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- PostInvestigating the effect of KEAP1 knockout and lactate treatment on histone lysine lactylation in KRAS-driven lung adenocarcinoma(2024) Karlsson, Elin; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Wittung-Stafshede, Pernilla; Raj , Dorota; Wiel, ClotildeLung cancer is a major global health concern, responsible for the highest number of deaths among all cancer types. Whereof, the subtype lung adenocarcinoma is the most common form, accounting for 40% of all lung cancer cases. Loss-of-function mutations in the gene KEAP1, found in 20% of lung adenocarcinoma patients, are associated with treatment resistance and lower overall survival, highlighting the importance of investigating this mutation in research. Additionally, KEAP1 loss of function are linked to metabolic reprogramming, a known hallmark of cancer. The Warburg effect, a metabolic reprogramming in cancer cells characterized by high rates of aerobic glycolysis , leads to elevated lactate concentrations in cancerous tissue compared to healthy tissue. Lactate, earlier considered to be a waste product, is now recognized for its role as a signaling molecule and its function in coordinating metabolism. Recent studies has identified histone lysine lactylation, a post-translational modification induced by lactate, which affects gene expression and chromatin structure. The aim of this thesis project was to investigate the effect of KEAP1 knockout and lactate treatment on histone lysine lactylation levels in lung adenocarcinoma. This study utilized mouse lung adenocarcinoma cell lines derived from genetically engineered mouse models with an activating mutation in the oncogene Kras. Using the protein detection method Western blot, the results demonstrated that histone lysine lactylation levels on H3 and H4 were lower in Keap1 knockout cells compared to Keap1 wild-type cells in one of the cell lines, while no definitive conclusion could be drawn for the other cell line. Inhibition of the KEAP1 protein without affecting the Keap1 gene directly, showed the same results. Additionally, lactate treatment induced histone lysine lactylation levels on H3 and H4 in Keap1 wild-type cells, but no increase was observed in Keap1 knockout cells. By measuring intracellular lactate concentration it was observed that the lactate treatment induced intracellular lactate levels for both Keap1 wild-type cells and Keap1 knockout cells. Indicating that there is no direct correlation between the intracellular lactate levels and histone lysine lactylation in cells with a Keap1 knockout.
- PostExploring the metabolism and mixture effects of the isothiazolinone biocides BIT and OIT in PLHC-1 cells(2024) Carvalho Nejstgaard, Aline; Blomqvist, Oskar; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Scheers, Nathalie; Celander, MalinThe powerful antimicrobial properties of isothiazolinones make them a common preservative in both industrial and consumer products. However, methylated deriva tives of isothiazolinones have been associated with concerning allergenic effects leading to stricter regulations. As a consequence, industries have turned to more lipophilic isothiazolinones, such as benzisothiazolinone (BIT) and octylisothiazoli none (OIT), which are less regulated. Similar effects have however been reported for exposures of BIT and OIT, highlighting the importance of expanding the knowledge about these compounds, to enforce stricter regulation and potentially controlling the isothiazolinones as a group. This study aimed to investigate the toxicodynamic and toxicokinetic responses in a fish liver cell model system, Poeciliopsis lucida hep atocellular carcinoma (PLHC-1) following exposure to BIT and OIT, alone and in mixture. Results from the ethoxyresorufin-O-deethylase (EROD) assay revealed no apparent induction of the common detoxification enzyme CYP1A after exposure to BIT and OIT. However, more data is needed for a more robust evaluation of whether the enzyme is involved in their metabolism. Analysis of cultured exposure media in high performance liquid chromatography (HPLC) demonstrated cellular biotransformation of BIT into a more polar metabolite. A rapid cellular uptake of OIT was observed, however no potential OIT metabolite could be detected, nei ther extracellularly nor intracellularly. A cytotoxic assessment was also conducted on exposed PLHC-1 cells investigating the mitochondrial activity and membrane in tegrity, common markers for cellular viability. Significant toxic effects were observed on both markers after exposure to 10 µM OIT, while no toxic effect was observed for the same concentration of BIT. However, co-exposure to these concentrations of OIT and BIT, displayed an even greater toxicity, indicating a synergistic mixture effect between the two. While this study provides valuable insights into the toxi codynamic and toxicokinetic profiles of BIT and OIT, highlighting the importance of considering mixture effects in assessing their potential health and environmental impacts, additional research is needed to fully understand their specific metabolic pathways and interaction mechanisms, to assess their long-term exposure effects.
- PostMachine Learning for Structural Predictions of PROTACs(2024) Källberg, Anders; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Wittung-Stafshede, Pernilla; Mercado, Rocío; Nittinger, Eva; Tyrchan, ChristianPROteolysis TArgeting Chimeras (PROTACs) are molecules that induce the degradation of targeted proteins by hijacking the ubiquitin–proteasome system in the cell. A PROTAC binds simultaneously to an E3 ligase and a protein of interest (POI), forming a ternary complex. The ubiquitin–proteasome system tags the POI with ubiquitin, marking it for degradation by the proteasome. The formation of a good ternary complex is essential for the ubiquitination and subsequent degradation of the POI. Being able to accurately model ternary complexes thus provides critical advantages in the development of PROTACs; however, data on PROTACs and their crystallized ternary complexes are limited. Accurate predictions of these structures are desirable, but current computational methods struggle to simulate the interactions between the PROTAC and both proteins simultaneously. AlphaFold, a machine learning tool, has been shown to accurately predict protein complexes. Yet, research on applying AlphaFold to predict ternary complexes is scarce. In the first part of this thesis, the ternary complex was modeled using AlphaFold by utilizing the sequences of both natural and artificially linked POIs and E3 ligase. Nevertheless, it was determined that AlphaFold was unable to accurately predict these complexes, reasonably because it was not able to take the PROTAC into account in the predictions. The second part of this thesis focused on generating data on PROTAC substructures, essential for the development of these molecules. Despite the availability of such data, obtaining high-quality data on substructures of specific PROTACs can be challenging and time-consuming. To address this, the PROTAC Splitter, a novel machine learning tool based on graph neural networks, was developed to predict these substructures. The PROTAC Splitter predicts 99.7% of PROTACs, with known substructures, to a maximal error of 6 atoms wrong between the boundaries of the ligands and linker. It generalizes to PROTACs with three unknown substructures, where 23.1% of these predictions satisfy the same criteria. The code for the PROTAC splitter is available at https://github.com/AndersKallberg/PROTAC_splitter. Although accurate predictions of ternary complexes remain challenging, the PROTAC Splitter makes the substructures easily accessible to anyone in this field of research. In summary, the work presented in this thesis answers scientific questions in two complementary areas of PROTAC development: (1) ternary (protein) structure prediction, and (2) PROTAC component prediction. This information is limited and valuable, and accurate predictions of these could accelerate the discovery of effective PROTACs and help in the fight against disease.
- PostNon-equilibrium stabilisation of proteins by chaperones - The Hsp70 molecular chaperone(2024) López Bautista, María Li; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Wittung-Stafshede, Pernilla; De Los Rios, PaoloWithin living organisms, proteins are essential components. They are responsible for carrying out almost every function in the cell. Proteins must fold into a specific three-dimensional shape to perform their diverse roles effectively. Indeed, improper folding is the root cause of many diseases. Not surprisingly, there is a specific group of proteins whose function is to assist and safeguard the folding process of other proteins. These are known as molecular chaperones. Here, we focus on the 70 kiloDalton heat shock protein, Hsp70, a molecular chaperone that has been under the spotlight of scientists for decades due to its ubiquitous presence across all living systems and its assistance in a wide range of cellular processes. It is well accepted that the mechanism of action of Hsp70 chaperones consists of a biochemical energy-consuming cycle, which allows them to drive the system out-of-equilibrium and escape the inherent limitations of equilib rium thermodynamics to perform their functions efficiently. Considering both the molecular details of chaperones and their client protein, along with a correct inclusion of the energy consumed in each step of the cycle and all relevant conformational transitions, we present a kinetic rate model for the description of the functional cycle of Hsp70 chaperones in protein folding that aims to elucidate the fundamental principles that govern their complex behaviour.
- PostDevelopment of digital PCR probe assays targeting SNPs to monitor co-cultured cell lines(2024) Frisk, Emma; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Westerlund , Fredrik; Ståhlberg, AndersCancer is driven by genetic alterations causing abnormal cell proliferation and invasion. Cancer heterogeneity refers to the diversity and variability observed within or between tumors, causing major issues in developing novel anti-cancer therapies targeting all cellular subtypes. To tackle this, more representative cancer models are developed to more accurately mimic the complex tumor environment and its diversity. This helps improve drug response studies and reveals important molecular mechanisms of cancer. However, downstream analytical methods for profiling the effects of these more complex models are costly and time-consuming and face practical challenges, limiting their feasibility in laboratory settings. This master’s thesis aimed to develop and evaluate a probe-based digital polymerase chain reaction (dPCR) setup with high sensitivity and specificity for monitoring various cell lines in a co-culture over time. The designed probes target cell line unique single nucleotide polymorphisms (SNPs) of each myxoid liposarcoma (MLS) and a fibrosarcoma human-derived cell line. In his thesis, a method was developed for monitoring co-cultures with up to four different cell lines with reliable quantification using dPCR analysis, proving to be both sensitive and efficient enough for our application. While this thesis project focused on MLS and fibrosarcoma cell lines, the workflow can be applied to other cell types, including immune cells or various cancer cell lines, as long as unique SNPs are identified. The method and workflow provide profiling of complex cell cultures and could be utilized for a deeper understanding of therapeutic responses across various culture model systems.