Browsing by study line "Molecular and Analytical Health Biosciences"

SerpinE2 is a serine protease inhibitor (serpin) family protein that inhibits several extracellular proteases, such as thrombin, urokinase-type plasminogen activator and trypsin. Proteases and their inhibitors are often involved in cancer. SerpinE2 transcripts are upregulated in several cancers and found to predict poor prognosis of cancer patients. However, such studies regarding protein levels of serpinE2 are scarce. In this study, serpinE2 protein was analysed in three urological cancers, with patient groups that address the greatest needs for clinical biomarkers. The major aim of this study was to examine the association of serpinE2 staining with patient survival and clinicopathological features in prostate, urinary bladder and kidney cancers, and to evaluate its usability as an immunohistochemical biomarker. Tissue microarray slides from cancer patient tissues were stained immunohistochemically for serpinE2. The staining intensity was scored with four-point scale from 0 (no staining) to 3 (very intensive staining). Prostate and kidney cancer patients had been treated surgically and some of the cancers had relapsed after the surgery. In bladder cancer, association of serpinE2 with treatment response to neoadjuvant chemotherapy was evaluated. SerpinE2 expression was also measured in two prostate cancer cell lines with quantitative PCR and Western blotting. The serpinE2 staining was observed both in cancer cells and epithelial structures of benign tissues. The results showed that cancer tissue serpinE2 is not associated with relapse, treatment response or survival in prostate and bladder cancer patients. However, serpinE2 staining was more pronounced in prostate cancer tissues compared with benign tissues adjacent to cancer, and, surprisingly, the staining in such benign tissues was stronger in tissues from patients who developed metastases after surgery as compared to those without detectable metastases during 10.3-year (median) follow-up (p = 0.017). In addition, higher serpinE2 staining intensity was observed in higher grade bladder cancers (p = 0.034). In kidney cancer, on the other hand, serpinE2 staining intensity was significantly lower in patients whose cancer relapsed (p = 0.048), and high intensity predicted favourable disease-specific survival (p = 0.013). To conclude, serpinE2 is worth of further investigation in urological cancers. In prostate cancer, the possible field effect of cancer on serpinE2 in adjacent benign tissues could be examined more closely. In kidney cancer, the impact of serpinE2 on patient survival was inverse compared to transcript data in the Cancer Genome Atlas/the Human Protein Atlas database, and most other cancers. Thus, further validation studies need to be performed, and if the results hold true, serpinE2 staining could be used as part of a prognostic model predicting kidney cancer-specific survival.

Prostate cancer is one of the most common cancers diagnosed in men. Somatic copy number alterations (SCNA), such as the deletion of PTEN or NKX3.1 and the amplification of MYC, have been associated with prostate cancer progression and could serve as potential biomarkers during diagnosis. One approach to utilize this information would involve screening a large number of prostate tissue sections for SCNAs and subsequently validating the findings using a secondary method. This process could enable more personalized treatment options for cancer patients. This thesis aimed to create a robust and reproducible workflow for SCNA identification. This was achieved by optimizing a chromogenic immunohistochemistry (IHC) protocol using immunostaining chambers and open-source 3D-printed laboratory hardware. The optimized protocol was then transferred to an automated liquid handling robot, and a panel of three antibodies for PTEN, NKX3.1, and MYC was developed for SCNA screening with IHC. Additionally, a chromogenic in situ hybridization (CISH) protocol was optimized to validate the results of the IHC. The immunostaining chambers required a lower antibody dilution to perform comparably to the manual IHC stainings. The automated protocol using the liquid handling robot produced high-quality stains with optimized dilutions. The optimized CISH protocol successfully identified the presence of the target gene, but unclear signals and merging of the signals obstructed detailed analysis. While complete deletion of PTEN was detectable, determining the number of gene copies per cell proved challenging due to signal variability and sample-dependent problems. Further optimization of the CISH protocol or development of an automated analysis workflow tailored to address these challenges is needed for more accurate analysis.

Pathogenic variants in BRCA-associated protein 1 (BAP1) cause BAP1 tumour predisposition syndrome (BAP1-TPDS) with increased risk of several cancers including uveal melanoma (UM). UM originates from the melanocytes of the choroid, iris or ciliary body in the eye. UM is a rare cancer with a high metastatic rate and is usually seen in Caucasian people. BAP1 is a deubiquitinating enzyme and to function as a tumour suppressor it needs to retain enzymatic activity. To retain its enzymatic activity, it needs a functioning UCH-domain and nuclear localisation signal. Loss of function variants in BAP1 are easily interpreted as pathogenic, however, many missense variants remain as unclear on their status of pathogenicity. We aimed to study the enzymatic activity of missense variants in the UCH domain of BAP1. We selected 22 missense variants near or in the BAP1 UCH domain (aa1-240). Nineteen were identified in patients with UM and three rare variants from gnomAD database detected in the general population. The variants were cloned to a bacterial expression vector and expressed as a GST-fusion protein. Then we assayed the purified proteins for their ability to cleave ubiquitin. Ten patient derived variants reduced the deubiquitinating activity of BAP1. Seven mutants with variants from patients with familial BAP1-TPDS, retained <20% of their activity. Three variants previously classified as Variant of unknown significance (VUS) and one pathogenic decreased the activity to half. The function of twelve variants was interpreted as normal (80-120%). Of these, two were previously interpreted as pathogenic. Functional studies are needed for accurate BAP1 missense variant classification. Although BAP1 variants are dominant, penetrance might be affected by variants effect on enzymatic activity and patients with pathogenic variants might not exhibit familial BAP1 -TPDS. If enzymatic activity is retained and patient exhibits familial BAP1-TPDS, further studies need to be conducted on effects on splicing and protein-protein interactions. However, if patient exhibits only familial UM and harbours a BAP1 VUS with normal activity, other genes predisposing to UM should be considered.

Vaccines have proven to be one of the most effective methods of mitigating the effects of infectious diseases. One of the most prominent hurdles facing widespread vaccine delivery is the cost and added time attributed to cold chain logistics. That is that vaccines need to be kept constantly below a certain temperature to retain their effectiveness, which in many cases is as low as -80°C. Application of specific biopolymers is one method of enhancing vaccine thermostability, diminishing the need for such cold chains by preservation at lower temperatures. This enhanced vaccine formulation is achieved through a technique known as microencapsulation, in which an active agent, in this case the vaccine, is protected through storage in a thin film of polymeric material. This preserves vaccine efficacy until later use and release from the protective film upon vaccine delivery. In the following study, a naturally derived biopolymer was investigated for its cryopreserving properties with the aim of exceeding the current benchmark of preservation at ambient temperature for 72 hours. This involved evaluation of the formulation, referred to as our designed technology (DT) across different stress parameters (4°C, 22°C and 37°C) for differing periods of time, applicability to different vaccine systems (adenovirus, envelope virus and mRNA based) and finally efficacy in both in vitro and in vivo settings. The results demonstrate that adenovirus based vaccines are able to withstand the physical, pH and temperature alterations of diverse stress tests when protected by biopolymer microencapsulation both in vitro and in vivo. This suggests that microencapsulation with the naturally derived biopolymer is a strong method of preserving adenovirus vaccine stability at temperatures up to 37°C for up to three weeks. As such, naturally derived biopolymer microencapsulation stands to drastically reduce the cost of vaccines by mitigating the need for cold chain logistics through preservation at temperatures up to 37°C.

Tiivistelmä – Referat – Abstract The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase is a master regulator of cell growth. In the presence of environmental cues, such as nutrients and growth factor, mTORC1 is transported to the lysosome where it is activated by a small GTPase Rheb. Dysregulation of mTORC1 has been linked to several diseases such as cancer and neurodegeneration. Despite our growing understanding of the nutrient-driven activation mechanism of mTORC1, we still do not fully understand how nutrients are transported out of the lysosome or how nutrient sensing is connected to nutrient transport. Recently, SLC38A9, a small lysosomal transmembrane protein, was identified as a mediator of the efflux of essential amino acids from the lysosome to the cytosol. It also acts as an amino acid sensor for mTORC1, playing a role in its activation. Due to poorly vascularized tumor cores, cancers such as pancreatic ductal adenocarcinoma, have access to very scarce amounts of free nutrients. Consequently, they rely on scavenging of protein macromolecules from the extracellular environment, followed by digestion inside lysosomes. The digested nutrients are released to the cytosol via transporters such as SLC38A9 and activate the mTORC1 pathway which carries out the growth processes. In fact, recent studies in mouse xenograft models have shown a severely slowed down growth of PDAC tumors with SLC38A9 knocked out. Blocking of SLC38A9 activity with pharmacologics or biologics would prevent the release of digested amino acids from the lysosomes, starving cancer cells of nutrients, while sparing normal cells that do not feed on extracellular proteins. However, SLC38A9 is still poorly understood, and development of selective inhibitors first requires mechanistic understanding of the protein and knowing what its binding pockets look like. In order to obtain this information, we aimed to determine the three-dimensional structure of SLC38A9 through cryogenic electron microscopy (cryo-EM). However, two significant challenges hindered our ability to obtain high-resolution images of this membrane protein: (i) its small size, and (ii) its constant conformational changes. To address this, I proceeded to develop a set of nanobodies that would bind SLC38A9 with high affinity and specificity. Nanobodies allow for locking of target proteins in specific conformational states, and they can also serve as chaperones for visualizing proteins in cryo-EM. To obtain these nanobodies, I used a library of 100 million unique nanobodies, displayed on the surface of yeast cells. Specific SLC38A9 binder nanobodies were obtained through multiple rounds of selection and sorting, using decreasing concentrations of fluorescently- labeled SLC38A9. After the final selection round, single colonies were picked and the strength of binding to SLC38A9 was evaluated. High-throughput screening results showed that we were able to obtain specific SLC38A9 binders and that there was variation in binding strength among the selected nanobodies. These nanobodies will enable the determination of the cryo-EM structure of SLC38A9 and also serve as tools to further dissect the function and mechanisms of SLC38A9 in amino-acid efflux from lysosomes to cytosol, providing further insights for the development of novel cancer therapeutics.

Clonal hematopoiesis is characterized by the accumulation of blood cells originating from a single mutated hematopoietic stem cell clone. Clonal hematopoiesis has been hypothesized to contribute to systemic inflammation, and by doing so increase the risk for inflammatory diseases. Despite the growing body of evidence from clinical datasets and murine levels linking clonal hematopoiesis and inflammation, the causative role of clonal hematopoiesis in promoting inflammation remains to be fully elucidated. The primary goal of this master's thesis is to investigate the potential causative relationship between clonal hematopoiesis (CH) and rheumatoid arthritis, one of the most common autoimmune diseases previously associated with clonal hematopoiesis. The analysis is done primarily by employing Mendelian randomization (MR) and multivariate logistic regression analyses to perform two-sample Mendelian randomization in a bidirectional manner to assess the strength of the analysis along with exploring potential pleiotropy. We leverage data from two large population-level cohorts: the FinnGen study and the UK Biobank project. We also employed multivariate logistic regression to validate the instrumental variables of the UK Biobank within the FinnGen data. Despite prior epidemiological and functional literature linking clonal hematopoiesis and rheumatoid arthritis, we observed no association between these two in these large-scale Mendelian randomization analyses. This may be due to differences in cohort characteristics and detection methods for clonal hematopoiesis between the two cohorts. The thesis discusses the strengths and weaknesses of Mendelian randomization in evaluating causative links between human phenotypes. This work contributes to the growing area of research on the genetic determinants of inflammatory diseases and opens avenues for further research into the role of CH in disease pathogenesis