[Truncated abstract] Olfactory ensheathing glia (OEG) have been demonstrated to improve functional and anatomical outcomes after injury to the nervous system and are currently being trialled clinically. This thesis presents the investigation of two important issues in OEG biology. The first study (Chapter 2) investigates effects of different members of the neuregulin (NRG) family of molecules on the proliferation of OEG, as a means of quickly obtaining large numbers of cells for clinical or experimental use. We report that NRG-1β, but not NRG- 2α or NRG-3, has a significant proliferative effect. Furthermore, we report for the first time that use of different mitogens (forskolin and pituitary extract) commonly used to expand these cells in vitro, can have a significant effect on the responsiveness of OEG to added NRG in subsequent mitogenic assays. OEG grown initially with forskolin and pituitary extract exhibited increased basal proliferation rates in comparison to OEG originally expanded without these factors, and this increased rate of proliferation was sustained for at least 6 days following their withdrawal from the culture medium. We also report for the first time the expression pattern of ErbB2, ErbB3 and ErbB4 receptors on p75-selected OEG, and investigate their contribution to the NRG mitogenic effect by the use of inhibitory ErbB antibodies. Our second study (Chapter 3) seeks to clarify the role of OEG in promoting myelination of central nervous system neurons. In this study we have investigated the myelinating ability of OEG derived from embryonic (EEG), postnatal (PEG) and adult tissue (AEG) both in vitro and in vivo. OEG selected by p75-immunopanning were co-cultured with dissociated cultures of TrkA-dependant embryonic dorsal root ganglion (DRG) neurons. EEG, but not AEG or PEG, successfully myelinated DRG neurons in the presence of serum and/or ascorbate. AEG also failed to myelinate GDNF-dependant embryonic DRG cultures, and growth factor-independent adult DRG cultures. Transplantation of OEG into lysolecithin demyelinated spinal cord demonstrated distinct ultrastructural differences between transplants of OEG derived from animals of different ages. Furthermore, we demonstrate that clearance of degraded myelin from the lesion site appears to be more effective when animals are transplanted with EEG rather than AEG or Schwann cell preparations. These results suggest that myelinating potential of OEG in vitro and behaviour of these cells following transplantation in vivo are developmentally regulated.

The diploma thesis deals with the effectiveness of specific regeneration on results of a test working capacity of mixed martial arts athletes. The effectiveness of Jacobson's progressive muscle relaxation, full body massage and sauna was tested on first proband. Schultz's autogenic training, whirlpool bath were used on second proband. Both probands performed the same compensatory exercises after MMA training session. Trainings were scheduled three times per week. Intervention programmes lasted for eight weeks. The thesis briefly summarizes the issues of MMA and subsequently the distribution of regeneration and its importance in relation to sport performance. Content analysis, synthesis and measurement method, were used. The research was conducted in the form of individual case studies. Using semi-structured interviews, case-histories of the probands were created. To determine the working capacity, the W170 test was used. The intervention program followed, after which, the W170 test, was performed again. According to the results of working capacity test, the level of working capacity increased, per 42,8 % at first proband and 16,07 % at second proband.

Indiana University-Purdue University Indianapolis (IUPUI) / After spinal cord injury (SCI), poor axonal regeneration of the central nervous system, which mainly attributed to glial scar and low intrinsic regenerating capacity of severely injured neurons, causes limited functional recovery. Combinatory strategy has been applied to target multiple mechanisms. Schwann cells (SCs) have been explored as promising donors for transplantation to promote axonal regeneration. Among the central neurons, descending propriospinal neurons (DPSN) displayed the impressive regeneration response to SCs graft. Glial cell line-derived neurotrophic factor (GDNF), which receptor is widely expressed in nervous system, possesses the ability to promote neuronal survival, axonal regeneration/sprouting, remyelination, synaptic formation and modulate the glial response. We constructed a novel axonal permissive pathway in rat model of thoracic complete transection injury by grafting SCs over-expressing GDNF (SCs-GDNF) both inside and caudal to the lesion gap. Behavior evaluation and histological analyses have been applied to this study. Our results indicated that tremendous DPSN axons as well as brain stem axons regenerated across the lesion gap back to the caudal spinal cord. In addition to direct promotion on axonal regeneration, GDNF also significantly improved the astroglial environment around the lesion. These regenerations caused motor functional recovery. The dendritic plasticity of axotomized DPSN also contributed to the functional recovery. We applied a G-mutated rabies virus (G-Rabies) co-expressing green fluorescence protein (GFP) to reveal Golgi-like dendritic morphology of DPSNs and its response to axotomy injury and GDNF treatment. We also investigated the neurotransmitters phenotype of FluoroGold (FG) labeled DPSNs. Our results indicated that over 90 percent of FG-labeled DPSNs were glutamatergic neurons. DPSNs in sham animals had a predominantly dorsal-ventral distribution of dendrites. Transection injury resulted in alterations in the dendritic distribution, with dorsal-ventral retraction and lateral-medial extension of dendrites. Treatment with GDNF significantly increased the terminal dendritic length of DPSNs. The density of spine-like structures was increased after injury and treatment with GDNF enhanced this effect.

published_or_final_version / Anatomy / Master / Master of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

In another study, hyperthermic treatment was applied to study whether HSP27 expression would be induced in un-injured RGCs, and whether this treatment performed after axotomy would have effects on HSP27 expression, RGC survival and/or regeneration into PN graft. Brief duration of heat shock that elevate the body temperature to 42°C did not up-regulate HSP27 in normal retina. About 8-10% increase in RGC survival in the hyperthermia group was observed compared to those received a 37°C treatment at one week post-axotomy and it depended on the number of post-injury heat treatments applied. At the same time, the number of HSP27-RGCs was also doubled, although the same increase occurred was irrespective of the number of hyperthermic treatments. Multiple heat shock application also significantly enhanced RGC regeneration into PN graft through increased the number of HSP27 regenerating RGCs. These results suggest that post-injury hyperthermic treatment enhance HSP27 induction in RGCs and lead to their successful regeneration into the PNG, whereas further studies are necessary to determine whether the protective effect on survival by heat shock is due to the increase in a subset of HSP27-RGCs. (Abstract shortened by UMI.) / In the second study, different neurotrophic factors were injected into the vitreous to enhance RGC survival and/or regeneration. Brain-derived neurotrophic factor (BDNF) significantly reduced RGC death transiently at 14 days after ON cut, but the expression of HSP27 was reduced compared to bovine serum albumin-injected controls. In peripheral nerve (PN)-grafted retina, BDNF suppressed RGC regeneration via reducing the number of HSP27-RGCs regenerating into the PN graft. In ciliary neurotrophic factor (CNTF)-injected group, although there was only a 10% increase in RGC survival, a 5-fold drastic increase in the number of RGCs which expressed HSP27 was observed, and some of these were found to undergo intra-retinal sprouting similar to VPN-transplanted retina. Combined treatment of intra-vitreal CNTF injection with PNG resulted in a 5 fold-increase in the number of regenerating RGCs as well as increasing the proportion of cells which expressed HSP27 from about 60% to about 80%. The data indicates that HSP27 participates in axonal regrowth especially under synergistic interaction of CNTF and PNG. Intra-vitreal injection of hepatocyte growth factor (HGF) significantly sustained RGC survival compared to BDNF at 28 days after axotomy, but the HSP27 expression in RGCs did not change correspondingly. In the PN-ON grafted retina, HGF promoted more RGCs regenerate without altering the number of HSP27-RGCs regrowing into the PNG. Such results indicate that some trophic factors can specifically enhance or suppress RGC regeneration by modulating HSP27 expression, while other trophic factors promote regeneration which is independent to HSP27. Therefore, it suggests that RGCs may regenerate through at least two different mechanisms. / In this study, the detailed in vivo expression of HSP27 in retinal ganglion cells (RGCs) of golden hamster following axotomy and regeneration stimulated by peripheral nerve grafting and neurotrophic factors have been examined. / In whole-mount normal retinas, HSP27 was constitutively expressed in astrocytes and blood vessels, but not in RGCs. Three days after optic nerve (ON) transection, a small subset of surviving RGC began to express HSP27, the number of which peaked at 7 days and dropped to a minimal level at two weeks post-axotomy. When axotomy was done more proximally to their cell bodies, RGCs survival was significantly decreased but HSP27 expression did not change. This suggests the HSP27 expression does not correlate with cell survival after axonal injury. When a viable peripheral nerve (VPN) was transplanted intravitreally into the eye after ON cut, it induced intra-retinal sprouting of RGCs. Although it did not promote RGC survival, VPN prolonged HSP27 expression up to 56 days after surgery and significantly increased the number of HSP27-RGCs. This protein was localized in the cell body, and especially, in dendritic sprouts and growth cones, indicating that it was transported to active growing sites where it may have a functional role associated with regenerative sprouting. / Wong, Wai Kai. / Adviser: Eric Cho. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3270. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 159-198). / 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.

Despite that the RGC axons closely associated with astrocytes, the role of astrocytes in RGC regeneration was uncertain. In view of this, the effect of cultured adult astrocytes on RGC regeneration through an extensive ON lesion segment was studied. Adult ON astrocytes were prepared by sub-culturing of cells migrating out of ON explants. A small hole in the ON was punctured by 27G needle and about 0.5 to 1.0mul (1000 cells) cultured astrocytes was injected into the extensive ON lesion segment. We found that cultured adult astrocytes promoted significant RGC axon regeneration in the extensive ON lesion. / Finally, co-transplantation of intravitreal PN followed by transplantation of astrocytes into the extensive lesion has a synergistic effect on the regrowth of RGC axons, as indicated by the maximum distance achieved by regenerating axons and integrated intensity of staining of the CTB-labeled axons. Transplanatation of VPN+AST, VPN+NAST and NPN+AST as 3.9, 2.5 and l.9 times more potent in inducing regeneration than that of NPN+NAST as shown by integrated intensity measurement. However, co-transplantation of PN and astrocytes could not enhance RGC survival. (Abstract shortened by UMI.) / In this study, we have established an extensive lesion paradigm to study the behavior of injured retinal ganglion cell (RGC) axons after ON crush in adult golden hamster. We found that RGC axons regenerated in the extensive lesion for 406.8mum at 1 week post-crush to 1174.0mum at 4 weeks post-crush. RGC axons were able to regenerate the entire lesion segment but they terminated precisely at the interface between the lesion and the distal segment of the ON. Regrowing axons were intimately associated with astrocytes which repopulated the lesion segment. Repopulated oligodendrocytes were scattered in the lesion segment and myelin debris was significantly decreased in the lesion segment with time. / It is commonly believed that central nervous system (CNS) neurons are unable to regenerate after injury. Recently, there have been several lines of evidence showing that damaged CNS neurons can undergo axonal regeneration under appropriate conditions. Since the retina and optic nerve (ON) are regarded as part of the CNS, therefore, they are used as a model to study CNS regeneration. / Kong Wai Chi. / "July 2005." / Adviser: Y.P. Cho. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3616. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 96-115). / 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.

Tse, Chi Lok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 116-131). / Abstracts in English and Chinese. / Declaration --- p.i / Abstract --- p.iii / Abstract in Chinese --- p.v / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.X / List of Tables --- p.xii / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- General Introduction / Chapter 1.1 --- Regenerative Medicine --- p.1 / Chapter 1.2 --- Embryonic Stem Cells and Reprogramming --- p.3 / Chapter 1.3 --- Transdifferentiation --- p.6 / Chapter 1.4 --- The Cerebellum --- p.7 / Chapter 1.4.1 --- Functions of the cerebellum --- p.7 / Chapter 1.4.2 --- Structure and organization of the cerebellum --- p.8 / Chapter 1.4.3 --- Principle cellular components in the cerebellum --- p.12 / Chapter 1.4.3.1 --- Purkinje cells --- p.12 / Chapter 1.4.3.2 --- Granule cells --- p.12 / Chapter 1.4.3.3 --- Mossy fibres --- p.13 / Chapter 1.4.3.4 --- Climbing fibres --- p.13 / Chapter 1.4.3.5 --- Deep cerebellar nuclei --- p.13 / Chapter 1.4.3.6 --- Other cerebellar neurons --- p.14 / Chapter 1.4.3.7 --- Neuroglia of the cerebellum --- p.16 / Chapter 1.4.4 --- Circuitry of the cerebellum --- p.17 / Chapter 1.5 --- Development of the Cerebellum --- p.21 / Chapter 1.5.1 --- Anatomical changes during cerebellar development --- p.21 / Chapter 1.5.2 --- Molecular control of cerebellar development --- p.25 / Chapter 1.5.2.1 --- Specification of the cerebellar region --- p.25 / Chapter 1.5.2.2 --- Neurogenesis from the ventricular zone --- p.26 / Chapter 1.5.2.3 --- Neurogenesis from rhombic lip --- p.29 / Chapter 1.6 --- Scope of the Thesis --- p.33 / Chapter CHAPTER 2 --- Materials and General Methods / Chapter 2.1 --- Materials for Molecular Biological Work --- p.35 / Chapter 2.1.1 --- Enzymes --- p.35 / Chapter 2.1.2 --- Chemicals and others --- p.35 / Chapter 2.1.3 --- Plasmid vectors and plasmid --- p.36 / Chapter 2.1.4 --- Solutions and media --- p.36 / Chapter 2.2 --- Materials for Tissue/Cell Culture --- p.38 / Chapter 2.2.1 --- Chemicals --- p.38 / Chapter 2.2.2 --- Culture media and solutions --- p.38 / Chapter 2.2.3 --- Culture cells --- p.39 / Chapter 2.3 --- Animals --- p.40 / Chapter 2.4 --- Materials for Immunocytochemistry --- p.40 / Chapter 2.5 --- Oligonucleotide Primers --- p.41 / Chapter 2.6 --- RNA Extraction --- p.44 / Chapter 2.7 --- Generation of cDNA from mRNA --- p.44 / Chapter 2.8 --- Preparation of Recombinant Plasmid DNA --- p.45 / Chapter 2.8.1 --- Small scale preparation of DNA --- p.45 / Chapter 2.8.2 --- QLAGEN plasmid midiprep kit method --- p.46 / Chapter 2.9 --- Preparation of Specific DNA Fragment from Agarose Gel --- p.46 / Chapter 2.10 --- Subcloning of DNA Fragments --- p.47 / Chapter 2.10.1 --- Preparation of cloning vectors --- p.47 / Chapter 2.10.2 --- Subcloning of DNA fragment --- p.48 / Chapter 2.10.3 --- Transformation of DNA into competent cells --- p.48 / Chapter 2.11 --- Preparation of Competent Cells --- p.48 / Chapter CHAPTER 3 --- Generation and Characterization of Induced Neurons / Chapter 3.1 --- Introduction --- p.50 / Chapter 3.2 --- Experimental Procedures --- p.51 / Chapter 3.2.1 --- Construction of expression vector --- p.51 / Chapter 3.2.1.1 --- Preparation of insert DNA --- p.51 / Chapter 3.2.1.2 --- Construction of entry vector --- p.52 / Chapter 3.2.1.3 --- Construction of destination vector --- p.52 / Chapter 3.2.1.4 --- Construction of expression vector --- p.52 / Chapter 3.2.2 --- Generation of induced neural cells --- p.57 / Chapter 3.2.2.1 --- Culture of mouse embryonic fibroblasts (MEF) --- p.57 / Chapter 3.2.2.2 --- Production of expression vector containing retroviruses --- p.57 / Chapter 3.2.2.3 --- Transfection and induction of neural fate of MEF --- p.57 / Chapter 3.2.3 --- Immunocytochemcial analysis --- p.58 / Chapter 3.2.4 --- Efficiency calculation --- p.59 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- A screen for cerebellar Purkinje and granule cell fate-inducing factors --- p.62 / Chapter 3.3.2 --- Characterization of the induced neurons --- p.67 / Chapter 3.3.2.1 --- Granule cell induction --- p.67 / Chapter 3.3.2.2 --- Purkinje cell induction --- p.71 / Chapter 3.4 --- Discussion --- p.102 / Chapter 3.4.1 --- Roles of inducing factors in Purkinje cells and granule cells development --- p.102 / Chapter 3.4.2 --- Mechanism of neural transdifferentiation --- p.107 / Chapter CHAPTER 4 --- Future Directions / Chapter 4.1 --- Complete Induction of Purkinje Cell Fate --- p.111 / Chapter 4.2 --- Induced Neurons of Different Subtypes --- p.112 / Chapter 4.3 --- Mechanism of Transdifferentiation --- p.114 / Chapter 4.4 --- Transdifferentiation and Regenerative Medicine --- p.114 / Bibliography --- p.116

Orientador: Fabio Papes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T05:41:39Z (GMT). No. of bitstreams: 1 Souza_MateusAugustodeAndrade_M.pdf: 6121419 bytes, checksum: a603ea19d560e8cfebddccca9b7d824a (MD5) Previous issue date: 2015 / Resumo: Em mamíferos, os neurônios sensoriais do Sistema Olfatório (OSNs) se encontram no interior da cavidade nasal, mas estão diretamente expostos ao ambiente externo. Por um lado, tal localização permite a esses neurônios o acesso imediato aos estímulos químicos ambientais, tomando vantagem do fluxo respiratório. Por outro lado, esses neurônios estão constantemente sujeitos a injúrias por agentes nocivos, como toxinas e patógenos, capazes de destruir essas células sensoriais. Sua perda constante, contudo, é contrabalanceada pela geração de novos OSNs durante toda a vida do indivíduo, fato que torna o Sistema Olfatório um dos poucos locais do organismo com neurogênese contínua na idade adulta. A regeneração dos OSNs tem atraído a atenção da comunidade científica tanto pelo seu potencial uso como modelo de estudo do Sistema Nervoso quanto pela sua potencial aplicação para o tratamento de doenças neurodegenerativas. Nesse sentido, muito conhecimento já foi produzido sobre a dinâmica de fatores de transcrição que acompanha a diferenciação dos progenitores neuronais olfatórios em OSNs. Porém, uma grande lacuna no conhecimento diz respeito a outros elementos capazes de coordenar esse processo, como os fatores moduladores da cromatina. Diante desse cenário, escolhemos como objeto de estudo as proteínas do Grupo Polycomb (PcG), que constituem uma maquinaria de controle transcricional relacionada a modificações na organização da cromatina. Neste trabalho, genes PcG selecionados foram caracterizados molecular e funcionalmente no epitélio olfatório principal de camundongos (MOE). Através de ensaios de hibridação in situ, cinco dos seis genes avaliados apresentaram expressão ubíqua por todo o epitélio (Cbx2, Cbx4, Phc2, Ezh1, Bcl6), enquanto um (Ezh2) mostrou-se expresso somente nos estratos basais do MOE. Em ensaios de colocalização, provamos que Ezh2 é expresso exclusivamente nos progenitores olfatórios, onde o processo de diferenciação se inicia, e em parte dos OSNs recém-diferenciados, ainda não funcionais. Esta foi a primeira vez que a expressão de um gene PcG foi analisada detalhadamente no Sistema Olfatório. O interessante perfil de expressão de Ezh2 foi sugestivo de um possível papel funcional relacionado à diferenciação dos progenitores olfatórios. Para investigar essa hipótese, utilizamos como ferramenta experimental a habilidade do MOE em se regenerar após a indução de injúrias específicas. Para isso, o MOE de camundongos foi lesionado quimicamente com o composto diclobenil, que leva à perda abrupta de OSNs, estimulando a proliferação e a diferenciação dos progenitores olfatórios para repovoar as regiões lesionadas. Os animais assim tratados receberam, por via intranasal, o fármaco GSK126, uma molécula inibidora específica da atividade da proteína EZH2. Acompanhando a regeneração subsequente do MOE, observamos que a inibição da atividade de EZH2 levou ao incremento de OSNs no epitélio, favorecendo a sua regeneração. Interessantemente, esse incremento também foi observado em MOEs não lesionados, mostrando que o efeito de GSK126 não é dependente da indução de injúrias prévias. Através dessa investigação molecular e funcional, buscamos contribuir para o melhor entendimento da diferenciação neuronal do MOE, e apontamos as proteína PcG como elementos importantes para esse processo / Abstract: In mammals, the olfactory sensory neurons (OSNs) are located inside the nasal cavity, but they are directly exposed to the external environment. Taking advantage of the respiratory flux, this location favors the access to the chemical stimuli presented by the environment. On the other hand, it leads OSNs to be continually damaged by pathogens and toxic substances carried by the inhaled air. However, the persistence of neuronal progenitors in the olfactory epithelium makes the constant reposition of the OSNs possible. This unique ability of regeneration makes the Olfactory System one of the few sites of neurogenesis throughout the adult life. Olfactory regeneration has attracted the attention scientific community because of its potential as a model of study of the Nervous System and application in the treatment of neurodegenerative diseases. A great amount of knowledge has been accumulated about the transcription factor dynamics that follows the differentiation of neuronal progenitors into OSNs. However, there is a great gap about other elements that could coordinate this process, such as chromatin modulator factors. In this scenario, we decided to study the Polycomb Group (PcG) proteins, a transcription control machinery involved in chromatin structure organization. In the present study, selected PcG genes were molecular and functionally analyzed in the mouse main olfactory epithelium (MOE). Using in situ hybridization assays, we characterized the expression of six PcG genes. Five of them were shown to be expressed throughout the MOE (Cbx2, Cbx4, Phc2, Ezh1, Bcl6), while one (Ezh2) was found only in the basal layers of this epithelium. Using colocalization strategies, we proved that Ezh2 gene is expressed exclusively in the olfactory progenitor cells, where the differentiation process begins, and in part of the newly differentiated OSNs that are still not functional. It was the first time that a PcG gene expression profile was finely analyzed in the Olfactory System. This interesting expression profile presented by Ezh2 suggested a possible involvement with the MOE neuronal progenitor differentiation. For this functional investigation, we used MOE¿s neuronal regeneration after specific injuries as an experimental tool. For this purpose, the MOE was chemically damaged by the compound dichlobenil, which causes a great loss of OSNs, stimulating proliferation and differentiation of neuronal progenitor cells, leading to the repopulation of the damaged tissue. Next, mice received by intranasal route the pharmacological inhibitor GSK126, which blocks EZH2 protein activity. The observation of the MOE regeneration that followed showed us that GSK126 application resulted in an increased number of OSNs, improving MOE regeneration. Interestingly, this increase was also found in intact MOEs, pointing that GSK126¿s effects do not depend on previous olfactory injuries. By this molecular and functional investigation, we aimed at a better understanding of olfactory neuronal differentiation, and we targeted the PcG proteins as relevant elements to this process / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

by Wai-Chi Kong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 83-92). / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations Frequently Used --- p.v / Chapter Chapter1 --- General Introduction --- p.1 / Chapter Chapter2 --- Long term culture of whole-mount retinal explant --- p.16 / Chapter Chapter3 --- Responses of retinal ganglion cells after peripheral nerve transplantation in vivo and in vitro --- p.46 / Chapter Chapter4 --- Effect of optic nerve or peripheral nerve explants on cultured whole-mount retinal explants --- p.62 / Chapter Chapter5 --- General Discussion --- p.78 / References --- p.83 / Tables --- p.93