Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A ocorrência de fenótipos multirresistentes de Corynebacterium pseudodiphtheriticum e sua associação a infecções graves, com elevada mortalidade em pacientes imunocomprometidos, aliados ao escasso conhecimento da virulência e patogenia destas infecções, motivou esta pesquisa, que teve como objetivo investigar mecanismos de virulência e resistência microbiana deste agente entre pacientes de um hospital universitário brasileiro. Um total de 113 amostras de C. pseudodiphtheriticum identificadas por métodos bioquímicos convencionais e sistema API-Coryne isoladas de pacientes de diferentes grupos etários. Os micro-organismos eram, em sua maioria, relacionados a infecções no trato respiratório (27,45%), urinário (29,20%) e sitios intravenosos (18,60%) e cerca de 32,70% das amostras foram provenientes de pacientes com pelo menos uma das condições predisponentes: insuficiência renal; transplante renal, tuberculose em paciente HIV+, câncer, cirrose hepática, hemodiálise e uso de cateter. As amostras testadas revelaram-se multirresistentes sendo a maioria resistente à oxacilina, eritromicina e clindamicina. A adesão das cepas ao poliestireno e ao poliuretano indicou o envolvimento de hidrofobicidade da superfície celular na fase inicial da formação de biofilmes. O crescimento subsequente conduziu à formação de microcolônias, agregados bacterianos densos incorporados na matriz exopolimérica rodeada por espaços vazios, típica de biofilmes maduros. Adicionalmente, a interação do micro-organismo com fibrinogênio e fibronectina humana indica o envolvimento destes componentes séricos na formação de biofilme, sugerindo a participação de diferentes adesinas neste processo e a capacidade deste agente formar biofilme in vivo. A afinidade por esses componentes e a formação de biofilme podem contribuir para o estabelecimento e disseminação da infecção no hospedeiro. Adicionalmente, as cepas de C. pseudodiphtheriticum isoladas de pacientes com infecções localizadas (ATCC10700/Pharyngitis) e sistêmicas (HHC1507/Bacteremia) exibiram um padrão de aderência agregativa-like a células HEp-2, caracterizado por aglomerados de bactérias com aparência de um "empilhado de tijolos". Através do teste FAS e ensaios de interação na presença de inibidores de citoesqueleto, demonstramos o envolvimento da polimerização de actina na internalização das cepas testadas. A internalização bacteriana e rearranjo do citoesqueleto pareceu ser parcialmente desencadeado pela ativação da tirosina-quinase. Finalmente, C. pseudodiphtheriticum foi capaz de sobreviver no ambiente intracelular e embora não tenha demonstrado capacidade de replicar intracelularmente, células HEp-2 foram incapazes de eliminar o patógeno completamente no ambiente extracelular no período de 24 horas. Todas as cepas estudadas foram capazes de induzir apoptose em células epiteliais 24 horas pós-infecção evidenciada pelo aumento significativo no número de células mortas e pela ocorrência de alterações nucleares reveladas através dos métodos de coloração pelo azul Trypan, pelo DAPI e microscopia electrônica de transmissão. Alterações morfológicas incluindo a vacuolização, a fragmentação nuclear e a formação de corpos apoptóticos foram observadas neste período. A citometria de fluxo demonstrou ainda uma diminuição significativa no tamanho das células infectadas e a utilização de dupla marcação (iodeto de propídio / anexina V) permitiu a detecção da ocorrência de necrose e apoptose tardia. Em conclusão, o conhecimento de tais características contribuiu para a compreensão de mecanismos envolvidos no aumento da frequência de infecções graves com elevada mortalidade em pacientes no ambiente hospitalar, por C. pseudodiphtheriticum, um patógeno rotineiramente subestimado em países em desenvolvimento. / The occurrence of multiresistant phenotypes and associated with severe infections, with high mortality in immunocompromised hosts due to Corynebacterium pseudodiphtheriticum, allied to little known about virulence and pathogenesis these infections, led to present investigation. The investigation aims to examine the virulence mechanisms and resistance to antimicrobial agents of C. pseudodiphtheriticum among patients with bacterial infections at a Brazilian teaching hospital. A total of 113 C. pseudodiphtheriticum strains identified by conventional biochemical methods and API-Coryne System were recovered from patients from different age groups. Micro-organisms were mostly related to infections in the respiratory tracts (27.45%), urinary (29.20%) and intravenous sites (18.60%) and approximately 32.70% samples were obtained of patients presenting at least one of the pre-disposing conditions: end-stage renal disease; renal transplant; AIDS and Mycobacterium tuberculosis infection; cancer, hepatic cirrhosis; haemodialysis and catheter use. Antimicrobial susceptibility tests identified multiresistant phenotypes. Most strains were resistant to oxacillin, erythromycin and clindamycin. Adherence to polystyrene and polyurethane indicated the involvement of cell surface hydrophobicity in the initial stage of biofilm formation. Further growth led to the formation of dense bacterial aggregates embedded in the exopolymeric matrix surrounded by voids, typical of mature biofilms. Data also showed C. pseudodiphtheriticum recognizing human fibrinogen (Fbg) and fibronectin (Fn) and involvement of these sera components in biofilm formation in conditioning films. These findings suggest that biofilm formation may be associated with the expression of different adhesins. C. pseudodiphtheriticum may form biofilm in vivo possibly by an adherent biofilm mode of growth in vitro currently demonstrated on hydrophilic and hydrophobic abiotic surfaces. The affinity to Fbg and Fn and the biofilm-forming ability may contribute to the establishment and dissemination of infection caused by C. pseudodiphtheriticum. Additionally, C. pseudodiphtheriticum strains isolated from patients with localized (ATCC10700/Pharyngitis) and systemic (HHC1507/Bacteremia) infections exhibited an aggregative adherence-like pattern to HEp-2 cells characterized by clumps of bacteria with a stacked-brick appearance. The fluorescent actin staining test demonstrated that actin polymerization is involved in the internalization of the C. pseudodiphtheriticum strains. Bacterial internalization and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 hours. All samples were able to induce apoptosis in HEp-2 cells 24 h post-infection, evidenced by significant increase in the number of dead cells and nuclear alterations were observed by the Trypan blue assay, DAPI and transmission electron microscopy. Morphological changes in HEp-2 cells observed 24 h post-infection included vacuolization, nuclear fragmentation and the formation of apoptotic bodies. Flow cytometry revealed an significant decrease in cell size of infected HEp-2 cells. Furthermore, a double-staining assay using Propidium Iodide/Annexin V gave information about the numbers of vital vs. early apoptotic cells and late apoptotic or secondary necrotic cells. In conclusion, these characteristics may contribute to understanding of mechanisms involved on increase of severe infection, with high mortality in nosocomial enviroment patients by C. pseudodiphtheriticum, a pathogen usually overlooked in emerging countries.

Made available in DSpace on 2016-06-02T20:18:12Z (GMT). No. of bitstreams: 1 3010.pdf: 1781268 bytes, checksum: 812b3af521e82fd881dcbddfcb1454b8 (MD5) Previous issue date: 2010-03-29 / Universidade Federal de Sao Carlos / The function of articular cartilage depends on the chondrocytes and on the components of the extracellular matrix, which in turn may be regulated by mechanical stimuli. Thus, changes in load support may affect its composition or its structure and interfere with their functional ability to sustain and distribute loads and minimize the stresses of contact. Thus, investigations of articular cartilage components, such as chondrocyte and or matrix components are essential for prevention and treatment of arthritic disease. A greater understanding of the relationship of use / disuse and degeneration as well as the consequences of situations such as shear stress, static load or unloading can generate in this tissue. The aim of this study was to evaluate the response of chondrocytes, proteoglycan, collagen and fibronectin in articular cartilage after application of a protocol of immobilization, stretching and joint remobilization. Material and Methods: We used 36 animals divided into six groups (n = 6): immobilized (I), immobilized and stretched seven days per week (IS7), immobilized and stretched three days per week (IS3), stretched seven days per week (S7), stretched three days per week (S3) and control (C). Groups I, IS3 and AS7 underwent four weeks of immobilization of the left hind limbs. Groups IS7 and IS3, after immobilization, were subjected to three weeks of the posterior muscle stretching of the left hind leg daily or three times per week, respectively. The S3 and S7 groups remained free in the cage for 4 weeks and subsequently underwent three weeks of posterior muscle stretching of the left hind limb daily or three times per week, respectively. After these procedures, the left ankle were collected, decalcified, processed in paraffin and stained with H&E, Safranin-O, Picrossiruius Red and immunostained with fibronectin and chondroitin sulfate 4 for further analysis. Two observers evaluated parameters such as chondrocyte cloning, loss of proteoglycan content, thin and thick fibrils collagen content, intensity of staining for fibronectin and chondroitin sulfate 4. For statistical analysis we used the following tests: Kruskal Wallis and post hoc Newman Keuls: cloning and the proteoglycan content of the different groups); Duncan multiple comparison: morphometric evaluation of cellularity; ANOVA and post hoc Tukey: proportion of thin and thick fibrils of collagen. For analysis of the immunohistochemistry reactions of fibronectin and chondroitin sulfate 4 it was used nonparametric test Kruscal Wallis and post hoc Newman Keuls. In all tests the significance level was p ≤ 0.05. Results: With respect to the cellularity IS7 group showed significant increase in cellularity compared to groups I and C. The IS3 group also showed significant celullar change with the formation of chondrocyte cloning compared to groups S7, S3 and C. The most significant loss of proteoglycan was in IS7 group compared to all other groups. The I group also lost significantly more proteoglycan than the others, except for IS7 group. With respect to collagen fibrils was observed that immobilization (I) significantly reduced the thin fibrils in relation to groups IS3, S7, S3 and C. The quantity of thick fibrils was influenced by mechanical overload, as there was a significant decrease of it in all groups compared to control. With respect to the findings of the fibronectin, the groups immobilized and stretched (IS3 and IS7) had significantly higher intensity staining of fibronectin than other groups. There was no statistical difference of chondroitin sulfate 4 immunostaining among the different groups. Conclusion: The protocols of muscle stretching after immobilization, applied on alternate days and daily provoked distinct adaptive responses in articular cartilage. The immobilization stimulated tissue atrophy that when stimulated by muscle stretching on alternate days, kept some matrix components, such as fine fibrils of collagen and proteoglycan, unlike the protocol used daily. Thus we can conclude that muscle stretching applied in previously immobilized joints should be applied with caution, on alternate days of mechanical stimulation. / A função da cartilagem articular é dependente do condrócito e dos componentes de sua matriz extracelular, que por sua vez, podem ser regulados por estímulos mecânicos. Assim, alterações no suporte de carga podem afetar sua composição ou sua estrutura e interferir na sua capacidade funcional de sustentar e distribuir cargas e minimizar os estresses de contato. Desta forma, investigações dos componentes da cartilagem articular, como o condrócito e ou componentes da matriz são essenciais para prevenção e tratamento de doenças articulares. É necessário um maior entendimento das relações de uso/desuso e degeneração, assim como das conseqüências que situações como estresse de cisalhamento, carga estática prolongada ou ausência de carga possam gerar neste tecido. O objetivo do presente estudo foi avaliar a resposta do condrócito, proteoglicana, colágeno e fibronectina da cartilagem articular, após aplicação de um protocolo de imobilização, alongamento e remobilização articular. Material e Métodos: foram utilizados 36 animais divididos em 6 grupos (n=6): imobilizado (I), imobilizado e alongado 7 dias por semana (IA7), imobilizado e alongado 3 dias por semana (IA3), alongado 7 dias por semana (A7), alongado 3 dias por semana (A3), e controle (C). Os grupos I, IA7 e IA3 foram submetidos a 4 semanas de imobilização da pata traseira esquerda. Os grupos IA7 e IA3, após a imobilização, foram submetidos a 3 semanas de alongamento da musculatura posterior da pata traseira esquerda diariamente ou 3 vezes por semana, respectivamente. Os grupos A7 e A3 permaneceram livres na gaiola por 4 semanas e posteriormente foram submetidos a 3 semanas de alongamento da musculatura posterior da pata traseira esquerda diariamente ou em dias alternados, respectivamente. Após esses procedimentos, os tornozelos esquerdos foram coletados, descalcificados, processados em parafina e corados com H&E, Safranina, Picrossiruius Red e imunomarcados para fibronectina e sulfato de condroitina 4 para posterior análise. Foram avaliados por dois observadores parâmetros como: celularidade, contagem de clones, perda de proteoglicanos, conteúdo de fibrilas finas e grossas de colágeno e expressão de fibronectina e sulfato de condroitina 4. Para comparação destes parâmetros entre os diferentes grupos foram utilizados os seguintes testes estatísticos: Kruskal Wallis com post hoc Newman Keuls: formação de clones e conteúdo de proteoglicanas; Comparações múltiplas de Duncan: avaliação morfométrica de celularidade e Anova com post hoc de Tukey: proporção das fibrilas finas e grossas de colágeno. Para análise das reações de imunohistoquímica para fibronectina e sulfato de condroitina 4 foi utilizado o teste não paramétrico de Kruscal Wallis e post hoc Newman Keuls. Em todos os testes o nível de significância foi de p≤0,05. Resultados: com relação a celularidade o grupo IA7 apresentou aumento significativo da celularidade em relação aos grupos I e C. O grupo IA3 também apresentou alteração celular significativa com formação de clones em relação aos grupos A7, A3 e C. A maior perda significativa de proteoglicanas foi do grupo IA7 em relação a todos os outros grupos. O grupo I também perdeu significativamente mais proteoglicanas que os demais, somente não com relação ao grupo IA7. Com relação às fibrilas colágenas foi observado que a imobilização (I) reduziu significativamente as fibrilas finas em relação aos grupos IA3, A7, A3 e C. Já a quantidade de fibrilas grossas sofreu influência da sobrecarga mecânica, pois que houve diminuição significativa das mesmas em todos os grupos em relação ao controle. Com relação aos achados de fibronectina, os grupos imobilizados e alongados (IA7 e IA3) apresentaram significativamente maior intensidade de marcação desta que os outros grupos. Não houve diferença estatística das imunomarcações para sulfato de condroitina 4 entre os diferentes grupos. Conclusão: Os protocolos de alongamento muscular, após imobilização, realizados em dias alternados e diariamente, provocaram respostas adaptativas distintas na cartilagem articular. A imobilização desencadeou um quadro de atrofia tecidual que quando estimulada por alongamentos musculares em dias alternados, manteve alguns componentes da matriz, como fibrilas finas de colágeno e proteoglicana. Esta resposta foi agravada quando o mesmo protocolo foi aplicado diariamente. Desta forma, podemos concluir que o alongamento muscular aplicado em articulações previamente imobilizadas deve ser aplicado com cautela, respeitando períodos intercalados de estímulo mecânico.

As células sangüíneas originam-se da medula óssea através da célula tronco que sofre processo de proliferação, diferenciação e maturação no microambiente hematopoiético. O microambiente hematopoiético é uma estrutura altamente organizada composta de células estromais, moléculas da matriz extracelular (MEC) e citocinas. A desnutrição protéico-energética diminui a produção de células sangüíneas e interfere na defesa do organismo. Neste trabalho estudamos os efeitos da desnutrição protéica (dieta contendo 4% de caseína) sobre a MEC da medula óssea em camundongos. Avaliamos a composição da MEC através de SDS-PAGE 7,5% e Western blot para Fibronectina (FN), laminina (LN) e trombospondina (TSP). Verificamos a capacidade da MEC aderir e sustentar proliferação da célula mielóide FDC-P1, na ausência e na presença de citocinas (GM-CSF e IL3). Avaliamos também a capacidade de ligação destas citocinas na MEC. O perfil eletroforético mostrou diferenças nas proteínas da MEC do animal desnutrido em relação ao controle. Através da densitometria dos géis observamos nas amostras obtidas do animal desnutrido, maior intensidade nas bandas de peso molecular 220, 182, 108 e 56 KDa em relação ao controle. Em 72 KDa a banda foi mais intensa nas amostras dos animais controles. A banda de 60 KDa foi evidenciada apenas nas amostras obtidas dos animais desnutridos. As bandas de 123 e 49 KDa foram evidenciadas apenas nas amostras dos animais controles. A expressão de FN, LN e TSP foi maior nas amostras obtidas dos animais desnutridos. Os ensaios de adesão e proliferação na presença e ausência de citocinas não apresentaram diferenças significativas entre as amostras. Quando avaliamos a capacidade da MEC ligar-se ao GM-CSF, houve maior interação com a MEC proveniente do animal desnutrido do que a MEC do animal controle. O teste de ligação para o IL3 não mostrou diferenças entre as amostras. Esses achados sugerem que a desnutrição protéica induz modificações na MEC, alterando o microambiente hematopoiético. / Blood cells have their origin at the bone marrow through the stem cell which undergoes a proliferation, differentiation and maturation process in the hematopoietic microenvironment. The hematopoietic environment is a highly organized structure formed by stromal cells, extracellular matrix (ECM) molecules, and cytokines. Protein-energy malnutrition reduces the production of blood cells, interfering with the defense of the organism. In the present work we have studied the effects protein malnutrition has on the ECM of bone marrow in mice. We have evaluated ECM composition by means of SDS PAGE 7,5% and Western blot for fibronectin (FN), laminin (LN) and thrombospondin (TSP). We assessed the capacity ECM has in adhesion and support of proliferation of the FDC-P1 myeloid cell both in the absence and in the presence of GM-CSF and IL3 cytokines. We have also measured the binding capacity of these cytokines in the ECM. The electrophoresis profile showed the existence of differences between the ECM proteins in the undernourished animal and the control. Using gel densitometry, we observed in samples from the undernourished animal a greater intensity of bands of 220, 182, 108, 60 and 56 KDa molecular weight as compared to control. At 72 KDa the band was more intense on samples from control animals. The 60 KDa band was evident only on samples taken from undernourished animals. The 123 and 49 KDa bands were evident on control animals only. Expression of FN, LN, and TSP was greater on samples from undernourished animals. Adhesion and proliferation assays, both in the presence and in the absence of cytokines, did not show significant differences among samples. When we evaluated the capacity ECM has to bind to GM-CSF, a greater interaction was seen with the ECM from the undernourished animal than the ECM from the control. Binding test for IL3 showed no differences existed among samples. Such findings suggest protein malnutrition causes alterations of the ECM, modifying the hematopoietic microenvironment.

O aparelho propulsor mandibular é utilizado na odontologia para modular o crescimento da cartilagem condilar, por meio de forças geradas pela alteração postural da musculatura. Neste estudo foi avaliado o papel de integrinas ligantes de fibronectina (FN) na transdução das forças mecânicas geradas pelo aparelho, em ratos. Por meio de imuno-histoquímica e PCR em tempo real verificou-se que, in vivo, o uso do aparelho modulou a expressão das subunidades <font face=\"symbol\">1, <font face=\"symbol\">5, e <font face=\"symbol\">v de integrinas, FN e PCNA, um marcador de proliferação celular. In vitro, forças distensivas cíclicas aplicadas sobre células da cartilagem condilar aumentaram a expressão de mRNA para FN, fatores de crescimento IGF-I e IGF-II e PCNA. A adição do peptídeo GRGDSP, que bloqueia a ligação de algumas integrinas à FN, inibiu todos os efeitos, exceto na expressão de IGF-II. Esses resultados sugerem que integrinas ligantes de FN desempenham papel importante na mecano-transdução neste sistema e contribuem para o entendimento das bases moleculares envolvidas na ortopedia funcional dos maxilares / The mandibular propulsor appliance is widely used in dentistry to modulate the growth of the condylar cartilage, through forces generated by postural changes in the orofacial musculature. The aim of this study was to evaluate the role of fibronectin (FN)-binding integrins in the transduction of mechanical forces generated by the appliance in rats. By immunohistochemistry and real time PCR it was observed that, in vivo, the appliance´s use modulated the expression of the integrin subunits <font face=\"symbol\">1, <font face=\"symbol>\"5, and <font face=\"symbol\">v, FN and PCNA, a cell proliferation marker. In vitro, the application of cyclic distension forces on condylar cartilage cells increased the expression of FN, IGF-I, IGF-II and PCNA mRNA. Addition of the peptide GRGDSP, which blocks the binding of some integrins to FN, inhibited all the effects except the increase in IGF-II mRNA. These results suggest that FN-binding integrins play an important role in mechanotransduction in this system, contributing to the understanding of the molecular basis involved in maxillary functional orthopedic therapy.

Der Erfolg neuer Strategien in der Regenerativen Medizin und im Tissue Engineering hängt maßgeblich von einem gut entwickeltem vaskulären Netzwerk ab, welches die auf den Implantaten wachsenden Zellen und Gewebe versorgen. Oberflächeneigenschaften der Implantate sowie die Präsentation verschiedener Liganden für extrazelluläre Matrixproteine spielen bei der Besiedlung der Implantate, als auch bei der Bildung versorgender Blutgefäße durch die Endothelzellen eine wesentliche Rolle. In dieser Arbeit konnte durch Variation der Anbindungsstärke (kovalent oder physisorptiv) des extrazellulären Matrixproteins Fibronektins an die MSA-Copolymere der Einfluss des Aufbaus der extrazellulären Matrix auf das Differenzierungsverhalten der Endothelzellen gezeigt werden. Auch die initiale Konzentration von Adhäsionsproteinen an der Substratoberfläche zeigte sich bedeutend für das Verhalten der Zellen. Optimal für eine gute Adhäsion, native Entwicklung und Kapillarbildung der Endothelzellen war die stabile (kovalente) Anbindung weniger Adhäsionsproteine (hier Fibronektin) an die Substratoberfläche, so dass die Zellen problemlos adhärieren konnten. Erfolgte die weiter Proteinadsorption an die Oberflächen in einem nativen Zustand (hier auf den hydrophilen Oberflächen) so waren die Endothelzellen in der Lage, die extrazelluläre Matrix zu reorganisieren und ein dem in vivo Zustand ähnlicher Aufbau der extrazellulären Matrix konnte realisiert werden. Dies ermöglichte den Zellen wiederum ein natürliches Verhalten. Die Ausbildung einer moderaten Anzahl von Adhäsionsstellen der Zellen, sowie der in vivo ähnliche Aufbau der Adhäsionspunkte ermöglichte den Zellen einen eher lockeren Kontakt zum Substrat. Daher waren sie sehr flexibel in ihrer Morphologieanpassung. Unter diesen Bedingungen war es möglich, dass die Endothelzellen bei Stimulierung der Angiogenese kapillarähnliche Strukturen ausbildeten. Die Verwendung dreidimensionaler Zellkulturträger zeigte eine Unterstützung der Kapillarbildung der Endothelzellen in Abhängigkeit unter den beschrieben Bedingungen. / The success of tissue engineering strategies using artificial scaffolds crucially depends on a controlled formation of well-developed vascular networks in growing tissues. The presentation of extracellular matrix ligands on scaffolds is often envisioned as an appropriate strategy to support capillary formation. We show that the control of primary coupling mode — covalent versus physisorbed — as well as of secondary interactions of cell-secreted extracellular matrix proteins have a strong impact on endothelial cell development. A set of maleic anhydride copolymer thin films was used as planar model substrates. They exhibit a switchable mode of primary matrix coupling combined with a gradation of secondary matrix–substrate interactions due to a variation of surface hydrophobicity and polarity. We found that the cells adhere in a more native state at a low amount of covalent primary coupled fibronectin ligands in conjunction with weak interactions of secondarily adsorbed adhesion ligands on hydrophilic surfaces. These substrates allow for a formation of capillary-like networks of endothelial cells. High ligand densities and strong secondary hydrophobic interactions inhibit a pronounced capillary formation. The composition and structure of the formed extracellular matrix correlates well with the specific integrin expression pattern. From these results it is concluded that the formation of blood capillaries in artificial scaffolds can be triggered by controlling primary and secondary coupling of cell adhesion ligands to implant materials. 2

Cells of the retinal pigment epithelium (RPE) are generated prenatally and generally survive the lifetime of the individual without undergoing proliferation or replacement. Therefore, the mechanisms promoting individual RPE cell survival and longevity in vivo may be distinct from, or a limited subset of, the mechanisms known to promote survival in proliferative cells in culture. To identify specific factors that sustain cell viability independent of effects on cell division, we studied RPE cells in low-density suspension culture, in which cell proliferation is inhibited. Single cells from Xenopus laevis eyes were plated onto a non-adhesive surface in protein-free medium, then assayed for survival using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell viability in these cultures was essentially undiminished over the initial 2 days. However, by approximately 1 week in culture, only an average of 53% of the cells remained alive. Plating cells on a fibronectin-coated substratum significantly enhanced survival, such that the number of cells alive at 1 week was 80-90% of the initial level. Essentially identical results were obtained with laminin- or collagen IV-coated substrata, or with insulin (5μg ml-1) in the medium. The absence of cell division in these cultures was confirmed by cell counting and BrdU incorporation experiments. Interestingly, in suspension cultures derived from monolayers previously established on microporous membrane filters, cells lost viability much faster (average of 80% dead at 3 days), and showed a relatively greater response to extracellular matrix proteins (five-fold increase in cell survival at 3 days). Enhanced RPE survival in response to fibronectin required spreading of the cell on a substratum, rather than mere adherence, as there was a high correlation between the percentage of spread cells and the percentage that were MTT-positive (r = 0·940). Cell spreading apparently enhanced survival by preventing the initiation of programmed cell death: unattached non-viable cells in culture exhibited morphological features expected of apoptosis, as well as positive staining by the TUNEL reaction. These studies demonstrate that, of several factors shown to maintain or increase cell number in proliferating cultures, some have their effect, at least in part, by promoting the survival of individual cells. The increased susceptibility of subcultured RPE to cell death has implications for clinical transplantation applications that may require manipulation of RPE in vitro.

<p>   </p> <p>Subcutaneous injection is a biotherapeutic drug delivery method that is currently growing due to low cost, better patient compliance, minimally invasive, and the convenience that it can be done at home. Common injection sites for subcutaneous injection include the upper outer arms, abdomen, buttocks, and upper outer thigh. Heterogeneity of the tissue exists between and within each of these locations. The subcutaneous tissue space is made up of adipose tissue, proteins, collagen, and blood vessels and each of these components has an impact on the mass transport of the injected biotherapeutics and how they are absorbed into the vascular system and then distributed to the body. The current methods used to model the subcutaneous tissue space are either very expensive and not feasible for multiple repetitions, cannot incorporate fibrillar proteins or cellular components, or model a more homogeneous tissue space. These limitations do not allow for these models to accurately represent the subcutaneous tissue space. The engineering objective for this project was to develop a platform with tunable matrix architecture and biochemical composition for evaluating mass transport. This project utilizes collagen and the primary matrix due to the large abundance of collagen in the body.  We explored the effects that a change in polymerization temperature of the collagen and collagen concentration had on the fiber architecture and pore diameter. The results showed that higher polymerization temperatures of the collagen gels resulted in smaller fiber and pore diameters and an increase in concentration resulted in an increase in fiber volume fraction and a decrease in pore diameter. Fibronectin (FN) and hyaluronic acid (HA) were added to the collagen gels to analyze the impact on the structure of collagen gels with a change in polymerization temperature and collagen concentration. The addition of FN did not strongly alter the collagen fiber architecture between polymerization temperatures and collagen concentrations. Through staining and imaging, we saw an aggregation of FN around the collagen fibrils due to their opposing charges causing them to bind. The addition of HA had moderate impact on collagen fiber architecture across all polymerization temperatures and between collagen concentrations. The collagen + FN gels were used for the mass transport study. The results showed that there was little to no difference between the recovery rates of macromolecules of different charges and size between the collagen and collagen + FN gels, indicating that the transport of molecules through both of the collagen gels was impacted by a steric effect rather than an effect in charge.</p> <p>  </p>

<p>  </p> <p>Metastases are responsible for approximately 90% of all cancer-related deaths, with metastatic breast cancer (BC) holding a 5-year survival rate of only 27%. Recent research has highlighted a complex dynamic between cancer cells and the tumor microenvironment as essential for the formation of macrometastases. Within this field, tissue stiffening through matrix accumulation and altered matrix organization at the primary tumor site were recently linked with sustained proliferation and increased migration of tumor cells. Separately, elevated levels of the glycoprotein, fibronectin, were correlated to poor patient survival in BC and were linked to enhanced seeding of disseminated tumor cells at metastatic sites. Through my doctoral work, we have identified several mechanisms through which accumulated fibronectin impacts the metastatic potential of BC cells. First, we identified a transient increase in extracellular fibronectin in the lungs, which peaked before overt metastasis, coupled with a non-transient increase in total lung volume. To better recapitulate physiological conditions, we then developed a novel magnetically-actuated platform with the ability to apply tensile strain on cells at various amplitudes and frequencies in a high-throughput multi-well culture plate using suspended fibrillar fibronectin for 3D cell culture that is not reliant on a synthetic substrate. Using this as a biomimetic lung model, we found that cyclic mechanical force acted as a suppressor of cancer cell growth in a biomimetic lung model, implicating the accumulation and reorganization of extracellular matrix as an attempt by the cancer cells to alter the mechanical properties of the lung tissue and resist entering dormancy. However, our results showed that BC cells could not organize extracellular fibronectin independently. Instead, BC cells altered the accumulation and architecture of fibronectin by conditioning fibroblasts through soluble factors and extracellular vesicles. We observed that the fibronectin produced by conditioned fibroblasts varied as an effect of both the method of conditioning and the phenotype of the BC cell as the conditioning source. Taken together, these results have increased our knowledge of the relationship between disseminated breast cancer cells, fibroblasts, and fibronectin architecture in the early metastatic lung niche that paves the way for further investigation on targeting disseminated BC cells during early disease intervention in order to inhibit later overt metastatic outgrowth.</p>

1) Aims To explore the ultrastructure of interstitial cells in the upper lamina propria of the human bladder, to describe the spatial relationships and to investigate cell-cell contacts. 2) Methods Focused ion beam scanning electron microscopy (FIB-SEM), 3-View SEM and confocal laser scanning microscopy were used to analyze the 3D ultrastructure of the upper lamina propria in male and female human bladders. 3) Results 3View-SEM image stacks as large as 59µm x 59µm x 17µm (xyz) at a resolution of 16nm x 16nm x 50 nm and high resolution (5nm x 5nm x 10nm) FIB-SEM stacks could be analyzed. Interstitial cells with myoid differentiation (mIC) and fibroblast like interstitial cells (fIC) were the major cell types in the upper lamina propria. The flat, sheet-like ICs were oriented strictly parallel to the urothelium sheet-like morphology. No spindle shaped cells were present. We furthermore identified one branched cell (bIC) with several processes contacting urothelial cells by penetrating the basal membrane. This cell did not make any contacts to other ICs within the upper lamina propria. We found no evidence for the occurrence of telocytes in the upper lamina propria. 4) Conclusions Comprehensive 3D-ultrastructural analysis of the human bladder confirmed distinct subtypes of interstitial cells. We provide evidence for a foremost unknown direct connection between a branched interstitial cell and urothelial cells of which the functional role has still to be elucidated. 3D-ultrastructure analyses at high resolution are needed to further define the subpopulations of lamina propria cells and cell-cell interactions.

<p>Metastasis of primary mammary tumors to vital secondary organs is the primary cause of breast cancer-associated death, with no effective treatment. Metastasis is a highly selective process that requires cancer cells to overcome multiple barriers to escape the primary tumor, survive in circulation, and eventually colonize distant secondary organs. One of the important aspects of metastatic cancers is the ability to undergo epithelial-mesenchymal transition (EMT) and the reverse process mesenchymal-epithelial transition (MET) process. Constant interconversion of tumor cells between these phenotypes creates epithelial-mesenchymal heterogeneity (EMH) and interaction between these tumor cell types and the stromal cell compartment is clearly important to metastasis. In healthy tissues, stromal cells maintain the composition and structure of the tissue through the production of extracellular matrix (ECM) proteins and paracrine signaling with epithelial cells. However, little is known about how EMH promotes changes in the ECM to promote breast cancer progression and metastasis. Cancer cells also secret exosomes, nano-size extracellular vesicles, to establish intercellular communication with distant organs in order to induce metastasis. These exosomes contain a plethora of different proteins including extracellular matrix proteins and matrix crosslinking enzymes. Fibronectin, an important ECM protein, plays an active role in tumor progression and is often crosslinked by tissue transglutaminase 2 (TGM2) to promote fibrosis in cancer. Both FN and TGM2 exist in exosomes and are expressed by heterogenous breast tumors. Although FN and TGM2 have been reported to play essential roles in cancer, their involvement in metastasis remains unclear. This work utilizes a variety of approaches to investigate the role of tumor heterogeneity and ECM proteins in promoting breast cancer metastasis. In this dissertation, we establish that mesenchymal cells expressing intracellular FN are held in a stable non-metastatic mesenchymal phenotype and produce cellular fibrils containing functionalized FN capable of supporting the growth of metastatic competent epithelial cells. We introduce a novel 3D culture system consisting of a tessellated scaffold which is capable of recapitulating cellular and matrix phenotypes <i>in vivo. </i>Further, we also demonstrate breast tumor cells secrete exosomes containing TGM2 crosslinked FN fibrils to promote premetastatic niche formation and induction of metastasis.<i> </i>Using genetic approaches, we establish TGM2 is essential and sufficient to drive metastasis. Finally, we demonstrate pharmacological inhibition of TGM2 offers a potential therapeutic strategy to treat metastatic breast cancer. Altogether, our research provides insights into the mechanism through which TGM2 promotes metastatic breast cancer. This work will help in developing new drugs to target TGM2 aimed at reducing breast cancer metastasis.<br></p>