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- PostA Step Toward Personalized Cancer Treatment Simultaneous Detection of Multiple Types of Chemotherapyinduced DNA Damage Using Single Molecule Imaging(2022) Foss, Ebba; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological Engineering; Westerlund, Fredrik; Akwasi Aning, ObedChemotherapy is commonly used to treat cancer today, either alone or more commonly as part of combination therapy. Response to a certain chemotherapeutic agent is highly individual, both in terms of treatment efficacy and the extent to which healthy cells are affected. For several drugs, induced DNA damage provides the main cytotoxic effect, and a method for evaluating this damage could therefore prove a powerful tool in treatment planning. In this thesis, a single molecule imaging approach is used to assess chemotherapy-induced DNA damage, allowing visualisation of damage sites on individual DNA strands. While previous studies have focused on one damage type, or collective damage without distinction between types, a novel modification to pre-existing techniques that allows for this distinction has recently been demonstrated. In this thesis, the alkylating agent temozolomide was used to illustrate how different damage types can be distinguished with a single molecule imaging approach. This is done using repair enzymes associated with different DNA repair pathways. The repair enzymes sequentially incorporate spectrally distinct fluorescent nucleotides at the damage site which are then visualized as fluorescent spots of two different colours on individual DNA molecules. This distinction could be shown with high repeatability in terms of colour ratio. While both enzymes used separately clearly repaired the treated DNA, there appeared to be an overlap when applying them sequentially. This could suggest a problem with enzyme specificity. Further exploration of this issue is needed to verify the feasibility of single molecule imaging for the purpose of simultaneous detection of chemotherapy-induced DNA damage types.
- PostDevelopment of a 3D in-vitro model for Alzheimer’s disease: Behavioral investigation of SH-SY5Y cells in brain-mimicking matrices(2017) Hardselius, Erik; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringWorldwide over 50 million people are suffering from Dementia and 60-70% of these cases are believed to be liked to Alzheies disease AD (1). Studies show that people suffering from repeated or severe head trauma (2), such as professional oes, ad people ith Dos sdoe(3, 4) ae oe poe to deelop Alzheies disease later in life. One common feature in these medical cases is an altered extracellular matrix in the brain due to either trauma or genetic factors. Understanding the mechanisms behind AD have has proven a challenge to the scientific community due to the lack of a functional disease model for AD. So far, the full AD progression has yet to be adequately modelled in a controlled setting, such as an in vitro or in vivo model, even though at least one promising candidate has been reported by Choi et al. utilizing 3D-cell culturing system that displays increased levels of both intracellular phosphorylated-tau proteins and extracellular amyloid β plaques compared to a 2D control (5). In this work a bare bones tuneable brain 3D-cell model protocol is presented and evaluated using three different cell lines of differentiated SH-SY5Y neuroblastoma cells. The most promising conditions in the matrix show neuron-like cells that exhibit comparable morphology to those presented by previous models but in a more controlled and well defined environment. The model makes promising use of hyaluronic acid in a 3D-matrix seeded with differentiated cells. Studies performed in the new model hints towards an ultimately cytotoxic interaction between Aβ and ECM components, which encourages further investigations of neuron/ECM/Aβ interactions in AD
- PostDirect cigarette smoke exposure to 3D epithelial cell cultures by an aerosol exposure system - Development and evaluation of an in vitro model for COPD with direct cigarette smoke exposure on small airway epithelial cells in the air-liquid interface by the Vitrocell smoking system(2021) Knudsen, Veronika; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Wittung Stafshede, Pernilla; Svärd, Rebecka; Horndahl, Jenny; Vdovikova, SvitlanaChronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory and progressive lung disease that causes obstructed airflow from the lungs. The Global Burden of Disease Study reports a prevalence of 251 million cases of COPD globally in 2016 and classify the disease as the third most common causes of deaths worldwide. Cigarette smoke exposure is one of the major risk factors for developing COPD. Up to 40% of smokers develop COPD over time, and continuous smoking drives the development and progression of the disease. The aim of this project was to develop and evaluate an in vitro model for direct whole cigarette smoke exposure on small airway epithelial cells (SAECs) with an automated aerosol system (Vitrocell smoking system). SAECs from three COPD donors and two healthy donor, cultured in the air-liquid interface (ALI), were used. To examine the effects of cigarette smoke on airway epithelium, cells were exposed to either one or three non-toxic doses of smoke, and samples were collected 24 and 48 hours post exposure. Cytotoxicity, barrier integrity, ciliary beat frequency, gene expression and cytokine expression were investigated. Cigarette smoke effectively increased the amount of inflammatory mediators IL-6 and IL-8 in supernatants from smoke-exposed samples, and up-regulated inflammatory gene expression of IL-8, and gene expression of antioxidants SRXN1 and HMOX1. This project was performed at AstraZeneca in the Bioscience COPD IPF Department within the Early Respiratory and Immunology R&D unit located in Gothenburg, Sweden.
- PostEngineering of Micro-Patterned Protein Scaffolds for the Study of Cancer Cell Behavior(2016) Latif, Arsalan; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringCancer is a leading cause of death in the developed world, where up to 90% of cancer related deaths are due to metastasis. Breast cancer accounts for 25% of all cancer related deaths. During metastasis, the cancer cells infiltrate tissues and blood vessels by migration. A better understanding of what drives cancer cells to migrate and how they could be stopped is needed in order to develop more effective drugs that prevent metastasis. It is known that cancer cell behavior is regulated by both physical and chemical external stimuli, and their behavior in two-dimensional (2D) environments is vastly different from three-dimensional (3D) surroundings. Both 2D and 3D surfaces are found in vivo, therefore it is important to evaluate the cell behavior in both 2D and 3D in vitro surfaces. In this study, we assess the behavior of invasive breast cancer cells in a 2D and a 3D matrix by creating nanowrinkled surfaces on and pores in tissue mimicking protein hydrogels. Collagen type I – Hyaluronic Acid (HA) and elastin-like polypeptide (ELP) hydrogels were modified with well-controlled topographical traits. Both the tissue mimicking hydrogels and cancer cell behaviour in their presence were evaluated with non-linear microscopy (NLM) techniques, namely, multi-photon excitation fluorescence (MPEF, autofluoresence and fluorescent stains), coherent anti-Stokes Raman scattering (CARS, chemical contrast based on vibrations) and second harmonic generation (SHG, collagen fibres). The fluorescent stains and MPEF microscopy reveal that the cells adopted either rounded or elongated cell morphology in the porous collagen type I scaffolds, being dependent on the pore diameter. On the contrary, the breast cancer cells did not thrive in the porous ELP scaffolds, possibly the large pore diameter of the porous ELP scaffold did not support the cells to attach fast enough or adverse reaction to the in situ crosslinking. The cells on the ELP nanowrinkled scaffolds adopted elongated morphology and aligned themselves in parallel to the wrinkles. Further, live-cell studies were conducted to observe the effect of the topographical traits on the migration mode of the cells. The morphology of cancer cells on different topographical traits emphasizes the plasticity of cancer cells, and hints towards specific cell migration modes dependent on topographical features.
- PostEstimation of Plasmid Lengths on ZEONEX®-Coated Cover Slips Using a Deposinator for Stretching of Single DNA Molecules(2017) Radenkovic, Alexander; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringAntibiotic resistance is one of the largest threats to global health, as it counters the way we treat bacterial infections. Antibiotic resistance genes can spread be-tween bacteria via plasmids. Simple and efficient methods for studying plasmids are thus important in the global fight against antibiotic resistance. This project optimizes and evaluates a novel molecular combing method for length estimation of plasmids – dragging a droplet of DNA solution across a ZEONEX®-coated cover slip followed by fluorescence microscopy. The method was optimized for pH, deposition speed and concentration using -DNA. A plasmid containing the resistance gene blaCTX−M−15 was linearized using either Cas9 or S1 nuclease, and the efficiency of both endonucleases was assessed. The plasmid was accurately estimated to 150 kbp by using -DNA as reference. The method shows promise, but further optimization is required to reduce noise that arise from broken DNA fragments.
- PostP27 deficiency accelerates the development of PTEN-deficiency-induced myeloproliferative disease(2015) Shao, Jingchen; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringPTEN acts as a phosphatase for PIP3 and negatively regulates the PI3K/AKT pathway, and CDKN1B (P27KIP1) is a cyclin-dependent kinase inhibitor that regulates G0 to S phase transitions by binding to and regulating the activity of cyclin-dependent kinases. Genetic alternations of Pten or Cdkn1b are common in hematological malignancies. Combined loss of PTEN and P27KIP1 expression is associated with tumor cell proliferation and poor prognosis in prostate cancer. However, it is not so clear how two mutations would cooperate in leukemogenesis. Here, we show that combined inactivation of PTEN or P27KIP1 in the hematopoietic compartment in mice results in a more severe myeloproliferative disease phenotype with shorter lifespan, lower hemoglobin and more enlarged spleen, lever comparted inaction of Pten or p27KIP1 alone.
- PostSelf-Adjusting Biochemically Controlled Nanoparticles - A Strategy to Achieve Precision Therapeutics(2023) Bardhi, Endri; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Stubelius, Alexandra; Erensoy, GizemDrugs such as DMARDs (Disease-Modifying Anti-Rheumatic Drugs), NSAIDs (Non-Steroidal Anti-Inflammatory Drugs), and glucocorticoids are used to manage acute inflammations and flares in chronic diseases, but their long-term use may result in side effects or drug tolerance. To address this, a drug delivery system was developed implementing polymeric nanoparticles (NPs). Although a conventional method such as nanoprecipitation was utilized, a microfluidic system was also established and optimized to evaluate its potential as a formulation technique. Dextran was chosen as a polymer base for its exceptional biocompatibility, biodegradability, and non-toxicity. Its acetalization provided a pH-responsive polymer that was utilized in the formulation of nanoparticles. The pH sensitivity of these nanoparticles was exploited, as inflamed tissues are associated with low pH levels, which led to the degradation of the nanoparticles and to a controlled release of the drug. The drug delivery system exhibited a lower release of the model drug at pH 7.4 than at pH 6.5 and 6.0, with the latter showing significantly higher release over time. Additionally, the drug delivery system displayed self-adjusting properties, as the release of the cargo could be initiated or stopped by altering the pH of NPs-containing buffers (ON/OFF/ON System). Nanoprecipitation enabled the formulation of a drug delivery system with controlled release and self-adjusting properties. This research highlighted the need for additional investigation to fully exploit the potential of the microfluidic system. The NPs offer alternative approaches to treat chronic inflammation diseases, as opposed to conventional therapies.
- PostThe Biological Implications of Sialic Acid Binding Nanoparticles in T cells(2023) de Carvalho, Viktoria; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Stubelius, Alexandra; Råberg, LoiseThe immune system is heavily regulated and affected by changes in the composition of cell surface molecules, such as glycans. One sugar usually located at the end of glycans is sialic acid (SA). Due to its negative charge and location, it is involved in many immune processes, such as immune cell migration and maturation, but many functions of SA are still unknown. The position and charge provide us with an easy target for engineered particles with SA affinity. Seeing that immune cells are sialylated, we can use this to target the immune system and possibly modulate it. This project has focused on polymeric SA affine nanoparticles (NPs) and T cells. The project primarily worked with the Jurkat E6-1 cell line of CD4+ T cells. Peripheral blood mononuclear cells (PBMCs) purified from buffy coat were also used. Cytotoxicity of NPs was investigated with the Alamar Blue assay. Activation markers and NP-binding to cells were investigated with flow cytometry and the cytokine profile was evaluated with enzyme-linked immunosorbent assay (ELISA). Confocal microscopy was used to qualitatively evaluate NP binding specificity. The results show that the NPs are not cytotoxic to T cells in tested concentrations ranging up to 500 μg/mL. Flow cytometry revealed that the NPs bind less to activated Jurkat cells and CD4+ primary cells, but more to activated CD8+ primary cells, compared to the respective unactivated cells. Confocal microscopy confirmed that NPs bind to unactivated cells to a higher extent. The NPs decreased the cellular expression of CD62L, which could be due to some activation asserted by the NPs, or steric hindering of the anti-CD62L antibody. ELISA showed that the IL-10 concentration in supernatant from PBMCs was lower for cells exposed to NPs, but further research is needed to confirm whether the NPs have a significant effect on the IL-10 profile of PBMCs. The IL-10 profile of Jurkats was not changed between particle concentrations. Finally, flow cytometry showed that the level of activation markers CD69 and CD25 were not affected by the particle concentrations, nor was the IL-8 concentration after evaluation with ELISA, leading to the conclusion that the activation level in T cells is not affected by the SA affine polymeric NPs. Thus, the NPs prove promising for future use in immunomodulation, with diagnostic or T cell activation-based targeting possibilities, and as a tool for understanding SA expression.