Life Sciences

<h4>"Exploring the metabolism and mixture effects of the isothiazolinone biocides BIT and OIT in PLHC-1 cells "</h4>
Abstract: The powerful antimicrobial properties of isothiazolinones make them a common preservative in both industrial and consumer products. However, methylated derivatives 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) &nbsp;and octylisothiazolinone (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 hepatocellular carcinoma (PLHC-1) following exposure to BIT and OIT, alone and in mixture. Results from the ethoxyresorufin-O-deethylase (EROD) assay, revealed no significant induction of the common detoxification enzyme, CYP1A, after exposure to BIT and OIT, &nbsp;thus not indicating involvement of this enzyme 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, neither extracellularly nor intracellularly. A cytotoxic assessment was also conducted on exposed PLHC-1 cells investigating the mitochondrial activity and membrane integrity, common markers for cellular viability. Significant toxic effects were observed on both markers after exposure to 10 µM OIT, while no 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 toxicodynamic 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.
 Examiner: Nathalie Scheers Supervisors: Malin Celander (Göteborgs universitet) &nbsp; Opponent: Johannes Panagiotidis &nbsp; Date and time: 24 May 2024, 10.30-11.30 Location: <a href="https://maps.chalmers.se/#61393995-42d3-4b0c-ba68-1fa7b4afe3c1" title="KA" data-anchor="#61393995-42d3-4b0c-ba68-1fa7b4afe3c1">KA</a>

<h4>"Using Bioluminescence Resonance Energy Transfer (BRET) Methodology to Investigate HCAR1 Signal Transduction"</h4>
 Examiner: Mikael Molin Supervisors: Simon Lind &nbsp; Opponent: Elisabeth Norberg &nbsp; Date and time: 24 May 2024, 13.15-14.15 Location:&nbsp;<a href="https://maps.chalmers.se/#77a73d20-5534-11e9-8dd5-9d5420a8a18d" title="Utsikten" data-anchor="#77a73d20-5534-11e9-8dd5-9d5420a8a18d">LIFE 7033 Utsikten</a>

<h4>"Development of a 3-D static wound model: Characterization and investigation of different stresses using Staphylococcus aureus wound isolates"</h4>
Abstract: Wounds have represented a big issue for centuries and a high cost to the health care system worldwide in recent years. Wound care, even though it is one of the biggest disciplines within medicine, has still several challenges to face. One of them: monitoring the wound healing process. Lately, multiple solutions have been developed enabling sensors using pH or temperature as sensing variables, but conventional methods are still on top. In this thesis, based on in vitro wound models, the aim was to find new biomarkers to be applied in monitoring the wound healing process. The thesis had two parts: the development of this model and the study of different stresses over the built model. The search of these biomarkers was decided to be done using omics techniques. After screening the biofilm formation capability and behaviour of seven wound isolates of S. aureus, a 3-D collagen-based wound model was implemented. The model was characterized by evaluating the bacterial growth on it at different time-points and through different microscopy techniques. Over this model, two different stresses were tested: iron starvation and oxidative stress. 2,2bipyridine and Chelex® 100 were used to induce iron starvation. The effect of 2,2bipyridine was visible evaluating the bacterial growth in E. coli (ATCC 25992), but only significant in S. aureus (ATCC 6538) when using the highest concentration. To induce oxidative stress, hydrogen peroxide (H2O2) was added over the model. The effect in bacterial growth in S. aureus (USA300 TCH-1516) was only visible at the highest concentration. Based on these results, the study of different stresses in S. aureus based on bacterial growth is not recommended due to the high adaptability seen in S. aureus. By including the measurement of new variables like pH, iron concentration or H2O2, the different scenarios of stress could be strengthened, together with the study of gene expression. Furthermore, adding specific genes related to each of the stresses in the gene expression study, these stresses could fit better a clinically relevant scenario.
Examiner: Ivan Mijakovic Supervisors: Shadi Rahimi, Leonarda Acha Alarcón, Erik Gerner &nbsp; Opponent: Sara Bauer &nbsp; Date and time: 27 May 2024, 10.00-11.00 Location: Seminar room 3039, <a href="https://maps.chalmers.se/#f20b23bb-5c36-4aad-be75-5d9d0b90e108" title="SysBio" data-anchor="#f20b23bb-5c36-4aad-be75-5d9d0b90e108">SysBio-floor 3</a>

<h4>"Machine learning for PROTACs: Predicting protein- and molecular structures"</h4>
Abstract: PROteolysis TArgeting Chimeras (PROTACs) are molecules that induce the degradation of targeted proteins and show potential as treatments for a range of diseases, including (but not limited to) neurodegenerative diseases, inflammatory diseases, cancers, and viral infections. However, PROTACs are challenging to develop due to their novel and complex mechanism of action, and current computational methods are unsuitable for studying PROTACs. This thesis investigates and evaluates the capacity of a machine learning tool, AlphaFold, for predicting PROTAC-protein structures. Furthermore, it presents the development of a novel machine learning tool that utilizes graph neural networks to identify molecular substructures of PROTACs. Accurate predictions of these structures would be useful in the design and development of new PROTACs, potentially shortening the time to discovery and consequently saving lives.
Examiner: Pernilla Wittung-Stafshede Supervisors: Rocío Mercado, Eva Nittinger (AstraZeneca), Christian Tyrchan (AstraZeneca) &nbsp; Opponent: Leo Andrekson
Date and time: 27 May 2024, 13.30-14.30 Location: <a href="https://maps.chalmers.se/#4749f4f0-8358-11e8-8cea-65be8f2c646c" title="GIGA" data-anchor="#4749f4f0-8358-11e8-8cea-65be8f2c646c">GIGA</a>
Online: <a href="https://chalmers.zoom.us/j/67678727142" title="Zoom link">Zoom</a> (Password: 90054)

<h4>"Learning meaningful representations of cells"</h4>
Abstract: Batch effects are a significant concern in single-cell RNA sequencing (scRNA-Seq) data analysis, where variations in the data can be attributed to factors unrelated to cell types. This can make downstream analysis a challenging task. In this study, a neural network model is designed utilizing contrastive learning and a novel loss function for creating an generalizable embedding space from scRNA-Seq data. When benchmarked against multiple established methods on scRNA-Seq integration, the model outperformed existing methods on creating a generalizable embedding space on multiple datasets. A downstream application that was investigated for the embedding space was cell type annotation. When compared against multiple well established cell type classifiers the model in this study displayed a performance competitive with top performing methods across multiple metrics, such as accuracy, balanced accuracy and F1 score. These findings motivates the meaningfulness contained within the generated embedding space by the model, highlighting its potential applications.
Examiner: Johan Bengtsson-Palme Supervisor: Rocío Mercado   Opponent: Anders Källberg   Date and time: 27 May 2024, 14.45-15.45 Location: <a href="https://maps.chalmers.se/#4749f4f0-8358-11e8-8cea-65be8f2c646c" title="GIGA" data-anchor="#4749f4f0-8358-11e8-8cea-65be8f2c646c">GIGA</a>

<h4>"Development of digital PCR-based probe assays targeting SNPs to monitor co-cultured cell lines."</h4>
 Examiner: Fredrik Westerlund Supervisors: Anders Ståhlberg, Daniel Andersson, Anna Gustafsson   Opponent: Thien Nguyen   Date and time: 29 May 2024, 10.00-11.00 Location: <a href="https://maps.chalmers.se/#e3838355-3672-4a2f-8615-15e94a32dd11" title="10:an" data-anchor="#e3838355-3672-4a2f-8615-15e94a32dd11">"10:an"</a>

<h4>"Identifying potential interacting partners of aqua-porin 4 via a high-throughput screening method of human&nbsp;brain cDNA."</h4>
Abstract: <span style="font-size: 12.0pt; font-family: 'Aptos',sans-serif; mso-fareast-font-family: 'Times New Roman'; mso-bidi-font-family: Aptos; color: black; mso-ansi-language: SV; mso-fareast-language: SV; mso-bidi-language: AR-SA;">Aquaporins (AQPs) are membrane proteins that play a crucial role in regulating water homeostasis in all living cells and they are involved in a variety of different physiological processes. Specifically, AQP4 has been implicated in many pathophysiological processes in the brain and has emerged as an important protein to study. Protein-protein interactions are important for biological processes to function, therefore investigating which proteins from the human brain interacts with AQP4 could prove useful in future development of diagnostics and therapies. A novel screening method utilizing bimolecular fluorescence complementation (BiFC) and fluorescence activated cell sorting (FACS) has been applied in this project, in hopes of identifying potential interacting partners of aquaporin 4 and improve on the screening method. We demonstrated that fresh yeast transformants are important for a higher fraction of fluorescent cells which would aid the determination of interacting candidates. However, the sorting gate parameters of the FACS machine and the transformation efficiency of BiFC fluorescent pairs in yeast remains undetermined due to inconsistent results. In the end, 14 sequences were sequenced yielding the six following unique potential interacting partners of AQP4, Leydig cell tumor 19 kDA protein homolog, B-cell receptor-associated protein 29, Serpin E3, ATP-binding cassette sub-family A member 6, Cytochrome c oxidase subunit 1 and Nicastrin. Their biological relevance and possibility of interaction with AQP4 is thoroughly discussed along with an outlook on future endeavors of AQP4 studies.
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Examiner: Cecilia Geijer Supervisor: Kristina Hedfalk
Opponent: Oskar Blomqvist and Aline Carvalho Nejstgaard
 Date and time: 30 May 2024, 15.00-16.00 Location: <a href="https://maps.chalmers.se/#77a73d20-5534-11e9-8dd5-9d5420a8a18d" title="Utsikten" data-anchor="#77a73d20-5534-11e9-8dd5-9d5420a8a18d">LIFE 7033 Utsikten</a>

<h4>"Investigating the effect of KEAP1 knockout and lactate treatment on histone lysine lactylation in KRAS-driven lung adenocarcinoma"</h4>
Abstract Lung 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.
Examiner: Pernilla Wittung-Stafshede Supervisors: Clotilde Wiel and Dorota Raj &nbsp; Opponent: Olivia Johnsson &nbsp; Date and time: 3 June 2024, 15.00-16.00 Location: <a href="https://maps.chalmers.se/#4749f4f0-8358-11e8-8cea-65be8f2c646c" title="GIGA" data-anchor="#4749f4f0-8358-11e8-8cea-65be8f2c646c">GIGA</a>

<h4>"Investigation of a secreted tannase from an unstudied acidophilic fungus: Biochemical characterization and insights into tannin-protein precipitation"</h4>
Examiner: Johan Larsbrink Supervisor: Tom Coleman
Opponent: Aleksandra Zagorska
 Date and time: 4 June 2024, 09.00-10.00 Location: <a href="https://maps.chalmers.se/#77a73d20-5534-11e9-8dd5-9d5420a8a18d" title="Utsikten" data-anchor="#77a73d20-5534-11e9-8dd5-9d5420a8a18d">LIFE 7033 Utsikten</a>

<h4>"Vessel-on-a-Chip Development to Understand Systemically Administered Nanoparticles"</h4>
Abstract:  Nanomedicine holds great promise for treating complex diseases, particularly due to its unique ability to sustain and target drug delivery. However, the validation method has been a great challenge to the development of nanodrugs. The main reason is the unpredictable delivery dosage and lack of adequate clinical trial methods that replicate the human biological environment for testing nanomedicine. This project introduced the organ-on-a-chip(OOC) technique to mimic the human vascular environment in vitro for studying the transportation of nanoparticles in human vessels. The build-up of this vessel-on-a-chip (VoC) was successfully achieved using a microfluidic chip from Ibidi manufacture along with a microfluidic set-up to stimulate the human umbilical vein endothelial cells(HUVEC) seeded in the chip. The cells formed an oriented endothelial cell (EC) monolayer mimicking the endothelium of a capillary under a 24-hour flow of 10dyn/cm2. This monolayer acted as a barrier preventing NPs from transmitting through during the experiment. 
In addition, another computational fluidic dynamic(CFD) simulation approach was conducted in the project to further enhance the study of NP transportation in the vascular system. The microfluidic simulation helped to explain unexpected phenomena during the flow experiment. Although the blood flow simulation had some limitations, it provides useful insights when evaluating the potential uptake amount of the NP in the vascular system. This integrated study of combining VOC with CFD simulation enhances our understanding of NP behavior in human vessels and requires further optimization.
Examiner: Alexandra Stubelius Supervisors: Elin Svensson   Opponent: Matilda Öjmertz   Date and time: 4 June 2024, 09:00-10.00 Location: <a href="https://maps.chalmers.se/#4749f4f0-8358-11e8-8cea-65be8f2c646c" title="GIGA" data-anchor="#4749f4f0-8358-11e8-8cea-65be8f2c646c">GIGA</a>

<h4>"Targeting metabolic dependencies in cancer cells in vitro"</h4>
Abstract: This study aimed to evaluate the efficacy of NCT-503 in inhibiting cancer cell viability and altering metabolic pathways in LUAD cell lines. Cell Titer Glow (CTG) assays were employed to assess cell viability across varying drug concentrations, while uptake assays measured the absorption and secretion of serine and glycine in treated and untreated cells. Experiments were conducted on two LUAD cell lines, A1 and Y2, representing elder and younger populations, respectively.
CTG assays revealed a dose-dependent response to NCT-503, with significant cell viability reduction at higher drug concentrations (200 µM). Interestingly, the A1 cell line showed higher sensitivity to lower concentrations (&lt;100 µM) compared to Y2. Uptake assays presented complex and varied responses; Y2 cells exhibited decreased serine secretion and glycine absorption post-treatment, while A1 cells showed increased secretion of these metabolites. Notably, the patterns of nutrient uptake were inconsistent across different trials, indicating potential variability in cellular responses to NCT-503.
The study confirmed that PHGDH inhibition by NCT-503 effectively hinders cancer cell growth, particularly in the A1 cell line. However, the discrepancies between CTG and uptake assays highlight the complexity of cellular metabolic responses. Variability in results suggests the need for further investigation to ensure reliability and reproducibility. Factors such as cell age, genetics, and experimental conditions may influence the drug’s efficacy. The study highlights the necessity for ongoing research, including repeated uptake assays, intracellular metabolite analysis, and in vivo trials, to validate NCT-503's therapeutic potential and ensure minimal side effects.
NCT-503 shows promise as a therapeutic agent against LUAD, particularly for elder patients, by effectively inhibiting cancer cell viability through PHGDH inhibition. However, the complexity of cellular responses and potential laboratory errors necessitate further research to confirm these findings and optimize treatment protocols.
Lung adenocarcinoma (LUAD) is a prevalent and aggressive form of lung cancer. Targeting metabolic pathways, particularly the serine and glycine biosynthesis pathways, represents a promising therapeutic strategy. Phosphoglycerate dehydrogenase (PHGDH), a key enzyme in serine biosynthesis, is frequently overexpressed in LUAD. NCT-503, a PHGDH inhibitor, has emerged as a potential therapeutic agent against LUAD.
 Examiner: Mikael Molin Supervisors: Samantha Alvarez, Volkan Sayin, Institute of Clinical Sciences, Sahlgrenska Center for Cancer Research
 Opponent: Liam Davidsson &nbsp; Date and time: 4 June 2024, 13.00-14.00 Location: only <a href="https://us04web.zoom.us/j/78999458002?pwd=Vp9SXbTOuzMiF7jLo51oklLZlP11Aa.1" title="Zoom link" data-anchor="?pwd=Vp9SXbTOuzMiF7jLo51oklLZlP11Aa.1">via Zoom</a>

<h4>"Unraveling the role of myeloid-derived reactive oxygen species on the epithelial-to-mesenchymal transition in metastasizing cancer"</h4>
Abstract: The epithelial-to-mesenchymal transition (EMT) allows epithelial cells in primary cancer tumors to adopt a more mesenchymal morphology, thus facilitating metastasis. This process is influenced by a multitude of transcription factors and pathways, including the p53 and NF-κB signaling pathways. Myeloid cells in the tumor microenvironment produce reactive oxygen species (ROS) via the NOX2 enzyme, and while inflammation previously has been linked to EMT, the role of myeloid-derived ROS remains unknown.
The long-term aim of this thesis was to understand the intracellular pathways connecting myeloid-derived ROS and EMT in breast cancer. To this end, breast cancer cell lines of varying invasiveness were treated with H2O2, a common ROS, together with different pathway activators/inhibitors. Differences in EMT-related genes and proteins were measured using quantitative PCR and western blot, respectively. It was observed that the SNAI family of EMT transcription factors was induced by the redox regulator Nrf2, while ROS-induced SERPINE1 was regulated by p53. SERPINE1 encodes plasminogen activator inhibitor-1 (PAI-1), a biomarker in several cancers that has also been linked to EMT and inflammation. The results were validated using CRISPR-Cas9 mediated knockout of the TP53 gene. In addition, bioinformatic and in silico analyses of publicly available datasets corroborated these results.
The links between H2O2 and EMT in breast cancer implied by the results of this thesis show potential targets for limiting EMT in breast cancer. Further studies are necessary to fully understand the involved mechanisms and their regulation.
 Examiner: Anna Karlsson-Bengtsson Supervisor: Anna Martner &amp; Mustafa Kaya
Opponent: Elin Karlsson
 Date and time: 5 June 2024, 13.15-14.45 Location: <a href="https://maps.chalmers.se/#4749f4f0-8358-11e8-8cea-65be8f2c646c" title="GIGA" data-anchor="#4749f4f0-8358-11e8-8cea-65be8f2c646c">GIGA</a>

<h4>"Patient-specific analysis of ctDNA as a biomarker for childhood Acute Lymphoblastic Leukemia"</h4>
Examiner: Annikka Polster Supervisors: Fani Pujol Calderón &amp; Martin Dalin
Opponent: Anna Andersson
 Date and time: 5 June 2024, 14.00-15.00 Location: <a href="https://maps.chalmers.se/#77a73d20-5534-11e9-8dd5-9d5420a8a18d" title="Utsikten" data-anchor="#77a73d20-5534-11e9-8dd5-9d5420a8a18d">LIFE 7033 Utsikten</a>

<h4>"Optimizing Acetate Utilization in S. Cerevisiae through gRNA-Landing Pads"</h4>
 Abstract:&nbsp; Lignocellulosic biomass, a residue from the agricultural and forestry industries, is the most abundant organic substance on the planet. For efficient utilization by Saccharomyces cerevisiae, the biomass must undergo a pretreatment process that also generates strong inhibitors, with acetate being the most prominent. This project served as a proof of concept and explored optimization of acetate utilization in Saccharomyces cerevisiae using a gRNA-landing pad strategy. This was done through a wide range of molecular biology methods such as the transformation of Escherichia coli and the Saccharomyces cerevisiae strain LX6, restriction cloning, the MoClo system, as well as in-silico design of plasmids and primers. A gRNA-landing pad was successfully assembled in-vitro, using in-silico custom-made MoClo parts, and transformed into Escherichia coli. Transformation of the gRNA-landing pad into LX6 presented a challenge, that as of yet is not overcome. Despite the challenges in transforming the yeast, the creation of an easy-to-assemble gRNA-landing pad that can be designed for integration anywhere in the Saccharomyces cerevisiae genome could enable a fast and reliable method for future research involving gRNA-landing pads. 
Examiner: Yvonne Nygård Supervisor: Arne Peetermans
Opponent: David Sandberg
 Date and time: 11 June 2024, 8.30-9.20 Location: <a href="https://maps.chalmers.se/#77a73d20-5534-11e9-8dd5-9d5420a8a18d" title="Utsikten" data-anchor="#77a73d20-5534-11e9-8dd5-9d5420a8a18d">LIFE 7033 Utsikten</a>

<h4>"Colorimetric monitoring of pathway flux"</h4>
 Abstract:&nbsp; Redirecting flux through the shikimate pathway necessitates an effective indicator corresponding to the pathway's flux. This project investigates whether violacein and prodeoxyviolacein (PDV), which exhibit purple and green colors respectively, can serve as such indicators. Genetic engineering techniques, including gene cloning, yeast and bacterial transformation, and integration of genetic constructs in four yeast strains (CEN.PK113-5D, IMX581, QL292 and QL32) using CRISPR-Cas9/ homologous recombination, were employed. The presence of violacein and PDV was subsequently measured using high-performance liquid chromatography (HPLC). Successful integration of genes coding for violacein was achieved in all four strains, while integration for PDV was successful in three strains. The distinct colors of violacein and PDV were visibly apparent in CEN.PK113-5D. While QL292 colonies were faint purple coloration, remaining strains were successfully transformed but did not produce a visual readout. Violacein and PDV showed potential as indicators of flux through the shikimate pathway, but further optimization is required to enhance visibility and measurement accuracy in various yeast strains. 
Examiner: Yvonne Nygård Supervisor: Elin Blick
Opponent: Asger Thorén
 Date and time: 11 June 2024, 9.20-10.10 Location: <a href="https://maps.chalmers.se/#77a73d20-5534-11e9-8dd5-9d5420a8a18d" title="Utsikten" data-anchor="#77a73d20-5534-11e9-8dd5-9d5420a8a18d">LIFE 7033 Utsikten</a>
Online: via <a href="https://chalmers.zoom.us/j/61542847064" title="Zoom link">Zoom</a> (Password: 382440)

<h4>"Inducible forward programming of human induced pluripotent stem cells into skeletal myocytes"</h4>
 Abstract:  Differentiation of human induced pluripotent stem cells into a desired phenotype is challenging. Previous attempts of hiPSC differentiation into skeletal myocytes using an in-house protocol has resulted in heterogeneous populations of cells. Resolving the heterogeneity of Skeletal myocyte differentiation is key for acquiring tissues and cell types suited for exploring novel therapies and mechanisms within muscle related diseases. Thus, the main objective of this thesis was to investigate if alterations in gene expression could resolve the heterogeneity of the differentiation process of Skeletal Myocytes. In this thesis, differentiation of hiPSCs into skeletal myocytes was explored through modulation of the transcription factors MYOD1 and OCT4 using the genome editing enrichment method "Xential". The addition of the HBB IVS2 intron sequence led to a 2-fold increase in MYOD1 expression, furthermore, adjustments to the seeding density led to an additional 2-fold increase of MYOD1. Knockdown efficiency of shRNA targeting POU5F1 is correlated with the seeding density. The downstream marker of myogenesis DES had a 15-fold increase when seeding density was adjusted. This correlation between differentiation efficiency and seeding density highlights the interplay between environmental pressures and gene expression. The HBB IVS2 intron sequence led to higher homogeneity in the cultures, this provides insights into future strategies to mitigate heterogeneity in hiPSC differentiation. 
Examiner: Yvonne Nygård Supervisor: Dr. Antje Rottner
Opponent: Albin Petrén
 Date and time: 11 June 2024, 10.30-11.20 Location: <a href="https://maps.chalmers.se/#77a73d20-5534-11e9-8dd5-9d5420a8a18d" title="Utsikten" data-anchor="#77a73d20-5534-11e9-8dd5-9d5420a8a18d">LIFE 7033 Utsikten</a>

<h4>"Development of novel high-throughput assays to measure compound binding to transcription factors in cells"</h4>
 Abstract:  Transcription factors (TFs) are proteins possessing domains that recognizes and bind to specific DNA sequences. These proteins also possess domains that recruit the transcriptional regulatory machinery, thus regulating the rate of transcription in nearby genes. Dysregulations of gene expression mediated by TFs is associated with numerous diseases, making them attractive targets for research. However, the understanding of the intricate mechanisms governing TF-DNA binding remains limited. This lack of knowledge complicates the study of TF-DNA binding, making it both labour-intensive and costly. To overcome these challenges, there is a need for high-throughput screening (HTS) methods that can efficiently identify TF-DNA interactions, characterize functional binding site motifs, and evaluate the impact of compounds on the TF-DNA binding.    This thesis project thereby aimed to develop a novel cell-based assay to measure TF-DNA interactions in high throughput by leveraging the split-reporter system NanoBiT®. This system comprises two subunits: the Large BiT (LgBiT) and Small BiT (SmBiT), that upon proximity forms an active enzyme that produces luminescence. The concept involved tagging dead Cas9 (dCas9) with a LgBiT, tagging various TFs with SmBiTs, and providing single guide RNA (sgRNA), directing dCas9 to target DNA sites. The study focused on two disease related TFs: signal transducers and activators of transcription 3 (STAT3) and androgen receptor (AR). Two distinct cell models were used: HEK-293 for STAT3 and LNCaP for AR. Plasmids expressing dCas9 linked to N- or C-terminus of the LgBiT were designed and synthesized. Following transfections with these constructs, the work of generating stable pools using geneticin antibiotic selection was initiated. Subsequently, TF-SmBiT plasmids were designed and synthesized, along with a range of sgRNAs. These guides were designed targeting promoter sequences of downstream genes associated with the TF. The established cell lines expressing dCas9-LgBiT were then transiently transfected with the TF-SmBiT plasmid and various sgRNAs, enabling subsequent measurement of the TF-DNA interaction.   In this project, stable pools of HEK-293 cells expressing dCas9-LgBiT-N/C-terminus were established and validated through western blot. Despite several attempts, generation of LNCaP cell lines stably expressing these constructs was never achieved. After generating the assay reagents, the NanoBiT® assay was developed and tested. Although specific interactions were not evident in the initial testing phase, the developed system holds potential for evaluating TF-DNA binding in a high-throughput manner, identifying transcription factor binding sites (TFBSs), and assessing the impact of compounds on TF-DNA interactions. Nonetheless, further validation of the assay is imperative to ensure its reliability and applicability in future studies.    Keywords: Transcription factor, High-throughput screening, Cell-based assay, Split-reporter system, dCas9, sgRNA, STAT3, AR, HEK-293, LNCaP 
Examiner: Yvonne Nygård Supervisor: Anna-Lina Cavallo and Sandra Stefanovic-Barrett
Opponent: Sigrid Tjernberg
 Date and time: 11 June 2024, 11.20-12.10 Location: <a href="https://maps.chalmers.se/#77a73d20-5534-11e9-8dd5-9d5420a8a18d" title="Utsikten" data-anchor="#77a73d20-5534-11e9-8dd5-9d5420a8a18d">LIFE 7033 Utsikten</a>

<h4>"Establishing a Transwell co-culture to model fibroblast-macrophage crosstalk relevant for progression of chronic kidney disease"</h4>
 Examiner: Nathalie Scheers Supervisor: Emelie Lassén &nbsp; Opponent: Ylva Öberg &nbsp; Date and time: 12 June 2024, 10.00-12.00 Location: <a href="https://maps.chalmers.se/#6ada37f8-0eae-443a-9e4a-181b6c83bc54" title="KB" data-anchor="#6ada37f8-0eae-443a-9e4a-181b6c83bc54">KB</a>

<h4>"B Cell-Activating Factor and Myeloid-Derived Suppressor Cells in Common Variable Immunodeficiency"</h4>
Examiner: Annikka Polster Supervisors: Jesper Waldenström and Anna Martner
Opponent: Emilia Gvozdenovic
 Date and time: 12 June 2024, 11.00-12.00 Location: <a href="https://maps.chalmers.se/#77a73d20-5534-11e9-8dd5-9d5420a8a18d" title="Utsikten" data-anchor="#77a73d20-5534-11e9-8dd5-9d5420a8a18d">LIFE 7033 Utsikten</a>

<h4>"Studying interactions of complex lipid vesicles with cell membrane mimics using neutron techniques"</h4>
 Examiner: Fredrik Höök Supervisors: Margaret Holme &nbsp; Opponent: Nastaran Fallah Randjbar &nbsp; Date and time: 12 June 2024, 13.00-14.00 Location: <a href="https://maps.chalmers.se/#e3838355-3672-4a2f-8615-15e94a32dd11" title="10:an" data-anchor="#e3838355-3672-4a2f-8615-15e94a32dd11">"10:an"</a>

<h4>"The Role of LigD in Non-Homologous End-Joining in Mycobacterium tuberculosis"</h4>
Examiner: Fredrik Westerlund Supervisors: Florian Morati and Evgeniya Pavlova &nbsp; Opponent: Tringa Berisha &nbsp; Date and time: 12 June 2024, 14.00-15.00 Location: <a href="https://maps.chalmers.se/#4749f4f0-8358-11e8-8cea-65be8f2c646c" title="GIGA" data-anchor="#4749f4f0-8358-11e8-8cea-65be8f2c646c">GIGA</a>

<h4>"Systemic inflammation in cariiovascular, renal, and metabolic diseases: Monitoring infiltration and interplay of monocytes with cardiac and kidney models on a microfluidic device."</h4>
Examiner: Annikka Polster Supervisor: Liisa Vilén (AstraZeneca)
Opponent: Emma Frisk
 Date and time: 12 June 2024, 13.30-15.30 Location: <a href="https://maps.chalmers.se/#77a73d20-5534-11e9-8dd5-9d5420a8a18d" title="Utsikten" data-anchor="#77a73d20-5534-11e9-8dd5-9d5420a8a18d">LIFE 7033 Utsikten</a>

<h4>"PNA-FISH for identification of pathogenic bacteria in neonates in low-resource settings"</h4>
 Examiner: Fredrik Westerlund Supervisor: Moa Wranne &nbsp; Opponents: Olivia Borg and Elin Allgén &nbsp; Date and time: 13 June 2024, 09.30-10.30 Location: <a href="https://maps.chalmers.se/#e3838355-3672-4a2f-8615-15e94a32dd11" title="10:an" data-anchor="#e3838355-3672-4a2f-8615-15e94a32dd11">"10:an"</a>

<h4>"Chromatin binding patterns of SWI/SNF chromatin remodeling complex components and interaction partners in FET sarcoma"</h4>
 Examiner: Fredrik Westerlund Supervisors: Malin Lindén/Anders Ståhlberg
Opponents: Samuel Lindroth and Moa Andersson &nbsp; Date and time: 13 June 2024, 11.00-12.00 Location: <a href="https://maps.chalmers.se/#e3838355-3672-4a2f-8615-15e94a32dd11" title="10:an" data-anchor="#e3838355-3672-4a2f-8615-15e94a32dd11">"10:an"</a>

<h4>"Identifying how ATF4-mediated regulatory networks influnece ferroptosis susceptibility via the integrated stress response in A549 lung cancer cells"</h4>
Examiner: Mikael Molin Supervisors: Viktor Garellick, Per Lindahl   Opponent: Selma Fornander   Date and time: 13 June 2024, 13.15-14.15 Location: <a href="https://maps.chalmers.se/#4749f4f0-8358-11e8-8cea-65be8f2c646c" title="GIGA" data-anchor="#4749f4f0-8358-11e8-8cea-65be8f2c646c">GIGA</a>

<h4>"Non-equilibrium stabilisation of proteins by chaperones"</h4>
Abstract: Within 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 equilibrium thermodynamics to perform their functions efficiently.&nbsp;
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. 
Examiner: Pernilla Wittung-Stafshede Supervisor: Paolo De Los Rios, Laboratoire de Biophysique Statistique (EPFL) &nbsp; Opponent: Wilma Franssila &nbsp; Date and time: 14 June 2024, 11.00-12.00 Location: <a href="https://maps.chalmers.se/#4749f4f0-8358-11e8-8cea-65be8f2c646c" title="GIGA" data-anchor="#4749f4f0-8358-11e8-8cea-65be8f2c646c">GIGA</a> and <a href="https://chalmers.zoom.us/j/66908497226" title="Zoom link">via Zoom</a> (Password: 1)

<h4>"DNA Microscopy: Establishing an immuno protocol"</h4>
Abstract: As we continue to explore and understand the world around us, the demands on our methods and techniques evolve too. DNA microscopy is a developing method that offers an alternative to the conventional light microscopy. Instead of relying on light and a convex lens, it utilizes nature's own building blocks, DNA, to create spatial images and describe different proteins vicinity to one another using sequencing. Björn Högberg's lab have previously shown the method to work in an artificial system and the project is now ready to start exploring one of the options for a cellular set up.
One possibility is to create a multiomic system that utilizes antibodies to deliver target strands to specified cells, mimicking the original artificial set up. This requires conjugation of designed DNA strands, targets, to the antibody. An appropriate protocol for which was developed and established in this thesis. The four cell lines used were A549, HeLa, MCF-7 and MDA-MB-231. In situ immunocytochemistry (ICC) was used to visually confirm relative specificity for the four cell types to the nine different antibodies. For the conjugation of targets both SiteClick and oYo-Link were trialed, using both ProFIRE and Amicon to purify the conjugates. The result showed that all four cell lines had general specificity to antibody CD44 and BASP1, but only HeLa and A549 to SLC38A1. A549 was the only cell line that showed specificity for ALDH3A1 and AKR1B10. For the conjugation protocol SiteClick in combination with ProFIRE and Amicon gave the best result for conjugate concentration and purity.
The end results of the thesis provides the beginning of a library with appropriate cell lines and antibodies to use in a cellular setup. It also establishes a protocol for the conjugation of target strands to antibodies. In order to fully realize a cellular version of the artificial system more study needs to be performed on appropriate cell lines and antibodies for a more complex set up. To further evaluate the conjugates effect on the DNA reactions the reaction requires sequencing and re-construction. 
Examiner: Fredrik Westerlund Supervisor: Björn Högberg, Karolinska institutet
Opponents: María López Bautista and Maya Awada
 Date and time: 14 June 2024, 14.30-15.30 Location: <a href="https://maps.chalmers.se/#24d8b7be-1f59-4388-be28-a07861b429b2" title="PJ-salen" data-anchor="#24d8b7be-1f59-4388-be28-a07861b429b2">PJ hall</a>

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Presentation of master thesis

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Presentation of master thesis

Master thesis presentations at Life Sciences

24 May 2024

10.30-11.30: Aline Carvalho Nejstgaard & Oskar Blomqvist, MPBIO

13.15-14.15: Emilia Gvozdenovic, MPBIO

27 May 2024

10.00-11.00: José Manuel Outomuro Ruiz, MPBME

13.30-14.30: Anders Källberg, MPBIO

14.45-15.45: Leo Andrekson, MPBIO

29 May 2024

10.00-11.00: Emma Frisk, MPBIO

30 May 2024

15.00-16.00: Johannes Panagiotidis, MPBIO

3 June 2024

15.00-16.00: Elin Karlsson, MPBIO

4 June 2024

09.00-10.00: David Sandberg, MPBIO

09:00-10.00: Fan Jia, MPBME

13.00-14.00: Julia Sahlén, MPBIO

5 June 2024

13.15-14.45: Olivia Johnsson, MPBIO

14.00-15.00: Wilma Franssila, MPBIO

11 June 2024

8.30-9.20: Erik Berg, MPBIO

9.20-10.10: Aleksandra Anna Zagorska, MPBIO

10.30-11.20: Emil Löfgren, MPBIO

11.20-12.10: Clara Johnsson, MPBIO

12 June 2024

10.00-12.00: Linnea Johansson, MPBIO

11.00-12.00: Elisabeth Norberg, MPBIO

13.00-14.00: Eleftheria Kosta, MPPHS

14.00-15.00: Selma Fornander, MPBIO

14.00-15.00: Thien Nguyen, MPBIO

13 June 2024

09.30-10.30: Moa Andersson & Maya Awada, MPBIO

11.00-12.00: Kajsa Grönkvist, MPBIO

13.15-14.15: Tringa Berisha, MPBIO

14 June 2024

11.00-12.00: Maria Li López Bautista, MPCAS

14.30-15.30: Anna Andersson, MPBIO