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Verlust der p53-Funktion als intrinsischer Resistenzmechanismus gegen das T-Zell-rekrutierende Antikörperkonstrukt AMG 330 zur Therapie der akuten myeloischen Leukämie
Get PDFEntwicklung und klinisch-experimentelle Evaluation von Ausbildungs- und Behandlungsstrategien bei degenerativen Erkrankungen der Lendenwirbelsäule mit besonderem Augenmerk auf die Spinalkanalstenose
Get PDFDevelopmental and activation pathways underlying mesenchymal stromal cell recruitment and differentiation in the context of neoplasias
Get PDFSolid tumors are a leading cause of mortality in adults across the world. Treatment options are often limited trough toxicity in non-target tissues. Mesenchymal stem cells (MSCs) present a tropism toward the inflammatory microenvironments present within solid tumors. Once there, they condition the tumor environment and differentiate into components of the tumor stroma. Our group had previously demonstrated the effectiveness of genetically engineered stem cells containing a therapeutic transgene, using the CCL5 promoter as a delivery vehicle for the treatment of solid tumors. CCL5/Rantes is a proinflammatory cytokine produced by a variety of cells including MSCs within tumor microenvironments. The CCL5 gene promoter can be activated by many factors including proinflammatory stimuli but varies with the cell type studied. The tumor-associated stimuli that activate the CCL5 promoter in MSCs was poorly understood. The aim of this thesis was to better understand the mechanisms of activation of CCL5 in stem cells, specifically in response to various signals present in the tumor microenvironment. A construct containing the CCL5 promoter driving a Gaussia luciferase reporter gene was created using Gateway cloning and a vector platform designed in house for the efficient stable integration of complex constructs into target cells. The CCL5 reporter construct was used to verify that the CCL5 promoter is activated by TNFα, but not significantly by IFN-γ; however, the combination of TNFα and IFN-γ showed a more than additive activity than the individual components. Neither hypoxia nor TGF-β, whether alone or in combination with other stimuli, activated the CCL5 promoter in the in vitro setting. We then developed synthetic variants of the promoter. 3AB, a synthetic promoter containing three tandem NFkB sequences taken from the immediate upstream region of the human CCL5 promoter [(R)AB region] and previously shown to be important for the functional activity of the promoter was generated. The 3AB synthetic promoter responded more effectively to TNFα and IFN- γ, with higher fold induction. It also showed a response to hypoxia that was increased in conjunction with TNFα and IFN-γ. TGF-β was not effective in the activation of 3AB. A second synthetic promoter MegaRantes, in which the R(AB) element in the native promoter was exchanged for the triplicate 3AB element, showed the same approximate pattern of activation as native CCL5, suggesting upstream regulating elements counteract any advantage derived from the 3AB modification. The results presented provide more complete information as to CCL5 is activation in MSCs and suggest that the synthetic construct 3AB, through its more specific activation, may represent an attractive candidate promoter for the delivery of therapeutic transgenes in the context of MSC-based tumor therapy
Development and validation of gene signatures predicting the prognosis for serous ovarian cancer patients
Get PDFKlinische und radiologische Bewertung von einteiligen vollkeramischen Dentalimplantaten aus Zirkoniumdioxid
Get PDFContribution of applied biology to mitigation of urban air pollution and prevention of its effects on health and environment
Get PDFAccording to World Health Organization (WHO), each year 7 million of people in the world die prematurely because of indoor and outdoor air pollutants. In recent years, fine and ultrafine particles (UFPs) are attracting more attention because of their health risks. UFPs, for example, due to their small size, can penetrate deeper into the lungs and part of them may translocate into the bloodstream.
The purpose of my PhD work is to evaluate the possible health effects on humans of air pollution with a specific focus on the nanoscale fraction. To do this, we used two approaches: the molecular approach using omics technologies and the epidemiological approach on specific blood biomarkers.
Using the molecular approach, we employed engineered nanoparticles (ENPs) to expedite the understanding of UFPs' toxicity mechanisms. We used Cadmium Sulfide Quantum Dots (CdS QDs) and, to better represent different routes of exposure, we conducted an in vitro study on HepG2 (liver hepatocellular carcinoma) and THP-1 (peripheral blood monocyte) cells. Both cell lines were exposed for 24 hours to sub-toxic dose of CdS QDs, and subsequently the RNA sequencing and miRNome profiling were performed. The transcriptomic data and miRNAs-mRNAs interactions analyses reveal for HepG2 the activation of RAS and Ca2+ signaling pathways, and for THP-1 points at JAK/STAT signaling and inflammatory pathways.
As part of the epidemiological approach, we developed a long-term population-scale study to assess the link between air pollutants (including UFPs) and blood biomarkers of inflammation and coagulation, namely fibrinogen, high-sensitivity C-reactive protein (hs-CRP), serum amyloid A (SAA), interleukin-6 (IL-6) and adiponectin. The air pollutants considered for this work were: PM(10; 2.5; coarse), NO(x; 2), O3 and ultrafine particles as PNC. We performed a multiple linear regression analysis adjusting for confounders. The results showed positive association between UFPs and fibrinogen and hs-CRP, and PM2.5 with IL-6.
In conclusion, we observed adverse effects of nanoparticles at molecular level and in a short-term exposure, as well as at population-scale in long-term exposure. Moreover, this study highlighted the association among inflammation and coagulation blood-biomarker with PM and gaseous pollutant, linking it directly with the increasing risk of cardiovascular disease
Structural and functional insights into the cytoplasmic RNA exosome and its cofactors
Get PDFIn eukaryotic cells, the RNA exosome serves as the primary 3' to 5' exonuclease responsible for degrading and processing various RNA substrates. To carry out its diverse functions, the exosome relies on compartment-specific cofactors that provide substrate specificity. In the cytoplasm, the conserved Ski238 complex, centered around the ATP-dependent RNA helicase Ski2, assists the exosome. Additionally, the bridging factor Ski7 in yeast, or its equivalent Hbs1L3 in higher eukaryotes, facilitates the interaction between the Ski238 complex and the RNA-degrading exosome. It is suggested that the Ski238 complex directly associates with the exosome to guide the RNA substrate towards the exosome's nuclease domain. However, molecular structures as proof for the direct channeling are not available and the precise coordination among these cofactors, which ultimately leads to RNA degradation, remains largely unknown.
This thesis presents a collection of structural snapshots that reveal the process of cytoplasmic 3' to 5' RNA degradation by the exosome and its cofactors. This series of complex structures provide unprecedented details regarding the interplay between different modules and reveal significant conformational changes within the Ski238 complex. These conformational rearrangements play a crucial role in coordinating the helicase and the nuclease activity during the degradation process. Based on this knowledge, for the first time, we were able to reconstitute a full Ski238-Ski7-exosome assembly and reveal its cryoEM structure. Remarkably, the interface between the exosome and its cytosolic helicase Ski2 resembles the nuclear complex where the exosome interacts with the helicase Mtr4.
In summary, the presented results offer a holistic understanding of the individual steps involved in 3' to 5' RNA degradation by the exosome in the cytoplasm of eukaryotic cells. These findings reveal both the similarities and differences between yeast and human complexes, serving as a foundation for further elucidation of cytosolic RNA quality control mechanisms
