In this work, we used atomic force spectroscopy to obtain the elastic modulus of melanoma and keratinocytes fixed cells, with the purpose to determine the initial conditions for studies of confluente cultures of these cells in the future. The cell lines used were HaCaT cells and WM1366 melanoma cell, the last one is derived from a radial growth melanoma and both were analyzed, parental WM1366 cells (WM1366 shSCR cells) and galectin-3 silenced WM1366 cells (WM1366 shGal3). Cells were located and images of them were obtained by AFM contact mode under liquid conditions. Single force curves acquired in the central region of cells were used to determine the elastic modulus by the Hertzian contact model for the pyramidal tip, allowing to establish a comparison patter between cancer and normal cells. It was found that the melanoma cell (21.8 ± 0.5 kPa) exhibit smaller elastic modulus than keratinocytes cells (31.9 ± 0.4 kPa). For WM1366 shGal3 was found a elastic modulus of 16.1 ± 0.6 kPa, therefore, we found that for large indentation depth it is possible to distinguish between the same melanoma cell line, which represents general alterations in the organization of the cytoskeleton induced by the presence or absence of the galectin-3 protein. On the other hand, to detect local elastic modulus variations along the cell and to identify subcellular regions characterized by specific stiffness associated with local structures, we took elasticity maps in which a single force curve is acquired in each probe position. In order to interpret these maps, the cell was sliced into several different heights, curves of each height section were analyzed and represented in histograms, adjusted by the binomial distribution function. It was observed that the gradient of elastic modulus in cells from the nuclear region towards the cell periphery is more pronounced in cells devoid of galectin-3 than parental cells. The increased elastic modulus in the pericellular region of cells devoid of galectin-3 suggests that the organization of the extracellular matrix in these areas is different than those observed around HaCaT and shSCR WM1366 cells. / Neste trabalho, utilizamos espectroscopia de força atômica para obtenção do módulo elástico de células fixadas de melanoma e queratinócitos, com o objetivo de determinar as condições iniciais de estudos a serem realizados futuramente de culturas de células confluentes do mesmo tipo. As linhagem celulares utilizadas foram as células HaCaT e as células de melanoma WM1366, sendo a última derivada de um melanoma de crescimento radial sendo analisadas tanto as células parentais (células WM1366 shSCR) e as células WM1366 silenciadas com galectina-3 (WM1366 shGal3). As células foram localizadas e imageadas no modo AFM contato em meio líquido. Curvas de força adquiridas na região central das células foram utilizadas para determinar o módulo elástico, a partir do modelo de contato hertziano por uma ponta piramidal, permitindo estabelecer um padrão para comparação entre células normais e cancerígenas. Verificou-se que a célula de melanoma exibe menor módulo de elasticidade (21.8 ± 0.5 kPa) do que as células de queratinócitos (31.9 ± 0.4 kPa). Para as células WM1366 shGal3 foi encontrado um módulo elástico de 16.1 ± 0.6 kPa. Portanto, verificou-se que, para grandes profundidades de indentação, é possível distinguir entre a mesma linhagem de melanoma, células que apresentam alterações gerais na organização do citoesqueleto induzidas pela presença ou ausência da proteína galectina-3. Por outro lado, para detectar variações locais do módulo elástico ao longo da célula e identificar regiões subcelulares, caracterizadas por rigidez específica associada a estruturas locais, foram obtidos mapas de elasticidade nos quais uma única curva de força é adquirida em cada posição da sonda. Para interpretar estes mapas, a célula foi dividida em regiões de diferentes alturas e curvas de cada seção de altura foram analisadas e representadas em histogramas, ajustadas pela função de distribuição binomial. Observou-se que o gradiente de módulo de elasticidade em células da região nuclear em direção à periferia celular é mais acentuado em células desprovidas de galectina-3 do que em células parentais. O aumento do módulo de elasticidade na região pericelular das células desprovidas de galectina-3 sugere que a organização da matriz extracelular nestas áreas é diferente das observadas em torno das células HaCaT e shSCR WM1366.

Telomere dysfunction is one mechanism of cellular and tissue ageing. Dysfunctional telomeres in fibroblasts are recognised as DNA double-strand breaks (DSBs) and trigger the DNA damage pathway of senescence. However, telomere uncapping in normal human epidermal keratinocytes, via expression of the dominant negative mutant of the telomere repeat-binding factor 2 (TRF2!B!M), resulted in a senescent-like arrest without a significant DNA damage response (DDR). This suggests that either keratinocytes are unusually sensitive to telomere uncapping and the low DDR is sufficient to induce senescence or that dysfunctional telomeres may also be signalled through an alternative pathway. Subsequent analysis revealed genes HIST2H2BE, ICEBERG, S100A7 and HOPX as potential markers for telomere dysfunction-induced senescence (TDIS) since they were induced by telomere uncapping and seemed to be regulated by telomerase. The aim of this project was to assess the specificity of these candidate markers for TDIS and to select the most promising for use as a biomarker. To this end, keratinocytes were exposed to doses of ionising radiation, capable of generating transient or permanent damage to the DNA, or transduced with retroviral constructs expressing p14ARF, p16INK4a, p53 or TRF2!B!M and the gene expression levels of the candidates assessed after a recovery period or at the early stages of senescence. Whilst S100A7, HOPX or ICEBERG were not induced by a transient or persistent DDR or by p16INK4a, ICEBERG and HOPX were induced by p53 and p14ARF when these were ectopically expressed at higher levels. Thus, S100A7 seems to be the most specific early marker for telomere dysfunction in keratinocytes since it was selectively induced by telomere uncapping via expression of TRF2!B!M and not by DSBs or by over expression of p14ARF, p53 or p16INK4a. S100A7 may have the potential to identify cells with telomere dysfunction in human epithelia and body fluids.

The skin-implant interface of percutaneous devices is generally weak and can fail when excessive loading disrupts the sealing of the interface by dermal and epidermal cells and tissue. As such, the formation of a stable implant-skin junction is a major factor in determining percutaneous implant success. In this study, we used functionalized self-assembled monolayers (SAMs) with discrete surface properties as model systems to assess the effects of biomaterial surface properties on controlling fibronectin (FN) adsorption and keratinocyte spreading and adhesion. The surface properties investigated were charge (positive and negative) and wettability (hydrophobic and hydrophilic). Gold slides prepared with SAMs were incubated with FN overnight. The cell binding sites were quantified on each surface using an antibody that targets the synergy binding site of the cell binding domains (HFN7.1) and the topography of the FN on the surfaces was evaluated with atomic force microscopy. The topography data demonstrated that the availability of cell binding domains is dependent on surface-mediated FN binding orientation. Cell spreading was assessed using a lipid membrane stain, maleimide. The cells were imaged by fluorescence microscopy and the cell area calculated. The percentage of cell adhesion was determined using a centrifugal force assay. Both keratinocyte assays suggested that the charge of the surface was the prominent factor in determining cell function on the surface over the surface wettability. The findings of this study strongly suggest that a positively charged implant surface with a FN coating will enhance the strength of the cutaneous seal around percutaneous implants over an unmodified surface.

Background: Staphylococcus aureus is an important trigger of flares in atopic dermatitis. The exact mechanisms by which S. aureus induces inflammatory responses and cell death in the skin epithelium is unclear. The aim of this thesis was to elucidate the cellular and molecular mechanisms by which S. aureus induces it's pathogenic effects on keratinocyte and fibroblast cell lines. Methods: Human keratinocytes (HEKa), and mouse embryonic fibroblasts (MEF) from the NC/Nga dermatitis prone mouse strain were used to investigate the induction of Th2-promoting cytokines (IL-33 and TSLP) and cell death by S. aureus. Cytokine levels were measured by ELISA and cytotoxicity by flow cytometry. Results: Live, but not killed S. aureus or other staphylococcal species, induced release of Th2-promoting cytokines (IL-33 and TSLP) and necrosis in both human and mouse cell lines. Cytokines were not induced by TLR2 ligands, and anti-TLR2 antibodies did not inhibit release, suggesting that the TLR2 pathway was not involved. By contrast, the release of cytokines was induced by a secreted, heat-labile factor/s and could be blocked by protease and PAR2 inhibitors, suggesting that the protease-PAR2 pathway was critical. NC/Nga mouse fibroblasts that lacked soluble IL-33 (sST2) receptor were more sensitive to the effects of S. aureus than control MEF. Conclusions: S. aureus is unique amongst staphylococcal species in it's ability to induce an inflammatory response and cytotoxicity in human keratinocytes and mouse fibroblasts. The protease-PAR2 pathway is critical to this bioactivity. Development of specific inhibitors of this pathway may provide novel therapies for treating S. aureus -induced eczema flares.

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Development leads unidirectionally towards a more restricted cell fate that is usually stable. However, it has been proven that developmental systems are reversible by the success of animal cloning of a differentiated somatic genome through somatic cell nuclear transfer (SCNT). Recently, reprogramming of somatic cells to a pluripotent embryonic stem cell (ESC)-like state by introducing defined transcripton factor has been achieved, resulting in the generation of induced pluripotent stem cells (iPSCs), which resemble ESCs. iPSC reprogramming is of great medical interest, as it has the potential to generate a source of patient-specific cells. However, the dangerous delivery method, low efficiency, and slow kinetics of the reprogramming process have hampered progress with this technology.

No description available.

Background. Keratinocytes are the first target of infection for lymphogranuloma venereum (LGV) Chlamydia trachomatis, yet they have been omitted from pathogenesis studies. We infect keratinocytes and cervical cells with C. trachomatis and hypothesize different growth and cytotoxicity profiles among the strains. Methods. HaCaT human keratinocytes and ME-180 cervical cells were infected with C. trachomatis (multiplicity of infection (MOI) 0.025) serovars L1, L2, L3, 3 LGV clinical isolates or serovar E and incubated at 37 or 33°C for 5 days. Cytotoxicity was quantified daily using the CytoTox96® Non-Radioactive Cytotoxicity Assay, cells stained with the MicroTrak C. trachomatis Culture Confirmation kit and growth quantified by area of 100X photographs covered by Chlamydia. HaCaT and ME-180 cervical cells were infected with C. trachomatis (MOI 0.25) serovar L2 or E, incubated at 37 or 33°C for 48 hours and viewed with a transmission electron microscope (TEM). Mitochondrial activity was quantified using the MTT assay. The DeadEndTM Colorimetric TUNEL System with C. trachomatis Culture Confirmation kit as a counter-stain was used to assess cell death in infected versus uninfected cells. The BioVisionTM CaspGLOW Fluorescein Caspase Staining Kit and Transwell® Permeable Supports was used to differentiate between apoptosis mediated by cell-to-cell contact or a secreted molecule. Results. Growth in ME-180 versus HaCaT cells at 37°C was similar, but slower at 33 versus 37°C in HaCaT cells (p < 0.05). By day 5 L2 had grown faster than other strains in HaCaT cells at 37°C (p < 0.05), faster than clinical isolates in ME180 cells (p < 0.01), and faster than serovar E, and 2 clinical isolates at 33°C (p < 0.01). After 5 days L2 induced cytotoxicty was 11% in ME180 cells, which was higher than the clinical isolates (p < 0.01). In HaCaT cells at 33°C L2 EB were identified in a non-membrane state in the cytoplasm but not in the inclusion at 48 hours post infection. Serovar E but not L2 caused mitochondrial swelling at 1 h post infection in HaCaT cells at 37°C. This corresponded with a 16% reduction in mitochondrial activity (p < 0.001). TUNEL assay analyses demonstrated numerous dead cells adjacent to chlamydial inclusions for strains L2 and L3 but not L1 and E. An elevated number of caspase positive cells was detected in uninfected cell monolayers exposed to both L2 and E at 37°C but not 33°C. Conclusions. 1. C. trachomatis infects human keratinocytes in vitro. 2. Fresh clinical isolates behaved differently to the L2 reference strain. This demonstrates the need for fresh clinical isolates in pathogenesis studies of LGV. 3. In HaCaT cells at 33°C serovar L2 EB leave the intact inclusion and migrate through the cytoplasm in a non-membrane bound state 4. C. trachomatis induces apoptosis in uninfected cells exposed to infected cells via a secreted molecule at 37°C. This is more marked with serovar L2 exposure than serovar E exposure. / Thesis (Ph.D)-University of KwaZulu-Natal, Durban, 2010.

Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisor: Robert M. Dyer, Dept. of Animal and Food Sciences. Includes bibliographical references.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.