The study of the electrophysiological properties of neurons has reached a new level thanks to recent techniques that combine knowledge from different fields of science. For a method such as all-optical electrophysiology, the quality of cell segmentation in the image has one of the critical roles since the accuracy of illumination and perturbation of cells depends on it. The task becomes challenging because neurons have a complex morphology, and therefore traditional image analysis methods cannot perform accurate segmentation.
This project focuses on building two AI-based models for neuron soma detection and mask prediction, as well as such an essential aspect of the experiment as the quality of the image recordings. Developed models demonstrate high performance and are ready to be applied to the images of cells with or without fluorescent labels, although expanding the training dataset is recommended for improving segmentation accuracy. In addition, the signal-to-noise ratio was measured for recordings with different parameters such as camera readout speed, illumination intensity, and frequency of laser switching. The project can be extended to detect and segment dendritic trees and spines to gain new insights into the subtle process of intercellular communication. ... Read more

Cancer remains one of the leading causes of death in today's world. Radiation is a widely used in the treatment of cancer. One of the relatively newer methods is proton therapy, which can spare healthy tissues better than traditional photon treatments due to the unique Bragg peak of protons. A promising improvement is FLASH proton therapy, which utilises ultra-high dose rates (40-100x higher than conventionally used). However, a lot is still unknown about this method. This research took first steps towards experimentally investigating FLASH proton radiation in living tissues. To achieve this, three types of experiments were performed. The first consisted of simulation with proton radiation, the second used proton to irradiation zebrafish, and the last experiment irradiated zebrafish with a gamma source. First, a virtual model of the irradiation conditions was built with TopasMC, using an 140 MeV proton beam propagating in a water tank containing model cells. The flux and number of protons were tallied at various positions. A new beam was created for each positing using the tallied data resembling the original beam. The new beam was used for small scale irradiation of a model cell. The damage was assessed by calculating DNA strand breaks.
The simulation showed a Bragg peak for dose measurements, in accordance with literature. The damages to DNA significantly increased as the LET increased, as expected. This forms a basis for future simulation into FLASH. Next an experimental setup was realised and a first experiment was preformed. The setup showed a field homogeneity with a maximum difference of 3\%, as well as a maximum dose difference of 2\% in the region of interest. This provided a solid foundation for accurate measurements of the experiment with living samples. Subsequently, zebrafish embryos were irradiated with protons with either 5 Gy or 20 Gy and a dose rate of approximately 1 Gy/min. A different batch of zebrafish embryos received gamma radiation with a dose rate of around 10 Gy/min, also to 5 Gy and 20 Gy dosages. The damage to the embryos was estimated via the detection of heart beats, and their length, directly after receiving radiation and after a day of growth. The results of the proton irradiation of the embryos showed no significant differences between the separate groups. For the gamma irradiation also no significant difference were found, either for the 0-day measurements. After a day of growth, a tangible difference was found, but this was with a control group that was not transported. Therefore, no direct link could be made to radiation as a sole source of the influence. Additionally, temperature had a large effect on the development of the embryos. The simulations showed the difference in biological effect of different LET coefficients of protons. The practical irradiation's do appear to reveal some discrepancy between the different samples, but the samples sizes are too small to draw definitive conclusions from, especially went comparing the results after a day of growth. Nevertheless, a solid base has has been build for future research into FLASH. A simulation capable of doing physical and biological measurement was constructed. Also, an accurate physical setup for proton irradiation was made. In addition, a design for the containment of the zebrafish during transport, irradiation, and processing was devised. Lastly, protocols for the transportation, handling, processing, and analysis of the samples were created. ... Read more

The presence of a stem cell source in the epicardium that can be stimulated by exogenous delivery of transforming growth factor-β (TGF-β) and migrate towards the myocardium can represent a new approach to achieve cardiac repair following myocardial infarction. However, injection of drugs always requires use of carriers which help stabilize the compound in the harsh and dynamic environment of the heart. To this extent, pH-responsive drug delivery systems represent an enthralling approach to ensure minimal invasiveness, instantaneous responsiveness, and targeted delivery of the incorporated compound. In this in vitro study we aimed to validate an Ureidopyrimidinone-based supramolecular hydrogel as TGF-β carrier for epicardial activation. The results demonstrated the capacity of the hydrogel to respond to pH changes and deliver the growth factor, which retained its ability to activate its signalling pathway and stimulate treated cells. ... Read more

Aortic Abdominal Aneurysm (AAA) is a chronic degenerative disease of the arterial wall. The aortic vessel wall abnormally dilates due to multiple possible causes and may eventually rupture. It is characterized by several factors leading to the extreme dilation due to the degeneration of the vessel wall extracellular matrix (ECM). It is often asymptomatic making it difficult to diagnose before rupture. The etiology of AAA is complex and not yet fully understood. Hallmarks of the disease are; loss of elastin and vascular smooth muscle cells, influx of inflammatory cells which produce proteases, increased stiffness due to higher collagen content, disturbed ECM network organization and an eventual destruction of the ECM leading to the vessel rupture. These changes may be related to environmental factors in combination with a genetic susceptibility, or by major defects in genes involved in the ECM homeostasis. Family history and genetic conditions play an important role in aneurysms. In approximately 20 % of aneurysm patients there is a familial disease or a family history of aneurysms, and in these families a major genetic defect is to be expected. Aneurysm patients show pathogenic variants in less than 5 % of the screened patients however, variants of unknown clinical significance in aneurysm genes occur at a much higher frequency. To determine the effect of these variants in aneurysm genes, functional assays which display a change in the function of the gene product needs to be established. Tissue engineering, in recent years, have become an alternative approach to animal models in developing tissue repair/replacement grafts or disease models. For this study, a cell-derived self-assembly tissue ring culture method was chosen and optimized for the use of patient cells with different pathogenic variants in aneurysm genes. After optimizing the culture conditions, the ring formation and structural ECM characteristics were compared for patient and control cases. The kinetics of ring formation showed differences in behavior and speed for patient and control cell lines. Patient lines took longer to settle into the ring shape and contract to their final thickness. The incidence of ring failure was higher in patient cases. The final patient rings were significantly thinner and non-uniform, additionally they showed a significantly lower area fraction of collagen compared to the controls. The tissue rings could not be mechanically tested due to their fragility. However, it was possible to identify the structural differences between the patient and control cases which were expected due to the pathogenic variants in the aneurysm genes of these patients. With further improvements, this method could be a potential functional assay for revealing the effects of variants of unknown significance in aneurysm genes, waiting to be identified. ... Read more

Titanium orthopaedic biomaterials to replace degenerated joint surface, improve bone regeneration and fixation are studied and used worldwide. However, not always biomaterial implantation is successful, and the main causes of implant failure remain implant associated infections (IAI) and poor osseointegration. In the recent years, additive manufacturing and titanium surface modification have been extensively researched in order to develop bone implants able to enhance bone formation at bone/material interface while reducing the risk of microbial adhesion. New bone formation at the implant site is an inflammation-driven process, since the implantation of a biomaterial in the human body is always followed by an immune response. Among the inflammatory cells involved in the inflammation stage following initial hemostasis after surgery, macrophages play a crucial role in regulating subsequent bone formation and remodelling secreting a wide range of factors. Therefore, in this study, human macrophage response to 3D printed Ti-6Al-4V surfaces was investigated. Ti-6Al-implants were modified by plasma electrolytic oxidation (PEO), during which an oxide layer containing electrolyte components were generated on implant surfaces. In order to generate an antimicrobial coating, silver nano-particles (Ag NPs) were also incorporated into the surface. Surface morphology and chemical composition were analysed by SEM. Human macrophages were cultured in the presence of SLM implants in a transwell culture or directly on the surface for 4 days. Macrophage morphology and viability were assessed with scanning electron microscopy (SEM) and fluorescence microscopy. Cell secreted factors were analysed performing an ELISA assay while gene expression analysis was performed using qPCR. Human mesenchymal stem cell (hMSC) response in terms of morphology and viability was also evaluated after 4 days of culture. PEO modification on SLM surfaces resulted in an additional TiO2 layer containing electrolyte components (Ca, P and Ag) and interconnected porosity. The ion release from SLM surfaces did not have an effect on human macrophage polarisation, while when cells where cultured directly on the implants, they secreted and expressed different amounts of pro- and anti-inflammatory factors. SLM NT (not PEO-treated) surfaces up-regulated macrophage pro-inflammatory cytokines compared to SLM PEO surfaces. However, cells cultured on SLM NT up-regulated also anti-inflammatory factors. SLM surfaces containing Ag NPs were cytotoxic for human macrophages but not for hMSCs. In general, both SLM NT and SLM PEO surfaces expressed anti-inflammatory and tissue repair-related macrophage factors, suggesting that either implant geometry achieved by 3D printing and surface biofunctionalization may have beneficial impact on promoting macrophage anti-inflammatory secretion and subsequent osteogenesis around the implant. ... Read more

Bioengineered skin was initially developed for clinical application in reconstructive surgery, but nowadays it is also considered as a suitable skin model for research. Research applications range from assessment of cosmetic and pharmaceutical products to the modeling of pathological skin conditions. For example, psoriasis is a complicated skin disease that strongly concerns the scientific community because of its unclear pathogenesis and the current inability to cure it. The Pharmacogenomics Lab of ETH has been working on the development of psoriatic skin models in order to investigate the driving mechanisms of the disease and to later implement high-throughput drug screening to explore treatment possibilities. However, the manual production into transwell inserts results in unstable, self-contracting and inconsistent skin models, pain points that the Pharmacogenomics Lab would like to eliminate. A recent collaboration between the Product Development Group and the Pharmacogenomics Lab has been recently initiated in an attempt to standardize the fabrication of the psoriatic skin models. For this purpose, this Master Thesis focused on the investigation of the potentials and limitations of automated injection molding, a method developed by the Product Development Group, in the generation of consistent and representative dermo-epidermal models for psoriatic skin.
After a series of experiments, it was seen that in-mold fabrication achieves homogeneous and consistent dermal hydrogels that maintain their dimensions throughout the whole cultivation period regardless of the collagen concentration employed for the dermal matrix, while automated injection molding allows the utilization of collagen concentrations higher than the unstable 5mg/ml, traditionally used in the manual fabrication method. All collagen concentrations exhibited similar biological performance, but the optimum fibroblasts viability was achieved in case of 5mg/mL, meaning that the whole injection molding process should be faster and even simpler to fully prevail over manual fabrication of skin models and better fit the requirements of skin disease research. Keratinocytes viability and differentiation was similar for all mold designs, all collagen concentrations and both fabrication methods. Development of a first psoriatic model, consisting of psoriatic fibroblasts and healthy keratinocytes, did not show any direct effect of diseased fibroblasts on keratinocytes proliferation and differentiation as the epidermal layer of the psoriatic model was very similar to this one of the healthy model.
Evaluation of the automated seeding of keratinocytes for further standardization of the process and elongation of the psoriatic model’s cultivation period for a more thorough study should be the next steps. In the long-term, incorporation of all the psoriasis-related cells and molecules in a simple and fast way, as well as further advancements in the mold design to facilitate a direct high-throughput drug screening should be considered.
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