Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2007. / Radhakrishna, Harish, Committee Member ; Zhu, Cheng, Committee Member ; LaPlaca, Michelle C., Committee Member ; Garca, Andrs J., Committee Chair ; Kowalczyk, Andrew P., Committee Member.

Thesis (Ph. D.)--Illinois State University, 1994. / Title from title page screen, viewed March 21, 2006. Dissertation Committee: H. Tak Cheung (chair), Herman E. Brockman, Lynne A. Lucher, Philip D. Morse, Anthony J. Otsuka. Includes bibliographical references (leaves 101-111) and abstract. Also available in print.

Thesis (Ph. D.)--Illinois State University, 1997. / Title from title page screen, viewed June 5, 2006. Dissertation Committee: H. Tak Cheung (chair), Sean Arkins, Herman E. Brockman, Paul A. Garris, Brian J. Wilkinson. Includes bibliographical references (leaves 113-121) and abstract. Also available in print.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on October 18, 2007) Vita. Includes bibliographical references.

Τα αστροκυττώματα αποτελούν τη συχνότερη ομάδα νεοπλασμάτων του κεντρικού νευρικού συστήματος. Συνιστούν μία ετερογενή ομάδα σε ότι αφορά τη βιολογική τους συμπεριφορά και την πρόγνωσή τους και παρά την πρόοδο που έχει σημειωθεί στην κατανόηση των παθογενετικών μηχανισμών τους, συνεχίζουν να αποτελούν μια από τις επιθετικότερες μορφές καρκίνου. Ένα από τα βασικότερα χαρακτηριστικά των αστροκυττωμάτων είναι η διηθητική τους ικανότητα δηλαδή η κυτταρική μετακίνηση (cell locomotion) και η αποδόμηση στοιχείων της εξωκυττάριας ουσίας (extracellular matrix degradation) που είναι απαραίτητη για να επιτευχθεί η διήθηση. Η ιδιότητα της κυτταρικής διήθησης καθιστά τα νεοπλάσματα αυτά ιδιαιτέρως καταστροφικά για το νευρικό ιστό και επιπλέον δυσχεραίνει τη θεραπεία τους. Τόσο η χειρουργική αντιμετώπιση όσο και η ακτινοθεραπεία καθίστανται μη επαρκείς για την ριζική αντιμετώπιση των όγκων αυτών. Η παρούσα μελέτη έχει ως σκοπό την μοριακή μελέτη του φαινομένου της κυτταρικής διήθησης και εντοπίζεται κυρίως στους μηχανισμούς διάσπασης της εξωκυττάριας ουσίας. Είναι γεγονός ότι τα τελευταία χρόνια μεγάλη ερευνητική προσπάθεια συντελείται προς την κατεύθυνση αυτή και πολλοί από τους μοριακούς μηχανισμούς που εμπλέκονται στη διαδικασία αποδόμησης της εξωκυττάριας ουσίας αρχίζουν να αποσαφηνίζονται. Όμως καθώς η έρευνα προχωρά όλο και περισσότερα δεδομένα αναδεικνύονται γεγονός που υποδηλώνει την πολυπλοκότητα του φαινομένου. Ένα σχετικά πρόσφατα ταυτοποιημένο μόριο η ογκοκατασταλτική πρωτείνη ING-4 (Inhibition of growth) πιστεύεται ότι είναι πιθανόν να εμπλέκεται στον έλεγχο της αποδόμησης στοιχείων της εξωκυττάριας ουσίας. Πρόσφατες μελέτες υποδεικνύουν τον ρυθμιστικό ρόλο του ING-4 στο μεταγραφικό παράγοντα NF-κB ο οποίος είναι γνωστό ότι αποτελεί κύριο ρυθμιστή της έκφρασης των ενζύμων διάσπασης της εξωκυττάριας ουσίας MMP-2, MMP-9 (matrix metalloproteases 2,9) και του ενεργοποιητή του πλασμινογόνου τύπου ουροκινάσης u-PA (urokinase-type plasminogen activator). Στη μελέτη αυτή γίνεται εκτίμηση των επιπέδων έκφρασης της πρωτείνης ING-4 της p65 υπομονάδας του NF-κB και των ενζύμων MMP-2, MMP-9 και του u-PA με τη χρήση της ανοσοιστοχημικής τεχνικής σε τομές παραφίνης ληφθείσες από 101 περιστατικά αστροκυττωμάτων του ανθρώπου όλων των βαθμίδων διαφοποίησης. Η γνώση του επιπέδου έκφρασης των μορίων αυτών στα αστροκυττώματα καθώς και η μεταξύ τους συσχέτιση αφενώς μεν θα συμβάλει στην κατανόηση των μηχανισμών που ελέγχουν την κυτταρική διήθηση, αφετέρου πιθανόν να αποτελέσει χρήσιμη γνώση για μελλοντικό σχεδιασμό θεραπειών περιορισμού του φαινομένου αυτού. / Inhibitor of growth 4 (ING-4) is a tumor suppressor gene that interacts with nuclear factorkappaB (NF-kB) and represses its transcriptional activity. Several lines of evidence suggest that the tumor suppressor gene ING-4, the transcription factor NF-kB and its target genes matrix metalloproteases MMP-2, MMP-9 and urokinase plasminogen activator (u-PA) are critically involved in tumor invasion. The aim of the present study was to investigate immunohistochemically the expression pattern of ING-4, NF-kB and the NF-kB downstream targets MMP-2, MMP-9 and u-PA in human astrocytomas from 101 patients. We found that ING-4 expression was significantly decreased in astrocytomas, and ING-4 loss was associated with tumor grade progression. Expression of p65, a NF-kB subunit, was significantly higher in grade IV than in grade III and grade I/II tumors, and a statistical significant negative correlation between expression of ING-4 and expression of nuclear p65 was noticed. MMP-9, MMP-2 and u-PA were overexpressed in human astrocytomas. Of note, astrocytomas of advanced histologic grades (grade III, IV) displayed significantly higher expression levels of these proteins compared to tumors of lower grades (grade I, II). Collectively, our data suggest an essential role for ING-4 in human astrocytoma development and progression possibly through regulation of the NF-kB-dependent expression of genes involved in tumor invasion.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O propósito do presente trabalho foi investigar a participação da proliferação celular e da expressão dos componentes da matriz extracelular na cascata de eventos do processo de reparo da fratura óssea, empregando as técnicas histológica, imunohistoquímica e morfométrica, em um modelo experimental padronizado para a indução da lesão na tíbia de ratos a partir do método empregado por Yuehuei e Friedman7. É importante padronizar um modelo de indução da fratura, para posterior investigação da participação das células e dos componentes da matriz extracelular no processo de reparo da fratura, considerando que o tempo de consolidação depende significantemente da natureza e do tipo da lesão produzida. Quarenta (n = 40) ratos Wistar foram submetidos a fratura . Os animais foram avaliados em oito (n = 8) grupos de cinco (n = 5) animais, cada grupo emperimental com 12, 24, 48, 72, 96, 144, 192 e 240 horas após a fratura (12h até 10 dias). As fraturas foram classificadas de acordo com o sistema de classificação internacional de fratura de Muller100, AO (Associação para Osteosíntese). Foram encontradas fraturas simples em 86% do total, sendo 68% de fraturas transversas e 18% de fraturas obliquas, 14% do total de fraturas foram complexas, sendo 8% de fraturas irregulares e 6% de fraturas segmentares. Esses resultados demonstram que o aparelho permite padronizar radiológicamente o tipo de fratura, caracterizado pela linha que separa os fragmentos ósseos. Os resultados qualitativos dos componentes da matriz extracelular para TGF-β, VEGF, colágeno I e II, osteopontina, proteoglicanos, fibras do sistema elástico com a coloração de resorcina funcsina de Weigert, e para proliferação celular pelo PCNA, assim como os resultados morfométricos, sugerem que a modulação da expressão dos componentes da matriz extracelular e a proliferação celular durante o processo de reparo da fratura não é homogênea para todos os componentes teciduais, dependendo significantemente das tensões locais geradas pelo tipo da linha de fratura que pode ser determinante no tempo de regeneração do osso e na qualidade da restauração das propriedades biomecânica. Nossos achados podem contribuir para melhor compreensão da reparo de fratura óssea e para novas abordagens terapêuticas que considerem as propriedades biomecânicas do tecido ósseo em reparo nas suas diferentes etapas / The purpose of this study was to investigate the role of cells proliferation and extracellular matrix components expression in the process of bone fracture repair. To do so it used histological techniques, immunohistochemistry and morphometric analysis as well as a standardized experimental model for the induction of injury to the tibia of rats as proposed by Yuehuei and Friedman7. It is important to standardize a model of fracture induction for further investigation of the involvement of cells and extracellular matrix components in the fracture repair process, whereas the healing time depends significantly on the nature and type of lesion produced. Forty (n = 40) Wistar rats were subjected to fracture. The animals were divided into eight (n = 8) groups of five (n = 5). Each subgroup was observed after 12, 24, 48, 72, 96, 144, 192 and 240 hours of fracture (12 to 10 days). Immediately afterwards, the fractures were classified according to the system of international classification of fracture by Muller100, AO (Association for Osteosynthesis). Simple fractures were found in 86% of the total, among them, 68% were transverse and 18% were oblique. Complex fractures were found in 14% of the cases, among them 8% were irregular and 6% were segmental. These results demonstrated that the device enables researchers to standardize the type of fracture by X-ray, marked by the line separating the bone fragments. The qualitative results of the cells and extracellular matrix components of TGF-β, VEGF, PCNA, collagen I and II, osteopontin, proteoglycans, elastic fibers system with resorcin funcsin of Weigert, as well as the morphometric results suggest that the repair process of the fracture is not homogeneous for all components. Expression of the extracellular matrix components and cell proliferation modulation significantly depends on the local stresses generated by the type of the fracture. Such type can be decisive in determining time duration for bone regeneration and quality of the biomechanical properties restoration. Our findings may contribute to better understanding of bone fracture repair and for new therapeutic approaches that consider the biomechanical properties of bone tissue in repair in its different stages

Les carcinomes évoluent dans un microenvironnement inflammatoire composé de cellules stromales (fibroblastes, cellules endothéliales et immunitaires) immergées dans une matrice extracellulaire (MEC). Les fibroblastes associés aux carcinomes (FACs) déposent et remodèlent la MEC dans le but de la rendre permissive à la croissance et l’invasion tumorale. Parmi les facteurs pro-inflammatoires responsables de l’activation des fibroblastes résidents, la cytokine Leukemia Inhibitory Factor (LIF) détient un rôle capital. En régulant l’activité de la chaîne légère de la myosine II (MLC-II), LIF induit la contractilité du cytosquelette d’actomyosine, générant des forces de tension et le remodelage de la MEC par les FACs. En revanche, les gènes régulés par LIF impliqués dans le phénotype pro-invasif des FACs ne sont pas connus. A l’aide d’un criblage phénotypique en trois dimensions, nous avons identifiés ICAM-1 comme régulateur majeur du remodelage de la MEC par les FACs. Nous démontrons qu’ICAM-1 est nécessaire et suffisant pour induire la réorganisation de la MEC indispensable à l’invasion collective des cellules de carcinome squameux. En effet, ICAM-1 est un régulateur de la contractilité cellulaire dépendante de la voie de signalisation RhoA-ROCK et de la kinase Src. De plus, la contractilité cellulaire régule l’expression d’ICAM-1, menant ainsi à une boucle de régulation positive. Nous proposons alors qu’ICAM-1 représente une cible thérapeutique afin de lutter contre l’invasion tumorale et la dissémination métastatique. / Acto-myosin contractility in carcinoma-associated fibroblasts leads to the assembly of the tumor extracellular matrix. The pro-inflammatory cytokine LIF governs fibroblast activation in cancer by regulating the myosin light chain 2 activity. So far, however, how LIF mediates cytoskeleton contractility remains unknown. Using phenotypic screening assays based on knock down of LIF-dependent genes in fibroblasts, we identified ICAM1 as a crucial regulator of stroma fibroblast proinvasive matrix remodeling. We demonstrate that ICAM1 is necessary and sufficient to promote inflammation-dependent extracellular matrix organization, which leads to cancer cell invasion. Indeed, ICAM1 mediates generation of acto-myosin contractility downstream of the Src kinases in stromal fibroblasts. Moreover, acto-myosin contractility regulates ICAM1 expression, establishing a positive feedback signaling. Thus, targeting stromal ICAM1 might constitute a possible therapeutic mean to counteract tumor cell invasion and dissemination.

Orientador: Marlise Inês Klein / Resumo: A cárie representa a doença humana mais prevalente no mundo, sua etiologia é dependente de biofilme e da dieta. Os biofilmes são comunidades altamente dinâmicas e estruturadas de células microbianas que se encontram embebidas em uma matriz extracelular tridimensional (MEC). Esta estrutura age como uma barreira que limita a difusão e fornece estabilidade e proteção aos microrganismos. Streptococcus mutans tem um potencial acidogênico e acidúrico, orquestra a construção do biofilme e modula sua virulência, produzindo ácidos lipoteicóicos (LTA), DNA extracelular (eDNA) e exopolissacarídeos (EPS), promovendo assim a adesão e coesão microbiana. Ao mesmo tempo a MEC produzida dificulta a difusão de metabólitos no biofilme. O objetivo do estudo foi determinar por meio de RT-qPCR a dinâmica da expressão de genes de S. mutans associados ao metabolismo de LTA (dltABCD, SMU_775c), eDNA (lytST, lrgAB, ccpA) e exopolissacarídeos (gtfBCD, gbpB, dexA), durante o desenvolvimento da MEC de biofilmes mistos em um estudo longitudinal. Biofilmes mistos de S. mutans UA159 (cepa parental) ou ΔgtfB (mutante com deleção do gene gtfB), Actinomyces naeslundii ATCC 12104 e Streptococcus gordonii DL-1 foram formados em discos de hidroxiapatita revestidos de película salivar, e cultivados a 37° C e 5% de CO2 em caldo triptona e extrato de levedura contendo 25% de saliva e alternando 0,1% de sacarose (escassez) e 0,5% de sacarose + 1% de amido (abundância). O pH do meio de cultura manteve-se ácido durante... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Dental caries represents the most prevalent human disease worldwide, its etiology is biofilm-diet dependent. Biofilms are highly dynamic and structured communities of microbial cells that are enmeshed in a three-dimensional extracellular matrix (ECM). This structure acts as a diffusion-limiting barrier providing stability and protection to the microorganisms. Streptococcus mutans is acidogenic, aciduric, and orchestrates the biofilm build-up process. It modulates the biofilm’s virulence by producing lipoteichoic acids (LTA), extracellular DNA (eDNA) and exopolysaccharides (EPS), thereby promoting microbial adhesion and cohesion. The resulting ECM hinders diffusion in the biofilm. The aim of the study was to determine via RT-qPCR the dynamics of expression of S. mutans genes associated with LTA (dltABCD, SMU_775c), eDNA (lytST, lrgAB, ccpA) and exopolysaccharides (gtfBCD, gbpB, dexA) metabolism, during ECM development in mixed-species biofilm by time-lapse studies. Mixed-species biofilms of S. mutans UA159 (parental strain) or ΔgtfB (mutant with deletion of the gene gtfB), Actinomyces naeslundii ATCC 12104 and Streptocsoccus gordonii DL-1 were formed onto saliva-coated hydroxyapatite discs. These biofilms were cultivated at 37°C and 5% CO2 in triptone with yeast extract containing saliva 25% and alternating 0.1% sucrose (scarcity) and 0.5% sucrose + 1% starch (abundance). The pH of the spent media remained acid during the experimental periods, and was lower after prolonged inc... (Complete abstract click electronic access below) / Mestre

Orientador: Ana Cláudia Pavarina / Abstract: Biofilmes formados por Candida estão relacionados a infecções bucais, como a candidíase. Embora a resistência do biofilme seja multifatorial, a proteção exercida por sua matriz extracelular (MEC) é importante para os altos níveis de resistência a drogas antifúngicas. O conhecimento dos princípios estruturais da MEC possibilita maior compreensão de como atuar para desorganizá-la e melhorar a difusão de agentes antifúngicos para que atinjam mais eficientemente o biofilme, além de, futuramente, possibilitar o desenvolvimento de terapias mais eficazes para o controle da formação de biofilmes. Sendo assim, os objetivos principais deste estudo foram: (1) verificar a influência da inativação de genes envolvidos na filamentação (EFG1 e TEC1) em características estruturais dos biofilmes e na produção de componentes da MEC; (2) verificar a influência do fluconazol (FLZ) na MEC de biofilmes de Candida albicans ATCC 90028 (susceptível a fluconazol- CaS), C. albicans ATCC 96901 (resistente a fluconazol- CaR), Candida glabrata ATCC 2001 (susceptível a fluconazol- CgS) e C. glabrata ATCC 200918 (resistente a fluconazol- CgR) e (3) estudar a ação de enzimas hidrolíticas (DNase, Dextranase e β-glucanase individualmente ou em diferentes combinações) sobre a MEC de biofilmes de CaS e CaR. Biofilmes maduros (48 horas) foram analisados através de contagem de unidades formadoras de colônia (ufc/mL), peso seco total, peso seco insolúvel e proteínas insolúveis. Os componentes da MEC- polissacarídeos... (Complete abstract click electronic access below) / Resumo: Biofilms formed by Candida are related to bucal infections, such as candidiasis. Although the biofilm resistance is multifactorial, the protection exerted by its extracellular matrix (ECM) is essential for its high levels of resistance to antifungals. The knowledge of the structural principles of the ECM permits a better understanding of how to disorganize the ECM and improve the diffusion of antifungal drugs to reach the biofilm. Moreover, the study of the ECM may enable the development of more effective therapies to control biofilm formation. Thus, the main objectives of this study were: (1) to verify the influence of the inactivation of genes involved in filamentation and structural characteristics of the biofilms (EFG1 and TEC1) on the production of ECM components; (2) to verify the influence of fluconazole (FLZ) on the biofilms' ECM of Candida albicans ATCC 90028 (fluconazole-susceptible: CaS), C. albicans ATCC 96901 (fluconazole-resistant: CaR), Candida glabrata ATCC 2001 (fluconazolesusceptible: CgS) and C. glabrata ATCC 200918 (fluconazole-resistant: CgR) and (3) to study the action of hydrolytic enzymes (DNase, Dextranase and β-glucanase individually or in different combinations) on the ECM of CaS and CaR biofilms. Mature biofilms (48 hours) were analyzed by colony counting forming units (cfu/mL), total dry weight, insoluble dry-weight and total proteins. ECM components- alkali-soluble polysaccharides (ASPs), water-soluble polysaccharides (WSPs), extracellular DNA (e... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Prostate cancer micrometastasis allows cancer cells to vacate their original tumor sites and migrate to distant parts of the body via the bloodstream, lymphatic system, or by direct extension. Cells synthesize and secrete matrix metalloproteinases (MMPs) that degrade proteins of the surrounding extracellular matrix (ECM); thus allowing them to escape into the lymphatic or circulatory systems to invade other tissues. Transforming growth factor β (TGF-β) induces the migration and invasion of cancer cells and the expression of matrix metalloproteinases (MMPs), specifically MMP-2, and -9 in several malignancies. In this study, we examined the role of MMP-7, a known activator of MMP-2 and MMP-9, in TGF-β signaling in cell proliferation, migration, and invasion in prostate cancer cells. Basal expression levels of MMP7 mRNA, protein, and secreted protein were determined using RT-PCR, western blot analysis, and ELISA, respectively. Our data show that MMP7 mRNA and proteins were differentially expressed in several cell line models representing different stages of prostate cancer. TGF-β1 induces MMP-7 gene expression and protein levels 24 and 48 hours after treatment in PC3 cells. Our data also show that TGF-β induces cell migration and invasion in PC3 and E006AA cells; however, the selective knockdown of MMP7 expression using siRNA resulted in a significant decrease in control and TGFβ-induced cell migration and invasion in both PC3 and E006AA cells. MMP-7 knockdown also caused significant reduction in cell proliferation in PC3 cells. Our data suggest that MMP7 is essential for cell migration and invasion in prostate cancer cells indicating that it may be required for TGFβ-induced cancer metastases.