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Browsing by study line "Cell- och utvecklingsbiologi"

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  • Butkovic, Rebeka (2020)
    Autophagy is a cellular recycling and quality control process that eliminates cellular material in a non-selective or selective fashion. Macroautophagy is non-selective, and degrades macromolecules or damaged organelles to sustain cellular homeostasis. The selective autophagy of dysfunctional or excess mitochondria is known as mitophagy. The clinical importance of functional degradation is exemplified by the lysosomal storage disorders (LSDs), where lysosomal hydrolytic enzymes are absent or dysfunctional. Previous investigations of a rare infantile LSD indicated a change in autophagy and decreased mitochondrial content. The aim of this MSc thesis was to quantitatively compare macroautophagy and mitophagy in a cellular model of this rare LSD, by generating fluorescent macroautophagy and mitophagy reporter-expressing cell lines from patient material. Fibroblasts derived from patients diagnosed with a rare infantile LSD were transduced with lentiviruses carrying either mCherry-GFP-LC3 or mito-QC reporters, for the microscopic analysis of autophagy and mitophagy, respectively. I also monitored autophagic flux by traditional biochemistry in untreated and starved cells, in the presence or absence of lysosomal inhibitors (bafilomycin A1). Basal and iron-depletion induced mitophagy was profiled using confocal microscopy, quantitative cell biology and biochemistry. My findings suggest differential autophagic turnover in LSD patient-derived fibroblasts, with a marked accumulation of non-acidified autophagic structures. Basal mitophagy was elevated in two out of three LSD patient cell lines compared to unaffected controls. LSD patient cells exhibited altered mitochondrial content and network architecture compared to controls. These phenotypes were accompanied by distinct changes in the endo-lysosomal system and increased cell size. The patient-derived cells exhibit a profound accumulation of lysosomes and autophagic structures. My findings are in accordance with previous research in the field, suggesting perturbed macroautophagy in this rare LSD. The observations of altered mitochondrial homeostasis in this LSD provide a basis for future investigation. The reporter-expressing cells, generated as part of this MSc thesis project, will enable future studies of mechanisms that underlie phenotypic changes, and will complement essential in vivo work in this area.
  • Lamichane, Nicole (2019)
    Over the past years sugar consumption has seen great increases worldwide, together with a rise in the prevalence of metabolic diseases. There is a growing need for a comprehensive characterisation of the genes involved in sugar metabolism, yet the mechanisms by which cells sense and respond to sugars in vivo have remained incompletely understood. Here, I analyse members of a protein family best known for their regulation of differentiation during development with regards to their role in sugar metabolism. The Hairy and Enhancer of Split (HES) protein family are a group of basic helix-loop-helix (bHLH) transcription factors that function as major downstream effectors of the Notch signalling pathway. In mammals, the HES proteins have mostly been studied for their role in cell differentiation, but HES1 has been implicated in metabolic control. Drosophila has several transcription factors belonging to the HES family, including Hairy and seven bHLH transcription factors located in the Enhancer of split complex (E(spl)-C). The E(spl)-C bHLH transcription factors display high homology and are considered to be genetically redundant, and therefore little is known about their individual functions. The other HES family members in Drosophila have not previously been linked to metabolic regulation, but Hairy has been shown to repress the tricarboxylic acid cycle. In light of the findings implicating HES1 and Hairy in the regulation of metabolism, I systematically investigated the role of the HES transcription factors in sugar metabolism. By using the GAL4/UAS system in Drosophila melanogaster, I knocked down gene expression of each of the family members, and raised the flies on diets varying in sugar content to identify possible sugar intolerance phenotypes. Here, I show that knockdown of one of the E(spl)-C bHLH genes led to severe sugar intolerance that affected both survival and organismal growth, but did not alter the levels of circulating carbohydrates and storage lipids as measured with colorimetric assays and lipid staining. Furthermore, I identify the tissues in which this transcription factor functions to provide sugar tolerance. Using analysis of publically available chromatin-immunoprecipitation sequencing data coupled with quantitative RT-PCR, I uncover mTOR target Thor/4E-BP as a putative target gene. Additionally, I show that Hairy is similarly required for complete sugar tolerance, but that the mechanism differs from the E(spl)-C bHLH transcription factor. Hairy binds to and positively regulates expression of genes involved in glycolysis and the pentose phosphate pathway, suggestive of a cooperation with earlier known regulators of sugar sensing. In conclusion, I have shown that only two HES family members are involved in the regulation of sugar metabolism and that their regulatory mechanisms are distinct, implying that the HES family members have more diverse roles than previously assumed.
  • Avdonin, Savva (2021)
    Tiivistelmä – Referat – Abstract ROS or Reactive Oxygen Species can be found throughout all living organisms on the planet. Without ROS, processes, which are essential for the sustainment of most living organisms, such as respiration would not be possible. On the other hand, uncontrolled ROS generation can cause severe damage to the cellular structure. The family of ROS includes multiple compounds, which share a common trait of high chemical activity. ROS can be produced on demand by specific enzymes which are localized within cellular structures, such as membranes. One group of enzymes is called NADPH (Nicotinamide adenine dinucleotide phosphate) oxidases. These enzymes possess common structure which is composed of transmembrane region with multiple loop helixes and usually two or more terminal motifs, which are devised into regulatory EF-hand motifs and catalytic motifs. NADPH oxidases are essential ROS producers and can be found throughout most clades of living organism and are widely represented in different cellular compartments and distributed across different tissues in multicellular organisms. As an example, Nox family of NADPH oxidases can be found in human tissues and immune cells. Another common group of NADPH oxidases is respiratory burst oxidase homologues (RBOH) can be found in plants. Members of this group play important role in plant immune defense against pathogens. One example is AtRBOHD, which is expressed in Arabidopsis genus of plants. Upon activation, these enzymes are known to produce hydrogen peroxide (H2O2) as mean of antibacterial defense. These host defense mechanisms are known to be driven by different signaling molecules. It has been determined that in some examples of NADPH Oxidases, including Nox5 and RBOHD, the state of activation can be induced through the effects of Ca2+ ions. Moreover, it has been determined, that ROS-producing state of these NADPH oxidases is achieved through change of conformation. This change in conformation is attributed to the different modes of interaction of motifs of oxidases, which are dependent on concentration of bivalent cation Ca2+. Previous research regarding intramolecular interactions within specific NADPH oxidase- Nox5β has been performed by multiple research teams and different sources appear to contradict each other on the exact mode of interaction of Nox5β EF-hand upon presence of Ca2+. Therefore the exact interaction model of terminals of Nox5β is unclear. In addition, the effect of presence of Ca2+ on the interaction terminals in another representative of NADPH oxidases- AtRBOHD, which possess highly analogous molecular structure of catalytic C-terminus to Nox5β, has never been thoroughly studied, as well as interactive cross-compatibility of the C and N terminals from these two distinct species of NADPH oxidases. The objectives of this research are to analyze intramolecular interactions of N- and C- terminals in Arabidopsis RBOHD and Human Nox5β upon presence of ionic calcium, compare Ca2+-induced terminals interactions in said oxidases and to establish possible cross-compatibility of terminals in these two distinct NADPH oxidase species. Practical aspects of this research included cloning the C- and N- cytoplasmic regions of Nox5β and AtRBOHD into bacterial expression vectors utilizing the PIPE cloning method, heterologous production of epitope-tagged tails of NOX5β and RBOHD in E. Coli BL21 and finally in-vitro pull-down assays to analyse the interactions of the tails upon the presence of Ca2+ as well as interactive cross-compatibility of these tails. By utilizing methods mentioned above, this research has demonstrated that interactions of terminals motifs both in Nox5β and AtRBOHD are possible even in calcium-deprived environment, which was achieved through use calcium-binding agent (EDTA) and the effect of calcium on interactions of terminals both in RBOHD and Nox5β is very limited if not insignificant. This research has also demonstrated that the cross-compatible interactions between terminals of Nox5β and AtRBOHD are possible. Results of this research indicate a strong structural conservation within NADPH oxidases, which indicates similar intramolecular interaction mechanisms within two highly diverged species. These findings may prove to be useful as a background for the future research regarding ROS producing enzymes and evolutional conservation in structures of oxidases.
  • Sket, Tina (2020)
    Endoplasmic reticulum (ER) stress is caused by the accumulation of unfolded proteins in the ER, which leads to the activation of unfolded protein response (UPR) through three transmembrane protein sensors located in the ER membrane. The sensors correspond to three branches of the UPR, namely protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) branches. Upon ER stress, IRE1 dimerizes and oligomerizes, and its endonuclease domain is activated. It specifically targets X-box-binding protein 1 (XBP1) mRNA, from which a 26 nt intron is spliced. This allows a complete translation of spliced XBP1 mRNA into a functional protein that acts as a transcription factor. Together with the other pathways, the UPR leads to a decrease in the protein folding load by causing a reduction in the general level of protein translation, and by inducing the expression of protein folding machinery. However, if the UPR is activated continuously for a long time, the apoptotic pathway will be triggered, and the cell will die. ER stress and UPR are associated with various disorders, such as some types of cancer, diabetes, chronic inflammatory syndromes, and particularly neurodegeneration. For example, in Parkinson’s disease, it was suggested that prolonged ER stress induces the extensive apoptosis of dopaminergic neurons in substantia nigra pars compacta region of the midbrain. This hinders the normal functioning of the nigrostriatal pathway, and hence results in the progressive development of Parkinson’s motor symptoms. In order to study the regulation or IRE1 branch of the UPR, and to identify the ER-stress-modulating compounds, a human luciferase reporter cell line (XBP1-NLuc) was created in this work. The reporter was expressed when IRE1 splicing was activated, since the XBP1 intron fragment was fused to the Nano luciferase gene. The expression of the reporter was observed with luciferase assay at several time points during treatments. The treatments were done with ER stress inducers thapsigargin and tunicamycin, and with IRE1 inhibitors KIRA6 and 4μ8c, or the combination of those. Quantitative PCR (qPCR) was used to validate the expression of the reporter and to monitor the expression of the other branches of the UPR. Additionally, the oligomerization of IRE1 was observed with IRE1-GFP cell line that was treated identically to the XBP1-NLuc cell line, fixed, stained for nuclei, and imaged with fluorescent microscopy. After imaging, the IRE1-GFP clusters were analysed and quantified with CellProfiller and CellAnalyst softwares. Both cell lines were used to test the effect of neurotrophic factors CDNF, MANF, and MANF mutant isomers on the UPR with and without tunicamycin treatment. Collectively, the experiments confirmed that XBP1-NLuc cell line was created successfully and that it accurately reports IRE1 splicing activity. As expected, ER stress treatment increased the reporter expression, while IRE1 inhibitors decreased the expression of the reporter. qPCR revealed that the other observed UPR markers were activated as well upon thapsigargin treatment, however, they were not decreased with the treatment with IRE1 specific inhibitors. In line with XBP1-NLuc cell line, the IRE1-GFP cell line demonstrated an increased oligomerization of IRE1 upon ER stress induction. The KIRA6 inhibitor of IRE1, which prevents IRE1 oligomerization, decreased the formation of IRE1-GFP clusters. Additionally, the IRE1-endonuclease-activity inhibitor 4μ8c induced the formation of IRE1-GFP clusters. Curiously, the distribution of the intensity of IRE1-GFP clusters was bimodal and could point to two manners of IRE1 clustering and/or activation. Together, the experiments done with cells transfected with CDNF, MANF or MANF mutants, suggested that the tested neurotrophic factors decreased IRE1 oligomerization and its activation. However, there were substantial problems in the quantification of viable cells, which should be considered in the interpretation of these results. No significant difference among the tested neurotrophic factors was observed. In conclusion, the XBP1-NLuc reporter cell line provided a reliable reporter of IRE1 endonuclease activity, whose expression is increased during the ER stress. Together with IRE1-GFP cell line, it revealed the amount of IRE1 oligomerization and activation under various treatments and at different time points relative to treatments. Due to the effectiveness and accuracy, the XBP1-NLuc cell line can be further used in studying the regulation and activation of IRE1, as well as for the identification of ER-stress modulating molecules, which can be used for development of novel treatments for ER stress associated diseases, such as Parkinson’s disease.
  • Muranen, Sampo (2019)
    Tree shoot architecture research is important due to its significance in fields such as timber production, fruit and nut production and aesthetics of common areas. Also, research on genetic factors that regulate shoot and root system architecture might provide novel methods to store more carbon in forests and, hence, mitigate global warming in the future. LAZY1 is one of the major genes that affects branch and tiller angle in herbaceous and woody species such as Arabidopsis, rice and peach tree. LAZY1 has been under scrutiny over a decade but its molecular function remains unknown. However, it is known that lazy1 mutation affects polar auxin transport. Here it is studied how LAZY1 affects initial branch angle, fiber length and reaction wood development in silver birch (Betula pendula). Also, transcript levels of few shoot architecture related genes were analyzed. LAZY phylogenetic analysis provided evidence of a duplication of LAZY1 in three studied tree species (Betula pendula, Prunus persica, Populus trichocarpa), duplicated genes are here named LAZY1a and LAZY1b. Plant material employed in this study was a segregating population (50:50) of back-cross 1 of weeping birch (B. pendula ´Youngii´) which has a truncated lazy1a. Histological samples of branches were prepared by cryo-sectioning, stained with carbohydrate binding Alcian Blue and lignin binding Safranin dyes to reveal patterns of tension wood development. Due to the large size of branch sections, samples were imaged with a microscope and the images were merged together in a Photoshop application. Branch angles were measured manually with a protractor (angle) tool from stem to the middle of a branch. The data was analyzed using mixed linear models due to the nature of used plant material. We could not use clones because of major issues in in vitro propagation. Branch samples were macerated, fibers imaged and measured by ImageJ software. LAZY1a gene expression levels were analyzed by RT-qPCR method. RNA-sequence analysis indicated that the expression pattern of LAZY1a and LAZY1b is similar in B. pendula. However, one should construct a promoter-reporter line to study with better resolution if their expression is spatially analogous. Initial branch angle was significantly different in wild type compared to lazy1a mutant. For future, one could generate single and double knock out lines of lazy1a/b to study if they have cumulative effect on the branch angle, an important factor in timber quality. Tension wood formation was difficult to quantify with the employed method, due to issues in segregating G-layered tension wood from thick-walled reaction wood. A chemical analysis of cellulose content might provide a more objective method to observe tension wood in branches. RT-qPCR method indicated that LAZY1a transcript levels are higher in wild type compared to mutant. A complementation or knock down experiment would provide sound evidence that lazy1a induces the weeping phenotype. X-ray diffraction method could be employed to study the orientation of cellulose microfibril angle in branches of the wild type vs. mutant. Generation of effective tensional stress requires a cellulose microfibril angle less than 10 and this angle is affected by auxin concentration. It is possible, that this angle is larger in lazy1a due to defect in polar auxin transport.
  • Amin, Al (2021)
    Wood development is a significant process with both financial as well as natural perspectives. Trees and wood are of highly significance in Finland where a huge part of the gross national income devises from the forestry area. Ecologically and commercially the Norway spruce (Picea abies) is one of the most common tree species in Europe. It covers about 30% of Finland's forest area. Norway spruce is frequently used in research to study many phenomena related specifically to the wood formation and lignification. The principal objective of my thesis work was to reveal an unknown step in the lignification process in developing xylem of Norway spruce, i.e. the initiation site(s) for lignification. To achieve this goal, the aim was to investigate the chemical identity of possible lignification initiation sites in the middle lamellae and cell corners of developing Norway spruce xylem, and to answer the question where in the cell wall soluble monolignols first emerge and lead to the start of lignin formation (polymerization). I was approaching this goal with immunolabeling technique for confocal microscopy and Raman spectroscopy to unravel this initiation site of lignification by using specific monoclonal antibodies for cell wall compounds and comparing the results with the initial lignin deposition sites. To detect the location/distribution of some important polysaccharides and lignin substructure for lignification initiation, monoclonal antibodies i.e. LM10, LM11, LM15, LM24 and antibody Dibenzodioxocin or DBD were applied for confocal microscopy and some monolignol specific spectra were applied for Raman microscopy. The xylan was detected by LM10 in secondary cell wall abundantly and few are in primary cell wall of Norway spruce. The LM11 against arabinoxylan was determined more in primary cell walls but less in secondary cell wall. The location of xyloglucan was identified in the middle lamellae, primary and secondary cell wall of Norway spruce by LM15. The LM24 against glycosylated xyloglucan was found in secondary cell walls, abundantly in cell corners but few in primary cell wall. The primary antibody Dibenzodioxocin or DBD for the lignin substructure revealed that these were present in the mature cells of secondary cell walls (S2 and S3 layers). The lignin substructures DBD were not found in youngest cells where secondary cell walls are absent. The developing xylem of Norway spruce was subjected Raman microscopy and which revealed the locations of cinnamyl alcohol, coniferyl alcohol and coniferyl aldehyde. The cinnamyl alcohol was abundantly found at cell corner and middle lamellae in most developing part of xylem. The coniferyl alcohol was determined only in developing xylem cell corners. The coniferyl aldehyde was observed at cell corners, middle lamella and primary cell walls of developing xylem. The coniferyl aldehyde was located more in mature cells than younger cells. So, the Confocal and Raman microscopy images revealed the possible bindings of monolignols to polysaccharide in young cell corners, cell wall layers and middle lamellae.
  • Vähäkangas, Eliisa (2020)
    The intestinal epithelium is one of the fastest renewing tissues in mammals. Intestinal stem cells (ISC) are responsible for producing all differentiated cell types of the intestinal epithelium, through transit amplifying generations. ISCs reside in the crypt domain of the intestine which are pit like structures located between villi protrusions. The ISCs are interspersed between Paneth cells, which along with cells of the surrounding mesenchyme act as the stem cell niche. ISCs have been reported to divide symmetrically to produce two identical daughter cells. However, the symmetry of these divisions has been concluded based on mathematical modelling which do not account for the possibility that a very small population of ISCs would divide asymmetrically or for qualitative asymmetry occurring in these divisions. Asymmetric cell division is a process by which daughter cells gain different amounts or different qualities of certain factors which lead to their differing fates. Asymmetric division can include asymmetric segregation of organelles such as mitochondria or peroxisomes, which have both been shown to be asymmetrically apportioned in yeast mitosis. Peroxisomes are single membrane enclosed organelles which function in many metabolic processes, most importantly in lipid and reactive oxygen species (ROS) metabolism. Mitochondria have been reported to be age selectively apportioned during cell division of mammary epithelial stem like cell. The same has been shown to occur for peroxisomes based on unpublished data from my host lab. This prior research of the lab also indicates that selective peroxisomal apportioning would require peroxisomes to be specifically gathered at the centrosomes from metaphase onwards to control their inheritance. In this thesis I will look into peroxisomal dynamics in the intestinal crypt. The first aim is to verify the Lgr5-EGFP-creERT2 x LSL-SNAP-tag-PTS1 mouse model, by checking that the SNAP-tag-PTS1 fusion protein properly localizes to peroxisomes. Secondly, I aim to look into the ages of peroxisomes in ISCs compared to differentiated cells, concentrating on Paneth cells. The third and final aim is to look into the apportioning of old and young peroxisomes during stem cell division. This aim includes looking into the peroxisomal localization at metaphase and checking how peroxisomes are expected to be inherited in later mitotic cells. The SNAP-tag-PTS1 construct adequately co-localizes with the peroxisomal membrane protein 70, also at the old SNAP labelling time point chosen for the following experiments. The SNAP-tag-PTS1 old labelling does not co-localize with the lysosomal associated protein Lamp1 to a high extent, indicating that the peroxisomes with the labelling are not in autolysosomes in amounts that would hamper with the results of the following experiments. There is no noticeable difference between the age contents of peroxisomes in stem cells versus Paneth cells. However, when moving up from base of the crypt to the transit amplifying zone there seems to be an increasing number of peroxisomes, as would be expected based on previous reports of peroxisomes in the intestinal epithelium. At metaphase it seems that approximately half of the cells have a tendency to gather peroxisomes at one centrosome to a higher extent than elsewhere in the cell. Interestingly this condensation was rarely seen at both centrosomes in a given cell. A large heterogeneity was observed when looking into the apportioning of old and young peroxisomes in anaphase or later on in mitosis. A majority of the dividing cells apportioned approximately equal amount of young and old peroxisomes to both daughter cells. Some divisions apportioned inequal amounts of peroxisomes to the daughter cells, with one daughter getting more peroxisomes overall. The daughter cell getting more peroxisomes was more likely to get significantly more of the old label than its pair. This indicates that there could be a small subpopulation of intestinal stem cells that divide their peroxisomes asymmetrically qualitatively as well as quantitatively, however, to definitively conclude this further research is required.
  • Neiro, Jakke (2019)
    In recent years, the two-spotted field cricket Gryllus bimaculatus has emerged as a central model for studies on insect development, regeneration and physiology. At the moment, G.bimaculatus has the most extensive molecular toolkit within the Exopterygota, making it the foremost model for evolutionary developmental biology and comparative physiology within the field of entomology. However, the postembryonic development of G. bimaculatus has received considerably less attention than embryonic development. In this thesis, I have studied the postembryonic development of G. bimaculatus to better understand the evolution and physiology of the understudied Exopterygota. My thesis encompasses five parts: postembryonic morphology, wing development, appendage regeneration, allometry, and growth. The postembryonic stages, the nymphal stages, have never been properly characterised in G. bimaculatus. By following postembryonic development daily at 30 C, 8 nymphal stages (instars) were identified. Size, coloration, sclerotisation of the thorax, and morphology of the wings, the hind tibia and the ovipositor were useful characters in distinguishing the stages. The Dpp/BMP signalling pathway patterns the wing venation in the endopterygotan insects Drosophila melanogaster and Athaliae rosae, but nothing is virtually known about wing development in exopterygotan insects. The wings and the wing venation pattern in different nymphal stages of G. bimaculatus were studied using the hydrogen peroxide clearing protocol along with both brightfield and fluorescence microscopy, while the role of the Dpp/BMP signalling pathway was studied using immunohistochemistry (IHC), in situ hybridisation (ISH), and RNA interference (RNAi). The longitudinal veins are patterned in the 3rd and 4th nymphal stages, while the secondary veins in the 8th stage. The IHC and ISH experiments displayed only non-specific staining, while the RNAi experiments did not produce any change in the phenotype, possibly because of molecular redundancy. The nymphal legs of G. bimaculatus are known to be highly regenerative, and the Dpp/BMP signalling pathway has been shown to provide positional information in leg regeneration. However, nothing is known about the regeneration of the other appendages in G. bimaculatus. Antennae and cerci were amputated in different nymphal stages, and the degree of final regeneration depended on the nymphal stage. RNAi experiments did not produce any change in the phenotype, possibly because of molecular redundancy. The interrelationship between static, ontogenetic and evolutionary allometry in insects is poorly understood. The allometry of hind femur length with respect to body length has been shown to be negative in Orthoptera (i.e. evolutionary allometry), but nothing is known about corresponding ontogenetic and static allometry. By measuring hind femur length and body length in G. bimaculatus in different nymphal stages, the ontogenetic allometry was determined to be slightly positive or isometric, while the static allometries of different stages tended to be negative but highly variable. This may indicate that allometric relationships constrain development in the microevolutionary perspective, but are nevertheless evolvable in a macroevolutionary perspective of millions of years. The growth conditions and rearing of crickets and other insects have been widely reported, but the shape of the growth curve itself has been less investigated. The exponential, the von Bertalanffy (VBGF), the West, Brown and Enquist (WBE), and the dynamic energy budget (DBE) models have been proposed as continuous models for insect growth. These models were t to growth data from G. bimaculatus and the DBE and was shown to be optimal with parameter values α=0 and pAm = 0.69. The insects have been thought to follow Dyar's law, i.e. that the growth ratio or moulting increment (MI) is constant throughout development, although numerous other competing moulting models have been devised for the crustaceans. By fitting different moulting models to head width data from G. bimaculatus, the log-linear model (Mauchline's model) turned out to explain the MI the best. Lastly, the oxygen-dependent induction of moulting (ODIM) model has been proposed to explain moulting patterns in insects, but the model has never been applied to exopterygotan taxa. By fitting the ODIM model to growth data from G. bimaculatus, the model could predict moulting mass but not instar durations, probably because of high postembryonic plasticity in G. bimaculatus.
  • Dawka, Sagarika (2021)
    Head and neck squamous cell carcinoma (HNSCC) is a diverse group of cancers defined by their localization in the head and neck region. These cancers are recognized for the heterogeneity between tumors from separate patients (inter-patient heterogeneity) as well as between cell types within individual tumors (intra-tumoral heterogeneity). Heterogeneity poses a major clinical challenge by making accurate diagnosis and selection of treatment options difficult. This study aims to improve precision of prognosis, quantify heterogeneity in HNSCC, and address its functional implications using two approaches: (1) profiling a set of HNSCC patient tumors using multiplexed immunohistochemistry and single-cell computational methods to identify a set of phenotypic descriptors correlating with differences in survival; and (2) using patient-derived cancer cell lines to investigate which cellular features correlate with relevant functional properties such as plasticity, invasiveness, clonogenicity and tumorsphere-forming abilities of cells. Epithelial-mesenchymal transition (EMT) as well as excistence of stem cell like states have been implicated in cancer aggressiveness and poor outcomes. We thus focused on identification of putative EMT, partial EMT (pEMT) and stem cell-like states. Based on a combination of morphometric analyses and stem cell- and EMT marker profiling, our computational method assigned patients into groups with different survival probabilities, and these patients’ tumors were found to differ in their expression of the stem cell transcription factor Sox2, the EMT transcription factor Slug, and in their morphometric parameters. Functional studies of patient-derived cell lines found that significant differences exist in protein expression, morphological features and cell behaviours between cell lines in vitro, and that inhibiting EMT promotes clonogenicity and can increase Sox2 expression. Thus, this study highlights important heterogeneous patient phenotypes and cellular behaviours in HNSCC, and implicates the need for a multimodal approach to diagnosis and therapy of this cancer.
  • Aung, July (2021)
    Epithelial cells line the surfaces of organs and tissues in a continuous and tightly packed manner, thereby functioning as a protective barrier between the tissue and the external environment known as the epithelium. During development, the epithelium undergoes a series of morphogenetic events which alters the shape and size of epithelial cells, enabling them to perform tissue specific functions in mature tissue. During morphogenesis, cells sense the mechanical forces and establish polarity through cell proliferation and rearrangement according to morphogenetic signalling pathways. This manoeuvre is achieved by the underlying actin cytoskeleton network which enables cells to resist the tension and stresses of morphogenesis via alteration of filament dynamics and network architecture. In vivo, numerous actin-regulatory proteins generate various polymerized forms of straight, branched, or contractile actin-myosin filaments, regulating dynamic actin filament turnover. The robust actin cytoskeleton provides the cell with protrusive and contractile forces that enable cells to migrate, maintain, and change its shape and form during morphogenetic events. Actin filament depolymerization is accomplished by ADF/cofilin (Drosophila homolog twinstar) binding to actin monomers (G-actin) and actin filaments. However, ADF/cofilin alone is not very efficient in promoting disassembly of actin monomers, especially in subcellular regions where ADF/cofilin is highly concentrated. AIP1 (Drosophila homolog flare) then enhances actin depolymerization via preferential binding to ADF/Cofilin rich regions in vitro. The aim of my thesis was to study the localization and roles of AIP1 and cofilin in follicular epithelium during Drosophila oogenesis. My results showed that Actin-Interacting-Protein-1 (AIP1) was expressed throughout oogenesis. AIP1 expression was increased in cell type-specific manner and AIP1 showed spatiotemporal localization in follicular epithelium during oogenesis. Silencing of AIP1 led to accumulation of ectopic F-actin aggregates, localization of which may reflect the cellular sites of dynamic actin reorganization in the follicular epithelium. My results also indicate that AIP1 may be indirectly responsible for maintaining epithelial integrity as its silencing resulted in formation of epithelial gaps throughout follicular epithelium. Also delays in border cell migration were observed. Considering the above, understanding how AIP1 functions in Drosophila morphogenetic events would therefore pave the way for a greater understanding of how this protein works in other organisms. The knowledge gained may also be used to extend the current understanding of the role of actin binding proteins in diseased states.
  • Sokka, Joonas (2019)
    Pluripotent stem cells (PSC) can exists in both primed and naïve states. The conventionally derived human PSCs represent the later primed state of pluripotency during embryo development, while the naïve state resembles the inner cell mass (ICM) of pre-implantation blastocyst. Primed human PSCs can be reverted chemically by transient histone deacetylase (HDAC) inhibition back to the naïve state in vitro. The reverted PSCs can then be characterized based on their morphology and expression of selected naïve markers using immunocytochemistry and RT-qPCR assays. Leucine twenty homeobox (LEUTX) is one of the genes expressed during the early stages of embryo development and is capable of activating the transcription of multiple genes, including pluripotency-associated genes, which are upregulated during the human embryonic genome activation (EGA). LEUTX expression could potentially improve the naïve reversion efficiency or the maintenance of naïve PSCs by driving the transcriptome of primed PSCs back towards the earlier cell stages of embryo development, potentially even to cell stages that precede the naïve state. The aim of this thesis was to setup the naïve reversion protocol and study the effects of LEUTX on the reversion by using the generated and tested H9 activator cell line for targeted activation of endogenous LEUTX expression. First, a conditionally stabilized CRISPRa activator cell line was generated for targeted activation of endogenous gene expression in H9 cells. Then sequence-specific guide RNAs (gRNA) targeting LEUTX for activation were introduced to the activator cell line. Using the generated activator cell line during the naïve reversions allows the targeted activation of specific genes, here LEUTX, and thus enables studying the effects of these genes on PSCs during the naïve reversion protocol. The induced activator cells expressing LEUTX managed to form four times as many naïve resembling colonies during the reversion compared to the controls, but most of these were lost after changing the medium conditions towards the end of the protocol. After the reversion was complete, the reverted PSCs were characterized as naïve PSCs based on their domed morphology and the high expression of naïve markers NANOG, KLF17, TFCP2L1 and DNMT3L when compared to the primed PSCs. The naïve reversion protocol was set up and optimized successfully and can now be used as a reliable way of obtaining human naïve PSCs for further experiments studying and modelling the earlier developmental stages during embryo development. Furthermore, the generated H9 activator cell line worked as intended and can be utilized for studying the effects of other targeted genes during the reversion or in the reverted naïve PSCs.
  • Lintala, Annika (2020)
    Bipotential gonads are precursor structures for testes and ovaries. Steroidogenic factor-1 (SF1) is one of the most important transcription factors in an embryo needed for the development and maintenance of bipotential gonads. If SF1 is not expressed, bipotential gonads fail to develop, and genitalia and kidneys are not formed. Later, SF1 expression persists high in testes, where it supports Sertoli and Leydig cell formation and development. If SF1 is not expressed enough in males, the bipotential gonads differentiate into ovaries. The factors activating and regulating SF1 are not currently fully known. By getting more knowledge of how SF1 is controlled, regulatory mechanisms behind normal fetal development of gonads and disorders of sex development (DSD) can be understood better. The aims of this thesis were to study whether growth factors, that naturally regulate differentiation of developing gonads, promote differentiation of human induced pluripotent stem cells (hiPSC) into Sertoli-like cells (SLCs) and whether SF1 expression is induced by the addition of these growth factors. For conducting the study, we used hiPSCs, which have an SF1 activation domain cassette previously introduced to the cells by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas9) method. SF1 could be activated by adding doxycycline (DOX) and trimethoprim (TMP). These hiPSCs were differentiated into intermediate mesoderm (IM) on the first four days according to the protocol published earlier by the group. After this, the differentiation to SLCs was guided by adding growth factors to the culture medium. Basic fibroblast growth factor (bFGF), fibroblast growth factor 9 (FGF9) and prostaglandin-2 (PGD2) were tested separately and in a combined cocktail also including follicle stimulating (FSH) and glial cell-derived neurotrophic factor (GDNF). In a control condition, cells were differentiated without additional growth factors. In all tested conditions, cells first differentiated into IM were further differentiated either in the presence or absence of DOX and TMP for 8 days. The differentiation medias were changed to the cells every day and lysis samples for quantitative PCR (qRT-PCR) were taken every other day. The relative gene expression levels of bipotential gonad, testis and steroidogenic gene markers from each condition were monitored with qRT-PCR and compared to the levels of the undifferentiated hiPSCs. Immunocytochemistry was performed to see the changes in protein production. Against our hypothesis and the previous studies by others, none of the tested growth factors induced the cells to differentiate into SLCs. However, SF1 expression was triggered by chemical induction with DOX and TMP. Also, the expression levels of bipotential gonadal and testicular gene markers increased in control conditions with/without chemical induction. PGD2 conditions were the only ones to resemble the gene expression and morphology of control conditions while the others differed. These results indicated that the addition of bFGF, FGF9, FSH and GDNF did not improve the differentiation of iPSCs into SLCs and in fact, bFGF and FGF9 hindered their differentiation into SLCs. As a future perspective the optimal concentrations for each growth factor and the duration of growth factor supplementation ought to be tested to refine the protocol.
  • Michalowski, Piotr (2019)
    The TrkB signaling pathway plays an important role in synaptic transmission and plasticity. Synaptic plasticity is disrupted in many neurological disorders, such as major depression and dementia. A number of studies indicate that TrkB (tropomyosin-related kinase B) signaling is required for the therapeutic effects of antidepressants. Both conventional and rapid-acting antidepressants encompass the TrkB pathway but the underlying mechanism of this remains unknown. Recent studies have, however, revealed an intriguing link between emergence of slow wave EEG activity (SWA) or sedation and the TrkB pathway. Notably, various anesthetics and sedatives (e.g. isoflurane and medetomidine) that increase SWA concomitantly induce TrkB signalling, and this seems to happen independently of BDNF (brain-derived neurotrophic factor), the primary ligand of TrkB. Given the ability of Src kinase to transactivate TrkB in vitro, we have examined the acute effects of medetomidine and isoflurane on SrcY416 and TrkBY816 phosphorylation in the adult rodent cortex and hippocampus by using Western blotting. Pyrazolopyrimidine 2 (PP2), a Src kinase inhibitor, was implemented in order to inhibit TrkB signalling pathway induced by medetomidine. The study was further extended to sleep deprivation experiments to investigate the effects of deep sleep on the Src and TrkB protein phosphorylation. Phosphorylation of GSK3βS9, another important molecular event coupled with antidepressant effects, was also investigated. The results indicate that both isoflurane and medetomidine activate Src kinase and TrkB signalling pathway. Such an effect was not, however, seen in the PP2 study and thus we failed to confirm the mechanistic connection between Src and TrkB. A trend in the phosphorylation of TrkB, Src and GSK3β was found in the brain samples collected after 15 minutes of recovery sleep, suggesting that TrkB signalling is also facilitated during physiological SWA. In conclusion, these results reinforce the hypothesis that SWA occurs simultaneously with TrkB signaling. Future studies are required to test the involvement of Src kinase in this phenomenon.
  • Prajapati, Biplu (2021)
    Transfer RNA (tRNA) is one of the most extensively modified RNA molecules. The role of tRNA modifications become apparent during physiological condition such as oxidative stress, where it serves as an adaptive response to the changing environment. These modifications are upregulated mainly at the wobble position of the tRNA to enhance the translational efficiency of the stress response genes through enhanced decoding rate and tRNA–mRNA interaction. Hence, tRNA modification has a crucial role in regulating translational fidelity, and such modifications can be utilized to fine-tune the translation for improved production of heterologous protein. Therefore, this study aimed to analyze the tRNA modification changes in two laboratory-significant E. coli strains (BL21 (DE3) and K12) during oxidative stress and utilize these modifications to enhance the production of heterologous protein using a defined cell-free protein synthesis system. Ultra-performance liquid chromatography-mass spectrometry was used to detect and quantify the tRNA modification changes in the hydrogen peroxide-treated E. coli cells. The results showed unique tRNA modification patterns and intensities between the two bacterial strains in response to oxidative stress. Modifications such as ac4c and m2,2G were upregulated in E. coli BL21 (DE3) following hydrogen peroxide treatment, whereas k2C and chm5U were increased in E. coli K12. Further analysis of the dataset revealed that most of the upregulated ribonucleoside modifications were predominant at the anticodon loop of the tRNAs, indicating the potentiality of these tRNA pools to impact on translation. Likewise, I optimized an E. coli-based cell-free protein synthesis system to investigate the effect of modified tRNA pools on translation. Hence, this study serves as a stepping stone to understand the tRNA modification landscape of E. coli and provides a platform to depict the function of post-transcriptional tRNA modifications in translation with the CFPS system.