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1
Junker, Johan. "Human Dermal Fibroblasts in Tissue Engineering." Doctoral thesis, Linköpings universitet, Cellbiologi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-19716.
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The loss or failure of tissues and/or organs is one of the most frequent problems in modern healthcare. The field of tissue engineering applies the principles of biology and engineering in order to develop functional substitutes for damaged tissues. Tissue engineering contains elements of medicine, material science and engineering with major components in focus being cells, biomaterials and soluble factors. All three components may be required for the development of clinical treatments. The usage of autologous tissue specific cells for clinical treatment is often not feasible due to poor growth kinetics or unstable phenotypes of the cells. Furthermore, lack of availability of healthy tissue that can be biopsied is a major problem in many applications. One approach to overcome this problem is to use adult stem cells which have the capacity to give rise to several different cell types. Although promising, adult stem cells have major impediments for use in several tissue engineering applications. The difficulties associated with harvest, culture and storage render problems in the development of clinically relevant procedures. During the last years, the inherent plasticity of differentiated somatic cells has been demonstrated. One of the easiest human cell types to obtain, expand and store is the dermal fibroblast. Recent reports indicate that dermal fibroblasts can be induced to differentiate towards several distinct mesenchymal lineages in vitro. The main aim of this thesis was to investigate the inherent stem cell plasticity of human dermal fibroblasts and explore their possible usefulness in tissue engineering applications. The papers included in this thesis employ routine and immunohistochemical staining, enzyme activity assay, analysis of low density lipoprotein incorporation, capillary-like network formation assay and full expression micro array analysis. Fibroblasts were shown to differentiate towards adipocyte, chondrocyte, endothelial and osteoblast-like cell types in vitro. The differentiation from fibroblasts to myofibroblasts in burn scar tissue upon stimulation by mechanical tension was also demonstrated. Adipogenic, chondrogenic and osteogenic induced fibroblasts display the upregulation of several genes associated with adipocytes, chondrocytes and osteoblasts.
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Junker, Johan P. E. "Human dermal fibroblasts in tissue engineering /." Linköping : Department of Clinical and Experimental Medicine, Linköping University, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-19716.
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Boright, Andrew Pepler. "Prolidase deficiency : studies in human dermal fibroblasts." Thesis, McGill University, 1988. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75956.
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Prolidase deficiency (MIM 26413), an autosomal recessive phenotype, is caused by rare alleles at a locus on chromosome 19cent.-q13.2. The clinical phenotype is pleiotropic (affecting skin, brain, etc.) and of variable expressivity (benign to early death). I established skin fibroblast cultures from 6 homozygous probands and 6 obligate heterozygotes, purified prolidase (E.C. 3.4.13.9, a homodimer) from normal human fibroblasts, raised a monospecific rabbit antiserum to the subunit, and studied its biosynthesis. Pulse-chase immunoprecipitation experiments showed that the subunit is synthesized in the cytosol as a 58 KDa. polypeptide and not processed further. Homozygous prolidase-deficient cell strains expressed 3 classes of mutant alleles which by complementation analysis mapped to one locus. The alleles were designated CRM$-$ (nul), CRM+ activity/size variant, and CRM+ activity variant. Heterozygotes carrying CRM$-$ alleles have heat stable prolidase (50$ sp circ$C, 1hr); heterozygotes carrying CRM+ variant alleles have heat labile enzyme. The finding implies that variant CRM+ allele(s) can confer negative allelic complementation on the dimeric enzyme (dominant relative phenotype). CRM$-$ homozygous cells contain varying amounts of an alternative imidodipeptidase-like activity. The variant prolidase allele (major gene) and amount of alternative "prolidase" activity (modifier gene) are apparently both determinants of the associated clinical phenotype in prolidase deficiency. I obtained and sequenced a tryptic peptide from human kidney prolidase for synthesis of oligonucleotide probes in the future.
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Mitchell, Stephen Andrew. "The radiation response of human dermal fibroblasts." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392193.
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Kashpur, Olga. "Oxygen-mediated basic fibroblast growth factor (FGF2) effects on adult human dermal fibroblasts." Digital WPI, 2015. https://digitalcommons.wpi.edu/etd-dissertations/546.
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This thesis investigates the effects of low oxygen culture conditions and fibroblast growth factor-2 (FGF2) on adult human dermal fibroblasts.
It was previously shown that low oxygen and FGF2 culture conditions lead to an extension of proliferative lifespan, low-level activation of stem cell genes, and global transcriptional changes in adult human dermal fibroblasts. Additionally, an increased in vivo tissue regenerative response can be observed when human muscle-derived fibroblasts grown with FGF2 and low oxygen are implanted into mouse muscle injury, leading to a decrease in collagen deposition and scar formation and increase of functional skeletal muscle regeneration, including formation of Pax7+ muscle stem cells.
These findings led to an analysis of key cellular oxygen sensors, hypoxia inducible factors (HIFs) and their role in this regenerative response. Directly linking these factors with the regenerative response, I have shown, with knockdown experiments, that HIF-2α is required for the increased proliferative capability and decreased senescence of human dermal fibroblasts (hDFs) induced by hypoxia. I have also determined that low oxygen causes an early and transient increase of HIF-1α and late and sustained increase of HIF-2α protein accompanied by increased nuclear translocation. Using overexpression and knockdown approaches via lent-virus, I determined that HIF-2α appears to modulate FGF2 signaling through the FGF receptors. First, under low oxygen conditions, exogenous FGF2 led to downregulation of endogenous FGF2, which can be mimicked by overexpression of HIF-2α. In ambient oxygen we didn't see this effect. Second, HIF-2α overexpression appears to lead to increases in FGFR1 phosphorylation and consequently increased ERK1/2 phosphorylation, and increases in the expression of heparan sulfate modifying enzymes (NDST1, NDST2, and EXTL2). Lastly, sustained supplementation with FGF2 in low oxygen inhibits receptor-mediated FGF2 signaling.
To understand these effects at the transcriptional level, using microarray technology, we identified oxygen-mediated FGF2 effects on genes involved in cell survival and proliferation.
Through bioinformatics analyses, I determined that genes involved in wound healing (extracellular matrix genes, adhesion molecules, cytokines) are upregulated in FGF2 treated fibroblasts grown under low oxygen. By utilizing a gain-of-function approach, we were able to assess the effects of altered HIF-2α activity on the expression of Oct4, Sox2, Nanog, Rex1, and Lin28 in adult hDFs. The results indicate that overexpression of the HIF-2α transcription factor increases Oct4 mRNA, but not Oct4 protein, levels, and had no effect on Nanog and Lin28 proteins. HIF-2α overexpression also mediated FGF2 induction of Sox2 and Rex1 proteins of higher molecular weight.
This thesis expands our knowledge about effects of low oxygen and FGF2 on adult human dermal fibroblasts and explains in part, how FGF2 under low oxygen conditions may lead to increased proliferation, extended life span, regenerative competency and increased developmental plasticity of adult hDFs.
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Al-Rikabi, Aaiad H. A. "Impaired Wound Healing and Inflammation: The Role of the Dermal Fibroblast. Phenotypic Changes in the Human Dermal Fibroblast with Inflammation; Potential Impact on Wound Healing." Thesis, University of Bradford, 2019. http://hdl.handle.net/10454/18331.
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Dermal fibroblasts positively contribute throughout the wounding response by
secreting a profile of pro- and anti-inflammatory cytokines in the wound milieu. However, a chronically inflamed environment will, cause detrimental effects on
the functional, secretory, and molecular properties of these cells. This study
aims to understand how the effect of the pro-inflammatory cytokine TNF-α
modulates both healthy and diabetic dermal fibroblast phenotype.
To mimic a chronic inflammatory environment and assess whether fibroblasts
respond similarly in different anatomical sites, donor-matched fibroblasts from
face and scalp were pre-incubated for 3 days with different concentrations (2.5,
25 or 250 ng/ml) of TNF-α. All concentrations significantly impaired proliferation
by day 14 in cells from both sites and stimulated (papillary) metabolic activity at
day 14.
However, this did not correlate with an increase in papillary cell senescence
since this did not appear until passage 17, and then only at a supra pathophysiological concentration.
Migration of dermal fibroblasts, assessed by the scratch assay. TNF-α
significantly inhibited the cells migration, particularly in diabetic fibroblasts,
suggesting they are more sensitive to TNF-α. Since TNF-α may stimulate the
secretion of soluble paracrine factors by dermal fibroblasts, conditioned medium
was collected to assess its effect on other dermal fibroblasts, however, this had
no significant effect on migration. However, using gelatin zymography, it was found that TNF-α did stimulate the
secretion of soluble paracrine factors that induce MMP activity in non-diabetic
fibroblasts, mirroring previous observations that a pro-inflammatory environment
can increase proteolytic activity, and indicating that diabetic fibroblasts were
again more sensitive than healthy. No difference was observed with MMP-9
activity and nor did the results with dermal fibroblasts reach statistical
significance, perhaps because of a relatively low n-number.
The ability of TNF-α to modulate the expression of genes associated with the
ECM (MMP-1, -2, -9, TIMP-1, and -2) and senescence (Sirt1 and 6) was
investigated. There was no change in Sirt1 and Sirt6 expression and no
evidence of paracrine effects (conditioned medium) on any of the genes. TNF-α
significantly induced mRNA expression of MMP-1 in healthy non-scratched and
scratched diabetic fibroblasts, and TIMP-1 in healthy non-scratched cells. There
was also considerable donor variability that prevented statistical significance
being achieved under the other conditions.
The secretion of various cytokines associated with inflammation was compared
in healthy and diabetic fibroblasts in the presence and absence of TNF-α.
Seven cytokines were secreted, by healthy and diabetic male and female
fibroblasts, although diabetic female fibroblasts did not secrete two of them.
TNF-α stimulated secretion of cytokines in healthy and diabetic, male and
female cells but the profiles of those released were different between the
different groups. There was no TNF-α induced paracrine effect on cytokine
secretion by healthy dermal fibroblasts.
In conclusion, changes in the microenvironment and the influx of pro inflammatory cytokines may significantly alter the dermal fibroblast phenotype.
Understanding these functional and molecular changes in response to
inflammatory cytokines will give a better understanding of the differences
between fibroblast activity in normal physiological wound healing and chronic or
diabetic non-healing wounds.
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Sommar, Pehr. "Differentiation of Human Dermal Fibroblasts and Applications in Tissue Engineering." Doctoral thesis, Linköpings universitet, Hand och plastikkirurgi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-60879.
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Tissue engineering applies principles of biology and engineering to the development of functional substitutes for damaged or lost tissues. Tools for the neo-generation of tissue in tissue engineering research include cells, biomaterials and soluble factors. One main obstacle in tissue engineering is the limited availability of autologous tissue specific progenitor cells. This has led to interest into using autologous cells with stem cell plasticity. Bone marrow derived stem cells were the first adult stem cells shown to have multilineage potential. Since, several reports have been published indicating that cells from other tissues; fat, muscle, connective tissue e.g., possess potential to differentiate into lineages distinct from their tissue of origin. The optimal cell type for use in tissue engineering applications should be easy to obtain, cultivate and store. The human dermal fibroblast is an easily accessible cell source, which after routine cell expansion gives a substantial cell yield from a small skin biopsy. Hence, the dermal fibroblast could be a suitable cell source for tissue engineering applications.The main aim of this thesis was to investigate the differentiation capacity of human dermal fibroblasts, and their possible applications in bone and cartilage tissue engineering applications. Human dermal fibroblasts were shown to differentiate towards adipogenic, chondrogenic, and osteogenic phenotypes upon subjection to specific induction media. Differentiation was seen both in unrefined primary cultures and in clonal populations (paper I). Fibroblasts could be used to create three-dimensional cartilage- and bone like tissue when grown in vitro on gelatin microcarriers in combination with platelet rich plasma (paper II). 4 weeks after in vivo implantation of osteogenic induced fibroblasts into a fracture model in athymic rats, dense cell clusters and viable human cells were found in the gaps, but no visible healing of defects as determined by CT-scanning (paper III). After the induction towards adipogenic, chondrogenic, endotheliogenic and osteogenic lineages, gene expression analysis by microarray and quantitative real-time-PCR found several master regulatory genes important for lineage commitment, as well as phenotypically relevant genes regulated as compared to reference cultures (paper IV). In conclusion, results obtained in this thesis suggest an inherent ability for controllable phenotype alteration of human dermal fibroblasts in vitro. We conclude that dermal fibroblasts could be induced towards adipogenic, chondrogenic, endotheliogenic or osteogenic novel phenotypes which suggest a genetic readiness of differentiated fibroblasts for lineage-specific biological functionality, indicating that human dermal fibroblasts might be a suitable cell source in tissue engineering applications.
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Fray, Timothy Richard. "Measuring single cell contractility : comparison of human dermal fibroblasts and myofibroblasts." Thesis, University of York, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298591.
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Karlsson, Lisa. "Differentiation of Human Dermal Fibroblasts : a New Tool in Vascular Tissue Engineering." Licentiate thesis, Linköpings universitet, Institutionen för klinisk och experimentell medicin, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-20319.
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Tissue engineering is an expanding field, which focuses on the development of func-tional substitutes for damaged tissues. A limitation in this field is difficulties with obtaining autologous cells. Recent research has shown the presence of cells with multilineage-potential within the connective stroma of the skin. In line with this, a potential plasticity inherent in human dermal fibroblasts has been demonstrated. The overall aim of this study was to investigate if human dermal fibroblasts can be used as a cell source for vascular tissue engineering. Differentiation towards an endothelial cell-like phenotype was induced by culturing dermal fibroblasts in endothelial growth medium. By utilizing in vitro cell culture models, the capacity of different types of serum and serum constituents in inducing a phenotypic shift in fibroblasts was investi-gated. To clarify the mechanisms behind this phenotypic shift and to eliminate the risk of having growth of residual endothelial cells in the cultures, both normal dermal fibroblasts and single-cell clone fibroblasts were used. Our results demonstrated that presence of human serum caused fibroblasts and single-cell clone fibroblasts to express vWf, to incorporate fluorochrome-labeled low-density lipoprotein, and to start form-ing capillary-like networks. As an initial step in using these cells in tissue engineering, their ability to endothelialize a surface in vitro was studied. Cells cultured in either fibroblast or endothelial growth medium were seeded on scaffolds. Differentiation was confirmed by western blotting and immunohistochemistry using antibodies directed towards vWf, ve-cadherin, eNOS, and bradykinin receptor B2. The results revealed that endothelial differentiated fibroblasts cultured on scaffolds showed histological resem-blance to endothelial cells, and expressed molecules indicative of an endothelial phenotype. In conclusion, the results presented in this study indicate a possibility to induce differentiation of human dermal fibroblasts towards an endothelial cell-like phenotype. Consequently, these data suggests that human dermal fibroblast may be a novel cell source for vascular tissue engineering.In the printed version the series number of this Licentiate thesis is 98. In the electronic version it has been corrected to 99.
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Rockwood, Jananie. "House Dust Mite Induced Gene Expression and Cytokine Secretion by Human Dermal Fibroblasts." Wright State University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=wright1347976529.
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Kwok, Hoi Hin. "The anti-aging effects of ginsenosides on human endothelial cells and dermal fibroblasts." HKBU Institutional Repository, 2011. http://repository.hkbu.edu.hk/etd_ra/1235.
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Rodrigues, Annelissa Zorzeto. "Avaliação in vitro do cultivo de fibroblastos gengivais humanos em matriz dérmica acelular." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/58/58132/tde-30062008-150532/.
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A matriz démica acelular, MDA, figura dentre os biomateriais que têm por objetivo restaurar defeitos mucogengivais. A correção de defeitos mucogengivais a partir de constituintes autógenos são os procedimentos mais comumente usados, no entanto, em decorrência da quantidade insuficiente de tecido doador, esses procedimentos se tornam limitados. Diante disso, o objetivo desse estudo foi avaliar, in vitro, diferentes aspectos relacionados ao cultivo prévio de fibroblastos gengivais humanos em MDA. Fibroblastos gengivais humanos foram cultivados pela técnica do explante a partir de amostras de tecido gengival queratinizado removido de três pacientes saudáveis. A MDA foi cultivada com esses fibroblastos por períodos de 14 e 21 dias para posterior análise dos eventos de: adesão celular, proliferação e viabilidade. Os resultados mostraram que em 7 dias, os fibroblastos estavam aderidos, espraiados e dispersos sobre a superfície externa da MDA, em 14 dias formavam monocamada de células de morfologia alongada e quiescentes (Ki-67 negativos) em sua maioria, sendo apenas ocasionalmente observadas no interior da MDA. Em 21 dias a monocamada exibia menor densidade celular. Os resultados sugerem que o cultivo de fibroblastos em MDA em períodos de 14 dias permite boas condições de adesão e espraiamento das células sobre a matriz, porém, a alta densidade de fibras colágenas parece ser um fator limitante à migração celular.Acellular Dermal Matrix, ADM, is a biomaterial that has been used in periodontal procedures to treat mucogingival defects. Mucogingival defects can be corrected by autogenous grafts that are the most common procedure used in periodontology, however, because of the limited source of donor\'s tissue this procedure became limited. The aim of this investigation was to verify, in vitro, different aspects related to human gingival fibroblasts seeding on to the ADM. Human gingival fibroblasts were established from explant cultures from the connective tissue of keratinized gingiva collected from three healthy patients. ADM was seeded with gingival fibroblasts for 14 and 21 days, and then cell adherence, proliferation and viability were analyzed. Results revealed that, at day 7, fibroblasts were adherent and spreading on the ADM surface, and were unevenly distributed, forming a discontinuous single cell layer, at day 14, a confluent fibroblastic monolayer lining ADM surface was noticed. At day 21, the cell monolayer exhibited a reduction in cell density. The results suggests that fibroblasts seeding on the ADM for 14 days can allow good conditions for cell adhesion and spread on the matrix, however, because of the high collagen fiber bundle density cell, migration inside the matrix was limited.
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Nguyen, Elise B. "Electrical Stimulation of Human Dermal Fibroblasts and the Quantification of Collagen, Collagenase, and Elastin." Thesis, California State University, Long Beach, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10263522.
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Electrical stimulation of tissues has been found to have many uses in pain management, antibacterial treatment, and wound healing. In vivo, it is known to stimulate epidermal migration and increase fibroblast cell proliferation. Here the effects of electrical field (EF) stimulation on collagen, elastin, and collagenase expression in human dermal fibroblasts are studied. The cells are stimulated in bioreactor using square wave voltage pulses controlled by potentiostat for up to 24 h period. The pulse voltage (0–10V), pulse bias (0, +,−), pulse time (10–1000 ms), rest time (0.1–10 s) was varied. The effects of EF stimulation is evaluated in terms of protein expression level and changes in cell morphology. The results show that the expressions of these proteins are correlated and are doubled when EF stimulation larger than 3V and positive bias is applied. The shorter pulse time stimulates the cells more effectively, while the rest time between pulses has smaller effect.
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Cunningham, Mary Jane. "The induction and inhibition of benzo(a)pyrene metabolism in human epidermal keratinocytes and dermal fibroblasts /." The Ohio State University, 1985. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487259125219762.
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Kole, Denis. "Role of Fibroblast Growth Factor 2 in Maintenance of Multipotency in Human Dermal Fibroblasts Treated with Xenopus Laevis Egg Extract Fractions." Digital WPI, 2014. https://digitalcommons.wpi.edu/etd-dissertations/207.
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Current usage of human embryonic stem cells (hES) and induced pluripotent stem cells (iPS) in clinical therapies and personalized medicine are limited as a result of ethical, technical and medical problems that arise from isolation and generation of these cells. Isolation of hES cells faces ethical problems associated with their derivation from human pre-implantation embryos. The most controversial aspect of hES cell isolation targets the generation of autologous hES cell lines which requires the transfer of a somatic-cell nucleus from the patient to an enucleated oocyte. While already established embryonic stem cell lines from IVF embryos can be used in a similar manner, lack of genetic identity can cause therapy rejection from the host, and prevent their use in personalized medicine. Induced pluripotent stem cells on the other hand, are generated from somatic cells that have been reprogrammed in vitro to behave like stem cells. While these cells can potentially be used for personalized medicine without the risk of rejection by the host system, derivation methods prevent their therapeutic use. The most efficient method used to generate iPS cells involves usage of viral particles which can result in viral DNA being integrated in the host cell’s genome and render these cells non-compliant for clinical therapies. Other methods not involving viral particles exist as well, but the reprogramming efficiency is too low and technical problems with generating large enough numbers of cells prevent these methods from being feasible approaches for clinical therapies. Direct reprogramming of a differentiated cell into a developmentally more plastic cell would offer alternatives to applications in regenerative medicine that currently depend on either embryonic stem cells (ES), adult stem cells or iPS cells. We hypothesize that Xenopus laevis egg cytoplasmic extract contains critical factors needed for reprogramming that may allow for non-viral, chemically defined derivation of human induced pluripotent/multipotent cells which can be maintained by addition of exogenous FGF2. In this thesis we investigated a new method for generation of multipotent cells through determining the ability of select fractions of Xenopus laevis egg extract to induce multipotency in already differentiated cells. We were able to identify select fractions from the extract that in combination with exogenously added FGF2 can reprogram and maintain the reprogrammed cells in an undifferentiated state. The findings of this work also determined that Xenopus laevis egg extract mRNA is required for achieving full reprogramming. The body of work presented in this thesis showed the ability of FGF2 isoforms to bind and activate select FGF receptor tyrosine kinases, act as extracellular mitogenic factors to support growth of hES cells in an undifferentiated state as well bind to nuclear DNA and affect expression of endogenous genes. Moreover, we showed that all FGF2 isoforms can induce expression of stem cell specific proteins in human dermal fibroblasts as well as extend lifespan of human dermal fibroblasts in vitro. In this work we identified HECW1, the gene coding for E3 ubiquitin ligase NEDL1, as a novel nuclear target for all FGF2 isoforms and showed that overexpression of recombinant FGF2 isoforms in human dermal fibroblasts can down regulate expression of HECW1 gene.
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Deing, Verena Daniela [Verfasser]. "Characterization of the oxytocin system in primary human dermal fibroblasts and keratinocytes / Verena Daniela Deing." Konstanz : Bibliothek der Universität Konstanz, 2013. http://d-nb.info/1043906886/34.
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Arikatla, Venkata Sravya. "Stress-Induced Senescence in Human Dermal Fibroblasts: Effects of Creatine and Nicotinamide Post Stress Treatment." Wright State University / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=wright1629899898596448.
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Dahlbom, Moa. "Gene Expression of CTGF in Human Dermal Fibroblasts When Exposed to TGF-β and IL-1α." Thesis, Örebro universitet, Institutionen för läkarutbildning, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-36966.
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Teves, Joji M. Y., Vedanshi Bhargava, Konner R. Kirwan, Mandi J. Corenblum, Rebecca Justiniano, Georg T. Wondrak, Annadurai Anandhan, et al. "Parkinson's Disease Skin Fibroblasts Display Signature Alterations in Growth, Redox Homeostasis, Mitochondrial Function, and Autophagy." FRONTIERS MEDIA SA, 2018. http://hdl.handle.net/10150/626553.
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The discovery of biomarkers for Parkinson's disease (PD) is challenging due to the heterogeneous nature of this disorder, and a poor correlation between the underlying pathology and the clinically expressed phenotype. An ideal biomarker would inform on PD-relevant pathological changes via an easily assayed biological characteristic, which reliably tracks clinical symptoms. Human dermal (skin) fibroblasts are accessible peripheral cells that constitute a patient-specific system, which potentially recapitulates the PD chronological and epigenetic aging history. Here, we compared primary skin fibroblasts obtained from individuals diagnosed with late-onset sporadic PD, and healthy age-matched controls. These fibroblasts were studied from fundamental viewpoints of growth and morphology, as well as redox, mitochondrial, and autophagic function. It was observed that fibroblasts from PD subjects had higher growth rates, and appeared distinctly different in terms of morphology and spatial organization in culture, compared to control cells. It was also found that the PD fibroblasts exhibited significantly compromised mitochondrial structure and function when assessed via morphological and oxidative phosphorylation assays. Additionally, a striking increase in baseline macroautophagy levels was seen in cells from PD subjects. Exposure of the skin fibroblasts to physiologically relevant stress, specifically ultraviolet irradiation (UVA), further exaggerated the autophagic dysfunction in the PD cells. Moreover, the PD fibroblasts accumulated higher levels of reactive oxygen species (ROS) coupled with lower cell viability upon UVA treatment. In essence, these studies highlight primary skin fibroblasts as a patient-relevant model that captures fundamental PD molecular mechanisms, and supports their potential utility to develop diagnostic and prognostic biomarkers for the disease.
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Mignon, Charles. "Photo-biomodulation of human skin fibroblast sub-populations : a systematic approach for the optimization of optical treatment parameters." Thesis, University of Bradford, 2017. http://hdl.handle.net/10454/16064.
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The thesis presents a rational path for the optimization of the selection of optical treatment parameters in photobiomodulation of human skin fibroblasts. The project begins with an extensive analysis of 90 bibliographic reports in photobiomodulation published between 1985 and 2015, and revealed major inconsistencies in optical parameters selected for clinical applications. Seeking greater clarity for optimal parameter choice, a systematic approach to disentangle the multiple factors underpinning the response of human dermal fibroblasts in vitro to visible and near-infra red (NIR) light was employed. Light-based devices were constructed to specifically and systematically screen the optical parameter window (i.e. wavelength, irradiance and dose) observed in literature. Additionally, critical culture and treatment conditions that have dramatic impact on the outcome of specific light treatment of these human skin dermal cells were identified. In particular, environmental oxygen concentration, cell confluency and serum concentration were all found to have a great effect on the response of dermal fibroblasts to light. In parallel, the induction of reactive oxygen species (ROS) by short visible wavelengths on two dermal fibroblast sub-populations or lineage, reticular and papillary, was monitored by live-cell imaging. The ROS species were found to be created in or close to mitochondria. Lastly, gene expression studies revealed a strong impact of short visible wavelengths, as compared to long and NIR wavelengths on both subpopulations of human dermal fibroblasts. In particular, blue light (450 nm) specifically down-regulated proliferation, metabolism and protein synthesis molecular pathways. At the protein level, 450-nm light inhibited the production of procollagen I in human reticular and papillary fibroblasts in a dose-dependent manner. Gene expression results were in agreement i.e., the same light parameter down-regulated collagen fiber genes, integrins and up-regulated collagenase MMP1. This thesis concludes with a chapter presenting a characterization of the accuracy of a potential translation tool for the prediction of optical photon density inside human skin.
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Jouret, Chantal. "Effects of matrix and phenotype on human dermal fibroblast attachment under laminar shear stress : implications for the development of tissue-engineered heart valves." Thesis, Georgia Institute of Technology, 1997. http://hdl.handle.net/1853/11251.
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Horobin, Adele Jayne. "Maggots and wound healing : the effects of Lucilia sericata larval secretions upon interactions between human dermal fibroblasts and extracellular matrix proteins." Thesis, University of Nottingham, 2005. http://eprints.nottingham.ac.uk/11516/.
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The introduction of necrophagous fly larvae (maggots) into chronic wounds for the purpose of inducing healing is an ancient practice that has recently undergone a renaissance in Western medicine. Through clinical observations, maggots are broadly recognised to debride the wound of necrotic tissue, cleanse the wound of infection and promote granulation tissue formation. Despite such recognition, little research at the biological level has been undertaken to identify the mechanisms by which maggots accomplish such feats. The dermal fibroblast is a major cellular component of granulation tissue and as such, its migration into the wound plays a vital role in new tissue growth. Fibroblast migration is directed by the composition of the extracellular matrix. Maggot secretions contain proteolytic enzymes that are active against a variety of extracellular matrix proteins which are present at the wound site. Hence, this thesis focused upon the effects of maggot secretions on human dermal fibroblast adhesion and migration in the presence of common extracellular matrix proteins. This was with the aim of elucidating the mechanisms by which maggots stimulate tissue formation within the wound and from there, developing new products that may be used to promote wound healing. Experiments showed that maggot secretions modulated fibroblast adhesion to tissue culture plastic surfaces and to surfaces coated with collagen and particularly fibronectin. Modification of the protein-coated surface by enzymes present within the secretion appeared to play a role. Fibroblast migration upon a fibronectin-coated surface was enhanced in the presence of maggot secretions. The same also occurred in the presence of a higher concentration of secretions when the cells were located within a three-dimensional environment comprising collagen gel and fibronectin. Evidence suggested that this may have been associated with enhanced matrix re-modelling.
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Gonzales, Christopher R. "3,3′,5′-triido-L-thyronine alters protein kinase B, phosphotase and tensin homolog and connective tissue growth factor expression in human dermal fibroblasts." Thesis, Boston University, 2012. https://hdl.handle.net/2144/12397.
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Thesis (M.A.)--Boston University
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Cutaneous tissue repair is complex and involves a variety of growth factors to regulate a balance of regeneration and fibrosis during healing. This process is divided into three sequential and overlapping phases: the inflammatory phase, the proliferative phase, and the remodeling phase. Fibroblasts are crucial during this process in that they help initiate inflammatory activity, deposit extracellular matrix proteins for granulation tissue and deconstruct granulation tissue to make way for mature scar formation. Previous studies on the effects of 3,3',5'-triiodo-L-thyronine (T3) on skin have revealed that healing tissue responds to T3 by accelerating skin cell proliferation and migration. These findings indicate that T3 offers potential as a therapeutic drug for individuals with extensive cutaneous damage, chronic skin maladies or retarded wound healing. The mechanisms underlying these changes are not clearly understood, however, elucidation of changes in protein expression patterns should be evaluated to appropriately judge the therapeutic potential of T3. This study aims to characterize T3 dose responsive expression of protein kinase B, phosphatase and tensin homolog, connective tissue growth factor and wnt5a. Western blot analysis and immunodetection revealed that wnt5a is not expressed in human dermal fibroblasts. Protein kinase B did not vary significantly with T3 concentration ranging from 1.0 nM-1.0 1µM, F(4,5)= 1.93, p > 0.05, nor did connective tissue growth factor, F(4,5) = 2.16, p > 0.05. In contrast, phosphatase and tensin homolog showed a statistically significant change in expression, F(4, 15) = 4.67, p less than 0.05. The results presented here provide insight into protein pathways and growth factors through which thyroid hormone produces its effects on the various cells of the integument and suggests that phosphatase and tensin homolog (PTEN) expression levels are responsive to varying concentrations of T3. Future studies should further evaluate the role of T3 on its various targets as a therapeutic option for skin disorders.
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Lee-Bellantoni, Margaret S. "Antioxidant defense and redox responses to telomere homolog oligonucleotides in human dermal fibroblasts: a model for investigating redox signaling responses to DNA damage." Thesis, Boston University, 2005. https://hdl.handle.net/2144/37162.
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Thesis (Ph.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
It has been demonstrated that oligonucleotides homologous to the 3' telomere repeat sequence TTAGGG (T-oligos) stimulate DNA damage responses that are also induced by disruption of the telomere loop structure. Adaptive defense against oxidative stress and UV or ionizing radiation has been reported, but adaptive antioxidant defense as a response to mimicking telomere loop exposure has not been described. The T-oligos pTT and pGTTAGGGTTAG were added to human dermal fibroblast cultures to investigate whether mimicking telomere loop disruption stimulates antioxidant defense. pTT stimulated mitochondrial superoxide dismutase protein levels within 72 hours. Cell yields were higher after H202 exposure in fibroblasts pretreated with pTT for 72 hours compared to diluent pretreated cells. Intracellular reactive oxygen species (ROS) levels, measured by flow cytometry and the dichlorofluorescein diacetate probe, increased during T-oligo treatment as compared to diluent and oligonucleotide controls. The time course and degree of ROS stimulation corresponded to the time course for activation and/or induction of p53 and p21/Cip1/Waf1. The NADPH oxidase inhibitor diphenyliodonium chloride abrogated this increase and fibroblasts retrovirally transduced to produce dominant negative p53 failed to display increased ROS, implicating that the T-oligos induced ROS through p53-responsive NADPH oxidases. A horseradish peroxidase assay for extracellular H20 2 showed no H20 2 release with pTT treatment. To determine whether there was induction of senescence, an endpoint response to increased ROS and prolonged T-oligo treatment in fibroblasts, the senescence-associated β-galactosidase assay was conducted in parallel with the DCF assay. Only the 11mer T-oligo treatment modestly increased the number of β-galactosidase positive cells by 72 hours (<30% of cells). This is the first report suggesting that antioxidant defense and ROS signaling are part of the broad adaptive response in mammalian cells presumably initiated by telomere loop disruption and mimicked by T-oligos. T-oligo treatment thus offers a new model for studies of ROS signaling in human dermal fibroblasts, allowing exploration of the relationships between DNA damage, ROS, oxidative stress, and the evolution of cellular defense mechanisms.
2031-01-01
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Castellano-Pellicena, Irene. "The role of photoreceptors in human skin physiology; potential targets for light-based wound healing treatments. Identification of opsins and cryptochromes and the effect of photobiomodulation on human skin and in cultured primary epidermal keratinocytes and dermal fibroblasts." Thesis, University of Bradford, 2017. http://hdl.handle.net/10454/16884.
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The positive effect of photobiomodulation in wound healing has previously been reported, however there is a considerable lack of knowledge regarding the molecular mechanisms involved, and no consensus on light parameters. Cytochrome c oxidase (CCO) is established as the main photoreceptor in cells, but light also induces nitric oxide (NO), production of reactive oxygen species (ROS) and activation of ion channels. Emerging new molecular targets include the GPCRs opsins (OPNs) and the circadian clock transcription factors, cryptochromes (CRYs).
Localisation of OPN1-SW, OPN3, OPN5, CRY1 and CRY2 was seen in female facial and abdominal human skin. Furthermore, expression of these photoreceptors was retained in primary epidermal keratinocytes and dermal fibroblasts in culture; both cell types expressed OPN1-SW, OPN3, CRY1 and CRY2, at the mRNA and protein level. OPN2 was only expressed in cultured dermal fibroblasts, while in line with in situ expression, OPN5 was only expressed in cultured keratinocytes. The photoreceptor-expressing cultured epidermal keratinocytes demonstrated a dose- and wavelength- dependent response in both metabolic activity and cell migration in a scratch-wound assay. Specifically, low dose (2 J/cm2) blue light (447 nm) increased metabolic activity, but it did not impact keratinocyte migration. In contrast, high dose (30 J/cm2) blue light had no effect on metabolism, but inhibited migration of epidermal keratinocytes. Red light (655 nm) at 30 J/cm2 stimulated metabolic activity but did not modulate migration, while a higher dose of 60 J/cm2 had no effect on keratinocyte metabolic activity.
In order to study OPN3 and CRY1 function, they were silenced in keratinocytes using siRNA; additionally 8 μM KL001 was used to stabilize CRY1. KL001 inhibited migration, and induced KRT1 and KRT10, an effect which was abrogated by knockdown of OPN3. Interestingly, knockdown of OPN3 upregulated CRY1 expression, while KL001 upregulated OPN3 expression, indicating a regulation by OPN3 of the molecular epidermal clock. Low levels of blue light increased early differentiation of epidermal keratinocytes, which was mediated by OPN3 and circadian clock mechanisms. However, low levels of blue light decreased keratinocyte DNA synthesis, which was mediated by circadian clock independently of OPN3. Translation of parameters ex vivo showed increasing re-epithelialisation and induction of OPN3 and CRY1 expression following exposure to 2 J/cm2 of blue light; however high doses of blue light inhibited re-epithelialisation. Red light, also increased re-epithelialisation, but had no effect on OPN3 or CRY1 expression. In conclusion, photoreceptors are expressed in human skin and they mediate DNA synthesis, migration and differentiation of epidermal keratinocytes. Furthermore, low dose of blue light interacts with OPN3 to induce epidermal differentiation, through the regulation of the circadian clock. A better understanding of the molecular mechanisms behind the photobiomodulation response in vitro will help to develop light based therapies for human wound healing. Interestingly, selected light parameters translated to human ex vivo skin showed a beneficial effect of low doses of blue (2 J/cm2) and red (30 J/cm2) light in re-epithelialisation.Marie Curie ... the CLaSSiC project
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Rusciani, Anthony. "Etude du mode de fonctionnement du complexe récepteur de l'élastine : modulation de la composition et de la dynamique de la membrane plasmique." Thesis, Reims, 2012. http://www.theses.fr/2012REIMS019/document.
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L'élastine est la protéine matricielle responsable de l'élasticité des tissus retrouvée dans des tissus soumis à de fortes contraintes mécaniques tels que les poumons, les artères ou la peau. La dégradation de cette protéine lors de processus physiopathologiques produit des peptides bologiquement actifs nommés peptides d'élastine portant le motif GXXPG essentiel à leur activité. Ces peptides régulent diverses fonctions biologiques telles que le chimiotactisme, la synthèse de protéases, la prolifération. Tous ces effets dépendent de la fixation des peptides d'élastine au complexe récepteur de l'élastine. Ce complexe est composé de trois sous-unités : une protéine périphérique de 67 kDa, l'Elastin Binding Protein (EBP), et deux protéines associées à la membrane, la Protective Protein/Cathepsin A (PP/CA) et la Neuraminidase-1 (Neu-1) de 55 et 61 kDa respectivement. L'activité sialidase de Neu-1 est responsable de l'activation de ERK 1/2 après fixation des peptides d'élastine au complexe récepteur de l'élastine.Dans cette étude, nous démontrons que l'EBP et les radeaux lipidiques sont colocalisés à la membrane plasmique. Nous montrons, de plus, que la déstructuration de ces microdomaines aussi bien que leur déplétion en glycolipides bloque la signalisation du récepteur. L'utilisation d'un anticorps monoclonal bloquant dirigé contre le GM3 montre qu'il est essentiel à la signalisation. Après traitement par les peptides d'élastine, le contenu cellulaire en GM3 diminue alors que celui en lactosylcéramide augmente suggérant une conversion du GM3 en lactosylcéramide. L'utilisation de lactose ou de siRNA Neu-1 bloque cette conversion ce qui tend à démontrer que le complexe récepteur de l'élastine est impliqué dans ce mécanisme. Une analyse par cytométrie en flux confirme cette production de lactosylcéramide induite par les peptides d'élastine.L'analyse par spectrométrie de masse mettrait en évidence deux lactosylcéramides (C23:0 et C24:1) potentiellement bioactifs dont la synthèse chimique a été entreprise. La purification des radeaux lipidiques par ultracentrifugation différentielle en gradient de saccharose ainsi que leur identification par Dot-blot couplé à la fluorescence montre un changement de densité de ces microdomaines après stimulation par les peptides d'élastine.L'évaluation biologique in vitro de ces lactosylcéramides montre qu'ils miment les effets des peptides d'élastine sur l'activation de ERK 1/2, la prolifération et la synthèse de MMP-1. Enfin, l'évaluation ex vivo des lactosylcéramides démontre une réduction de la zone de tissu cardiaque nécrosé suggérant un rôle cardioprotecteur de ces molécules. Ce travail propose un mécanisme original de transduction du signal à la membrane plasmique et nous laisse envisager le complexe récepteur de l'élastine, les peptides d'élastine et le lactosylcéramide comme de nouveaux agents thérapeutiques potentielsElastin is the matrix protein responsible for the elasticity of tissues where resilience is required such as lung, arteries or skin. Elastin degradation during physiopathological processes produces biologically active peptides named elastin peptides bearing the GXXPG pattern essential for their activity. These peptides regulate various biological functions such as chemotaxis, proteases synthesis and proliferation. These effects are dependent of elastin peptide binding to the elastin receptor complex (ERC). This complex is composed of three subunits: a peripheral protein of 67 kDa called elastin binding protein (EBP) and two membrane-associated proteins, protective protein/cathepsin A (PP/CA) and neuraminidase-1 (Neu-1) of 55 and 61 kDa, respectively. The sialidase activity of Neu-1 is responsible for ERK 1/2 pathway activation following binding of elastin peptide on the elastin receptor complex.In this study, we demonstrate that EBP and lipid rafts colocalize at the plasma membrane. We also show that the disruption of these microdomains and their depletion in glycolipids block the receptor signaling. The use of a monoclonal anti-GM3 blocking antibody shows that this glycosphingolipid is essential for signaling. Following elastin peptide treatment, cellular GM3 level decreases while the lactosylceramide one increases consistently with a GM3/LacCer conversion. The use of lactose or Neu-1 siRNA blocks this process suggesting that the elastin receptor complex is involved in this mechanism. Flow cytometry analysis confirms this elastin peptide-driven LacCer generation.Mass spectrometry analysis of elastin peptide-stimulated cell membrane extracts identified two potentially bioactive lactosylceramides (C23:0 and C24:1) and their synthesis has been realized. Lipid rafts purification by differencial ultracentrifugation in sucrose gradient shows a variation of the microdomains density as well as their identification by fluorescence linked-Dot-blot following elastin peptide stimulation.In vitro biological evaluation of these lactosylceramides shows that they mimic the elastin peptide effects on ERK 1/2 activation, proliferation and MMP-1 synthesis. Finally, ex vivo lactosylceramides evaluation demonstrates a decrease of cardiac tissue necrosis area suggesting that these molecules could be cardioprotective agents. This work proposes an original mechanism of signal transduction at the plasma membrane and let us foresees the elastin receptor complex, elastin peptides and lactosylceramide as new potential therapeutical targets
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Gsib, Olfat. "Synthèse et caractérisation d’hydrogels de fibrine et de polyéthylène glycol pour l’ingénierie tissulaire cutanée." Thesis, Compiègne, 2018. http://www.theses.fr/2018COMP2416.
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Depuis plus d’une cinquantaine d’années, de formidables avancées ont été initiées dans le domaine de l’ingénierie tissulaire cutanée menant à la reconstruction in vitro de substituts de peau. La plupart sont des substituts dermiques destinés à être utilisés comme aide à la cicatrisation des plaies aigües et chroniques en complément des traitements de greffes conventionnels ainsi que pour l’augmentation des tissus mous. Bien qu’un nombre croissant de patients aient pu bénéficier de ces matrices dermiques, leur application clinique reste encore restreinte, en raison de leur coût élevé mais également à cause de résultats cicatriciels parfois peu satisfaisants. Par conséquent, il reste un défi de taille, celui de développer des substituts dermiques stimulant activement la cicatrisation, présentant un faible coût de production, sans propriétés antigéniques et possédant des propriétés mécaniques adaptées. Dans ce cadre, les hydrogels à base de fibrine constituent des candidats prometteurs, en particulier en raison du rôle central de cette protéine dans la cicatrisation. Le principal inconvénient est qu’à concentration physiologique, ces hydrogels sont faibles mécaniquement, ce qui les rend difficilement manipulables. L’objectif de cette thèse a été la mise au point ainsi que la caractérisation de différents hydrogels destinés à être utilisés comme substituts dermiques. Ces derniers présentent l’avantage d’associer les propriétés biologiques de la fibrine avec les propriétés mécaniques d’un polymère synthétique, le polyéthylène glycol dans une architecture de réseaux interpénétrés de polymères (RIP). Les résultats obtenus ont permis : - de confirmer les propriétés physico-chimiques des RIP développés initialement par nos collaborateurs de l’université de Cergy-Pontoise, - de valider en trois étapes (in vitro, ex vivo puis in vivo) la biocompatibilité de ces nouvelles matrices, destinées à être utilisées comme supports de culture 2D et pour l’augmentation des tissus mous, - d’élaborer et de caractériser des matrices macroporeuses, optimisées pour la culture 3D de fibroblastes de dermes humainsOver the past five decades, we assisted in extraordinary advances in the field of skin tissue engineering which led to the in vitro reconstruction of a wide range of skin substitutes. Most of them are dermal substitutes: Their clinical application ranges from treating acute and chronic wounds to soft tissue augmentation. Although increasing numbers of patients have been treated with dermal substitutes, their clinical application has been limited by their substantial cost and some poor healing outcomes. Hence, there is still a challenge to produce a dermal substitute which enhance sufficiently wound healing. To this end, the substitute should exhibit suitable properties for enabling the repair process. Other requirements such as excellent biocompatibility, minimal antigenicity, ease to handle and cost-effective production are also essential. In this context, fibrin hydrogels constitute promising candidates for skin tissue engineering since fibrin fibers form a physiological and provisional backbone during wound healing. However, the poor mechanical properties of fibrin-based hydrogels at physiological concentration are an obstacle to their use. In this study, our aim was to design and characterize mechanically reinforced fibrin-based hydrogels by combining the intrinsic properties of a fibrin network with the mechanical features of a polyethylene glycol network using an interpenetrating polymer network (IPN) architecture. They are intended to be used as dermal scaffolds. The results obtained in this thesis: - Confirmed the suitable physico-chemical properties of IPN, first developed by our partner of the University of Cergy-Pontoise. - Validated their biocompatibility using a three-step approach (in vitro, ex vivo and in vivo assays). - Led to the synthesis and characterization of a new type of fibrin-based macroporous matrices, optimized for 3D dermal fibroblast culture
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Grella, Alexandra R. "A mechanism for the FGF2-mediated down-regulation of integrin alpha-11 identified through studying altered adhesome of human dermal fibroblasts undergoing early Mesenchymal-to-Epithelial Transition." Digital WPI, 2015. https://digitalcommons.wpi.edu/etd-dissertations/53.
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Work in our lab has resulted in the development of a novel approach to creating a more developmentally plastic human dermal fibroblast (hDF) phenotype that allows for the study of molecular mechanisms involved in cell-fate conversion. Culturing hDF under defined culture conditions (5% O2 and supplementation with fibroblast growth factor FGF2) induces induced the regeneration competent (iRC) phenotype that is characterized by stem cell gene expression, and increased life-span in vitro. The work presented in this thesis further characterizes the system, and describes an overall shift in extracellular matrix and adhesion molecules in human dermal fibroblasts (hDF) undergoing the transition to a more developmentally plastic phenotype (iRC). This work suggests that we create the initiation phase of Mesenchymal-to-Epithelial Transition (MET) during conversion to the iRC phenotype. This transition is marked by loss of integrin alpha-11 (α11) and its binding partner Collagen-I (COL-I). Moreover, we describe the mechanism for the down-regulation of α11 that is mediated by FGF2 activation of ERK1/2 through systematic investigation of several potential molecular mechanisms. The body of work presented here shows that the ERK 1/2 mediated down-regulation of α11 is independent of activation of TGF-β1-mediated regulation of α11. In addition to down-regulation of α11, an overall shift in the transcript levels of other adhesion molecules is observed, which demonstrates that iRC are most likely transitioning their attachment to a laminin and fibronectin-based matrix. These results suggest that iRC may be producing a more “pro-regenerative matrixâ€�. We hypothesize that the changes in integrin expression profile and interaction with ECM serve as a feedback loop during the iRC phenotype shift. Our findings suggest that this “pro-regenerativeâ€� shift in attachment of iRC as well as the ERK 1/2 mediated down-regulation of α11 could be exploited in wound healing biology and fibrosis research. Manipulation of the dynamic relationship between TGF-β1 and FGF2 has the potential to reduce scar deposition. Further identification of molecular mechanisms controlling this phenotype conversion will allow development of strategies for in situ manipulation of wound healing outcomes.
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Schneider, Sabine [Verfasser], Jean [Akademischer Betreuer] Krutmann, and William [Gutachter] Martin. "Characterization of molecular mechanisms involved in intrinsic and extrinsic skin aging of in situ aged normal human dermal fibroblasts / Sabine Schneider ; Gutachter: William Martin ; Betreuer: Jean Krutmann." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2021. http://d-nb.info/1241824169/34.
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Alase, Adewonuola Adelodi. "The role of interleukin-10 family members in inflammatory skin diseases : understanding the mechanism of action of interferon lambda and interleukin-22 on human primary keratinocytes and dermal fibroblasts with a focus on healing responses in inflammatory skin diseases." Thesis, University of Bradford, 2015. http://hdl.handle.net/10454/14303.
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Cutaneous lupus erythematosus (CLE) is an autoimmune disease that resolves with or without permanent scars depending on the subtype. Interferons (IFNs), including the skin specific IFNλ mainly activate STAT1, which results in inflammation in CLE and may play a significant role in scar formation in chronic discoid CLE. IL-22 activates STAT3 and it is emerging as a mediator with significant impact on normal wound repair, epidermal hyperproliferation and prevention of fibrosis. This work focussed on understanding the regulation and functional impact of IL-22 and IFNλ on skin cells. The counter-regulatory effect of IL-22 on the activities of IFNλ was assessed through downstream interferon stimulated genes (ISGs) expression in healthy and CLE keratinocytes. Cell proliferation and gap closure were investigated in skin resident cells using cell trace dye and scratch assay. Dermal fibroblasts were assessed for the presence of IFNλR1 and IL-22R1, downstream activities of the receptors. Results showed that IL-22 accelerated “scratch” closure in keratinocytes while IFNλ caused a delay in closure. IL-22 significantly downregulated IFNλ-induced chemokines expression in healthy, but not CLE keratinocytes. Reduced IL-22R1 expression and “STAT3 signature genes” was observed in CLE keratinocytes. A key finding of this project is that dermal fibroblasts respond to both IFNλ and IL-22. This work shows that IL-22 can reduce the damaging effect of IFNs in inflamed skin and also identifies dermal fibroblasts as important cells in skin immune responses. In conclusion, IL-10 family members can have both beneficial and destructive effects on the skin organ depending on the micro milieu and cell-type involved. Manipulating the balance of IL-10 family members in the skin may offer new therapeutic approach for both psoriasis and CLE.
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Alase, Adewonuola A. "The Role of Interleukin-10 Family Members in Inflammatory Skin Diseases. Understanding the mechanism of action of interferon lambda and interleukin-22 on human primary keratinocytes and dermal fibroblasts with a focus on healing responses in inflammatory skin diseases." Thesis, University of Bradford, 2015. http://hdl.handle.net/10454/14303.
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Cutaneous lupus erythematosus (CLE) is an autoimmune disease that resolves with or without permanent scars depending on the subtype. Interferons (IFNs), including the skin specific IFNλ mainly activate STAT1, which results in inflammation in CLE and may play a significant role in scar formation in chronic discoid CLE. IL-22 activates STAT3 and it is emerging as a mediator with significant impact on normal wound repair, epidermal hyperproliferation and prevention of fibrosis.
This work focussed on understanding the regulation and functional impact of IL-22 and IFNλ on skin cells. The counter-regulatory effect of IL-22 on the activities of IFNλ was assessed through downstream interferon stimulated genes (ISGs) expression in healthy and CLE keratinocytes. Cell proliferation and gap closure were investigated in skin resident cells using cell trace dye and scratch assay. Dermal fibroblasts were assessed for the presence of IFNλR1 and IL-22R1, downstream activities of the receptors.
Results showed that IL-22 accelerated “scratch” closure in keratinocytes while IFNλ caused a delay in closure. IL-22 significantly downregulated IFNλ-induced chemokines expression in healthy, but not CLE keratinocytes. Reduced IL-22R1 expression and “STAT3 signature genes” was observed in CLE keratinocytes. A key finding of this project is that dermal fibroblasts respond to both IFNλ and IL-22.
This work shows that IL-22 can reduce the damaging effect of IFNs in inflamed skin and also identifies dermal fibroblasts as important cells in skin immune responses. In conclusion, IL-10 family members can have both beneficial and destructive effects on the skin organ depending on the micro milieu and cell-type involved. Manipulating the balance of IL-10 family members in the skin may offer new therapeutic approach for both psoriasis and CLE.University of Bradford and Centre for Skin Sciences
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Salamito, Mélanie. "Le facteur de transcription antioxydant NRF2 comme nouveau régulateur de la matrice extracellulaire des fibroblastes de peau humaine." Thesis, Lyon, 2020. http://www.theses.fr/2020LYSEN058.
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Le facteur de transcription nuclear factor erythroid-2-related factor (NRF2) est connu dans différentes espèces pour son rôle dans la défense cellulaire contre les stress oxydatifs et xénobiotiques. SKN-1, l'homologue de NRF2 chez C.elegans, est un important régulateur de la longévité du nématode qui, dans certaines conditions métaboliques spécifiques, agit de manière surprenante par l'activation de l'expression des collagènes en plus des gènes de détoxification. Les fibroblastes sont les principaux producteurs et organisateurs de tissus riches en collagène et, à ce titre, jouent un rôle clé dans l'homéostasie du derme. Nous avons étudié le nouveau rôle potentiel de NRF2 dans la régulation de l'expression de la matrice extracellulaire (MEC) dans les fibroblastes cutanés humains. La dérégulation de NRF2 a été réalisée en utilisant siRNA et shRNA. Une analyse transcriptomique globale des fibroblastes cutanés humains siNrf2, effectuée par RNA-seq, a révélé qu’en plus des cibles NRF2 connues, les gènes du matrisome et du tissu-squelette étaient les catégories de gènes les plus affectées, comprenant certains gènes clés de la MEC. L'analyse de la production et de l'organisation de la MEC a ensuite été réalisée en utilisant une combinaison de microscopies (SHG, confocal, TEM et AFM) dans des fibroblastes shNrf2 en culture. La sous-expression à long terme de NRF2 dans les fibroblastes (shNRF2) affecte les niveaux d’expression du collagène I, ainsi que la formation des fibrilles de collagènes, probablement dû au ratio collagène I/collagène V perturbé. L’analyse transcriptomique a également permis d’identifier en tant que nouvelle cible de NRF2, un second facteur de transcription, décrit comme régulateur de l'expression des collagènes et impliqué dans une maladie du tissu conjonctif. Le marquage de ce facteur par immunofluorescence a révélé que sa localisation subcellulaire, et en particulier sa translocation, est affectée par la sous-expression de NRF2 (siRNA et shRNA). Nos résultats montrent ainsi que la sous-expression de NRF2 dans les fibroblastes cutanés humains impacte les gènes de la MEC et en particulier l’expression des collagènes. Le second facteur de transcription identifié pourrait être un intermédiaire ou un co-facteur spécifique de NRF2 impliqué dans la régulation de l’expression des gènes de la MEC. NRF2 peut ainsi être considéré comme nouveau régulateur des gènes de la MEC dans les fibroblastes cutanés humains et représente une nouvelle cible pour maintenir l'homéostasie du dermeThe nuclear factor-erythroid 2-related factor 2 (NRF2) is a transcription factor involved in cell defense against oxidative and xenobiotic stresses. SKN-1, the nematode homologue of NRF2 is a master regulator of longevity that, under specific metabolic conditions, surprisingly acts through the activation of collagens expression. Fibroblasts are the major producers and organizers of collagen-rich tissues and, as such, play a key role in dermis homeostasis. Therefore, we investigated the potential new role of NRF2 in regulating extracellular matrix (ECM) expression in human skin fibroblasts. Dysregulation of NRF2 was realized using siRNA and shRNA. A global transcriptomic analysis of siNrf2 human skin fibroblasts performed by RNA-seq revealed that, in addition to known NRF2 targets, matrisome and tissue skeleton genes were the most represented gene sets, including some key ECM genes. Analysis of ECM production and organization was further conducted in cultured shNrf2 fibroblasts using a combination of microscopies (SHG, confocal, TEM and AFM). Long-term effect of silencing NRF2 in fibroblasts (shNrf2) resulted in defects in collagen expression and fibril formation, likely due to a disturbed collagen I to collagen V ratio. Interestingly, a transcription factor involved in connective tissue disease and described as a regulator of collagen expression was identified as a novel target of NRF2. Immunofluorescence staining of silenced NRF2 fibroblasts (siRNA and shRNA) strikingly revealed that NRF2 downregulation impacts its translocation rate into the nucleus. Our results demonstrate that silencing NRF2 impacts ECM and especially collagens in human skin fibroblasts. A transcription factor known to regulate collagen expression, could act as a specific cofactor of NRF2 in the regulation of ECM gene expression. NRF2 can thus be considered as a novel regulator of ECM genes in human skin fibroblasts and represents a new target to maintain dermis homeostasis
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Kamala, Ola. "A Comparison of Cultured Human Dermal Fibroblasts Derived from Terminal and Vellus Hair Bearing Skin. Differences in the expression of inhibitors of apoptosis proteins, oestrogen receptors, and responses to oestradiol under normal and wound induced conditions." Thesis, University of Bradford, 2014. http://hdl.handle.net/10454/13841.
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Wounds heal better in skin with terminal hair follicles (large and pigmented) as opposed to those with vellus hair follicles (small and unpigmented), while dermal fibroblasts from different anatomical regions also exhibit phenotypical differences. Tissue repair requires a tight control of cell proliferation, migration and apoptosis, and recent studies have shown the importance of inhibitors of apoptosis proteins (IAPs), which are proteins that prevent the process of apoptosis via their interaction with caspase molecules in wound healing. Oestrogens improve the rate and quality of wound healing, but their relationship with IAPs in human skin has not been studied. Therefore, terminal (scalp) and vellus (facial) hair bearing skin from the same donor was compared in situ and matching primary cultures of dermal fibroblasts were established from terminal (DF(T)) and vellus (DF(V)) hair bearing skin. Using immunofluorescent staining, the expression of IAPs and their antagonists was compared at different stages of the hair cycle following depilation using a murine model and then in terminal and vellus hair bearing human skin. The size and granularity of matching DF(T) and DF(V) cultures was compared by FACS analysis and mRNA and protein expression of Apollon, cIAP2, NAIP and XIAP and their antagonists DIABLO and Xaf1 analysed by qRT-PCR and immunocytochemistry in unwounded and mechanically wounded fibroblast cultures. Differences in proliferation, migration, viability and caspase 3 activity in the presence of 17β-oestradiol and changes in mRNA expression of the oestrogen receptors (GPR30, ERα and ERβ) were compared between the two cell types. IAP protein expression was generally found higher during mid anagen of the hair cycle in murine skin and hair follicles. Overall, expression was slightly higher in human terminal hair bearing skin compared to corresponding vellus hair bearing skin. IAP protein expression was similar in unwounded DF(T) and DF(V) cells with the exception of Apollon which was higher in DF(V) cells. With the exception of XIAP and its direct antagonist Xaf1, mRNA expression was higher in DF(V) cells compared to corresponding DF(T) cells. FACS analysis demonstrated that DF(V) cells were more granular than matching DF(T) cells and proliferated faster. 17β-oestradiol accelerated migration of DF(T) cells only. Mechanical wounding decreased XIAP mRNA in DF(T) and increased it in DF(V) cells, while simultaneously decreasing Xaf1 expression. In unwounded cells, 17β-oestradiol stimulated the expression of XIAP mRNA in both DF(T) and DF(V) cells, but in scratched monolayers, while it also increased expression in DF(T) cells it decreased it in DF(V) cells. A XIAP inhibitor reduced cell viability in both DF(T) and DF(V) cells, which was rescued by 17β-oestradiol in unwounded and mechanically wounded DF(T) cells, but only in unwounded DF(V) cells. 17β-oestradiol decreased caspase 3 activity in the presence of a XIAP inhibitor only in DF(T) cells. These results demonstrate significant differences between dermal fibroblasts cultured from terminal and vellus hair bearing skin of the same individual. The correlation between an increase in XIAP in response to 17β-oestradiol and a higher number of viable cells, along with a reduction in caspase 3 activity suggests that the protective effect of 17β-oestradiol may be modulated via the regulation of XIAP. Further elucidation of these different signalling pathways in dermal fibroblasts from hair bearing skin may lead to improved therapies for chronic non-healing wounds, particularly in postmenopausal females.
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Tay, Jing Q. "The roles of vitamin D in cutaneous wound healing: In vitro and ex vivo studies of the effect of 1,25(OH)2D3 and its precursors on human dermal fibroblasts and epidermal keratinocytes in cutaneous wound healing." Thesis, University of Bradford, 2018. http://hdl.handle.net/10454/17350.
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In humans, the epidermis is the main site for the synthesis of Vitamin D3 (cholecalciferol) from 7-dehydrocholesterol. Cholecalciferol undergoes further hydroxylation in the liver and kidney to produce the active form of the circulating hormone 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). In target cells, 1,25(OH)2D3 interacts with the specific intracellular vitamin D receptor (VDR), a member of the nuclear receptor superfamily. However, epidermal keratinocytes, in addition to being target cells, have enzymes required for autocrine production of 1,25(OH)2D3. They can convert cholecalciferol to 1,25(OH)2D3 via 25-hydroxylase (CYP2R1) and 1α-hydroxylase (CYP27B1). Another enzyme, 24-hydroxylase (CYP24A1), regulates local levels by inactivating 1,25(OH)2D3. While recent studies have shown that absence of VDR or 1,25(OH)2D3 impairs formation of granulation tissue during wound healing in mice, little is known about the autocrine and paracrine regulation of biologically active vitamin D3 by human dermal fibroblasts during cutaneous wound healing.
Primary cultures of human keratinocytes and fibroblasts expressed VDR and all the cytochrome enzymes necessary for autocrine production of vitamin D. The relative expression of VDR mRNA was higher in dermal fibroblasts than donor-matched keratinocytes. In contrast, epidermal keratinocytes had a higher mRNA expression of vitamin D3 metabolising enzymes. A scratch wound assay confirmed that 1,25(OH)2D3 stimulated keratinocyte migration, but paradoxically inhibited fibroblast migration as early as 4h, yet neither cholecalciferol nor 25-hydroxyvitamin D3 had any effect. VDR knockdown using small interfering RNA (siRNA) abolished the inhibitory effect of 1,25(OH)2D3 on fibroblast migration, demonstrating the requirement for the VDR in this response.
Immunofluorescent staining revealed that 1,25(OH)2D3 increased nuclear VDR protein expression, without a corresponding increase in VDR mRNA transcription only in mechanically wounded dermal fibroblasts, indicating activation of the receptors. Incubation with either 1,25(OH)2D3, cholecalciferol or 25(OH)D3 up-regulated CYP24A1 transcription. This response was most pronounced with 1,25(OH)2D3, suggesting a tightly regulated feedback control on 1,25(OH)2D3 bioavailability within the dermis. In addition, cholecalciferol also increased CYP2R1 and CYP27B1 mRNA expression in scratched dermal fibroblasts, providing evidence for autocrine regulation of 1,25(OH)2D3 by dermal fibroblasts.
Expression of α-SMA protein was up-regulated in cultured dermal fibroblasts following scratching, which was down-regulated in the presence of 1,25(OH)2D3. These observations suggest that 1,25(OH)2D3 may restrict differentiation of wounded dermal fibroblasts into pro-fibrotic myofibroblasts. 1,25(OH)2D3 also down-regulated MMP-2 secretion and collagen type I to III ratio in scratched dermal fibroblasts. Using a human ex vivo wound healing model, it was demonstrated that 1,25(OH)2D3, but not cholecalciferol, stimulated the rate of wound closure.
In summary, this study has confirmed that human dermal fibroblasts express the transcriptional machinery for autocrine production of 1,25(OH)2D3, and a higher VDR expression suggests they are more responsive than keratinocytes. Changes in CYP and VDR expression in the presence of cholecalciferol, 25-hydroxyvitamin D3 or 1,25(OH)2D3 indicate fine-tuning of the bioavailability of vitamin D in the dermis after wounding. Down-regulation of α-SMA, MMP-2 secretion and the collagen type I to III ratio by 1,25(OH)2D3 highlight an important role for 1,25(OH)2D3 in modulating wound healing and the scarring process.
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Al-Aasswad, Naser M. I. "An examination of the bioactive lipids involved in skin cell inflammation and in response to ultraviolet radiation : effect of n-3 polyunsaturated fatty acid supplementation on red blood cell and human dermal fatty acid and production of eicosanoids by HaCaT keratinocytes and 46BR.1N fibroblasts following exposure to UVR." Thesis, University of Bradford, 2013. http://hdl.handle.net/10454/14844.
Full textAbstract:
Ultraviolet radiation (UVR) in solar light is important for skin biology. It is involved in the development acute and chronic skin inflammation, aging and cancer, causing erythema, tanning and local or systemic immunosuppression. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are considered anti- inflammatory and could reduce the damage caused by overexposure to UVR. Although, n-3 PUFA have been considered as photoprotective agents, their exact mechanisms of action is not completely understood. The aim of the work is to determine the effect of UVR and the n-3 PUFA eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) on human skin cells (in vitro study), specifically on: cell viability, apoptosis and their metabolism through the cyclooxygenase and lipoxygenase pathways. Also, to study the cellular incorporation and effect of n-3 PUFA on the fatty acid profile of skin cells. A clinical study was undertaken to assess the incorporation of n-3 PUFA supplements in human skin. A clinical study was performed in 40 healthy women (active group) supplemented with 4g/day of EPA (70%) and DHA (10%) and 40 healthy women (placebo group) supplemented with 4g/day of glyceryl tricoprylate coprate (GTCC). After 3 months, both blood samples and skin punch biopsies were collected and analysed for fatty acids by gas chromatography (GC). HaCaT keratinocytes and 46BR.1N fibroblasts were cultured and treated with 10 and 50μM of either EPA, or DHA or oleic acid (OA) for 72h and exposed to 15 and 50 mJ/cm2. Cell viability was measured by the MTT assay and cell apoptosis by a colorimetric method, at 24h post UVR. Cells and culture media were analysed by GC and liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) to assess cellular fatty acids and production of eicosanoids. The clinical a study showed that in RBC saturated fatty acids (SFA) (44.27±7.43%) were the main fatty acid group followed by n-6 PUFA (29.61±5.53%). While in dermal tissue monounsaturated fatty acids (MUFA) (58.90±9.80%) was the main fatty acid group followed by SFA (27.06±6.78%). A significant increase in EPA, DHA and docosapentaenoic acid (DPA) was observed in RBC but only EPA was significantly increased in the dermis post n-3 PUFA supplementation. . The viability of HaCaT keratinocytes and 46BR.1N fibroblasts decreased post UVR and this was further reduced post PUFA treatment. Cell apoptosis increased when cells were exposed to UVR and further increased when cells were treated with EPA and DHA. . In HaCaT keratinocytes MUFA (54.22±8.82%) was the main fatty acid group followed by FAS (37.11±.9.16%), while SFA (51.94±8.68%) was the main group followed by MUFA (27.07±4.79) in 46BR.1N. Treated both cells with EPA and DHA showed significant increased in cellular EPA, DPA and DHA. 46BR.1N fibroblasts produced higher levels of prostaglandins (PG) compared to HaCaT keratinocytes: PGE2 and PGD2 were the main PG in both HaCaT (7.96±3.18 and 1.48±1.19 pg/million cell; respectively) and 46BR.1N with (44.2±23.00 and 17.1±9.71 pg/million cell; respectively). Significant increase in PGE1 and PGE2 occurred when cells were exposed to 15mJ/cm2 UVR. Treatment with n-3 PUFA decreased the level of PGE1 and PGE2, and increase production PGE3 at the baseline and post UVR. Both cell lines produced hydroxy fatty acids and the concentration of these mediators was higher in 46BR.1N than HaCaT. The concentrations of these mediators were significant increased post UVR: treatment with n-3 PUFA decreased the level of HODE and HETE, and increase production of HEPE and HDHA at baseline and post UVR. Overall, n-3PUFA treatment led to increases in the content of EPA and DHA on RBC, dermal tissue and human skin cell lines. EPA and DHA in skin cell lines appear to offer protection by increasing cellular apoptosis, decreasing inflammatory mediators specifically PGE2 and 12-HETE, and increasing anti-inflammatory mediators such as PGE3, 15-HEPE and 17-HDHA.
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Hill, Rebecca Philomena. "Human dermal fibroblast responses to inflammatory stress." Thesis, University of Sheffield, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422116.
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Maximilien, Jacqueline. "Studies of the impact of core-shell polystyrene nanoparticles on cell membranes and biomimetic models." Thesis, Compiègne, 2015. http://www.theses.fr/2015COMP2180.
Full textAbstract:
L’objectif de ce projet est d’étudier l’interaction de nanoparticules polymères avec les membranes, soit directement sur des cellules entières ou grâce à des modèles membranaires biomimétiques, dans l’optique de valider leur utilisation dans le cadre d’applications biologiques. Des nanoparticules (NPs) polymères cœur/enveloppe avec un diamètre inférieur à 100 nm ont été synthétisés. Cette taille a été choisie afin de leur permettre de pénétrer à travers les membranes plasmiques. Des nanoparticules ayant la même composition chimique mais avec un diamètre hydrodynamique supérieur, de l’ordre de 250 nm, ont été également préparées afin de mettre en évidence l’effet de la taille des particules sur le processus d’internalisation cellulaire. Dans cette thèse, une méthode innovante de synthèse monotope a été développée pour obtenir des NPs coeur-enveloppe, compatibles en milieu aqueux et présentant à leur surface des résidus iniferter. Le coeur est composé de polystyrène avec une taille d’environ 30 nm. Un large éventail de fonctionnalités peut être greffé sur la surface du coeur par polymérisation radicalaire contrôlée en faisant varier différents types de monomères. L’épaisseur de l’enveloppe peut être ajustée en fonction de la concentration en monomère et du temps de polymérisation. Les nanoparticules synthétisées ont été caractérisées par diffusion dynamique de la lumière, par spectroscopie infrarouge à transformée de Fourier, par analyse micro-élémentaire et par microcopie à transmission électronique. Les interactions des NPs à coeur polystyrène et avec des enveloppes de charge neutre et négative ont été étudiées avec des cellules kératinocytes épidermiques humaines néonatales (NHEK), des fibroblastes primaires humains et les cellules HACaT de kératinocytes humains. Les études de cytotoxicité réalisées en utilisant un marquage à l’iodure de propidium et un test à la lactate déshydrogénase n’ont relevé aucune toxicité sur les lignées testées. Cependant, le suivi de la prolifération cellulaire par impédance électrique de substrats cellulaires a indiqué que les nanoparticules anioniques induisent une forte diminution de la prolifération des kératinocytes. L’internalisation cellulaire des NPs a été confirmée par microscopie confocale qui n’indique pas leur colocalisation avec les endosomes précoces, les lysosomes et l’actine. De plus, les données obtenues par triage cellulaire par cytofluorométrie soutiennent qu’un mécanisme énergétiquement-dépendant est mis en œuvre pour l’internalisation des NP neutres, ce qui semble être moins le cas pour les nanoparticules négatives. Les membranes biomimétiques ont été employées afin d’étudier les spécificités des interactions entre nanoparticules et lipides dans des conditions contrôlées. L’étude sur des modèles de vésicules géantes couplée à de la spectroscopie de fluorescence a révélé que les nanoparticules coeur/enveloppe sont capables d’interagir profondément dans la région hydrophobe de la membrane, mais uniquement quand la bicouche lipide est en phase fluide désordonnée. Le mode de pénétration des NPs au travers de la bicouche des vésicules semblent engendrer la formation de pores. Un effet plus prononcé de rigidification de la bicouche a pu être observé lors de l’interaction de nanoparticules chargées négativement avec les bicouches de phosphatidycholines. Cet effet pourrait être attribué à un changement de l’orientation des têtes phosphocholines du à des interactions électrostatiques. En conclusion, les nanoparticules polymère que nous avons synthétisées apparaissent être des outils polyvalents pour les études d’interaction cellulaire et d’imagerie. Ces nanomatériaux peuvent être éventuellement être employés pour la délivrance de médicaments en incorporant les molécules actives dans une enveloppe polymère thermosensible par exempleThis project’s aim was to study polymeric nanoparticle-membrane interactions using both live cells and biomimetic models with the idea to validate such nanoparticles for use in bio-applications. Core-shell polymeric nanoparticles below 100 nm, as this small size is capable of penetrating plasma membranes, were synthesised. Nanoparticles (NPs) with the same chemical composition but with hydrodynamic diameters of ~250 nm, were also prepared in an effort to highlight any effect of NP size on cell internalisation. In this thesis, an innovative method is presented for the synthesis of water-compatible, iniferter-bound polystyrene core shell NPs (~30 nm) using a one-pot synthetic method. A plethora of functionalities could be added to the nanoparticles via shell grafting from the surface of the polystyrene core in the presence of additional monomers via controlled living radical polymerisation. Shell thickness could be tuned as a function of monomer’s concentration and polymerisation time. The nanoparticles were fully characterised by dynamic light scattering, Fourier transform infra-red spectroscopy, microelemental analysis and transmission electron microscopy. Further, the interactions of polystyrene core NPs possessing neutral and anionic shells were investigated using neonatal human epidermal keratinocytes (NHEK), human primary fibroblasts and HaCaT cells. Cytotoxicity studies performed using propidium iodide and lactate dehydrogenase indicated no evidence of cytotoxicity in either cell line. However, cell proliferation monitored by electric cell substrate impedance sensing (ECIS) protocols indicated that anionic nanoparticles induced a dramatic decrease in cell proliferation in keratinocytes. The cellular internalisation of NPs was confirmed by confocal microscopy and no co-localisation was found with early endosomes, lysosomes or actin. Additionally, fluorescence activated cell sorting (FACS) data support the theory that an energy-dependent mechanism is employed for neutral NP internalisation but less so for negatively charged NPs. Biomimetic membrane models were used to investigate specific nanoparticle-lipid interactions under controlled conditions. Employing giant vesicles coupled with fluorescent spectroscopy techniques revealed that core-shell nanoparticles interact deep in the hydrophobic region of bilayers only when the membrane is in the fluid phase. Their mode of entering artificial cells (i.e giant vesicles) appears to cause the formation of pores. Anionic nanoparticles interact with the choline moiety of phosphatidylcholine and confer a rigidifying effect on phosphocholine containing bilayers. Therefore we conclude that the polymeric nanoparticles that we synthesized are versatile tools for cell interaction and imaging studies. These nanomaterials could eventually be applied to drug delivery studies by incorporation of the drug in for instance a thermoresponsive polymeric shell. Furthermore, it is clear that NPs coated with anionic and neutral polymeric shells present a lower toxicity profile than previously reported cationic nanoparticles. Both nanoparticles increase the order lipid bilayer vesicles composed of POPC (the most common glycerophospholipid) in animal and plants. Anionic nanoparticles in particular exhibit a rigidifying effect on POPC lipid bilayers and their mode of entry into cells may be due to the formation of pores which was determined to not induce cell death
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Souto, Luis Ricardo Martinhão. "Modelo de pele humana (derme + epiderme) reconstruida in vitro." [s.n.], 2005. http://repositorio.unicamp.br/jspui/handle/REPOSIP/313309.
Full textAbstract:
Orientador: Maria Beatriz Puzzi, Maria Helena Stangler KraemerDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-04T03:54:34Z (GMT). No. of bitstreams: 1 Souto_LuisRicardoMartinhao_M.pdf: 2402921 bytes, checksum: a79b6ae181ce1b24d01ec608815d8bf7 (MD5) Previous issue date: 2005
Resumo: A obtenção de uma pele humana que apresente derme e epiderme, reconstruída a partir de células isoladas de pacientes, possibilita a realização de enxertos autólogos de pele reconstruída em laboratório (in vitro) em pacientes com áreas doadoras escassas além de permitir ensaios com substâncias químicas e drogas in vitro e não mais in vivo. A partir da cultura de fibroblastos humanos, é possível obter um número suficiente de células que podem ser injetadas em uma matriz de colágeno bovino tipo I que, mantida imersa em meio de cultura, específico para fibroblastos, permite a formação de uma derme humana reconstruída in vitro. Sobre essa derme, através de cultura de queratinócitos e melanócitos humanos, forma-se uma epiderme diferenciada levando à formação de uma pele humana reconstruída in vitro, constituída de derme e epiderme associadas. Essa pele humana formada é, histologicamente, semelhante à pele humana in vivo. Na derme, identifica-se o tecido colágeno, com suas células, e a matriz extracelular organizados paralelamente à epiderme. Esta se desenvolve em várias camadas. Não há distinção entre derme e epiderme no experimento controle, onde não foi utilizado o colágeno bovino tipo I
Abstract: The technique to obtain human skin presenting dermis and epidermis reconstructed from cells isolated from patients allows the performance of autologous grafts of skin reconstructed in laboratory (in vitro) on patients with scarce donor sites, in addition to permitting trials with chemical substances and drugs no more in vivo, but in vitro. It is possible to obtain a sufficient number of cells from human fibroblast culture that can be injected in bovine collagen type I matrix and kept submerged in a specific culture medium for fibroblasts. This will permit the formation of human dermis reconstructed in vitro. On this dermis, through culture of human keratinocytes and melanocytes, a differentiated epidermis is formed, leading to the creation of human skin reconstructed in vitro, composed of associated dermis and epidermis. This human skin is histologically formed in the same way as human skin in vivo. Collagen tissue can be identified in the dermis, with its cells and extracellular matrix organized in parallel to the epidermis, which is developed in several layers
Mestrado
Patologia Clinica
Mestre em Ciências Médicas
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Bain, Peter A., and n/a. "Gene Expression Profiling of Cylindrospermopsin Toxicity." Griffith University. School of Biomolecular and Physical Sciences, 2007. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20080404.145834.
Full textAbstract:
Cylindrospermopsin (CYN) is a toxic alkaloid produced by several freshwater cyanobacterial species, the most prevalent in Australian waters being Cylindrospermopsis raciborskii. The occurrence of CYN-producing cyanobacteria in drinking water sources worldwide poses a potential human health risk, with one well-documented case of human poisoning attributed to the toxin. While extensive characterisation of CYN-induced toxicity has been conducted in rodents both in vivo and in primary cell cultures, little is known about mechanisms of toxicity in human cell types. This thesis describes studies undertaken to further define the molecular mechanisms of CYN toxicity in human cells. Concentration-response relationships were determined in various cultured human cell types using standard toxicity assays. As expected, CYN caused dose-dependent decreases in the growth of three cell lines, HepG2, Caco-2 and HeLa, and one primary cell type, human dermal fibroblasts, according to tetrazolium reduction assays. CYN treatment did not disrupt cellular membranes according to the lactate dehydrogenase release assay in HepG2 or Caco-2 cells after 24, 48 or 72 h exposure, but did cause membrane disruption in fibroblasts after 72 h exposure to relatively high concentrations of the toxin. Apoptosis occurred more readily in HeLa cells than HepG2 cells or fibroblasts, with 72 h exposure to 1 &mug/mL required before statistically significant rates of apoptosis occurred in the latter cell types. CYN did not appear to directly affect the structure of actin filaments or microtubules under the conditions used in the present study. The major portion of the work presented in this thesis comprises a large-scale interrogation of changes in gene expression induced by the toxin in cultured cells. To assess the effects of CYN on global gene expression, relative messenger RNA (mRNA) levels in human dermal fibroblasts and HepG2 cells after 6 h and 24 h exposure to 1 &mug/mL CYN were determined using oligonucleotide microarrays representing approximately 19 000 genes. Overall, the number of transcripts significantly altered in abundance was greater in fibroblasts than in HepG2 cells. In both cell types, mRNA levels for genes related to amino acid biosynthesis, carbohydrate metabolism, and protein folding and transport were reduced after CYN treatment, while transcripts representing genes for apoptosis, RNA biosynthesis and RNA processing increased in abundance. More detailed data analyses revealed the modulation of a number of stress response pathways—genes regulated by NF-&kappaB were induced, DNA damage response pathways were up-regulated, and a large number of genes involved in endoplasmic reticulum stress were strongly down-regulated. Genes for the synthesis and processing of mRNA, tRNA and rRNA were strongly up-regulated, indicating that CYN treatment may increase the turnover of all forms of cellular RNA. A small group of genes were differentially expressed in HepG2 cells and fibroblasts, revealing cell-specific responses to the toxin. Selected changes in transcript level were validated using real-time quantitative reverse transcriptase PCR (qRT-PCR). The modulation of stress response pathways by CYN, indicated by microarray analysis, was further investigated using other methods. The role of tumour suppressor protein p53 in CYN-mediated gene expression was confirmed by measuring the expression of known p53-regulated genes following CYN treatment of HepG2 cells and human dermal fibroblasts using qRT-PCR. Western blotting of protein extracts from CYNtreated cells showed that p53 protein accumulation occurred in HepG2 cells, providing additional evidence of the activation of the p53 pathway by CYN in this cell line. The immediate-early genes JUN and FOS were found to be induced by CYN in a concentration-dependent manner, and MYC was induced to a lesser extent. The mitogen-activated protein kinase c-Jun NH2-terminal kinase, implicated in the ribotoxic stress response initiated by damage to ribosomal RNA, appeared to become phosphorylated in HeLa cells after CYN exposure, suggesting that ribotoxic stress may occur in response to CYN in at least some cell types. The expression of a reporter gene under the control of a response element specific for NF-&kappaB was induced at the mRNA level but inhibited at the protein level. This shows that while transcription factors such as p53 and NF-&kappaB are apparently activated in response to the toxin, transactivation of target genes may not necessarily manifest a corresponding increase at the protein level. The current work contributes significantly to the current understanding of cylindrospermopsin toxicity in human-derived cell types, and provides further insight into putative modes of action.
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Bain, Peter A. "Gene Expression Profiling of Cylindrospermopsin Toxicity." Thesis, Griffith University, 2007. http://hdl.handle.net/10072/367068.
Full textAbstract:
Cylindrospermopsin (CYN) is a toxic alkaloid produced by several freshwater cyanobacterial species, the most prevalent in Australian waters being Cylindrospermopsis raciborskii. The occurrence of CYN-producing cyanobacteria in drinking water sources worldwide poses a potential human health risk, with one well-documented case of human poisoning attributed to the toxin. While extensive characterisation of CYN-induced toxicity has been conducted in rodents both in vivo and in primary cell cultures, little is known about mechanisms of toxicity in human cell types. This thesis describes studies undertaken to further define the molecular mechanisms of CYN toxicity in human cells. Concentration-response relationships were determined in various cultured human cell types using standard toxicity assays. As expected, CYN caused dose-dependent decreases in the growth of three cell lines, HepG2, Caco-2 and HeLa, and one primary cell type, human dermal fibroblasts, according to tetrazolium reduction assays. CYN treatment did not disrupt cellular membranes according to the lactate dehydrogenase release assay in HepG2 or Caco-2 cells after 24, 48 or 72 h exposure, but did cause membrane disruption in fibroblasts after 72 h exposure to relatively high concentrations of the toxin. Apoptosis occurred more readily in HeLa cells than HepG2 cells or fibroblasts, with 72 h exposure to 1 µg/mL required before statistically significant rates of apoptosis occurred in the latter cell types. CYN did not appear to directly affect the structure of actin filaments or microtubules under the conditions used in the present study. The major portion of the work presented in this thesis comprises a large-scale interrogation of changes in gene expression induced by the toxin in cultured cells. To assess the effects of CYN on global gene expression, relative messenger RNA (mRNA) levels in human dermal fibroblasts and HepG2 cells after 6 h and 24 h exposure to 1 µg/mL CYN were determined using oligonucleotide microarrays representing approximately 19 000 genes. Overall, the number of transcripts significantly altered in abundance was greater in fibroblasts than in HepG2 cells. In both cell types, mRNA levels for genes related to amino acid biosynthesis, carbohydrate metabolism, and protein folding and transport were reduced after CYN treatment, while transcripts representing genes for apoptosis, RNA biosynthesis and RNA processing increased in abundance. More detailed data analyses revealed the modulation of a number of stress response pathways—genes regulated by NF-?B were induced, DNA damage response pathways were up-regulated, and a large number of genes involved in endoplasmic reticulum stress were strongly down-regulated. Genes for the synthesis and processing of mRNA, tRNA and rRNA were strongly up-regulated, indicating that CYN treatment may increase the turnover of all forms of cellular RNA. A small group of genes were differentially expressed in HepG2 cells and fibroblasts, revealing cell-specific responses to the toxin. Selected changes in transcript level were validated using real-time quantitative reverse transcriptase PCR (qRT-PCR). The modulation of stress response pathways by CYN, indicated by microarray analysis, was further investigated using other methods. The role of tumour suppressor protein p53 in CYN-mediated gene expression was confirmed by measuring the expression of known p53-regulated genes following CYN treatment of HepG2 cells and human dermal fibroblasts using qRT-PCR. Western blotting of protein extracts from CYNtreated cells showed that p53 protein accumulation occurred in HepG2 cells, providing additional evidence of the activation of the p53 pathway by CYN in this cell line. The immediate-early genes JUN and FOS were found to be induced by CYN in a concentration-dependent manner, and MYC was induced to a lesser extent. The mitogen-activated protein kinase c-Jun NH2-terminal kinase, implicated in the ribotoxic stress response initiated by damage to ribosomal RNA, appeared to become phosphorylated in HeLa cells after CYN exposure, suggesting that ribotoxic stress may occur in response to CYN in at least some cell types. The expression of a reporter gene under the control of a response element specific for NF-?B was induced at the mRNA level but inhibited at the protein level. This shows that while transcription factors such as p53 and NF-?B are apparently activated in response to the toxin, transactivation of target genes may not necessarily manifest a corresponding increase at the protein level. The current work contributes significantly to the current understanding of cylindrospermopsin toxicity in human-derived cell types, and provides further insight into putative modes of action.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Faculty of Science, Environment, Engineering and Technology
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Marí, Buyé Núria. "Design and development of biomimetic surfaces and three-dimensional environments to study cell behavior." Doctoral thesis, Universitat Ramon Llull, 2012. http://hdl.handle.net/10803/81111.
Full textAbstract:
La biomimètica o biomimetisme són termes que simbolitzen el concepte “aprendre de la naturalesa”, és a dir, aprendre dels seus sistemes, processos i models, a fi d’utilitzar la natura com a font d’inspiració per solucionar problemes de l’home. El biomimetisme és actualment un concepte recurrent en l’àrea d’enginyeria de teixits i d’ell en sorgeixen idees per obtenir plataformes més elegants i sofisticades que puguin imitar millor les interacciones entre les cèl•lules i el seu ambient. Aquesta tesi pretén desenvolupar models, en dues i en tres dimensions, mitjançant la recreació d’un o més factors característics de l’ambient natural de la cèl•lula i que juguen un paper important en el comportament cel•lular.
Se sap que tant les propietats químiques com les mecàniques de la matriu extracel•lular influeixen sobre les funcions cel•lulars. És per això que es va dissenyar un nou film polimèric que pogués combinar un hidrogel, amb propietats mecàniques variables, amb un monòmer reactiu capaç d’immobilitzar biomolècules. Degut a la complexitat del polímer dissenyat, va ser necessari recórrer a una tècnica de polimerització superficial molt versàtil com és la deposició química iniciada en fase vapor (més coneguda pel seu acrònim en anglès iCVD). Els polímers varen ser àmpliament caracteritzats i es va corroborar que podien ser modificats amb petites biomolècules com ara pèptids senyalitzadors. Les superfícies resultants són bioactives i permeten l’adhesió de cèl•lules endotelials.
Unes altres superfícies biomimètiques, rellevants en l’àmbit de l’enginyeria de teixits d’os, es varen obtenir a partir d’una hidroxiapatita sintetitzada pel mètode de sol-gel submergint-la en diferents medis fisiològics. La dissolució i posterior reprecipitació dels ions proporcionen una capa d’apatita amb una composició similar a la que es troba in vivo. Els experiments evidencien la importància de partir d’un material relativament soluble. És per això que la hidroxiapatita pura no és capaç d’induir la precipitació d’aquesta apatita biomimètica in vitro. Diversos investigadors han relacionat la capacitat de formar apatita amb la bioactivitat del material, entenent bioactivitat com l’habilitat d’aquests materials de promoure la unió amb l’os.
Per a l’enginyeria de teixits, però, és necessari un ambient tridimensional per tal de generar un teixit artificial. S’ha desenvolupat un nou model basat en l’ús d’un gel molt tou per tal d’obtenir un teixit dur com el de l’os. Malgrat que aquests dos conceptes poden semblar contradictoris, les cèl•lules adquireixen l’habilitat d’allargar-se ràpidament i crear una densa xarxa cel•lular dins d’aquest ambient poc restrictiu des d’un punt de vista mecànic. La consegüent contracció del sistema acaba formant un constructe més petit i resistent. Aquest és un sistema biomimètic ja que promou una gran interacció cel•lular i també la condensació de les cèl•lules, esdeveniments que tenen lloc també durant el desenvolupament de l’os i el cartílag. El model es va caracteritzar extensament amb cèl•lules ostoprogenitores MC3T3-E1 que es diferenciaren amb inducció química. A més a més, es va demostrar que l’ambient tridimensional podia promoure l’expressió espontània de marcadors osteogènics. Degut a les interessants propietats del sistema, el mateix model es va utilitzar per induir la diferenciació condrogènica de fibroblastos dermals humans. Aquests tipus cel•lular no ha estat gaire explorat en l’àmbit de l’enginyeria de teixits, malgrat que ofereix un gran potencial en teràpia regenerativa. Aquest treball proporciona proves de la capacitat condrogènica d’aquestes cèl•lules en el sistema tridimensional prèviament desenvolupat.La biomimètica o biomimetismo son términos que simbolizan el concepto “aprender de la naturaleza”, es decir, aprender de sus sistemas, procesos y modelos, y utilizarlos como fuente de inspiración para solucionar problemas del hombre. El biomimetismo es actualmente un concepto recurrente en el área de ingeniería de tejidos y de este surgen ideas para obtener plataformas más elegantes y sofisticadas que puedan mimetizar mejor las interacciones entre las células y su ambiente. La presente tesis se centra en desarrollar modelos, tanto en dos como en tres dimensiones, mediante la recreación de uno o más factores que caracterizan el ambiente natural de la célula y que tienen su rol importante en el comportamiento celular. Se conoce que tanto las propiedades químicas como mecánicas de la matriz extracelular influyen en las funciones celulares. Debido a esto, se diseñó un nuevo film polimérico que pudiera combinar un hidrogel, con propiedades mecánicas variables, con un monómero reactivo, capaz de inmovilizar biomoléculas. Debido a la complejidad del polímero diseñado, fue necesario recurrir a una técnica de polimerización superficial muy versátil como es la deposición química iniciada en fase vapor (más conocida por su acrónimo en inglés iCVD). Los polímeros fueron ampliamente caracterizados y se corroboró que podían ser modificados con pequeñas biomoléculas como péptidos señalizadores. Las superficies resultantes son bioactivas y permiten la adhesión de células endoteliales. Se obtuvieron otro tipo de superficies biomiméticas relevantes en el ámbito de la ingeniería de tejidos de hueso, a partir de una hidroxiapatita sintetizada por el método sol-gel sumergiéndolas en diferentes medios fisiológicos. La disolución y posterior reprecipitación de los iones proporcionan una capa de apatita con una composición similar a la que se encuentra in vivo. Los experimentos evidencian la importancia de partir de un material relativamente soluble. Precisamente debido a esto la hidroxiapatita pura no es capaz de inducir la precipitación de esta apatita biomimética in vitro. Varios investigadores han relacionado la capacidad de formar apatita con la bioactividad del material, entendiendo bioactividad como la habilidad de estos materiales de promover la unión con el hueso. De todos modos, en ingeniería de tejidos, es necesario un ambiente tridimensional para generar un tejido artificial. Se ha desarrollado un nuevo modelo basado en el uso de un gel blando para obtener tejido duro como el del hueso. Aunque estos conceptos pueden parecer contradictorios, las células adquieren la habilidad de estirarse rápidamente y de formar una densa red celular dentro de este gel tan poco restrictivo desde un punto de vista mecánico. La consiguiente contracción del sistema acaba formando un constructo mucho más pequeño y resistente. Este es un sistema biomimético ya que promueve una gran interacción celular y también la condensación de las células, eventos que también ocurren durante el desarrollo de hueso y cartílago. El modelo se caracterizó extensamente con células osteoprogenitoras MC3T3-E1 que se diferenciaron bajo inducción química. Además, se demostró que el microambiente tridimensional podía promover la expresión espontánea de marcadores osteogénicos. Debido a las interesantes propiedades del sistema, el mismo modelo se usó para inducir la diferenciación condrogénica de fibroblastos dermales humanos. Este tipo celular no ha sido demasiado explorado en ingeniería de tejidos, a pesar de que puede tener un gran potencial en terapia regenerativa. Este trabajo proporciona pruebas de la capacidad condrogénica de estas células en el sistema tridimensional previamente desarrollado.
Biomimetics or biomimicry are terms that imply “learning from nature”, from its systems, processes and models, in order to use nature as inspiration to solve human problems. In tissue engineering, biomimetics is nowadays a recurrent term and a source of ideas to obtain more elegant and sophisticated platforms that could better mimic the interactions between cells and their environment. This thesis is focused on developing models both in two- and three-dimensions by recreation of one or more factors of the cell natural environment that are known to play an important role in cell behavior. Since both the chemical and mechanical properties of the extracellular matrix are known to effectively influence cell function, an innovative polymeric thin film was designed combining a hydrogel with tunable mechanical properties and a reactive molecule, capable to immobilize biomolecules. Due to the complexity of the polymers, a versatile technique such as initiated chemical vapor deposition (iCVD) was required for the synthesis. Extensive characterization revealed that nanostructured hydrogels were obtained and that small biomolecules, such as signaling peptides, could be attached on the surface. The final surfaces are bioactive and support endothelial cell attachment. Relevant biomimetic surfaces for bone tissue engineering could also be obtained from a sol-gel synthesized hydroxyapatite after immersion in different physiological media. The dissolution and posterior reprecipitation of the ions rendered a final apatite layer with a composition similar to that found in vivo. The experiments evidenced the importance of starting from a rather soluble material and, thus, pure hydroxyapatite was not able to promote apatite precipitation in vitro. This capacity has been related to the material bioactivity by many researchers in terms of its ability to bond to bone in tissue engineering applications. However, for tissue engineering a three-dimensional environment is required to build tissue-like constructs. A new model was developed based on the use of a very soft gel to obtain hard tissue. Although the concepts might seem to work in opposite directions, cells gain the ability to rapidly elongate and form a dense cellular network within this unrestrictive environment. Subsequent contraction of the whole system rendered a smaller and stronger final tissue-like construct. This system was considered biomimetic as it promotes high cell-cell interaction and cellular condensation, which are events that occur in bone and cartilage development. This system was extensively characterized with osteoprogenitor MC3T3-E1 cells that could undergo full osteogenic differentiation under chemical induction. More interestingly, the three-dimensional microenvironment was also able to promote by itself spontaneous expression of bone-related markers. Due to the interesting properties of this system, the same model was used to induce chondrogenic differentiation of human dermal fibroblasts. This cell type has been poorly explored for tissue engineering applications, but it might have great potential in future therapeutic platforms. This work provides proof of concept of chondrogenic potential of these cells in this three-dimensional system.
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Carne, Naomi Angharad. "The effect of endoplasmic reticulum and reductive stress on the human dermal fibroblast proteome." Thesis, Durham University, 2018. http://etheses.dur.ac.uk/12794/.
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Dermal fibroblasts are responsible for the secretion of extracellular matrix (ECM) components that support the structural integrity of the skin. Alterations to the ECM have been implicated in many skin diseases including systemic sclerosis and fibrotic disorders, as well as wrinkle formation and wound healing in the aged phenotype. The endoplasmic reticulum (ER) is responsible for the production and quality control of secreted proteins, and perturbations to its correct function could therefore lead to aberrant ECM deposition from dermal fibroblasts. ER stress occurs when homeostasis of this organelle is imbalanced, which can be prompted by the effects of redox agents that disrupt the careful redox balance within the ER lumen. Previous research has often focused on the effects of oxidising agents that lead to oxidative stress within the cells, however little is known about the effects of reductants (and therefore reductive stress). Reductants are present in pollutants, depilatory creams and some cosmetics yet relatively little is known about their potential effects on the skin. This thesis aims to investigate the effect of reductive stress on dermal fibroblasts, looking first at signalling responses and then investigating changes that occur at the proteomic level. In the final chapter a comparison is made between the proteomic response to reductive stress by DTT and oxidative stress by UV-A radiation. The implications of these findings are discussed in the context of fibroblast functions in the skin.
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Wang, Yongliang. "Human dermal fibroblast activation under pulsed electrical stimulation via conductive fabrics : signalling pathways and potential benefit for wound healing." Doctoral thesis, Université Laval, 2015. http://hdl.handle.net/20.500.11794/26437.
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Lors de la cicatrisation, plusieurs types cellulaires dont les kératinocytes et les fibroblastes ainsi que plusieurs facteurs de croissance jouent d’importants rôles. La cicatrisation cutanée peut aussi être activée par des facteurs exogènes, dont la stimulation électrique (SE). La SE peut moduler les fonctions fibroblastiques durant la cicatrisation. Le fibroblaste contribue de façon active à la cicatrisation en sécrétant différentes protéines (collagène, fibronectine, élastine) pour favoriser le comblement tissulaire. Les fibroblastes adoptent aussi un phénotype contractile en exprimant l’α-actine contribuant à la fermeture de la plaie. Notre hypothèse est que certaines de ces fonctions fibroblastiques pourraient être modulées par une stimulation électrique. Pour vérifier cette hypothèse nous avons utilisé une membrane biocompatible et conductrice à base de polyethylene terephthalate (PET) recouvert de polypyrrole (PPy). Les fibroblastes dermiques humains ont été cultivés sur ces membranes conducteurs, puis exposés ou non à un courant pulsé (PES) selon deux régimes : soit 10s PES suivi de 1200s de repos, ou 300s PES suivi de 600s de repos, durant 24 h. Deux intensités électriques ont été étudiées, 50 et 100 mV/mm. Nos travaux démontrent que la SE favorise l’adhésion, la prolifération et la migration des fibroblastes dermiques. Ces activités cellulaires sont consolidées par une sécrétion importante de FGF2 et d’α-SMA. Il est important de noter que l’effet de la SE favorise le changement phénotypique des fibroblastes en myo-fibroblastes grâce à la voie des Smad et de TGFβ/ERK. Nous avons aussi démontré que l’effet de la SE est maintenue à long terme et est transférable de la cellule mère vers les cellules filles. En effet après sous-culture les cellules expriment toujours de façon importante l’α-SMA. En conclusion, nous avons démontré que la stimulation électrique pulsée module positivement les fonctions cicatricielles des fibroblastes humains. Ces travaux démontrent pour la première fois les voies de signalisation (Smad et TGFβ/ERK) sollicitées par la SE pour activer les fibroblastes lors de la cicatrisation. Ces travaux suggèrent l’utilisation de la SE pour favoriser la guérison/cicatrisation des plaies.During skin wound healing, cutaneous cells particularly fibroblasts and keratinocytes as well as several growth factors play important roles. Wound healing can be activated by exogenous factors, including electrical stimulation (ES). ES can also modulate fibroblast functions. Fibroblasts contribute to healing by secreting structural proteins (collagen, fibronectin, elastin) to repair the wound area. Fibroblasts also adopt a contractile phenotype expressing α-actin contributing to wound closure. The hypothesis of the thesis is that fibroblasts proliferate and transdifferentiate into myofibroblasts by sensing pulsed electrical signals and adjusting relevant signalling pathways. To test this hypothesis we used biocompatible polyethylene terephthalate (PET) fabrics coated with electrically conductive polypyrrole (PPy). Human dermal fibroblasts were cultured on these conductive fabrics and exposed to the optimized pulsed ES: either 10s PES in a period of 1200s, or 300s PES in 600s period, for a total of 24 hours. Two electric intensities were studied, 50 and 100 mV/ mm. Our work showed that the PES promoted the adhesion, proliferation and migration of dermal fibroblasts. These cellular activities were consolidated by an elevated level of fibroblast growth factor 2 (FGF2) and the high expression of α-smooth muscle actin (α-SMA). Important findings were that PES promoted the phenotypic change of fibroblasts to myofibroblasts, and such change was coordinated through the Smad and TGFβ/ERK pathways. It also demonstrated that the effect of PES was able to maintain for a long period of time after the end of stimulation, and was transferable from the mother cells to the daughter cells. Following subculture, the electrically stimulated fibroblasts still expressed significant amount of α-SMA. In conclusion, this thesis demonstrates that PES through conductive fabrics can activate the wound healing functions in human dermal fibroblasts. This work revealed for the first time that Smad and TGFβ/ERK pathways are required by the PES-induced fibroblasts-to-myofibroblasts differentiation. This work also demonstrated that the PES activated cells can survive in vivo. These studies suggest the application of the PES in promoting tissue regeneration and wound healing.
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Groult, Vanina. "Interactions des fibroblastes de derme humain en culture avec l'élastine." Paris 12, 1992. http://www.theses.fr/1992PA120013.
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Nous nous sommes intéressés aux interactions des fibroblastes de derme humain avec l'élastine. Dans un premier temps, nous avons etudié l'interaction des cellules avec l'élastine fibreuse. Cette interaction nécessite une synthèse et une sécrétion de protéines. Dans une seconde partie, nous avons etudié la fixation cellulaire des peptides d'élastine: ils se fixent à une protéine de 67 kDa différente de l'ABS, tout comme la laminine. La dernière partie de notre travail a consisté en l'étude de la stimulation par les peptides d'élastine de l'adhésion de l' élastine fibreuse. Le PDGF, le fMLP et la laminine exercent la même action.
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Zague, Vivian. "Influência da suplementação com colágeno hidrolisado no metabolismo da matriz extracelular e proliferação de fibroblastos dérmicos humanos derivados de áreas fotoprotegida e fotoexposta, cultivados em monocamada e equivalente dérmico." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/42/42134/tde-08122015-202409/.
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Este trabalho investigou, pela primeira vez, a influência do CH na modulação do metabolismo e proliferação de fibroblastos dérmicos humanos (FDHs) derivados de áreas fotoprotegida e fotoexposta, cultivados em modelo de monocamada. Além disto, foram investigados os efeitos da suplementação com CH na secreção de colágeno tipo I, em modelo de cultura 3D de equivalente dérmico, derivado de matriz produzida exclusivamente por FDHs. O tratamento com CH não influenciou a proliferação celular dos fibroblastos derivados de ambas as áreas, porém modulou expressivamente o metabolismo dos FDHs cultivados em monocamada, elevando o conteúdo de pró-colágeno I e colágeno I e diminuindo a atividade de metaloproteinases de matriz (MMP) 1 e 2. Concentrações menores de CH foram suficientes para estimular as células de área fotoexposta, sugerindo efeitos mais pronunciados do CH nestas células. Este estudo é uma contribuição importante para compreensão dos efeitos biológicos do CH nas células da pele e viabilidade do seu uso como ingrediente funcional de suplementos alimentares.This study investigated, for the first time, the influence of CH on the extracellular matrix metabolism and proliferation of human dermal fibroblasts (HDFs) derived from sun-protected and sun-exposed body sites, cultured in monolayer in vitro model. Moreover, CH effects on the secretion of type I collagen were investigated in dermal equivalent 3D model derived from dermal matrix produced exclusively by HDFs. CH treatment did not affect cellular proliferation of either cell cultures, but notably modulated cell metabolism in monolayer model, increasing the content of procollagen I and collagen I and decreasing metalloproteinase activity (MMP) 1 and 2. These effects were confirmed in the human dermal equivalent model. Lower concentrations of CH were enough to stimulate sun-exposed-derived HDFs, suggesting more pronounced effect in these cells. This study presents an important contribution to understanding the biological effects of CH in skin cells and viability of its use as a functional ingredient in food supplements.
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El-Sheemy, Mohamed Adbo. "The effects of extracts from the human dermis on the ability of the human fibroblasts to cause contraction in vitro." Thesis, University of Aberdeen, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323397.
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The starting point of this investigation was the clinical observation that raw areas of the body resurfaced by split-thickness skin grafts contract markedly, whereas those covered by thick grafts (e.g. Wolfe grafts) contract little or not at all. The reason for this behaviour is unknown. The present work was an attempt to investigate this behaviour using fibroblasts cultured in a hydrated collagen lattice (FHCL) as a laboratory model for wound contraction in vivo. Sequential extracts of normal human dermis were prepared in water, 0.15M sodium chloride, 1M sodium chloride, 0.15M citrate buffer (pH 3.5) and 6M urea, and their effects on FHCL contraction were examined. Only citrate buffer dermal extract had a marked inhibitory effect on FHCL contraction. The effect was reversible, concentration-dependent and lasted throughout the course of the 96 hours' experiments. There was no toxic effect on the cells although their proliferation within the lattices was also inhibited. Furthermore the extracts from the deeper parts of the dermis have more inhibitory effect on the FHCL contraction than those from the more superficial layers. Preliminary attempts to characterise the citrate buffer dermal extract biochemically showed the presence of protein and proteoglycans or glycosaminoglycans with a range of molecular weights showed by gel electrophoresis, and the presence of collagen was excluded by amino acid analysis. This study shows that there is a biochemical factor (factors) present in the dermis which inhibits the ability of fibroblasts to cause lattice contraction. This factor (s) can be extracted by citrate buffer. The inhibitory effect of the citrate buffer dermal extract on lattice contraction is due to inhibition of both fibroblasts proliferation and migration.
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Tupet-Defrance, Armelle. "Modulation de la fonctionnalité des integrines par les rayonnements ultraviolets A dans les fibroblastes du derme humain." Paris 7, 1999. http://www.theses.fr/1999PA077240.
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Grandemange, Stéphanie. "Bases moléculaires de l'induction de la transformation des fibroblastes du derme humain après surexpression du récepteur mitonchondrial de la triiodothyronine." Montpellier 2, 2005. http://www.theses.fr/2005MON20086.
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Renaud-Salis, Valérie. "Alterations fonctionnelles induites par irradiation gamma a faibles doses du fibroblaste de derme humain, au cours du vieillissement in vitro (collagene, fibronectine, metalloproteinases, metalloendopeptidase)." Paris 11, 1991. http://www.theses.fr/1991PA112264.
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L'irradiation gamma aigue, therapeutique ou accidentelle, favorise l'evolution d'une fibrose cicatricielle sous-cutanee. Des travaux effectues in vivo chez le porc ont souleve deux questions: a) des fibroblastes de derme faiblement irradies peuvent-ils etre impliques dans l'initiation d'une reponse fibrotique? b) des facteurs de croissance comme le tgf sont-ils capables de moduler le phenotype collagenique des fibroblastes irradies? dans ce but, nous avons irradie a des doses faibles des fibroblastes de peau humaine et nous avons etudie jusqu'au stade de senescence tardive l'evolution de leurs expressions en collagene, fibronectine et metalloproteinases. L'influence du tgf a ete egalement testee sur l'expression de ces phenotypes. Les resultats montrent, d'une part qu'il existe quatre types de remaniements chromosomiques majeurs sans selection clonale induite in vitro pour ces doses d'irradiation, et d'autre part une expression de type fibrosant qui apparait au cours de la phase de senescence du fibroblaste irradie. Cette expression est caracterisee par: a) une augmentation specifique et dose dependante, de la quantite et de la biosynthese du collagene, en particulier de l'isotype trois, b) une moindre mais specifique augmentation de la synthese de fibronectine, c) une diminution de l'activite des metalloproteinases degradant les composants macromoleculaires de la matrice extracellulaire. L'adjonction de tgf dans les cultures de fibroblastes montre que les fibroblastes irradies sont sensibilises a l'action des facteurs de l'environnement susceptibles de moduler une expression de type fibrosant. Ces resultats suggerent donc, que l'irradiation seule puisse directement initier l'apparition d'un phenotype fibrosant par le fibroblaste. L'expression d'un tel phenotype in vivo, serait modulee par des mediateurs de l'inflammation tels que le tgf et aboutirait alors a la constitution finale d'une fibrose tissulaire radio-induite
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Kiezel-Tsugunova, Magdalena. "Elucidating the metabolism of n-3 polyunsaturated fatty acids and formation of bioactive lipid mediators in human skin." Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/elucidating-the-metabolism-of-n3-polyunsaturated-fatty-acids-and-formation-of-bioactive-lipid-mediators-in-human-skin(773abedd-c726-4dab-890a-694a96b1c074).html.
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Human skin has distinct lipid metabolism and production of bioactive lipid mediators that can be modulated by nutritional supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFA), of which eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids exert anti-inflammatory effects. The aims of this project were to gain better understanding of their individual mechanisms in human epidermis and dermis. HaCaT keratinocytes, 46BR.1N fibroblasts, primary human epidermal keratinocytes and dermal fibroblasts were treated with EPA or DHA for 72h and then sham-irradiated or exposed to 15 mJ/cm2 ultraviolet radiation (UVR). Viability was measured by the MTT assay. The expression of cyclooxygenase-2 (COX-2), microsomal prostaglandin synthase-1 (mPGES-1) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) proteins was explored by western blotting. Human skin explants (n=4 donors) were cultured for 3 or 6 days and supplemented with EPA, DHA or vehicle. Culture media were collected to evaluate tissue damage and PUFA cytotoxicity (lactate dehydrogenase assay). Epidermal and dermal lipid profiles were assessed by gas chromatography and liquid chromatography coupled to tandem mass spectrometry. Primary keratinocytes were treated with fatty acids and various lipid mediators for 48h. Their effect was determined by the scratch assay and transepithelial electrical resistance. UVR upregulated COX-2 in HaCaT and primary epidermal keratinocytes, but did not affect mPGES-1 and 15-PGDH protein expression. UVR upregulated COX-2 and mPGES-1 in 46BR.1N fibroblasts but had no effect on 15-PGDH expression. The same UVR dose did not alter the expression of COX-2, mPGES-1 and 15-PGDH in primary dermal fibroblasts. Only EPA attenuated COX-2 expression in HaCaT and primary keratinocytes and either EPA or DHA had any effect in 46BR.1N and primary fibroblasts. Skin explants showed initial post-biopsy tissue damage. EPA and DHA supplementation augmented cellular levels of the corresponding fatty acids in both epidermis and dermis to a different extent. Increased uptake of DHA in the dermis was accompanied by reduced arachidonic acid levels. EPA treatment stimulated the production of PGE3 and various HEPE in epidermis, while DHA treatment caused high levels of HDHA species in dermis. N-3 PUFA and their derivatives delayed wound healing, cell migration and epidermal barrier permeability, while n-6 PUFA lipids showed the opposite effect. Overall, these findings suggest that EPA and DHA differently affect skin cells and skin, with EPA preference in epidermis and DHA in the dermis. These results highlight the importance of differential skin responses that could be important in skin health and disease.