The relevance of the Akt kinase for the viability of tumor cells and as a therapeutic target in multiple myeloma

01_Einleitung 02_Material und Methoden 03_Ergebnisse 04_Diskussion 05_Zusammenfassung 06_Literaturverzeichnis 07_AnhangZusammenfassung: Der PI3K/Akt Signalweg scheint wesentlich zum verbesserten Überleben und der malignen Entwicklung des Multiplen Myeloms beizutragen. Da der Großteil der Daten aus Experimenten mit pharmakologischen Inhibitoren stammt, ist unklar, ob sie der Inhibition von Akt zuzuordnen sind oder unspezifische Effekte widerspiegeln. Die Akt-Kinase-Familie besteht aus drei homologen Isoenzymen, die trotz ihrer strukturellen Ähnlichkeit individuelle Funktionen haben könnten. In dieser Arbeit wurden genetische "knock- down"-Experimente mit Akt-siRNA-Konstrukten durchgeführt. Dabei bestand die Möglichkeit, die Isoenzyme einzeln und in Kombinationen auszuschalten und die Effekte auf die Viabilität der MM Zellen zu untersuchen. Diese Untersuchungen wurden durch eine ausführliche Analyse des Aktivierungsstatus und der funktionellen Bedeutung der Akt-Kinase für die Viabilität in primären MM Zellen vervollständigt (n=30). Die Experimente mit siRNAs zeigten, dass in MM.1S, einer MM Zelllinie mit konstitutiver Akt Phosphorylierung, Akt1 und Akt2 zum Überleben beitragen, wohingegen Akt3 von geringerer Bedeutung war. Im Gegensatz dazu wurde die Viabilität von AMO-1, einer MM Zelllinie ohne erkennbare Akt Phosphorylierung, von einer Ausschaltung von Akt1 und Akt2 nicht beeinträchtigt. Die Behandlung dieser Zelllinien mit dem Akt1 und Akt2 spezifischen Inhibitor Akti-1/2 blockierte die Phosphorylierung von Akt und seines Substrates FoxO1 bei einer Konzentration von 10 μM. Das löste in MM.1S, aber nicht in AMO-1 Zellen, Apoptose aus und spiegelte so das Ergebnis der siRNA-Experimente wider. Im Folgenden wurde die Akt-Aktivität in einer größeren Anzahl MM Patientenproben mittels immunhistochemischer Analyse auf phospho-Akt und intrazellulärer phospho-Akt Färbung und Durchflusszytometrie bestimmt. Eine konstitutive Akt-Aktivierung wurde in ungefähr 58% der MM Patientenproben festgestellt. Dieses konstitutive Akt-Signal wurde von dem Akt-Inhibitor Akti-1/2 in primären MM Zellen sowohl in Kokultur mit Knochenmarkstromazellen als auch ohne inhibiert und führte in 50% der Proben zu deutlicher Apoptose. Die Patientenproben mit konstitutivem Akt-Signal reagierten größtenteils sensitiv auf Akt-Inhibition, wohingegen alle ohne sichtbare Akt-Aktivierung resistent waren. Um die Ursache für die konstitutive Akt Aktivierung zu bestimmen, wurden MM Zellen von 20 Patientenproben auf eine beschriebene Akt1 Mutation untersucht. Diese wurde nicht gefunden. Da Deletionen des Tumorsuppressors PTEN aufgrund ihres selteneren Vorkommens nur in einer geringen Anzahl von Fällen für die Akt Aktivierung verantwortlich sein können, wird es Bestandteil weiterer Untersuchungen sein, die Ursache für die konstitutive Akt-Aktivität zu finden. Diese Arbeit zeigt grundlegende Unterschiede in der Akt Aktivierung von MM Zellen und definiert Untergruppen, die entweder abhängig oder unabhängig von der Aktivität von Akt überleben. Dabei scheinen Akt1 und Akt2 die im MM wichtigen Isoformen zu sein.Summary: The PI3K/Akt pathway has been reported to critically contribute to survival and malignant growth of multiple myeloma (MM). Because most of these data are based on pharmacologic inhibition it is not clear if the effects are due to Akt inhibition or off-target effects. Furthermore, the Akt family of kinases consists of three highly homologous isoforms, that may, nonetheless, display individual functional properties. We therefore conducted siRNA experiments to knock-down the isoforms individually and in combinations to assess their role for the viability of MM cells. This was complemented with extensive analyses into the functional and signaling properties of the Akt kinase in primary MM cells (n = 30). Our knock-down experiments revealed that in MM.1S, an MM cell line with constitutive phospho-Akt signaling, Akt1 and Akt2 both contributed to MM cell survival whereas Akt3 seemed to be of less relevance. Conversely, survival of MM cell line AMO-1 which has no constitutive phospho-Akt signal was completely unaffected. Treatment of these MM cell lines with the Akt1 and Akt2 specific inhibitor Akti-1/2 showed that this drug totally abolished the phospho-Akt signal in MM.1S at a concentration of 10 μM. Again, MM.1S cells underwent apoptosis whereas AMO-1 cells were resistant. Next, we analyzed Akt signaling in a large panel of primary MM samples. Phosphorylated Akt was determined with immunohistochemical staining in bone marrow biopsies and with intracellular staining and flow cytometry analysis in primary tumor samples and could be detected in about 58% of MM cases. This constitutive signal could be blocked with Akti-1/2 in the presence and absence of bone marrow stromal cells. Pharmacologic inhibition of Akt led to strong induction of cell death in 50% of primary MM samples, whereas the rest was largely resistant to Akt inhibition. The samples sensitive to Akt inhibition were mostly identical to those that displayed a constitutive phospho-Akt signal. Of interest, Akt1 mutation could be excluded as reason for constitutive Akt activation. Because the rare occurance of PTEN deletions in MM there have to be performed further investigations to clarify the cause for the activation of Akt. This analysis indicates substantial heterogeneity in MM cells that defines Akt dependent and Akt independent MM subgroups. Akt1 and Akt2 proved relevant for the survival of subsets of MM cell lines and primary samples. Taken together, with this comprehensive functional and molecular signaling analysis of primary MM samples it was possible to identify novel functionally defined myeloma subgroups

Enantioselektive Totalsynthese, biologische Aktivität und Strukturvariation des antitumoralen Alkaloids (-)-Dibromphakellstatin aus dem Meeresschwamm Phakellia mauritiana

Pyrrole-imidazole alkaloids represent a unique family of natural products which are exclusively found in marine sponges, mainly the Agelasidae, Axinellidae, and Halichondridae families. The pyrrole-imidazole alkaloids are fascinating due to thier biogenetic relationship leading to a large, strucurally diverse family based on a common key metabolite, oroidin. Some of the pyrrole-imidazole alkaloids have interesting biological activities, e. g., potent antitumor or antibacterial activities. One of these chellenging alkaloids is dibromophakellstatin which was first isolated from Phakellia mauritiana. In this work an efficient five step synthesis of dibromophakellstatin is described providing the natural product in an overall yield of 10%. Based on a broad investigation of the reactivity of dipyrrolopyrazinone-type enamides, a novel three component imidazolidinone anellation was exploited as the key step of the total synthesis. With this findings the overall yield of the synthesis could increased up to 18%. Based on these results it was possible to develope the first enantioselective total synthesis of (-)-dibromophakellstatin in a chiral pool approach (10 steps, 3.5% overall yield). In a collaboration effort the antitumor activity of dibromophakellstatin was confirmed. It was found that only the natural enantiomer shows biological activity by an unknown mechanism. Encouraged by the biological activity of (-)-dibromophakellstatin, different cyclopropane variations of the tetracyclic phakellin- and isophakellin scaffolds were obtained by reaction of dichlorocarbene with tri- and bicyclic precursors. Some of these compounds werde found to have interesting new anti proliferative activities

N-[2-(3-Methyl-1-oxo-1,2-dihydro­pyrrolo­[1,2-a]pyrazin-2-yl)eth­yl]methane­sulfonamide

In the title compound, C11H15N3O3S, the dihedral angle between the five- and six-membered rings is 1.13 (18)°. The ethyl­methane­sulfonamide group is in a (+)synclinal conformation. In the crystal, inter­molecular N—H⋯O and C—H⋯O hydrogen-bond inter­actions link mol­ecules into zigzag ribbons parallel to the b axis. The ribbons are further connected by C—H⋯π inter­actions

Адаптация опыта использования компьютерных программ-симуляторов для оценки компетенций студентов в процессе обучения

В рамках магистерской диссертации проведено исследование возможности адаптации компьютерных программ-симуляторов для оценки компетенций студентов в процессе обучения посредством изучения существующего международного и отечественного опыта использования программ-симуляторов в образовательной сфере, анализа нормативно-правовой базы, интервьюирования экспертов и проведения экспертного семинара.Within the framework of the master's thesis, the possibility of adapting computer simulators for assessing the competencies of students in the learning process was studied by studying the existing international and domestic experience in the use of simulator programs in the educational sphere, analyzing the regulatory framework, interviewing experts and holding an expert seminar

Recruitment and Baseline Characteristics of Participants in the AgeWell.de Study: A Pragmatic Cluster-Randomized Controlled Lifestyle Trial against Cognitive Decline

Targeting dementia prevention, first trials addressing multiple modifiable risk factors showed promising results in at-risk populations. In Germany, AgeWell.de is the first large-scale initiative investigating the effectiveness of a multi-component lifestyle intervention against cognitive decline. We aimed to investigate the recruitment process and baseline characteristics of the AgeWell.de participants to gain an understanding of the at-risk population and who engages in the intervention. General practitioners across five study sites recruited participants (aged 60-77 years, Cardiovascular Risk Factors, Aging, and Incidence of Dementia/CAIDE dementia risk score ≥ 9). Structured face-to-face interviews were conducted with eligible participants, including neuropsychological assessments. We analyzed group differences between (1) eligible vs. non-eligible participants, (2) participants vs. non-participants, and (3) between intervention groups. Of 1176 eligible participants, 146 (12.5%) dropped out before baseline; the study population was thus 1030 individuals. Non-participants did not differ from participants in key sociodemographic factors and dementia risk. Study participants were M = 69.0 (SD = 4.9) years old, and 52.1% were women. The average Montreal Cognitive Assessment/MoCA score was 24.5 (SD = 3.1), indicating a rather mildly cognitively impaired study population; however, 39.4% scored ≥ 26, thus being cognitively unimpaired. The bandwidth of cognitive states bears the interesting potential for differential trial outcome analyses. However, trial conduction is impacted by the COVID-19 pandemic, requiring adjustments to the study protocol with yet unclear methodological consequences

Compression as a universal principle of animal behavior

A key aim in biology and psychology is to identify fundamental principles underpinning the behavior of animals, including humans. Analyses of human language and the behavior of a range of non-human animal species have provided evidence for a common pattern underlying diverse behavioral phenomena: words follow Zipf's law of brevity (the tendency of more frequently used words to be shorter), and conformity to this general pattern has been seen in the behavior of a number of other animals. It has been argued that the presence of this law is a sign of efficient coding in the information theoretic sense. However, no strong direct connection has been demonstrated between the law and compression, the information theoretic principle of minimizing the expected length of a code. Here we show that minimizing the expected code length implies that the length of a word cannot increase as its frequency increases. Furthermore, we show that the mean code length or duration is significantly small in human language, and also in the behavior of other species in all cases where agreement with the law of brevity has been found. We argue that compression is a general principle of animal behavior, that reflects selection for efficiency of coding.Comment: This is the pre-proofed version. The published version will be available at http://onlinelibrary.wiley.com/journal/10.1111/%28ISSN%291551-670

COVID-19 government measures and their impact on mental health: a cross-sectional study of older primary care patients in Germany

BackgroundWith the outbreak of COVID-19, government measures including social distancing and restrictions of social contacts were imposed to slow the spread of the virus. Since older adults are at increased risk of severe disease, they were particularly affected by these restrictions. These may negatively affect mental health by loneliness and social isolation, which constitute risk factors for depressiveness. We aimed to analyse the impact of perceived restriction due to government measures on depressive symptoms and investigated stress as mediator in an at-risk-population in Germany.MethodsData were collected in April 2020 from the population of the AgeWell.de-study, including individuals with a Cardiovascular Risk Factors, Aging, and Incidence of Dementia (CAIDE) score ≥9, using the depression subscale of the Brief Symptom Inventory (BSI-18) and the Perceived Stress Scale (PSS-4). Feeling restricted due to COVID-19 government measures was surveyed with a standardized questionnaire. Stepwise multivariate regressions using zero-inflated negative binomial models were applied to analyse depressive symptoms, followed by a general structural equation model to assess stress as mediator. Analysis were controlled for sociodemographic factors as well as social support.ResultsWe analysed data from 810 older adults (mean age = 69.9, SD = 5). Feeling restricted due to COVID-19 government measures was linked to increased depressiveness (b = 0.19; p < 0.001). The association was no longer significant when adding stress and covariates (b = 0.04; p = 0.43), while stress was linked to increased depressive symptoms (b = 0.22; p < 0.001). A final model confirms the assumption that the feeling of restriction is mediated by stress (total effect: b = 0.26; p < 0.001).ConclusionWe found evidence that feeling restricted due to COVID-19 government measures is associated with higher levels of depressive symptoms in older adults at increased risk for dementia. The association is mediated by perceived stress. Furthermore, social support was significantly associated with less depressive symptoms. Thus, it is of high relevance to consider possible adverse effects of government measures related to COVID-19 on mental health of older people