Browsing by master's degree program "Translationaalisen lääketieteen maisteriohjelma (Translational Medicine)"

Extracellular matrix components such as laminins have important roles in supporting the mammary gland epithelium and guiding its development and homeostasis. Adhesion to laminin alpha-5 subunit (Lama5), notably secreted by the hormone receptor positive luminal epithelial cells, promotes luminal epithelial differentiation and cellular identity, as well as controls mammary progenitor activity, contributing to physiological growth of the mammary epithelium. Lama5 loss in luminal mammary epithelial cells results in abnormal epithelial differentiation, aberrated ductal development, and diminished mammary epithelial growth in mice. Breast cancer is one of the most common forms of cancer, and the most common subtypes are hormone receptor positive luminal breast cancers. While expression of other epithelial laminin alpha subunits is often lost, Lama5 is commonly overexpressed in human breast cancer cells, notably in luminal cancer subtypes, as opposed to basal-like and other cancers subtypes. However, the role of Lama5 in mammary tumor growth and identity has not been experimentally studied in neither mice nor humans. In this thesis, the role of Lama5 in the growth and identity of mammary tumors was studied using both in vivo mouse and in vitro human approaches. Lama5 deletion in luminal epithelial cells of tumor-bearing mice was shown to result in five-fold decrease in the amount of tumorous growth in mouse mammary glands in vivo. In vitro LAMA5 downregulation of MCF-7 luminal human breast cancer cells was shown to lower the proliferation rate and increase the doubling time in 2D culture, decrease their mammosphere forming capacity, as well as decrease total growth in 3D culture, while no effects were observed in triple-negative basal-like MDA-MB-231 human breast cancer cells with LAMA5 downregulation. Additionally, downregulation of LAMA5 was shown to promote the expression of basal-like breast cancer and EMT markers vimentin and fibronectin in luminal MCF-7 cells, while the expression of luminal identity markers was not altered. No changes in the expression of luminal or basal cytokeratin markers CK8 and CK14 were seen in mammary tumors in vivo on mice with luminal laminin alpha-5 deletion. This thesis provides the first set of experimental evidence of the role of laminin alpha-5 as a factor promoting mammary tumor growth in both mice and human cells, especially in hormone receptor positive luminal cancer types. Lama5 contribution to tumor identity, and the exact mechanisms require further studies.

Myelin is a lipid-rich substance wrapped around nerve axons that can be adaptively modified in response to neuronal activity and experience. Recent research has revealed myelination of parvalbumin (PV) inhibitory interneurons, critical for brain oscillations and balance. Defects in PV interneuron myelination have been linked to psychiatric disorders, like schizophrenia. Tropomyosin receptor kinase B (TrkB) signaling has been shown to be important for myelination. Moreover, fluoxetine, an antidepressant, binds to TrkB receptors in PV interneurons, enhancing plasticity. While previous studies support the importance of PV interneuron mediated TrkB signaling for anti-depressant induced neural plasticity, its effect on PV interneuron myelination remains unexplored. The objective of this thesis was to investigate whether TrkB signaling, and fluoxetine affect the overall and PV-interneuron specific myelination in the medial prefrontal cortex (mPFC) in mice. Using immunohistochemical analysis, we assessed myelin changes through node of Ranvier morphology and myelin immunostaining intensity in control and in mice with heterozygous conditional TrkB deletion in PV interneurons (hereafter referred to as TrkB KO), with or without fluoxetine. We found that fluoxetine increases node length in TrkB KO mice, while reduced TrkB signaling shortens paranodes in PV neurons compared to controls. Our findings also depict that fluoxetine and PV-mediated TrkB signaling do not alter the overall myelination of the mPFC. The findings of this work provide mechanistic insights into PV interneuron myelination in the mPFC, with potential implications for demyelinating and psychiatric conditions where PV myelination plays a role.

MELAS syndrome is a multi-organ disorder with a wide range of clinical manifestations, including hearing loss, cardiomyopathy, retinopathy, and stroke-like events. It can also be associated with type 2 diabetes. Eighty per cent of MELAS Syndrome cases have a 3243A>G mutation in the MTTL1 gene, which codes for tRNA (Leu-UUR). Although the exact cause of MELAS syndrome remains unknown, microvascular angiopathy and endothelial dysfunction, which result in reduced microvascular perfusion, are assumed to have a role in the reported phenotype. Yet, it is unclear how important this depicted angiopathy is in general and how it relates to the disease-specific stroke-like episodes in particular. In this thesis, I employed MELAS-patient-derived human induced pluripotent stem cell (hiPSC)-derived endothelial cells (EC) under flow as a disease model to investigate the possibility of an altered endothelial cell phenotype or function that could account for the disease phenotype. I used hiPSCs from healthy controls and patient-derived iPS-cell lines from MELAS patients with the 3243A>G mutation with different levels of heteroplasmy in all the experiments. Transcriptional patterns reflecting the flow response were observed, demonstrating that the MELAS hiPSC-EC cells respond to a flow stimulus. In the high-heteroplasmy cell lines, there was a significant downregulation of genes related to one carbon folate metabolism and serine biosynthesis, especially in response to flow, and these changes were also indicated in basal conditions. This is interesting as the same metabolic pathways have been shown dysregulated in other mitochondrial diseases. This study showed no appreciable difference in the mitochondrial oxygen respiration rate between MELAS-patient and control hiPSC-EC. These findings could link the disease phenotype to this web of pathways and explain the mechanism underlying the pathophysiology of MELAS syndrome.

Antibiotic-resistant bacteria present a severe threat to global health. The future treatment of common bacterial infections relies on the identification of novel antibiotics and targets in the present. One area of antimicrobial research is the study of bacteriophage (Petrovic Fabijan et al.) mechanisms and the identification of phage-derived antimicrobials. Sequenced phage genomes are largely (>70%) annotated as “hypothetical proteins of unknown function” (HPUFs) and investigation into HPUFs with a toxic effect on host bacteria (toxHPUFs) aims to reveal new antibacterial targets and antimicrobials. Next-generation sequencing and plating-based toxicity screening of Staphylococcus phage Stab21 HPUFs identified nine HPUFs that incurred toxicity to Escherichia coli. In this study, the tightly controlled tetracycline-inducible plasmid pRAB11N was used as a shuttle vector and verified the toxicity of five out of nine HPUFs to E. coli and revealed that no HPUFs caused toxicity to the Stab21 natural target and clinically relevant Staphylococcus aureus. These results suggest that screening for toxHPUFs should be carried out in closely related bacterial species or the phages’ natural host. The five toxHPUFs of E. coli were further characterised by protein function and structural predictions. Only one toxHPUF, g024, returned a reliable model with homology to Bacillus phage SPO1 homing endonuclease I-HmuI, yet the role of this DNase in bacterial host toxicity is still unknown. To determine the bacterial targets of the toxHPUFs, spontaneous toxin-insensitive mutants of the five toxHPUFs were investigated. For three toxHPUFs, the toxin insensitivity was ascribed to the elimination of the toxin-encoding gene. However, toxin-insensitive g172 and g187 sequences revealed mutations in the tetR gene of pRAB11N that led to the inability of tetracycline binding and thus no induction of gene expression and did not aid in identifying the bacterial targets of these toxHPUFs. This study highlights the experimental complexities of phage-derived antimicrobial research. It also maintains the value of this research strategy, with the verification of HPUFs with a toxic effect on E. coli and accompanied future studies of bacterial target determination having the potential to uncover novel antimicrobial mechanisms that can be exploited for therapeutic application.