruppen unterteilt. Gruppe A1: Endometriumbioptate (n=12) von vier klinisch-gynäkologisch gesunden fertilen Rindern in definierten Zyklusphasen; Gruppe A2: Endometriumbioptate (n=36) von 36 klinisch genitalgesunden Rindern mit mindestens einer Abkalbung und Gruppe B: Uterusquerschnitte (n=69) von 69 sub-/ infertilen Rindern. Die Proben werden anhand der von HOFFMANN (2006) definierten Kriterien auf histopathologische Veränderungen hin untersucht und charakterisiert. Das histomorphologische Erscheinungsbild der involvierten periglandulären Stromazellen erlaubt die Einteilung der Endometrose in eine aktive, inaktive und gemischte Fibrose, die, je nach der Integrität des Drüsenepithels, einen destruierenden oder nicht destruierenden Charakter aufweist. Zur Charakterisierung der Endometroseformen werden neben histomorphologischen Kriterien die Intermediärfilamente Desmin, Vimentin und Zytokeratin sowie die Expression von α-Aktin und Laminin berücksichtigt. Die deskriptive statistische sowie die Inferenzstatistik-Auswertung erfolgen unter Zuhilfenahme der Software SPSS 18. Eine aktive Fibrose tritt bei 96,2 % der Rinder auf; 1,9 % weisen eine inaktive und 1,9 % eine gemischte Endometrose auf. Ein nicht destruierendes Erscheinungsbild der Endometrose kann bei 88,6 % der untersuchten Rinder nachgewiesen werden. Bei 11,4 % der untersuchten Rinder liegt eine Endometrose mit destruierendem Charakter vor. Die Endometrose betrifft bei 81 % der Rinder Einzeldrüsen und bei 19 % Drüsennester. Drüsennester sind häufiger bei mittel- und hochgradigen Endometrosen nachweisbar. 20 % der Proben mit nicht destruierender Endometrose und 58 % der Proben mit destruierender Endometrose weisen eine entzündliche Infiltration der Drüsen auf, wobei ein Zusammenhang zwischen der entzündlichen Infiltration der endometrotischen Drüsen und dem destruierenden Charakter der Endometrose festgestellt werden kann. Zusätzlich zur Endometrose zeigen 61 % der Rinder eine Endometritis, 12,4 % eine Perivaskulitis und 66 % eine interkarunkuläre und/oder intrakarunkuläre Angiosklerose. Insgesamt findet sich nur bei 24 % der Fälle ausschließlich eine Endometrose, bei 10,5 % eine Endometrose und zugleich eine Endometritis, bei 16 % liegt eine Endometrose zusammen mit einer Angiosklerose vor. Eine Kombination von Endometrose, Angiosklerose und Endometritis ist bei 49,5 % der Proben nachweisbar. Insgesamt bestehen jedoch keine erkennbaren statistischen Zusammenhänge zwischen den einzelnen Befundkombinationen. Aufgrund der immunhistologischen Untersuchung kann konstatiert werden, dass die periglandulären Stromazellen innerhalb der Endometrose eine stromale Koexpression von Desmin, Vimentin und α-Aktin aufweisen, welche ein für Myofibroblasten charakteristisches Merkmal ist. Ein kleiner Prozentsatz der Drüsenepithelzellen in der destruierenden Endometrose reagiert multifokal positiv mit dem Vimentinantikörper. Dies ist möglicherweise Ausdruck einer Fehldifferenzierung zur Stabilisierung der Zelle oder Anzeichen einer intensivierten (pathologischen) Proliferation. Die Lamininexpression der Basallamina der endometrotisch veränderten Drüsen ist, insbesondere bei der destruierenden Endometrose, diskontinuierlich und geht mit einer Auffaserung der Basallamina einher. Vermutlich lassen sich die umfangreichen Basallaminaalterationen auf von Myofibroblasten sezernierte Enzyme zurückführen. Bei Rindern kann kein Zusammenhang zwischen der Endometrose und dem Alter der Rinder oder der Anzahl der Kalbungen festgestellt werden. In der vorliegenden Arbeit dominiert die geringgradig aktive nicht destruierende Endometrose gegenüber den anderen bovinen Endometroseformen. Die sub-/ infertilen Kühe (Gruppe B) zeigen häufiger eine schwerere und destruierende Endometrose als die klinisch gesunden Rinder (Gruppe A1, A2). Die klinisch-gynäkologisch gesunden Rinder in definierten Zyklusphasen weisen variable Endometroseformen oder Endometrosegrade auf. Die sub-/ infertilen Rinder zeigen eine höhere Güstzeit (252,82 ± 163,83 Tage) als die klinisch-gynäkologisch gesunden Tiere mit mindestens einer Abkalbung (94 ± 28,4 Tage). Die längere Güstzeit bei den sub- und infertilen Rindern könnte somit eine Folge des insgesamt in Charakter und Grad stärker geschädigten Endometriums bei dieser Gruppe sein. Somit kann unter Berücksichtigung der vorliegenden Ergebnisse angenommen werden, dass die aufgeführten Alterationen die Fertilität des Rindes negativ beeinflussen. Die Ergebnisse ermöglichen eine histomorphologische und immunhistologische Charakterisierung der Endometrose beim Rind. Anhand der Ergebnisse der hier durchgeführten detaillierten Untersuchungen ist es möglich, eine präzisere Deskription degenerativer endometrialer Befunde vorzunehmen. In Hinblick auf die Fertilität bei Vorliegen einer bovinen Endometrose wird somit die Grundlage für zukünftige prognostische Bewertungen gelegt.

TGF-β isoforms are important signalling molecules in wound repair in the skin. Transforming growth factor β3 (TGF-β3) has been implicated in scarless healing. In both animal and human models the application of exogenous TGF-β3 causes a reduction in the inflammatory response and improves the architecture of the neodermis. Research into the influence of TGF-β on scarring has tended to focus on fibroblasts. However, keratinocytes play a major role in scarring both indirectly, as a result of their influence over the behaviour of fibroblasts and also by directly influencing wound contraction. Thus, experiments were carried out to investigate the influence of TGF-β3 on the behaviours of a keratinocyte cell line (HaCaT). Incubation with TGF-β3 increased cell spreading and appeared to reduce cell-surface contacts indicated by both SPR imaging and a detachment assay. TGF-β3 also caused a decreased cell alignment response to microcontact printed protein patterns, in part due to the deposition of laminin which is associated with the TGF-β induced cell migration. There is evidence that TGF-β isoforms differentially influence the outcome of wound healing. Similar to the results produce following addition of exogenous TGF-β3, the neutralisation of TGF-β1 and 2 has been shown to reduce scar formation in the adult wounds. During reepithelialisation keratinocytes experience a dynamic environment. Both extracellular matrix proteins and growth factors influence the progression of wound repair which includes both cell migration and proliferation. Few studies have examined collective cell behaviour in response to TGF-β isoforms and ECM coated substrates. Thus both wound closure and cell proliferation assays were conducted for different ECM proteins fibronectin, laminin and collagen type I and for TGF-β1, 2 and 3. Rates of wound closure were significantly reduced on laminin coated substrates while cell proliferation rates were increased. TGF-β2 and 3 induced significant increases in wound closure rates. This appeared to correspond with an increase in the number of cells independently migrating out from the wound margins. Only TGF-β3 caused a significant decrease in cell proliferation over a 4 day period. Laminin332 deposition is central to the reepithelialisation process and is known to be induced in response to TGF-β. Thus experiments were carried out to investigate HaCaT cell laminin332 deposition in response to TGF-β1, 2 and 3. Both an immunofluorescence staining technique and an ELISA based semi-quantification method was used. Following 4 day incubation all TGF-β isoforms significantly increased laminin332 deposition; however TGF-β2 and 3 caused the most significant increases. Integrin receptors enable cell-matrix interactions during wound repair. TGF-β is known to influence the expression of integrin subunits. Thus, experiments were carried out to compare the influence of each TGF-β isoform on the expression of subunits β3, β2, β5, β1 and β4. All TGF-β isoforms significantly increased all subunit expression. TGF-β3 caused the most significant increase in β4 and both TGF-β2 and 3 caused the most significant increase in β2. While there were differences in cell responses to each isoforms, TGF-β3 did not stand out from the other two isoforms. Interestingly, TGF-β2 shared more similarities with TGF-β3 than it did with TGF-β1, in its role in enhancing wound closure and LN332 deposition. These comparative studies have shown that differences exist in the way TGF-β isoforms influence HaCaT cell behaviour, namely migration, laminin deposition and integrin expression.

TGF-β isoforms are important signalling molecules in wound repair in the skin. Transforming growth factor β3 (TGF-β3) has been implicated in scarless healing. In both animal and human models the application of exogenous TGF-β3 causes a reduction in the inflammatory response and improves the architecture of the neodermis. Research into the influence of TGF-β on scarring has tended to focus on fibroblasts. However, keratinocytes play a major role in scarring both indirectly, as a result of their influence over the behaviour of fibroblasts and also by directly influencing wound contraction. Thus, experiments were carried out to investigate the influence of TGF-β3 on the behaviours of a keratinocyte cell line (HaCaT). Incubation with TGF-β3 increased cell spreading and appeared to reduce cell-surface contacts indicated by both SPR imaging and a detachment assay. TGF-β3 also caused a decreased cell alignment response to microcontact printed protein patterns, in part due to the deposition of laminin which is associated with the TGF-β induced cell migration. There is evidence that TGF-β isoforms differentially influence the outcome of wound healing. Similar to the results produce following addition of exogenous TGF-β3, the neutralisation of TGF-β1 and 2 has been shown to reduce scar formation in the adult wounds. During reepithelialisation keratinocytes experience a dynamic environment. Both extracellular matrix proteins and growth factors influence the progression of wound repair which includes both cell migration and proliferation. Few studies have examined collective cell behaviour in response to TGF-β isoforms and ECM coated substrates. Thus both wound closure and cell proliferation assays were conducted for different ECM proteins fibronectin, laminin and collagen type I and for TGF-β1, 2 and 3. Rates of wound closure were significantly reduced on laminin coated substrates while cell proliferation rates were increased. TGF-β2 and 3 induced significant increases in wound closure rates. This appeared to correspond with an increase in the number of cells independently migrating out from the wound margins. Only TGF-β3 caused a significant decrease in cell proliferation over a 4 day period. Laminin332 deposition is central to the reepithelialisation process and is known to be induced in response to TGF-β. Thus experiments were carried out to investigate HaCaT cell laminin332 deposition in response to TGF-β1, 2 and 3. Both an immunofluorescence staining technique and an ELISA based semi-quantification method was used. Following 4 day incubation all TGF-β isoforms significantly increased laminin332 deposition; however TGF-β2 and 3 caused the most significant increases. Integrin receptors enable cell-matrix interactions during wound repair. TGF-β is known to influence the expression of integrin subunits. Thus, experiments were carried out to compare the influence of each TGF-β isoform on the expression of subunits α3, α2, α5, β1 and β4. All TGF-β isoforms significantly increased all subunit expression. TGF-β3 caused the most significant increase in β4 and both TGF-β2 and 3 caused the most significant increase in α2. While there were differences in cell responses to each isoforms, TGF-β3 did not stand out from the other two isoforms. Interestingly, TGF-β2 shared more similarities with TGF-β3 than it did with TGF-β1, in its role in enhancing wound closure and LN332 deposition. These comparative studies have shown that differences exist in the way TGF-β isoforms influence HaCaT cell behaviour, namely migration, laminin deposition and integrin expression. / EPSRC and DTA grant