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Potentiell inadäquate Medikation forensisch bedeutsamer Todesfälle im Krankenhaus von 2011-2015
Get PDFIm Jahr 2010 wurde die Priscus-Liste herausgegeben, die Empfehlungen zu 83 Arzneistoffen enthält, die im höheren Alter vermieden oder mit Vorsicht eingesetzt werden sollten. Ziel dieser Arbeit war es, an einem Kollektiv von Patienten des Institutes für Rechtsmedizin in München festzustellen, inwiefern die Einnahme von Priscus-Arzneistoffen am Todeseintritt oder an der Krankenhausaufnahme beteiligt war und ob die Einnahme in der Vormedikation erfolgte oder der Arzneistoff erst im Krankenhaus verabreicht wurde.
Hierfür wurden alle Patienten ausgewählt, die ≥ 65 Jahre alt waren, zwischen Januar 2011 und Dezember 2015 in einem Krankenhaus verstorben waren, am Institut für Rechtsmedizin in München obduziert worden waren und bei denen postmortal eine toxikologische Untersuchung durchgeführt worden war. Für diese insgesamt 109 Patienten wurde mithilfe der forensischen Gutachten, Ermittlungsunterlagen, Medikationspläne von Hausärzten sowie der Krankenhausunterlagen die Medikation vor und während des Krankenhausaufenthaltes erfasst. In Hinblick auf die Einnahme von Priscus-Arzneistoffen konnten in Hinblick auf Alter, Geschlecht, Krankenhausaufenthaltsdauer, Stürze, Suizide und Reanimationen keine Zusammenhänge zwischen der Einnahme von Arzneistoffen und insbesondere Priscus-Arzneistoffen mit der Krankenhausaufnahme bzw. Todesursache festgestellt wurden.
Polymedikation - definiert als die Einnahme von mindestens fünf Arzneistoffen - war unabhängig vom Verabreichungsort (Vormedikation, Krankenhaus) bei den Priscus-positiven Patienten öfter vorhanden als bei den Priscus-negativen Patienten. Insgesamt betraf Polymedikation in diesem Kollektiv über 74% der Patienten.
Eine Fragestellung, die sich im Verlauf dieser Untersuchung, ist der Einfluss QT-Zeit verlängernder Arzneistoffe auf die Todesursachen wegen deren häufigen Vorkommens. In dieser Untersuchung waren solche bei 75% der Patienten nachweisbar.
Am häufigsten von den Priscus-Arzneistoffen waren Lorazepam, Haloperidol, Diphenhydramin, Dimenhydrinat, Zolpidem und Zopiclon nachweisbar.
Zusammenfassend lässt sich ausführen, dass in dieser Untersuchung trotz des hohen Anteils an Priscus-positiven Patienten (61,5%) Priscus-Arzneistoffe nicht häufiger als andere Arzneistoffe für den Krankenhausaufenthalt oder Tod der Patienten ursächlich waren
On the importance of symbol grounding and top-down processes in computer vision
Get PDFIn the past decade, feedforward artificial neural networks have stormed the field of artificial intelligence and shown impressive results in many domains. Nevertheless, one of the challenges in artificial intelligence is connecting the differentiable feature space in deep learning to the rich world of object-based, symbolic knowledge. For example, in computer vision, images consist of different features, such as edges and curves at a lower level, while at a higher level, they include objects and relations. Even though it is not feasible to describe the low-level features using the natural language, the attributes and relations between objects can be represented by symbols and are well-documented throughout human literature. Therefore, developing novel and effective architectures that can learn and utilize symbolic knowledge within the differentiable deep learning framework is essential. To this end, in this dissertation we argue for methods that map symbols to image-grounded representations such that they share the same representation space as images.
Furthermore, we discuss the key role of top-down processes in utilizing object-level knowledge; top-down signals have been shown to play a significant role in the human brain for overcoming challenges such as occlusion. For example, even though there might not be enough pixels from a truck's wheel in an image, after detecting the truck itself within the top layers of a neural network, we can use the higher-level knowledge to recognize a small area in a corner that corresponds to the wheel. Nevertheless, current feedforward neural networks lack effective inductive biases for top-down processing. We show that grounding symbols in images and employing top-down mechanisms not only improves the scene understanding but also allows us to benefit from the massive pool of human-written symbolic knowledge in addition to image annotations.
In summary, this dissertation introduces significant advances in the artificial intelligence domain, particularly computer vision and modeling commonsense. We propose models that utilize (1) structured knowledge, (2) unstructured text, and (3) 3d information to improve scene understanding, and through large-scale experiments, we show that our models significantly improve state-of-the-art results
Singel-cell characterisation of ageing and disease
Get PDFOver the last decade, emerging single-cell technologies triggered a revolution in our perception of biological systems. While new library generation methods flourished and precipitated a deeper understand- ing of the mechanisms of life, novel computational analyses saw the light, permeabilising even further the boundaries of the field.
Systemic approaches, by the large scope and complexity that they aim to cover, particularly benefited from this leap. Unravelling the heterogeneity of cells identities is key to understanding their states dynamic and how they influence each others with regards to the host phenotype.
In this cumulative thesis, we present two articles that highlight ageing immune mechanisms as fertile soil for the emergence of neu- rodegeneration. We demonstrated the critical influence of the cell location on the gene expression and cellular state, especially in the white-matter, a lipid-rich region of the parenchyma that is particu- larly susceptible to ageing and neurodegeneration.
The first article focuses on a microglial population found in the ageing white matter: the white matter associated microglia (WAM). We show that WAM formation is dependent on TREM2 but not APOE. WAMs present pathways significantly enriched in lipid processing and antigen presentation. They cluster in nodules and are engaged in clearing the myelin degradation accumulating in ageing.
The second article complements the WAM study by exploring fur- ther the antigen presentation mechanisms. It also focuses on the ques- tion of white-matter volume loss more directly. We show that CD8 T cells, by crossing the blood-brain barrier, and entering the immuno- logically priviledged central nervous system, play a role in oligoden- drocyte degeneration, notably by secretion of interferon-γ, that we confirm to be detrimental specifically in the aged brain.
Overall, we deliver new arguments to enrich the dichotomic de- bate on the good and bad role of microglia in ageing and disease. Fur- thermore, we illustrate the current trajectory of single-cell-enriched research at the interface of the neuroscience and immunology fields. We provide revolutionary insights revealing the importance of the lo- cation, signalling and environmental context of a cell in the shaping of its identity and the functions that it carries. Identifying further the mechanisms of ageing draws an optimistic horizon for the future of medical treatment and life improvement in ageing and disease
Establishment and function of nucleosome organization at eukaryotic chromosome replication origins
Get PDFThe whole genome has to be replicated only once per cell cycle and only during S phase. DNA replication initiates at specific sequences within the genome referred as origins of replication, and the number of origins correlates with the size of the genome. Problems with replication are associated with genomic instability and replication stress, both hallmarks of cancer and/or growth defects.
The budding yeast S. cerevisiae has been the preferred model organism to study DNA replication in detail because (a) its origins are well defined, (b) decades of genetics have painted a pretty clear picture of the process, and importantly, (c) replication has been reconstituted with purified components.
Replication origins contain an ARS that harbors an AT-rich conserved motif, known as the ACS, which is only present in yeast. ORC binds specifically to this motif and, together with other loading factors, recruits and loads the replication helicase, the MCM or Mcm2-7/Cdt1, as a double-hexamer. The chromatin structure at yeast origins is characterized by an NFR with flanking nucleosomal arrays of regular spacing. ORC has been shown to influence nucleosome positioning at origins of replication. However, prior to our work, the precise mechanism was unknown. Further, it was unclear if this stereotypical chromatin structure is functionally important for replication.
By screening ORC and seventeen purified chromatin factors via genome-scale in vitro reconstitution, we were able to determine the factors that establish this chromatin structure at origins. We found that ORC works together with the spacing remodelers INO80, ISW1a, ISW2 and Chd1 to generate nucleosome arrays at origins. Moreover, by testing different mutations of the Orc1 subunit of ORC, we were able to dissect ORC’s chromatin function at origins of replication by uncoupling it from its canonical function as the MCM loader. We found that nucleosome array generation depends on Orc1’s ability to hydrolyze ATP and on the BAH and IDR domains. These mutations were lethal in vivo and lost their arrays in vitro. Most importantly, this hindered DNA replication in in vivo and in vitro.
To replicate chromatinized DNA, the replisome requires the assistance of chromatin factors. It is known that some chromatin remodelers and histone chaperones enhance replication rates. In this context, we characterized Yta7 as a new type of chromatin remodeler namely chromatin segregase, that is different from the classical SF2 chromatin remodelers. One major difference is that its motor subunit belongs to the AAA+-ATPase superfamily. We found that Yta7 is activated during the S phase by S-CDK, which phosphorylates it in close proximity of the ATPase domain. Interestingly, phosphorylation causes stimulation of the ATPase activity, activation of its chromatin segregase function and strongly facilitated chromatin replication in vitro.
Finally, we collaborated with the Duderstadt lab at the MPI Biochemistry, to study how FACT, a two-subunit complex composed of Spt16 (SuPpressor of Ty’s 16) and Pob3 (Pol1 Binding 3), engages with the replisome to enhance replication as previously reported. We found that Spt16’s N-terminus is required for FACT’s direct interaction with the replication machinery while the C-terminus of both Spt16 and Pob3 is required for nucleosome interaction ahead of the replication fork
TGFβ differentially specifies T follicular helper versus Th17 cell fates of murine CD4+ T cells
Get PDFThe generation of high-affinity antibodies is of most importance for the clearance of pathogens and the efficacy of vaccines. Potent antibody responses require interactions between B cells and T follicular helper (Tfh) cells, which are specialized in providing cognate help to B cells. However, despite Tfh cells being first described in the year 2000, no robust and reliable protocol is available for in vitro Tfh cell differentiation of murine CD4+ T cells.
In this thesis, we challenged the longstanding theory of the inhibitory effect of TGFβ on murine Tfh cell differentiation, by identifying TGFβ as a critical driver of murine Tfh cell differentiation in vitro. TGFβ was required to initiate CXCR5 protein expression, the characteristic chemokine receptor expressed by Tfh cells, and to aid in its maintenance. By going against the common practice of including a TGFβ-neutralizing antibody into murine Tfh cell cultures, we first established a robust and reproducible in vitro protocol to generate Tfh cells from naïve mouse CD4+ T cells with plate-bound anti-CD3 and anti-CD28 stimulation. The in vitro TGFβ-induced CXCR5+ T cell population exhibited transcriptional and functional features of in vivo-generated Tfh cells. The TGFβ signaling pathway was biologically relevant, as the disruption of this pathway by knockout of the TGFβ receptor significantly reduced CXCR5 expression and also reduced the Tfh cell population in an in vivo immunization setting. An important optimization step of the Tfh cell culture was the reduction of cell density, which reduced paracrine IL-2 signaling, thereby strongly enhancing Tfh cell differentiation.
Tfh and Th17 cells show substantial plasticity between the two T helper subsets. Interestingly, we next discovered that in the in vitro model system a mixture of both Tfh-like and Th17-like cells were generated. To identify potential transcription factors that drive the divergence between Tfh and Th17 cells, we performed bulk RNA-seq analyses of sorted Tfh and Th17 cells. We next analyzed a selection of promising targets in an arrayed CRISPR/Cas9 screen and identified c-Maf as a transcription factor regulating Tfh versus Th17 cell fate as a molecular switch. Ablation of Maf strongly shifted the balance from Tfh towards Th17 cells. Finally, we confirmed in an acute LCMV setting that c-Maf also regulated Tfh versus Th17 differentiation in vivo.
Taken together, we established a robust and reproducible in vitro protocol to differentiate murine Tfh cells. This protocol provides a versatile platform for studying Tfh cell differentiation and plasticity in more detail. By using this culture, we identified c-Maf driving the divergent differentiation of Tfh and Th17 cells. We also debunked the longstanding concept of the inhibitory effect of TGFβ on murine Tfh cells. Since TGFβ can also induce human Tfh cells, our data indicate that human and mouse Tfh biology may actually be closer related than previously believed.Die Produktion von hochaffinen Antikörpern ist von großer Bedeutung sowohl für die Abwehr von Krankheitserregern als auch für die Wirksamkeit von Impfungen. Eine effektive Antikörperantwort erfordert das Zusammenspiel von B-Zellen und follikulären T-Helfer (Tfh)-Zellen, eine auf die Hilfe von B-Zellen spezialisierte Population von CD4+ T-Zellen. Obwohl Tfh-Zellen bereits im Jahre 2000 zuerst beschrieben wurden, fehlt bis heute ein robustes und reproduzierbares Protokoll für deren in vitro Differenzierung aus murinen CD4+ T-Zellen.
In der hier vorliegenden Arbeit wurde TGFβ als ein essentieller Faktor für die in vitro Tfh-Zell-Differenzierung identifiziert. TGFβ wurde nicht nur für die Initiierung sondern auch für die Aufrechterhaltung der Proteinexpression des für Tfh-Zellen charakteristischen Chemokinrezeptors CXCR5 benötigt. Zuerst wurde ein robustes und reproduzierbares Protokoll für die Tfh-Zell-Generierung aus naiven Maus-CD4+ T Zellen mit anti-CD3 und anti-CD28 Stimulation etabliert. Diese in vitro-generierte, TGFβ-induzierte CXCR5+ Zellpopulation wies transkriptionelle und funktionale Eigenschaften von in vivo-generierten Tfh-Zellen auf. Wir zeigten, dass der TGFβ Signalweg von biologischer Relevanz für die Tfh-Differenzierung in vivo ist. Ein Knockout des TGFβ Rezeptors in CD4+ T-Zellen verhinderte die Expression von CXCR5 und resultierte in einer signifikanten Reduktion der Tfh-Zellen in einem Protein-Immunisierungs-Szenario. Eine weitere Optimierung der in vitro Tfh-Zell-Differenzierung basierte auf der Reduktion der Zelldichte in der Zellkultur, wodurch die parakrine Signalweiterleitung von IL-2 reduziert und dadurch eine Verstärkung der Tfh-Zell-Differenzierung erreicht werden konnte.
Im weiteren Verlauf wurde das neuartige in vitro-Zellkulturprotokoll angewendet, um die Plastizität von Tfh- und Th17-Zellen zu untersuchen. So konnte gezeigt werden, dass in klassischen Th17-Zellkulturen sowohl Th17- als auch Tfh-Zellen induziert werden. Mittels RNA-Sequenzierung wurden potentielle Transkriptionsfaktoren identifiziert, die im weiteren Verlauf der Arbeiten funktional mittels CRISPR/Cas9 getestet wurden. c-Maf wurde dabei als ein molekularer Schalter für die Zell-Schicksalsentscheidung zwischen Tfh und Th17 identifiziert und sowohl in vitro als auch in vivo im akuten LCMV-Model validiert.
Zusammengefasst wurde in dieser Arbeit ein robustes und reproduzierbares Protokoll für die Tfh-Zell-Differenzierung aus naiven Maus-CD4+ T-Zellen entwickelt und validiert. Dieses Protokoll schafft eine vielseitige Plattform, die es erlaubt, die Tfh-Zell-Differenzierung und Plastizität in Zukunft noch viel detaillierter untersuchen zu können. Mit Hilfe dieser Zellkultur wurde c-Maf als ein treibender Faktor für die beobachtete Divergenz zwischen Tfh- und Th17-Zellen identifiziert. Darüber hinaus widerlegten wir das bisher gängige Konzept des hemmenden Effekts von TGFβ auf die Maus-Tfh-Zell-Differenzierung. Da TGFβ auch humane Tfh-Zellen induzieren kann, deuten unsere Daten darauf hin, dass die Unterschiede zwischen Mensch und Maus in Bezug auf die Tfh-Zell-Biologie womöglich kleiner sind als vorher angenommen
