Мотиваційний аспект діяльності учителя при вивченні фізики в школі

(uk) У статті розглянуті проблеми активізації пізнавальної діяльності учнів при вивченні фізики в середній школі.(en) The problems of students knowledge activity while learning physics in secondary school are reviled in the article

UVA/B induced redox alterations and apoptosis in human melanocytes

Malignant melanoma is one of the most rapidly increasing cancers and accounts for about three-quarter of all skin cancer deaths worldwide. Despite compelling evidence that ultraviolet (UV) irradiation causes melanoma the knowledge how various wavelength spectra affect the balance between proliferation and apoptosis controlling the homeostasis of the melanocyte population is still limited. The aim of this thesis was to elucidate the regulation of UVA/B induced apoptotic signaling in human epidermal melanocytes in vitro in relation to redox alterations and antioxidant photoprotection. UVA irradiation induced changes in plasma membrane stability, decreased cell proliferation and increased apoptosis. In comparison, melanocyte plasma membrane was markedly resistant to UVB irradiation although apoptosis was triggered. Thus, UVA irradiation should not be overlooked as an etiologic factor in melanoma development. Further, after irradiation with UVA/B we found alterations in redox state manifested by a reduction of intracellular GSH levels, translocation of nuclear factor-κB from the cytosol to the nucleus, an increase of γ-glutamylcysteine synthetase, the rate-limiting enzyme in GSH synthesis, and an increased apoptosis frequency. α-Tocopherol provided photoprotection through several modes of action affecting redox alterations and signaling, stabilizing the plasma membrane, and decreased proliferation and apoptosis rate, while β-carotene did not show the same protective capacity. Altogether, α-tocopherol might be a useful substance in protecting melanocytes from UV induced damage. We demonstrate UVA/B irradiation to activate the intrinsic pathway of apoptosis in melanocytes where translocation of Bcl-2 family proteins to the mitochondria modulates the apoptosis signal. Interestingly, the anti-apoptotic Bcl-2 family proteins generally thought to be attached to membranes, were localized in the cytosol before UV irradiation and translocated to the mitochondria in the surviving population, which might be a critical event in preventing apoptotic cell death. Lysosomal cathepsins were released to the cytosol acting as pro-apoptotic mediators upstream of activation and translocation of Bax to the mitochondria. When melanocytes were exposed to UVA, p53 participated in apoptosis regulation through interaction with Bcl-2 family proteins, while UVB induced p53-transcriptional activity and apoptosis involving lysosomal membrane permeabilization. Thus, depending on the UV wavelength p53 mediated apoptosis in melanocytes by transcriptional dependent or independent activity. These results emphasize p53 as an important pro-apoptotic component in the regulation of apoptosis. This thesis gives new insight in the harmful and various effects of different wavelengths within the UV spectrum on human melanocytes in vitro. Improved knowledge of the apoptosis regulatory systems in melanocytes might lead to a better understanding of the formation of pigment nevi and malignant melanoma and, in the future, provide better strategies to prevent and eliminate tumor development and progression

Linköping University Medical Dissertations

UVA/B induced redox alterations and apoptosis in human melanocyte

apoptosis in UVA-irradiated keratinocytes

Lysosomal exocytosis and caspase-8-mediate

Redox-Dependent Translocation of p53 to Mitochondria or Nucleus in Human Melanocytes after UVA- and UVB-Induced Apoptosis

The p53 protein is an important transcription factor and tumor suppressor that is induced in response to many forms of cellular stress. UVA irradiation of human melanocytes caused generation of reactive oxygen species, which altered the intracellular redox balance and was accompanied by translocation of p53 to mitochondria. In contrast, UVB did not affect the redox status and p53 was translocated to the nucleus. Although different intracellular location of p53, UVA/B induced apoptosis through the intrinsic pathway detected as translocation of Bax to mitochondria, release of cytochrome c, and activation of caspases. These events were all prevented by inhibition of p53 with pifithrin-α. Furthermore, inhibition of p53 prevented lysosomal membrane permeabilization, detected as translocation of cathepsins to the cytosol, after UVB exposure, whereas UVA-induced lysosomal release was unaffected by inhibition of p53. In control cells, p53 coimmunoprecipitated with the antiapoptotic proteins Bcl-2 and Bcl-xL and upon UVA exposure the interaction was replaced by binding to the proapoptotic proteins Bax, Noxa, and Puma. Our findings suggest that UVA-induced apoptosis is caused by extensive oxidative damage leading to p53-regulated mitochondrial release, whereas UVB induces DNA damage and apoptosis signaling upstream of lysosomal membrane permeabilization

Cell fate regulated by nuclear factor-κB- and activator protein-1-dependent signalling in human melanocytes exposed to ultraviolet A and ultraviolet B.

BACKGROUND: Ultraviolet (UV) radiation constitutes an important risk factor for malignant melanoma, but the wavelength responsible for the initiation of this disease is not fully elucidated. Solar UV induces multiple signalling pathways that are critical for initiation of apoptotic cell death as a cellular defence against malignant transformation. OBJECTIVES: To evaluate the involvement of the transcription factors nuclear factor (NF)-κB and activator protein (AP)-1 in the signalling pathways induced by UVA or UVB irradiation in human melanocytes. METHODS: Primary cultures of normal human melanocytes were irradiated with UVA or UVB, and the concomitant DNA damage and redox alterations were monitored. The resulting activation of the NF-κB and AP-1 signalling pathways and subsequent apoptosis were studied. RESULTS: UVB irradiation causes DNA damage detected as formation of cyclobutane pyrimidine dimers, while UVA induces increased levels of 8-hydroxydeoxyguanosine and lipid peroxidation. UVA and UVB initiate phosphorylation of c-Jun N-terminal protein kinase and extracellular signal-regulated kinase, and the apoptosis signalling pathways converge into a common mechanism. Downregulation of c-Jun suppresses AP-1-mediated signalling and prevents apoptosis upstream of lysosomal and mitochondrial membrane permeabilization, whereas inhibition of NF-κB by SN50 increases apoptosis. CONCLUSIONS: We conclude that AP-1 induces proapoptotic signalling, whereas NF-κB is a key antiapoptotic/prosurvival factor in both UVA- and UVB-induced cellular damage in human melanocytes, which might in turn impact melanoma development and progression.Funding text:This study was supported by the Swedish Research Council, the Swedish Cancer Society, the County Council of Ostergotland, Konung Gustav V och Drottning Victorias Frimurarestiftelse and the Welander-Finsen Foundation.</p

Sunbathing : What’ve lysosomes got to do with it?

Solar radiation is an important risk factor for skin cancer, the incidence of which is increasing, especially in the fair-skinned populations of the world. While the ultraviolet (UV)B component has direct DNA damaging ability, UVA-induced effects are currently mainly attributed to the production of reactive oxygen species. In our recent study, we compared the effects of UVA and UVB radiation on human keratinocytes and found that UVA-induced plasma membrane damage was rapidly repaired by lysosomal exocytosis, which was detected based on the expression of lysosomal membrane associated protein-1 (LAMP-1) on the plasma membrane of non-permeabilized cells. Later, the keratinocytes died through caspase-8 mediated apoptosis. In contrast, the plasma membranes of keratinocytes exposed to UVB showed no LAMP-1 expression, and, although the cells died by apoptosis, no initial caspase-8 activity was detected. We have also demonstrated the occurrence of UVA-induced lysosomal exocytosis in reconstructed skin and shown the relocation of lysosomes from the center of cells to the vicinity of the plasma membrane. Thus, we suggest that lysosomal exocytosis also occurs in keratinocytes covered by the stratum corneum following exposure to UVA. Our findings provide new insight into the mechanism of UVA-induced skin damage.Article Addendum to: H Appelqvist, P Waster, I Eriksson, I Rosdahl, K Ollinger. Lysosomal exocytosis and caspase-8-mediated apoptosis in UVA-irradiated keratinocytes. Journal of Cell Science 2013; 126: 5578- 5584. DOI: 10.1242/jcs.130633</p

Ultraviolet exposure of melanoma cells induces fibroblast activation protein-alpha in fibroblasts: Implications for melanoma invasion

Fibroblast activation protein-alpha (FAP-alpha) promotes tumor growth and cell invasiveness through extracellular matrix degradation. How ultraviolet radiation (UVR), the major risk factor for malignant melanoma, influences the expression of FAP-alpha is unknown. We examined the effect of UVR on FAP-alpha expression in melanocytes, keratinocytes and fibroblasts from the skin and in melanoma cells. UVR induces upregulation of FAP-alpha in fibroblasts, melanocytes and primary melanoma cells (PM) whereas keratinocytes and metastatic melanoma cells remained FAP-alpha negative. UVA and UVB stimulated FAP-alpha-driven migration and invasion in fibroblasts, melanocytes and PM. In co-culture systems UVR of melanocytes, PM and cells from regional metastases upregulated FAP-alpha in fibroblasts but only supernatants from non-irradiated PM were able to induce FAP-alpha in fibroblasts. Further, UV-radiated melanocytes and PM significantly increased FAP-alpha expression in fibroblasts through secretory crosstalk via Wnt5a, PDGF-BB and TGF-beta 1. Moreover, UV radiated melanocytes and PM increased collagen I invasion and migration of fibroblasts. The FAP-alpha/DPPIV inhibitor Gly-ProP(OPh)(2) significantly decreased this response implicating FAP-alpha/DPPIV as an important protein complex in cell migration and invasion. These experiments suggest a functional association between UVR and FAP-alpha expression in fibroblasts, melanocytes and melanoma cells implicating that UVR of malignant melanoma converts fibroblasts into FAP-alpha expressing and ECM degrading fibroblasts thus facilitating invasion and migration. The secretory crosstalk between melanoma and tumor surrounding fibroblasts is mediated via PDGF-BB, TGF-beta 1 and Wnt5a and these factors should be evaluated as targets to reduce FAP-alpha activity and prevent early melanoma dissemination

Extracellular vesicles are transferred from melanocytes to keratinocytes after UVA irradiation

Ultraviolet (UV) irradiation induces skin pigmentation, which relies on the intercellular crosstalk of melanin between melanocytes to keratinocytes. However, studying the separate effects of UVA and UVB irradiation reveals differences in cellular response. Herein, we show an immediate shedding of extracellular vesicles (EVs) from the plasma membrane when exposing human melanocytes to UVA, but not UVB. The EV-shedding is preceded by UVA-induced plasma membrane damage, which is rapidly repaired by Ca2+-dependent lysosomal exocytosis. Using co-cultures of melanocytes and keratinocytes, we show that EVs are preferably endocytosed by keratinocytes. Importantly, EV-formation is prevented by the inhibition of exocytosis and increased lysosomal pH but is not affected by actin and microtubule inhibitors. Melanosome transfer from melanocytes to keratinocytes is equally stimulated by UVA and UVB and depends on a functional cytoskeleton. In conclusion, we show a novel cell response after UVA irradiation, resulting in transfer of lysosome-derived EVs from melanocytes to keratinocytes.Funding Agencies|Swedish Research Council; Swedish Cancer Society; Welander-Finsen Foundation; County Council of Ostergotland; Stiftelsen Olle Engkvist Byggmastare; Konung Gustav V och Drottning Victorias Frimurarestiftelse; Ostgotaregionens Cancerfond</p

Lysosomal exocytosis and caspase-8-mediated apoptosis in UVA-irradiated keratinocytes

Ultraviolet (UV) irradiation is a major environmental carcinogen involved in the development of skin cancer. To elucidate the initial signaling during UV-induced damage in human keratinocytes, we investigated lysosomal exocytosis and apoptosis induction. UVA, but not UVB, induced plasma membrane damage, which was repaired by Ca2+-dependent lysosomal exocytosis. The lysosomal exocytosis resulted in extracellular release of cathepsin D and acid sphingomyelinase (aSMase). Two hours after UVA irradiation, we detected activation of caspase-8, which was reduced by addition of anti-aSMAse. Furthermore, caspase-8 activation and apoptosis was reduced by prevention of endocytosis and by the use of cathepsin inhibitors. We conclude that lysosomal exocytosis is part of the keratinocyte response to UVA and is followed by cathepsin-dependent activation of caspase-8. The findings have implications for the understanding of UV-induced skin damage and emphasize that UVA and UVB initiate apoptosis through different signaling pathways in keratinocytes.The previous status of this article was manuscript with the title Lysosomal exocytosis repairs the plasma membrane after UVA and is followed by caspase-8 induced apoptosis.</p