M. Tech. / Stem cells are regarded as undifferentiated cells that are capable of selfrenewal, proliferation, production of a great number of differentiated progeny, and regeneration of tissues (Blau et al., 2001). The therapeutic potential of multilineage stem cells for tissue engineering (TE) applications is vast. Two general types of stem cells are potentially useful for this application: embryonic stem cells (ESCs) and adult (autologous) stem cells (Zuk et al., 2001). Traditionally, ESCs are isolated from the inner cell mass (ICM) of blastocysts, however harvesting of these cells results in the death of the embryo, which has led to ethical, religious and political issues (Moore, 2007). In contrast, adult stem cells, by virtue of their nature, are immunocompatible and have no ethical issues associated with their use (Zuk et al., 2001). Subcutaneous adipose tissue is an active and highly complex tissue composed of several different cell types, and is derived from the mesodermal germ layer and contains a supportive stromal vascular fraction (SVF) that can be easily isolated. This SVF contains a heterogeneous mixture of cells including preadipocytes (Raposio et al., 2007; Schäffler and Büchler, 2007; Jurgens et al., 2008). The preadipoctyes are considered as the multipotent stem cells termed adipose derived stem cells (ADSCs) that have similar properties to bone marrow mesenchymal stem cells (BM-MSCs) (Fraser et al., 2006). ADSCs are idyllic for cellular therapy applications due to various factors: they can be harvested, multiplied and handled easily, efficiently and noninvasively, they have a pluripotential and proliferative capacity comparable to BM-MSCs, and morbidity to donors is considerably less, requiring only local anaesthesia and a short wound healing time. Human ADSCs (hADSCs) can be expanded in an undifferentiated state and have multipotential differentiation capacity along the classical mesenchymal lineages of adipogenesis, osteogenesis, chondrogenesis and myogenesis (de Villiers et al., 2009).

O estudo da distribuição e propagação a luz em tecidos biológicos é importante para diversas aplicações em fototerapias e diagnóstico. Os tecidos biológicos são considerados meios túrbidos, onde existe uma combinação de absorção e espalhamento, e a determinação do comportamento da luz dentro deles tem sido estudado através da aplicação de modelos que tem suas limitações. Então, determinar e manipular a distribuição para entrega da melhor dose de luz é crucial para o sucesso dos tratamentos. Para casos de Terapia Fotodinâmica, em particular, os tratamentos de lesões planas lisas, em grande parte dos casos, são bem sucedidos. Este sucesso deve-se ao fato de que existe uma boa possibilidade de distribuição uniforme da luz dentro do tecido da lesão tratada. Por outro lado, para tumores espessos de superfícies irregulares, a iluminação é dificultada devido à presença de sombras, rachaduras entre outras imperfeições sobre a lesão. Deste modo, a entrega da dose de luz inadequada e a iluminação não homogênea, resultam em regiões de necroses parciais e recidiva. Portanto, é decisivo melhorar o perfil da distribuição dentro do tecido. Neste trabalho avaliamos o acoplamento da luz utilizando um gel posicionado entre a fonte ( &#955= 630 nm) e a superfície de um phantom sólido. O acoplamento do feixe foi melhorado incorporando baixas concentrações de espalhadores ao gel. Imagens do perfil de distribuição da luz foram coletadas e transformadas em matrizes de intensidades, e posteriores curvas de isointensidades. Nossos resultados mostraram uma grande melhora na uniformidade da distribuição. De fato, utilizando um meio acoplador, conseguimos entregar um feixe mais homogêneo promovendo uma melhor iluminação. Esta técnica elimina drásticamente os efeitos da rugosidade da superfície do phantom dentro do volume tratado. Acreditamos que esse trabalho mostra uma possibilidade de melhora na dosimetria para fototerapias. / The light propagation and distribution studies in biological media are importante for several phototherapy and diagmostic aplications. The biological tissues are considered turbida medias, where there is absortion and scattering combination, and determinate the light behavior inside this tissues have been studied by models that has some limitations.Then, to determine and control the light distribution to improve the light dose delivery is important for the treatment successful. For particular cases, such as Photodynamic Therapy (PDT) applied on smooth planar lesions treatments is appropriated, since there is a good possibility of uniform distribution of light within the tissue of the treated lesion. On the other hand, tumors with more thickness may not receive the needed doses of energy to cause its death. For an efficient treatment by photodynamic therapy is required an optimal coupling light inside the lesion tissue. Shadow effects, slits or physical irregularities in the lesion can lead a nonhomogeneous light distribution inside the tissue. The results can be a partial necrosis regions and tumor recurrence. Therefore it is crucial improving the light profile inside the tissue to overcome these problems. In this study, we measured the light profile inside the phantom after the light passing through a gel as coupler. We used a solid phantom as biological tissue model and was used a red laser (&#955 = 630 nm) as light source with an optical fiber to direct illumination. The coupling is controlled by introducing a gel with low concentration of scatters between the fiber and de phantom. Was collected pictures of light profile with a camera and the data were processed with MatLab software. Our results shows a strong improvement in the light distribution when the gel with scatters is positioned between fiber and tissue. In fact, a more homogeneous laser bean is delivered to tissue promoting a better light distribution. This technique eliminates drastically the roughness effect of the phantom surface in the bulk. We believe that this work shows a possibility of dosimetry improvement for phototherapy.

Orientador: Josimeri Hebling / Banca: Pedro Paulo Chaves de Souza / Banca: Adriano Fonseca de Lima / Resumo: Objetivo: Avaliar o efeito do LED infravermelho (855 nm), aplicado em diferentes doses de energia, na produção e expressão de mediadores inflamatórios por células pulpares humanas (HDPC) em cultura. Metodologia: HDPC obtidas de dentes decíduos foram semeadas (105 células/well) e após 24 h foram colocadas em contato com LPS (10 μg/mL α-MEM) ou TNF-α (25 ng/mL α-MEM). Em seguida, as células foram submetidas a uma única irradiação com LED infravermelho (855 nm) nas diferentes doses de energia: 2, 4, 8, 15 ou 30 J/cm2. Gupos não irradiados, com e sem LPS/TNF-α, serviram como controles. Decorridas 24 h, as células estimuladas com LPS foram avaliadas quanto à produção de óxido nítrico (NO), de espécies reativas de oxigênio (EROs) e viabilidade celular (ensaio de MTT) (n=8 por grupo). As células estimuladas com TNF-α foram avaliadas quanto à produção de citocinas pelos métodos de antibody array e q-PCR (n=5 por grupo). Os dados foram submetidos aos testes de Kruskal-Wallis e Mann-Whitney (α=0,05). Resultados: O contato com LPS resultou em aumento significativo da produção de NO sem causar qualquer dano ao metabolismo respiratório das células. Independentemente da dose de energia aplicada, a produção de NO foi significativamente reduzida quando as células foram irradiadas. O melhor efeito foi observado para a dose de 15 J/cm2. A irradiação também promoveu uma diminuição na produção de EROs, enquanto o metabolismo das HDPC não foi alterado. Nas células em contato com TNF-α verificou-se que as citocinas mais produzidas foram: GROα, IL-6, IL-8, TNF-α, MCP-1 e Serpin E1. Na análise por q-PCR redução foi observada para a expressão de IL-1 β, quando as células foram irradiadas com 4 J/cm2. Conclusão: O estresse oxidativo de HDPC pode ser biomodulado por uma única dose de irradiação com LED infravermelho. Quanto a expressão de citocinas inflamatórias... / Abstract: Aim: To investigate the effect of infrared light emitting diode (LED) irradiation (855nm), at different energy doses, on the production and expression of inflammatory mediators by cultured human dental pulp cells (HDPC). Methodology: HDPC were harvested from sound, near-exfoliation primary teeth. Cells were seeded (105 cells/well) using α-MEM supplemented with 10% FBS and after 24h, were exposed to LPS (10 μg/mL α-MEM) or TNF-α (25ng/mL α- MEM). Then, HDPC were submitted to a single irradiation with an infrared LED (855 nm) delivering different doses of energy: 0, 2, 4, 8, 15 or 30 J/cm2. Nonirradiated cells, with and without LPS/TNF-α, served as controls. Followed 24h, the cells stimulated with LPS were tested for nitric oxide (NO) quantification, cell viability (MTT assay), and reactive oxygen species (ROS) production (n=8 per group). Cells stimulated with TNF-α were analyzed regarding cytokine production and expression using antibody array and q-PCR (n=5 per group). Data were submitted to Kruskal-Wallis and Mann-Whitney tests (α=0.05). Results: LPSinduced stress resulted in significant increase in NO production by HDPC without causing any damage to cell respiratory metabolism. Irrespective of the energy dose delivered, NO production was significantly reduced when LPS-stressed cells were irradiated. The best effect was observed when 15 J/cm2 were delivered to cells. Infrared LED irradiation also promoted decrease in ROS production, while HDPC metabolism was not significantly affected. The most produced cytokines in cells stimulated with TNF-α were: GROα, IL-6, IL-8, TNF α, MCP-1 and E1 Serpin. qPCR analysis showed a decrease in expression of IL- 1 β when the cells were irradiated with the dose of 4 J/cm2. Conclusion: The oxidative stress of HDPC can be biomodulated by a single irradiation of an infrared LED. Regarding the expression of inflammatory cytokines, the delivery of 4 J/cm2 was... / Mestre

O estudo da distribuição e propagação a luz em tecidos biológicos é importante para diversas aplicações em fototerapias e diagnóstico. Os tecidos biológicos são considerados meios túrbidos, onde existe uma combinação de absorção e espalhamento, e a determinação do comportamento da luz dentro deles tem sido estudado através da aplicação de modelos que tem suas limitações. Então, determinar e manipular a distribuição para entrega da melhor dose de luz é crucial para o sucesso dos tratamentos. Para casos de Terapia Fotodinâmica, em particular, os tratamentos de lesões planas lisas, em grande parte dos casos, são bem sucedidos. Este sucesso deve-se ao fato de que existe uma boa possibilidade de distribuição uniforme da luz dentro do tecido da lesão tratada. Por outro lado, para tumores espessos de superfícies irregulares, a iluminação é dificultada devido à presença de sombras, rachaduras entre outras imperfeições sobre a lesão. Deste modo, a entrega da dose de luz inadequada e a iluminação não homogênea, resultam em regiões de necroses parciais e recidiva. Portanto, é decisivo melhorar o perfil da distribuição dentro do tecido. Neste trabalho avaliamos o acoplamento da luz utilizando um gel posicionado entre a fonte ( &#955= 630 nm) e a superfície de um phantom sólido. O acoplamento do feixe foi melhorado incorporando baixas concentrações de espalhadores ao gel. Imagens do perfil de distribuição da luz foram coletadas e transformadas em matrizes de intensidades, e posteriores curvas de isointensidades. Nossos resultados mostraram uma grande melhora na uniformidade da distribuição. De fato, utilizando um meio acoplador, conseguimos entregar um feixe mais homogêneo promovendo uma melhor iluminação. Esta técnica elimina drásticamente os efeitos da rugosidade da superfície do phantom dentro do volume tratado. Acreditamos que esse trabalho mostra uma possibilidade de melhora na dosimetria para fototerapias. / The light propagation and distribution studies in biological media are importante for several phototherapy and diagmostic aplications. The biological tissues are considered turbida medias, where there is absortion and scattering combination, and determinate the light behavior inside this tissues have been studied by models that has some limitations.Then, to determine and control the light distribution to improve the light dose delivery is important for the treatment successful. For particular cases, such as Photodynamic Therapy (PDT) applied on smooth planar lesions treatments is appropriated, since there is a good possibility of uniform distribution of light within the tissue of the treated lesion. On the other hand, tumors with more thickness may not receive the needed doses of energy to cause its death. For an efficient treatment by photodynamic therapy is required an optimal coupling light inside the lesion tissue. Shadow effects, slits or physical irregularities in the lesion can lead a nonhomogeneous light distribution inside the tissue. The results can be a partial necrosis regions and tumor recurrence. Therefore it is crucial improving the light profile inside the tissue to overcome these problems. In this study, we measured the light profile inside the phantom after the light passing through a gel as coupler. We used a solid phantom as biological tissue model and was used a red laser (&#955 = 630 nm) as light source with an optical fiber to direct illumination. The coupling is controlled by introducing a gel with low concentration of scatters between the fiber and de phantom. Was collected pictures of light profile with a camera and the data were processed with MatLab software. Our results shows a strong improvement in the light distribution when the gel with scatters is positioned between fiber and tissue. In fact, a more homogeneous laser bean is delivered to tissue promoting a better light distribution. This technique eliminates drastically the roughness effect of the phantom surface in the bulk. We believe that this work shows a possibility of dosimetry improvement for phototherapy.

Thesis (Ph.D.)--University of Alberta, 1997. / Submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Department of Medical Sciences. Also available online.

Made available in DSpace on 2014-08-13T14:50:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-12-16Bitstream added on 2014-08-13T18:01:37Z : No. of bitstreams: 1 000741517_20141216.pdf: 699498 bytes, checksum: 2434505afb00e9d5ad399ba36a38470a (MD5) Bitstreams deleted on 2014-12-19T18:32:49Z: 000741517_20141216.pdf,Bitstream added on 2014-12-19T18:33:36Z : No. of bitstreams: 1 000741517.pdf: 1188792 bytes, checksum: 76b9d56cb27251ea24b1fcd75de2ea6e (MD5) / Objetivo: Avaliar o efeito do LED infravermelho (855 nm), aplicado em diferentes doses de energia, na produção e expressão de mediadores inflamatórios por células pulpares humanas (HDPC) em cultura. Metodologia: HDPC obtidas de dentes decíduos foram semeadas (105 células/well) e após 24 h foram colocadas em contato com LPS (10 μg/mL α-MEM) ou TNF-α (25 ng/mL α-MEM). Em seguida, as células foram submetidas a uma única irradiação com LED infravermelho (855 nm) nas diferentes doses de energia: 2, 4, 8, 15 ou 30 J/cm2. Gupos não irradiados, com e sem LPS/TNF-α, serviram como controles. Decorridas 24 h, as células estimuladas com LPS foram avaliadas quanto à produção de óxido nítrico (NO), de espécies reativas de oxigênio (EROs) e viabilidade celular (ensaio de MTT) (n=8 por grupo). As células estimuladas com TNF-α foram avaliadas quanto à produção de citocinas pelos métodos de antibody array e q-PCR (n=5 por grupo). Os dados foram submetidos aos testes de Kruskal-Wallis e Mann-Whitney (α=0,05). Resultados: O contato com LPS resultou em aumento significativo da produção de NO sem causar qualquer dano ao metabolismo respiratório das células. Independentemente da dose de energia aplicada, a produção de NO foi significativamente reduzida quando as células foram irradiadas. O melhor efeito foi observado para a dose de 15 J/cm2. A irradiação também promoveu uma diminuição na produção de EROs, enquanto o metabolismo das HDPC não foi alterado. Nas células em contato com TNF-α verificou-se que as citocinas mais produzidas foram: GROα, IL-6, IL-8, TNF-α, MCP-1 e Serpin E1. Na análise por q-PCR redução foi observada para a expressão de IL-1 β, quando as células foram irradiadas com 4 J/cm2. Conclusão: O estresse oxidativo de HDPC pode ser biomodulado por uma única dose de irradiação com LED infravermelho. Quanto a expressão de citocinas inflamatórias... / Aim: To investigate the effect of infrared light emitting diode (LED) irradiation (855nm), at different energy doses, on the production and expression of inflammatory mediators by cultured human dental pulp cells (HDPC). Methodology: HDPC were harvested from sound, near-exfoliation primary teeth. Cells were seeded (105 cells/well) using α-MEM supplemented with 10% FBS and after 24h, were exposed to LPS (10 μg/mL α-MEM) or TNF-α (25ng/mL α- MEM). Then, HDPC were submitted to a single irradiation with an infrared LED (855 nm) delivering different doses of energy: 0, 2, 4, 8, 15 or 30 J/cm2. Nonirradiated cells, with and without LPS/TNF-α, served as controls. Followed 24h, the cells stimulated with LPS were tested for nitric oxide (NO) quantification, cell viability (MTT assay), and reactive oxygen species (ROS) production (n=8 per group). Cells stimulated with TNF-α were analyzed regarding cytokine production and expression using antibody array and q-PCR (n=5 per group). Data were submitted to Kruskal-Wallis and Mann-Whitney tests (α=0.05). Results: LPSinduced stress resulted in significant increase in NO production by HDPC without causing any damage to cell respiratory metabolism. Irrespective of the energy dose delivered, NO production was significantly reduced when LPS-stressed cells were irradiated. The best effect was observed when 15 J/cm2 were delivered to cells. Infrared LED irradiation also promoted decrease in ROS production, while HDPC metabolism was not significantly affected. The most produced cytokines in cells stimulated with TNF-α were: GROα, IL-6, IL-8, TNF α, MCP-1 and E1 Serpin. qPCR analysis showed a decrease in expression of IL- 1 β when the cells were irradiated with the dose of 4 J/cm2. Conclusion: The oxidative stress of HDPC can be biomodulated by a single irradiation of an infrared LED. Regarding the expression of inflammatory cytokines, the delivery of 4 J/cm2 was...

Made available in DSpace on 2014-06-11T19:27:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-25Bitstream added on 2014-06-13T19:15:18Z : No. of bitstreams: 1 turrioni_aps_me_arafo.pdf: 6913344 bytes, checksum: d860b5c16f62f7513a0953411968f01f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Pesquisas recentes demonstraram que a irradiação transdentinária da polpa pode resultar em aumento na síntese de matriz de dentina e redução na resposta inflamatória local. Entretanto, os mecanismos que regem estes processos permanecem desconhecidos. Assim, o objetivo desta pesquisa foi investigar o efeito transdentinário do LED, em três comprimentos de onda (CO: 455nm, 630nm e 850nm) e duas doses de energia (DE: 4J/cm2 e 25 J/cm2), sobre células odontoblastóides MDPC-23 cultivadas em discos de dentina (molares humanos) com 0,2 mm de espessura. Foram realizadas análises do metabolismo celular (SDH), proteína total (PT) e fosfatase alcalina (ALP), através dos ensaios de MTT, Read Northcote e Ponto Final, respectivamente. O ensaio de RT-PCR foi aplicado para avaliação da expressão dos genes que codificam para colágeno tipo I (Col-1), fibronectina (FN) e fosfatase alcalina (ALP). Além disso, foi realizada a análise da morfologia celular em Microscopia Eletrônica de Varredura (MEV). Para a produção de PT, os resultados não apontaram diferença estatisticamente significante entre os grupos irradiados e controle (Mann-Whitney, p>0,05). Entretanto, para o metabolismo celular, o grupo irradiado com LED no CO de 630 nm, na DE de 25 J/cm2, obteve melhores resultados (aumento de 21,8 %), com diferença significante quando comparado ao grupo controle (Mann-Whitney, p<0,05). Para a produção de fosfatase alcalina, houve aumento significante pra todos os parâmetros utilizados (Mann-Whitney, p<0,05), com exceção da luz azul na dose de 4 J/cm2 (Mann-Whitney, p>0,05). Na análise por RT-PCR, houve maior expressão de Col-1 para o LED infravermelho na dose de 4 J/cm2. Um maior número de células MDPC-23 com morfologia normal aderidas aos discos de dentina, semelhante ao grupo controle, foi... / Several studies have demonstrated that the transdentinal irradiation of dental pulp may increase the dentin matrix synthesis as well as decrease the local inflammatory reaction. However, the mechanisms that regulate these processes remain unknown. Therefore, the objective of this in vitro study was to investigate the transdentinal effect of LED irradiation at three different wavelengths (λ= 455, 630 and 850 nm) and two doses (4J/cm2 and 25 J/cm2) on odontoblast-like cells seeded on 0.2-mm-thick dentin disks obtained from sound human molars. Cell metabolism (MTT), alkaline phosphatase expression (ALP), total protein synthesis, and cell morphology (MEV) were evaluated. The expression of genes that encode for collagen type-1 (Col-1), fibronectin (FN) and alkaline phosphatase (ALP) was analyzed by RT-PCR. For total protein synthesis, the results showed no statistical difference among irradiated and control groups (Mann-Whitney test, p>0.05). However, for cellular metabolism, the group irradiated with 630 nm LED (dose of 25 J/cm2) showed better results (21,8% increase), with significant difference when compared to control group (Mann-Whitney test, p<0.05). For alkaline phosphatase activity, a significantly increase was observed for all parameters (Mann-Whitney test, p<0.05), except for the blue light at a dose of 4 J/cm2 (Mann-Whitney test, p>0.05). RT-PCR showed a higher expression of Col- 1 for the infrared LED at a dose of 4 J/cm2. A larger number of MDPC-23 cells with normal morphology adhered to the dentin discs was observed after irradiation with 25 J/cm2 when compared to 4 J/cm2, for all wavelengths evaluated. It may be concluded that LED irradiation was effective for transdentinal cell biostimulation and the cellular response was dose and wavelength-dependent.

Prof. H. Abrahamse

M.Tech. / Low level laser therapy, commonly known as LLLT or biomodulation, is a form of phototherapy which involves the application of low power monochromatic and coherent light to injuries and lesions to stimulate healing. In the medical field, lasers are classified as high power or surgical lasers and low level lasers which are used to stimulate cellular responses. Phototherapy has been successfully used for pain attenuation and induction of wound healing in non healing defects. Even though phototherapy has been found to be beneficial in a wide variety of therapeutic applications, it has been shown that phototherapy can induce DNA damage; however this damage appears to be repairable (Houreld and Abrahamse, 2008). DNA repair is vital to cells to avoid mutation. Literature reports show that red light or phototherapy up or down regulates genes involved in DNA repair (Zhang et al., 2003). N-methylpurine DNA glycosylase (MPG) is involved in DNA repair by catalysing the excision of a variety of modified bases. The exact mechanism by which phototherapy works is still poorly understood. Several authors have demonstrated that phototherapy enhances cell proliferation and migration. However, these cellular responses seem to confuse scientists as to whether wound healing is due to cell proliferation or migration or both. To determine the effect of phototherapy on cell proliferation or migration, a mini project was conducted (Zungu et al., 2008). Thus, cell proliferation was arrested using 5 mM hydroxyurea (HU) which is an antiproliferative drug. Wounded (W) human skin fibroblast cells (WS1, ATCC iii CRL 1502) were irradiated with 5 J/cm2 using a Helium-Neon (He-Ne) laser with a wavelength (λ) of 632.8 nm on day 1 and 4. Cell morphology, viability and proliferation were measured 24 h post irradiation. Reports indicate that several cell culture studies have used HU to control proliferation (Cai et al., 2000; Hamuro et al., 2002). Thereafter, the main study which was aimed at determining the effects of phototherapy on DNA damage and gene activation related to repair using 5 or 16 J/cm2 on W human skin fibroblast (WS1) cells was performed. Both studies involved growing WS1 cells aseptically in complete minimum essential medium (MEM) with Earle’s balanced salt solution and incubated at 37 °C in 5% CO2 and 85% humidity. Normal (N) and W cell cultures were irradiated with 5 or 16 J/cm2 30 min and 72 h (day 1 and 4) post wounding. Non irradiated cells (0 J/cm2) served as controls, while irradiated cells were the experimental groups. A wound was simulated by creating a central scratch across a monolayer of cells using a sterile 1 ml pipette. A 3 mW/cm2 He-Ne laser, λ 632.8 nm, was used to irradiate cells. After a repair time of 1 or 24 h on day 4, cell morphology (microscopy), cell viability (Trypan blue exclusion test and ATP luminescent assay), proliferation (XTT assay) and DNA integrity (alkaline comet assay with and without Formamidopyrimidine glycosylase [Fpg]) were assessed. The up or down regulation of the DNA repair gene, MPG, and regulation of three reference genes namely; beta Actin (ACTB), Glyceraldehyde 3 phosphate dehydrogenase (GPDH) and Ubiquitin c (UBC) were assessed by real time reverse transcriptase polymerase chain reaction (real time RT-PCR). iv Non irradiated HU treated cells had a reduced number of cells in the central scratch compared to non irradiated non treated cells, suggesting that HU inhibited cellular proliferation. Irradiated HU treated cells showed an increased number of cells in the central scratch compared to non irradiated treated cells. This observation proved that this increase was due to the stimulatory effect of irradiation with 5 J/cm2. The addition of HU had no significant effect on cell viability. The Trypan blue exclusion test showed no significant difference in percent viability between treated and non treated cells. Irradiated non treated cells showed a significant increase in the formazan dye, which is as a result of cleavage of XTT by the mitochondrial succinate dehydrogenase in actively proliferating cells, compared to non irradiated non treated cells (P=0.01). W cells, which were not irradiated, showed incomplete wound closure at both 1 and 24 h, while W cells irradiated with 5 J/cm2 showed complete wound closure. Similarly, W cells irradiated with 16 J/cm2 showed incomplete wound closure at 1 and 24 h. Cell viability, proliferation and DNA integrity assays showed that irradiated and non irradiated N cells were not significantly affected at both 1 and 24 h post irradiation. W cells (1 h) irradiated with 5 J/cm2 showed a significant increase in percentage cell viability and ATP compared to non irradiated W cells (1 h), (P=0.05 and P=0.04 respectively), while irradiation with 16 J/cm2 showed a significant decrease (P=0.014 and P=0.02 respectively). W cells (24 h) irradiated with 5 J/cm2 also showed a significant increase in percentage cell viability and ATP when compared to non irradiated W cells (24 h), (P=0.006 and P=0.04 respectively). Contrary, irradiation with 16 J/cm2 showed a significant decrease (P<0.001 and P=0.003 respectively). v Cell proliferation results showed that irradiation with 5 J/cm2 was stimulatory while 16 J/cm2 was inhibitory. The comet assay demonstrated that N cells irradiated with 5 or 16 J/cm2 exhibited an insignificant change in DNA damage at both 1 and 24 h when compared to their respective controls. This finding is in agreement with Karu et al., (2003) who observed that phototherapy does not alter the biological activity of cells which at the time of irradiation are functioning normally. W cells (1 and 24 h) irradiated with 16 J/cm2 showed a significant increase in DNA damage compared to their respective controls. However, there was a significant decrease in damage at 24 h compared to 1 h incubation due to the activation of DNA repair mechanisms. Though not significant, comet assay with Fpg (modified comet assay) showed more DNA damage compared to comet assay without the enzyme (conventional comet assay). It can be explained that the modified comet assay detected and cleaved oxidised bases in addition to single strand breaks, which the conventional comet assay detected, suggesting that the modified comet assay is more sensitive than the conventional comet assay. After validation of the three reference genes, ACTB was chosen to be the gene with which to normalise MPG expression in WS1 cells. It was found to be the least variable; its expression was consistent in W cells as well as cells exposed to a He-Ne laser at a fluence of 5 or 16 J/cm2. It produced an acceptable correlation coefficient (R2 >0.999) and PCR efficiency (94%). Conversely, other primers like GAPDH produced a low PCR efficiency (82%), while UBC produced a low R2 (0.898). Wang et al., (2006) recommends the value of R2 to be more than 0.995 and a PCR efficiency of between 90 and 100% for PCR results to be reliable. Other researchers have not supported the use of ACTB as a reference gene, stating that it is highly regulated (Wang et al., 2006), however this study showed that ACTB was not regulated by laser irradiation (632.8 nm at 5 or 16 J/cm2). The cell culture conditions and vi laser irradiation in this study did not induce MPG expression; perhaps an alternative repair pathway might have been induced, and hence repaired the DNA damage. In conclusion, the mini project demonstrated that HU is able to inhibit cell proliferation through its cytostatic effect without affecting the viability of W WS1 cells. This study also showed that irradiation of W cells with 5 J/cm2 using the correct parameters enhances cell migration and proliferation as evidenced by the presence of more cells in the central scratch in HU treated cells, and a significant increase in cell proliferation as shown by the XTT assay in non treated cells respectively. Thus, migration and proliferation are the direct result of phototherapy as both are involved in wound closure. This study further confirmed that irradiation of W cells with 5 J/cm2 stimulated ATP production, and hence cellular viability, as well as cell proliferation and migration. Irradiation of cells with higher fluences such as 16 J/cm2 is damaging to DNA and inhibitory to cell proliferation, migration and possibly to MPG expression. The study further showed that N cells are not stimulated by phototherapy, supporting the notion that lasers stimulate compromised cells. Thus, if they are growing normally there is nothing to stimulate. This understanding helps to clarify why N cells irradiated with 5 or 16 J/cm2 had insignificant responses. Cell culture conditions, fluence and duration of exposures are important parameters that can affect gene expression, and hence documentation of all experimental conditions needs to be emphasised and published if reproducibility is to be achieved.

M.Tech. / Low Level Laser Therapy (LLLT), also known as photo-biostimulation or simply phototherapy, has widely been used in the treatment of wounds, with its history dating back to the early 1960s (Ohshiro and Calderhead, 1991). Despite some literature reporting negative and non-existent cellular responses to LLLT, a growing body of literature reports the positive and beneficial effects of LLLT. LLLT has proved to be efficient in speeding and improving the quality of wound healing. Stressed cells respond more favourably to LLLT by recovering to their most natural state and functional capability (Bernett, 1998; Karu, 1998). When healing appears to be impaired, these tissues respond positively to the appropriate doses of light, especially light that is within 600 to 1,000 nm wavelengths (Enwemeka et al., 2004). Cellular responses to LLLT include changes in mitochondrial intracellular calcium ion (Ca2+) levels, Mitochondrial Membrane Potential (MMP), Adenine Triphosphate (ATP) concentration, and cyclic 5’, 3’ Adenosine Monophosphate (cAMP) (Karu, 1998). The mitochondrion is the power house of a cell and the major location of cellular ATP synthesis (Bayens and Dominiczak, 1999). ATP is an energy rich molecule that drives processes responsible for cell growth or proliferation (Klug et al., 2003). LLLT alters intracellular pH which is related to activation of ATPase leading to an increase in ATP production in the mitochondria of the cell (Alexandratou et al., 2002; Karu, 1998). However the mechanisms by which the beneficial effects are attained by cells in stress or injury state are not clear.