Faculty of Biological and Environmental Sciences
Recent Submissions
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(2024)Degeneration of dopaminergic neurons in the substantia nigra is a primary pathological feature of Parkinson’s disease (PD). Current treatment strategies available are not disease-modifying but rather symptom-alleviating due to limitations in understanding the molecular event(s) that trigger the degeneration of dopaminergic neurons. While several animal and cellular models exist to study the disease progression and pathophysiology, their translational relevance to humans is debatable. In this thesis, our goal was to replicate a published protocol designed to differentiate SH-SY5Y human neuroblastoma cell lines into dopaminergic neurons. Our study shows successful differentiation and characterization of SH-SY5Y cells into dopaminergic neurons, but also the limitations in reproducibility. This finding highlights the challenges of technical variability, but also the reliability of existing protocols by emphasizing the need for more consistent reproducible methods as it could be significant for improving this cellular model.
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(2024)Aging is characterized by a gradual decline in cellular functions and the emergence of various cellular phenotypes that negatively affect lifespan. However, the molecular mechanisms behind these aging phenotypes remain unclear. One potential factor is the decline in proteostasis, characterized by increased protein misfolding and reduced efficiency of the proteostasis network. A key regulator of this network is Heat shock protein 90 (Hsp90), a molecular chaperone that ensures the proper folding and stability of hundreds of client proteins. Many of these clients are metastable signal transduction and gene expression regulators, such as kinases and transcription factors. By controlling the functional conformation of its clients, Hsp90 plays a central role in modulating the activities of many cellular processes. During environmental stress conditions, when Hsp90's folding capacity is exceeded, new phenotypes arise due to the altered folding of Hsp90 clients. I hypothesize that the age-related rise in protein misfolding similarly limits Hsp90’s availability to assist in the folding of client proteins, resulting in the emergence of aging phenotypes. To address this, I investigated whether Hsp90's functional capacity declines with age and how this decline is linked to proteome alterations and consequent phenotypic changes. To assess how Hsp90 function changes with age I utilized a β-galactosidase-based Hsp90 availability reporter. Measurements in young and aged cells demonstrate that Hsp90 activity declines with age. To assess the consequences of Hsp90's functional decline on the proteome of aged cells I employed quantitative proteomics. Results identified Hsp90-sensitive targets associated with cell cycle regulation, supporting previous findings linking Hsp90 inhibition with cell cycle defects and cellular elongation. Indeed, the progeny of aged mother cells exhibited elongated phenotypes compared to the progeny of young cells, but this trait was restored by Hsp90 overexpression. Furthermore, high-level expression of an exogenous Hsp90 client, a glucocorticoid receptor, which competes for Hsp90 availability, exacerbated elongation effects even in young cells. Altogether, these findings suggest that the availability of Hsp90 declines with age, correlating with the onset of aging phenotypes due to changes in the client proteome. Given Hsp90's role in suppressing cellular senescence in myoblasts and its association with brain aging and neurodegenerative diseases, these insights may have broader implications for understanding the emergence of aging phenotypes at a molecular level.
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(2024)Double homeobox 4 (DUX4) is a transcription factor normally repressed in somatic cells but expressed in early human embryo and involved in embryonic genome activation. Before transcription starts from its own genome, the embryo uses maternally stored transcripts and proteins, which undergo selective degradation as development progresses. DUX4 is transcribed from a repetitive region called D4Z4, along with another gene, DBE-T, that encodes a long non-coding RNA involved in the de-repression of DUX4. DBE-T partially overlaps with the D4Z4 region and shows some sequence similarity with DUX4. DUX4 regulation in early embryo is currently not well understood. The aim of this study was to use updated RNA in situ hybridization technology (RNAscopeTM) to observe DUX4 transcripts in doxycycline-inducible DUX4-TetOn human embryonic stem cells and ovarian samples. DUX4 RNA target probes were first validated in doxycycline-treated DUX4-TetOn human embryonic stem cells. Doxycycline-treated cells express DUX4 protein as observed by immunofluorescence staining. After confirming that the DUX4 target probes were visible in DUX4-expressing stem cells, the same probes were used in human ovarian tissue samples. DUX4 probes showed signals in both somatic granulosa cells and oocytes in primordial and primary follicles in the ovarian samples. However, due to the sequence similarity between DUX4 and DBE-T, there is possible cross-detection of DBE-T with the DUX4 target probes. It is not possible to say with confidence whether the detected signal is derived from DUX4 or DBE-T. These results indicate that D4Z4-related transcription activity occurs in the immature oocytes and further studies are needed to determine which transcripts are expressed in the oocyte.
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(2024)Anxiety disorder is the most common mental health disorder and often comorbid with sleep disturbances. However, anxiety disorder and its link to sleep features is not well-studied. In this study, we hypothesized that anxiety and depression can affect sleep architecture in different ways. Polysomnographic (PSG) recordings of 203 subjects from two different studies were analysed to characterize changes of electroencephalographic (EEG) activity between patient groups and healthy controls during sleep. Results show that sleep patterns and EEG spectral power densities in patients with anxiety and depression follow similar patterns when compared to healthy controls. Stage N1 sleep increased in all patient groups, while REM sleep decreased compared to healthy control group. Moreover, REM sleep duration increased, and REM latency decreased in all patient groups following the exclusion of individuals under antidepressant medication. In addition, decreased EEG delta (0.4-4 Hz) and increased EEG beta (15-32 Hz) power were seen in all patient groups. These results demonstrate that sleep quality (quantified as increased N1 sleep) in patients with anxiety and depression decreases and that both disorders affect sleep in a similar way. Observing specific sleep alterations as a whole could prove more efficient than solely analysing individual sleep parameters. The use of diagnostic EEG data provides new opportunities for sleep research with relatively large sample size while also taking into account age, antidepressant use, and comorbidity with depression.
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(2024)Antigen presenting dendritic cells (DC) are important mediators between innate and adaptive immunity. They constantly patrol for foreign and self-antigens in peripheral tissues. Capture of an antigen initiates activation cascade in DCs resulting in the upregulation of chemokine receptor CCR7. Endothelial chemokine CCL21, ligand of CCR7, guides activated DCs from the peripheral tissues to the vicinity of the lymphatic vessels (LV) and drives the subsequent transmigration into LV lumen. From LV lumen, DCs are transported to lymph nodes where they activate T cells by presenting captured antigens to them. Despite the importance of DC migration from peripheral tissues to lymph nodes, the molecular mechanisms underlying the guidance of DCs to the transmigration sites on lymphatic endothelium are poorly understood. In this thesis I asked whether DCs have preferential transmigration sites on the lymphatic endothelium and if lymphatic endothelial chemokine CCL21 guides DCs to the transmigration sites. To seek answers to the proposed questions, I employed primary cell co-culture model where wild-type or CCR7-/- mouse bone marrow derived DCs were placed on top of the primary human lymphatic endothelial cell (LEC) monolayer overexpressing CCL21. The DC migration and transmigration events on the LEC monolayer were live imaged on epifluorescence wide field microscope and analysed with image analysis tools (ImageJ and IMARIS). Prior to transmigration wild-type DCs probed the monolayer with their dendrites and polarised them towards the LEC monolayer at the transmigration sites. Majority of wild-type DCs arrested and transmigrated at multicellular junctions (junctions where more than two cells meet). To investigate the role of CCL21 in DC transmigration, I compared the arrest site distributions and migration patterns of wild-type and CCR7-/- DCs on lymphatic endothelium. Again, majority of wild-type DCs found multicellular junctions whereas the arrest sites of CCR7-/- DCs were distributed evenly between multicellular, bicellular (junction where two cells meet) and non-junctional sites. Moreover, the wild-type DCs had straighter migratory paths to the arrest sites, and they were less prone of subsequent detachment than CCR7-/- DCs. In conclusion, in this thesis I define multicellular junctions, for the first time, as preferential DC transmigration sites across lymphatic endothelium. In addition, I show that lymphatic endothelial chemokine CCL21 guides DCs on the LEC monolayer to the multicellular junctions and thus promotes the identification of preferential transmigration sites by DCs. Future studies should focus on characterization of multicellular junctions of lymphatic endothelium and validation studies in vivo. Taken together, these findings improve the understanding of molecular mechanisms in DC transmigration across lymphatic endothelium and give a platform for development of improved vaccines and cancer therapies.