La inflamación hepática crónica es un fenómeno crucial en la patogénesis del carcinoma hepatocelular (HCC). En los últimos años, el proceso inflamatorio desencadenado por la activación del complejo del inflamasoma se ha asociado con diferentes enfermedades hepáticas. Sin embargo, su papel en el desarrollo de HCC está prácticamente sin explorar. En esta tesis, se estudia y analiza el impacto de diferentes componentes del inflamasoma, incluyendo la proteína caspasa 1 y los receptores AIM2 (del inglés absent in melanoma 2) y NLRP3 (del inglés NOD-like receptor family pyrin domain containing 3), en el desarrollo de HCC inducido por dietilnitrosamina (DEN) en ratones. La inactivación genética de AIM2, pero no de NLRP3, reduce el daño hepático y el desarrollo de HCC en este modelo. La deficiencia de AIM2 está asociada a una disminución en la activación del inflamasoma, la inflamación hepática y la respuesta proliferativa durante la fase de iniciación del HCC. También se identificó que AIM2 está altamente expresado en células de Kupffer, y que la producción de interleucina (IL)-1β, mediada por AIM2, en estas células se ve aumentada tras el daño hepático inducido por DEN. Además, la inhibición de IL-1β resultó en menor daño hepático, inflamación y proliferación durante la fase inicial de hepatocarcinogénesis; sin embargo, esta inhibición no fue suficiente para frenar la progresión de tumores hepáticos ya establecidos. Así, los datos de esta tesis indican que AIM2 promueve el proceso inflamatorio durante el daño hepático carcinogénico, contribuyendo al desarrollo genotóxico de HCC en ratones y, por tanto, reconociendo AIM2 como una posible diana terapéutica en esta enfermedad.

The area of data imputation, which is the process of replacing missing data with substituted values, has been covered quite extensively in recent years. The literature on the practical impact of data imputation however, remains scarce. This thesis explores the impact of some of the state of the art data imputation methods on HCC survival prediction and classification in combination with data-level methods such as oversampling. More specifically, it explores imputation methods for mixed-type datasets and their impact on a particular HCC dataset. Previous research has shown that, the newer, more sophisticated imputation methods outperform simpler ones when evaluated with normalized root mean square error (NRMSE). Contrary to intuition however, the results of this study show that when combined with other data-level methods such as clustering and oversampling, the differences in imputation performance does not always impact classification in any meaningful way. This might be explained by the noise that is introduced when generating synthetic data points in the oversampling process. The results also show that one of the more sophisticated imputation methods, namely MICE, is highly dependent on prior assumptions about the underlying distributions of the dataset. When those assumptions are incorrect, the imputation method performs poorly and has a considerable negative impact on classification.

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Resistance spot welding is the main welding method used in the automotive industry to weld thin sheet metal. Today adaptive control systems have been developed for RSW, which means it can adjust the parameters in the weld process automatically during welding. The control systems can register the parameters and properties of the weld in real-time and from that calculate with algorithms how to adjust to give optimal weld conditions. This project is performed at Scania CV AB, Oskarshamn. Conducted in the part of body in white, where an adaptive control system called HCC is used in all weld processes. In this project, HCC was compared to the fixed control system CCR and another adaptive control system named Master mode. First step in the comparison was to create a weld schedule for each control system and test them on two different material combinations. The aim was to quantify gains and benefits that adaptive resistance spot welding systems have on the welding process. Benefits are quantified by examining the parameters and factors such as: weld time, expulsion, robustness, electrode wear and parameters in the control system. The tests were performed by welding as many approved spot welds as possible without tip-dressing the electrode. The experiment followed the requirements from international standards and the Scania standard for resistance spot welding.  The results from the experiment showed that HCC was the most robust process and the spot welds never decreased in size, which CCR and Master mode did. It is possible to weld several different material combinations with HCC, it increases flexibility in production and reduces the time needed to develop new weld schedules. The same schedule can handle many combinations with the same thickness. HCC allows the process to use several pulses and each pulse adds in time. Therefore, the weld schedule should be well developed and optimized to avoid waste in terms of long weld times. The results will give Scania knowledge about the processes and how to further optimize the welding processes in production. The result can also be used as foundation for selection of products or future investments. / Motståndspunktsvetsning är den huvudsakliga svetsmetoden som används inom fordonsindustrin för att svetsa tunn plåt. Idag har adaptiva styrsystem utvecklats för RSW vilket innebär att de automatiskt kan justera parametrarna i svetsprocessen under svetsning. Styrsystemen kan registrera parametrarna och egenskaperna hos svetsen i realtid och därmed beräkna med algoritmer hur de bör justeras för att ge optimala svetsförhållanden. Detta projekt är resultatet av ett examensarbete på Scania CV AB, Oskarshamn. Det utfördes i den nya karossfabriken, där ett adaptivt styrsystem som heter HCC används i alla svetsprocesser. I projektet jämfördes HCC med ett konstantströms styrsystem CCR samt ett annat adaptivt styrsystem kallat Master mode. Den primära metoden var att skapa ett svetsschema för varje styrsystem och testa dem på två olika materialkombinationer. Syftet var att kvantifiera vinster och fördelar som adaptiva punktsvetssystem har på svetsprocessen. Testerna utfördes genom att svetsa så många godkända punkter som möjligt utan att formera elektroden. Fördelarna kvantifieras genom att man undersökte parametrarna och faktorerna svetstid, sprut, robusthet, elektrodslitage och parametrar i styrsystemen. Experimentet följde kraven i enighet med internationella standarder och Scania-standarden för punktsvetsning. Resultaten från experimentet visade att HCC var den mest robusta processen och punkterna minskade aldrig i storlek, vilket CCR och Master mode gjorde. Det är möjligt att svetsa flera olika materialkombinationer med HCC, det ökar flexibiliteten i produktionen och minskar den tid som krävs för att utveckla nya svetsscheman eftersom samma schema kan hantera många kombinationer med samma tjocklek. HCC tillåter processen att använda flera pulser, och varje puls adderar tid och svetsschemat bör därför vara välutvecklat och optimerat för att undvika slöseri med avseende på långa svetstider. Resultaten kommer att ge Scania mer kunskap om processerna och hur man kan optimera processerna ytterligare i produktionen. Resultatet kan också användas som grund för val av produkter eller framtida investeringar.

Transcription factor LSF is an oncogene in Hepatocellular Carcinoma (HCC). HCC is the sixth most common cancer worldwide and the second highest cause of cancer-related death globally. LSF is overexpressed in human HCC cell lines, compared to normal hepatocytes, and expression levels show significant correlation with the stage and grades of the disease. Current treatments for HCC are insufficient, especially given the frequency of late stage diagnoses. Therefore, it is necessary to understand the molecular mechanism of HCC disease to aid in targeted and effective treatments. Most investigations of the regulation of LSF activity have focused on its post-translational modifications in response to cellular proliferation and signal transduction. Chromatin modifications and epigenetic mechanisms of LSF-mediated gene regulation had not been investigated. Given that alterations of epigenetic writers or readers have been demonstrated in a large fraction of HCC patient samples, I examined the connection between LSF and epigenetic regulators. In particular, LSF is shown to interact with DNA methyltransferase 1 (DNMT1) and Ubiquitin like with PHD and Ring Finger Domains (UHRF1), with consequences for global DNA methylation and transcription patterns. Additionally, I identified unexpected, pairwise associations between LSF, histone methyltransferase SET8, and tubulin, both in vitro and in vivo. The interactions were identified by proteomics analyses, co-localization, co-immunoprecipitation, and direct protein-protein interaction studies in vitro. Strikingly, both LSF and SET8 associate with microtubules, leading to the discovery that SET8 methylates α-tubulin at several novel, specific lysines. This suggests parallels between regulation of chromatin by the histone code and regulation of microtubule function by the tubulin code. Surprisingly, LSF enhances tubulin methylation by SET8 in vitro and FQI1, a specific LSF small molecule inhibitor, reduces tubulin methylation. Furthermore, LSF promotes, and FQI1 inhibits, tubulin polymerization in vitro. Taken together, these findings suggest that SET8 is a microtubule-associated methyltransferase that LSF recruits to microtubules to enhance tubulin modification. The results indicate that both LSF and SET8 have cellular implications beyond their roles in gene transcription and histone methylation. Finally, this discovery of the dual functions for LSF and SET8 set up the possibility for connections between epigenetic and cytoskeleton modifications in cancer. / 2021-12-24T00:00:00Z

Background and Aims: In the treatment of hepatocellular carcinoma (HCC), response prediction to transarterial chemoembolization (TACE) based on serum biomarkers is not established. We have studied the association of circulating Dickkopf-related protein 1 (DKK-1) with baseline characteristics and response to TACE in European HCC patients. Methods: Patients with HCC treated with TACE from 2010 to 2018 at a tertiary referral hospital were retrospectively enrolled. Levels of DKK-1 were measured in serum samples collected before TACE. Response was assessed according to mRECIST criteria at week 12 after TACE. Results: Ninety-seven patients were enrolled, including seventy-nine responders and eighteen refractory. Before TACE, median DKK-1 serum levels were 922 [range, 199–4514] pg/mL. DKK-1 levels were lower in patients with liver cirrhosis (p = 0.002) and showed a strong correlation with total radiologic tumor size (r = 0.593; p < 0.001) and with Barcelona Clinic Liver Cancer stages (p = 0.032). Median DKK-1 levels were significantly higher in refractory patients as compared to responders (1471 pg/mL [range, 546–2492 pg/mL] versus 837 pg/mL [range, 199–4515 pg/mL]; p < 0.001), and DKK-1 could better identify responders than AFP (AUC = 0.798 vs. AUC = 0.679; p < 0.001). A DKK-1 cutoff of ≤1150 pg/mL was defined to identify responders to TACE with a sensitivity of 78% and specificity of 77%. DKK-1 levels were suitable to determine response to TACE in patients with low AFP serum levels (AFP levels < 20 ng/mL; AUC = 0.843; 95% CI [0.721–0.965]; p = 0.003). Conclusion: DKK-1 levels in serum are strongly associated tumor size and with response to TACE in European HCC patients, including those patients with low AFP levels.

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Die vorliegende Arbeit beschäftigt sich mit einem neuartigen proteinbasierten, suizidgentherapeutischen Ansatz zur sicheren und effektiven Behandlung von soliden Tumoren. Verwendet wurden zellpermeable Fusionsproteine auf der Grundlage des bakteriellen Enzyms Cytosin Desaminase, welches spezifisch die Umsetzung der inaktive, nichttoxische Substanz (Prodroge) 5-Fluorcytosin in den hochwirksamen, stark toxischen Wirkstoff 5-Fluoruracil katalysiert. Dieser bewirkt die selektive Zerstörung von Tumorzellen. Durch die Fusion der bakteriellen Cytosin Desaminase (bCD) mit der Sequenz des Zellpermeabilität vermittelnden Peptides HBV-Translokationsmotiv (TLM) des Hepatits B-Virus (HBV) wurden zunächst zellpermeable E.coli Cytosin Desaminase Suizidfusionskonstrukte generiert. Für die bakteriell synthetisierten HBV-TLM-Fusionsproteine konnten eine Hexamerisierung sowie eine spezifische enzymatische Aktivität bei der Umsetzung von Cytosin zu Uracil als strukturelle und funktionelle Voraussetzungen für einen Einsatz in der Suizidgentherapie nachgewiesen werden, die vergleichbar mit dem wt-Protein waren. Bei Versuchen zur Internalisierung der zellpermeablen Fusionsproteine wurde für die Fusionsproteine mit C-terminal fusioniertem HBV-TLM (bCD-HBV-TLM) eine Aufnahme in das Zytoplasma von Hepatomzellen mittels konfokaler Laserscanmikroskopie und differentieller Zellfraktionierung nachgewiesen, nicht jedoch für Fusionsproteine mit N-terminalem HBV-TLM (HBV-TLM-bCD). Die gezeigte Internalisierung des Proteins HBV-TLM-bCD erfolgte effizient und schnell und war unabhängig vom endosomalen Aufnahmeweg. Bei der nachgewiesenen Translokalisation blieb die enzymatische, suizidgentherapeutische Aktivität des zellpermeablen Suizidproteins (HBV-TLM-bCD), d.h. die katalytische Wirkung bei der Umsetzung der Prodroge 5-Fluorcytosin vollständig erhalten, so dass sich dieses Fusionsprotein für einen therapeutischen Einsatz in der Suizidgentherapie eignet. Zusätzlich zur antitumoralen Wirkung können durch einen gezielten, lokal begrenzten therapeutischen Einsatz der vorgestellten zellpermeablen bCD-HBV-TLM-Fusionsproteine starke Nebenwirkungen, wie sie bei einer konventionellen Chemotherapie zu beobachten sind, weitgehend vermieden werden. / This work investigates the application of protein based therapeutic suicide enzyme/prodrug approaches providing novel means for both safe and effective local therapeutic regimes in solid tumors. The concept of the used suicide gene therapy system is based mainly on the transfer of the cell permeable bacterial suicide enzyme cytosine deaminase which specifically convert the inactive, non-toxic prodrug 5-fluorocytosine into the toxic metabolite 5-fluorouracil finally executing the efficient destruction of tumor cells. Employing a novel cell permeable peptide, known as the translocation motif (TLM) of hepatitis B virus (HBV), E.coli cytosine deaminase (bCD) suicide fusion proteins were generated. HBV-TLM fusion proteins formed hexamers (as do parental wt bCD) and retained the specific enzymatic activity of cytosine conversion to uracil also being comparable to parental wtbCD protein. However, only bCD-HBV-TLM fusion proteins, but not HBV-TLM-bCD fusion proteins were found to be taken up to the cytoplasm of target hepatoma cells as demonstrated both by confocal laser scanning microscopy and cell fractionation. Uptake of bCD-HBV-TLM worked both efficiently and rapidly and was found to be independent from the endosomal pathway. Since bCD-HBV-TLM fusion proteins completely retained their suicide enzymatic activity in the course of translocation across the plasma membrane their usage as profound inducers of chemo-sensitivity to 5-fluorocytosine strongly is suggested. Future therapeutic local application of cell permeable bCD-HBV-TLM fusion proteins together with a systemic 5-fluorocytosine prodrug application could result in profound antitumor activities without apparent side effects.

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.