Dysregulated microRNAs in osteoclasts could cause many skeletal diseases. The therapeutic manipulation of these pathogenic microRNAs necessitates novel, efficient delivery systems to facilitate microRNAs modulators targeting osteoclasts with minimal off-target effects. Bone resorption surfaces characterized by highly crystallized hydroxyapatite are dominantly occupied by osteoclasts. Considering that the eight repeating sequences of aspartate (D-Asp8) could preferably bind to highly crystallized hydroxyapatite, we developed a targeting system by conjugating D-Asp8 peptide with liposome for delivering microRNA modulators specifically to bone resorption surfaces and subsequently encapsulated antagomir-148a (a microRNA modulator suppressing the osteoclastogenic miR-148a), i.e. (D-Asp8)-liposome-antagomir-148a. Our results demonstrated that D-Asp8 could facilitate the enrichment of antagomir-148a and the subsequent down-regulation of miR-148a in osteoclasts in vivo, resulting in reduced bone resorption and attenuated deterioration of trabecular architecture in osteoporotic mice. Mechanistically, the osteoclast-targeting delivery depended on the interaction between bone resorption surfaces and D-Asp8. No detectable liver and kidney toxicity was found in mice after single/multiple dose(s) treatment of (D-Asp8)-liposome-antagomir-148a. These results indicated that (D-Asp8)-liposome as a promising osteoclast-targeting delivery system could facilitate clinical translation of microRNA modulators in treating those osteoclast-dysfunction-induced skeletal diseases.

Dissertation submitted in partial compliance with the requirements for the Master's Degree in Technology: Homoeopathy at Technikon Natal, 1999. / The purpose of this placebo-controlled study was to determine the morbid symptomcomplex, in healthy people, produced by the administration of Sceletium tortuosum (a South African herb) in the 6 CH potency, so that it may be prescribed according to the Law of Similars, as required by homoeopathic principles. / M

University of Macau / Institute of Chinese Medical Sciences

M.Tech. / Allergic rhinitis is on the increase around the world affecting between 15-20% of the global population and is the most common chronic condition of children in South Africa. Luffeel® is a propriety complex homeopathic product manufactured by Biologische Heilmittel Heel. It is available in nasal spray and tablet form. These two forms are designed to work in combination with each other in the treatment of allergic rhinitis. The aim of this study is to determine the effect of a combination of Luffeel® nasal spray and Luffeel® tablets on allergic rhinitis using Phadiatop® tests, RAST Inhalant screens and subjective evaluation on symptoms such as nasal itching, sneezing, congestion, discharge, ocular redness and itching as indicators. This study was a double-blind, placebo-controlled study conducted at the University of Johannesburg, Doornfontein campus over a period of eight weeks. Thirty participants were recruited by poster advertisement. It can be concluded that Luffeel® nasal spray and Luffeel® tablets decrease the symptoms of allergic rhinitis, when compared to the placebo, thus giving persons suffering from allergic rhinitis an alternative treatment with no rebound or lethargic effects. It can also be concluded that this study that Luffeel® nasal spray and Luffeel® tablets has no significant effect on Phadiatop® and RAST inhalant screens over an eight week period. The results are inconclusive due to the small sample size and short time frame of the study.

M.Sc. / Background: The continuous emergence of drug resistant strains of HIV as a result of errors made by reverse transcriptase coupled with undesirable side effects of available drugs, latency problems, cost etc, warrants the continuous search for new drug candidates. Chrysotherapy which is the use of gold compounds for the treatment of various ailments has been practiced since 2500 BC. The use of gold compounds such as auranofin for the treatment of rheumatoid arthritis has lead to remission of this disease. Gold compounds such as auranofin not only prevented the progression of arthritis but also increased the CD4+ count of an HIV positive patient who was not on antiretrovirals. These compounds have been implicated in the treatment of cancers, autoimmune diseases and microorganism infections. Objectives: In this work, novel gold compounds were evaluated with the aim of identifying lead compound(s) that can eventually serve as anti-HIV agents. Materials and Methods: Eleven gold (I) phosphine complexes, four of their corresponding ligands (compound without gold atom), and a gold (III) complex were tested for the ability to inhibit reverse transcriptase (RT) and protease (PR) in direct enzyme assays. Uptake of the compounds by host cells was evaluated with inductively coupled plasma atomic emission spectrometry (ICP-AES). Potential toxicity of the gold compounds was screened for by viability dyes and flow cytometry assays. To determine inhibition of whole virus by other mechanisms in addition to RT or PR, p24 production by infected cells was evaluated. Prior to all these analysis, stability of compounds in solution was determined by 31P nuclear magnetic resonance (NMR) and UV-visible spectroscopy. Results: The compounds were shown to be stable in solution over a one week period and were taken up by both continuous cell lines and primary cells. Eight of the gold compounds significantly inhibited HIV-1 reverse transcriptase at concentrations of 25 and 250 μM while four compounds and the four ligands did not. In a fluorogenic assay against HIV-1 PR, four of the gold compounds demonstrated inhibitory activity. The gold compounds were toxic to cells lines but not to primary cells. One of the complexes (EK231) significantly reduced p24 (p=0.0042) production at a concentration of 25 μM. Conclusion: Data provided here suggests that the therapeutic benefits of these gold containing compounds as potential HIV-1 reverse transcriptase and protease inhibitors should be considered.

Prof. H. Abrahamse

M.Tech. / The homoeopathic medicine Arnica montana is often prescribed in cases of trauma, before and after surgery and in cases where there is bleeding. Many conventional medical practitioners, however, do not advise its use for the above complaints due to the herbally prepared Arnica montana mother tincture containing coumarin derivatives which are said to have an anti-coagulant effect and cause a potential risk of bleeding. The aim of this particular study was to investigate the in vivo effect of the complex remedy Arnica montana composing of potencies 6C, 30C and 200C on coagulation and bleeding. This study forms part of a three part in vivo study to determine the effect of homoeopathic Arnica montana in various potencies on blood coagulation. The effect of Arnica montana on blood coagulation was evaluated by measuring the Bleeding Time (BT), activated Partial Thromboplastin Time (aPTT) and Prothrombin Time (PT). This is a double blind, placebo controlled trial with a total sample group of eighty healthy participants between the ages of eighteen to thirty five. As this study forms part of a three part study the total sample group was shared. Twenty participants were allocated to the placebo group and received 20% ethanol. Twenty participants were allocated to the experimental group and received the complex homoeopathic preparation of Arnica montana 6C, 30C and 200C in 20% ethanol. The Bleeding Time was measured by a trained medical technologist and blood samples underwent coagulation tests comprising of aPTT and PT. The study was conducted over a period of two weeks at the UJ Doornfontein Campus Homoeopathy Health Centre. After two weeks another venous sample was drawn by the phlebotomist and sent away for the same coagulation studies as described above. The technologist again measured the Bleeding Time. Results obtained from the Prothrombin Time, activated Partial Thromboplastin Time and Bleeding Time tests pre- and post medication were compared and v analysed. Analysis of data was done using SPSS 15.0 (Statistical Package for Social Sciences). Results showed that complex remedy Arnica montana 6C, 30C and 200C had no significant effect on blood coagulation and bleeding in vivo.

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.

M. Tech. / Stem cells are defined as undifferentiated cells that can proliferate indefinitely and have the capacity of both self-renewal and differentiation to one or more types of specialised cells. Traumatic tissue injury and age-related degenerative diseases are a major problem in South Africa and worldwide. Stem cells could be used for tissue engineering and reconstructive surgery. In treating these conditions, the main principle of stem cell therapy is the replacement of damaged and dead cells in injured tissues and organs with new healthy ones expanded in vitro from stem cells (Orlic et al., 2002). These cells can be isolated from adipose tissue in significant numbers and exhibit stable growth and proliferation kinetics in culture and could be differentiated into bone, fat, cartilage and muscle when treated with established lineage-specific factors (Zuk et al., 2002). Low Level Laser Therapy (LLLT) is currently applied in the treatment of numerous diseases and pathological conditions (Gasparyan et al., 2004). LLLT produces positive effects on irradiated cells and tissues such as proliferation of cells, capillary growth and adenosine triphosphate (ATP) activation (Schindl et al., 1998). Low level laser radiation at different intensities has been shown to stimulate as well as to inhibit cellular processes (Moore et al., 2005). Epidermal growth factor (EGF) is a growth factor that plays important roles in the regulation of cell growth, proliferation and differentiation. This study investigated the effect of low level laser radiation alone as well as in combination with EGF on adult adipose derived stem cells (ADSCs) isolated from human adipose tissue. ADSCs were isolated from human adipose tissue through collagenase digestion and cultured in DMEM-F12 containing 10% FBS and antibiotics and incubated at 37°C in a humidified atmosphere of 5% CO2 (Zuk et al., 2001). iii Semi-confluent monolayers of ADSCs were exposed to low level laser at 5 J/cm2 using 636 nm diode laser with a power density of 12.1 mW/cm2 at room temperature in the dark. Cell morphology was monitored at 0, 24 and 48 h using an inverted light inverted microscope. Cell viability was evaluated at 0, 24 and 48 h using the Trypan Blue exclusion test and an adenosine triphosphate (ATP) luminescence assay. bFGF (basic fibroblast growth factor) indirect ELISA and optical density assays were used to monitor cell proliferation at 0, 24 and 48 h post irradiation. In addition the expressions of stem cell markers, β1-integrin and Thy-1, were monitored by immunocytochemical live cell surface labelling and Western blot analysis. Cells were incubated with EGF to enhance proliferation and differentiation and the cell morphology, viability and proliferation were monitored as well as the expressions of stem cell markers, β1-integrin and Thy-1. Morphology of the cells was not altered by irradiating them with 5 J/cm2 using diode laser at 0, 24 and 48 h. Cell viability and proliferation showed an increase at 24 and 48 h post irradiation. At 0 h, there was no significant difference between irradiated and non-irradiated cells in cell viability and proliferation. There was an increase in the expression of β1-integrin and Thy-1 after irradiation as shown by Western blot analysis and immunocytochemical live cell surface labelling. Cell viability and proliferation showed a significant increase at all time points post irradiation with the addition of EGF. There was no noticeable change in cellular morphology at any time point. Low level laser irradiation of human ADSC’s at 636 nm with 5 J/cm2 and 12.1 mW/cm2 increased the viability and proliferation of these cells in vitro. Furthermore, low level laser irradiation appeared to increase the expression of stem cell markers, β1-integrin and Thy-1. In addition, laser irradiation did not alter the morphology of the cultured cells. The addition of EGF to the cells also increased their viability and proliferation as well the expression of the markers, β1-integrin and Thy-1. The study showed that laser irradiation stimulates two important cellular responses namely cell viability and proliferation which indicates that ADSCs may be suitable for tissue engineering and future cell differentiation studies.