This thesis describes the development of artificial disc technology for the replacement of intervertebral discs in the human lumbar spine. The clinical problem is back pain. There may be a relationship between certain forms of back pain and disc degeneration. The mechanical properties of human intervertebral discs are examined in detail. The genetic basis of disc degeneration is presented. The hypothesis is that such degeneration leads to a loss of normal stiffness in the segments affected leading to abnormal mechanical behaviour which in turn leads to pain. The evidence for this is presented. The development of surgical solutions to relieve back pain, from fusion through first generation mechanical artificial discs to elastomeric designs, is traced. The author‘s personal contributions to this area of knowledge are set out. The appreciation of the requirement for a restoration of physiological stiffness is argued throughout, showing where fusion and first generation discs have not met the clinical aim of pain relief, because they have not restored physiological stiffness. The path to an elastomeric, viscoelastic, polyhydrocarbon, rubber solution in the form of the “Freedom“ disc has filled 17 years of the author‘s academic pursuits. It will be shown that this technology may represent a possible solution to the clinical problem. Failure is part of all new advancement and this too is presented, to show how that has influenced thinking, producing original ideas to overcome these failures. Providing lessons are learned from these failures then our patients in the future will benefit.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Myxomatous mitral valve degeneration (MMVD) is the single most common cardiac disease of the dog, and is analogous to Mitral Valve Prolapse in humans. Very little is known about the aetiopathogenesis of this disease or the changes in valvular interstitial cell populations in diseased valves. The aim of this study was to identify morphological, cellular and molecular changes associated with MMVD. Mitral valve leaflets from both normal and varying grades (Whitney’s 1-4) of diseased dogs were subject to image analysis, immunophenotyping, proteomics and RT-PCR. Image analysis - leaflet thickening due to accumulation of glycosaminoglycan was significant in this disease. MMVD is associated with loss of connective tissue, reduction in cell numbers but no change in cell shape in the overtly myxomatous area. Near the surface, increase in valvular interstitial cells (VIC) towards the damaged endothelium in concert with destruction of collagen and building up of ground substance was manifested during the disease process. Immunophenotyping - activated myofibroblasts were increased and fibroblast-like VICs were reduced without any change in desmin and myosin expression in MMVD compared to clinical normal dogs. In addition, other cell types like macrophage, adipocyte, chondrocyte, mast cell, and stem cell were identified and their possible role in MMVD is discussed. Proteomics - a protein expression profile was established, with 64 proteins being positively identified from dog’s mitral valve using 1-D SDS PAGE LC/MS. Amongst them 44 proteins were differentially expressed comparing normal and severely diseased. Two actin binding proteins, tropomyosin alpha and myosin light chain-2 were found to be differentially expressed in the normal but down regulated in the diseased. RT-PCR was used to assess the expression of 8 genes of interest. Their expression was compared with 3 different housekeeping genes.

<p>Complement factor H (CFH) is a major susceptibility gene for age-related macular degeneration (AMD); however, its impact on AMD pathobiology is unresolved. Here, the role of CFH in the development of AMD pathology in vivo was interrogated by analyzing aged Cfh+/- and Cfh-/- mice fed a high fat, cholesterol-enriched diet. Strikingly, decreased levels of CFH led to increased sub-retinal pigmented epithelium (RPE) deposit formation, specifically basal laminar deposits, following high fat diet. Mechanistically, our data show that deposits are due to CFH competition for lipoprotein binding sites in Bruch’s membrane. Interestingly and despite sub-RPE deposit formation occurring in both Cfh+/- and Cfh-/- mice, RPE damage accompanied by loss of vision occurred only in old Cfh+/- mice. We demonstrate that such pathology is a function of excess complement activation and C5a production, associated with monocyte recruitment, in Cfh+/- mice versus complement deficiency in Cfh-/- animals. Due to the CFH dependent increase in sub-RPE deposit height we interrogated the potential of CFH as a novel regulator of Bruch’s membrane lipoprotein binding and show, using human Bruch’s membrane explants, that CFH removes endogenous human lipoproteins in aged donors. Interestingly, although the CFH H402 variant shows altered binding to BrM, this does not affect its ability to remove endogenous lipoproteins. This new understanding of the complicated interactions of CFH in AMD-like pathology provides an improved foundation for the development of targeted therapies for AMD.</p> / Dissertation

Ackmann, Charlotte Osmotische Induktion des Komplementfaktors C9 in retinalen Pigmentepithelzellen Universität Leipzig, Dissertation 98 Seiten, 208 Literaturangaben, 28 Abbildungen, 8 Tabellen Die altersbedingte Makuladegeneration (AMD) ist die häufigste Ursache für Erblindung bei Erwachsenen in den industrialisierten Ländern. Die AMD ist unter anderem eine chronisch entzündliche Erkrankung, bei der die Aktivierung der alternativen Komplementkaskade eine Rolle spielt. Daneben erhöht Bluthochdruck, der auch durch eine salzreiche Ernährung getriggert wird, das Risiko an einer AMD zu erkranken. Untersucht wurde die Genexpression des Komplementfaktors C9 unter verschiedenen pathologischen Bedingungen in humanen retinalen Pigmentepithel (RPE)-Zellen sowie deren Wirkung auf die physiologischen Eigenschaften der Zellen. Gezeigt wird, dass die Expression des C9 Gens in humanen RPE-Zellen spezifisch durch Hyperosmolarität, Hypoxie und oxidativen Stress induziert wird. Die Menge an C9 Protein wurde durch Hyperosmolarität leicht aber signifikant erhöht. Die hyperosmotische Induktion der C9 mRNA ist abhängig von der Aktivierung der Signalproteine p38 MAPK, ERK1/2, JNK, PI3K, sowie der Transkriptionsfaktoren STAT3 und NFAT5 während für die Hypoxie-induzierte C9 mRNA Expression nur eine Beteiligung des Transkriptionsfaktors STAT3 nachgewiesen wurde. Die Aktivierung verschiedener Signalwege durch Hyper-osmolarität und Hypoxie lässt vermuten, dass eine hohe Kochsalzaufnahme auch unter normoxischen Verhältnissen die Eigenschaften RPE-Zellen verändert. Hyperosmolarität hemmt die Proliferation und Migration der RPE-Zellen, während chemische Hypoxie nur die Proliferationsrate verringert. Die Wirkung einer erhöhten extrazellulären NaCl-Konzentration auf die C9 mRNA Expression wird über zwei Mechanismen vermittelt: über die Erhöhung der extrazellulären Osmolarität und über die Veränderung des NaCl-Gradienten über der Plasmamembran. Die NaCl Wirkung über den veränderten NaCl-Gradienten lässt vermuten, dass eine übermäßige Aufnahme von Kochsalz nicht nur über die Erhöhung des Blutdruckes die Pathogenese der AMD stimuliert, sondern dass Kochsalz auch eine direkte stimulierende Wirkung auf RPE-Zellen besitzt. Diese Vermutung könnte erklären, weshalb hoher Blutdruck ein Risikofaktor der AMD ist, aber Medikamente zur Behandlung des Bluthochdruckes das Risiko der AMD nicht verändert.

Despite surgical innovation, the sensory and motor outcome after peripheral nerve injury is incomplete. In this thesis, the biological pathways potentially responsible for the poor functional recoveries were investigated in both the distal nerve stump/target organ, spinal motoneurons and dorsal root ganglia (DRG). The effect of delayed nerve repair was determined in a rat sciatic nerve transection model. There was a dramatic decline in the number of regenerating motoneurons and myelinated axons found in the distal nerve stumps of animals undergoing nerve repair after a delay of 3 and 6 months. RT-PCR of the distal nerve stumps showed a decline in expression of Schwann cells (SC) markers, with a progressive increase in fibrotic and proteoglycan scar markers, with increased delayed repair time. Furthermore, the yield of SC which could be isolated from the distal nerve segments progressively fell with increased delay in repair time. Consistent with the impaired distal nerve stumps the target medial gastrocnemius (MG) muscles at 3- and 6-months delayed repair were atrophied with significant declines in wet weights (61% and 27% compared with contralateral sides). The role of myogenic transcription factors, muscle specific microRNAs and musclespecific E3 ubiquitin ligases in the muscle atrophy was investigated in both gastrocnemius and soleus muscles following either crush or nerve transection injury. In the crush injury model, the soleus muscle showed significantly increased recovery in wet weight at days 14 and 28 (compared with day 7) which was not the case for the gastrocnemius muscle which continued to atrophy. There was a significantly more pronounced up-regulation of MyoD expression in the denervated soleus muscle compared with the gastrocnemius muscle. Conversely, myogenin was more markedly elevated in the gastrocnemius versus soleus muscles. The muscles also showed significantly contrasting transcriptional regulation of the microRNAs miR-1 and miR-206. MuRF1 and Atrogin-1 showed the highest levels of expression in the denervated gastrocnemius muscle. Morphological and molecular changes in spinal motoneurons were compared after L4-L5 ventral root avulsion (VRA) and distal peripheral nerve axotomy (PNA). Neuronal degeneration was indicated by decreased immunostaining for microtubule-associated protein-2 in dendrites and synaptophysin in presynaptic boutons after both VRA and PNA. Immunostaining for ED1-reactive microglia and GFAPpositive astrocytes was significantly elevated in all experimental groups. qRT-PCR analysis and Western blotting of the ventral horn from L4-L5 spinal cord segments revealed a significant upregulation of apoptotic cell death mediators including caspases-3 and -8 and a range of related death receptors following VRA. In contrast, following PNA, only caspase-8 was moderately upregulated. The mechanisms of primary sensory neuron degeneration were also investigated in the DRG following peripheral nerve axotomy, where several apoptotic pathways including those involving the endoplasmic reticulum were shown to be upregulated. In summary, these results show that the critical time point after which the outcome of regeneration becomes too poor appears to be 3-months. Both proximal and distal injury affect spinal motoneurons morphologically, but VRA induces motoneuron degeneration mediated through both intrinsic and extrinsic apoptotic pathways. Primary sensory neuron degeneration involves several different apoptotic pathways, including the endoplasmic reticulum.

The aim of this thesis was to identify genetic factors involved in frontotemporal lobar degeneration (FTLD), a neurodegenerative disorder clinically characterised by a progressive change in personality, behaviour and language. FTLD is a genetically complex disorder and a positive family history is found in up to 40% of the cases. In 10-20% of the familial cases the disease can be explained by mutations in the gene encoding the microtubule associated protein tau (MAPT). In the first study we describe the clinical and neuropathological features of a Finnish family with FTLD caused by a mutation in MAPT. We also provide evidence that the pathogenic mechanism of this mutation is through altered splicing of MAPT transcripts. Recently, mutations in the gene encoding progranulin (PGRN) were identified as a major cause of FTLD. In the second study we describe a Swedish family with FTLD caused by a frameshift mutation in PGRN. We provide a clinical and neuropathological description of the family, as well as evidence that the pathogenicity of this mutation is through nonsense-mediated decay of the mutant mRNA transcripts and PGRN haploinsufficiency. In the third study we describe a novel PGRN splice site mutation and a previously described PGRN frameshift mutation, found in a mutation screen of 51 FTLD patients. We describe the clinical and neuropathological characteristics of the mutation carriers and demonstrate that haploinsufficiency is the pathogenic mechanism of the two mutations. In the fourth study we investigate the prevalence of PGRN and MAPT gene dosage alterations in 39 patients with FTLD. No gene dosage alterations were identified, indicating that variations in copy number of the PGRN and MAPT genes are not a common cause of disease, at least not in this FTLD patient collection.

No description available.

<p>Age-related macular degeneration is one of the leading causes of vision loss in the world. While identification of various environmental risk factors including but not limited to smoking, ethnicity, and diet have been reported to contribute to the complex etiology of AMD, age and genetics remain the largest susceptibility factors in its pathogenesis. Initially, with the identification of the common Y402H variant in CFH, approximately 35% of the genetic determinants of AMD had been identified with the majority remaining unknown. Therefore, we set forth to A) identify additional AMD susceptibility genes that contribute to AMD through the use to next generation sequencing technologies and B) to assess associated alleles for pathogenicity in the attempt to interpret their functional contributions to AMD outcome as observed via patient serum and zebrafish analysis. In doing such, we have identified both common and rare variants that contribute to the heritability of AMD. Additionally, we report one of the first instances of a rare variant significantly increasing disease onset and a gene with increased rare mutational burden in AMD patients. All together adding to our understanding of the genetics of AMD and potentially leading to putative therapeutic targets.</p> / Dissertation

Vision provides a critical interface with the physical world. This work examines visual development and vision loss in mice to glean the influence of the retinal state on visual connections. I first assessed the impact of retinal activity on the eye-specific segregation of retinal afferents in the lateral geniculate nucleus (LGN) of young Gβ5 -/- mice. Gβ5 is the fifth member of the β subfamily of heterotrimeric G proteins. Gβ5 binds and stabilizes the R7 family of regulators of G-protein signaling (RGS), which accelerate Gi/o GTP hydrolysis. Gβ5 -/- mice, which lack R7RGS activity, have malformed synapses in the outer plexiform layer (OPL) and impaired OPL transmission. Altered spontaneous retinal activity in Gβ5-/- mice at P7, P12, P14, and P28 correlates with impaired eye-specific segregation of retinal afferents in the LGN at corresponding timepoints. However, Gβ5-/- mice exhibit a normal transition from cholinergic to glutamatergic drive that corresponds with a temporary recovery of refinement at P10. Thus the abnormal-normal-abnormal pattern of activity in the retina is coupled with abnormal-normal-abnormal segregation. This activity-segregation profile suggests activity may instruct early retinogeniculate development. nob mice, which also exhibit impaired OPL transmission, have aberrant retinal waves that align with loss of segregation. nobxGβ5-/- mice have similar levels of segregation as Gβ5-/- at P21, but activity only similar P14 nobxGβ5-/- and Gβ5-/- RGCs. This suggests that the critical period of eye-specific segregation closes shortly after P14 and that R7RGS activity is critically important to postnatal RGCs. Next, I investigated the aged visual system via the retinofugal projections of mice with retinal remodeling after photoreceptor degeneration (PD). ΔCT mice, with mild remodeling, and TG9N mice, with aggressive remodeling, retain gross anatomical and physiological connectivity in the presence of attenuated visual activity compounded by organic remodeling. However, the magnitude of pupillary light responses in PD mice was diminished. Reduced melanopsin signal in the retina, not downstream anomalies, explains this functional deficiency. These observations suggest that changes to eye-specific segregation are limited once projections are established, regardless of retinal activity or remodeling. These observations bode well for future retina-based treatments of vision loss.