Eine retrospektive Studie über das Progressionsrisiko asymptomatischer früher Altersabhängiger Makuladegeneration in die symptomatische Spätphase überzugehen. Anhand von Lipofuszin-Mustern, die per Fundusautofluoreszenzmessung erkannt werden können, lassen sich Risikopatienten identifizieren. Diese Methode könnte zukünftig im klinischen Alltag als nicht-invasive, kostengünstige Screening-Methode zur Anwendung kommen, um Risikopatienten einer intensivierten Therapie unterziehen zu können, bevor sie eine Beeinträchtigung ihrer Sehkraft erfahren.

Background: Systemic hypertension is a risk factor of age-related macular degeneration (AMD), a chronic inflammatory disease. Acute hypertension is caused by increased extracellular osmolarity after intake of dietary salt (NaCl). We determined in cultured human retinal pigment epithelial (RPE) cells whether high extracellular NaCl alters the gene expression of inflammasome-associated proteins, and whether autocrine/paracrine purinergic (P2) receptor signaling contributes to the NaCl-induced NLRP3 gene expression. Methodology/Principal Findings: Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Gene and protein expression levels were determined with real-time RT-PCR and Western blot analysis, respectively. IL-1β and IL-18 levels were evaluated with ELISA. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. High extracellular NaCl induced NLRP3 and pro-IL-1β gene expression, while the gene expression of further inflammasome-associated proteins (NLRP1, NLRP2, NLRP6, NLRP7, NLRP12, NLRC4, AIM2, ASC, procaspase-1, pro-IL-18) was not altered or below the detection threshold. The NaCl-induced NLRP3 gene expression was partially dependent on the activities of phospholipase C, IP3 receptors, protein kinase C, the serum and glucocorticoid-regulated kinase, p38 MAPK, ERK1/2, JNK, PI3K, and the transcription factors HIF-1 and NFAT5. Pannexin-dependent ATP release and P2Y1 receptor activation is required for the full induction of NLRP3 gene expression. High NaCl induced a transient increase of the NLRP3 protein level and a moderate NLRP3 inflammasome activation, as indicated by the transient increase of the cytosolic level of mature IL-1β. High NaCl also induced secretion of IL-18. High extracellular NaCl induces priming of the NLRP3 inflammasome in RPE cells, in part via P2Y1 receptor signaling. The inflammasome priming effect of NaCl suggests that high intake of dietary salt may promote local retinal inflammation implicated in the development of AMD.

Fam84b is a largely unstudied protein, where its function in eukaryotic cells is unclear. This thesis work presents a FAM84B knockout mouse model and characterises the resulting retinal phenotype in detail. FAM84B KO mice were morphologically assessed by optical coherence tomography and histological processing, revealing dynamic changes stemming from the photoreceptor and pigmented epithelial layers. This potent phenotype progresses with age, spreading inwards towards the inner retinal layers, as well as laterally to adjacent retinal regions. Comparative localisation of standard retinal cell markers demonstrates that FAM84B KO retinal layering becomes increasingly disorganised, together with deformation of the retinal macrostructure. Due to this, KO mice experience reducing responses to light, as demonstrated by electroretinography, where overall retinal efficiency falls. Fam84b shows homology to the HRASLS enzyme family, which are capable of attenuating Ras-associated signalling. To investigate whether Fam84b possesses a similar function, the level of phosphorylated and activated downstream Ras effectors were compared between wild type and FAM84B KO mouse retinal lysates. A reduction of pERK1 (pY204) in KO lysates suggests that Fam84b holds some function related to this pathway downstream of Ras....

Parkinson's disease (PD) is the second most common neurodegenerative disease. With the rapid global increase in population aging, neurodegenerative diseases are considered a primary threat to human health. As dopaminergic neuronal cell death and dysfunction are the main pathogenic mechanisms of PD, neural stem cell (NSC) replacement therapy has been identified as a potentially effective and indeed ideal therapeutic strategy. However, current in vitro stem cell culture methods, which require various chemical growth factors (GFs), are unsafe and relatively inefficient. To solve this problem, we developed two strategies for enhancing the proliferation and differentiation of NSCs in vitro based on extracellular nanomatrices and natural active ingredients. First, we developed novel nanomatrices comprising biomaterials used for promoting NSCs proliferation and differentiation without requiring additional GFs. We developed two types of inorganic sculptured extracellular nanomatrices comprising SiO2 (iSECnMs) which deposited by glancing angle deposition (GLAD). The physiological properties of nanomatrices mediate the activation of multiple bio-signaling pathways. Accordingly, iSECnMs, especially those sculptured in zigzag forms, can significantly promote the proliferation and specific neuronal phenotypic differentiation of NSCs without requiring additional GFs. The differentiated neurons survived well in vivo and achieved outstanding therapeutic effects in a rat model of 6-OHDA-induced parkinsonism. Second, 20(S)-protopanaxadiol (PPD) and oleanolic acid (OA), two crucial active ingredients derived from ginseng, significantly enhanced the proliferation and neuronal differentiation of NSCs through activating Wnt/GSK-3β/β-catenin pathway. This research is expected to promote significant developments in the induction of NSCs and provide insights into stem cell therapies for PD without undesirable prognoses

Amyloids are fibrous protein aggregates associated with age-related diseases, such as Alzheimer’s disease and Parkinson’s disease. The role of amyloids in the etiology of neurodegeneration is debatable, but genetic and molecular evidence supports a causative relationship between amyloidogenesis and disease. Amyloidogenic proteins are constitutively expressed throughout the lifespan of an organism, and yet only become pathogenic in certain situations. This led to a hunt to understand how amyloidogenic proteins could be modified in order to become aggregation-prone. One possibility that has garnered attention is phosphorylation, primarily because several amyloid aggregates such as tau and α-synuclein are often highly phosphorylated in disease. However, the contribution of phosphorylation to disease progression remains unclear.While amyloid aggregates are typically described as irreversible and pathogenic, some cells utilize reversible amyloid-like structures that serve important functions. One example is the RNA-binding protein Rim4 which forms amyloid-like assemblies that are essential for translational control during S. cerevisiae meiosis. If Rim4 is unable to translationally repress its mRNA targets, cells mis-segregate chromosomes during meiosis resulting in aneuploid gametes. Importantly, Rim4 amyloid-like assemblies are disassembled in a phosphorylation-dependent manner at meiosis II onset which allows previously repressed transcripts to become translated. In Chapter 1, I describe the significance and complexity of protein phosphorylation as it relates to disease-associated amyloids and why Rim4 is an ideal model for studying this phenomenon. The objective of this thesis is to examine the mechanisms underlying clearance of Rim4 amyloid-like assemblies. The work described in Chapter 2 focuses on identifying co-factors that mediate clearance of amyloid-like assemblies in a physiological setting. I demonstrate that yeast 14-3-3 proteins, Bmh1 and Bmh2, bind to Rim4 assemblies and facilitate their subsequent phosphorylation and timely clearance. Furthermore, distinct 14-3-3 proteins play non-redundant roles in facilitating phosphorylation and clearance of amyloid-like Rim4. In Chapter 3, I explore the mechanism underlying 14-3-3 contribution to Rim4 amyloid-like disassembly. I find that 14-3-3 proteins are critical for the interaction between Rim4 and its primary kinase Ime2, thus facilitating downstream multi-site phosphorylation of Rim4. In Chapter 4, I explore additional roles for 14-3-3 proteins in general protein aggregate homeostasis. I find that 14-3-3 mutants exhibit greater protein aggregate burdens. Additionally, 14-3-3 mutants accumulate ubiquitinated proteins and are sensitized to proteasome mutations, suggesting a role for 14-3-3 proteins in proteasome function. Collectively, the studies described in this thesis support a protective role for 14-3-3 proteins in protein aggregation that may have implications for amyloid biology in human disease.

The inability to repair axonal damage is a feature of neurological impairment after injury and in neurodegenerative diseases. Axonal repair after injury depends in part on intrinsic factors. Several genes cell-autonomously regulate both axon regeneration and degeneration in response to injury. Recently, Sarm1 has emerged as a key regulator of neurodegeneration. Whether Sarm1 plays a role in axon regeneration is unknown. In this thesis, I identified a role for the C. elegans homolog of Sarm1, tir-1, as a negative regulator of axon regeneration. Investigating the genes which regulate axon regeneration and degeneration has been hindered by technical difficulties in visualizing and manipulating both of these processes in vivo simultaneously. To circumvent this challenge, I developed a new model of axon injury, where both axon regeneration and degeneration can be monitored in vivo with single neuron resolution in C. elegans. I found that the C. elegans homolog of Sarm1, tir-1, strongly inhibits axon regeneration in response to injury. I found that TIR-1 functions cell-intrinsically and that its subcellular localization is dynamically regulated in response to injury. To regulate both axon regeneration and degeneration after injury, I found that TIR-1 function is determined by interaction with two distinct genetic pathways. Together, this work reveals a novel role for tir-1/Sarm1 in axon regeneration, increases our understanding of the injury response, provides new avenues of investigation for studies of TIR-1/SARM1, and inspires candidate approaches to repair the injured nervous system.

Medfödda hjärtfel drabbar ca 1% av alla barn. Vid vissa typer av hjärtfel saknas adekvat förbindelse mellan höger kammare och lungartären, antingen pga stenosering, obstruktion eller total avsaknad. Dessa hjärtfel krävs operation i form av rekonstruktion av förbindelsen (RVOT). För rekonstruktionen används vanligen mänskliga hjärtklaffar, så kallade homograft. Homograft används fördelaktigt pga dess goda hemodynamiska prestanda samt att mottagaren besparas behandling med blodförtunnande mediciner. Syftet med projektet var att genomföra en hållbarhetsstudie av homograft som bevarats i antibiotikalösning över tid, tidsintervall upp till två månader. Vilket besvaras utifrån hur homograftet cell- och vävnadsstruktur påverkas av antibiotikalösningen, och om det skiljer sig mellan aorta- och pulmonaliskärl samt om och hur homograftets mottaglighet för histologisk infärgning påverkas av antibiotikalösningen över tid? Studien omfattade kärlmaterial från både aorta- och pulmonaliskärl från 20 donerade homograft, där samtliga donatorer är upp till 65 år och har samtyckt till att homograften används till studien. Kärlmaterialet processas morfologiskt och färgas in med histofärgerna Azan, Erytrosin-Saffran samt Van Gieson Elastin för att visualisera kärlväggens cell- och vävnadsstrukturer. Resultat av studien påvisar att homograften påverkas negativt av antibiotikalösningen över tid med avseende på cell- och vävnadsstruktur. Det går även att påvisa adekvata skillnader i hur aorta- och pulmonaliskärlen påverkas. Däremot påvisades ingen signifikant antbiotikapåverkan med avseende på homograftets mottaglighet för histofärgningarna.

Eine lokale Aktivierung des Komplementsystems im RPE ist ein pathogener Faktor der AMD. Neben der Wirkung von angiogenen Faktoren wie VEGF könnte eine Aktivierung des Blutgerinnungssystems im RPE dazu beitragen, dass sich aus einer trockenen eine feuchte AMD entwickelt. Dies könnte auf mehreren Ebenen geschehen: Gerinnungsfaktoren könnten die Expression der Komplementfaktoren und der angiogenen Faktoren regulieren sowie Wirkungen auf die Proliferation und Migration der RPE-Zellen besitzen. Eine Stimulierung der Proliferation und Migration der RPE-Zellen trägt zur Ausbildung von CNV-Membranen bei. Es ist aber bis jetzt nichts darüber bekannt, ob RPE-Zellen Faktoren des Blutgerinnungssystems exprimieren und ob z.B. Thrombin (als zentrale Protease des Blutgerinnungssystems) die Genexpression von Komplementfaktoren und von VEGF im RPE beeinflusst. Die Ziele der vorliegenden Dissertation waren daher: ● Nachweis der mRNA-Expression von Blutgerinnungs- und Komplementfaktoren im RPE; ● Nachweis der Wirkung von Thrombin auf die Expression von VEGF und von Komplementfaktoren, sowie auf die Proliferation und Migration der RPE-Zellen; und ● Nachweis der Wirkung der Komplementfaktoren C5a und C9 auf die Sekretion von VEGF und die Proliferation und Migration der RPE-Zellen.

Gymnastics is noted for involving highly specialized strength, power, agility and flexibility. Flexibility is perhaps the single greatest discriminator of gymnastics from other sports. The extreme ranges of motion achieved by gymnasts require long periods of training, often occupying more than a decade. Gymnasts also start training at an early age (particularly female gymnasts), and the effect of gymnastics training on these young athletes is poorly understood. One of the concerns of many gymnastics professionals is the training of the spine in hyperextension—the ubiquitous ‘arch’ seen in many gymnastics positions and movements. Training in spine hyperextension usually begins in early childhood through performance of a skill known as a back-bend. Does practising a back-bend and other hyperextension exercises harm young gymnasts? Current information on spine stretching among gymnasts indicates that, within reason, spine stretching does not appear to be an unusual threat to gymnasts’ health. However, the paucity of information demands that further study be undertaken.

Following a cortical injury, neurons in areas near and connected to the site of injury begin to degenerate. The observed neuronal death may contribute to the severity of the observed behavioral impairments. The purpose of the present study was to examine if progesterone, a hormone known for its effectiveness at reducing cerebral edema, could protect against secondary neuronal death and facilitate the acquisition of an avoidance learning task in an ablation model of cortical injury. Rats served as sham controls or received bilateral ablation of the medial prefrontal cortex followed by a 10-day regimen of progesterone (4 mg/kg) or oil vehicle (1 ml/kg) beginning 1 h after cortical lesions. Progesterone-treated lesion rats showed a significant facilitation of avoidance learning compared to oil- treated lesion controls. In addition, progesterone-treated lesion animals did not differ from either progesterone- or oil-treated sham controls in avoidance learning. Anatomical analysis revealed that progesterone treatment decreased the amount of neuronal death seen in the striatum and the mediodorsal nucleus of the thalamus. The findings are consistent with the notion that progesterone is an effective neuroprotective agent and suggest that the hormone can reduce the behavioral impairments associated with frontal cortical ablation injury.