Made available in DSpace on 2014-10-09T12:50:01Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:02:03Z (GMT). No. of bitstreams: 1 10430.pdf: 2501583 bytes, checksum: d9bc55d07c6e6e0db459c7c5d10481e7 (MD5) / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

M.A. (Philosophy) / South Africa has endured many hardships; the most prominent of all is the apartheid regime and effects thereof. Almost twenty years after democracy and freedom from this system of oppression, the hurt and pain generated through the traumatic events during this period linger on. Possibly the most significant effort to overcome these effects and deal with the hurt was the Truth and Reconciliation Commission (TRC). The TRC had many successful accounts of victims confronting their torturers. During these confrontations, the complete truth about an act was revealed, an apology was offered to the victims and the family of the victims, and many times, forgiveness was granted. Forgiveness that was granted was often given in order to help these victims and families of the victims move past the trauma that they experienced. However, one can ask, can victims truly promptly forgive perpetrators of severe violence and murder after hearing the complete truth and hearing the perpetrators apologise? What has happened to the hurt and pain victims experienced during the traumatic event if they have not dealt with it? Does the memory of the event not still stand out in the victims’ mind? It can be said that traumatic memories, if ignored and left unaddressed, would not only affect individuals in various areas of life, but also impact negatively on society as a whole. In all probability, the individual will remain haunted by the effects of a traumatic event and as a result, would subsequently fail to enjoy a higher quality life. If numerous individuals continue to experience persistent, unresolved or untreated after-effects of trauma, one can imagine a society that has become a populace of mourning, anxious, moping and mistrustful citizens. Hence it can be said that there appears to be a need in society at large to address the issue of traumatic memories. These may include, memories associated with typical traumatic events such as rape or murder, and for the purposes of this study in particular, mass murder, for example the Holocaust or severe abuse committed against a group of individuals based on ethnicity and/or race, for example the apartheid system. In defining the term ‘trauma’ it is understood to be a violation against an individual, in the form of an injury. I believe that it is acceptable to state that physical trauma will necessitate psychological trauma. Versteeg (2012:115) has aptly described trauma as follows: It is – literally- a wound: an injury to living tissue, caused by a cut, blow or other impact, typically one in which the skin is cut or broken.

Chronic wounds represent a severe socio-economic burden and a key area of unmet clinical need. PKCα is ubiquitous in the skin, particularly the epidermis and functions in numerous pathways that are fundamental to wound repair. By utilising a global PKCα-/- mouse we have identified PKCα-regulated processes both in unwounded skin and during wound healing. PKCα-/- mice display considerably delayed wound healing with a dramatic reduction in re-epithelialisation. By analysing the ultrastructure of the epidermis, I have shown that this delay directly correlates with a failure of wound edge desmosomes to switch to a their adhesive properties. A major risk factor for the development of chronic wounds is age. Crucially, this delay in modulating cell adhesion is conserved in human chronic wounds and aged murine skin. Furthermore, manipulation of PKCα using an inducible bitransgenic mouse containing epidermal specific constitutively active PKCα can accelerate the modulation of desmosome adhesion and subsequently improve re-epithelialisation. Global gene expression analysis of PKCα-/- skin and wounds revealed further defects. Upon wounding, we observed a failure to correctly regulate expression of key collagen and Wnt signalling genes that are essential for correct and timely wound healing. Finally, intrinsic gene expression changes were identified in the skin of PKCα-/- mice, specifically a downregulation of multiple extracellular matrix genes. Of note was the downregulation of small leucine-rich proteoglycans which led to alterations to dermal collagen structure and skin tensile strength. These changes render the PKCα-/- skin susceptible to breaking and wound development. To conclude, we have identified multiple roles for PKCα intrinsically in the skin and also during cutaneous wound healing. Importantly, these intrinsic changes appear to predispose PKCα-/- skin to the development of cutaneous wounds and altered wound-specific processes that manifest in a delayed healing phenotype.

Abstract The effects of perioperative filgrastim (rhG-CSF) and surgery per se on the postoperative acute phase reaction were studied by assessing leukocyte functions, cytokine levels and tenascin-C (Tn-C) and procollagen propeptide (PINP, PIIINP) concentrations in different body fluid compartments in patients undergoing gastrointestinal surgery. Thirty consecutive patients were randomized to receive either filgrastim or placebo for five days, starting 12 hours before colorectal surgery. Filgrastim treatment led to marked neutrophilia with decreased neutrophil migration in peripheral blood but not in peritoneal fluid 48 hours postoperatively. Neutrophil phagocytosis and bacterial killing did not differ between the groups. Filgrastim caused increased postoperative expression of neutrophil CD11b/CD18 in blood but not in peritoneal fluid or wound fluid. CD11b/CD18 expression was higher in both wound fluid and peritoneal fluid than in blood in the placebo group. The expression of neutrophil CD62L was higher in blood than in peritoneal fluid or wound fluid in both groups. The serum concentration of interleukin (IL)-8 was lower in the filgrastim group 5 hours postoperatively. The concentrations of IL-1β, IL-6, transforming growth factor (TGF)-β and IL-10 did not differ between the groups. The cytokine levels were markedly higher locally in the wound and in the peritoneal cavity compared to circulating blood. No adverse events attributable to filgrastim were seen. Leukocyte counts, neutrophil and monocyte functions and the levels of IL-6, IL-8 and granulocyte colony-stimulating factor (G-CSF) were measured from 18 patients before and after colorectal surgery. Surgery caused an increase in neutrophil and monocyte counts along with lymphocytopenia. Neutrophil phagocytosis was decreased 4 and 24 hours postoperatively, but normalized after that. A distinct systemic cytokine response was seen postoperatively. In a study with 24 patients, Tn-C concentration increased in wound fluid during the first postoperative week after abdominal surgery. The Tn-C level was markedly higher in wound fluid than in serum.

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.

Magister Legum - LLM / This thesis aimed to explore the various legislative provisions that have affected traditional health practice in South Africa. The discussion is limited to the period dating from 1891 to 2004. The relevance of this period is that during the 19th and early 20th centuries traditional healing has gone through some turmoil as the governments of that period promulgated legislation, which curtailed the practice of traditional healing. This study focused on the art of traditional healing in a legislative context. / South Africa

Epithelia act as protective barriers and it is therefore essential that wounded epithelia are rapidly repaired to maintain barrier function. Cells surrounding epithelial wounds become motile following wounding, which involves generating dynamic actin structures that drive closure of the wound. These actin structures include filopodia which are important in the final stage of epithelial closure in which the opposing epithelial edges are joined together. The molecular mechanisms that trigger wound edge cells to become motile are not well understood. Using Drosophila wound healing and the morphogenetic process dorsal closure as models, we find that phosphatidylinositol 3,4,5-triphosphate (PIP3) regulates epithelial closure by promoting the formation of filopodia at epithelial edges. PIP3 accumulates at epithelial edges and genetically depleting PIP3 results in reduced filopodia and defects in epithelial closure. We demonstrate that the GTPase Rac and guanine nucleotide exchange factor Myoblast City function downstream of PIP3 to promote filopodia formation. We also demonstrated that the scaffolding protein Par3/Bazooka and the lipid phosphatase PTEN are responsible for restricting the localisation of PIP3 and consequently the downstream signals to the epithelial leading edge, so acting to determine the location of filopodia formation. This project reveals a novel mechanism by which actin protrusions, required for epithelial closure, are formed in response to epithelial damage. Additionally, we have identified an additional role for PIP3 in regulating the extrusion of cells from epithelial sheets in the Drosophila embryo. This finding implicates PIP3 in the regulation of tissue homoeostasis, and could contribute to our understanding of tumour initiation as unregulated tissue growth can result in the formation of tumours.

The complex process of wound repair becomes disrupted in the elderly with a profound effect on patient morbidity and huge financial implications for the NHS. While age itself is a risk factor for delayed healing recent work implicates estrogen decline, rather than intrinsic ageing per se, as the critical regulator of delayed healing in elderly subjects. In women estrogen levels fall dramatically post-menopause and with increasing life expectancy most women in the developed now world spend at least a third of their lives in a state of estrogen deprivation. Estrogen replacement can reverse this delay, but unfortunately long term estrogen treatment (HRT) increases breast cancer risk such that steroidal estrogen is now listed as a carcinogen. The aim of this study has been to functionally dissect the role of estrogen signalling during repair at the molecular, cellular and physiological levels. New data presented within this thesis reveal estrogen to be a global regulator of healing with pleiotropic effects on multiple wound cell types. By combining pharmacological manipulation and genetic ablation my data reveals novel diametrically opposed roles for the two estrogen receptor isoforms, ERalpha and ERbeta, during healing. I have further exploited this to demonstrate the in vivo therapeutic potential of compounds with receptor selective agonistic/antagonistic activity. Additionally, I have further investigated the mechanism of action of estrogen and these selective compounds implicating the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) in beneficial effects on healing. This research leads the way toward translation into human studies with the ultimate aim of developing targeted therapeutics for the treatment of delayed acute and chronic wounds, particularly in the elderly.