Synthese und Cytotoxizität membranpermeabler Phallotoxine

Die Phallotoxine, eine Familie F-Aktin stabilisierender, giftiger Peptide aus dem Grünen Knollenblätterpilz (Amanita phalloides), lassen sich aufgrund ihrer schlechten Membran-permeabilität nur bedingt zur Untersuchung lebender Zellen verwenden. Wie diese Arbeit zeigt, können jedoch durch Kopplung an verschiedene Liganden Phallotoxin-Derivate mit hoher Membranpermeabilität erhalten werden. Insbesondere drei Strategien verbesserten die Translokation der Toxinderivate in das Zellinnere ohne Verlust der biologischen Aktivität: (1) Die Erhöhung der Hydrophobizität der Toxine durch Veresterung mit Fettsäuren; (2) die Bindung von kationischen Polymeren wie Polylysin; und (3) die Kopplung an trojanische Peptide, wie HIV-Tat-Peptid oder Arginin8, über Disulfid-haltige Linker. Alle drei Strategien lieferten Phallotoxin-Derivate mit Cytotoxizitäten, die bis zu 1000-mal höher waren als die des natürlichen Phalloidins. Die Cytotoxizität wurde anhand der Proliferationshemmung in Mausfibroblasten-Kulturen bestimmt, wobei für Oleoyl-Phalloidin, Tat-SS-Phalloidin und Poly-L-Lysin-Phalloidin IC50-Konzentrationen von 2.5 µM, 3.8 µM bzw. 3.1 µM erhalten wurden (Phalloidin >1000 µM). Da die Affinität von Oleoyl-Phalloidin und Tat-SS-Phalloidin für F-Aktin gering ist (bis zu 40-mal geringer als Phalloidin), kann man von einer hydrolytischen Abspaltung des Toxins durch zelluläre Esterasen im Oleoyl-Phalloidin, bzw. einer Reduktion der Disulfidbrücke im Tat-SS-Phalloidin ausgehen. Fluoreszenz-mikroskopische Untersuchungen zeigten, daß die Aufnahme bei allen Derivaten über eine nicht-spezifische, adsorptive Endocytose erfolgt. Dabei wurden die modifizierten Phallotoxine an die Zellmembran gebunden, entweder über hydrophobe oder elektrostatische Wechselwirkungen, anschließend in Vesikeln internalisiert und gespalten, und schließlich über einen unbekannten Transportweg ins Cytosol transportiert. Es stellte sich heraus, daß die vieldiskutierte, permeationsvermittelnde Wirkung der sogenannten trojanischen Peptide weitgehend auf deren kationischer Natur, d.h. auf elektrostatischer Wechselwirkung mit der Membran, beruht. Morphologisch führte die Vergiftung durch membrangängige Phallotoxine zu charakteristischen Veränderungen der Cytoskelett-Struktur der Fibroblasten, wie einer Kumulation des Aktins in "Patches", einer starken Verkleinerung der Zellen und zu häufiger Doppelkernigkeit. Der Nachweis charakteristischer Apoptose-Marker in Jurkat-Zellen deutet darauf hin, daß die toxinbedingte Stabilsierung des Aktin-Cytoskeletts zur Apoptose führt. Eine therapeutische Verwendung F-Aktin-stabilisierender Toxine zur Proliferationhemmung schien aufgrund der Erfolge Tubulin-stabilisierender Toxine in der Tumorbehandlung interessant. Eine gezielte Vergiftung von Tumor-Zellinien durch Kopplung spezifischer Liganden wie Folsäure, Polyethylenglykole und Albumin an Phallotoxine konnte jedoch an Zellkulturen nicht gezeigt werden. Gleichwohl erscheinen für Folsäurederivate und Albumin-konjugierte Phallotoxine weitere Studien im Tiermodell vielversprechend

Neurofilament as a blood marker for diagnosis and monitoring of primary progressive aphasias

Objective: To assess the utility of serum neurofilament for diagnosis and monitoring of primary progressive aphasia (PPA) variants. Methods: We investigated neurofilament light chain (NF-L) levels in blood of 99 patients with PPA (40 with nonfluent variant PPA [nfvPPA], 38 with semantic variant PPA [svPPA], 21 with logopenic variant PPA [lvPPA]) and compared diagnostic performance with that reached by CSF NF-L, phosphorylated neurofilament heavy chain (pNF-H), b-amyloid (Ab(1)-42), tau, and phosphorylated tau. The longitudinal change of blood NF-L levels was measured and analyzed for correlation with functional decline and brain atrophy. Results: Serum NF-L is increased in PPA compared to controls and discriminates between nfvPPA/svPPA and lvPPA with 81% sensitivity and 67% specificity (cutoff 31 pg/mL). CSF NF-L, pNF-H, tau, phosphorylated tau, and Ab1-42 achieved similar performance, and pNF-H was the only marker for discrimination of nfvPPA from svPPA/lvPPA. In most patients with nfvPPA and svPPA, but not lvPPA, serum NF-L increased within follow-up. The increase correlated with functional decline and progression of atrophy of the left frontal lobe of all patients with PPAs and the right middle frontal gyrus of patients with nfvPPA and svPPA. Conclusions: Blood level of NF-L can aid the differential diagnosis of PPA variants, especially in combination with CSF pNF-H. Because serum NF-L correlates with functional decline and atrophy in the disease course, it qualifies as an objective disease status marker. Extended follow-up studies with cases of known neuropathology are imperative

Serum neurofilament light chain in behavioral variant frontotemporal dementia

Objective To determine the association of serum neurofilament light chain (NfL) with functional deterioration and brain atrophy during follow-up of patients with behavioral variant frontotemporal dementia (bvFTD). Methods Blood NfL levels from 74 patients with bvFTD, 26 with Alzheimer disease (AD), 17 with mild cognitive impairment (MCI), and 15 healthy controls (Con) at baseline and follow-up were determined and analyzed for the diagnostic potential in relation to functional assessment (Clinical Dementia Rating Scale Sum of Boxes [CDR-SOB], frontotemporal lobar degeneration-related CDR-SOB, Mini-Mental State Examination [MMSE]) and brain volumetry. Results At baseline, serum NfL level correlated with CSFNfL (bvFTD r = 0.706, p < 0.0001;AD/MCI r = 0.666, p = 0.0003). Highest serum levels were observed in bvFTD (p < 0 0.0001 vs Con and MCI, p = 0.0078 vs AD, respectively). Discrimination of bvFTD from Con/MCI/AD was possible with 91%/74%/74% sensitivity and 79%/74%/58% specificity. At follow-up, serum NfL increased in bvFTD and AD (p = 0.0039 and p = 0.0006, respectively). At baseline and follow-up, NfL correlated with functional scores of patients with bvFTD (e.g., CDR-SOB [baseline] r = 0.4157, p = 0.0006;[follow-up] r = 0.5629, p < 0.0001) and with atrophy in the gray and white matter of many brain regions including frontal and subcortical areas (e.g., frontal lobe: r = -0.5857, p < 0.0001;95% confidence interval -0.7415 to -0.3701). For patients with AD/MCI, NfL correlated with the functional performance as well (e.g., CDR-SOB [baseline] r = 0.6624, p < 0.0001;[follow-up] r = 0.5659, p = 0.0003) but not with regional brain volumes. Conclusions As serum NfL correlates with functional impairment and brain atrophy in bvFTD at different disease stages, we propose it as marker of disease severity, paving the way for its future use as outcome measure for clinical trials. Classification of evidence This study provides Class III evidence that for patients with cognitive problems, serum NfL concentration discriminates bvFTD from other forms of dementia

Longitudinal Diffusion Tensor Imaging Resembles Patterns of Pathology Progression in Behavioral Variant Frontotemporal Dementia (bvFTD)

Objective: Recently, the characteristic longitudinal distribution pattern of the underlying phosphorylated TDP-43 (pTDP-43) pathology in the behavioral variant of frontotemporal dementia (bvFTD) excluding Pick's disease (PiD) across specific brain regions was described. The aim of the present study was to investigate whether in vivo investigations of bvFTD patients by use of diffusion tensor imaging (DTI) were consistent with these proposed patterns of progression. Methods: Sixty-two bvFTD patients and 47 controls underwent DTI in a multicenter study design. Of these, 49 bvFTD patients and 34 controls had a follow-up scan after ~12 months. Cross-sectional and longitudinal alterations were assessed by a two-fold analysis, i.e., voxelwise comparison of fractional anisotropy (FA) maps and a tract of interest-based (TOI) approach, which identifies tract structures that could be assigned to brain regions associated with disease progression. Results: Whole brain-based spatial statistics showed white matter alterations predominantly in the frontal lobes cross-sectionally and longitudinally. The TOIs of bvFTD neuroimaging stages 1 and 2 (uncinate fascicle—bvFTD pattern I; corticostriatal pathway—bvFTD pattern II) showed highly significant differences between bvFTD patients and controls. The corticospinal tract-associated TOI (bvFTD pattern III) did not differ between groups, whereas the differences in the optic radiation (bvFTD pattern IV) reached significance. The findings in the corticospinal tract were due to a “dichotomous” behavior of FA changes there. Conclusion: Longitudinal TOI analysis demonstrated a pattern of white matter pathways alterations consistent with patterns of pTDP-43 pathology

FDG-PET underscores the key role of the thalamus in frontotemporal lobar degeneration caused by C9ORF72 mutations

C9ORF72 mutations are the most common cause of familial frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). MRI studies have investigated structural changes in C9ORF72-associated FTLD (C9FTLD) and provided first insights about a prominent involvement of the thalamus and the cerebellum. Our multicenter, F-18-fluorodeoxyglucose positron-emission tomography study of 22 mutation carriers with FTLD, 22 matched non-carriers with FTLD, and 23 cognitively healthy controls provided valuable insights into functional changes in C9FTLD: compared to non-carriers, mutation carriers showed a significant reduction of glucose metabolism in both thalami, underscoring the key role of the thalamus in C9FTLD. Thalamic metabolism did not correlate with disease severity, duration of disease, or the presence of psychotic symptoms. Against our expectations we could not demonstrate a cerebellar hypometabolism in carriers or non-carriers. Future imaging and neuropathological studies in large patient cohorts are required to further elucidate the central role of the thalamus in C9FTLD

Atrophy in the Thalamus But Not Cerebellum Is Specific for C9orf72 FTD and ALS Patients - An Atlas-Based Volumetric MRI Study

Background: The neuropathology of patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS) due to a C9orf72 mutation is characterized by two distinct types of characteristic protein depositions containing either TDP-43 or so-called dipeptide repeat proteins that extend beyond frontal and temporal regions. Thalamus and cerebellum seem to be preferentially affected by the dipeptide repeat pathology unique to C9orf72 mutation carriers. Objective: This study aimed to determine if mutation carriers showed an enhanced degree of thalamic and cerebellar atrophy compared to sporadic patients or healthy controls. Methods: Atlas-based volumetry was performed in 13 affected C9orf72 FTD, ALS and FTD/ALS patients, 45 sporadic FTD and FTD/ALS patients and 19 healthy controls. Volumes and laterality indices showing significant differences between mutation carriers and sporadic patients were subjected to binary logistic regression to determine the best predictor of mutation carrier status. Results: Compared to sporadic patients, mutation carriers showed a significant volume reduction of the thalamus, which was most striking in the occipital, temporal and prefrontal subregion of the thalamus. Disease severity measured by mini mental status examination (MMSE) and FTD modified Clinical Dementia Rating Scale Sum of Boxes (FTD-CDR-SOB) significantly correlated with volume reduction in the aforementioned thalamic subregions. No significant atrophy of cerebellar regions could be detected. A logistic regression model using the volume of the prefrontal and the laterality index of the occipital subregion of the thalamus as predictor variables resulted in an area under the curve (AUC) of 0.88 while a model using overall thalamic volume still resulted in an AUC of 0.82. Conclusion: Our data show that thalamic atrophy in C9orf72 mutation carriers goes beyond the expected atrophy in the prefrontal and temporal subregion and is in good agreement with the cortical atrophy pattern described in C9orf72 mutation carriers, indicating a retrograde degeneration of functionally connected regions. Clinical relevance of the detected thalamic atrophy is illustrated by a correlation with disease severity. Furthermore, the findings suggest MRI volumetry of the thalamus to be of high predictive value in differentiating C9orf72 mutation carriers from patients with sporadic FTD

Historical evolution view on the substance of the light

The principle of the seminar work is to create the skilled text which could be used such as educational material for other students to they can better understand to the evolution of opinions on the substance of the light. The work is composed from the five parts. Each part is enplaining the view of the scientists on the substance of the light from the certain period. The first part is connected to the era of the antiquity {--} the period of the first contentions about the substance of the light. The second part is intent on the era from the fifth century (Optical theory) to the nineteen century (Young theory ). The third part refers to the 20th century. The fourth part apply to the present, to the newest researches and work in the sphere of the light. The fifth part is created by the biographies of the most meaningful people who have been working on the question about the substance of the light

Construction and Realization of Automatic Battery Charger for Emergency Illumination

The thesis is focusing on the construction and realization of automatic source charger of emergency illumination. The work introduces the question of emergency lighting systems and backup power sources briefly. The main part of this work is concentrating on the concrete designing and realizing projects of the charging system. The output of this work is a specific technical solution and realization of an automatic charger in practice