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Transforming growth factor-βs (TGF-βs) are highly expressed in neural development but why the adult nervous system continues to express them is unclear. TGF-β2 is concentrated at mature neuromuscular junctions (NMJs) of mammalian skeletal muscle fibres, and the nerve terminal expresses TβR-II receptors. To test the role of TGF-β2 at mammalian NMJs, I performed four experiments. The first study tested whether TGF-β2 acutely modulates synaptic transmission at mature mammalian NMJs. Second, I asked if chronically reduced TGF-β2 expression disrupts synaptic transmission. Third, I asked if TGF-β2’s effects differ in terminals adapted to different activity patterns in vivo. Lastly, I asked whether TGF-β1, a related peptide to TGF-β2, is distinct in terms of its effects on transmitter release. Using single electrode potential recording, I found TGF-β2 significantly increased the amplitude of spontaneous released single neurotransmitter vesicles (miniature endplate potentials, MEPPs) and nerve stimulation evoked multi-vesicular release (endplate potentials, EPPs). These effects were blocked by L-vesamicol, a vesicular acetylcholine transporter inhibitor, and bafilomycin, a proton pump inhibitor, suggesting the increase in MEPP/EPP amplitude is due to increased vesicle filling presynaptically. These effects were also blocked by the MARK inhibitors, UO126 and PD98059, suggesting TGF-β2 acts via a MARK-dependent pathway. Postsynaptically, two electrode recording showed postsynaptic potential amplitude was enhanced by an increased fibre input resistance, suggesting TGF-β2 also acts postsynaptically. TGF-β2 reduced the number of vesicles released per stimulus (quanta content, QC) but this was blocked by atropine, showing this was indirect through autoreceptor negative feedback. Voltage clamp recording showed TGF-β2 significantly increased the miniature end plate currents (MEPCs), but not the end-plate currents (EPCs), supporting my initial hypothesis that TGF-β2 acts mainly presynaptically to increase vesicle filling. In TGF-β2+/- mice, I found greater MEPP amplitude variability. This supports my previous findings that TGF-β2 modulates vesicle filling. Unexpectedly, there was an excess in larger MEPP sizes (>0.88 mV), perhaps reflecting upregulation of either presynaptic signalling or another synaptic mediator. Two MEPP amplitude populations were induced in TGF-β2-treated TGF-β2+/- mouse NMJs, similar to the bimodal vesicle population in electroplaques. The extensor digitorum longus (EDL, ~95% fast fibres) and soleus (SOL, ~95% slow fibres) were used to investigate whether the TGF-β2-mediated effect differed between fibre types. Overall, TGF-β2 increased the quantal size (MEPP amplitude) in NMJs of both muscles, suggesting this effect is not fibre-type specific and, together with results in mice, that the TGF-β2-mediated increase in vesicle filling is common to all mammalian neuromuscular terminals. With respect to EPP amplitude and QC, the results differed between muscles. In EDL, the EPP amplitude was not significantly changed, whereas it increased in SOL. In EDL, QC was reduced but not in SOL. These difference compared to diaphragm perhaps do reflect muscle fibre-type dependent differences. TGF-β1, at 0.1 ng/ml, significantly reduced quantal size – the opposite of TGF-β2 at any concentration. One explanation would be that a receptor inhibition by TGF-β1 at low concentration interferes with endogenous TGF-β2 binding/receptor activation at the NMJ. However, when the TGF-β1 concentration was raised to 1 ng/ml, like TGF-β2, it significantly increased MEPP amplitude. This suggests that perhaps sufficient binding of TGF-β1 results in the receptor activation of TGF-β2 like signalling. Overall, I conclude that TGF-β2 enhances the size of spontaneous synaptic potentials in all types of muscle fibres, and this is much more rapid (1 hr vs 1 day) than at central neurone synapses in culture. Detailed study in the rat diaphragm shows it increased the evoked EPP amplitude, reduced QC and increased postsynaptic input resistance. Together, TGF-β2 would therefore enhance the postsynaptic depolarisation increasing synaptic strength, and by reducing QC, increase the efficiency of neurotransmission at mammalian NMJs. While unimportant for single stimuli in healthy terminals, by conserving vesicle use, it may help maintain release during stimulus trains, especially during neuromuscular disease.

The release of genome sequence information is revolutionising the study of helminth parasites by providing important datasets for comparative genomics, allowing the comprehensive analysis of signalling pathways that regulate nematode development. Much of the current knowledge of nematode signalling pathways is based on studies of the free-living model Caenorhabditis elegans. The recent availability of the Trichinella spiralis genome sequence presented an opportunity to study signalling pathways of this, and other, parasitic nematodes, providing a phylum-wide overview of a given signalling pathway. The transforming growth factor-β (TGF-β) ligands are a superfamily of structurally related polypeptides that regulate a wide range of cellular processes in animal tissues. Since the discovery that the TGF-β daf-7 regulates the developmentally-arrested dauer stage in C. elegans, there is the potential for TGF-β signalling to regulate developmental arrest, parasite development and even host-parasite communication in T. spiralis and other nematodes. In the present study, thirteen genes encoding putative TGF-β signalling components, from T. spiralis, have been identified and characterised. Phylogenetic analysis suggests that daf-7 is not conserved beyond C. elegans and that functional extrapolation from C. elegans biology to distantly-related nematodes is difficult. Furthermore, the analysis herein shows a high level of divergence among parasitic nematode TGF-βs. Since the last common ancestor of T. spiralis and C. elegans was the ancestor of the entire nematode phylum, these comparisons allow speculation on the TGF-β signalling networks of the ancestral nematode and provide information on the emergence of TGF-β signalling in animals. ES products from T. spiralis are shown to be capable of interacting directly with mammalian cell receptors and utilise their receptors to control gene expression in vitro. This presents the possibility that these TGF-β ligands may play a part in the formation and maintenance of the host-parasite complex.

Tendon, ligament, and joint capsular injuries represent 45% of the 32 million musculoskeletal injuries each year in the United States. Tendon injuries are especially common, requiring surgical repair for the shoulder’s rotator cuff tendons (51,000 per year), the Achilles tendon (44,000 per year), and the patellar tendon (42,000 per year). Tissue engineering provides an alternative in the treatment of tendon lesions through replacement of an injured tendon segment. The purpose of this study was to develop a tendon construct in vitro for clinical reconstructive surgery. Human tenocytes were isolated from hamstring tendons of patients who had undergone anterior cruciate ligament (ACL) surgeries. These tenocytes were cultured with culture media (α-MEM) supplemented with various concentrations of foetal bovine serum (FBS) (0%, 1%, 5% and 10%) and in the presence of different growth factors such as PDGFBB (0, 5, 10 and 50ng/ml), basic FGF (0, 5, 10 and 50ng/ml), IGF-1 (0, 10 and 50ng/ml) and TGFβ-3 (0, 1 and 10ng/ml). Fractional factorial design was utilized to select the combinations of growth factors that supported the following criteria: (1) the maximal cell proliferation with a minimum differentiation of the tenocytes in the presence of the least concentration of FBS possible and (2) maintaining cell survival and promoting tenocyte differentiation in FBS free culture media. The results have shown that: (i) The tenocyte cell number when cultured for 14 days in media supplemented with 1% FBS, 50ng/ml PDGFBB and 50ng/ml bFGF matched that of the positive control (10% FBS-treated cells). Not only was the collagen synthesis significantly reduced in these growth factor-treated cultures compared to positive control tenocytes, but also a significant inhibition of the mRNA expression of various tenocyte differentiation markers (Scleraxis, Tenomodulin, Collagen type I and Decorin) was evident. IGF-1 did not promote significant cell proliferation under low serum conditions but did induce tenocyte differentiation in vitro. Examination of the cell morphology confirmed that tenocytes were capable of less differentiation when cultured with 1% FBS, 50ng/ml PDGFBB and 50ng/ml bFGF, this culture condition was termed “the expansion phase”; (ii) The cell survival was maintained for up to 14 days in serum free culture media supplemented with 50ng/ml IGF-1 and 10ng/ml TGFβ-3 whilst cell differentiation was enhanced and evident by the increase in collagen synthesis and cell morphology. Furthermore, mRNA expression of the aforementioned cell differentiation markers were also significantly increased, this culture condition was termed “the differentiation phase”; (iii) By combining the culture condition optimized for the expansion and differentiation phase sequentially, it was possible to maintain a long term 2-D tenocyte culture in vitro for up to 28 days. In these cultures, the presence of dense collagen formation was clearly evident whereas in positive control group (10% FBS group) such observation was not noted even after prolonged culturing period of up to 45 days. These results suggested that the sequential treatment of tenocytes with growth factors identified for the expansion and differentiation phases was significantly more superior than the standard 10% FBS treatment; (iv) By combining the expansion and differentiation phases optimized for the 2-D cultures, it was possible to maintain human tenocytes in a 3-D scaffold (Bombix silk) for up to 28 days. The tendon like constructs that were formed, macroscopically and microscopically resembled the human hamstring tendon. This observation was confirmed by using H&E staining, scanning electron microscopy and by detecting collagen type I immunohistochemically; (v) It was possible to further validate these findings using in vivo animal models. This was undertaken by implanting the tenocytes cultured sequentially in the defined culture media described above, into the quadriceps of Balb/c nude male mice for up to 30 days. The nature and specificity of the tendon like structure that was formed after this implantation was investigated by H&E staining and immunohistochemistry. It was revealed that the culture conditions that were optimized during the expansion and differentiation phases were suitable for generating a human tendon reconstruct; a finding which is of significance due to its potential for tendon reconstructive surgery.

Sy Chun Choi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 123-160). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENT --- p.vi / Chapter 1. --- Introduction / Chapter 1.1 --- General review of the testis --- p.1 / Chapter 1.1.1 --- Structure and function of the testis --- p.1 / Chapter 1.1.2 --- Testicular vasculature --- p.2 / Chapter 1.1.3 --- Testicular angiogenesis --- p.3 / Chapter 1.2 --- Vascular endothelial growth factor (VEGF) --- p.4 / Chapter 1.2.1 --- Discovery of VEGF --- p.4 / Chapter 1.2.2 --- Organization of VEGF --- p.4 / Chapter 1.2.3 --- Properties of the VEGF isoforms --- p.5 / Chapter 1.2.4 --- VEGF receptors --- p.6 / Chapter 1.2.5 --- Functions of VEGF --- p.8 / Chapter 1.3 --- VEGF in the testis --- p.10 / Chapter 1.3.1 --- Localization of VEGF and VEGF receptors in the testis --- p.10 / Chapter 1.3.2 --- Postulated functions of VEGF in the testis --- p.11 / Chapter 1.4 --- Regulation of VEGF --- p.11 / Chapter 1.4.1 --- "VEGF, hypoxia and the testis" --- p.11 / Chapter 1.4.2 --- "VEGF, nitric oxide and the testis" --- p.14 / Chapter 1.4.3 --- Cadmium-induced testicular toxicity --- p.16 / Chapter 1.4.4 --- "VEGF, glucocorticoids and the testis" --- p.17 / Chapter 1.4.5 --- Hormonal regulation of VEGF expression 226}0ؤimportance of LH --- p.19 / Chapter 1.4.6 --- VEGF and Leydig cell - macrophage interaction --- p.21 / Chapter 1.5 --- Aims of the present study --- p.24 / Chapter 2. --- Materials and methods / Chapter 2.1 --- Animals --- p.26 / Chapter 2.1.1 --- Spermatic cord torsion --- p.26 / Chapter 2.1.2 --- Cadmium chloride treatment --- p.27 / Chapter 2.1.3 --- Leydig cell depletion and cadmium chloride treatment --- p.28 / Chapter 2.1.4 --- Dexamethasone pretreatment and cadmium chloride injection --- p.28 / Chapter 2.1.5 --- hCG injection --- p.29 / Chapter 2.2 --- Immunohistochemistry --- p.29 / Chapter 2.2.1 --- Perfusion fixation of the testes --- p.29 / Chapter 2.2.2 --- Processing of the testes for histological section --- p.29 / Chapter 2.2.3 --- Immunohistochemical staining for VEGF --- p.30 / Chapter 2.2.4 --- Photomicrograph --- p.32 / Chapter 2.3 --- Cell cultures --- p.32 / Chapter 2.3.1 --- Cell lines of mouse TM3 Leydig cells and TM4 Sertoli cells --- p.32 / Chapter 2.3.2 --- Tumour cell line of mouse MLTC-1 Leydig cells --- p.33 / Chapter 2.4 --- Cell treatments --- p.33 / Chapter 2.4.1 --- Hypoxic treatment --- p.34 / Chapter 2.4.2 --- Cadmium chloride treatment --- p.36 / Chapter 2.4.3 --- hCG treatment --- p.37 / Chapter 2.4.4 --- Activators of second messenger systems --- p.37 / Chapter 2.4.5 --- Effect of pro-inflammatory cytokines and angiogenic growth factors --- p.38 / Chapter 2.5 --- Preparation of cDNA probes --- p.39 / Chapter 2.5.1 --- VEGF cDNA probe preparation --- p.39 / Chapter 2.5.2 --- P-actin cDNA probe preparation --- p.42 / Chapter 2.5.3 --- Purification of PCR products --- p.44 / Chapter 2.5.4 --- Confirmation of PCR products --- p.45 / Chapter 2.5.5 --- cDNA probe labeling --- p.46 / Chapter 2.6 --- RNA extraction --- p.46 / Chapter 2.6.1 --- Extraction of total RNA from testicular cell lines --- p.46 / Chapter 2.6.2 --- Extraction total RNA from testicular tissues --- p.50 / Chapter 2.7 --- Northern blot analysis --- p.51 / Chapter 2.7.1 --- Measurement of total RNA concentration --- p.51 / Chapter 2.7.2 --- RNA gel electrophoresis --- p.52 / Chapter 2.7.3 --- Transfer of RNA to membrane --- p.53 / Chapter 2.7.4 --- Hybridization with [α-32P]dCTP-labelled probes --- p.53 / Chapter 2.7.5 --- Autoradiography and densitometric quantification --- p.54 / Chapter 2.8 --- Data and statistical analysis --- p.55 / Chapter 3. --- Results / Chapter 3.1 --- Effects of hypoxia and cobalt chloride treatment on VEGF expression in TM3 and TM4 cells --- p.57 / Chapter 3.2 --- Effects of testicular torsion on VEGF expression in adult rat testes --- p.61 / Chapter 3.3 --- Antagonism of hypoxic induction of VEGF expression in TM3 cells by nitric oxide --- p.66 / Chapter 3.4 --- Effect of cadmium on VEGF expression in TM3 and TM4 cells --- p.66 / Chapter 3.5 --- Effect of dexamethasone on Cd-induced increase in VEGF expression in TM3 cells --- p.73 / Chapter 3.6 --- Effect of cadmium treatment on VEGF expression in the adult rat testes --- p.73 / Chapter 3.7 --- Effect of Leydig cell depletion on basal and Cd-induced expression of VEGF in adult rat testes --- p.76 / Chapter 3.8 --- Effect of dexamethasone on basal and Cd-induced expression of VEGF in adult rat testes --- p.76 / Chapter 3.9 --- "Effects of hCG,forskolin and phorbol ester on VEGF expression in TM3 and TM4 cells" --- p.79 / Chapter 3.10 --- Effect of hCG on VEGF expression in MLTC-1 cells --- p.92 / Chapter 3.11 --- "Effect of EL-lα, IL-1β, IL-6, TNF- α and TNF- β on VEGF expression in TM3 cells" --- p.92 / Chapter 3.12 --- Effect of bFGF and TGF- β1 on VEGF expression in TM3 cells --- p.102 / Chapter 4. --- Discussion / Chapter 4.1 --- Upregulation of VEGF expression in TM3 and TM4 cells by hypoxia and cobalt chloride --- p.108 / Chapter 4.2 --- Effect of testicular torsion on VEGF expression in adult rat testes --- p.110 / Chapter 4.3 --- Antagonism of hypoxic induction of VEGF expression in TM3 cells by nitric oxide --- p.111 / Chapter 4.4 --- "Effect of cadmium on VEGF mRNA levels in TM3 and TM4 cells, and in adult rat testes" --- p.113 / Chapter 4.5 --- "Effect of hCG,forskolin and phorbol ester on VEGF expression in TM3 and TM4 cells" --- p.116 / Chapter 4.6 --- Effect of cytokines and growth factors on VEGF expression in TM3 cells --- p.119 / Chapter 5. --- References --- p.123

Liu Lin. / "January 2006." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 117-135). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.