Browsing by Subject "biomedicine"

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  • Lavilla-Alonso, Sergio (Helsingin yliopisto, 2012)
    Despite the rapid advance of cancer research and improvement of conventional therapeutic regimes like chemotherapy, surgery, immunotherapy or radiotherapy, cancer remains a leading cause of death worldwide. Metastatic disease normally represents the most deadly stage and leads to the poorest prognosis. The disseminated location of metastases makes it even more difficult nowadays for existing drugs to target tumors without damaging healthy tissues. The work presented in this Doctoral Thesis is aimed at developing targeted adenoviral vectors and regimes that can be applied to the treatment of cancer, especially colorectal cancer. Gene therapists have explored widely interactions of the viruses to cancer and normal cells and have proved that molecular modifications in the capsid can unleash striking differences in viral tropism. In this Doctoral Thesis, the utility of arginine-glycine-aspartic acid (RGD) targeting αvβ integrins substituted for the lysine-lysine-threonine-lysine (KKTK) domain of the fiber shaft or inserted in the HI-loop of adenovirus serotype 5 (Ad5) was evaluated for increased tumor targeting and antitumor efficacy. Both modifications increased gene transfer efficacy in colorectal cancer cell lines and improved the tumor to-normal ratio after systemic administration of the vector. Furthermore, antitumor potency was not compromised with RGD modified viruses suggesting that an increased safety profile did not involve any loss of therapeutic effect. Treatments based on adenovirus vectors should not have negative effects on tumor progression or metastases. In order to evaluate this possibility, we designed a novel murine model of human colorectal cancer (CoCa) to test our treatments. To this end, we have developed a readily imageable mouse model of colorectal cancer featuring highly reproducible formation of spontaneous liver metastases derived from intrasplenic primary tumors. We optimized several experimental variables, and found that the correct choice of cell line and genetic background of the recipient mice as well as their age, were critical for the establishment of a useful animal model. A magnetic resonance imaging (MRI) protocol was optimized for use with this mouse model, and demonstrated to be a powerful method for analyzing the antitumor effects of an experimental therapy. Poor spreading of the virus through tumor tissue is one of the major issues limiting efficacy of oncolytic adenoviruses, even after local administration by intratumoral injection. In this study, ECM-degrading proteases relaxin, hyaluronidase, elastase, and macrophage metalloelastase (MME) were used to increase oncolytic adenovirus spreading. Moreover, MME improved the overall antitumor/antitumour efficacy of oncolytic adenovirus in subcutaneous HCT116 xenografts. In a liver metastatic colorectal cancer model, intratumoral treatment of HT29 primary tumors with MME monotherapy or with oncolytic adenovirus inhibited tumor growth. Combination therapy showed no increased mortality in comparison to monotherapies. In addition, our work demonstrated for the first time in a metastatic animal model that MME, as a monotherapy or in combination with an oncolytic virus, does not increase tumor invasiveness. Co-administration of MME and oncolytic adenovirus may be a suitable approach for further optimization of metastatic colorectal cancer treatment. To summarise, we described how RGD moieties inserted in the fiber protein are capable of improving tumor targeting of wild-type or capsid-modified adenovirus vectors. We also showed that MME is a safe coadjuvant to be used in combination with oncolytic adenoviruses for intratumoral administration and we presented a highly optimized mouse model for liver metastasis of colorectal cancer.
  • Weldatsadik, Rigbe Gebremichael (Helsingin yliopisto, 2021)
    Proteogenomics is an emerging field that combines genomic (transcriptomic) and proteomic data with the aim of improving gene models and identification of proteins. Technological advances in each domain increase the potential of the field in fostering further understanding of organisms. For instance, the current low cost and fast sequencing technologies have made it possible to sequence multiple representative samples of organisms thus improving the comprehensiveness of the organisms’ reference proteomes. At the same time, improvements in mass spectrometry techniques have led to an increase in the quality and quantity of proteomics data produced, which are utilized to update the annotation of coding sequences in genomes. Sequencing of pooled individual DNAs (Pool-seq) is one method for sequencing large numbers of samples cost effectively. It is a robust method that can accurately identify variations that exist between samples. Similar to other proteogenomics methods such as the sample specific databases derived from RNA-seq data, the variants from Pool-seq experiments can be utilized to create variant protein databases and improve the completeness of protein reference databases used in mass spectrometry (MS)-based proteomics analysis. In this thesis work, the efficiency of Pool-seq in identifying variants and estimating allele frequencies from strains of three β-hemolytic bacteria (GAS, GGS and GBS) is investigated. Moreover, in this work a novel Python package (‘PoolSeqProGen’) for creating variant protein databases from the Pool-seq experiments was developed. To our knowledge, this was the first work to use Pool-seq for sequencing large numbers of β-hemolytic bacteria and assess its efficiency on such genetically polymorphic bacteria. The ‘PoolSeqProGen’ tool is also the first and only tool available to create proteogenomic databases from Pool-seq data. For organisms such as the β-hemolytic bacteria GAS, GBS and GGS that have open pangenomes, the sequencing and annotation of multiple representative strains is paramount in advancing our understanding of these human pathogens and in developing mass spectrometry databases. Due to the increasing use of MS in diagnostics of infectious diseases, this in turn translates to better diagnosis and treatment of the diseases caused by the pathogens and alleviating their devastating burdens on the human population. In this thesis, it is demonstrated that Pool-seq can be used to cost effectively and accurately identify variations that exist among strains of these polymorphic bacteria. In addition, the utility of the tool developed to extend single genome based databases and thereby improve the completeness of the databases and peptide/protein identifications by using variants identified from Pool-seq experiments is illustrated.
  • Jokinen, Riikka (Helsingin yliopisto, 2016)
    Mitochondrial DNA (mtDNA) is a small extra-nuclear genome present in all nucleated cells of the body and important for mitochondrial function. The mtDNA is a present in hundreds to thousands of copies per cell and therefore arising mutations cause heteroplasmy: the co-existence of two or more distinct mtDNA variants in the same cell. Because of these features mtDNA variants segregate mitotically in the tissues of an individual, which can lead to time-dependent changes in the relative proportions of the mtDNA variants. Mutations in the mtDNA cause diseases and most pathogenic mtDNA mutations are heteroplasmic. In heteroplasmic situations a certain threshold proportion of the mutant mtDNA must be exceeded prior to onset of symptoms. Somatic mtDNA segregation of mtDNA mutations affect whether the threshold is exceeded, and can thus be a factor in determining disease onset and severity. Some pathogenic mtDNA mutations exhibit tissue-specific mtDNA segregation patterns, but the genes and mechanisms involved in this process are unknown. The aim of this thesis was to uncover genetic regulators of tissue-specific mtDNA segregation and study their properties to gain insight into the mechanisms involved in this process. We investigated tissue-specific mtDNA segregation in a mouse model that segregates two neutral mtDNA variants. These mtDNA variants display tissue-specific mtDNA segregation in three tissue types: the liver, kidney and hematopoietic tissues. In these tissues there is selection for one mtDNA variant over the other. Using this mouse model we identified and verified Gimap3 as a modifying gene for mtDNA segregation in the hematopoietic tissues. In a follow-up study we further studied Gimap3 and a functionally related gene Gimap5. We uncovered a novel subcellular localization to the endoplasmic reticulum for the Gimap3 protein. Moreover we established Gimap5, which encodes a lysosomal protein, as another genetic modifier of mtDNA segregation in hematopoietic tissues. Taken together these results demonstrated the involvement of other organelles in the segregation of mtDNA. To study tissue-specific mtDNA segregation from another aspect we investigated the role of mitochondrial fission in this process. Mitochondrial fission has been implicated to play a role in mtDNA segregation in yeast. We utilized a dominant-negative mouse model for Dnm1l, a master regulator of mitochondrial fission. We demonstrated that expression of the dominant-negative Dnm1l modulated the mtDNA segregation specifically in the hematopoietic tissues. In conclusion, we were able discover the first genetic modifiers for tissue-specific mtDNA segregation in mammals. These findings can be utilized to guide future research aiming to uncover the molecular mechanisms of this process, which can ultimately elucidate the genetics of pathogenic human mtDNA mutations.
  • Holm, Matilda (Helsingin yliopisto, 2021)
    Colorectal cancer (CRC) is the third most common cancer worldwide, accounting for around 10% of the global cancer burden, and is second only to lung cancer in terms of cancer death. For patients with CRC, the overall 5-year survival rate is around 65%, although it is highly dependent on stage at diagnosis. Biomarkers are molecules that can be used for the detection of a disease, follow-up after radical surgery, monitoring of progression, detecting or assessing the risk of recurrence, and predicting prognosis. Glycans are carbohydrates that play important physiological roles in processes such as cell signaling, growth, and motility. Changes in glycans are seen during cancer and glycans are directly involved in different aspects of cancer such as proliferation, invasion, and metastasis. The proteome, which consists of all the proteins expressed in a cell, tissue, or organism, is dynamic due to factors such as posttranslational modifications, regulation of gene expression, and differential splicing of mRNAs. Changes in the proteome are also seen during cancer. The plasma proteome is a good candidate for studies due to the multitude of proteins it contains, the ease of obtaining samples, and the way it reflects the condition of the host. Mass spectrometry is often used to study the glycome and proteome in order to discover candidates for new biomarkers and to obtain new knowledge of how the levels of glycans and proteins change during cancer. The aim of this study was the glycomic and proteomic profiling of CRC with a focus on studying differences in glycan and protein levels depending on factors such as tumor location, stage, and patient outcome. Glycans were isolated from CRC tissue samples and healthy colonic tissue and analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Separately, preoperative plasma samples were processed and analyzed using Ultra Performance Liquid Chromatography-Ultra Definition Mass Spectrometry. All samples were obtained from patients who underwent surgery at Helsinki University Hospital. The results were analyzed using methods including principal component analysis, Mann-Whitney U and ANOVA tests, hierarchical clustering, pathway analysis, the Kaplan-Meier method, log-rank tests, and Cox proportional hazards regression analysis. The main differences in glycan levels seen were between acidic glycans, several of which had significantly different levels between stage II and III CRC. Surprisingly, the only significant differences in glycan levels between samples from patients with cancer in the right and left colon were seen when samples were divided according to both tumor location and stage. Many significant differences were seen in the levels of glycans when compared between healthy colon and tumor tissue samples. Multiple plasma proteins were identified whose levels differed significantly between stage II and III CRC. The levels of 13 proteins were found to differ between stage II and III CRC regardless of primary tumor location. Significant differences in plasma protein levels were also observed depending on primary tumor location. Samples from patients with rectal cancer separated from samples from patients with colon cancer solely on the basis of plasma protein expression when analyzed using principal component analysis and hierarchical clustering. Several plasma proteins were identified of which altered levels were independently linked to significant differences in long-term outcome. Higher plasma levels of proteins such as ceruloplasmin and fetuin-B were linked to significantly improved long-term survival rates for stage II CRC patients, while higher plasma levels of signal-induced proliferation-associated 1-like protein 1 and the CNK3/IPCEF1 fusion protein were linked to significantly poorer long-term survival rates for stage III CRC patients. In conclusion, these glycomic and proteomic studies provide new knowledge of CRC at the molecular level and show that significant differences can be seen in the levels of specific glycans and plasma proteins depending on factors such as tumor stage, location, and patient outcome. These results lend further support to the notion that it may be important to consider colon and rectal cancer as separate entities. While multiple plasma proteins that could be of value for predicting cancer progression and, separately, patient outcome were identified, validation of their clinical utility is still needed. Further studies to investigate why the levels of certain glycans and plasma proteins differ depending on CRC stage, tumor location, or patient outcome are also warranted.
  • Jian, Ching (Helsingin yliopisto, 2022)
    It has been estimated that one in five adults is affected by metabolic syndrome globally, characterized by obesity concomitant with several metabolic derangements such as insulin resistance or non-alcoholic fatty liver disease. The current treatment options for metabolic syndrome-associated conditions are mainly based on lifestyle modification, such as dietary strategies. However, there is substantial interindividual heterogeneity in dietary response. A wealth of evidence derived from animal models suggests that the gut microbiota, i.e., the collection of intestinal microbes is causally involved in the diet-induced metabolic alternations. In contrast, the diet-microbiota-host interplay is still poorly understood in humans. In the present dissertation, the gut bacterial microbiota was characterized and compared by qPCR and Illumina sequencing of the 16S rRNA gene amplicons before and after two dietary intervention studies, overfeeding and calorie restriction, that induced metabolic alternations in opposite directions in overweight and obese adults. The associations between dietary response and the pre-intervention gut microbiota were explored. In addition, a qPCR-based experimental quantitative approach for microbiome profiling was developed. The results showed that overfeeding was associated with macronutrient-specific changes in individual bacterial taxa, but not the overall community. The baseline levels of genus Bilophila were positively associated to overfeeding-induced liver fat increases independently of the macronutrient composition. On the other hand, caloric restriction by a low-energy diet was associated with remodeling of the gut microbiota, with large decreases in the relative abundances of butyrate-producing bacteria and Bifidobacterium and an increased proportion of mucus-utilizing Akkermansia. These changes in the gut bacterial microbiota correlated with changes in host metabolic variables. Importantly, the pre-intervention gut bacterial microbiota was predictive of diet-induced metabolic worsening or improvements. Quantitative microbiota profiling was shown to be easy to perform and palliate the caveats to data interpretation when using relative abundance data. The present work identified alterations in the human gut bacterial microbiota associated with quantity- and quality-specific dietary changes that may have context-dependent effects on host metabolic health. The present findings also suggest that pre-intervention participant stratification based on the gut microbiota holds potential for improving intervention response.
  • Bao, Jie (Helsingin yliopisto, 2022)
    Non-small cell lung cancer (NSCLC) constitutes over 85% of lung cancer. Histologically, NSCLC can be broadly classified into adenocarcinoma (AC), squamous cell carcinoma (SCC), large cell carcinoma (LCC), and adenosquamous carcinoma (ASC). AC represents about 65% of all NSCLC cases, and it can be further subdivided based on tumor size and primary growth patterns, such as papillary, acinar, and mucinous. The formation of NSCLC histotypes is orchestrated by cells of origin, genetic alterations, and microenvironmental properties. Although NSCLC carries significant heterogeneity, some genetic mutations, functional phenotypes, and therapeutic responses are associated with specific NSCLC histotypes. Therefore, understanding histotype-selective etiology becomes essential for mechanistic studies and therapeutic applications in the NSCLC research field. Image-based tissue phenotyping has been commonly used for histological classification. It also allows the direct visualization of the distribution and expression of functional molecules. Quantifying such in situ phenotypes can be applied to hypothesis-based functional studies or data-driven correlative analyses. The first part of this thesis developed a spatial image analysis tool package. The making of Spa-RQ, an open-source tool package for image registration and quantification, reflected on the need to perform spatial phenotyping using serial tissue sections in a standardized laboratory workflow. Subsequently, we applied Spa-RQ to identify the histotype-selective, rather than genetically defined activation of MAPK, AKT, and mTOR signaling pathways in murine and human NSCLC samples. The diverse co-activation patterns between these pathways in different tissue compartments, measured by marker expression overlapping using Spa-RQ, may associate with heterogeneous responses towards combinatorial targeted therapies. The second part of this thesis work investigated the histotype-selective functions of a potential therapeutic target. The lung developmental transcription factor SOX9 is silenced in normal adult lung epithelia while it is re-expressed in NSCLC tissues. Its oncogenicity is widely acknowledged but has thus far not been confirmed in NSCLC subtypes. Analyzing the correlation between SOX9 expression and histotype-specific clinical staging, survival, and invasiveness revealed a clinical significance for increased SOX9 expression only in non-mucinous ACs, despite its broad expression in ASC, SCC, and mucinous AC. Supporting this, by comparing the histotype spectra in mouse models following Sox9 loss, we identified a critical role of SOX9 in promoting lung papillary AC progression. On the other hand, its expression was not required for developing squamous and mucinous structure tissues. Finally, using spatial phenotyping, we explained such opposing roles of SOX9 in NSCLC subtypes by the different cells of origin and microenvironmental properties: SOX9 expression was required to form advanced AC from the lung alveolar progenitor cells; on the contrary, its expression was dispensable for SCC development and even interfered with squamous metastasis. Therefore, this work exposed SOX9 as a potential drug target specific to a subgroup of lung AC. In summary, the identification of histotype-selective functional oncogenic phenotypes, as achieved in this thesis, contributes to understanding the heterogeneous nature of tumorigenesis, cancer progression, and drug sensitivities.
  • Rantanen, Ville (Helsingin yliopisto, 2015)
    Images provide invaluable information to Biomedicine. Especially, microscopy as an information source has been providing knowledge for research and clinical diagnostics. We have moved away from simply looking at the images to quantifiable computerized image analysis. Over the last decades, image analysis developers have prepared algorithms and software to address various scientific enquiries using images. These software are often created for a single purpose. Naturally, not even the most generic software can include all the algorithms ever created. From an image analysis developer point of view, the choice of software creates limitations. It limits the developer to the algorithms included and to the language it was developed in. Even if the software is modular and extendable, a specific language is required and the earlier algorithm implementations would have to be ported. This thesis presents an integration platform for image analysis: Anima. It is capable of using existing software and including them in analysis workflows. Since image analysis is very case specific, custom processing commands are frequently needed. Anima comes with a large number of data and image analysis components developed directly for the platform, as well as components that send custom commands to the integrated software. All of the components can be executed in a single analysis pipeline. Anima itself is built on top of Anduril, another software, inheriting its software architecture. Anduril gives Anima the power of parallel processing and rerun prevention mechanism, speeding up the development cycle of new algorithms. The usability of Anima for method development is shown by implementing new segmentation algorithms and visualization tools. The tools and methods are all suited to large data sets. To display the modularity, the tools are published as separate programs that are then integrated in Anima. The usefulness of the platform is shown by applying it in different biomedical research settings. The settings include different organisms: human, rat and nematode; different sample material: brain tissue, lymphatic nodes and serum; and different medical interests: cerebral ischemia, cancer and allergy. Anima is a versatile open-source image analysis platform, that encourages the use of best practices of programming habits. It makes the development of analysis workflows and individual algorithms more efficient.
  • Wasik, Anita Agnieszka (Helsingin yliopisto, 2014)
    Diabetic nephropathy (DN) is a major microvascular complication of diabetes and a common cause of end-stage renal disease. Mechanisms leading to the development of DN are not fully understood, but podocyte injury is involved. Interestingly, in respect to glucose uptake podocytes are uniquely insulin sensitive cells. Insulin rapidly induces remodeling of the actin cytoskeleton and leads to glucose uptake via glucose transporters GLUT1 and GLUT4. Defects in the trafficking of the glucose transporters may affect the insulin sensitivity of podocytes. Thus, regulators of glucose transporter trafficking may provide suitable targets to enhance insulin sensitivity of podocytes and prevent the development and progression of DN. However, the precise mechanisms regulating glucose transporter trafficking and glucose uptake into podocytes are largely uncharacterized. To identify changes in the expression of glomerular proteins at an early stage of DN we performed quantitative proteomic profiling of glomeruli isolated from rats with streptozotocin-induced diabetes and controls. Ezrin was found to be downregulated in diabetic glomeruli. In cultured podocytes depletion of ezrin increased glucose uptake by increasing translocation of GLUT1 to the plasma membrane. Loss of ezrin also induced actin remodelling, which involved cofilin-1. Phosphorylated cofilin-1 was upregulated in diabetic glomeruli suggesting altered actin dynamics. Furthermore, reduced expression of ezrin was found in the podocytes of human patients with diabetes. We found that the filament-forming septin 7 forms a complex with CD2AP and nephrin, both of which are essential for glomerular ultrafiltration. We showed that septin 7 negatively regulates GLUT4 storage vesicle (GSV) trafficking by forming a physical barrier between the vesicles and the plasma membrane. The novel interaction partner of septin 7, nonmuscle myosin heavy chain IIA (NMHC-IIA), was found to positively regulate insulin-stimulated glucose uptake into podocytes. Loss of NMHC-IIA reduced formation of the SNARE complex involved in GSV exocytosis. Furthermore, we presented that insulin regulates the association of septin 7 and phosphorylated RLC (pp-RLC), a part of myosin hexameric complex, with a plasma membrane SNARE, SNAP23. pp-RLC is upregulated in diabetic glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with Type 1 diabetes. Our findings indicate that ezrin, septin 7 and NMHC-IIA regulate glucose uptake into podocytes and may play a role in the development of the renal complication in diabetes by regulating glucose transport and organization of the actin cytoskeleton in podocytes.
  • Chen, Ping (Helsingin yliopisto, 2016)
    Transcriptome, defined by the collection of all RNA molecules in a cell, acts as a central bridge that transfers genetic information into molecular functions. Transcriptome regulates the biological characteristics in all living organisms, thus it is one of the most important research subjects in biology. RNAs are transcribed at different levels tightly controlled by cellular conditions. This produces great diversity in cellular transcriptome dynamics, introducing a lot of complexity to the transcriptomic research. Though tremendous challenges exist, the study of transcriptome dynamics is essential to the understanding of the complex systems within the cells and cellular behavior. The dynamics of transcriptome can be investigated by high-throughput technologies, such as microarrays and RNA-sequencing. The large amounts of data introduces challenges to data management, analysis and interpretation. To generate biologically testable and conclusive results, efficient computational methods are urgently needed. This thesis includes theoretical and methodological research. The theoretical part of the research comprehensively studies the characteristics of gene expression, the splicing of ancient and novel exons during the evolution by comparative analysis on transcriptomic data of nine tissues from five species. The methodological research includes new methods developed to solve the research questions related to the study of transcriptomic dynamics in evolution and cancer. The main methods developed in this thesis are 1) exon age classifier, which is able to classify exons according to their evolutionary time, providing the basis for the theoretical study in this thesis; 2) MEAP, a new exon array preprocessing method for expression quantification at multi-levels; 3) PSFinder, a new approach to identify patient prognostic subgroups from treatment naive tumor samples based on their transcriptomic profiles and associated clinical survival times. The theoretical part gives a comprehensive view on the mechanisms of dynamic changes during the evolution of the transcriptome, which provides a solid theoretical basis to the methodological part. The application of MEAP and PSFinder to high-grade serous ovarian cancer revealed a small set of isoform markers with distinct expression profiles for patient prognosis stratification. In combination with experimental validation, the results demonstrate the applicability of these methods in the quantification and stratification of tumor transcriptome dynamics, which provides new insights to the clinical diagnosis and precision medicine for human cancers.
  • Zheng, Wei (2013)
    Blood vessels deliver nutrients and oxygen to tissues, whereas lymphatic vessels collect interstitial fluid and absorb lipids. Both types of vessels are involved in immune surveillance. Malformation or malfunction of either vasculature is closely associated with various diseases. Although the molecular mechanisms regulating the growth, development and function of these vessels have begun to emerge in recent decades, many questions remain. This study aimed to understand if and how the growth of new blood and lymphatic vessels (angiogenesis and lymphangiogenesis, respectively) is regulated by the endothelial signaling molecules BMX, VEGFR-3, notch 1 and angiopoietin-2. BMX, a non-receptor tyrosine kinase, is upregulated in some types of cancer and promotes cell survival, migration and proliferation. Under physiological conditions, it is also highly expressed in blood endothelial cells (BECs). Thus, we hypothesized that BMX might contribute to tumor growth by promoting tumor angiogenesis. Using multiple tumor implantation and spontaneous tumor models in Bmx gene-deleted mice, we found that tumor angiogenesis and growth were significantly reduced in the absence of BMX. Conversely, when BMX was overexpressed in epithelial keratinocytes, chemical carcinogen led to increased tumor angiogenesis and growth. VEGFR-3 is a growth factor receptor on the surface of endothelial cells (ECs). Here, we characterized a blocking antibody that inhibits VEGFR-3 signaling by a novel mechanism. In contrast to conventional blocking antibodies that prevent ligand-receptor binding, this new antibody inhibited dimerization of two identical VEGFR-3 molecules, thereby impairing signaling activation and sprouting of lymphatic endothelial cells (LECs). These inhibitory effects persisted even at ligand concentrations so high that conventional blocking antibodies were no longer effective. Importantly, concurrent treatment with both types of antibodies yielded combined additive benefits. Notch signaling regulates angiogenic sprouting. We found in this study that Notch inhibitors enhance VEGF-induced lymphatic sprouting in 3-dimensional EC sprouting assays in vitro. In vivo, VEGF alone induced only lymphatic dilation but not sprouting. However, concomitant treatment with a Notch inhibitor induced both vascular events. In addition, a mosaic-sprouting assay showed that Notch signaling also determined the fates of tip/stalk cells (the cell leading a vessel sprout and the following cells, respectively) during lymphatic sprouting. Angiopoietin-2 (Ang2) is an endothelial growth factor required for proper lymphatic remodeling during development, but the exact mechanisms of this process have previously remained unclear. In contrast to the zipper-like pattern of cell-cell junctions (zippers) in collecting lymphatic vessels, mature lymphatic capillaries have a distinct pattern of button-like junctions (buttons), which transform from zippers during development. We found in this study that Ang2 is indispensable for such transformation of the junctional patterns during embryogenesis. At the molecular level, Ang2 is required for phosphorylation of the adherens junction marker vascular endothelial cadherin (VE-cadherin) at the tyrosine residue 685. Ang2 blocking antibody treatment of mouse neonates disrupted cell-cell junctions in the mesenteric lymphatic vessels, causing leakage of chyle (milky fluids containing fat droplets and lymph). Ang2 blockade also inhibited lymphatic valve formation and maturation and resulted in abnormal smooth muscle recruitment. Together, these results provide new insights into the molecular mechanisms of angiogenesis and lymphangiogenesis. Elucidation of these mechanisms is important in developing new drugs for the treatment of vessel-related diseases, including cancer and pathological inflammation. Moreover, these findings introduce the reorganization of cell-cell junctions as a novel parameter for evaluating lymphatic development and call for future research into this new field.
  • Ferro, Claudio; Florindo, Helena; Santos, Hélder A. (2021)
    Selenium (Se) is an essential element to human health that can be obtained in nature through several sources. In the human body, it is incorporated into selenocysteine, an amino acid used to synthesize several selenoproteins, which have an active center usually dependent on the presence of Se. Although Se shows several beneficial properties in human health, it has also a narrow therapeutic window, and therefore the excessive intake of inorganic and organic Se-based compounds often leads to toxicity. Nanoparticles based on Se (SeNPs) are less toxic than inorganic and organic Se. They are both biocompatible and capable of effectively delivering combinations of payloads to specific cells following their functionalization with active targeting ligands. Herein, the main origin of Se intake, its role on the human body, and its primary biomedical applications are revised. Particular focus will be given to the main therapeutic targets that are explored for SeNPs in cancer therapies, discussing the different functionalization methodologies used to improve SeNPs stability, while enabling the extensive delivery of drug-loaded SeNP to tumor sites, thus avoiding off-target effects.
  • Ruottu, Maria Eveliina (2007)
    This thesis is a descriptive study about the plural healthcare system on Siberut Island, Indonesia. The study explores the theoretical concepts of medical systems and medical pluralism in relation to the case study of Siberut Island. For the situation encountered on the island, Arthur Kleinman’s model of health care systems and Tapio Nisula’s term medical culture are appropriate. Because the study concentrates on describing the traditional shamanic medical system of Siberut on one hand, and the relatively recent biomedical healthcare on the other, the theoretical chapter includes discussion on ethnomedicine and the medical anthropological study of biomedicine. In this study, biomedicine is viewed as one ethnomedicine among others. Its basis in the Cartesian dualism and how this shows in biomedical theory and practice is discussed. This thesis is written in the spirit of critical medical anthropology (CMA), paying attention to macro-level processes such as national politics and policies, economy, history, questions of identity etc. The thesis is based on data collected during a seven month field work period in 2003, out of which approximately three months were spent in the small village of Ugai on Siberut. Siberut belongs to the Mentawai Archipelago, on the western side of Sumatra. Siberut is the biggest island of the archipelago, and has until recently remained fairly untouched by outside influences. In the last two decades, it has become increasingly known internationally due to its unique nature and indigenous culture. The field data was collected through participant observation, discussions with villagers and people working in healthcare, and some interviews. In addition, available literature on Siberut and on healthcare seeking and medical pluralism has been used. After the theoretical considerations, the thesis first describes Siberut and its history, then the traditional cosmology and healing practices related to it. The history and functioning of biomedical healthcare on the island is discussed next. The last two chapters discuss the plural health care system as a whole, describing what factors affect healthcare seeking. Seven case histories are presented to give the reader an understanding of the lived reality of Siberut’s plural healthcare system. Using Kleinman’s model, three sectors can be identified in the Siberutan health care system: 1) the folk sector (of self-medication with herbal remedies and over the counter biomedical products), 2) the traditional sector (of shamans, herbalists and traditional midwives), and 3) the professional sector (of biomedical doctors and nurses). The sectors are overlapping and complementary. People on Siberut Island use the plural medical system flexibly according to their needs and depending on their situation. They do not view the different kinds of healthcare as separate bounded systems, and see no contradiction in using e.g. shamanic healing and biomedical healthcare simultaneously or consequtively. Especially the providers of biomedical healthcare formulate stricter boundaries between the systems. Still collaboration between the different kinds of healthcare providers has increased and further cooperation is hoped for the future.
  • Kuryk, Lukasz (Helsingin yliopisto, 2016)
    Despite major advances in conventional cancer treatments by surgery, chemotherapy, radiotherapy and their combination, the outcome remains partially ineffective against numerous cancer types, for example mesothelioma, lung cancer, and colon cancer. Furthermore, due to resistance factors and the subsequent loss of response, which may occur rapidly during the conventional treatments regimes, new anti-cancer agents, presenting new mechanisms of action and lacking cross-resistance to commonly used therapies, are in high demand. Oncolytic virotherapy is a promising anti-cancer strategy, and the approval of the first oncolytic virus, Imlygic (T-Vec, talimogene laherparepvec), in Western world by US Food and Drug Administration (FDA) and European Medicines Agency (EMA) has opened up new perspectives for improved treatment of cancer. Single therapy is rarely successful in treating cancer, particularly in metastatic or advanced cancer, and survival rates with monotherapies alone are generally poor. The combination of multiple therapies to treat cancer has already shown significant results in the standard care of cancer. This strategy utilizes the combination of both conventional and novel therapies that can bring the future promise of cancer treatment. In this thesis it has been hypothesized that by combining oncolytic adenoviruses (oAd) with chemotherapeutic drugs and a biological agent we could improve anti-cancer efficacy through synergistic effect against cancer. Therefore, we have tested various treatment regimes with the overall goal being the improvement of oncolytic virotherapy efficacy. Secondly, since safety issues concerning gene therapy and viral vectors are tremendously important, we have performed studies on safety issues of adenoviral vectors. In brief, we have evaluated the anti-cancer activity of combination treatment with standard of care (SoC) chemotherapy (Pemetrexed, Cisplatin, Carboplatin) and Ad5/3-d24-GM-CSF (ONCOS-102) in vitro and in a xenograft BALB/c model of human malignant mesothelioma (MM). We could show improved anti-tumor effects when ONCOS-102 was combined with SoC chemotherapy regimens over chemotherapy and virus alone. Combination therapy resulted in synergistic anti-cancer effect improving the therapeutic outcome. In a subsequent study we tested anti-cancer properties of the dipeptide L-Carnosine complexed with an oncolytic adenovirus (virus-L-Carnosine complex). The complex demonstrated improved anti-tumor efficacy both in vitro and in vivo in tested cancer models. In HCT116 colon and A549 lung cancer cells, the virus-L-Carnosine complex presented a higher transduction level and infectious titer over uncoated oncolytic adenovirus. The in vivo efficacy of the virus-L-Carnosine complex was tested in two cancer models: i) lung and ii) colon cancer xenograft mice models. It exhibited a significant reduction in tumor growth compared to other tested groups. Additionally, we investigated the molecular mechanism underlying the effects of the complex on tumor growth reduction. Safety assessment of viral vectors was performed in animal studies. Extensive studies on toxicity and bio-distribution of ONCOS-102 in Syrian hamsters and experiments in BALB/c nude mice indicated no side effects of repeated administration of oncolytic adenovirus. The side effects were evaluated by assessment of body weight, food consumption, hematology, clinical chemistry, histopathology and bio-distribution. We concluded that combinatory studies utilizing oncolytic viruses with standard of care chemotherapy and an experimental virus-L-Carnosine complex showed synergistic anti-cancer efficacy, thus providing a strong rationale for clinical testing of such combinations in mesothelioma, lung and colon cancer. Additionally, our studies suggested that adenovirus could be used in future studies for delivery of other bioactive drugs as a novel strategy in cancer therapy.
  • Scifo, Enzo (Helsingin yliopisto, 2014)
    Neuronal ceroid lipofuscinoses (NCL) are common inherited childhood brain disorders. Since 1995, 13 known NCL causative genes (CLN1-8, CLN10-14) have been identified. Despite progress in the NCL field, the primary function and physiological roles of most NCL proteins remain unresolved. In this thesis, we utilized various techniques, such as: functional proteomics, bioinformatics, and mouse disease models, in an effort to clarify disease pathways associated with congenital (CLN10), infantile, late-Infantile (CLN1, CLN5) and juvenile NCL (CLN3) in the human brain. Firstly, we examined the synaptic proteome in a cathepsin D (Ctsd / Cln10) knockout mouse model of congenital NCL, where the synaptic pathology resembles that of patients. Quantitative mass spectrometry analysis of mouse brain synaptosomes, showed that 43 proteins were differentially expressed in the Ctsd knockout mice brains. We used protein-protein interaction databases to generate a network of differentially expressed proteins and checked individual proteins for involvement in processes, pathways or disease phenotypes. This work highlighted defects in migratory functions of cathepsin D deficient cells that were attributed to downregulation of cytoskeletal proteins. We also aimed to map the CLN3-CLN5 protein interactome in the brain by identifying their associated proteins. Protein complexes from CLN3 or CLN5 expressing SH-SY5Y stable cells were analysed by mass spectrometry and processed by bioinformatics, to unravel molecular mechanisms underlying CLN3 and CLN5 diseases. Novel CLN3 and CLN5 interacting partners (42 and 31, respectively) were identified in this study. The extent of crosstalk amongst CLN3 and CLN5, suggests that the mechanisms leading to the functional deficits are shared between them. CLN3 was implicated in new roles of G-protein signalling and protein folding / sorting in the ER. Finally, we analysed protein complexes from human PPT1 (CLN1) expressing SH-SY5Y stable cells as described above. The goal of this study was to identify in vivo PPT1 substrates that could provide insight on the onset and progression of CLN1 disease. Our findings suggest putative new roles of PPT1 in neuronal migration and dopamine receptor mediated signalling pathway.
  • Belitškin, Denis (Helsingin yliopisto, 2022)
    Proteolytic enzymes play a significant role in normal physiology and pathophysiology by regulating protein stability, processing, and activation of growth factors. The central role of proteolysis and the potential for pharmacological interventions make the enzyme class an attractive target for therapy in cancer. However, the regulatory mechanisms acting downstream of many proteolytic enzymes are poorly understood, which caused significant failures in clinical trials targeting matrix metalloproteinases in the past. This thesis describes the up- and downstream pathways of the Type II Transmembrane Serine Protease Hepsin. Hepsin is overexpressed in most prostate, ovarian, and breast cancers. Studies in mouse models suggest a role in promoting metastatic spread and tumor growth. However, the signaling pathways around hepsin remain largely unknown. We determined the upstream oncogenic signaling that upregulates hepsin protein in breast cancer. The hepsin overexpression in breast cancer is regulated by posttranslational mechanisms and involves the stabilization of hepsin by Heat Shock Proteins following RAS transformation. Hepsin upregulation causes loss of epithelial junctions and the basement membrane. To study the hepsin regulated pathways, we generated an inducible hepsin overexpression model, which allows upregulation of hepsin in cell lines and in vivo models of breast cancer. Ectopic hepsin overexpression experiments demonstrated that hepsin overactivation can explain the disruptive effects of RAS on epithelial integrity. Hepsin overexpression also increases pro-HGF processing. To study the pathways downstream of hepsin, we generated a hepsin knockout mouse strain via gene editing. The knockout mouse model revealed a novel mammary branching phenotype and a connection between hepsin and the TGFβ pathway in mammary gland development and cancer. Hepsin activates the TGFβ pathway by releasing the latent TGFβ from the extracellular matrix via the degradation of fibronectin. By using in vitro and mouse models, we found that fibronectin is a direct substrate of hepsin. The hepsin driven TGFβ signaling induces cell proliferation in 3D culture models, which can be suppressed with existing hepsin antibody inhibitors. Genetic inhibition of hepsin reduces primary and metastatic mouse tumor growth, providing proof of principle that targeting hepsin can be a strategy for reducing the growth of breast tumors.
  • Cervera Taboada, Alejandra (Helsingin yliopisto, 2020)
    Cancer is a collection of diseases that combined are one of the leading causes of deaths worldwide. Although great strides have been made in finding cures for certain cancers, the heterogeneity caused by both the tissue in which cancer originates and the mutations acquired in the cell’s DNA results in unsuccessful treatments for some patients. The genetic alterations caused by carcinogenics or by random mutations acquired during normal cell division promotes changes in the cell’s metabolism. These changes are usually reflected in abnormal gene expression that can be studied to understand the underlying mechanisms giving rise to cancer as well as suggest treatments that can exploit each tumor’s specific vulnerabilities. RNA-Seq is a technology that allows the identification and quantification of the genes that are being expressed inside the cell in a given moment. RNA-Seq has several characteristics and advantages that allow a diversity of applications to exist. For example, apart from quantifying gene expression, it can be used to detect different variants of the same gene, has base pair resolution which is informative of the gene sequence, and can also be used to quantify other RNA molecules besides messenger RNA (mRNA), such as microRNAs. The two main aims of this work are to provide computational methods for data analysis of RNA-Seq and to show specific applications of RNA-Seq that can shed light into cancer mechanisms. In Publications I and IV we developed the Sequence Processesing Integration and Analysis (SePIA) and the Fusion Gene Integration (FUNGI) toolsets that facilitate the creation of reproducible pipelines for investigating different aspects of the cancer transcriptome. SePIA’s utility is showcased with the analysis of datasets from two public data repositories. One of the analysis shows a standard RNA-Seq analysis, while the second one produced a pipeline for mRNA-microRNA integration. The second toolset, FUNGI, is aimed specifically at finding reliable gene fusions with oncogenic potential. To demonstrate FUNGI’s features, we analyzed 107 in-house samples and processed over 400 public samples from a public data repository. FUNGI allowed us to detect fusions in ovarian cancer with a higher prevalence than previously recognized. Additionally, we identified a fusion gene that has not been reported before in ovarian cancer, but that can be targeted with a drug currently in clinical trials. In Publication II we investigated the role of alternative splicing in diffuse large B-cell lymphoma and were able to show that isoform-level instead of gene-level is better at discriminating between subtypes. Additionally, specific isoforms, such as APH1A, KCNH6, and ABCB1, were correlated with survival. In Publication III, we used RNA-Seq to complement the phasing of genetic variants with somatic mutations in tumor suppressor genes. In this study we found enrichment of haplotype combinations that suggest that haploinsufficiency of tumor suppressor genes is enriched in cancer patients. SePIA and FUNGI are tools that can be used by the community to explore their datasets and contribute to the acquisition of knowledge in the field of cancer genetics with next generation sequencing. The applications of RNA-Seq studies included in this dissertation showed that RNA-Seq can be effectively used to aid in the classification of cancer subtypes, and that RNA-Seq can be used in combination with DNA sequencing to explore gene expression mediated by genetic variation in cancer.
  • Roos, Annikki (Hanken School of Economics, 2016)
    Economics and Society – 298
    Information practices are human activities that are related to seeking, managing, giving, using, and producing information in context. This thesis concentrates on the information practices of the researchers in the scientific domain of biomedicine. The object of this study has been to understand the special nature of the information related work and practices as a part of the biomedical research work. It is argued that to be able to build efficient tools and advantageous information services for researchers in the biomedical domain, these efforts should be based on the understanding of knowledge creation processes and work practices in this domain. The domain analytical approach forms an alternative view to those models, which try to identify similarities in patterns of seeking and use of information across the research domains. In this study, this approach has been used as an alternative to the generalizing model. The findings of the thesis support the arguments, which oppose the general view of information needs and uses. In information science, the study of information practices is quite a new research orientation. There are no previous studies, where the domain of biomedicine would have been in focus. Another important contribution of this study is the use of the activity theory as a theoretical research frame in the study of information practices. The activity theory appeared to be very helpful in setting information practices in the context. When implementing the activity theoretical research framework, information practices are comprehended as one mediating tool in the activity system of the research work. It aids the researcher to achieve the objectives of the research work.
  • Kavalakatt, Sina (Helsingin yliopisto, 2022)
    Diabesity is a major health burden worldwide, particularly in Kuwait, where several epidemiologic studies reveal the parallel escalation of the prevalence of obesity and diabetes. There is still a lack of complete understanding of the pathways and their interactions triggering the development of obesity-related co-morbidities. Urocortin 3 (UCN3) is a novel neuropeptide implicated in the regulation of food intake, energy homeostasis, cardioprotection, and identified as a regulator of insulin secretion and as a marker for functional pancreatic β-cells. Hence, UCN3 could be a potential therapeutic target for managing metabolic diseases. In this thesis we have assessed UCN3 expression in two cross-sectional populations living in Kuwait: adults with obesity and type 2 diabetes (T2D) and children with overweight and obesity. In addition, we have examined associations of UCN3 with metabolic markers and stressors and assessed the effect of UCN3 overexpression on signaling pathways of insulin, glucose uptake, endoplasmic reticulum (ER) stress, and heat shock response in an adipocyte cell model. In study (I), we investigated the circulating UCN3 levels in the plasma of adults with obesity and T2D. The effect of inflammatory microenvironment in adipose tissue on the expression of UCN3 and the effect of physical exercise training on UCN3 expression was also investigated. We demonstrated that UCN3 expression levels were impaired in response to an inflammatory microenvironment, obesity, and T2D. In study (II), the effect of UCN3 overexpression on apoptotic, ER, and heat-shock stress response pathways was studied in 3T3L1 adipocytes. We showed that the overexpression of UCN3 attenuated markers of apoptosis, inflammation, ER stress, and heat shock response. These events were associated with improved glucose uptake and insulin signaling. We showed that the levels of UCN3 and the other corticotropin-releasing factor (CRF) family are impaired with obesity both in plasma and peripheral blood mononuclear cells obtained from children. In studies (III) and (IV), the circulating levels of UCN3 were assessed in children with different levels of adiposity. Metabolic stressors such as palmitic acid and high glucose concentrations differentially modulated the neuropeptide levels in human monocytic cell line (THP1) cells depending on the duration of the exposure. The homeostasis of energy balance and metabolism is regulated by the central nervous system and peripheral mechanisms. The progression of obesity and T2D is associated with disturbances in this homeostasis, dysregulation of insulin secretion, and alterations in inflammatory and stress response pathways. Most of the thesis findings were shown for the first time and highlighted the role of UCN3 in metabolic dysregulations in obesity and T2D. UCN3 might be a promising marker in future approaches to monitor the progression of obesity, T2D, and related metabolic co-morbidities.
  • Zarkada, Georgia (2014)
    Blood and lymphatic vessels form extensive networks throughout the body, which function in order to deliver oxygen and nutrients to the tissues, to remove extravasated fluid and to absorb dietary lipids. The formation of new blood and lymphatic vessels (termed angiogenesis and lymphangiogenesis) is critical during embryonic development and in the adult, and is regulated by multiple signaling pathways. Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGFRs), as well as the Notch signaling system, are key governors of blood and lymphatic endothelial cell fate, and regulate angiogenesis and lymphangiogenesis in health and disease. Despite the numerous recent advances in the field of vascular biology, many steps in the complex processes of angiogenesis and lymphangiogenesis remain unclear. In this study we investigated the role of VEGFR3 signaling in blood endothelial cells, tip cell specification, as well as the interplay of the receptor with the VEGFR2 and the Notch signaling pathways during angiogenesis. VEGFR3 is a tyrosine kinase receptor that is mainly expressed in lymphatic endothelial cells in the adult. We observed VEGFR3 expression in sprouts that guide the blood vascular endothelium in angiogenic conditions. VEGFR3 blockade with a monoclonal antibody displayed synergistic properties with simultaneous VEGFR2 targeting in reducing angiogenesis and inhibiting tumor growth. Furthermore we found that Notch signaling suppresses VEGFR3 expression in endothelial cells, identifying VEGFR3 as a novel tip cell marker, which is normally repressed by Notch activation. In the next step we employed a combination of genetic and in vitro models to show that loss of VEGFR3 results in a hypervascular phenotype, accompanied by loss of Notch signaling. VEGFR3 could be stimulated by VEGFC and activate Notch in blood endothelial cells. Our results point towards a mechanism where VEGFC produced by macrophages at the vascular front acts via VEGFR3 to activate Notch and turn tip into stalk cells; thus promoting the formation of stable vascular loops. Furthermore we identified the transcription factor FOXC2 as the downstream target of the VEGFC/VEGFR3/Notch signaling cascade. These data reinforce the idea that VEGFR3 has two distinct signaling modalities, one ligand-dependent and one ligand-independent, and that different perturbations in VEGFR3 expression and function result in diverse vascular phenotypes. Subsequently we investigated the interplay of VEGFR2 and VEGFR3 in postnatal angiogenesis and lymphangiogenesis, using a genetic approach of conditional mutagenesis. Various combinations of genetic ablation of VEGFRs and pharmacological inhibition of Notch showed that VEGFR2 is irreplaceable during sprouting angiogenesis, also in endothelial cells with low Notch signaling, and that it acts upstream of VEGFR3 expression in angiogenic settings. On the other hand VEGFR3 suppressed VEGFR2 expression in a negative feedback loop. Finally we employed for the first time lymphatic endothelial specific deletion of VEGFRs in postnatal conditions and found no significant role for VEGFR2 in lymphatic vessel growth and remodeling, while VEGFR3 signaling was indispensable. Our results unravel previously unknown roles for VEGFR3 in sprouting angiogenesis and provide new insight into the signaling cross-talk of the receptor with other important regulators of blood vessel development. Increasing our understanding of the molecular mechanisms underlying this process is crucial in order to comprehend the pathophysiology of angiogenesis-related diseases, characterized by excessive or insufficient blood vessel growth, and promote the emergence of novel angiogenic therapies.