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Regulation of soluble adenylyl cyclase in the sea urchin <em>Arbacia punctulata</em>
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The role of the purinergic receptor P2Y12 in Alzheimer's disease
No full textAlzheimer’s disease (AD), the most common cause of dementia, shows a significant rise in prevalence due to increased global life expectancy and represents a major public health, social, and economic burden. Microglia, the resident immune cells of the CNS, have recently moved into prime focus of AD research due to the discovery of risk loci and genes associated with immune mechanisms in microglia. Extracellular ATP or ADP activates the purinergic receptor P2Y12, which is highly expressed in microglia and is critical for several microglial functions (chemotaxis, blood-brain barrier closure, synaptic plasticity, interaction with neurons and phagocytosis). As many patients with cardiovascular diseases worldwide are prescribed anticoagulant P2Y12R antagonists (e.g clopidogrel) on a daily or chronic basis and blood brain barrier impairment is known to preceed AD development, this thesis investigates the role of the P2Y12 receptor in AD, microglial barrier formation around Aβ plaques and its impact on plaque pathology. Impaired microglial phagocytic clearance of Aβ and impaired plaque barrier formation upon P2Y12 dysfunction may affect AD progression. To conduct the study, 3D reconstructed confocal images of brain tissue from female transgenic APP/PS1 mice with and without genetic deletion of the P2Y12 receptor were digitally analyzed. In addition, whole-slide imaging and super-resolution microscopy were used to quantify astrocytosis, plaque or dystrophic neurite load and synaptic density. Our results suggest that P2Y12R may influence the microglia-plaque interaction. Not only a reduction of microglia-plaque coverage, particularly in the hippocampus, was detected in the absence of the P2Y12 receptor, but also an increase in astrocyte reactivity and a reduction in the area of dystrophic neurites near the plaques in the cortex. This may represent a synergistic or compensatory effect on astroglia in neuroprotection. However, significant changes in plaque-associated microglia ramification or cell volume, total plaque burden, plaque morphology and synaptic density were not observed. Experimental limitations require cautious interpretation related to the question if P2Y12 activity may have beneficial or detrimental effects on the AD progression. Further investigation with larger sample size, different mouse models and advanced techniques will be needed to elucidate implications in the field of neuroprotection and to develop new therapeutic strategies in early stages of AD
Antagonistic Nanobodies Reveal Mechanism of Gasdermin D Pore Formation and Unexpected Therapeutic Potential
No full textActivation of various inflammasomes converges on the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases, followed by oligomerization of the N-terminal domain (GSDMDNT) and the assembly of pores penetrating target membranes. As methods for live cell investigations of the GSDMD pore formation process are limited, the order of conformational changes, oligomerization, and membrane insertion remained unclear. Moreover, specific inhibitors of GSDMD have not yet been identified.
To deal with these shortcomings, we raised twenty-three nanobodies (VHHs) specific for human GSDMD and created a large and diverse repertoire of tools to study and perturb GSDMD in living cells at a molecular level. We found that cytosolic expression of VHHGSDMD-1 or VHHGSDMD-2 specifically abrogates GSDMD-mediated pyroptosis and IL-1β release in macrophage-like cell lines and human primary macrophages. This could be thoroughly confirmed upon both NLRC4- and NLRP3-induced inflammasome activation, and also showed potential upon the activation of the non-canonical inflammasome as well as the AIM2 inflammasome.
Binding of the antagonistic nanobodies to GSDMDNT interferes with its oligomerization, while inflammasome assembly and GSDMD processing itself were not affected. The nanobody-stabilized monomers of GSDMDNT partitioned into the plasma membrane, suggesting that monomeric GSDMDNT is able to directly insert into the plasma membrane and build up the pore monomer by monomer within target membranes. The formation of pre-pores, i.e. oligomerization before membrane insertion, is thus no prerequisite for GSDMD pore formation.
Inhibition of GSDMD pore formation switches cell death from pyroptosis to apoptosis, which still required ASC specks and the activation of caspase-1. We propose a novel layer of regulation of the caspase-1 activity by GSDMD pores, as the enzymatic activity was substantially augmented in presence of the antagonistic nanobodies. Caspase-1 therefore seems to be the key regulator that activates apoptotic mediators such as caspase-3, caspase-7, caspase-8, caspase-9, and BID in the absence of GSDMD pores. GSDME cleavage was observed as well, but no GSDME-mediated pyroptosis could be detected.
Furthermore, we revealed the unexpected therapeutic potential of antagonistic GSDMD nanobodies, as recombinant nanobodies added to the extracellular space prevented inflammatory cell death. The nanobodies thereby enter through the first sublytic pores, after which they curtail the assembly of additional pores and prevent inflammatory cell death. GSDMD nanobodies may thus exhibit the features necessary to treat the evergrowing list of diseases caused by activation of (non-) canonical inflammasomes
Chromatin Architecture Shapes the Immune System : Chromatinopathies Perspective
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FVIII-albumin fusion proteins enhance the induction of immune tolerance towards coagulation factor VIII in haemophilia A mice
No full textHaemophilia A (HemA) is an X-linked recessive disease caused by the lack of functional coagulation factor VIII (FVIII) causing life-threatening prolonged bleeding events due to a coagulation incapacity. HemA is treated by prophylactic or on-demand infusions of recombinant FVIII (rFVIII), according to the grade of disease severity. Individuals affected by severe haemophilia A are characterised by large deletions or intron inversions in the fviii gene and therefore present a significantly different portion of FVIII compared with the wildtype protein present in healthy individuals. Therefore, intravenously-injected exogenous rFVIII is recognised as a foreign antigen. FVIII-specific B cells are activated upon immunisation and trigger the production of anti-drug neutralising antibodies, known as inhibitors. Presently, the only known method to induce tolerance towards FVIII is the Immune Tolerance Induction regime, known as ITI, which consist of repetitive high-dose FVIII infusions pursued until the inhibitor levels are abrogated. The limitations and poor accessibility of the ITI therapy open the door to alternative strategies to enhance tolerance induction and improve the quality of the individuals’ life.
This work aimed to induce immune tolerance towards exogenous FVIII by the administration of FVIII-albumin conjugates in a haemophilia A mouse model. Here, FVIII-albumin fusion proteins were generated by chemical cross-linking of FVIII to albumin in a 1:1 or 1:5 molar ratio, referred to as FP(1:1) and FP(1:5), respectively. Unlike the ITI models, the fusion proteins were injected intravenously in singular weekly intervals and aligned with FVIII control group. The mechanisms underlying the FVIII-specific immune response were elucidated in vivo by flow cytometry, confocal microscopy and ELISA and by the establishment of an in vitro HemA splenocytes cultures.
Collectively, the data indicated that FVIII-albumin impairs the development of FVIII-specific B cells into activated subtypes reducing dramatically the inhibitor production compared with FVIII alone, while being coagulative active. Fusion protein is transported to the liver where it could be internalised by professional antigen-processing cells, including dendritic cells and potentially by macrophages. Nevertheless, fusion protein treatments affect the co-stimulation of APCs causing the disruption of follicular CD4+ T helper cell activation. CD25+ regulatory T cells may contribute to the suppression of epitope-specific T helper cells. In vitro co-cultures indicated that the Fas/FasL and PD-1/PD-L1 axes may be employed as a potential FVIII-specific B cell suppressive Treg-mediate mechanism. Overall, FVIII-albumin stimulation in vitro failed to stimulate FVIIIspecific B cells and TFH in comparison with FVIII stimulation.
To explore the membrane trafficking of albumin and FVIII-albumin, an uptake assay was established in RAW264.7 macrophages. Here, I evaluated the compartmentalisation of albumin within the recycling or degrading endosomes and its segregation into recycling tubules by confocal microscopy and live cell imaging. FVIII-albumin fusion protein, similar to albumin alone, segregated from FVIII-positive endosomes into tubular structures which may undergo membrane trafficking and recycling.
Overall, FVIII-albumin fusion proteins seem to be a promising therapeutic tool to enhance induction of immune tolerance protocols towards FVIII
Regulation of FVIII-specific immunity in Hemophilia A mice
No full textThe major complication of Hemophilia A treatment is the formation of inhibitors that neutralize the administered FVIII. Immune tolerance induction (ITI) is the most frequently used therapy in order to establish long-lasting tolerance towards FVIII in patients with inhibitors. However, predicting ITI success is challenging and the underlying cellular and molecular mechanisms are poorly described, thus therapy stability and duration is individualized, and might be influenced by several risk factors. Here, I demonstrate that upon successful tolerization of HemA mice, a PR8 infection in the lung triggered a systemic alteration of the inflammatory state. FVIII-specific B cells, responsible for the anti-FVIII humoral alloimmune response, are increased upon infection, while the FVIII-specific Tregs and their PD-L1 expression, induced during ITI in vivo, were reduced. Additionally, the FVIII-specific T cell compartment changed into a pro-inflammatory phenotype with an increase of FVIII-specific Th17 and Tfh cells. The inter-organ crosstalk between lung and spleen, was mediated by IFN-g, reactivating FVIII-specific B cells, which exhibited a significantly elevated IFN-gR expression. Consequently, these FVIII-specific B cells shifted their phenotype, by increasing their CD80 expression and an initiated IL-6 production. This shift contributed to a loss of the previously established tolerogenic environment towards FVIII achieved through the ITI protocol by fostering T cell dedifferentiation upon IL-6 exposure. Finally emphasizing the importance of the IL-6 signaling in tolerance disruption, the inhibition of IL-6R proved that the tolerized phenotype was restored upon application. Furthermore, other external elements such a high salt diet (HSD) did not impact the ITI outcome. Nevertheless, the HSD in wildtype mice induced elevated corticosterone levels in the bloodstream which modified the uptake behavior of splenic red pulp macrophages and seemed to promote a shift towards a pro-inflammatory phenotype. This was accompanied by a reduction of the FVIII levels in the bloodstream, disturbing the equilibrium of the FVIII homeostasis. Overall, these results may pave the wave for novel therapeutic intervention strategies to improve ITI success and the hemostatic FVIII abundance through a more detailed understanding of the interplay between FVIII, immune and tolerance responses and external factors
Seneca über Selbsterkenntnis und Selbstbewusstsein
No full textDie vorliegende Arbeit beschäftigt sich mit Senecas Überlegungen zum Selbstbewusstsein. Genauer gesagt wird die Frage behandelt, ob und inwieweit Seneca über einen Begriff des Selbst verfügt, der aus der Innenperspektive des Bewusstseins formuliert ist. Das Selbstbewusstsein im Sinne der Innenperspektive ist aus der geläufigen philosophiehistorischen Sicht stark durch Descartes‘ cogito (ergo) sum-Argument geprägt. Daher scheint jeder Versuch, ein personales Selbst bereits bei Seneca zu identifizieren, auf einen Anachronismus hinauszulaufen. Im Mittelpunkt der vorliegenden Arbeit steht daher die Frage, ob die senecanische Idee des Selbstbewusstseins – wenn es diese denn gibt – eine Innovation im Vergleich zu den Ansätzen seiner Vorgänger darstellt, die ihre Vorstellungen vom Selbst eher aus einer objektivistischen Perspektive innerhalb eines intellektualistischen Modells entwickelten.
Meine These besagt, dass dies in der Tat so ist: Bei Seneca handelt es sich um einen wichtigen Übergangsautor vom intellektualistischen zum bewusstseinstheoretischen Selbstverständnis. Entsprechend vertrete ich die Meinung, dass im senecanischen Werk Indizien für einige zentrale Komponenten vorliegen, die in den Rahmen einer personalen Konzeption des Selbst gehören. Ich skizziere den Selbstbewusstseinsbegriff anhand von neun Kriterien: 1. Subjektivität, 2. Reflexion, 3. Einheit, 4. Unmittelbarkeit, 5. Individualität, 6. Privatheit, 7. Kontinuität, 8. Willensfreiheit und 9. das Fremdpsychische, und versuche, Belege für diese Merkmale bei Seneca zu identifizieren. Daher kann meine Sammlung zahlreicher Beobachtungen zu Seneca dazu dienen, unser Bild des frühkaiserzeitlichen Stoikers deutlich zu verändern. Das Ziel dieser Arbeit besteht aber insofern in einer gemäßigten Interpretation, als Seneca nicht zum Urheber und theoretischen Begründer einer vollständigen Bewusstseinstheorie gemacht werden soll. Vielmehr ist er m.E. eine herausragende Figur des Stoizismus, die auf eine interessante Weise der späteren Bewusstseinskonzeption annähert
Pharmacological and structural investigations of the ATP-gated ion channel receptor P2X4
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Mechanicalstress and Proteostasis : analysis of the effect of mechanical stress at various levels of cellular organization
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