Under det här kandidatexamensarbetet har protein-nanofibers påverkan på material undersökts genom att jämföra fibrillerade filmer med ofibrillerade. Sojaproteinisolat fibrillerades under förhållandena pH 2 och 85 ◦C under minst ett dygn och de syntetiserade nanofibrerna analyserades med Thioflavin T (ThT) fluorescens och atomkraftsmikroskopi (AFM). Spektra från analysmetoden ThT fluorescens indikerade på förekomsten av β-flak och analyserna med AFM visade på att fibrerna hade en morfologi som är karakteristisk för protein-nanofibrer. Resultaten antyder att de parametrar som påverkar morfologin hos fibrerna är fibrilleringstid och typ av protein. De gjutna filmerna från fibrillära respektive ofibrillära proteiner var sammanhängande bortsett från vissa sprickor. Värdena på E-modulen från AFM visade att det fibrillerade materialet var mer heterogent än det ofibrillerade. Filmer med sammanhängande yta erhölls vid tillsats av det mjukgörande additivet glycerol. Slutligen, material av både fibrillär och ofibrillär form kan framställas, däremot krävs vidare forskning för att optimera materialens egenskaper. / During this bachelor thesis project, the impact of protein nanofibers on materials has been analysed by comparing films made from fibrillar and non-fibrillar protein. Fibrillation of soy protein isolate was performed during at least 24 hours at pH 2 and a temperature of 85 ◦C. Analysis of the nanofibers was made with Thioflavin T (ThT) fluorescence and atomic force microscopy (AFM). The spectra from ThT Fluorescens indicated the presence of β-sheets and AFM confirmed that the fibrils had a morphology that is characteristic of protein nanofibers. The results indicated that heating time and protein type were the parameters which had the largest impact on the morphology of the fibrils. The synthesised films from both fibrillar and non-fibrillar protein were coherent with exception of some cracks. The elastic modulus from AFM indicated that the fibrillar film was more heterogeneous compared to the non-fibrillar film. To attain coherent films, the plasticising agent glycerol was added. To summarise, both fibrillar as well as non-fibrillar materials were successfully synthesised, however, further research is necessary to optimise the properties of the material.

No description available.

Introduction: Alzheimer's disease is a neurodegenerative disease that initially manifests itself primarily as impaired short-term memory and impaired language ability. The course of the disease is mainly due to an atrophy in the brain that can be attributed to the protein amyloid B and tau. Monoclonal antibodies that target Alzheimer's disease often have a high rate of cerebral edema, where proteinaceous fluid leaks into the extracellular space of the brain and creates edema. Some of the most common symptoms for amyloid-related imaging abnormalities (ARIA-E) are headache, dizziness, and blurred vision. In a few cases, patients with ARIA-E need to be hospitalized for observation, but most show a decline in ARIA-E within one to two months. Objective: To investigate the frequency of ARIA-E in clinical studies of monoclonal antibodies to patients with Alzhiemer's disease and to investigate the role of the ApoE4 allele in the development of ARIA-E. Method: Literature review of five RCT studies based on four different monoclonal antibodies. PubMed was used to search for the RCT-studies. Results: ARIA-E varies between different types of antibodies. ARIA-E usually occurs early in treatment when the degree of amyloid b is highest in the brain. Most cases are asymptomatic and treatment resumes within 1-2 months. Conclusion: Aria-E frequency correlates strongly with dose strength as well as APOE4 -status and most of the incidences are asymptomatic. With the right titration and individually selected drugs as well as individual dosages a safe care can be established for patients with Alzheimer's disease. If treatment is initiated at an early stage, the risk of side effects is reduced and more neurons can be saved from atrophy. The combination of several different types of medicine will further reduce the risk of ARIA-E.

No description available.

Einleitung: Bei Fohlen ist bisher wenig bekannt über die Eisenparameter im Blut. Das Spurenelement Eisen ist für Säugetiere essenziell, seine Hauptaufgabe ist der Sauerstofftransport. Adulte Pferde nehmen das benötigte Eisen mit dem Raufutter auf, welches in der Regel ausreichend hohe Eisengehalte aufweist. Das Hauptmolekül zur Eisenspeicherung ist Gewebe-Ferritin. Das im Serum vorkommende Serum-Ferritin spiegelt die Gesamteisenmenge des Körpers wider. Im Rahmen einer systemischen Entzündung sinkt der Serum-Eisenspiegel, da Eisen ins Gewebe umverteilt wird und in das Gebiet der Entzündung abwandert. Für die Parameter des Eisenstoffwechsels gibt es keine Referenzwerte für Fohlen, für adulte Pferde gibt es lediglich einen Referenzbereich für Serum-Eisen von 1,00-3,61 mg/l. Es gibt keine Studien, bei denen die Eisenparameter von Fohlen im Alter von ein bis sechs Monaten bestimmt wurden. Ziel der Studie: Ziel der vorliegenden Arbeit war es, die Parameter des Eisenstoffwechsels im Serum bei akuter systemischer Entzündung bei ein bis sechs Monate alten Fohlen zu untersuchen. Außerdem sollte überprüft werden, inwiefern diese Veränderung des Eisenstoffwechsels zu einem Abfall der Serum-Eisenwerte führt. Tiere, Material und Methoden: In dieser prospektiven Studie wurden EDTA-Vollblut- und Serumproben von 66 gesunden Fohlen und 84 Fohlen mit einer akuten systemischen Entzündung in Form einer abszedierenden Bronchopneumonie genommen. Die Fohlen waren im Median 109 Tage alt. Die Diagnose 'Bronchopneumonie' wurde mittels Ultraschalldiagnostik gestellt, die Fohlen hatten am Tag der Probennahme einen sonografisch ermittelten Abszesscore von über 15 cm sowie eine rektal gemessene Körperinnentemperatur über 39,0 °C. Die gesunden Fohlen wiesen eine unauffällige klinische Allgemeinuntersuchung sowie Lungenultraschalluntersuchung am Tag der Probennahme sowie in der vorherigen und darauffolgenden Woche auf. Im Labor wurde der Serum-Amyloid-A-Gehalt bestimmt. Die gesunden Pferde wiesen SAA-Werte innerhalb des Referenzbereiches auf, bei den kranken Pferden bestätigte ein Wert > 7µg/ml die Diagnose einer akuten systemischen Entzündung. Aus der EDTA-Vollblutprobe wurde innerhalb von einer Stunde mittels Durchflusszytometrie die Blutleukozytenzahl bestimmt. Die Analyse der Serumproben erfolgte im Labor LABOKLIN mit dem Analysesystem Cobas 8000 von Roche. Die statistische Auswertung der Daten erfolgte mit dem Statistikprogramm SPSS (IBM Statistics 27). Die Daten der Gesamtpopulation (n = 150) waren nicht normalverteilt. Es wurde ein Mann-Whitney-U-Test als nicht parametrischer Test für zwei unabhängige Stichproben durchgeführt, um signifikante Unterschiede zwischen den Gruppen zu ermitteln. Das Signifikanzniveau wurde auf p < 0,05 festgelegt. Ergebnisse: Bei der Auswertung der Probanden ergab sich für die gesunden Fohlen ein medianer SAA-Wert von 3,50 µg/ml, der der kranken Fohlen liegt mit 541 µg/ml hochsignifikant darüber. Die Bestimmung des Serum-Ferritin-Wertes ergab für die Gruppe der gesunden Fohlen einen Median von 3,80 µg/l (IQR 2,00-4,40 µg/l), der Median für die Gruppe der kranken Fohlen ist mit 4,60 µg/l (IQR 2,48-6,40 µg/l) signifikant (p = 0,003) höher. Die Serum-Eisenwerte der gesunden Fohlen lagen innerhalb des laborinternen Referenzbereichs für adulte Pferde (Median 1,57 mg/l; IQR 1,21-1,79 mg/l), auch die weiteren Bluteisenwerte entsprachen denen adulter Pferde. Die kranken Fohlen zeigten signifikant niedrigere Serum-Eisenwerte (Median 0,550 mg/l; IQR 0,368-0,778 mg/l), signifikant höhere Ferritin- und UIBC-Werte sowie eine signifikant geringere Eisensättigung. Die Auswertung der Leberenzymaktivitäten aus dem Serum ergab für die kranken Fohlen bei allen gemessenen Parametern im Durchschnitt niedrigere Werte als für die gesunden Fohlen. Bei dem Spurenelement Kupfer liegt der Median der gesunden Fohlen mit 18,8 µmol/l (IQR 16,4-20,1 µmol/l) im oberen Bereich der Referenzwerte von 7,9-21 µmol/l, der Median der kranken Fohlen liegt mit 25,7 µmol/l (IQR 23,1-29,0 µmol/l) signifikant über den Werten der gesunden Fohlen (p < 0,001). Schlussfolgerung: Durch eine akute systemische Entzündung kommt es zu einem Abfall des Serum-Eisens durch Umverteilung ins Gewebe und Abwanderung ins Entzündungsgebiet. Da es jedoch sowohl in der Literatur als auch in den Ergebnissen dieser Studie keine Hinweise auf einen absoluten Eisenmangel beim Fohlen gibt, sollte von einer Eisensupplementation in jedem Fall abgesehen werden.:1 Einleitung 1 2 Literaturübersicht 2 2.1 Vorkommen und chemische Eigenschaften von Eisen 2 2.2 Eisenmetabolismus 2 2.2.1 Absorption 2 2.2.2 Transport 3 2.2.3 Speicherung, Mobilisierung 4 2.2.4 Exkretion, Verlust 5 2.3 Physiologische Funktionen 6 2.4 Feststellung des Eisenstatus 7 2.5 Eisenbedarf des Pferdes 9 2.6 Eisengehalt von Futtermitteln 10 2.7 Eisenmangel 11 2.8 Supplementation von Eisen 12 2.9 Eisenüberschuss, Toxizität 12 2.10 Bakterieller Eisenstoffwechsel 13 2.11 Entzündung 14 2.11.1 Abszedierende Bronchopneumonie 15 2.12 Eisenstoffwechsel bei systemischer Entzündung 16 2.12.1 Ferritin als Akute-Phase-Protein 17 2.12.2 Serum-Eisen als Entzündungsmarker 17 3 Publikation 19 3.1 Vergleich des Eisenstatus im Serum von gesunden Fohlen und Fohlen mit Bronchopneumonie 19 4 Diskussion 30 5 Zusammenfassung 34 6 Summary 36 7 Literaturverzeichnis 38 Danksagung 47 / Introduction: Little is known about iron parameters in the blood of foals. The trace element iron is essential for mammals, its main task is the transport of oxygen. Horses take up the required iron with roughage, which has sufficiently high iron contents. The main molecule for iron storage is tissue ferritin. The serum ferritin found in the serum reflects the total amount of iron in the body. In the context of systemic inflammation, serum iron levels decrease as iron is redistributed to the tissues and migrates to the area of inflammation. There are no reference values for the parameters of iron metabolism for foals, for adult horses there is only a reference range for serum iron from 1.00-3.61 mg/L. There are no studies in which the iron parameters of foals were determined at the age of one to six months. Aim of the study: The aim of the present work was to investigate the parameters of serum iron metabolism in acute systemic inflammation in one- to six-month-old foals. In addition, it was to be determined to what extent this change in iron metabolism leads to a drop in serum iron levels. Animals, material, and methods: In this prospective study, EDTA whole blood and serum samples were collected from 66 healthy foals and 84 foals with acute systemic inflammation in the form of abscessed bronchopneumonia. The median age of the foals was 109 days. The diagnosis 'bronchopneumonia' was made by ultrasound diagnosis, the foals had a sonographically determined abscess score of more than 15cm as well as a rectally measured internal body temperature of more than 39.0 °C on the day of sampling. The healthy foals had an unremarkable clinical general examination and lung ultrasound on the day of sampling and in the previous and following week. In the laboratory, the serum amyloid A content was determined, a value > 7µg/mL confirmed the diagnosis of acute systemic inflammation. From the EDTA whole blood sample, the blood leucocyte count was determined within one hour by flow cytometry. The serum samples were analyzed in the LABOKLIN laboratory using the analyzer Cobas 8000 from Roche. Statistical analysis of the data was performed with SPSS (IBM Statistics 27). The data of the total population (n = 150) were not normally distributed. A Mann-Whitney U test was performed as a non-parametric test for two independent samples to determine significant differences between the groups. The significance level was set at p < 0.05. Results: The evaluation of the blood samples showed a median SAA level of 3.50 µg/mL for the healthy foals, the median of 541 µg/mL for the sick foals was significantly higher than in healthy foals. The determination of serum ferritin showed a median of 3.80 µg/l (IQR 2.00-4.40 µg/l) for the group of healthy foals, the median for the group of sick foals was significantly (p = 0.003) higher at 4.60 µg/l (IQR 2.48-6.40 µg/l). The serum iron values of the healthy foals were within the laboratory reference range for adult horses (median 1.57 mg/l; IQR 1.21-1.79 mg/l). The other blood iron values also corresponded to those of adult horses. The sick foals showed significantly lower serum iron levels (median 0.550 mg/l; IQR 0.368-0.778 mg/l), significantly higher ferritin and UIBC levels and significantly lower iron saturation. The evaluation of liver enzyme activities from the serum showed lower values on average for the sick foals than for the healthy foals for all measured parameters. For the trace element copper, the median of the healthy foals with 18.8 µmol/l (IQR 16.4-20.1µmol/l) is in the upper range of the reference values of 7.9-21µmol/l, the median of the sick foals with 25.7 µmol/l (IQR 23.1-29.0 µmol/l) is significantly higher than the values of the healthy foals (p < 0.001). Conclusion Acute systemic inflammation causes a drop in serum iron due to redistribution into the tissues and migration into the area of inflammation. However, since there is no evidence of an absolute iron deficiency in foals, either in the literature or in the results of this study, iron supplementation should be avoided in any case.:1 Einleitung 1 2 Literaturübersicht 2 2.1 Vorkommen und chemische Eigenschaften von Eisen 2 2.2 Eisenmetabolismus 2 2.2.1 Absorption 2 2.2.2 Transport 3 2.2.3 Speicherung, Mobilisierung 4 2.2.4 Exkretion, Verlust 5 2.3 Physiologische Funktionen 6 2.4 Feststellung des Eisenstatus 7 2.5 Eisenbedarf des Pferdes 9 2.6 Eisengehalt von Futtermitteln 10 2.7 Eisenmangel 11 2.8 Supplementation von Eisen 12 2.9 Eisenüberschuss, Toxizität 12 2.10 Bakterieller Eisenstoffwechsel 13 2.11 Entzündung 14 2.11.1 Abszedierende Bronchopneumonie 15 2.12 Eisenstoffwechsel bei systemischer Entzündung 16 2.12.1 Ferritin als Akute-Phase-Protein 17 2.12.2 Serum-Eisen als Entzündungsmarker 17 3 Publikation 19 3.1 Vergleich des Eisenstatus im Serum von gesunden Fohlen und Fohlen mit Bronchopneumonie 19 4 Diskussion 30 5 Zusammenfassung 34 6 Summary 36 7 Literaturverzeichnis 38 Danksagung 47

Master of Science / Biochemistry and Molecular Biophysics / Jianhan Chen / Proteins are fundamental building blocks of life in an organism, and to function properly, they must adopt an appropriate three-dimensional conformation or conformational ensemble. In protein aggregation diseases, proteins misfold to incorrect structures that allow them to join together and form aggregates. A wide variety of proteins are involved in these aggregation diseases and there are multiple theories of their disease mechanism. However, a common theme is that they aggregate into filamentous structures. Therapies that target the process by which the aggregating proteins assemble into these similar fibril-like structures may by effective at countering aggregation diseases. This requires models that can accurately describe the assembly process of the fibrils. An analytical theory was recently described where fibrils grow by the templating of peptides onto an existing amyloid core and the kinetics of the templating process is modeled as a random walk in the backbone hydrogen bonding space. In this thesis, I present my work integrating molecular simulation with this analytical model to investigate the dependence of fibril growth kinetics on peptide sequence and other molecular details. Using the Aβ16-22 peptide as a model system, we first calculate the rate matrix of transitions among all possible hydrogen bonding microscopic states using numerous short-time simulations. These rates were then used to construct a kinetic Monte Carlo model for simulations of long-timescale fibril growth. The results demonstrate the feasibility of using such a theory/simulation framework for bridging the significant gap between fibril growth and simulation timescales. At the same time, the study also reveals some limits of describing the fibril growth as a templating process in the backbone hydrogen bonding space alone. In particular, we found that dynamics in nonspecifically bound states must also be considered. Possible solutions to this deficiency are discussed at the end.

The classification of neurodegenerative disorders is based on the major component of the protein aggregates in the brain. The most common altered proteins associated with neurodegeneration are Hyperphosphorylated tau (HPt), beta amyloid (Aβ), alpha-synclein (αS) and transactive response DNA binding protein 43 (TDP43). In this study we assessed the incidence and the neuroanatomical distribution of proteins associated with neurodegeneration in the brain tissue of cognitively unimpaired subjects. We demonstrated the early involvement of the Locus Coeruleus (LC) with HPt pathology in cognitively unimpaired mid aged subjects, a finding which supports the notion that LC is an initiation site of HPt pathology. This may suggest that development of clinical assessment techniques and radiological investigations reflecting early LC alterations may help in identifying subjects with early stages of neurodegeneration. Furthermore, we studied a large cohort of cognitively unimpaired subjects with age at death ≥50 years and we applied the National Institute on Aging –Alzheimer’s disease (AD) Association (NIA-AA) guidelines for the assessment of AD related neuropathological changes. Interestingly, a considerable percentage of the subjects were classified as having an intermediate level of AD pathology. We also showed that the altered proteins;  HPt , Aβ, αS, and TDP43 are frequently seen in the brain of cognitively unimpaired subjects with age at death ≥50 years, the incidence of these proteins increased significantly with age. This finding suggests that neurodegeneration has to be extensive to cause functional disturbance and clinical symptoms. Moreover, we investigated the correlation between AD related pathology in cortical biopsies, the AD / cerebrospinal fluid (CSF) biomarkers and the Mini Mental State examination (MMSE) scores in a cohort of idiopathic Normal Pressure Hydrocephalus (iNPH) patients. We demonstrated that AD/ CSF biomarkers and MMSE scores reflect AD pathology in the cortical biopsies obtained from iNPH patients.  In conclusion, this study shows that the altered proteins associated with neurodegeneration are frequently seen in the brain tissue of cognitively unimpaired aged subjects. This fact should be considered while developing diagnostic biomarkers for identification of subjects at early stages of the disease, in order to introduce therapeutic intervention prior to the occurrence of significant cognitive impairment.

Alzheimer’s disease (AD) is a neurodegenerative disorder, with extensive evidence implicating the misfolding, aggregation and deposition of the amyloid-β (Aβ) peptide as central to the pathogenesis. Heparan sulfate (HS) is an interactive glycosaminoglycan, attached to core proteins as HS proteoglycans (HSPGs). HSPGs are present on cell surfaces and in the extracellular matrix where they facilitate multiple signaling functions, but HS is also consistently present in all amyloid deposits, including those of AD. In amyloidosis HS has been studied as an aggregation template, promoting fibril formation and serving a scaffold function in the resulting deposits. The objective of this thesis was to assess how cell surface HS is potentially implicated in Aβ amyloidosis and the associated neuroinflammation of AD.   In AD brain we determined that HS predominantly accumulated in Aβ deposits with dense cores and found glial-expressed HSPGs within these deposits. Aβ elevated HSPG levels in primary glial cultures, implicating activated glia as one source of the Aβ-associated HS. Next, we determined that microglial HSPGs are critical for the upregulation of interleukin-1β and tumor necrosis factor-α following exposure to lipopolysaccharide, an established inflammatory insult. Together these results raise the possibility that Aβ-induced expression of microglial HSPGs may promote neuroinflammation.   Multiple mechanisms of Aβ toxicity have been proposed and different Aβ assemblies exert their toxicity through alternative routes. We found that three different preparations of Aβ aggregates all exhibited HS-dependent cytotoxicity, which in part correlated with Aβ internalization. Furthermore, heparin treatment attenuated Aβ cytotoxicity and uptake. In Aβ-positive AD microvasculature, HS deposited with Apolipoprotein E (ApoE) and its receptor, the low density lipoprotein receptor-related protein 1 (LRP1). In cell culture, HS and LRP1 co-operated in Aβ interactions and the addition of ApoE increased the levels of cell-associated Aβ in a HS- and LRP1-dependent manner. This ApoE-mediated increase in cell-associated Aβ may promote toxicity and vascular degeneration, but equally HS-mediated internalization of Aβ could represent a clearance route across the blood-brain-barrier. The findings presented here illustrate multiple roles for cell-surface HSPGs in interactions relevant to the pathogenesis of AD.

Cerebral hypoperfusion can occur in normal ageing and is proposed to underlie white matter disturbances observed in the ageing brain. Moreover, cerebral hypoperfusion and white matter attenuation are early events in the progression of Alzheimer’s disease (AD). White matter mostly consists of myelinated axons which have distinct protein architecture, segregated into defined regions; the axon initial segment (AIS), the node of Ranvier, paranode, juxtaparanode, and internode. These sites are essential for action potential initiation and/or propagation and subsequently effective brain function. At the outset of the studies in the thesis there was evidence that the different regions within the myelinated axons are vulnerable to injury and disease. Thus it is hypothesised that in response to normal ageing and/or cerebral hypoperfusion these structures are altered and associated with cognitive impairment and that these effects are exacerbated in a transgenic mouse model (APPSw,Ind, J9 line) which develops age-dependent amyloid-β (Αβ) pathology. The first study aims to investigate the effect of normal ageing and Aβ deposition on myelinated axons and on learning and memory. To address this, the effects of normal ageing on the integrity of the AIS, nodes of Ranvier, myelin, axons, synapses and spatial working memory are examined in young and aged wild-type and TgAPPSw,Ind mice. A significant reduction in the length of nodes of Ranvier is demonstrated in aged wild-type and TgAPPSw,Ind mice. In addition, the length of AIS, is significantly reduced in the aged wild-type animals while the young TgAPPSw,Ind have significantly shorter AIS than the young wild-type mice. These effects are not influenced by the presence of Aβ. Myelin integrity is affected by age but this is more prominent in the wild-type animals whilst axonal integrity is intact. Moreover, there is an age-related decrease of presynaptic boutons only in the TgAPPSw,Ind mice. Contrary to the original hypothesis, working memory performance is not altered with age or influenced by increasing Aβ levels. The second study aims to examine the effects of cerebral hypoperfusion in combination with Αβ pathology and/or ageing on cognitive performance and the structure of myelinated axons. To address this, the effects of surgically induced cerebral hypoperfusion on the integrity of the nodes of Ranvier, paranodes, myelin, axons and spatial working memory performance are investigated in young and aged wild-type and TgAPPSw,Ind mice. A decrease in nodal length is observed in response to hypoperfusion in young and aged animals. This effect is shown to be exacerbated in the young TgAPPSw,Ind animals. Moreover, the disruption of the nodal domain is shown to occur without any gross alterations in myelin and axonal integrity. It is also demonstrated that in response to hypoperfusion, spatial working memory performance is defected in young and aged animals of both genotypes. This deficit is exacerbated in the young TgAPPSw,Ind. The observed changes in the nodal structure are associated with poor working memory performance indicating functional implication for the nodal changes. These data highlight that structures within myelinated axons are vulnerable to ageing and cerebral hypoperfusion. Therefore, the development of strategies that minimize injury or drive repair to these regions is necessary together with therapeutic approaches against the vascular insults that induce hypoperfusion and lead to white matter attenuation and cognitive decline. In the future, it would be interesting to investigate how alterations at the AIS/nodes of Ranvier affect neuronal excitability.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy