Until the beginning of this decade the antioxidant, melatonin, had been considered as little more than a tranquilizing hormone, responsible for regulating certain circadian and circannual rhythms. However, it is the discovery of melatonin as a free radical scavenger that has generated the most interest in recent years. The reduction of melatonin with age has been associated with neurodegenerative diseases such as Alzheimer’s disease (AD)and therefore, melatonin has been implicated to have an important clinical role in neuroprotection. Thus, for several years melatonin has attracted increasing attention from the general press with many advertisements touting this indoleamine to act as an aphrodisiac, rejuvenator, protector against diseases and a general wonder drug. However, melatonin formulations appear with no labelling for the correct storage conditions, dosage and side effects, as well as no control for purity and self-medicating with an unregulated product. In addition, there is much controversy surrounding the antioxidative properties of the indolemaine, 6-hydroxymelatonin (6-OHM). Therefore, the first part of this study aims to elucidate the physico-chemical and various stability characteristics of the pineal antioxidant, melatonin, while the second part is devoted to investigating the neuroprotective properties of the primary hepatic metabolite of melatonin, 6-OHM. The physical properties of melatonin were determined using various chemical techniques. This information served to both characterize and confirm the identity of melatonin raw material used in this study. In addition, this information serves to be essential as the physical properties of melatonin have not been reported in detail in literature, to date. Thereafter, using a validated high performance liquid chromatography (HPLC) method, the various physico-chemical and stability characteristics of melatonin were determined. Melatonin was shown to be extremely lipophilic, while the hygroscopic study indicates that melatonin raw material is extremely hygroscopic at temperatures above 40°C, whereas melatonin tablets are hygroscopic when left out of the original container. This study highlights the need for consumers to be aware of the proper storage of melatonin tablets to improve the stability and ensure long term integrity of the compound. Since, melatonin is most often administered orally, thus exposing it to a large variations in pH, within the gastrointestinal tract, it was decided to investigate the stability of melatonin over a range of pH’s and temperatures. The findings imply that melatonin is relatively stable at body temperature when ingested orally and that orally administered slow release preparations of melatonin should be relatively stable and therefore exhibit favourable bioavailability. However melatonin was shown to be unstable in solution. This provides important information and a challenge to the formulators of this drug substance in a liquid dosage form. An assessment of the photostability of melatonin dosage forms using International Committee on Harmonization (ICH) conditions revealed melatonin to be light sensitive and thus indicates a need for careful consideration of the packaging of these drug products. In addition a detailed assessment of the photochemistry and photoproducts formed during the UV photodegradation of melatonin is reported. Melatonin is shown to rapidly degrade in the presence of UV light, with the presence of oxygen accelerating the photodegradation. N1-acetyl-N2-formyl-5-methoxykynurenamine(AFMK) and 6-OHM were identified as the major photoproducts formed and these agents have been shown previously to retain antioxidant activity. One of the concerns of using melatonin in sunscreens is its photostability. However, it is reported in this study that the degraded solution of melatonin still possesses equipotent free radical scavenging ability as melatonin, despite the absence of melatonin in solution. In addition, melatonin is shown to reduce UV-induced oxidative stress in rat skin homogenate. Thus, these results make melatonin a likely candidate for inclusion in sunscreen preparations. Neuronal damage due to oxidative stress has been implicated in several neurodegenerative disorders. 6-OHM is not only formed as the major hepatic metabolite of melatonin, but also when melatonin reacts with toxic radicals as well as UV light. Thus the second part of the study aims to elucidate and further characterize the mechanism behind 6-OHM’s neuroprotection. The results show 6-OHM to be a more potent singlet oxygen and superoxide anion scavenger than melatonin. In addition, the results show 6-OHM to offer protection against, oxidative stress and lipid peroxidation induced by several neurotoxins in the rat brain and hippocampus. The hippocampus is an important region of the brain responsible for the formation of memory and any agent that induces stress in this area has detrimental effects and could lead to various types of dementia. Such agents include quinolinic acid (QA) and iron (II). Histological studies undertaken reveal that 6-OHM is able to protect hippocampal neurons against QA and iron (II) induced necrotic cell death. Immunohistochemical investigations showed that QA moderately induces apoptotic cell death in the hippocampus which is inhibited by both melatonin and 6-OHM. The study sought to elucidate possible mechanisms by which 6-OHM exerts its neuroprotective capabilities and the results show 6-OHM to inhibit the action of cyanide on the mitochondrial electron transport chain (ETC), one of the most common sources of free radicals. In addition, 6-OHM treatment alone, increased ETC activity above basal control levels and the results show 6-OHM to increase complex I activity in the mitochondrial ETC. Electrochemical, ultraviolet/visible spectroscopy (UV/Vis) and HPLC assessment show that an interaction exists between 6-OHM and iron (III) and 6-OHM is able to reduce iron (III) to a more biologically usable form viz. iron (II) which can be incorporated into important biomolecules such as heme. One dire consequence of this interaction is the ready provision of iron (II) to drive the Fenton reaction. However the biological and histological assessments show 6-OHM to prevent iron (II)-induced lipid peroxidation and necrotic cell death and thus, provide evidence of its antioxidant properties. The results also show 6-OHM to promote Hsp70 induction in the hippocampus. Heat shock proteins, especially Hsp 70 plays a role in cytoprotection by capturing denatured proteins and facilitating the refolding of these proteins once the stress has been relieved. 6-OHM treatment alone and together with QA was shown to increase the level of expression of Hsp70, both inducible and cognate forms of the protein. This suggests that 6-OHM helps to protect against cellular protein damage induced by any form of stress the cell may encounter. Melatonin treatment alone and in combination with QA was shown to prevent increases in the level of Hsp70 in the hippocampus, indicating that melatonin was able to reduce oxidative stress induced by QA such that Hsp70 expression was not required. The discovery of neuroprotective agents, such as melatonin and 6-OHM, is becoming important considering the rapid rise in the elderly population and the proportionate increase in neurological disorders. The findings of this study indicate the need for important information regarding the correct storage conditions and stability characteristics of melatonin dosage forms. In addition, the results indicate that 6-OHM has a definite role to play as an antioxidant. Thus further research may favour the use of these agents in the treatment of several neurodegenerative disorders.

Axons use a conserved program to actively drive their own destruction after injury. Axon degeneration is present in many neurological disorders and an axon death program could be a major pharmaceutical target to preserve neuronal function. This intrinsic signaling cascade activates pro-degenerative dSarm/Sarm1, rapidly depletes axonal stores of NAD+, and terminates in cytoskeletal breakdown. Conversely, loss of dSarm/Sarm1, maintenance of NAD+ levels or its biosynthetic enzyme Nmnat, result in long-term morphological perseveration of severed axons. Exactly how dSarm/Sarm1 and loss of NAD+ execute axon death remains poorly defined. We sought to uncover novel regulators of axon death and maintenance by performing a deficiency screen and a forward genetic mutagenesis screen in axotomized Drosophila wing sensory neurons. We identified a BTB domain protein enriched in neurons, we named Axundead (Axed), which is specifically required for axon death. Severed axons harboring loss of function mutations in axed, similar to dSarm mutants, remain preserved for 50 days post axotomy. Spontaneous neurodegeneration induced by activated dSarm or dNmnat depletion are both suppressed in axed mutants, but not in dSarm mutant alleles. Additionally, severed axed mutant axons also expressing activated dSarm or lacking Nmnat are preserved. These results indicate that dSarm acts upstream of dNmnat loss, and both events precede essential Axed function and axon destruction. Thus, the axon death pathway converges on Axed function.

Age-related macular degeneration (AMD) is the most common cause of vision loss in America for people over the age of 60. Due to damage to the retina, symptoms normally include blurred central vision, difficulty reading, and seeing shadows. While there is no cure for the disease, there are treatments that slow its progression and can restore vision. The treatments explored in this paper are: anti-vascular endothelial growth factor (VEGF) drugs, photodynamic therapy (PDT) and steroids. All three require invasive eye procedures that carry their own risks. The possibility of more effective treatments by combining these therapies is being tested through clinical trials. Studies of combined PDT and anti-VEGF, combined PDT and steroids, and anti-VEGF monotherapy were reviewed, comparing changes in average visual acuity, foveal thickness, and number of injections administered. PDT and anti-VEGF was concluded to be the most efficient of the three, requiring fewer injections while showing an increase in visual acuity similar to anti-VEGF monotherapy.

No description available.

Understanding how different regions of the central nervous system (CNS) are affected by genetic insults is critical to advancing the study of CNS pathologies. The cerebellum is one such region which is disproportionately hypoplastic in the majority of cases of CASK gene mutation in humans. CASK is an enigmatic multi-domain scaffolding protein which plays a vital role in organizing protein complexes at the pre-synapse through interactions with both active zone proteins and trans-synaptic adhesion molecules such as liprins-α and neurexins. Mutations in the X-linked CASK gene in humans are largely post-natally lethal in the hemizygous condition and result in microcephaly with pontine and cerebellar hypoplasia (PCH) and also optic nerve hypoplasia (ONH) in heterozygous mutations. Herein, I used various molecular and genetic strategies to uncover the role of the CASK protein in brain function and pathogenesis of cerebellar hypoplasia associated with CASK mutations/deletions. First, using the face- and construct-validated heterozygous CASK knockout (Cask+/-) murine model, I conducted bulk RNA-sequencing and proteomics experiments from whole brain lysates to uncover changes in the Cask+/- brain. RNA-sequencing revealed the majority of changes to be broadly categorized into metabolic, nuclear, synaptic, and extracellular-matrix associated transcripts. Proteomics revealed the majority of changes cluster as synaptic proteins, metabolic proteins, and ribosomal subunits. Thus, absence of CASK in half of brain cells seems to affect synaptic protein content, cell metabolism, and protein homeostasis. Extending these observations, I conducted GFP-trap immunoprecipitation followed by tandem mass spectroscopy to reveal protein complexes in which CASK participates. Commensurate with proteomic changes, CASK was found to complex with synaptic proteins, metabolic proteins, cytoskeletal elements, ribosomal subunits, and protein folding machinery. Next, in order to investigate the pathogenesis of CASK-linked cerebellar hypoplasia, I utilized a human case of early truncation wherein the 27th arginine of CASK is converted to a stop codon. Immunohistochemical analysis of this brain revealed an upregulation of glial fibrillary acidic protein, a common marker for degenerative cell death. To mechanistically test the hypothesis that cerebellar hypoplasia results from cell death rather than developmental failure, I created a murine model wherein CASK is deleted from the majority of cerebellar cells post-development using Cre recombinase driven by the Calb2 promoter. Deleting CASK from all cerebellar granule neurons post-migration indeed leads to degeneration of the cerebellum via massive depletion of granule cells while sparing Purkinje cells. Overall, the cerebellum shrinks by approximately half in cross-sectional area and degeneration is accompanied by a collapsing of the molecular layer and of Purkinje cell dendrites. In addition, cerebellar degeneration presents with a profound locomotor ataxia. In conclusion, CASK seems to be affecting brain energy homeostasis and synaptic connections via interactions with metabolic proteins, synaptic proteins, and protein homeostatic elements. Further, alterations in brain volume associated with CASK-linked disorders is the result of degenerative cell death rather than developmental failure as previously posited. / Doctor of Philosophy / One of the main challenges facing modern neuroscience is the question of how constitutive mutations in genes present in every cell can cause different effects on different parts of the brain. CASK is one such gene which is expressed in every cell of the brain and, when mutated, typically results in an overall smaller brain volume. However, the cerebellum is one region of the brain involved in motor coordination which is disproportionately smaller than the rest of the brain. Through this gene, I investigate here two questions principally: (1) what is the role of the CASK protein in cells? And (2) how is the cerebellum differentially affected? Firstly, I conduct a molecular investigation into what changes in the brain of a mouse model of CASK deletion which recapitulates the majority of human cases found in girls. This genetic model results in half of cells in the body lacking CASK and leads to smaller brain volume with disproportionate reduction in cerebellum size, as in the human subjects. Using a variety of molecular and biochemical methods, I uncover that several classes of proteins are changed in this brain, primarily those associated metabolism and cell-to-cell communication. Further, my experiments indicate that CASK interacts with many of these proteins. Next, I use human cases as well as a novel mouse model to uncover the trajectory of CASK-linked reduction in cerebellar size. The human case indicates molecular signatures of cell death, a surprising finding given that CASK-linked disorders are thought to result from developmental failure. Investigating this mechanistically in a mouse model, I uncover that when CASK is deleted after development, cerebellar cells still die and the cerebellum actually shrinks. Thus, my work herein elucidates potential roles for the CASK molecule in cells and shows, for the first time, that CASK-linked cerebellar size diminishment is degenerative in nature rather than developmental. This degeneration of the cerebellum occurs very early on in infancy and so was missed until now. The most important implication is that a degenerative process could be halted with therapies other than relying exclusively on genetic therapies.

Objectives: To investigate the feasibility of real-time three-dimensional (3D) echocardiographic analysis of right ventricular (RV) function in healthy dogs and to compare conventional and 3D echocardiographic (3DE) indices of RV function in dogs with various stages of myxomatous mitral valve disease (MMVD), classified per the guidelines of the American College of Veterinary Internal Medicine, to those from healthy dogs. Animals: Twenty-two healthy dogs and 56 dogs with MMVD Methods: All dogs underwent conventional and 3D echocardiographic examinations. Measurements of 3DE RV function indices including RV end-diastolic volume (EDV), RV end-systolic volume (ESV), RV stroke volume (SV), and RV ejection fraction (EF) were recorded. Measurements of conventional indices of RV function were also obtained. RV EDV, ESV, and SV were indexed to bodyweight (BW) and analyzed using commercially available software. Results: Three-dimensional RV datasets could be acquired and analyzed in all dogs. Intra- and inter-observer coefficents of variation were > 20% for all 3D RV indices. Right ventricular EDV and ESV were decreased and RV EF was increased in dogs with advanced MMVD when compared to controls. Several conventional echocardiographic indices of RV function also differed between the control group and various MMVD groups. Conclusions: Real-time 3DE RV assessment is feasible in normal dogs with acceptable intra- and inter-observer variability. Several 3DE indices of RV systolic function differ between dogs with advanced MMVD when compared to normal dogs. Further investigation is required to determine if these differences have clinical implications. / Master of Science

Liu, Ke. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 175-198). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Tam, Oi Sin Pancy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 101-128). / Abstracts in English and Chinese. / Table of Contents / Title page --- p.i / Abstract --- p.iii / 摘要 --- p.vi / Acknowledgements --- p.viii / Table of Contents --- p.ix / List of Figures --- p.xiii / List of Tables --- p.xv / Abbreviations --- p.xvii / Publications related to the work of this thesis --- p.xx / Conference Presentations related to this thesis --- p.xxi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- AMD --- p.1 / Chapter 1.2 --- Epidemiology --- p.4 / Chapter 1.3 --- Classification --- p.5 / Chapter 1.3.1 --- Dry AMD --- p.6 / Chapter 1.3.2 --- Wet/Exudative AMD --- p.9 / Chapter 1.4 --- Etiology and risk factors of AMD --- p.10 / Chapter 1.4.1 --- Gender and Ethnicity --- p.10 / Chapter 1.4.2 --- Smoking and vascular factors --- p.11 / Chapter 1.4.3 --- Genetic Factor --- p.11 / Chapter 1.5 --- Molecular Genetics of AMD --- p.12 / Chapter 1.5.1 --- Linkage studies --- p.12 / Chapter 1.5.2 --- Candidate genes search --- p.15 / Chapter 1.5.3 --- Genome-Wide Association --- p.18 / Chapter 1.5.3.1 --- Complement Factor H --- p.20 / Chapter 1.5.3.2 --- LOC387715 --- p.22 / Chapter 1.6 --- Statistical Analysis --- p.23 / Chapter 1.6.1 --- Genotyping --- p.23 / Chapter 1.6.2 --- Quality Assessment of Genetic Data --- p.24 / Chapter 1.6.3 --- Association Analysis --- p.26 / Chapter 1.6.4 --- Population Stratification --- p.26 / Chapter 1.6.5 --- Haplotype Analysis of Multiple SNPs --- p.26 / Chapter 1.6.6 --- Population Attributable Risk --- p.27 / Chapter 1.6.7 --- Interaction analysis --- p.28 / Chapter 1.7 --- Objectives --- p.28 / Chapter Chapter 2 --- Materials and Method --- p.30 / Chapter 2.1. --- Materials --- p.30 / Chapter 2.1.1. --- Proteins --- p.30 / Chapter 2.1.2. --- Chemicals --- p.30 / Chapter 2.1.3. --- Solutions and Buffers --- p.31 / Chapter 2.1.4. --- Reagents and Kits --- p.31 / Chapter 2.1.5. --- Apparatus --- p.32 / Chapter 2.1.6. --- Softwares --- p.32 / Chapter 2.2. --- Methods --- p.32 / Chapter 2.2.1. --- Study Subjects --- p.33 / Chapter 2.2.2. --- AMD atients --- p.33 / Chapter 2.2.3. --- Control Subjects --- p.34 / Chapter 2.2.4. --- DNA Extraction and Quantification --- p.34 / Chapter 2.2.5. --- Whole genome wide SNP scanning --- p.34 / Chapter 2.2.6. --- HTRA1 Genotyping --- p.38 / Chapter 2.2.6.1. --- Serial Polymerase Chain Reactions --- p.38 / Chapter 2.2.6.2. --- Cycle sequencing --- p.40 / Chapter 2.3. --- Statistical analysis --- p.40 / Chapter 2.3.1. --- Hardy-Weinberg Equilibrium Test --- p.40 / Chapter 2.3.2. --- Association Analysis: Linkage disequilibrium --- p.42 / Chapter 2.3.3. --- Haplotype Analysis --- p.43 / Chapter 2.3.4. --- Interaction Analysis --- p.43 / Chapter Chapter 3 --- Results --- p.46 / Chapter 3.1. --- Genome-wide Association Study of Exudative AMD --- p.46 / Chapter 3.1.1. --- Genotyping and Association Analysis --- p.46 / Chapter 3.1.2. --- Haplotype Analysis --- p.50 / Chapter 3.2. --- HTRA1 Genotyping --- p.57 / Chapter 3.2.1. --- Association Analysis --- p.57 / Chapter 3.2.2. --- Haplotype Analysis --- p.68 / Chapter 3.2.3. --- rsl 1200638 - Smoking Interaction --- p.68 / Chapter 3.2.4. --- rsl 1200638 - rs800292 Interaction --- p.74 / Chapter Chapter 4 --- Discussion --- p.79 / Chapter 4.1. --- Genome-wide Association Study of Exudative AMD --- p.79 / Chapter 4.1.1. --- Limitations and Concerns of Genome-Wide Association Study --- p.84 / Chapter 4.2. --- HTRA1 Genotyping --- p.85 / Chapter 4.2.1 --- Association and Haplotype Analysis --- p.85 / Chapter 4.2.2. --- HTRA1 --- p.87 / Chapter 4.2.3. --- Gene-Environment Interaction --- p.93 / Chapter 4.2.4. --- Gene-Gene Ineraction --- p.94 / Conclusions and Future Aspects --- p.97 / Electronic-Database Information --- p.100 / References --- p.101

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

<p><strong>Introduktion:</strong> Synen är det viktigaste mediet genom vilket människan uppfattar omgivningen. Degenerativa processer som försämrar synen kan förbindas till ärftligt betingade eller degenerativa processer och mutationsfaktorer eller multifaktoriella degenerativa näthinnesjukdomar: retinitis pigmentosa, glaukom och åldersrelaterad makula degeneration. Forskarna har försökt förhindra utveckling av näthinnedegenerationen genom användning av stam- eller progenitorceller. Under differentiering och självförnyande processer utsöndrar dessa celler olika endogena tillväxtfaktorer: substanser som är aktiva vid cellens mognads- och reparationsprocesser. Att rädda och skydda celler från apoptos eller återskapa nya fotoreceptorer eller nervceller är forskarnas mål. Under de senaste 10 åren har man provat att ersätta förlorad syn med synproteser (biomimetisk utrustning).</p><p><strong>Syfte:</strong> Syftet med den här studien var att jämföra olika typer av terapier och beskrivna forskningsmetoder över stamcellsterapi vid diverse sjukliga tillstånd i ögat.</p><p><strong>Resultat:</strong> Stam- och progenitorceller kan skydda fotoreceptorer och nervceller från apoptos. De kan ha förmildrande effekt vid olika degenerativa tillstånd i ögat. Det finns fortfarande osäkerhet över möjligheten att transplanterade celler kan bilda tumörliknande strukturer. Det krävs mer forskning för att utesluta detta. I samodling med humana neurala progenitorceller (hNPC) var yttre nukleära lagret (ONL) mellan 10 % och 40 % tjockare, antalet fotoreceptorer som har överlevt signifikant större i båda testade grupper än vid kontrollen. Fotoreceptorers dödlighet har minskat med 30 % till över 50 % . Det är enormt svårt att kunna skapa från stamceller neuronceller som t.ex. Amakrina celler, bipolära celler och ganglionceller. Humana embryonala stamceller (hESC) differentierar bättre än vad musembryonala celler gjorde. Med synproteser som baseras på kvarstående näthinnemöjligheter har man kunnat återskapa en del av synen.</p><p><strong>Diskussion:</strong> Terapin med stamceller kan ha förmildrande effekter vid glaukomatösa förluster av ganglionceller. Närvaro av humana neurala progenitorceller (hNPC) minskar signifikant dödlighet hos fotoreceptorer och neuronceller men möjligheterna att återskapa redan förlorade näthinneceller är begränsade. Det finns fortfarande risk att transplanterade celler bildar tumörliknande strukturer i ögat. Plats och utvecklingsfas där transplantat hamnar spelar stor roll vid lyckad behandling. Det krävs mer forskning innan dessa terapier blir klinisk tillåtna.</p>