No description available.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Parkinson’s disease (PD) is a progressive and debilitating neurodegenerative disorder, characterized by a distinct motor phenotype and the selective loss of dopaminergic neurons in the substantia nigra. While the etiology of PD is not fully understood, it is thought to involve a combination of different genetic, cellular and environmental factors that independently or concurrently contribute to neurodegeneration. To date, several PD-causing genes have been identified, and investigations of their function have provided novel insights into the pathobiology of disease. Particularly interesting among the known PD genes is parkin, mutations in which are the most common genetic cause of early onset PD. Parkin is an E3 ligase that ubiquitinates protein substrates and targets such substrates for degradation via the ubiquitin proteasome system (UPS). Therefore, the loss of parkin may result in the deleterious accumulation or dysregulation of parkin substrates and neurotoxicity. Parkin’s enzymatic activity has also been implicated in the maintenance of mitochondrial health, and mitochondrial dysfunction is commonly reported in cellular and animal models of parkin deficiency. This study aimed to investigate parkin and its role in PD on various levels. Initially, genetic screening approaches were used to assess the contribution of parkin mutations to PD in a group of 229 South African patients. It was concluded that parkin mutations are rare in the South African PD population, being present in only seven (3.1%) patients in the study group. Interestingly, this study identified two of only three Black African PD patients with mutations in a known PD-causing gene to date. The low frequency of known PD genes raises the interesting possibility that the unique South African ethnic groups may harbor mutations in novel PD-causing genes. Although many parkin-interacting proteins have been identified in the literature, it is anticipated that novel, pathologically-relevant parkin substrates remain to be discovered. Hence, this study used a yeast two-hybrid (Y2H) approach to identify novel parkin interactions. This yielded 29 putative parkin interactors, of which four, namely ATPAF1, SEPT9, actin and 14-3-3η, were prioritized for verification by co-localization and co-immunoprecipitation experiments. Interestingly, two of the parkin interactors (ATPAF1 and SEPT9) were found to accumulate in the absence of parkin, supporting their role as authentic parkin substrates. The identification of these two intriguing proteins implicates parkin in the regulation of mitochondrial ATP synthase assembly and septin filament dynamics, which may be of significant relevance to our understanding of processes underlying neurodegeneration. Moreover, it was aimed to assess various markers of mitochondrial function in a parkin-deficient cellular model, as previous studies had reported conflicting results regarding mitochondrial impairments in patient-derived cells with parkin mutations. Hence, dermal fibroblasts were obtained from PD patients with homozygous parkin mutations, after which cell growth and viability, mitochondrial membrane potential, respiratory rates and the integrity of the mitochondrial network were assessed. Surprisingly, it was found that cell growth was significantly higher in the parkin-mutant fibroblasts compared to wild-type controls fibroblasts under basal conditions (p=0.0001), while exhibiting a greater inhibition of cell growth in the presence of the mitochondrial toxin CCCP (p=0.0013). Furthermore, whereas the mitochondrial networks of patient-derived fibroblasts were more fragmented than controls (p=0.0306), it was found that mitochondrial respiratory rates were paradoxically higher in the patients (p=0.0355). These unanticipated findings are suggestive of a compensatory response to the absence of parkin. The parkin-deficient cellular model was also used in a pilot study of the functional effects of vitamin K2 treatment, which has recently been identified as a promising PD therapeutic modality. It was found that treatment with vitamin K2 resulted in more interconnected mitochondrial networks (p=0.0001) and enhanced respiratory rates (p=0.0459) in both parkin-mutant and wild-type control cells. While these results need to be studied further, it suggests that vitamin K2 supplementation may be of use as a general promoter of mitochondrial integrity and function. In conclusion, this dissertation highlights some novel interactions of the parkin protein and some interesting phenotypes of parkin deficiency. It is hoped that further investigation of parkin and its role in PD will, ultimately, aid in the development of therapeutic strategies to treat this debilitating and poorly-understood disorder. / AFRIKAANSE OPSOMMING: Parkinson se siekte (PS) is 'n progressiewe en aftakelende neurodegeneratiewe kondisie, wat gekarakteriseer word deur 'n kenmerkende bewegingsfenotipe en die selektiewe afsterwing van dopaminergiese neurone in die substantia nigra. Terwyl die etiologie van PS nie ten volle verstaan is nie, behels dit waarskynlik 'n kombinasie van verskillende genetiese, sellulêre en omgewings-faktore wat onafhanklik of gelyktydig lei tot senuwee-afsterwing. Tot op hede is daar al verskeie PS-veroorsakende gene geïdentifiseer, en die bestudering van hul funksie het nuwe insigte in die patobiologie van hierdie siekte verskaf. Onder meer hierdie PS gene is parkin van besondere belang, aangesien mutasies in parkin die mees algemene genetiese oorsaak van vroeë-aanvang PS is. Parkin is 'n E3 ligase-ensiem wat proteïen substrate ubiquitineer en teiken vir degradasie via die ubiquitien proteasoomstelsel (UPS). Dus kan die verlies van parkin lei tot die beskadigende opeenhoping of wanregulasie van parkin substrate en senuwee-afsterwing. Parkin se ensiematiese aktiwiteit is ook betrokke by die instandhouding van mitokondriale gesondheid, en mitokondriale afwykings word dikwels gerapporteer in sellulêre en diermodelle van parkin tekort. Hierdie studie het gepoog om parkin en sy rol in PS op verskillende vlakke te ondersoek. Aanvanklik is genetiese siftingsbenaderinge gebruik om die bydrae van parkin mutasies tot PS in 'n groep van 229 Suid-Afrikaanse pasiënte te evalueer. Die gevolgtrekking is bereik dat parkin mutasies skaars is in die Suid-Afrikaanse PS bevolking, aangesien dit teenwoordig is in net sewe (3.1%) pasiënte in die studie groep. Interessant genoeg, hierdie studie het twee van slegs drie gevalle van Swart Afrika-pasiënte met mutasies in 'n bekende PS geen to op datum geïdentifiseer. Die lae frekwensie van bekende PS gene versterk die stimulerende moontlikheid dat die unieke Suid-Afrikaanse sub-populasies dalk mutasies in nuwe PS-veroorsakende gene mag koester. Alhoewel baie parkin proteïen-interaksies reeds in die literatuur geïdentifiseer is, word daar verwag dat nuwe, patologies-relevante parkin substrate nog wag om ontdek te word. Dus het hierdie studie 'n gis twee-hibried (G2H) benadering gebruik om nuwe parkin interaksies te identifiseer. Hierdie het 29 vermeende parkin interaktors opgelewer, waarvan vier, naamlik ATPAF1, SEPT9, aktien en 14-3-3η, geprioritiseer is vir verifikasie deur mede-lokalisering en mede-immunopresipitasie eksperimente. Interessant genoeg, daar is gevind dat twee van die parkin interaktors (ATPAF1 en SEPT9) ophoop in die afwesigheid van parkin, wat hul rol as werklike parkin substrate ondersteun. Die identifisering van hierdie twee interessante proteïene impliseer parkin in die regulering van mitokondriale ATP sintase vervaardiging en septienfilament dinamika, wat moontlik van beduidende belang is vir ons begrip van die onderliggende prosesse wat senuwee-afsterwing veroorsaak. Verder is daar daarop gemik om verskeie aanwysigings van mitokondriale funksie in 'n parkin-gebrekkige sellulêre model te evalueer, aangesien vorige studies teenstrydige resultate rapporteer rakende mitokondriale afwykings in pasiënt-selle met parkin mutasies. Dus is daar dermale fibroblaste verkry van PS pasiënte met homosigotiese parkin mutasies, waarna sel-groei en lewensvatbaarheid, mitokondriale membraanpotensiaal, respiratoriese tempo en die integriteit van die mitokondriale netwerk geëvalueer is. Daar is verbasend gevind dat sel-groei aansienlik hoër is die parkin-mutante fibroblaste in vergelyking met wilde-tipe kontrole fibroblaste onder basale kondisies (p=0.0001), terwyl hulle 'n groter inhibisie van sel-groei in die teenwoordigheid van die mitokondriale toksien CCCP ondergaan (p=0.0013). Verder, terwyl die mitokondriale netwerke van pasiënt fibroblaste meer gefragmenteer is as die van kontroles (p=0.0306), is daar gevind dat mitokondriale respiratoriese tempo’s, paradoksaal-gewys, hoër is in die pasiënte (p=0.0355). Hierdie onverwagte bevindinge is suggestief van die aanskakeling van 'n vergoedende respons-proses in die afwesigheid van parkin. Die parkin-gebrekkige sellulêre model is ook gebruik in 'n voorlopige studie van die funksionele effekte van vitamiene K2 behandeling, wat onlangs geïdentifiseer is as 'n belowende terapeutiese moontlikheid vir PS. Daar is gevind dat sel-behandeling met vitamiene K2 lei tot meer geïnterkonnekteerde mitokondriale netwerke (p=0.0001) en verbeterde respiratoriese fuksie (p=0.0459) in beide parkin-mutante en wilde-tipe kontrole selle. Terwyl hierdie resultate verder bestudeer sal moet word, dui dit daarop dat vitamiene K2-aanvulling moontlik gebruik kan word as 'n algehele promotor van mitochondriale integriteit en funksie. Ten slotte, hierdie verhandeling beklemtoon ‘n paar nuwe interaksies van die parkin proteïen en 'n paar interessante fenotipes van parkin tekort. Daar word gehoop dat verdere ondersoek van parkin en parkin se rol in PS sal, uiteindelik, steun in die ontwikkeling van terapeutiese strategieë om hierdie aftakelende en swak-verstaande wanorde beter te behandel.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Aspergillus flavus is the most common causal agent of aflatoxin contamination of food and feed. However, aflatoxin-producing potential varies widely among A. flavus genotypes with many producing no aflatoxins. Some non-aflatoxigenic genotypes are used as biocontrol agents to prevent contamination. Aflatoxin biosynthesis genes are tightly clustered in a highly conserved order. Gene deletions and presence of single nucleotide polymorphisms (SNPs) in aflatoxin biosynthesis genes are often associated with A. flavus inability to produce aflatoxins. In order to identify mechanisms of non-aflatoxigenicity in non-aflatoxigenic genotypes of value in aflatoxin biocontrol, complete cluster sequences of 35 A. flavus genotypes from Africa and North America were analyzed. Inability of some genotypes to produce aflatoxin resulted from deletion of biosynthesis genes. In other genotypes, non-aflatoxigenicity originated from SNP formation. The process of degeneration differed across the gene cluster; genes involved in early biosynthesis stages were more likely to be deleted while genes involved in later stages displayed high frequencies of SNPs. Comparative analyses of aflatoxin gene clusters provides insight into the diversity of mechanisms of non-aflatoxigenicity in A. flavus genotypes used as biological control agents. The sequences provide resources for both diagnosis of non-aflatoxigenicity and monitoring of biocontrol genotypes during biopesticide manufacture and in the environment.

The bidirectional influence of parenchymal cells and cells of the immune system, especially of antigen-presenting and CD8\(^+\) T cells, in situations of putative auto- immune pathogenicity and degeneration was the main topic of this thesis. In the first part, the influence of human muscle cells on antigen-presenting cells was investigated. In inflammatory myopathies prominent infiltrates of immune cells containing T cells and antigen-presenting cells like macrophages and dendritic cells are present. The hypothesis was that human myoblasts have an inhibiting influence on these antigen-presenting cells under homeostatic conditions. A dysfunction or impairment under inflammatory circumstances might contribute to the development of myopathic conditions. The surface analysis of dendritic cells cocultured with myoblasts showed that immature dendritic cells could be driven into a reversible semi- mature state with significantly elevated levels of CD80. These dendritic cells were additionally characterized by their inhibiting function on T-cell proliferation. It was also shown that the lysates of healthy myoblasts could strongly enhance the phagocytic ability of macrophages, which could help with muscle regeneration and which might be disturbed in myositis patients. The second part of this thesis was about the clonal specificity of CD8\(^+\) T cells in a mouse model with genetically induced over-expression of PLP in oligodendrocytes. Here, we could show that the cytotoxic T lymphocytes, which had previously been shown to be pathogenic, were clonally expanded in the CNS of the transgenic mice. The amino acid sequences of the corresponding receptor chains were not identical, yet showed some similarities, which could mean that these clones recognize similar antigens (or epitopes of the same antigen). The knockout of PD-1 in this setting allowed for an analysis of the importance of tissue immune regulation. It became evident that the absence of PD-1 induced a larger number of clonal expansions in the CNS, hinting towards a reduced threshold for clonal disturbance and activation in these T cells. The expansions were, however, not pathogenic by themselves. Only in the presence of tissue damage and an antigenic stimulus (in our case the overexpression of PLP), the PD-1 limitation exacerbated the immune pathogenicity. Therefore, only in the presence of a “tissue damage signal”, the dyshomeostasis of T cells lacking PD-1 achieved high pathogenetic relevance. Finally, we investigated the pathogenetic role of CD8 T cells in Rasmussen encephalitis, a rare and chronic neurological disease mainly affecting children. The analysis of the T-cell receptor repertoire in Rasmussen encephalitis patients in the peripheral CD4\(^+\) and CD8\(^+\) T-cell compartments as well as the brain revealed the involvement of T cells in the pathogenicity of this disease. Many clonal expansions in the brain matched CD8\(^+\) T-cell expansions in the periphery on the sequence level. These putatively pathogenic clones could be visualized by immunohistochemistry in the brain and were found in close proximity to astrocytes and neurons. Additionally, the expanded clones could be found in the periphery of patients for at least one year. / Der Einfluss von Parenchymzellen auf Immunzellen und umgekehrt, im Besonderen von Antigen-präsentierenden Zellen und CD8\(^+\) T-Zellen, im Zusammenhang von auto- immuner Pathogenese und Degeneration war das Hauptthema dieser Dissertation. Im ersten Teil wurde der Einfluss menschlicher Muskelzellen auf Antigen- präsentierende Zellen untersucht. In entzündlichen Myopathien kommt es zu massiven Infiltraten von Immunzellen, die T-Zellen und auch Antigen-präsentierende Zellen wie Makrophagen und dendritische Zellen enthalten. Die Hypothese war, dass menschliche Myoblasten einen hemmenden Einfluss auf die Antigen-präsentierenden Zellen unter homöostatischen Bedingungen haben. Eine Störung dieses Einflusses oder eine Beeinträchtigung unter entzündlichen Rahmenbedingungen könnte eventuell zur Entwicklung eines myopathischen Zustands beitragen. In der Oberflächenanalyse der dendritischen Zellen, die mit Myoblasten kultiviert wurden, zeigte sich, dass unreife dendritische Zellen in einen halb-reifen Zustand versetzt werden konnten, der sich beispielsweise durch stark erhöhte CD80 Expression kennzeichnet. Diese dendritischen Zellen wurden weiterhin charakterisiert über ihre hemmende Funktion auf die T-Zell Proliferation. Außerdem wurde gezeigt, dass Zelllysate gesunder Myoblasten die Phagozytoserate von Makrophagen enorm verstärken, was die Regeneration des Muskelgewebes erhöhen und möglicherweise in Myositispatienten gestört sein könnte. Im zweiten Teil der Dissertation ging es um die klonale Spezifität von CD8\(^+\) T-Zellen in einem Mausmodell mit genetisch induzierter Überexpression von PLP in Oligodendrozyten. Hier konnte gezeigt werden, dass die zytotoxischen T-Zellen, deren Pathogenität Gegenstand früherer Arbeiten war, im ZNS der transgenen Mäuse klonal expandiert waren. Die Aminosäuresequenzen der TCRβ Kette der expandierten Klone waren nicht identisch, zeigten jedoch einige Ähnlichkeiten, die darauf hinweisen könnten, dass diese Klone ähnliche Antigene (oder Epitope des gleichen Antigens) erkennen. Die genetisch induzierte Abwesenheit von PD-1 ermöglichte es, in diesem Zusammenhang den Einfluss von spezifischer Immunregulation im Gewebe zu untersuchen. Es zeigte sich, dass die Deletion von PD-1 eine erhöhte Anzahl von klonalen Expansionen im ZNS der Mäuse erzeugte, was auf eine herabgesetzte Schwelle für klonale Störungen und Aktivierung schließen lässt. Diese Expansionen 
 waren jedoch für sich genommen nicht pathogen. Nur in der Anwesenheit eines Gewebeschadens und eines zusätzlicher Antigenstimulus (in unserem Fall in Form der PLP Überexpression) konnte man die erhöhte Pathogenität durch die PD-1 Deletion erkennen. Deswegen erreichten die PD-1 deletierten T-Zellen nur in der Gegenwart eines „Gewebeschaden-Signals“ hohe pathogenetische Relevanz. Schließlich untersuchten wir die pathogenetische Rolle von CD8\(^+\) T-Zellen in der Rasmussen Enzephalitis, einer seltenen, chronischen Erkrankung des Gehirns, die hauptsächlich in Kindern vorkommt. Die Analyse des T-Zell-Rezeptor Repertoires in Rasmussen Enzephalitis Patienten in peripheren CD4\(^+\) und CD8\(^+\) T-Zell Populationen und im Gehirn zeigte die Beteiligung von T-Zellen in der Pathogenese dieser Krankheit auf. Viele klonale Expansionen waren zwischen Gehirn und der peripheren CD8\(^+\) Population bis hin zur Aminosäuresequenz identisch. Diese vermutlich pathogenen Klone konnten in Gehirnbiopsien von Rasmussenpatienten histochemisch nachgewiesen werden und wurden in enger Nachbarschaft zu Astrozyten und Neuronen gefunden. Zusätzlich konnten diese expandierten Kone in der Peripherie von Patienten für die beobachteten Zeiträume (mindestens ein Jahr) nachgewiesen werden.

University of Macau / Institute of Chinese Medical Sciences

The lens is a crystallin tissue of the anterior part of the eye that focuses light onto the retina. Aged-related cataract, which is the result of loss of lens transparency, is the most common cause of blindness in the world. Being constantly exposed to UV-light, lens is significantly affected by its UVA spectrum. UV-light exposure has been shown to result in apoptosis of lens cells which can lead to cataract formation. This suggests the need for molecular mechanisms to remove apoptotic debris from the lens. In the set of experiments it was proven that integrin αvβ5-mediated pathway is involved in phagocytosis of apoptotic cell debris in the ocular lens, thus contributing to its homeostasis. Additionally, it was shown that exposure to UV-light plays role in cataract formation by influencing integrin αvβ5-mediated phagocytosis function. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

Chiu Kin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 100-116). / Abstract also in Chinese. / TABLE OF CONTENTS --- p.I / ACKNOWLEDGEMENTS --- p.V / LIST OF FIGURES --- p.VI / LIST OF ABBREVIATIONS --- p.VIII / ABSTRACT --- p.1 / Chapter 1. --- INTRODUCTION --- p.5 / Chapter 2. --- LITERATURE REVIEW --- p.7 / Chapter 2.1 --- RETINAL ISCHEMIA --- p.7 / Chapter 2.1.1 --- INDUCTION OF RETINAL ISCHEMIA --- p.7 / Chapter 2.1.2 --- MECHANISMS OF RETINAL ISCHEMIA/REPERFUSION DAMAGE --- p.8 / Chapter 2.1.2.1. --- Free radical --- p.8 / Chapter 2.1.2.2 --- Excitotoxicity --- p.9 / Chapter 2.1.3 --- APOPTOSIS IN RETINAL ISCHEMIA/REPERFUSION INJURY --- p.10 / Chapter 2.2 --- RETINAL DYSTROPHIC ROYAL COLLEGE OF SURGEONS (RCS) RAT --- p.15 / Chapter 2.3 --- BCL-2 FAMILY MEMBERS --- p.16 / Chapter 2.3.1 --- FAMILY MEMBERS AND THEIR INTERACTIONS --- p.16 / Chapter 2.3.2 --- SUBCELLULAR LOCALIZATION --- p.18 / Chapter 2.3.3 --- PHYSICAL STRUCTURE AND PORE FORMATION --- p.19 / Chapter 2.3.4 --- BIOLOGICAL EFFECTS OF BCL-2 --- p.20 / Chapter 3. --- OBJECTIVES --- p.24 / Chapter 4. --- MATERIALS AND METHODS --- p.27 / Chapter 4.1. --- RETINAL ISCHEMIA AND REPERFUSION INDUCED LOSS OF INNER RETINAL ELEMENTS --- p.27 / Chapter 4.1.1. --- TISSUE RESPONSES IN THE RAT RETINAS AFTER TRANSIENT ELEVATED INTRAOCULAR PRESSURE INDUCED RETINAL ISCHEMIA/REPERFUSION INSULT --- p.27 / Chapter 4.1.1.1. --- Induction of retinal ischemia/reperfusion insult with transient elevated intraocular pressure (IOP) --- p.27 / Chapter 4.1.1.2. --- Animal experiments --- p.28 / Chapter 4.1.1.3. --- Histopathology and measurement of inner retinal thickness (IRT) --- p.28 / Chapter 4.1.1.4. --- Flat preparation of the retinas and retinal ganglion cell counts (RGCCs) --- p.29 / Chapter 4.1.2. --- INTERNUCLEOSOMAL DNA FRAGMENTATION AND IN SITU NICKED DNA DETECTIONS AT DIFFERENT TIME AFTER REPERFUSION IN THE RAT RETINAS --- p.30 / Chapter 4.1.2.1. --- Enzyme-linked immunosorbent assay (ELISA) of mono- and oligonucleosomes --- p.30 / Chapter 4.1.2.2. --- In-situ terminal deoxynucleotidyl transferase (TdT)-mediated biotin- dUTP nicked end labelling (TUNEL) --- p.31 / Chapter 4.1.3. --- "IMMUNOHISTOCHEMISTRY OF BCL-2, BAX AND P53" --- p.32 / Chapter 4.1.4. --- "DOUBLE LABELLING OF BCL-2, BAX AND TUNEL" --- p.33 / Chapter 4.1.5. --- IN-SITU REVERSE TRANSCRIPTASE - POLYMERASE CHAIN REACTION OF BCL-2 AND BAX --- p.34 / Chapter 4.1.5.1. --- Primers design and specificity test --- p.34 / Chapter 4.1.5.2. --- In-situ RT-PCR --- p.36 / Chapter 4.2. --- LOSS OF INNER RETINAL ELEMENTS IN THE RETINAL DYSTROPHIC ROYAL COLLEGE OF SURGEONS (RCS) RATS --- p.38 / Chapter 4.2.1. --- HISTOPATHOLOGY --- p.38 / Chapter 4.2.2 --- MORPHOMETRY OF CELLS IN THE RETINAL GANGLION CELL LAYER (RGCL) AND THE INNER NUCLEAR LAYER (INL) --- p.39 / Chapter 4.2.3. --- IMMUNOHISTOCHEMISTRY OF BCL-2 AND BAX --- p.39 / Chapter 5. --- RESULTS --- p.40 / Chapter 5.1. --- RETINAL ISCHEMIA AND REPERFUSION INDUCED LOSS OF INNER RETINAL ELEMENTS --- p.40 / Chapter 5.1.1. --- TISSUE RESPONSES IN THE RAT RETINAS AFTER TRANSIENT ELEVATED INTRAOCULAR PRESSURE INDUCED ISCHEMIA/ REPERFUSION INSULT --- p.40 / Chapter 5.1.1.1. --- Histopathology --- p.40 / Chapter 5.1.1.2. --- Morphometry of inner retinal thickness --- p.40 / Chapter 5.1.1.3. --- Retinal ganglion cell counts (RGCCs) --- p.41 / Chapter 5.1.2. --- INTERNUCLEOSOMAL DNA FRAGMENTATION AND IN SITU NICKED DNA DETECTION AT DIFFERENT TIME AFTER REPERFUSION IN THE RAT RETINAS --- p.41 / Chapter 5.1.2.1. --- Enzyme-linked immunosorbent assay (ELISA) of mono- and oligonucleosomes --- p.42 / Chapter 5.1.2.2. --- In situ TUNEL --- p.42 / Chapter 5.1.3. --- BCL-2 AND RETINAL ISCHEMIA/REPERFUSION INJURY --- p.42 / Chapter 5.1.3.1. --- Immunohistochemistry of Bcl-2 --- p.42 / Chapter 5.1.3.2. --- Double labelling of Bcl-2 and TUNEL --- p.43 / Chapter 5.1.3.3. --- In situ RT-PCR for bcl-2 mRNA --- p.43 / Chapter 5.1.4. --- BAX AND RETINAL ISCHEMIA/REPERFUSION INJURY --- p.44 / Chapter 5.1.4.1. --- Immunohistochemistry of Bax --- p.44 / Chapter 5.1.4.2. --- Double labelling of Bax and TUNEL --- p.45 / Chapter 5.1.4.3. --- In situ RT-PCR for bax mRNA --- p.45 / Chapter 5.1.5. --- P53 IMMUNOREACTIVITY AT VARIOUS TIME AFTER REPERFUSION --- p.46 / Chapter 5.2. --- LOSS OF INNER RETINAL ELEMENTS IN THE RETINAL DYSTROPHIC ROYAL COLLEGE OF SURGEON (RCS) RATS --- p.47 / Chapter 5.2.1. --- HISTOPATHOLOGY --- p.47 / Chapter 5.2.2. --- MORPHOMETRY OF CELLS IN THE RGCL AND INL --- p.47 / Chapter 5.2.3. --- IMMUNOHISTOCHEMISTRY OF BCL-2 AND BAX --- p.47 / Chapter 5.2.3.1. --- Bcl-2 --- p.47 / Chapter 5.2.3.2. --- Bax --- p.48 / Chapter 6. --- DISCUSSION --- p.49 / Chapter 6.1. --- RETINA ISCHEMIA AND REPERFUSION INDUCED LOSS OF RETINAL ELEMENTS --- p.51 / Chapter 6.1.1 --- REPERFUSION TIME DEPENDENT TISSUE RESPONSES IN RAT RETINAS --- p.51 / Chapter 6.1.2 --- ISCHEMIA/REPERFUSION INDUCED APOPTOSIS IN RAT RETINAS --- p.52 / Chapter 6.1.3 --- BCL-2 AND RETINAL ISCHEMIA/REPERFUSION INSULT --- p.53 / Chapter 6.1.4 --- BAX AND RETINAL ISCHEMIA/REPERFUSION INSULT --- p.58 / Chapter 6.1.5 --- P53 AND RETINAL ISCHEMIA/REPERFUSION INJURY --- p.60 / Chapter 6.2. --- LOSS OF INNER RETINAL ELEMENTS IN THE RETINAL DYSTROPHIC ROYAL COLLEGE OF SURGEON (RCS) RAT --- p.61 / Chapter 6.2.1. --- HISTOPATHOLOGY AND MORPHOMETRY --- p.62 / Chapter 6.2.2. --- BCL-2 --- p.63 / Chapter 6.2.3. --- BAX --- p.64 / Chapter 7. --- CONCLUSION --- p.65 / APPENDIX A FIGURES --- p.66 / APPENDIX B REFERENCES --- p.100