As the name suggests, synthetic biology designs new biology using human power, knowledge, and creativity. Biology is vast, complicated, and all-inclusive, and so is synthetic biology. I believe synthetic biology is the Utopia of biologists, chemists, physicists, material scientists, engineers,and computer scientists. It is a newly emerged and vastly growing field that can impact and improve our lives in many aspects. I dare to say that anything you see that is done by biology can, in the future, be done better by synthetic biology since, on top of having biology as a teacher and as a template, synthetic biology has the benefit of creative and rational design provided by the human brain. In a way, it is the next step in evolution. In this thesis, we worked on some yeast synthetic biology applications. We used engineered yeasts to create bandages to enhance and accelerate the healing of diabetic wounds, make biosensors for pathogenic bacteria and a small molecule metabolite (glucose) important in diabetic patients, and design a community of cells that could contain artificial intelligence. Chapter 1 gives a short introduction and background information regarding diabetes, wound healing, and advanced healing therapies. Chapter 2 is focused on engineering yeasts to secrete wound-healing proteins and in vitro and cell-based studies on the engineered yeasts and secreted recombinant proteins. Chapter 3 presents two wound dressings that contain engineered live yeasts as active ingredients. This chapter includes further in vitro and cell-based studies to assess the functionality of the designed dressings. Chapter 4 focuses on in vivo experiments to study the wound-healing properties of the designed live yeast dressings. Finally, Chapter 5 presents two other projects: one on live yeast biosensors and one on designing modular smart yeast communities that can do computation based on neural network algorithms.

Ligament tears are more prevalent than all other knee injury pathologies, and contribute significantly to musculoskeletal joint pain and disability reported worldwide. Despite current soft tissue reconstruction techniques, the injured ligament fails to regenerate due to dysregulated cell-extracellular matrix (ECM) interactions that culminate in scar formation. Hallmarks of scar formation, or fibrotic healing include disorganized ECM, pathological stiffness or tissue rigidity, and the accumulation and persistence of myofibroblasts. A primary driver of fibrosis, myofibroblasts are characterized by high contractility, excessive deposition of collagen type I, coupled with inflammatory and fibrotic signaling. Notably these cells are critical early on in the response to injury, by aiding in the contracture of the wound bed and depositing collagen to repair the injury site. However, myofibroblasts are not capable of fully regenerating the native ligamentous matrix, and resolution of the phenotype is necessary in order to cue surrounding cells, prevent chronic inflammation and aberrant tissue remodeling. Persistence of the myofibroblast phenotype thus leads to a ligament scar that is functionally weaker than the healthy tissue matrix, characterized by significantly different histological, biochemical, and biomechanical properties. The consequential instability of this scar disrupts load distribution within the knee joint and increases the risk of subsequent injury, osteochondral degeneration, and ultimately, the development of post-traumatic osteoarthritis. Therefore, there is a critical need for strategies that target the inflammatory and fibrotic myofibroblast phenotypes for soft tissue healing. It follows that the overarching goal of this thesis is to engineer an immunomodulatory matrix to regulate myofibroblast activation and downstream fibrogenic signaling. To this end, models of soft tissue fibrotic repair are explored in order to test the central hypothesis that cues from the repairing ECM play an important role in regulating myofibroblast activation and persistence. Specifically, this thesis will compare myofibroblast differentiation and signaling in three in vitro models of tissue repair: 1) 2D on tissue culture polystyrene (TCPS), and two 3D models namely 2) collagen hydrogel and 3) electrospun collagen fiber matrices. As expected, on the 2D model, a persistent myofibroblast phenotype could be generated over time with an optimized transforming growth factor beta 1 (TGF-β1) stimulation protocol. To create repair-relevant 3D matrix models, we engineered collagen hydrogels with controlled mechanical properties, as well as electrospun fiber platforms that isolate key matrix factors including, collagen content, stiffness, fiber diameter, and alignment. These models emulate the connective tissue repair process via recapitulating the increasing matrix stiffness and fiber assembly of the early (granulation tissue), proliferative, and remodeling stages of the repair. Myofibroblast differentiation potential, parallel inflammatory and fibrotic cytokine secretion, as well as matrix remodeling potential were observed to be dependent on matrix model parameters. Moreover, single-cell resolution RNA sequencing revealed heterogenous myofibroblast populations within the context of response to engineered collagenous substrates. Specifically, myofibroblast accumulation was observed on hydrogel substrates that recapitulate the pathologically stiff mechanics and disorganization of fibrotic scar tissue while architectural cues of engineered fiber substrates prevented myofibroblast differentiation in a diameter and alignment-dependent manner. Moreover, nanoscale fibers elicited the greatest anti-fibrotic and anti-inflammatory properties compared to microscale fibers and stiff collagen-based hydrogels. Throughout, this thesis also explores the contribution of NF-κB signaling to myofibroblast plasticity and persistence using engineered collagen-based platforms, highlighting the dynamic role of myofibroblasts as critical immunoregulating cells. The NF-κB signaling pathway is implicated in a broad array of fibrotic and chronic inflammatory conditions, and more recently has been associated with survival of persistent myofibroblast populations in soft-tissue fibrosis and tendon degeneration models. In this thesis, NF-κB activation was seen to be related to the persistent myofibroblast phenotype and increase over time in both 2D TCPS and 3D collagenous hydrogel matrices that mimic pathologically stiff scar tissue, while a temporally dependent activation pattern was observed in electrospun collagen fiber-based models. At the transcriptional level, NF-κB survival signaling was significantly enriched in myofibroblast populations supported by TCPS and stiff collagen-based hydrogels but downregulated on soft hydrogels and fibers with decreasing fiber diameter that prevented robust myofibroblast differentiation at single cell resolution. Building upon these new insights regarding matrix cues that drive myofibroblast activation, we designed an immunomodulatory matrix that mediates small molecule release targeting NF-κB inhibition. The immunomodulatory matrix achieved robust amelioration of the myofibroblast phenotype as well as reduced the secretion of key inflammatory and fibrotic cytokines by these cells. Moreover, a similar anti-fibrotic response was seen for human ligament fibroblasts treated with these matrices. Collectively, this thesis work presents a systematic evaluation of myofibroblast plasticity and persistence within the context of 2D (TCPS), 3D (collagen-based hydrogels), and finally 3D with defined microarchitectural cues (electrospun collagen-based fibers) that recapitulate the progressive stages of scar-mediated healing, and reveals NF-κB as a promising target for reducing myofibroblast persistence. Moreover, the immunomodulatory control of myofibroblast plasticity and persistence via matrix cues coupled with NF-κB inhibition informs future strategies for true ligament healing.

Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2007. / SUPERFICIAL digital flexor tendon injury has a serious negative impact on the competitive horse industry. Injured horses require up to a year of rest for recovery and likelihood of re-injury upon return to normal activity is as high as 80 %. Tendon healing requires (a) production of collagen by fibroblasts, to provide tensile strength and elasticity to the tendon, (b) minimisation of restrictive fibrosis, which compromises tendon gliding function and (c) minimisation of peritendinous adhesions. We review conventional treatments for tendon healing before exploring stem cell application as a therapeutic alternative. We promote the use of hematopoietic and mesenchymal stem cells derived from adult peripheral blood - as opposed to bone marrow-derived stem cells or embryonic stem cell sources - and review published research output in this regard. In conclusion, we outline our research objectives and present and discuss our results in the chapters that follow. Mononuclear cells - consisting of hematopoietic stem cells, mesenchymal stem cells and leucocytes – were isolated from the peripheral blood of sheep and horses through red blood cell lysis and blood plasma extraction. Cell counts and propidium iodide dye exclusion viability tests were conducted on the cell pellets. Sheep sub samples were tested for CD45 expression and horse sub samples for CD4 and CD11a/18 cell surface markers by flow cytometry for characterisation purposes. In both cases, separate sub samples were incubated with matched immunoglobulin (IgG) isotypes, conjugated to fluorescein isothiocyanate (FITC), to serve as controls. For the culture of mononuclear cells, 4.5 x 106 cells were selected for autologous sheep injections, 3 x 106 CD45- cells for allogeneic sheep injections (the latter excluding leucocytes that may induce an immune response) and 72 x 106 cells for horse injections. These cells were incubated with bromo-deoxyuridine (BrdU), cultured and subsets were extracted for a second round of cell counts and viability tests before being resuspended in blood plasma. For the horse samples an additional 1 x 106 mononuclear cells were incubated until reaching 60 % confluence and tested for myogenic differentiation. Low cell mortality and lack of fluorescence from IgG-FITC controls reflected effective protocols and a lack of false positive results. The fact that the equine cell population differentiated into myotubes verified the presence of mesenchymal stem cells in injections. We tested whether surgical incisions or collagenase injections best mimicked naturally occurring tendon injuries and compiled macroscopic and microscopic descriptions of tendon injury sites at seven weeks post-injury. The superficial digital flexor tendons of 27 sheep received an incision, a collagenase injection or a saline control injection. After one week a number of sheep were sacrificed while the remainder received further saline treatment and were sacrificed after another seven weeks. Tendons were examined through clinical observations, image analysis of maximum tendon diameter, mechanical testing and histological sectioning of affected tissues. Collagenase-induced injury resembled tendonitis more closely than surgically-induced injury. Collagenase-injured tendons (a) induced lengthier lameness in affected limbs, (b) were more swollen and difficult to palpate, (c) assumed the bow appearance characteristic of natural injury, (d) experienced extensive haemorrhage due to collagen lysis, (e) had decreased elasticity and capacity to carry loads and stress, (f) displayed decreased stiffness due to collagen fibre disruption and (g) developed severe inflammation. After seven weeks injured tendons displayed increased vascularisation in the areas of haemorrhage and in the adjacent collagen matrix. High inflammation rates and low collagen levels however still persisted. Collagenase injections were used to induce tendonitis in the superficial digital flexor tendons of 27 sheep. After one week these tendons received treatment with a control saline solution, autologous peripheral blood mononuclear cells (MNCs) or allogeneic peripheral blood CD45- MNCs. Healing rates were compared after a further seven week period by conducting ultrasonographic evaluations, clinical observations, image analyses of maximum tendon diameter, mechanical tests and histological investigations. Tendons treated with MNCs displayed an improvement in echogenicity and fibre linearity, higher and more organised collagen levels, stronger mechanical properties and less swelling. Although these improvements were not always significant, they provided strong evidence to suggest marked healing benefits over a longer time period. Collagenase injections were used to induce tendonitis in the superficial digital flexor tendons of four horses. After one week these tendons received treatment with either a control saline solution or autologous peripheral blood mononuclear cells (MNCs). Healing rates were compared after a further seven week period by conducting ultrasonographic evaluations, clinical observations, image analysis of maximum tendon diameter and histological investigations. Tendons treated with MNCs displayed significant improvements in fibre linearity in the direct vicinity of the lesion, as well as recovery rate thereof, and experienced less swelling when compared with their untreated counterparts. Healing trends suggested that, given a longer period of observation post-injury, more significant improvements may become apparent. Human adipose tissue is known be an easily accessible and high yielding source of multipotent mesenchymal stem cells. These stem cells could potentially be used for therapeutic advancement of tendon regeneration. Our first goal was to examine the in vitro myogenic differentiation potential of adipose-derived, adherent mononuclear cells (MNCs) from six adult sheep. The second goal was to characterise the population of cells isolated through various available ovine specific, non-mesenchymal stem cell surface markers, namely, CD1, CD31, CD34 and CD45. After incubation, only four of the six MNC cultures started to proliferate. These four cultures all exhibited high myogenic differentiation ability. The isolated cell populations did not express any of the non-mesenchymal stem cell specific cell surface markers. In conclusion, our data suggests that peripheral blood stem cells and adipose-derived stem cells are important candidate cell types for therapeutic application to improve tendon repair in horses and sheep. Sufficient time must be allowed following injury and prior to stem cell treatment (at least one month) and a controlled exercise program should be followed posttreatment. A larger sample size is required and at least six months of recovery before macroscopic and histological repair can be analysed more accurately and conclusively. Ultrasonography should be carried out on a continuous basis, as it is a non-invasive method of monitoring change over time.

[Truncated abstract] There is limited intrinsic potential for repair in the adult human central nervous system (CNS). Dysfunction resulting from CNS injury is persistent and requires prolonged medical treatment and rehabilitation. The retina and optic nerve are CNSderived, and adult retinal ganglion cells (RGCs) and their axons are often used as a model in which to study the mechanisms associated with injury, neuroprotection and regeneration. In this study I investigated the effects of a variety of strategies on promoting RGC survival and axonal regeneration after optic nerve injury, including the use of reconstructed chimeric peripheral nerve (PN) grafts, gene therapy, and intraocular application of pharmacological agents and other factors . . . C3 transferase is an enzyme derived from Clostridium botulinum that inactivates Rho GTPase. Because SC myelin contains MAG and PN also contains CSPGs, I tested the effects of intraocular injection of a modified form of C3 (C3-11), provided by Dr Lisa McKerracher (CONFIDENTIAL data, under IP agreement with Bioaxone Therapeutic, Montreal) on RGC axonal regeneration into PN autografts. My results showed that there was significantly more RGC survival and axonal regeneration in PN autografts after repeated intraocular injection of C3. I also tested whether intraocular injections of CPT-cAMP and/or CNTF can act in concert with the C3 to further increase RGC survival and/or regeneration. Results showed that the effect of C3 and CPT-cAMP plus CNTF were synergistic and partially additive. The use of combination therapies therefore offers the best hope for robust and substantial regeneration. The overall results from my PhD project will help determine how best to reconstruct nerve pathways and use pharmacological interventions in the clinical treatment of CNS injury, hopefully leading to improved functional outcomes after neurotrauma.

Establishment of animal model for studying treatment efficacy of low-intensity pulsed ultrasound stimulations for accelerating bone-tendon repair. Standard partial patellectomy was conducted in the 18-week old rabbits that were then divided into the LIPUS treatment and control groups. The animals were followed for 2, 4, 8, and 16 weeks for various tissue analyses. LIPUS was applied to the experimental animals from postoperative day 3 to 16 weeks. We demonstrated that the healing process of PPT junction was initiated through endochondral ossification. The results showed that the size and length of newly formed bone, and its bone mineral content (BMC), but not its bone mineral density (BMD) were correlated with the failure load, ultimate strength and energy at failure. Using radiographic, biomechanical, histomorphologic and biomechanical methods, it was found that LIPUS had significant accelerating effect on PPT junction repair. We validated our study hypothesis in that LIPUS enhances bone-tendon junction healing by stimulating angiogenesis, chondrogenesis and osteogenesis. / Establishment of in vitro model for mechanism study on effects of low-intensity pulsed ultrasound stimulations. An in vitro model of osteoblast-like cell line (SaOS-2 cells) was studied using cDNA microarray to explore the molecular mechanism mediated by LIPUS. This microarray analysis revealed a total of 165 genes that were regulated at 4 and 24 hours by LIPUS treatment in osteoblastic-like cells. These genes belonged to more than ten protein families based on their function and were involved in some signal transduction pathways. This study has validated the hypothesis that LIPUS can regulate a number of critical genes transient expressions in osteoblast cell line Saos-2. / Keywords. partial patellectomy model; bone-tendon junction repair; low intensity pulsed ultrasound stimulations (LIPUS); gene expression; complementary DNA microarray; rabbit. / This study explored the intact morphology, regular healing and the augmented healing under the effects of low intensity pulsed ultrasound stimulations (LIPUS) on the patella-patella tendon (PPT) junction in a rabbit partial patellectomy model. To probe its possible mechanism, the key genes involved in regulating osteogenesis mediated by LIPUS were identified using the state-of-the-art methods---complementary DNA microarray. / Lu Hongbin. / "June 2006." / Advisers: Ling Qin; Kwok Sui Leung. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1548. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 259-288). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

A "Job Coach" model was developed based on sports medicine and rehabilitation principles for athletes, and individual placement and supported employment for people with psychiatric disabilities. This is a biopsychosocial model, emphasising workplace-based intervention. / Background. Occupational musculoskeletal soft tissue injuries represent a major source of work disability. There has been a gradual rise in the occurrence of occupational musculoskeletal injuries of the upper extremity, including both acute injuries and more chronic health problems. Return to work following an occupational injury is a multifactoral process, although traditional clinic-based rehabilitation programmes do not appreciate the importance of contextual factors. / Conclusion. Workplace work hardening programmes are a further development of work rehabilitation programmes. The therapeutic use of actual work facilities and the work environment can effectively facilitate the successful return to work process of the injured worker. More importantly, many of the psychosocial problems associated with separation from the work routine, peer group and/or the employer are minimised by the presence of the Job Coach. The results of this study confirm that workplace-based rehabilitation intervention is more effective than conventional clinic-based rehabilitation programmes in terms of prevention of further work disability, improvement in functional capabilities and decrease in perceived pain and disability. / Keywords. Job coach, workplace-based rehabilitation, rotator cuff injury, work disability, return to work intervention. / Methodology. A randomised controlled trial was conducted on 94 workers recruited from Workers' Compensation insurers. These workers had all sustained occupational rotator cuff injury and had lost more than 90 workdays. The workers were randomly assigned into control or experimental groups. The control group received a traditional work hardening programme and the experimental group received a workplace work hardening programme using the Job Coach model. The return to work outcomes of the two groups were compared. Areas of comparison included return to work rate after training, job retention ability and impact on earning capacity. Other outcome measures included change in active range of motion of the shoulder joint, eight basic functional capacities and the worker's perception of shoulder pain and disability based on the Shoulder Pain and Disability Index (SPADI). / Results. After one-month of the training programme, a higher return to work rate was obtained in the experimental group compared to the control group (71.4% against 37%, chi2=11.095, p=0.001). For job retention ability, 93.5% of the workers in the experimental group were still at work compared to 72.9% of workers in the control group (chi 2=7.031, p=0.008). No obvious salary change was noted between the two groups. / The SPADI was statistically significantly lower in the experimental group than in the control group (p=0.032), meaning that workers in the experimental group had fewer shoulder problems after training. Other significant improvements were noted in active shoulder flexion (p=0.001), arm lift strength (p=0.01), high-near lift strength (p=0.014), dynamic carrying strength (p=0.007) and overhead work tolerance (p=0.032), all of which were found to be statistically significantly higher in the experimental group than in the control group. / Cheng Shu-kei. / "July 2006." / Adviser: Leung Kim Hung. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1568. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 216-238). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Traumatic brain injury (TBI) is the signature injury of modern military conflicts due to widespread use of improvised explosive devices (IEDs) and modern body armor. However, the exact biophysical mechanisms of blast-induced traumatic brain injury (bTBI) and its pathological effects on the blood-brain barrier (BBB) – a structure essential for maintaining brain homeostasis – remain poorly understood. The specific aims of this thesis are to: 1) determine a threshold for primary blast-induced BBB dysfunction in vitro; 2) determine the effect of repeated blast on BBB integrity in vitro; 3) improve BBB recovery in vitro as a potential therapeutic strategy for mitigating effects of blast; and 4) quantify the time course and pore-size of BBB opening in vivo. In this work we utilized a shock tube driven by compressed gas to generate operationally relevant, ideal pressure profiles consistent with IEDs. By multiple measures, the barrier function of an in vitro BBB model was disrupted after exposure to a range of blast-loading conditions. Trans-endothelial electrical resistance (TEER) decreased acutely in a dose-dependent manner that was most strongly correlated with impulse, as opposed to peak overpressure or duration. Significantly increased hydraulic conductivity and solute permeability post-injury further confirmed acute alterations in barrier function. Compromised ZO-1 immunostaining identified a structural basis for BBB breakdown. These results are the first to demonstrate acute disruption of an in vitro BBB model after primary blast exposure; defined tolerance criteria may be important for development of novel helmet designs to help mitigate effects of blast on the BBB. After determining that exposure to a single primary blast caused BBB disruption, we hypothesized that exposure to two consecutive blast injuries would result in exacerbated damage to the BBB in vitro. However, contrary to our hypothesis, repeated mild or moderate primary blast delivered within 24 or 72 hours did not significantly exacerbate reductions in TEER across a brain endothelial monolayer compared to sister cultures receiving a single exposure. Single blast exposure significantly reduced immunostaining of ZO-1 and claudin-5 tight junction proteins, but subsequent exposure did not cause additional damage to tight junctions. The second injury delayed recovery of TEER and hydraulic conductivity in BBB cultures. Extending the inter-injury interval to 72 hours, the effects of repeated injury on the BBB were independent given sufficient recovery time between consecutive exposures. Investigation of repeated blast on the BBB will help identify a temporal window of vulnerability to repeated exposure. Restoration of the BBB after blast injury has emerged as a promising therapeutic target. We hypothesized that treatment with dexamethasone (DEX) after primary blast would potentiate recovery of an in vitro BBB model. DEX treatment resulted in complete recovery of TEER and hydraulic conductivity 1 day after injury, compared with 3 days for vehicle-treated injured cultures. Administration of RU486 (mifepristone) inhibited effects of DEX, confirming that barrier restoration was mediated by glucocorticoid receptor signaling. Potentiated recovery with DEX treatment was accompanied by stronger ZO-1 tight junction immunostaining and expression, suggesting that increased ZO-1 expression was a structural correlate to BBB recovery. This is the first study to provide a mechanistic basis for potentiated functional recovery of an in vitro BBB model due to glucocorticoid treatment after blast injury. Using an in vivo bTBI model, systemic administration of sodium fluorescein (NaFl; 376 Da), Evans blue (EB; 69 kDa when bound to serum albumin) and dextrans (3 – 500 kDa) was used to estimate the pore-size of BBB opening and time required for recovery. Exposure to blast resulted in significant acute extravasation of NaFl, 3 kDa dextran, and EB. However, there was no significant acute extravasation of 70 kDa or 500 kDa dextrans, and minimal to no extravasation of NaFl, dextrans, or EB 1 day after exposure. This work is the first to quantify the time course and size of BBB opening after bTBI, suggesting that the BBB recovered 1 day post-injury. This study supports our hypothesis that transient opening of the BBB may permit serum-components to infiltrate the brain parenchyma and contribute to pathological secondary cascades. This research has shown that BBB damage, demonstrated in vitro and in vivo, is a major mechanism contributing to vascular and neuronal pathology of bTBI at exposure levels above a critical threshold. Compared with published studies on blast-induced damage to the BBB, we have developed primary blast injury tolerance criteria by precisely controlling the biomechanical initiators of injury and measuring resulting alterations to the structure and function of an in vitro BBB model by methods not possible in vivo. We have also developed a potential glucocorticoid treatment to rapidly restore the BBB after injury, which may lead to more promising therapeutic strategies to treat TBI-related pathologies. This work will also guide the development of novel armor designs to protect service members and civilians in order to more effectively address the burden to society of bTBI.

影響全球數百萬的患者的慢性傷口，以其持續性過度發炎，纖維細胞增殖放緩，及血管生成受損為表徵。藥用草藥地黃已證明在大鼠糖尿足模型上有顯著傷口癒合作用。然而，關於地黃的炮製及其活性成分對此等傷口癒合的活動主要是未知的。 / 我們首先在地黃的炮製中，以抗一氧化氮（NO）和纖維細胞增殖實驗，確定了乾地黃表現出有效的傷口癒合活動。採用多方位生物活性導引分離（BGF），我們進一步研究乾地黃在抗炎，纖維細胞增殖，血管生成的活性成分，分別以抗NO產生，纖維細胞增殖，和TG(fli1:EGFP)y1/+(AB)斑馬魚芽血管生成模型為生物測定。此等具傷口癒合效果的活性成分將會作進一步研究。此外，我們會以電子細胞基質阻抗判斷(ECIS)的技術，對名為NF3(含RR的中草藥配方)在人類血管內皮細胞(HECV)上作體外血管生成及其信息的研究。 / 通過抗NO測定導引分離，我們證明活性萃取物C3比其粗提物擁有100倍更有效的抗炎效果。C3中含地黃苦甙元。地黃苦甙元可顯著地抑制NO的產生。C3中地黃苦甙元的存在可能是其具抗炎的原因。C3進一步以抑制一氧化氮合酶(iNOS)，環氧合酶-2(COX-2)和細胞白介素第六因子(IL-6)的基因，蛋白質，及/或中介物的表達，證明其舒緩炎症的作用。因此，C3可能對慢性傷口癒合中的炎症有治療作用。此外，我們發現先從水提的RR粗提物，再以乙酸乙酯提取的活性萃取物C2-B4，證明此萃取物在纖維細胞增殖中具最有效及劑量依存作用。 / 斑馬魚芽誘導模型導引分離顯示，C1-1萃取物在RR中具有最有效的血管生成作用。為斑馬魚芽誘導度身設計的30個血管生成相關基因顯示，C1-1廣泛地引發血管生成中的基因差異表達，特別在生長因子和血管穩定方面。就促進血管生成，C1-1進一步以體外人類微血管內皮細胞的血管生成檢測進行研究，結果發現C1-1具細胞移動及類血管生成能力。此外，降毛荚醛(norviburtinal)，在地黃提取物中首次被發現和具有血管生成作用。如此，C1-1和降毛荚醛為RR中血管生成作用的活性成分。 / 此外，應用ECIS技術，含RR配方的NF3能通過磷酯肌醇激酶(PI3K)及WiskottAldrich氏症候群神經元蛋白 (N-WASP) 路徑在人類血管內皮細胞（HECV）上誘導內皮細胞粘附，遷移及類血管生成。西方墨點法分析表明，NF3 在HECV上激活Akt和有絲分裂活化蛋白質激酶(MAPKs)的表達。這些誘導在各方面促進血管生成。因此，這顯示NF3能在分子及功能上激活血管生成作用的複雜性。此外，我們進一步支持ECIS在血管內皮細胞篩選傷口癒合劑中的高靈敏度。 / 總括而言，通過靶向抗炎，纖維細胞增殖增長和改善血管生成，各地黃生物活性導引化合物和萃取物，及其含RR的配方，可以在治療慢性傷口癒合上發揮功效。 / Chronic wounds, which influence millions of patients worldwide, are manifested with its sustained hyperinflammation, slackened fibroblast proliferation, and impaired angiogenesis. Agents retrieving these activities could facilitate the healing. Medicinal herb, Rehmanniae Radix (RR) demonstrated profound wound healing effect in rat diabetic foot model. However, the subtypes and the active components behind RR for such wound healing activities were largely unknown. / Here we firstly identified that dried RR, among its subtypes, exhibited potent wound healing activities through nitric oxide (NO) anti-inflammatory and fibroblast proliferation assays. Using multi-directional bioassay-guided fractionation (BGF), we further studied the active component(s) of dried RR in anti-inflammation, fibroblast proliferation, and angiogenesis, respectively, by anti-NO production, fibroblast proliferation, and TG(fli1:EGFP)[superscript y]¹/+(AB) zebrafish sprout angiogenesis model. Active component(s) of such wound healing effects were further characterized. Furthermore, with a RR-containing herbal formula, NF3, the in vitro angiogenic activities and its underlying signaling of NF3-treated human vascular endothelial cells (HECV) were studied using electric cell-substrate impedance sensing (ECIS) technology. / Via anti-NO assay-guided fractionation of dried RR, we demonstrated that the sub-fraction C3, possessed 100-fold more potent anti-inflammatory effect than that of the crude extract. Characterization of C3 showed that the anti-inflammatory activity could be partly due to the presence of rehmapicrogenin, which could significantly inhibit NO production. C3 was further demonstrated in blocking inflammation by inhibiting gene, protein, and/or mediator expression of inducible NO synthase, COX-2 and IL-6. Hence, C3 could be useful in treating inflammation in chronic wound healing. Additionally, we revealed that an active sub-fraction, C2-B4, from the ethyl acetate extract of the aqueous extract of RR, demonstrated the most potent and dose-dependent fibroblast proliferative effect. / Zebrafish sprout-inducing model-guided fractionation suggested C1-1 sub-fraction possessed the most potent angiogenesis effect in RR. A 30 tailor-made angiogenesis-associated gene panel designed for zebrafish sprout angiogenesis revealed that C1-1 triggered differential gene expression across wide angiogenic events, particularly concerned with those of growth factors and vessel stabilization. The pro-angiogenic activity was further supported by in vitro human microvascular endothelial cell-based angiogenesis assays, with C1-1 being pro-motogenic and tubule inducing. Also, norviburtinal was, for the first time, found in the extract of RR and possessed novel angiogenesis effect. Thus, C1-1 and norviburtinal were the active components responsible for the pro-angiogenesis effect of RR. / Moreover, RR-containing formula (NF3), which induced endothelial cell attachment, migration, and tubule formation in human vascular endothelial cell (HECV), could be mediated through PI3K and N-WASP pathways. Activated Akt and MAPK kinases expression in western blot analysis were also demonstrated in NF3-treated HECV. These inductions would promote angiogenesis at various levels. Hence, the complexity of angiogenesis effect activated by the NF3 treatment molecularly and functionally was shown, and we further supported the high sensitivity of ECIS in the screening of wound healing agents with endothelial cells. / In conclusion, through targeting anti-inflammation, elevated fibroblast proliferation and improved angiogenesis, our respective bioassay-guided active fractions and compounds in Rehmanniae Radix, and the RR-containing formula, could play beneficial uses in treating chronic wound healing. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Cheuk Lun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 221-249). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Table of Contents --- p.i / Abstract (in English) --- p.vi / Abstract (in Chinese) --- p.ix / Statement of Originality --- p.xiii / Acknowledgements --- p.xiv / Publications --- p.xiv / List of Tables --- p.xvii / List of Figures --- p.xviii / List of Abbreviations --- p.xxi / Chapter Chapter 1: --- Literature Review and Study Objectives / Chapter 1.1 --- Overview on wound healing / Chapter 1.1.1 --- Normal wound healing --- p.1 / Chapter 1.1.2 --- Chronic wound healing / Chapter 1.1.2.1 --- Venous ulcers --- p.8 / Chapter 1.1.2.2 --- Diabetic ulcers --- p.11 / Chapter 1.1.2.3 --- Mechanism of chronic wound healing --- p.13 / Chapter 1.1.2.4 --- Current treatments for chronic wound healing --- p.19 / Chapter 1.2 --- Rehmanniae Radix (RR) overview / Chapter 1.2.1 --- RR and its subtypes --- p.29 / Chapter 1.2.2 --- Chemistry of RR --- p.34 / Chapter 1.2.3 --- Pharmacology of RR --- p.38 / Chapter 1.2.3.1 --- RR and chronic wound healing / Chapter 1.3 --- Study objectives --- p.42 / Chapter Chapter 2: --- comparison of wound healing effect of the subtypes of rr / Chapter 2.1 --- Introduction --- p.43 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Preparation and authentication of subtypes of RR --- p.46 / Chapter 2.2.2 --- RAW 264.7 murine macrophage culture and sample treatment protocol, and cell viability test --- p.47 / Chapter 2.2.3 --- Nitric oxide inhibitory assay --- p.47 / Chapter 2.2.4 --- Hs27 human fibroblast culture and sample treatment protocol --- p.48 / Chapter 2.2.5 --- Fibroblast proliferation assay --- p.48 / Chapter 2.2.6 --- Statistical analysis --- p.49 / Chapter 2.3 --- Results --- p.63 / Chapter 2.3.1 --- Nitric oxide anti-inflammatory effect of the subtypes of RR --- p.49 / Chapter 2.3.2 --- Fibroblast proliferative effect of the subtypes of RR --- p.52 / Chapter 2.4 --- Discussion --- p.54 / Chapter Chapter 3: --- Fibroblast proliferative effect of RR / Chapter 3.1 --- Introduction --- p.57 / Chapter 3.2 --- Methods / Chapter 3.2.1 --- Preparation of aqueous extracts of RR --- p.61 / Chapter 3.2.2 --- Hs27 human fibroblast culture and sample treatment protocol --- p.61 / Chapter 3.2.3 --- Hs27 human fibroblast proliferation assay --- p.61 / Chapter 3.2.4 --- Bioassay-guided fractionation of RR --- p.61 / Chapter 3.2.5 --- LC-MS analysis of bioassay-guided fraction, C2-B4 --- p.65 / Chapter 3.2.6 --- Statistical analysis --- p.66 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Fibroblast proliferative effect of RR aqueous crude extract and its bioassay-guided fractions --- p.67 / Chapter 3.3.2 --- Chemical structure of the isolated compounds --- p.70 / Chapter 3.3.3 --- LC-MS analysis of bioassay-guided fraction, C2-B4 --- p.72 / Chapter 3.4 --- Discussion --- p.73 / Chapter Chapter 4: --- Anti-inflammatory effect and its underlying mechanism of RR / Chapter 4.1 --- Introduction --- p.77 / Chapter 4.2 --- Methods / Chapter 4.2.1 --- Preparation of aqueous extracts of RR --- p.82 / Chapter 4.2.2 --- RAW 264.7 murine macrophage culture and sample treatment protocol, and cell viability test --- p.82 / Chapter 4.2.3 --- Assay for nitric oxide inhibitory effect using RAW264.7 cells --- p.83 / Chapter 4.2.4 --- Bioassay-guided fractionation of RR --- p.83 / Chapter 4.2.5 --- Ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS) analysis of sub-fraction, C3 --- p.85 / Chapter 4.2.6 --- Prostaglandin E2 (PGE2) and interleukin-6 (IL-6) assay --- p.87 / Chapter 4.2.7 --- Real-time PCR --- p.87 / Chapter 4.2.8 --- Western blot analysis --- p.89 / Chapter 4.2.9 --- Statistical analysis --- p.91 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Nitric oxide inhibitory effects of L-NMMA, RR aqueous crude extract and its bioassay guided fractions --- p.92 / Chapter 4.3.2 --- LC-MS analysis of bioassay-guided fraction, C3 --- p.96 / Chapter 4.3.3 --- Suppression of inflammation-related mRNA expression level in macrophages by C3 --- p.97 / Chapter 4.3.4 --- Suppression of protein expression of inducible nitric oxide synthase and COX-2 in macrophages by C3 --- p.99 / Chapter 4.3.5 --- Inhibition of release of inflammatory cytokine in macrophages by C3 --- p.102 / Chapter 4.4 --- Discussion --- p.103 / Chapter Chapter 5: --- Angiogenesis effect and its underlying mechanism of RR / Chapter 5.1 --- Introduction --- p.111 / Chapter 5.2 --- Methods / Chapter 5.2.1 --- Preparation of aqueous extracts of RR --- p.113 / Chapter 5.2.2 --- Zebrafish culture --- p.113 / Chapter 5.2.3 --- Collection of zebrafish embryos and herbal treatment protocol --- p.115 / Chapter 5.2.4 --- Microinjection of vascular endothelial growth factor (VEGF) to zebrafish embryos --- p.116 / Chapter 5.2.5 --- Screening of zebrafish embryos using fluorescence microscopy --- p.116 / Chapter 5.2.6 --- Bioassay-guided fractionation of RR / Chapter 5.2.7 --- Isolation and structure elucidation of compound C2A --- p.120 / Chapter 5.2.8 --- Gas chromatographymass spectrometry (GC-MS) analysis of bioassay-guided fraction of RR, C1-1 --- p.120 / Chapter 5.2.9 --- Detection of zebrafish mRNA expression level by real-time PCR (RT-PCR) --- p.122 / Chapter 5.2.10 --- Human microvascular endothelial cell (HMEC-1) culture and sample treatment protocol --- p.125 / Chapter 5.2.11 --- HMEC-1 proliferation assay --- p.125 / Chapter 5.2.12 --- HMEC-1 scratch assay --- p.126 / Chapter 5.2.13 --- HMEC-1 tubule formation assay --- p.126 / Chapter 5.2.14 --- Statistical analysis --- p.127 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Angiogenesis effects of RR aqueous crude extract, its bioassay-guided fractions and isolated compound, in zebrafish model --- p.128 / Chapter 5.3.2 --- Chemical structure and angiogenesis effect of the isolated compound C2A, norviburtinal --- p.133 / Chapter 5.3.3 --- Components of C1-1 from GC-MS analysis --- p.135 / Chapter 5.3.4 --- Angiogenesis effect of C1-1 in angiogenesis-related mRNA expression level in zebrafish --- p.137 / Chapter 5.3.5 --- Effect of endothelial cell proliferation of C1-1 in HMEC-1 cell --- p.142 / Chapter 5.3.6 --- Cell migration effect of C1-1 in HMEC-1 cell --- p.143 / Chapter 5.3.7 --- Tubule formation of C1-1 in HMEC-1 cell --- p.145 / Chapter 5.4 --- Discussion --- p.147 / Chapter Chapter 6: --- Angiogenesis effect and its underlying mechanism of RR AND AR-containing two-herbs formula, NF3 using ecis model / Chapter 6.1 --- Introduction --- p.165 / Chapter 6.2 --- Methods / Chapter 6.2.1 --- Preparation and authentication of aqueous extracts of NF3 --- p.176 / Chapter 6.2.2 --- Human vascular endothelial cells (HECV) culture --- p.177 / Chapter 6.2.3 --- HECV cell proliferation assay --- p.178 / Chapter 6.2.4 --- Scratch assay --- p.178 / Chapter 6.2.5 --- Tubule formation assay --- p.179 / Chapter 6.2.6 --- Electric cell-substrate impedance sensing (ECIS)-based cell attachment and motility assay --- p.180 / Chapter 6.2.7 --- Western blot analysis --- p.181 / Chapter 6.2.8 --- Statistical analysis --- p.182 / Chapter 6.3 --- Results / Chapter 6.3.1 --- LC-MS analysis of NF3 --- p.184 / Chapter 6.3.2 --- Effects of NF3, AR and RR on cell viability and migration of HECV --- p.185 / Chapter 6.3.3 --- Tubule formation effect of NF3, AR and RR in HECV cell --- p.188 / Chapter 6.3.4 --- Effects of NF3, AR, and RR on HECV cell attachment using ECIS model --- p.190 / Chapter 6.3.5 --- Effects of NF3, AR and RR on HECV cell migration using ECIS model --- p.191 / Chapter 6.3.6 --- Effects of NF3 on HECV for MAPK and Akt protein activation --- p.195 / Chapter 6.4 --- Discussion --- p.197 / Chapter Chapter 7: --- General Discussion and Conclusions / Chapter 7.1 --- General discussion and conclusions --- p.206 / Chapter 7.2 --- Limitation of the study --- p.215 / Chapter 7.3 --- Future work --- p.215 / Appendix --- p.218 / References --- p.221

Spinal cord injury (SCI) is a devastating disease which poses health problems in human and veterinary medicine. SCI causes neurological disability, with loss of motor, sensory and autonomic function. This study investigated the efficacy of local treatment with IKVAV-peptide on spinal cord regeneration following compression injury at T12 vertebra in Balb-c mice. IKVAV-peptide is a membrane spanning peptide known to have a long half-life and the peptide motif IKVAV. Thirty Blab-c female mice were used. Hemilaminectomy was performed at T12 and spinal cords were compressed using extradural application of a 24 g modified aneurysm clip for 1 min in the treatment groups. After 24 hours mice were treated with one of 4 different treatments including isoleucine-lysine-valine-alanine-valaine(IKVAV), IKVAVpeptide, peptide and mannitol (vehicle). Functional improvement was assessed every day using Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale. 28 days later, the mice were euthanized, and spinal cord segments were studied histologically. Statistical analysis, one-way and two-way analysis of variance (ANOVA) and linear regression model were used to measure some parameters and describe the outcome after SCI. Over a 4weeks period, IKVAV-peptide group demonstrated statistical and histological evidence of cellular reconstruction and behavioral improvement. The BBB score in the IKVAV-peptide group increased by 5.4 (25%) points, the IKVAV and peptide groups by approximately 1 point (5%) and the mannitol group by 4 points (19%). The number of protoplasmic astrocytes in the IKVAV-peptide group was significantly increased compared to IKVAV, mannitol and normal groups but not with the peptide group (p<0.001). Neuron and muscle bundle size were also increased significantly (p<0.05 and p<0.007, resp.) in the IKVAV-peptide group compared to other treatment groups. The treated control groups showed cellular and gross damages including neuron inactivation and muscle atrophy, gliosis and inability of movement. / Graduation date: 2013

With over 50,000 deaths and 80,000 disorders annually in the United States resulting from traumatic brain injury (TBI), there is a demand for improved therapeutic strategies. Cell transplantation offers the potential to treat TBI by targeting multiple mechanisms in a sustained fashion. However, efforts are needed to improve survival and integration of transplanted cells, and ultimately enhance functional recovery. Using tissue engineering strategies, we aimed to mimic key aspects of fetal tissue grafts by combining neural stem cells with a fibronectin or laminin based scaffold that could be delivered to the injured brain in a minimally invasive fashion. We found that the incorporation of extracellular matrix proteins into a cell transplantation paradigm led to improved donor cell survival and restored cognitive ability for treated animals. To begin to examine how fibronectin and laminin mediate these improvements, we first examined the endogenous role of these two proteins in the injured brain. Using a clinically-relevant model of TBI, we found both proteins are increased in the injured brain at acute time points. The spatial localization of fibronectin and laminin with specific support cells in the brain suggests a role for these proteins in repair, warranting further investigation. Using conditional plasma fibronectin knockout animals, we found that fibronectin is neuroprotective to the traumatically injured brain. Specifically, injured fibronectin knockout animals had more severe motor and cognitive deficits, increased cell death, and decreased retention of phagocytic cells compared to injured wild type animals. Thus, we have identified novel therapeutic treatments for TBI which utilize tissue engineered transplants and/or exploit endogenous repair mechanisms for fibronectin.