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Coming dissertations at Uppsala university

  • Additive Manufacturing and Mesoporous Materials for Pharmaceutical Applications Author: Christos S. Katsiotis Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-523234 Publication date: 2024-03-22 08:54

    Additive Manufacturing (AM), over the past decade, has evolved into a versatile technology with significant applications in pharmaceutical research. This technology enables the production of drug formulations tailored to individual patients, offering customization in both dosage and dissolution profiles. While challenges in mass production persist, 3D printing, particularly through techniques like Fused Deposition Modeling (FDM) and Semi Solid Extrusion (SSE), proves ideal for crafting smaller batches of personalized dosage forms.

    A prevalent issue in drug development revolves around poor water solubility, impacting bioavailability upon oral administration. To combat this, the integration of mesoporous materials emerges as a promising strategy to enhance the dissolution of poorly water-soluble drugs. Here, the applicability of mesoporous materials is explored, as well as their incorporation with various AM techniques. Overall, the thesis dives into the investigation of combinatorial formulations, incorporating at least one 3D printed component to address specific requirements in drug delivery. 

    By combining FDM with Selective Laser Sintering (SLS), a hybrid two-compartmental formulation is developed. The durable FDM-printed shell regulates buffer medium access to the contained SLS-produced inserts loaded with the drug. Varying printing parameters and insert combinations within the shell showcase the adjustability and flexibility of this hybrid approach.

    Tablets with different infill percentages, containing drug-loaded mesoporous materials, are developed. Poorly water-soluble drugs are successfully amorphized within mesoporous material pores, formulated into filaments through Hot Melt Extrusion (HME), and printed via FDM. These tablets exhibit improved dissolution compared to the crystalline drug, with the dissolution behavior regulated also by the infill percentage.

    The study explores the impact of drug-loaded mesoporous materials on HME-produced filament properties, studying their effect on maximum tensile strength and Young’s modulus. The relationship between these properties and filament printability is investigated. Additionally, a protective effect of mesoporous materials on drugs from thermal degradation is revealed.

    For Semi Solid Extrusion (SSE) manufactured formulations, a paste is developed, comprising mesoporous material loaded with a poorly water-soluble drug and an excipient. This paste demonstrates favorable rheological properties and easy extrudability via a syringe. The formulation proves versatile for printing dosage forms for both oral and rectal administration, with the printed tablet and suppository exhibiting effective drug release.

    In conclusion, this work presents valuable strategies for developing patient-tailored dosage forms, addressing specific pharmaceutical challenges like poor solubility. The integration of mesoporous materials and various 3D printing techniques showcases a promising direction for personalized medicine in the pharmaceutical field.

  • Pitfalls in β-cell ion imaging with fluorescent indicators and their use for real-time detection of somatostatin secretion Author: Mingyu Yang Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-523019 Publication date: 2024-03-22 08:24

    Fluorescent ion indicators have become indispensable tools in cell physiology. Dye indicators can easily be loaded into most types of cells, while genetically encoded indicators are advantageous in allowing specific cellular or subcellular targeting. Comparing responses of dye and protein-based indicators may provide useful insights into indicator properties and cellular processes. Here, a few genetically encoded Ca2+ indicators and fluorescent Ca2+ dyes were compared in insulin-secreting β-cells. Recordings of depolarization-triggered changes of the cytoplasmic Ca2+ concentration ([Ca2+]i) beneath the plasma membrane with total internal reflection fluorescence microscopy demonstrated distinct [Ca2+]i spikes with protein-based indicators, while the dyes mainly reported stable [Ca2+]i elevations. The spikes reflected Ca2+ release from the endoplasmic reticulum, triggered by autocrine purinergic receptor activation from exocytotic release of ATP. The indicator-dependent differences were unrelated to Ca2+ binding affinity and buffering and probably reflected slower Ca2+ dissociation kinetics of the protein indicators. In glucose-stimulated mouse islets, the dye Fura-2 reported the characteristic [Ca2+]i response with an initial lowing followed by rapid increase, which was abolished by hyperpolarization with the K+-channel opener diazoxide. The simultaneously present genetically encoded indicator R-GECO1 failed to detect the lowering and reported a spurious [Ca2+]i elevation also in the presence of diazoxide, responses that could be ascribed to pH sensitivity of the indicator. Recordings with fluorescent H+ indicators demonstrated that glucose increases cytoplasmic pH in β-cells. Elevations of [Ca2+]i counteracted the alkalinization and [Ca2+]i oscillations in glucose-stimulated islets were associated with anti-phasic oscillations of [Ca2+]i and pH. A [Ca2+]i imaging-based reporter cell assay for real-time detection of the islet hormone somatostatin was generated by transfecting HeLa cells with somatostatin receptor 2, the G-protein Gα15 and R-GECO1. The reporter cells detected somatostatin secretion from islets imaged in the same view-field as dose-dependent [Ca2+]i elevations. Mouse and human islets released somatostatin in response to high K+, glucose, and the hormones GLP-1 and ghrelin. In glucose-stimulated mouse islets, bursts of somatostatin release were synchronized with islet [Ca2+]i oscillations. Analyses of islets from human donors indicated that type 2 diabetes is associated with hypersecretion of somatostatin. In conclusion, this thesis highlights potential pitfalls with fluorescent ion indicators in β-cell signalling studies and provides new insights into β-cell regulation of [Ca2+]i and pH. Moreover, it introduces an assay for real-time detection of somatostatin secretion from islets that holds promise for studies of the role of this hormone under normal conditions and in diabetes.

  • Prognostic and Predictive Factors in Metastatic Colorectal Cancer Author: Emerik Österlund Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-522642 Publication date: 2024-03-22 07:34

    The outcome for metastatic colorectal cancer (mCRC) patients has improved substantially in recent decades. This has chiefly been observed in study populations, and predominantly in left-sided primary tumours, which is why we wanted to study if and how survival has improved in the background population. It has also been seen that certain molecular subtypes are more common in population-based materials, and, thus, we studied the prevalence and effects of different molecular alterations.

    Paper I is a national population-based material of all 19 566 Swedish patients with a diagnosis of mCRC 2007-2016, 55% were male and 70% had synchronous metastases. Median overall survival (OS) for all patients was 14.0 months. An improvement could be seen over time, also in stratified analyses. OS was influenced by presentation of metastases, age, primary tumour location, and sex. All except sex remained statistically significant in a multivariable analysis. Differences of about one month in median OS were seen between healthcare regions, but these diminished over time.

    Paper II included all 765 patients from the Uppsala Region with a mCRC diagnosis 2010-2020. Right colon primary tumours were seen in 38%, left colon in 27% and rectum in 34%. BRAF-V600E mutations (mt) and deficient mismatch repair (dMMR) had a poor OS and were more common in right colon primary tumours. Primary tumour location did not affect OS in subgroups according to mutations in RAS or BRAF, nor in a multivariable analysis. Molecular alterations seem to be more important than primary tumour location for prognosis.

    Paper III studied KRAS-G12Cmt in three population-based and one real-world material. KRAS-G12C was seen in 2-4% of all tested and in 4-8% of all KRASmt. No differences in patient characteristics were observed between KRAS-G12C and other KRASmt. No differences in OS were seen between KRAS-G12C and other KRASmt, neither for all patients, nor in different treatment groups.

    Paper IV studied atypical BRAFmt (aBRAFmt) in two population-based and one real-world cohort. aBRAFmt was seen in 1-4% of the adequately tested patients in the different cohorts. aBRAFmt patients were predominantly male, had dMMR less often, more rectal primary tumours, and less peritoneal metastases compared with BRAF-V600Emt. Serrated adenocarcinomas were seen in about half of the aBRAFmt. OS was significantly better for aBRAFmt than in BRAF-V600Emt, but worse than for RASmt and RAS&BRAFwt patients. Nine aBRAFmt received epidermal growth factor receptor inhibitors without responses.

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