Strontium (Sr) is not only widely studied for its compound as a drug for treating osteoporosis, but there is also a growing interest of its addition in orthopaedic biomaterials. Over the years, the development of orthopaedic biomaterials has already advanced to a new era in the search of resorbable and/or bioactive materials. Due to its anabolic and anti-resportive properties of Sr on bone regeneration as a drug, strontium has been extensively investigated for its potential in other orthopaedic applications. The purposes of this study were to investigate strontium containing hydroxyapatite (Sr-HA) bone cement for the delivery of gentamicin and the effects of Sr incorporation in coral and borosilicate glass as bone engineering scaffolds. 　　　 　　　Three types of Sr incorporated materials are reported here, in which these include an applied study of the drug elution property of previously published bone cement and two initial studies of the biological properties of newly developed coral and borosilicate scaffolds. Firstly, the gentamicin release, bioactivity and mechanical property of bioactive bone cement filler based on Sr-HA were compared to a commercially available gentamicin-loaded poly(methyl methacrylate) (PMMA). Over the study period of 30 days, the cumulative gentamicin release from Sr-HA bone cement was much greater than PMMA bone cement (+ 34%); better bioactivity of Sr-HA was also confirmed with the apatite formation after simulated body fluid immersion. Goniopora, a highly interconnected porous coral, was hydrothermally converted to coralline hydroxyapatite (CHA) or coated with hydroxyapatite and incorporated with Sr. As the first report of incorporating Sr into coral with the structure remained, about 4-16% Sr was detected on CHA. Sr-HA coated coral was studied in vitro and in vivo (ovariectomized rat model) resulting in better cell proliferation and higher scaffold volume retention (+40%). Finally, the development of Sr incorporated borosilicate (SrB) glass scaffold explored a new material for bone tissue engineering, but more importantly, it introduced a phenomenal idea of the stimulatory effect of a local alkaline microenvironment on bone regeneration. Detections of an exceedingly high pH (~ pH 8.6) condition on the material surface and release of Sr, Si and B ions during the degradation of scaffold SrB were confirmed to stimulate osteoblasts and facilitate apatite formation. Although new bone was observed on both scaffolds, higher bone area/tissue area (B.Ar/T.Ar) on scaffold SrB indicated more new bone formation over borosilicate scaffold without Sr addition. 　　　 　　　The significance of this study is to explore and develop three orthopaedic biomaterials advancing the stimulatory effects of Sr on bone regeneration. The drug elution properties of Sr-HA bone cement provides a fascinating alternative for treating osteomyelitis. Furthermore, by incorporating Sr into CHA and borosilicate scaffold, it brings out the importance on the readiness of the Sr release of the materials in order to deliver the stimulatory effects. Subsequently, a localized pH micro-environment arisen by material degradation is emphasized as a controlling factor in bone regeneration on biomaterials. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Although clinical outcomes of some cancer have been greatly improved by advancements made in surgery, chemo or radiotherapy and development of novel strategies such as molecular targeted therapy, Traditional Chinese Medicine (TCM) is particularly appreciated for cancer therapy in China based on its 5,000-year-old history, well established theoretical system and numerous exciting case reports. However, due to lack of quality assurance, laboratory evidences and well-designed clinical trials, TCM always encounters much skepticism and pessimism by the West. The study aims to identify the bioactive compounds in a Chinese herb Spatholobus suberectus (SS, 雞血藤) by targeting on lactate dehydrogenase A (LDH-A) in breast cancer. Glycolysis inhibition has been considered as important strategy to block cancer energy metabolism and therefore suppressing cancer growth. LDH-A has been demonstrated to be up-regulated in various cancer cells. In our study, 46 breast cancer specimens were collected to study the relation between LDH-A expression and clinicopathological characteristics including menopause, tumor size, node involvement, differentiation and pathological subtypes classified by estrogen receptor (ER), progesterone receptor (PR) and Her-2. LDH-A expression was found to be correlated significantly with breast cancer size and independent with other clinicopathological factors. LDH-A silencing in breast cancer cell lines MDA-MB-231 and MCF-7 resulted in an inhibited breast cancer cell proliferation, elevated intracellular oxidative stress, induction of mitochondiral pathway apoptosis and limited tumorigenic ability, indicating that LDH-A inhibition might offer a promising therapeutic strategy for breast cancer. SS is historically recommended to invigorate blood circulation and has been prescribed to treat diseases relating to blood stasis syndromes including menstrual abnormalities, anemia, numbness of the limbs, arthritis and cancer, etc. Our following study revealed that SS aqueous extracts could significantly inhibited breast cancer LDH-A expression and activity in both in vitro and in vivo models built by MDA-MB-231 and MCF-7 cell lines. Bioactivity guided fractionation based on LDH-A activity, apoptosis and LDH-A expression further identified epigallocatechin (EGC) as the key compound responsible for the inhibited LDH-A expression. Mechanistic studies found that the inhibitory effect of EGC on LDH-A expression was mainly through promoting Hypoxia inducible factor-1α (HIF-1α) proteasome degradation rather than inhibiting transcription activity, which might be correlated with decreased binding between HIF-1α and Hsp90. Consistent with in vitro findings, EGC was also demonstrated effecitve in suppressing breast cancer growth in vivo correlating to down-regulation of LDH-A, HIF-1α and triggerment of apoptosis. This study provides preliminary laboratory evidences for applying SS in breast cancer therapy. However, further research is needed to evaluate its metabolism, the synergistic effects with chemotherapeutic agents and clinical efficiency. / published_or_final_version / Chinese Medicine / Doctoral / Doctor of Philosophy

Bioactive peptides constitute a major class of signaling molecules in animals and have been shown to play a role in diverse physiological processes, including hypertension, appetite and sleep. As a result, knowing the identity of these molecules and understanding the mechanisms by which they are regulated has basic and medical significance. In this dissertation, I describe the development and application of novel methods for discovering bioactive peptides and the molecular pathways that control their activity. Recent analyses of mammalian RNAs have revealed the translation of numerous short open reading frames (sORFs). However, it is unknown whether these translation events produce stable polypeptide products that persist in the cell at functionally relevant concentrations. In Chapter 1, I describe a study in which we used a novel mass spectrometry-based strategy to directly detect sORF-encoded polypeptides (SEPs) in human cells. This analysis identified 115 novel SEPs, which is the largest number of mammalian SEPs discovered in a single study by more than a factor of 25. We observed widespread translation of SEPs from non-canonical RNA contexts, including polycistronic mRNAs and sORFs defined by non-AUG start codons. We also found that SEPs possess properties characteristic of functional proteins, such as stable expression, high cellular copy numbers, post-translational modifications, sub-cellular localization, the ability to participate in specific protein-protein interactions and the ability to influence gene expression. Taken together, these findings provide the strongest evidence to date that coding sORFs constitute a significant human gene class. In chapter 3, I describe a study in which we combine quantitative in vivo peptidomics, classical biochemical experiments and pharmacological studies in animal models to elucidate the metabolism of the neuropeptide substance P in the spinal cord. We identified two physiological substance P metabolites: the N- terminal fragments SP(1-9) and SP(1-7). Focusing our efforts on the SP(1-9)- producing pathway, we determined that an activity sensitive to the inhibitor GM6001 is the dominant SP(1-9)-generating activity in the spinal cord. We also show that GM6001 treatment causes a nearly three-fold increase in endogenous substance P levels in the spinal cords of mice, highlighting the functional relevance of the pathway blocked by this inhibitor.

Στην παρούσα εργασία η ενασχόλησή μας αφορούσε τη σύνθεση και τον χαρακτηρισμό βιοενεργών γυαλιών. Οι συνθέσεις που πραγματοποιήθηκαν ήταν καθαρής SiO2, μικτών γυαλιών SiO2–ΜΟ (Μ=Ca, Mg) αλλά επίσης και γυαλιών σύστασης SiO2 –CaΟ–P2O5. Πραγματοποιήθηκαν παρασκευές με διάφορα ποσοστά τροποποιητών (Ca, Mg). Μετά την παρασκευή των υλικών ακολούθησε ο φυσικοχημικός χαρακτηρισμός τους με διάφορες τεχνικές όπως: προσδιορισμός ειδικής επιφάνειας και όγκου πόρων (ΒΕΤ), ηλεκτρονική μικροσκοπία σάρωσης (SEM), θερμοσταθμική ανάλυση (TGA), περίθλαση ακτίνων Χ (XRD) και φασματοσκοπία απορρόφησης υπερύθρου (IR). Μετά ακολούθησε μελέτη της βιοενεργότητας με εμβάπτιση σε διάλυμα SBF. Τα αποτελέσματα έδειξαν ότι τα γυαλιά που παρασκευάστηκαν είχαν μεσοπορώδη δομή ενώ όσον αφορά τη βιοενεργότητα τα γυαλιά σύστασης SiO2–CaO and SiO2–CaΟ–P2O5 προκάλεσαν σχηματισμό απατίτη στην επιφάνειά τους. / In the present thesis our work was focused on the study of the synthesis and characterization of bioactive glasses. Pure SiO2 and mixed glasses composed of SiO2–ΜΟ (Μ=Ca, Mg) and SiO2–CaΟ–P2O5 were prepared using different amounts of modifiers (Ca, Mg). Next the prepared materials were characterized by using various techniques such as: BET, SEM, TGA, XRD and IR. Finally their bioactivity was studied in vitro after immersion in SBF solution. It was found that the prepared materials showed a mesoporous structure. Regarding the bioactivity glasses with compositions of SiO2–CaO and SiO2–CaΟ–P2O5 induced the formation of apatite layer on their surface.

Lactic acid bacteria (LAB) are of interest because of their potential to modulate immune responses. The effects of LAB range from regulation to stimulation of the immune system. It has been reported that LAB affect health via two main mechanisms: directly through physical interactions between LAB and cells of the immune system, and indirectly through the products of these bacteria. The studies presented in this thesis examine the direct and indirect effects of LAB on the immune system specifically on murine dendritic cells (DCs). Mouse DCs (in form of the DC2.4 cell line) were treated in vitro with a fraction of bovine milk fermented with Lactobacillus helveticus-2 (LH-2) or three synthetic peptides identified within the fermented milk fraction. Cell culture supernatants were analyzed for presence of tumor necrosis factor (TNF)-α and interleukin (IL)-6 to determine the effects of LAB on DC activation. The results of this study showed that the ability of the milk derived fraction and the synthetic peptides to induce DC activation and production of pro-inflammatory cytokines was limited, suggesting that these peptides may induce regulatory immune responses. A series of studies was performed in vitro to investigate the effects of six LAB species and strains, (LH-2), Lactobacillus acidophilus-5 (La-5), Lactobacillus acidophilus-115 (La-115), Lactobacillus acidophilus-116 (La-116), Lactobacillus acidophilus-14 (La-14), and Lactobacillus salivarius, on maturation and activation of DC2.4. Production of TNF-α, IL-6 and IL-10 by DCs was determined after treating cells with live LAB. The expression of DC maturation markers, CD80 and CD40, was also measured using flow cytometry after stimulation with LAB. In addition, the expression of toll-like receptors (TLRs) 2, 4 and 9 by DCs stimulated with LAB was measured. Our results revealed that LAB act differentially on pro-inflammatory and anti-inflammatory cytokine production and induction of co-stimulatory molecules by DCs. Specifically, L. salivarius was found to be the most effective LAB to induce pro-inflammatory cytokine production and expression of co-stimulatory molecules. Moreover, La-14, La-116 and La-5 induced moderate maturation and activation of DCs. On the other hand, LH-2 and La-115 are the least likely lactobacilli to induce DC response. In conclusion, various strains and species of LAB can differentially regulate DC activation and maturation, raising the possibility that these microbes can influence and steer immune responses of the host.

The functional food industry has expanded yet research into consumer perceptions of functional foods is limited. Among consumers, older adults could benefit from functional foods due to age-related health concerns. This thesis aimed to generate information about the consumption of functional foods among community-dwelling older adults (>60 years old, n=200) using a researcher-administered questionnaire. Prevalence of functional food consumption was 93.0% and yogurt with probiotics (56.0%) was the top product consumed. The primary functional food matrix consumed was yogurt (51.5%) and dietary fibre was the primary functional food bioactive consumed (79.5%). Most participants (86.2%) consume functional foods to improve health and osteoporosis/bone health (67.5%), heart disease (61.0%) and arthritis (55.0%) were the primary health areas identified. Participants wanted more information about functional foods (63.5%) with preferred sources being newspapers/magazines/books (68.5%) and food labels (66.1%). These results inform stakeholders regarding the potential of functional foods to promote improved health among older adults. / Canadian Foundation for Dietetic Research

Submitted in fulfillment for the Degree of Master of Technology (Biotechnology) in the Department of Biotechnology and Food Technology, Durban University of Technology, Durban, 2009. / Information regarding the nutritional value of wild food plants in Africa and current information varies from source to source. Prior to commercialization of wild foods the nutritional, ethnobotanical, medical, chemical, anthropological and toxicity requires investigation. Plants from the Amaranthaceae family were chosen because the family is characterized by several species which are used by indigenous communities as a source of nutrition in different plants of the world. The focus of this study was to investigate the nutritional and biological activities of three plants from the Amaranthaceae family viz. Achyranthes aspera, Alternanthera sessilis and Guilleminea densa that are considered famine plants. This study aimed to determine the nutritional value (proximate, minerals and vitamins), biological activity, toxicity and potential of a tissue culture system for three species from the family Amaranthaceae. Nutritional analysis comprised of determining moisture, ash, protein, fat, carbohydrate, dietary fibre and energy. Mineral analysis of calcium, copper, iron, magnesium, manganese, phosphorus, sodium and zinc was performed by microwave digestion and then analyzed by ICP Spectrophotometry. Vitamin A, Vitamin B1, Vitamin B2, Vitamin B3 and Vitamin C were also analyzed. For biological and safety analyses aqueous and methanolic extracts were prepared. Anti-oxidative and anti-inflammatory properties of the extracts were tested; antimicrobial activity was tested by evaluating the bactericidal, fungal effect and minimum inhibitory concentration on selected bacteria and fungi using the agar disk diffusion method. Anti mosquito potential was determined by setting up repellency, larvacidal assay and insecticidal assay. The safety and toxicity analysis was carried out by measuring cytotoxicity, toxicity and mutagenicity. The potential of an in vitro tissue culture system of A. aspera, A. sessilis and G. densa was determined using micropropagation. A. aspera indicated significant amounts moisture, ash, dietary fibre, protein, vitamin B1, vitamin B2, magnesium and manganese. Plant extracts of A. aspera had antibacterial activity against the Gram negative bacteria Esherichia coli, Pseudomas aeroginosa and Salmonella typhi; Gram positive bacteria Staphylococcus epidermis and Staphylococcus aureus. The methanolic extract had antifungal activity against Sacchromyces cerevisiae and exhibited significant free radical scavenging activity as well as 85% repellency against Anopheles arabiensis. The aqueous extract stimulated the growth of the K562 (Chronic Myclogenous Leukaemia) cell line and the plant extracts showed no mutagenicity or toxicity. A. sessilis indicated significant levels of ash, dietary fibre, protein, energy, vitamin A, vitamin B1, vitamin B2, vitamin B3, iron, magnesium and manganese present. Plant extracts of A. sessilis had antibacterial activity against Gram negative bacteria P. aeroginosa and Gram positive bacteria S. epidermis. The plant also showed antifungal activity against the yeasts S. cerevisiae and Candida albicans. The methanolic plant extract showed excellent antioxidant activity. The aqueous plant extract stimulated the growth of the K562 cell line and the plant extracts possessed no mutagenicity or toxicity. This plant grew well in a tissue culture system where it was propagated from callus to a fully grown plant able to survive in environmental conditions. G. densa has ash and dietary fibre, vitamin B2, vitamin B3 and iron. The plant extracts had antibacterial activity against Gram negative bacteria E. coli, P. aeroginosa and Klebsiella. oxytoca; Gram positive bacteria Baccilus stereathermophilus and S. aureus. The plant also has antifungal activity against C. albicans and significant repellency activity against A. arabiensis where it showed 100% repellency. This plant was not found to be mutagenic or toxic. The results obtained from this study show promising potential for the plants to be exploited as famine food plants. The nutritional value, biological activity and ability to micropropagate A. aspera, A. sessilis and G. densa indicates a good potential for purposes of harnessing biotechnological products.

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2009. / Dichrostachys cinerea, Carpobrotus dimidiatus, Capparis tomentosa and Leonotis leonurus are all plants that are indigenous to South Africa. These plants are used in traditional medicine to treat various ailments. However, there is little or no scientific data to justify these traditional uses. Furthermore, it is difficult to reconcile traditional knowledge with scientific evidence because of the overwhelming targeting of signal-responsive systems by plant defensive compounds, multiple sites of action and the connectedness of the signaling pathways, which provide many cures and have pleiotropic effects. In order to evaluate the action spectrum of these plants, and validate its widespread use, this research evaluated the antibacterial, antioxidant, anti-inflammatory, anti-mosquito and immunomodulatory properties of these plants. Antimicrobial activity of the extract was determined by evaluating the bactericidal and fungicidal action using the agar disc diffusion assay. Anti-oxidative properties of the extracts were tested using the DPPH photometric assay. Anti-inflammatory properties were carried out using the 5-lipoxygenase assay. The larvicidal, repellency and insecticidal assay was determined against A.arabiensis. The safe use of these plant extracts was determined by evaluating toxicity, a brine shrimp lethality assay and an in vitro cell culture system using human myelogenous leukemia cell line. Potential carcinogenic activity was evaluated using the Ames Salmonella Mutagenecity assay. The immunomodulatory activity of the extracts on human peripheral blood mononuclear cells 6 was evaluated on freshly harvested lymphocytes using the MTT assay. Cytokine response was evaluated by measuring the secretion of interferon-gamma and interleukin-10. Elucidation of the B cells, T cells, activated T cells, CD 4+, CD 8+ and NK cells was performed by flow cytometry. The extracts showed anti-microbial activity against Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca, Salmonella typhimurium, Serratia marcescens, Bacillus cereus and Tricoderm sp. The highest activity was shown by methanolic and aqueous extracts of L. leonurus leaves followed by methanolic and aqueous extracts of D. cinerea. Extracts of C. tomentosa and D.cinerea demonstrated a higher degree of free radical scavenging than rutin, which was used as a standard indicating that these plants have strong antioxidant properties. None of the plants showed significant anti-inflammatory activity when compared to NDGA. In the anti-mosquito assays, the extracts showed strong repellency and insecticidal activity. L. leonurus extracts demonstrated the highest insecticidal and repellency activity against the mosquito, and was also found to cause ‗knockdown‘ and mortality. The extracts display no toxicity, cytotoxicity and mutagenicity. The immunological studies for immune modulation showed that the methanol extracts of these plants induce a Th1- predominant immune response because they significantly suppressed the secretion of IL-10 and augment IFN-γ production, which are hallmarks used to indicate a stimulation of the innate immune response. This study also provides new information, with respect to the potential use of these plants in producing a mosquito repellent and an immunostimulant.

Food protein-derived peptides have increasingly become important sources of ingredients for the formulation of therapeutic products. The main aim of this work was to study the in vitro and in vivo bioactive properties of structurally diverse group of peptides produced through enzymatic hydrolysis of flaxseed proteins (FP). Hydrolysis of FP with seven proteases followed by fractionation into low-molecular-weight (LMW) cationic fractions yielded multifunctional peptides that inhibited angiotensin converting enzyme (ACE) and renin activities, which are molecular targets for antihypertensive agents. The LMW peptides also exhibited antioxidant properties by scavenging free radicals and inhibiting amine oxidase activity. The peptide fractions showed inhibition of calmodulin-dependent phosphodiesterase, an enzyme that has been implicated in the pathogenesis of several chronic diseases. Moreover, FP hydrolysis with thermolysin and pronase followed by mixing with activated carbon yielded branched-chain amino acids (BCAA)-enriched multifunctional peptide mixture (Fischer ratio of 23.65) with antioxidant properties and in vitro ACE inhibition; Fischer ratio of 20.0 is considered minimum for therapeutic purposes. The BCAA-enriched peptide product can be used in clinical nutrition to treat muscle wasting symptoms associated with hepatic diseases. Furthermore, an arginine-rich peptide mixture (31% arginine versus 11% in the original flaxseed protein) was produced by hydrolysis of FP with trypsin and pronase followed by separation using electrodialysis-ultrafiltration. Arginine plays important physiological roles especially as precursor to vasodilator, nitric oxide. The arginine-rich peptide mixture exhibited in vitro ACE and renin inhibition and led to decreased systolic blood pressure (–17.9 and –11.7 mmHg, respectively at 2 and 4 h) after oral administration to spontaneously hypertensive rats. For the first time in the literature, we showed that arginine peptides have superior physiological effects when compared to the amino acid form of arginine. Lastly, quantitative structure-activity relationship studies using partial least squares (PLS) regression yielded two predictive models for renin-inhibiting dipeptides with z-scales amino acid descriptors. The PLS models indicated that hydrophobic and bulky side chain-containing amino acids contribute to renin inhibition if present at the amino- and carboxyl-terminal of dipeptides, respectively. Based on this study, Ile-Trp was discovered as potent renin-inhibiting dipeptide, and may serve as a useful template for the development of potent antihypertensive peptidomimetics.

Peppers are a rich source of diverse bioactive compounds with potential health-promoting properties. The levels of bioactive compounds and antioxidant activity can be affected by analytical methods, pre-harvest factors, and the quality of peppers. In order to understand the nutrient composition and antioxidant activity in peppers, determination of factors influencing the contents of bioactive compounds is important. The overall objectives were to determine the efficient conditions of sample preparation and the impact of pre-harvest factors affecting bioactive compounds and antioxidant activity. Optimal extraction procedures were developed, and HPLC methods were validated for bioactive compounds in peppers. The highest flavonoids were extracted in ethanol, while myricetin was extracted using N-N-dimethylformamide. Optimized conditions for flavonoids were obtained during 3 h of extraction time and hydrolysis in M HCl for 60 min at 95 degrees C. Capsaicinoids and ascorbic acid were simultaneously separated and extracted using a solvent mixture consisting of 3% metaphosphoric acid: ethanol (2:8) after 30 min of sonication. To determine the relationship between bioactive compounds and antioxidant activities in pepper extracts from different solvent properties, bioactive compounds were analyzed, and the antioxidant activities were assayed by 2,2,-Diphenyl-1-picryl hydrazyl (DPPH), reducing power, and degradation of deoxyribose. Hexane extracts had the highest levels of capsaicinoids and carotenoids, while methanol extracts had the highest levels of flavonoids. Strong DPPH scavenging activity and reducing power were found in lipophilic extracts, while hydrophilic extracts were appropriate for inhibition of deoxyribose degradation. Variation in content of ascorbic acid, capsaicinoids, and flavonoids was evaluated at immature and mature stages of pepper cultivars in different locations over two years. Mature peppers contained the highest levels of capsaicinoids and ascorbic acid. Flavonoids were variable at different maturity stages. Interactions between pre-harvest factors and bioactive compounds were highly significant. This study demonstrated the efficient sample preparation methods and simultaneous separation of bioactive compounds, which reduces analysis time and leads to reduced cost. The antioxidant properties were strongly associated with the concentration of bioactive compounds based on selective pepper extracts. The pepper quality can be improved by using appropriate pre-harvest conditions that increase the levels of bioactive compounds in peppers.