Cordyceps cicadae, a parasitic fungus, has been used as a traditional Chinese herb for treatment of illnesses related to immune dysfunction. However, mechanistic actions are not entirely clear. The specific aim of the present study is to investigate the immunological mechanisms of C. cicadae on human T lymphocytes in vitro. The two main objectives carried out in the present study in order to achieve the aim are: i. to investigate the immunological effects of the water extract of C.cicadae on human resting and proliferating T lymphocytes; 2. to investigate the adjuvant property of C.cicadae with the immunosuppressant Cyclosporin A (CsA) on the control of human T cells proliferation, cell death and cytokines secretion. C. cicadae has demonstrated a dual effect of enhancing the activity of resting Tlymphocytes and inhibiting that of PHA-stimulated cells. In resting T cells, C. cicadae increased cell activity, cytokines secretion, expression of IL-2α receptor (CD25) and the population of Th17 as well as Treg cells without affecting the cell cycle progression. In the viability and cell death test, C. cicadae has been found not to exert toxicity on T cells. These results reveal the role of C. cicadae as an invigorant to improve the immunity as a whole in healthy individuals. In PHA-stimulated proliferating T-lymphocytes, C. cicadae inhibited the cell proliferation by arresting them at G0/G1 phase and decreasing both S and G2/M phase cell populations. The inhibitory effect was not through the induction of cell death as both PHA- and CsA-induced cell apoptosis and necrosis were reduced by C. cicadae treatment. It was believed that C. cicadae affects the progression of inflammatory responses by selectively suppressing the secretion of Th1 cytokines and enhancing that of Th2 cytokines, but it seemed unable to influence the CD25 expression in PHA- and CsA-treated T cells. An increased population of Treg cells by C. cicadae was also observed in PHA-stimulated T cells. C. cicadae adjuvant to CsA showed greater inhibition of cell proliferation and Th1 cytokines production. However, C. cicadae antagonized the effect of CsA in the secretion of IL-10, a Th2 and Treg cytokine. These results suggest a potential role of C. cicadae as an adjuvantive agent with CsA in the therapy of autoimmune diseases and organ transplantation. Furthermore, C. cicadae demonstrated superior immunomodulatory properties over CsA as it can act selectively between resting and proliferating T cells, and also protect them from apoptosis and necrosis, alleviating the toxic effect caused by CsA. In summary, this study showed the potential of C. cicadae as an immunomodulator from which both healthy individuals and inflammatory disease patients can benefit. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Sonoporation, being an ultrasound-induced membrane perforation phenomenon, has received considerable interest in view of its therapeutic potential and is rapidly emerging as a promising approach to facilitate drug delivery. This event generally occurs when acoustic cavitation develops in the vicinity of living cells, as the mechanical interactions between ultrasound and microbubble would exert a force that is substantial enough to create pores on the cell membrane. The resulting increase in cell membrane permeability is transient in nature, and short-term survival of sonoporated cells is generally assumed. However, it remains unclear as to whether sonoporation would affect the cell fate in the long run. In particular, the contemporary mechanistic understanding of sonoporation has lacked account of the cellular response at a subcellular level. This inherently raises concerns on the general therapeutic applicability of sonoporation in mediating drug delivery. This thesis first addressed the question of whether cell fate may be affected on time-lapse basis as a result of sonopopration. As observed our analysis of DNA contents and cytoplasmic signaling proteins, some cells were found to commit apoptosis (programmed cell death) after sonoporation while the remaining viable cells may enter into cell-cycle arrest that disrupted normal cell proliferation. These findings should carry two major implications from a drug-delivery standpoint. First, cellular protection strategies should be developed when using sonoporation for drug delivery in cases where cell viability should be maintained. Second, for cancer therapy where cell death is required, the cytotoxic impact of sonoporation may represent a complementary factor that can be leveraged upon in facilitating the delivery of anti-cancer drugs. Further investigations were conducted to gain insight into the intermediate transduction mechanism in which sonoporation has entailed to bring about various cytoplasmic signaling changes that promote cell-cycle arrest and apoptosis. Our results reveal a transient enhancement of intracellular Ca2+ concentration in sonoporated cells. This bioelectrical disruption event is often recognized as a central messenger to instigate a series of cell-fate regulation pathways. In addition, observations on cell membrane repair revealed an exocytotic patching mechanism, accumulation of internal vesicles and increased activities in the Golgi apparatus. Given that the elevated Ca2+ level were observed in sonoporate cells, a follow-up study was conducted to investigate the potential role of endoplasmic reticulum (ER) and mitochondria in sonoporation-induced bioeffects. These two organelles were found to be activated in succession and in ways connected to the initiation of pro-apoptotic signaling. In particular, stress response was found to be active in the ER, and this in turn induced the dysfunction of mitochondria. Also, our time-lapse observations on the mitochondrial membrane potential have confirmed that this organelle is involved in facilitating sonoporation-induced apoptosis. In summary, investigations of time-lapse dynamics of cellular and subcellular responses mediated by sonoporation are so important in elucidating the fate of the sonoporated cells and understanding the mechanism in which sonoporation has entailed to instigate the sequential signaling pathways that bring cells into such conditions, thereby refining the therapeutic role of this biophysical phenomenon and making it more efficient in facilitating drug delivery. / published_or_final_version / Electrical and Electronic Engineering / Doctoral / Doctor of Philosophy

The purpose of this study was to design and develop anti-hyperglycemic functional foods containing phenolic carbohydrate absorption modulator. Physical, chemical, structural and digestive aspects of the reactions of carbohydrates in the presence of phenolic extracts of traditional Chinese medicines were investigated. Water extracts of 14 traditional Chinese medicinal (TCM) plants with reputation of usefulness in treating diabetes were examined for total phenolic contents, antioxidant activities and α-amylase inhibitory activities through in vitro assays. A positive and significant linear relationship between total phenolic content and antioxidant capacity was found (R2=0.99), indicating that the phenolic compounds are the major antioxidant components in the tested materials. Extract of Chinese galls from Rhus chinensis exhibited highest total phenolic content, antioxidant capacity and α-amylase inhibitory activities. The results of this study demonstrate that the extract of Rhus chinensis would be the most effective carbohydrate modulator among the samples tested. Swelling, pasting and thermal properties of wheat and potato starch were studied in the presence of aqueous extract of Galla chinensis (GAC), also known as Chinese galls. The swelling factor was reduced in both starches after GAC extract addition. The extract increased peak viscosity and cold paste viscosity of wheat starch, but decreased those of potato starch. Increased breakdown has shown for wheat starch, but that for potato starch decreased. GAC extract also delayed gelatinization of both starches with higher onset temperature (To). The results of this study demonstrated that GAC extract supplementation can alter the physicochemical properties of wheat and potato starch. To examine the potential application of Galla chinensis (GAC) extract supplementation in Asian white salted noodles, the effects of various GAC extract addition levels on microstructure and in vitro starch digestibility of noodles were investigated. Scanning electron microscopy (SEM) indicated that the gluten protein network was improved (more compact and smooth) by GAC extract in both raw and cooked noodles. The estimated glycemic index (GI) was significantly decreased from 100 to 90.6 after 0.5% GAC extract was added. This indicated that GAC extract supplementation could reduce starch digestibility and blood glucose response. This feature might make GAC extract-enriched noodles sufficiently attractive to health-conscious consumers. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Sonoporation, referring to transient membrane permeation phenomenon generated by acoustic cavitation, has spurred significant scientific interests for its potential applications in facilitating uptake of drugs and genes into living cells. With an increasing level of technical maturity in realizing sonoporation, scientists are trying to gain a deeper understanding of the cellular responses related to this biophysical phenomenon from the standpoint for drug/gene delivery. However, challenges and difficulties remain to be overcome including providing direct evidences for the microbubble-cell wave matter interaction mechanism, obtaining controllable sonoporation at the desired locations on the cell membrane, maintaining the viability of the sonoporated cells with high efficiency delivery outcomes and so on. Such a lack of scientific foundations has been recognized as a fundamental obstacle in substantiating the application merit of sonoporation. In this study, the overall objective is to stepwise unravel the cellular stress induced by microbubble-mediated sonoporation after resealing. To achieve it, two kinds of well-calibrated ultrasound exposure platforms are designed. One of them can be used for the in situ observation of the wave matter interaction ways during sonoporation via the confocal microscope. The other ultrasound exposure setup can be used for the studies of the sonoporation induced bio-effects which need many cells for analysis. With these designed and well calibrated ultrasound exposure platforms, new insights for the cellular impacts induced by sonoporation are provided. As demonstrated in vitro, sonoporation may inadvertently induce repressive cellular features even whilst enhancing exogenous molecule uptake. Both suspension-type (HL-60) and adherence-type (ZR-75-30) cells were employed in this investigation. They were routinely exposed to 1 MHz pulsed ultrasound with calibrated acoustic field profile and in the presence of microbubbles. The post-exposure morphology and the intracellular actin cytoskeletons dynamics of sonoporated cells were examined in situ using confocal microscopy. Furthermore, the cell-cycle progression kinetics of the viable sonoporated cells were analyzed using flow cytometer. Results show that, for both investigated cell types, viable sonoporated cells would exhibit membrane and nucleus shrinkage, intracellular lipid accumulation and actin deploymerization over a two hours period. On the other hand, as compared to the sham control cells, the deoxyribonucleic acid (DNA) synthesis duration of sonoporated cells is significantly lengthened as indicative of a delay in cell-cycle progression. These features are known to be characteristics of a cellular stress response, suggesting that sonoporation indeed constitutes itself as a cellular stress to living cells even after the cells are resealed. In terms of the implication of this work, this study has shown that sonoporation can be a significant cellular stress both short term and long term after ultrasound exposure. In particular, the intracellular homeostasisis found disrupted even with membrane resealing. Therefore, if sonoporation is to be used for drug delivery, efficiency may be a problem that really needs to be solved in optimizing sonoporation for drug/gene delivery purposes. On the other hand, it raises opportunities for developing other therapeutic applications via sonoporation. / published_or_final_version / Electrical and Electronic Engineering / Doctoral / Doctor of Philosophy

Therapeutic ultrasound employs the acoustic energy carried by high-frequency mechanical wave to induce beneficial effects on living systems. This therapeutic approach is advantageous in that its energy could be remotely focused on the targeted tissue in a non-invasive manner. Although ultrasound therapy has been shown to be feasible and effective in both laboratory experiments and clinical trials, its safety and efficacy are still challenged by the lack of fundamental knowledge of how ultrasound wave exerts physical effects on the cell system and how the cell functionally responds to the ultrasound stimulation. Motivated by the above insight, this thesis aims to provide direct experimental evidence for illustrating the biophysical details of how ultrasound wave (alone or combined with microbubble) interacts with live cells. An acoustic experimental platform with well-calibrated ultrasound field and live-cell imaging modality was developed to observe ultrasound-cell interaction. Based on this platform, a series of single-cell studies was then conducted to monitor the structural and functional changes of the live cell as well as its fluorescently-labelled components over the course of ultrasound exposure. Results obtained in this thesis provided image-level evidence for characterizing the ultrasound-cell interactions in the following three aspects. First, it was found that low-intensity ultrasound pulsing could directly perturb the plasma membrane, the cytoskeletal network and the inner nucleus of live neuroblastoma cells. This cytomechanical perturbation would result in reversible and structural alternations of subcellular components. Second, low-intensity pulsed ultrasound, when applied on neuronal cells, could exert morphological impact through inducing neurite retraction and cell body displacement, and electrophysiological impact in the form of membrane depolarization and calcium influx. This finding verified the potential of ultrasound in modulating neuronal development and excitability. Last, the cell membrane perforation and resealing dynamics induced by the ultrasound-activated microbubble were visualized and characterized. The subsequent cellular responses to this ultrasound-induced sonoporation were also identified at both membrane and cytoskeleton levels. The significance of this study is to provide direct and solid experimental evidence for understanding the biophysical interactions between ultrasound wave and live cell. This advanced scientific interpretation is definitely crucial for establishing the cellular mechanisms of therapeutic ultrasound and for providing technical insights into ultrasound treatment. / published_or_final_version / Electrical and Electronic Engineering / Doctoral / Doctor of Philosophy

Anxiety is one of the most common psychological disorders in school-age children, which has detrimental impact on behavioral, social, and emotional functioning. Cognitive-behavioral therapy has demonstrated effectiveness in treating youth with anxiety disorders; however, little is known about how or why this intervention is beneficial. This study explores the mechanisms of therapeutic change in anxious children and adolescents by evaluating clients’ perspectives through semi-structured interviews. A qualitative research method will be used to assess the thoughts, feelings, and experiences of youth and parents who participate in a cognitive-behavioral intervention program with a parent-training component. Specifically, grounded theory will be used to collect and analyze data and generate a theory, which explains the mechanisms of positive therapeutic change. / text

published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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