Väitöskirjat

Recent Submissions

  • Hiitiö, Heidi (Helsingin yliopisto, 2018)
    Mastitis is the most common and economically damaging production disease of dairy cows. In the majority of cases it is induced by intramammary infection with a pathogen. Identification of mastitis-causing pathogens enables treatments to be targeted appropriately, avoiding unnecessary antimicrobial treatments, and enabling design of the best possible herd level management protocols. Diagnosis of bovine IMI has been traditionally based on microbial culture of aseptically taken quarter milk samples. Due to the requirements of modern dairy production and development of molecular technologies, milk samples can be examined for target pathogens with DNA-based methods such as multiplex real-time PCR assays. Despite several years of routine use of PCR assays in some countries, scientific knowledge concerning interpretation of the results is sparse. This dissertation focuses on providing new information for diagnosing bovine mastitis using multiplex real-time PCR. In order to accomplish that, first a commercial multiplex real-time PCR assay was compared with the current reference method, conventional culture (I). It showed that PCR was a reliable diagnostic method for Staphylococcus aureus, but less reliable for diagnosing true non-aureus staphylococci infections. In an experimental challenge study (IV), elimination of Staphylococcus epidermidis and S. simulans was followed with PCR in addition to conventional culture. The results showed that PCR yielded target-positive results for several days after the culture results were negative. Subsequently, the time of sampling related to the onset of inflammation may be of importance when interpreting the PCR results. Secondly, quarters with suspected mastitis were sampled using two experimental techniques, in addition to the conventional aseptic technique (II, III), and examined with multiplex real-time PCR. In the needle technique, the whole teat and teat area were bypassed. With the cannula technique, the teat orifice and the teat canal were bypassed. The results showed that samples taken with alternative techniques were of better hygienic quality. We also reported that over half of the non-aureus staphylococci findings in our PCR results originated from sites other than the mammary gland. A diagnosis of a true IMI or mastitis, with possible outcome of an antimicrobial treatment course for the animal, cannot be based solely on a PCR result, but must be combined with all the available information about a cow`s history, symptoms, changes in milk and indicators of inflammation. Some microbes detected with PCR can be considered to be contaminants, especially when present with low levels of DNA. If the signs of the cow or the degree of udder inflammation do not fit the usual signs caused by the pathogen detected, the possibility of contamination should be considered.
  • Leino, Teppo (Helsingin yliopisto, 2018)
    Orexin peptides, orexin-A and orexin-B, and orexin receptor 1 and orexin receptor 2 form the orexin signaling system. The most studied part of the orexin system is its key role in sleep-wake regulation, although it is linked also to other physiological functions, such as addiction and nociception. To date, a large number of orexin receptor antagonists have been developed with one, suvorexant, having reached the market in the treatment of insomnia. However, much less attention has been paid to the development of small-molecular agonists of the orexin receptors, and only a few are known in the literature. Agonists might be beneficial for patients with narcolepsy or certain types of cancers. The aim of this thesis was to develop small molecules based on the azulene scaffold for targeting orexin receptors. Azulene is an unexplored ring structure in medicinal chemistry, however, it resembles other bicyclic aromatics such as indole and naphthalene, which are frequently found in drug molecules. The small number of existing general synthetic routes for azulenes possessing three or more substituents has most likely hindered the use of azulene-based compounds in medicinal chemistry. Due to this, the study was initiated by developing two different synthetic routes to access 1,3,6-trisubstituted azulenes. In the developed methods, the azulene scaffold was first synthesized from simple, readily available and inexpensive starting materials. Then the scaffold was functionalized via versatile synthetic handles, such as a halogen atom or a formyl group, which allow a facile generation of compound series. The efficiency of the synthetic routes was demonstrated with test substances, which gave good overall yields. The developed methods were used in the synthesis of azulene-based compounds, whose biological activity was assessed on the orexin receptors. The first series of compounds was based on the results from virtual screening of the library of 70 000 synthetically accessible azulene-based compounds. The second series was designed based on the results from the biological evaluation of the first series. With this approach, novel azulene-based ligands for orexin receptors were identified. The two most promising binders had Ki values in the low micromolar range and five other compounds acted as weak orexin receptor agonists. In addition, compounds potentiating the response of orexin-A to OX1 receptors in a concentration-dependent manner were discovered. These novel azulene-based compounds offer an interesting starting point for further development of antagonists, agonists and potentiators for orexin receptors.
  • Ollikainen, Eliisa (Helsingin yliopisto, 2018)
    Objective Saccular intracranial aneurysm (IA) rupture causes subarachnoid hemorrhage, an acute intracranial bleeding with high mortality (30-40%) and morbidity. Unruptured IAs are common in the population (2-3%), but their rupture rate escapes prediction. Risk factors are smoking, female sex, and hypertension. Hypercholesterolemia, a risk factor for atherosclerosis, plays an unknown role in IAs. IA walls show histological changes that resemble those in atherosclerotic lesions. Altered hemodynamics are hypothesized to contribute to IA pathogenesis. Role of atherogenic mechanisms in IAs remains, however, unestablished. The aim of this thesis was to discover the role of atherosclerotic and flow-related changes as a potential trigger of IA rupture and to discuss potential tools for imaging those changes. Methods In total, 55 (25 unruptured and 30 ruptured) intraoperatively resected IA fundus specimens were evaluated by histological and immunohistochemical methods. 36 IAs underwent analysis for wall remodeling, neovascularization, lipids and apolipoproteins, erythrocyte remnants, and infiltrations of CD163+ and CD68+ macrophages, CD3+ T lymphocytes, and mast cells. Preoperative CT-angiography images provided the data source for hemodynamic simulations, compared with histology in 20 IAs. To correlate histology with MRI, 11 IAs underwent 4.7T MRI ex vivo. Results and Discussion Patient age, sex, hypertension or smoking did not associate with IA rupture or degenerative wall changes. Of 41 of the IAs, 9 showed an intact endothelium and linear smooth muscle cells (SMCs; type A, 22%), 17 showed SMC proliferation and disorganization (type B, 41%), 13 showed a hypocellular wall with a myointimal hyperplasia and/or organized thrombus (type C, 32%), and 2 showed an extremely thin, thrombosed wall (type D, 5%). Neovascularization occurred in 28/36 (78%) walls and associated with wall degeneration (type C). Hemosiderin adjacent to neovessels indicated microhemorrhages from neovessel leakage. Accumulation of lipids and apolipoproteins of HDL and LDL occurred in all 36 IAs, suggesting atherosclerotic processes, independent of plasma lipid levels. Lipid accumulation was associated with IA wall inflammation and degeneration, suggesting proinflammatory effect of lipids. Extracellular adipophilin was most extensive in ruptured IAs, reflecting death of lipid-laden cells. Cytotoxic effect of lipid may thus contribute to wall rupture. Degenerated and ruptured IAs showed a massive accumulation of erythrocytes (glycophorin A+), mainly in old thrombus. Erythrocytes associated with oxidized lipids and hemoglobin-phagocytozing macrophages. Hemoglobin may thus promote oxidative stress and inflammation in the IA wall. Glycophorin A and hemosiderin associated with signal-intensity changes ex vivo MRI, although their histological staining pattern remained unidentified in MRI. Flow models of IAs showed the association of wall shear stress with wall inflammation and remodeling, suggesting that hemodynamic simulations, as well, could serve as a tool in detection of rupture-prone human IA walls. Conclusion Aneurysm walls show a variety of degenerative remodeling changes similar to those in extracranial atherosclerosis, changes which may predispose to IA wall rupture. Experimental models are warranted to verify the suggested mechanisms. Nevertheless, the present study provides important clarification of IA-wall pathogenesis which may prove useful in development of preventive treatment for low-risk IAs and better diagnostic methods to reveal high-risk IAs.
  • Leino, Viljami (Helsingin yliopisto, 2018)
    The Standard Model of particle physics provides the most precise description of elementary particle physics. However, the Standard Model is not a complete theory and many theoretical questions still remain unanswered. For example, the exact nature of dark matter, baryogenesis, and neutrino masses remain unknown. Therefore, there is still need for beyond the Standard Model physics. Many interesting beyond the Standard Model scenarios rely on strong inter- actions. For example, the extended technicolor mechanism could explain the fermion masses and the electroweak symmetry breaking without elementary scalar fields. However, perturbative calculations at large couplings are not reliable and non-perturbative methods are needed. Of special interest are theories which have a vacuum phase structure indicating an existence of an infrared fixed point. Such conformal theories form a basic building block of many beyond the Standard Model scenarios, including the aforementioned extended technicolor theory. In this thesis I present the results of multiple studies aiming to understand the infrared behavior and conformal window of the SU (2) gauge model with six and eight massless Dirac fermions transforming in the fundamental representation of the gauge group. Multiple theoretical estimates indicate that the conformality sets in between five and eight fermions in the SU (2) model, rendering six and eight fermion cases interesting for non-perturbative study. However, previous lattice studies on the six and eight fermion cases have been inconclusive. Using clover improved fermion action and smeared Wilson gauge action, we have performed a gradient flow study of the coupling and scheme invariant quantities: the mass anomalous dimension γm and the leading irrelevant exponent of the coupling γg . Our results indicate that both of the models under the study have infrared fixed points and are therefore within the conformal window.
  • Turkki, Riku (Helsingin yliopisto, 2018)
    The aim of this dissertation was to investigate the use of computer vision for tissue characterization and patient outcome prediction in cancer. This work focused on analysis of digitized tissue specimens, which were stained only for basic morphology (i.e. hematoxylin and eosin). The applicability of texture analysis and convolutional neural networks was evaluated for detection of biologically and clinically relevant features. Moreover, novel approaches to guide ground-truth annotation and outcome-supervised learning for prediction of patient survival directly from the tumor tissue images without expert guidance was investigated. We first studied quantification of tumor viability through segmentation of necrotic and viable tissue compartments. We developed a regional texture analysis method, which was trained and tested on whole sections of mouse xenograft models of human lung cancer. Our experiments showed that the proposed segmentation was able to discriminate between viable and non-viable tissue regions with high accuracy when compared to human expert assessment. We next investigated the feasibility of pre-trained convolutional neural networks in analysis of breast cancer tissue, aiming to quantify tumor-infiltrating lymphocytes in the specimens. Interestingly, our results showed that pre-trained convolutional neural networks can be adapted for analysis of histological image data, outperforming texture analysis. The results also indicated that the computerized assessment was on par with pathologist assessments. Moreover, the study presented an image annotation technique guided by specific antibody staining for improved ground-truth labeling. Direct outcome prediction in breast cancer was then studied using a nationwide patient cohort. A computerized pipeline, which incorporated orderless feature aggregation and convolutional image descriptors for outcome-supervised classification, resulted in a risk grouping that was predictive of both disease-specific and overall survival. Surprisingly, further analysis suggested that the computerized risk prediction was also an independent prognostic factor that provided information complementary to the standard clinicopathological factors. This doctoral thesis demonstrated how computer-vision methods can be powerful tools in analysis of cancer tissue samples, highlighting strategies for supervised characterization of tissue entities and an approach for identification of novel prognostic morphological features.
  • Hirvonen, Jonni (Helsingin yliopisto, 2018)
    The interplay between senses and actions is one of the most crucial processes that takes place in the brain. The successful course from perception of a stimulus to a meaningful action requires coherent communication between different cortical areas. In humans, these events can be measured non- invasively outside the skull, for example by recording electric or magnetic fields that are produced by neuronal population activity on the cortex, with electroencephalography and magnetoencephalography (EEG and MEG). By combining MEG and EEG with simultaneous behavioural experiments, it is possible to extract neuronal activities that are correlated with perception and action. In this thesis, MEG recordings combined with advanced data-analysis techniques were used to study the role of cortical oscillations –brain rhythms – in coordinating conscious perception and action as well as their deficits in chronic schizophrenia. In Study I and Study II, I investigated what the local and large-scale neuronal correlates of conscious somatosensory perception are, respectively. Healthy subjects were stimulated at their index fingers with somatosensory stimuli, adjusted individually at the threshold of detection, so that around half of the time the stimulus was detected. Concurrent MEG recordings and subsequent source-modelling revealed in Study I that perceived trials were correlated with strengthened evoked responses (ERs), phase-locking to stimulus onset (SL), and induced oscillation amplitude modulations. The most robust and widespread of these was SL that was sustained in the low-alpha (6-10 Hz) band. The strength of SL and to a lesser extent that of ER predicted conscious perception in the somatosensory, lateral and medial frontal, posterior parietal, and in the cingulate cortex. In Study II, I investigated the role of large-scale synchronization in the conscious somatosensory perception. Perceiving and reporting of weak somatosensory stimuli were correlated with sustained strengthening of large-scale synchrony, concurrently in delta/theta- (3-7 Hz) and gamma- (40-60 Hz) frequency bands. In a data-driven network localization, I found this synchronization to dynamically connect the task-relevant, i.e. the frontoparietal, sensory and motor systems. The strength and temporal pattern of interareal synchronization were also correlated with the response times. These data showed that a rapid phase-reorganization and concurrent oscillation amplitude modulations in the specific areas play a key role in the emergence of a conscious decision-making, and subsequent actions. Furthermore, this study showed that perception is dependent on transient large-scale phase synchronization in the delta/theta and gamma bands. In the third study, I investigated whether aberrant large-scale synchronization or dysconnectivity could underlie perceptual deficits in patients suffering from schizophrenia. To this end, I analysed MEG data from chronic schizophrenia patients and healthy control subjects recorded during a visual perception closure task. In schizophrenia patients, a reduction in gamma-band (30–120 Hz) oscillation amplitudes, accompanied by a pronounced deficit in large-scale synchronization at gamma-band frequencies characterized visual processing compared to healthy control subjects. Synchronization was reduced within visual regions, as well as between the visual and frontal cortex. Additionally, the reduction of synchronization correlated positively with clinical disorganization scores. Accordingly, these data imply that schizophrenia is associated with a profound disruption of transient synchronization. This observation provides critical support for the notion that the core aspect in the pathophysiology of schizophrenia arises from an impairment in coordination of distributed neural activity.
  • Hiltunen, Sini (Helsingin yliopisto, 2018)
    The aim of this study was to investigate clinical characteristics of cerebral venous thrombosis (CVT), risk-factors, and factors associated with poor outcome. We established retrospectively a database on all CVT patients treated at the Helsinki University Hospital from 1987 to 2015, and patients were invited for a follow-up visit. We formed collaboration with the Academic Medical Centre in Amsterdam, and Sahlgrenska University Hospital in Gothenburg. Helsinki CVT registry collected data on 243 patients. Patients aged from 15 to 82 years (median 42), and 60% were women. In the study investigating sinus recanalization, 43 of 91 patients had complete, 31 had partial, and 17 had no recanalization of the sinuses at follow-up. Poor recanalization was associated with older age, male gender, and absence of known risk factors or causes. In multivariate analysis recanalization was not associated with functional recovery. Fibrin D-dimer was measured in 71 out of 138 patients before initiation of anticoagulation. D-dimer <0.5mg/l was measured in 12.7%, 0.5-2.9mg/l in 52.1%, and >3mg/l in 35.2%. Levels of were lower in patients with longer symptom duration and higher when more sinuses were thrombosed. Hyperglycemia was investigated in 308 patients (169 from Amsterdam and 139 from Helsinki). Hyperglycemia (plasma glucose >7.8mmol/l) was present in 21% of the patients at admission. In multivariate analysis hyperglycemia was independently associated with recovery and mortality. Role of cancer as CVT risk was investigated in 594 cases (243 from Helsinki, 224 from Amsterdam, and 128 from Gothenburg) and 6278 controls. In the first year after cancer diagnosis the risk was clearly elevated in solid cancer, and very high in hematological cancer. The study investigating long-term outcome after CVT included 161 patients, with a mean follow-up of 39 months. Mortality was 11%, with 4% due to CVT. Good functional recovery (mRS 0-1) was observed in 83%, however residual symptoms were reported by 68% of patients. Vocational status analysis included 121 working-aged patients; 23% were unemployed, and 16% were on permanent disability pension. Major stroke symptoms, and compulsory education only were associated with both functional outcome and vocational status in multivariate analysis. In conclusion, our CVT cases in Helsinki are in demographically similar to other CVT series from high-income countries. Recanalization occurred less often in patients with known factors of poor outcome, but the importance of recanalization to recovery needs further studies. Fibrin D-dimer measurements cannot be reliably used to exclude CVT. We established hyperglycemia as a factor affecting outcome. Hyperglycemia should be treated, but optimum level needs investigation. Newly diagnosed cancer is a major risk factor for CV was confirmed. Functional outcome after CVT is generally good, however residual symptoms are common. Patients are often young, so even mild residual symptoms that affect working ability are of large importance.
  • Penttilä, Sini (Helsingin yliopisto, 2018)
    The aim of this study was to find and describe the genetic background of a new form of spinal muscular atrophy (SMA). Late-onset spinal motor neuronopathy (LOSMoN), later named spinal muscular atrophy, Jokela type (SMAJ), is a relatively benign autosomal dominant form of a lower motor neuron disorder. Disease onset is after the age of 30-40 years, and SMAJ is characterized by initial painful cramps and fasciculations affecting the proximal and distal muscles of the upper and lower limbs. The disease is slowly progressive, resulting in weakness and mild to moderate muscle atrophy later in life. SMAJ was originally identified in two families in Eastern Finland. The genome-wide scan study performed for these families showed that the disease is linked to chromosome 22q11.2-q13.2. The disease-associated haplotype was identical in both families, suggesting a founder effect. The founder hypothesis was also confirmed later, as several other unrelated patients carrying the same haplotype were identified. The disease-causing mutation, c.197G>T p.G66V in CHCHD10, was detected by whole-genome sequencing. The mutation was present in all then identified 55 SMAJ patients belonging to 17 families. At the same time, other dominant mutations in CHCHD10 were described to cause a wide range of neurological disorders. CHCHD10 was the first SMA-causing gene identified that encodes for a mitochondrial protein. The prevalence and clinical outcome of CHCHD10 mutations in Finnish neuromuscular disease patients were clarified in a screening study. The only detected mutation was c.197G>T p.G66V, and all patients carrying this mutation had a phenotype restricted to SMAJ. The prevalence of c.197G>T p.G66V was estimated to be around 4/100 000 in Finland, i.e. approximately 200 symptomatic SMAJ patients. The results of this study confirm that SMAJ is a genetically distinct entity caused by a dominant mutation c.197G>T p.G66V in CHCHD10. This finding enables genetic testing of SMAJ, providing patients with an accurate diagnosis and prognosis. According to our genotyping results, c.197G>T p.G66V is a founder mutation in Finland, all SMAJ patients having common ancestry. SMAJ was shown to be relatively common in Finland. It is clearly the most common CHCHD10-related disease reported, and in Finland it may be the most common form of SMA. Because SMAJ seems to be absent from other populations, it can be considered a part of the Finnish disease heritage.
  • Weltner, Jere (Helsingin yliopisto, 2018)
    Somatic cells can be reprogrammed to pluripotent state by ectopic expression of a defined set of transcription factors. These induced pluripotent stem cells (iPSC) hold great potential for biomedical applications, such as disease modelling, drug discovery and cell therapies. The derivation of iPSCs is a complex multistep process that can commonly result in inefficient or incomplete conversion of the cells. The reprogramming efficiency and the quality of the reprogrammed cells can be affected by various components of the reprogramming method, including reprogramming vectors, starting cell populations and the choice of reprogramming factors. The aim of this thesis was to explore novel approaches for improving the pluripotent reprogramming outcome. The particular aims of this thesis were to investigate the use of recombinant Adeno-associated virus (rAAV) as a gene transfer vector for cellular reprogramming, characterization of the effects of old donor age and long term passaging on the reprogramming of fibroblasts, and development of reprogramming methods based on CRISPR/Cas9-mediated activation of endogenous reprogramming factors. In this study, rAAV-mediated transduction of mouse embryonic fibroblasts with OCT4, SOX2, KLF4 and C-MYC was found to successfully induce reprogramming to pluripotency. Unlike initially expected, the AAV vectors were integrated with high efficiency into the host genome during the reprogramming process, resulting in all analyzed iPSCs containing vector integrations. Both donor age and the culture time of the donor fibroblasts correlated with reduction in pluripotent reprogramming efficiency. This effect was found to be associated with upregulation of cellular P21 expression and reduction in cell proliferation. Downregulation of P21 expression by siRNA treatment was able to promote reprogramming of late passage senescent fibroblasts. By optimizing the reprogramming factor guide composition, CRISPR/Cas9-mediated gene activation (CRISPRa) could be used to derive iPSCs reprogrammed fully by targeted activation of endogenous genes. The efficient reprogramming of somatic cells by CRISPRa was found to be dependent on the inclusion of additional guides targeting an embryonic genome activation enriched Alu-motif. Due to the direct targeting of endogenous loci and the high multiplexing capacity of CRISPRa, the reprogramming approach has a high potential for mediating comprehensive and specific reprogramming. Overall, this thesis provides a number of novel tools and insights into the pluripotent reprogramming process. The results of this work can be used to develop more robust reprogramming methods and to improve the quality of reprogrammed cells.
  • Cairns, Johannes (Helsingin yliopisto, 2018)
    Antibiotics often occur in different environments at concentrations insufficient to inhibit the growth of susceptible bacteria. Subinhibitory concentrations can, nevertheless, select for chromosomal antibiotic resistance mutations and mobile genetic elements carrying antibiotic resistance genes, such as conjugative plasmids. However, it has been unclear whether this occurs in typical natural habitats where bacteria live in multispecies communities and interact with viral and protozoan consumers. In such settings, similar selection can also occur on other bacterial traits, potentially interfering with antibiotic resistance evolution. In this thesis, the combined effect of low antibiotic concentrations and ecological interactions on antibiotic resistance evolution was investigated. The thesis consists of four studies. In the first and second study, antibiotic and bacteriophage resistance evolution was examined at the phenotypic or whole-genome level when a bacterial population was exposed to one versus both factors. In the third study, the effect of antibiotics and protozoan predation on the spread and maintenance of a conjugative antibiotic resistance plasmid in a bacterial population was determined. The fourth study examined the effect of a low antibiotic concentration, protozoan predation, and spatial layout of habitat on community composition and horizontal transfer of a conjugative plasmid in a multispecies bacterial community. Antibiotic resistance evolution was shown to be promoted by low antibiotic concentrations independent of the presence of ecological interactions. The presence of protozoan predation also promoted the spread and maintenance of a resistance plasmid even in the absence of antibiotics. This demonstrates that ecological factors other than antibiotics can also play a role in the horizontal transfer of antibiotic resistance genes. Moreover, a low antibiotic concentration altered bacterial community composition, diversity, and the strains that received a resistance plasmid, although predation and spatial layout of habitat were equally strong or stronger drivers. Since the community effects of low antibiotic concentrations can be weaker or similar in strength to the effects of other ecological drivers, knowledge of antibiotic alone may be insufficient for predicting changes in the structure or diversity of a community.
  • Leppänen, Juho (Helsingin yliopisto, 2018)
    The thesis statistical properties of non-stationary dynamical systems with intermittency concerns two types of non-stationary dynamical systems: sequential compositions of interval maps with a neutral fixed point (Pomeau-Manneville maps) and intermittent quasistatic systems. Both systems are non-uniformly expanding and time-dependent, and (typically) lack invariant measures. The evolution of states under a sequential system is described by a sequence of varying self-maps of a phase space. Such constructions are motivated by applications to non-equilibrium processes in nature, where the map describing how a state evolves should depend on time. Quasistatic systems on the other hand draw inspiration from thermodynamics and model situations where the observed system transforms (infinitesimally) slowly with time due to external influence. At any given time the system is at an equilibrium, but over a long time span the equilibrium slowly changes. The thesis consists of an introduction and three scientific articles. The first and third article are about quasistatic systems, while the second article deals with sequential systems. The main result of the second article is a functional correlation bound widely useful for showing limit theorems in the sequential settting. We prove the result by modifying a technique of Liverani, Saussol, and Vaienti, which is based on a probabilistic approximation of the deterministic system. We present two applications of the result for a single Pomeau-Manneville map, by showing that the bound implies the correlation-decay conditions of the normal approximation methods due to Pène-Rio and Stein. Both methods yield a multivariate central limit theorem with an estimate on the rate of convergence. The rate produced by the former method is optimal with respect to the Kantorovich (or Wasserstein) metric. The latter method is suitable also for normal approximation in non-stationary settings. In the first article we introduce the intermittent quasistatic system and obtain several tools for further analysis of its statistical properties, including L^1-perturbation estimates for the transfer operators. The main result is an almost sure ergodic theorem for the time-averages of the model. The proof, which is partly based on a general theory developed by Stenlund, makes extensive use of the polynomial memory loss bound shown recently by Aimino et al. The third article builds on the results of the first two articles. By solving a well-posed martingale problem, we show that limiting distributional behavior of intermittent quasistatic systems can be characterized by a stochastic diffusion process. The result extends that shown by Dobbs and Stenlund for a class of uniformly expanding quasistatic systems.
  • Kentala, Henriikka (Helsingin yliopisto, 2018)
    ABSTRACT OSBP-related proteins (ORPs) are lipid binding and transport proteins expressed ubiquitously in eukaryotic cells. ORPs localize at specific organelle interfaces designated membrane contact sites (MCSs). They possess a characteristic ligand-binding domain with specificity for oxysterols, cholesterol and phospholipids. Some ORPs have the capacity to mediate bi-directional transfer and exchange of two different lipids over MCSs. However, a number of findings suggest that a majority of the ORP family members act as lipid sensors with cell signalling properties rather than actual lipid transporters. ORP2 localizes on the surface of cellular lipid droplets. Earlier finding suggests that ORP2 contributes in triglyceride (TAG) and cholesterol homeostasis but the comprehensive knowledge of the cellular function of ORP2 is deficient. In my thesis I investigated the sterol regulation of the subcellular localization ORP–VAP complexes and the role of ORP2 in cellular physiology by generating a stable ORP2 knock out (KO) cell line in HuH7 hepatocytes. I demonstrated that the subcellular distribution of OSBP–, ORP4L– and ORP2–VAP-A complexes at MCSs are regulated by ORP oxysterol liganding and by the cellular cholesterol status. Moreover, ORP2-KO impaired a diversity of cellular functions. The ORP2-KO affected mRNAs of >2000 genes, including TAG and glucose processing metabolic enzymes, components of several cell signalling processes and actin cytoskeleton regulators. Analysis of ORP2 interaction partners revealed novel connections of ORP2 with PI3K/Akt and RhoA signalling, important regulators of cell metabolic processes and the actin cytoskeleton. Consistently, the ORP2-KO impaired the hepatocellular energy metabolism by inhibiting the synthesis of TAGs and glycogen, and by reducing the rates of glucose uptake and glycolysis, suggesting an extensive contribution of ORP2 to cellular bioenergetics. In addition, I present novel clues of ORP2 function beyond the lipid and energy metabolism. In migrating cells, ORP2 was found to localize to lamellipodial cell surface protrusions, and the ORP2-KO, on the other hand, was found to impair the lamellipodia formation. Moreover, ORP2-KO resulted in an inhibition of cell adhesion, migration and proliferation, important cell physiological processes involving function of the actin cytoskeleton. The results point at a novel function of ORP2 as a lipid-sensing regulator of the actin cytoskeleton, which importantly involves the integration of cellular metabolism with growth, migration and cell signalling.
  • Saeed, Khalid (Helsingin yliopisto, 2018)
    The thesis addresses the impact of androgen deprivation in combination with gene functions in prostate cancer (PCa), as well as the development of ex vivo cancer models. The studies extended to assess of drug efficacies that may employed in future strategies for precision therapies in PCa. The patient-derived-cells (PDCs) from kidney cancer or renal cell carcinoma (RCC) patients further provided opportunities to study clonal evolution pathways, and to evaluate, how the intra-tumor heterogeneity may influence drug resistance at subclonal level. The biology of PCa revolves around androgen receptor (AR) signaling, thus, it remains the key target for therapeutic strategies. However, development of combinatorial therapies is of critical importance, since targeting AR alone is not enough for long-term benefits of patients. The two projects in the thesis address different avenues of finding novel treatment strategies for PCa. In the first project, RNA interference (RNAi) screens with 2068 human genes, was design to understand the combinatorial impact of individual gene functions and environmental challenges posed by androgen depletion in PCa. The screen identified several known genes, as well as novel ones, such as HSPBAP1, that inhibited proliferation exclusively in androgen-deprived PCa cells. HSPBAP1 was found to interact with AR in the cell nuclei, and its inhibition led to reduced AR-mediated transcription. The data suggested that the AR-interacting protein HSPBAP1 could be a potential target for intervention in combination with androgen-deprivation in PCa cells. Our second approach to discover treatment avenues for PCa was to generate novel PCa cell models from patient-derived material for ex vivo evaluation of drug efficacies. The PDCs from PCa patients displayed an AR-negative predominantly basal/transit-amplifying phenotype. The cancer culture retained cancer-specific copy number changes and exhibited a distinct drug response when profiled with 306 oncology compounds. The drugs included taxanes, bexarotene, tretinoin, oxaliplatin, mepacrine and navitoclax function independently of AR signaling, and most of these have already been explored in clinical trials for treating advanced PCa. Next, we took the strategy applied in PCa to RCC. Since RCC is well-recognized to be genetically heterogeneous disease, we explored the impact of intra-tumor heterogeneity and clonal evolution on drug efficacies based on multiple PDCs generated from the patients. The PDCs were sensitive to established drugs already applied in clinical practice for RCC treatment, such as the mTOR-inhibitor temsirolimus and multi-kinase-inhibitor pazopanib. The individual PDC models from the same patient exhibited diverse drug response patterns suggesting that clonal evolution of cancer directly contributes to differences in drug response. Furthermore, cure in advanced RCC will depend on the design of combinatorial treatments that block each such clone. In summary, in our first project on RNAi-based functional screening in PCa, we established that knockdown of the HSPBAP1 gene will synergize with androgen-depletion in inhibiting PCa cell growth. We then generated comprehensive drug sensitivity testing (DST) on ex vivo models of prostate and renal cancer cells with the aim to discover additional treatment options. DST profiles measured from patient-specific cancer models could in the future pave the way for exploring new indications for existing drugs, to prioritize drug leads and to allocate individualized therapies to cancer patients.
  • Samad, Samia (Helsingin yliopisto, 2018)
    Unlike annual plants, perennials have repeated cycling between the vegetative and generative stages. Studying the balance between these two phases would enable breeders to produce higher quality crops. The woodland strawberry is used as a model to study developmental patterns in perennials because it has a wide geographical distribution, a small sequenced genome, and a number of available natural mutants, which provide excellent resources for physiological, molecular and genetic studies. This thesis investigated the genetic and environmental coordination of shoot apical meristem (SAM) and axillary meristem (AXM) fates in woodland strawberry. In woodland strawberry, SAM forms an inflorescence after flower induction, whereas AXMs can differentiate either into runners or branch crowns that are able to form additional inflorescences. Genetic mapping and the experiments using transgenic lines and natural accessions with contrasting environmental responses showed that a number of genes regulated the balance between vegetative and generative development in woodland strawberry. In general, cool temperature or short days (SD) induced flowering and promoted AXM differentiation to branch crowns, while warm temperature and long days (LD) promoted runner formation. High levels of FvTERMINAL FLOWER1 (FvTFL1) expression in FvTFL1 overexpression lines and NOR1 accession inhibited flowering at temperatures of 10-22°C in both SD and LD, but the environmental control of AXM fate was not affected in these plants indicating that environment influenced AXM differentiation irrespective of flowering. In the seasonal flowering genotype, FvSUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (FvSOC1) was observed to quantitatively increase runner formation. The photoperiodic control of flowering and AXM fate was studied in more detail using FvCONSTANS (FvCO) and FvFLOWERING LOCUS T1 (FvFT1) transgenic lines. These studies showed that FvCO controls the expression of FvFT1, and they both have a major role in the control of the balance between the vegetative and generative development in SD and LD. Genetic mapping studies under differing environments identified five QTLs that, together, explained about half of the observed flowering time variance in the mapping population, and two additional QTLs were identified for the number of branch crowns explaining about 20% of variance. The flowering time QTL on LG6 colocalized with FvTFL1, and one of the QTL regions on LG4 that controlled both flowering time and AXM fate was close to the PFRU, a previously identified locus in the commercial strawberry. Among the previously unknown loci, two flowering time QTLs on LG7 colocalized with putative flowering time genes FvEARLY FLOWERING 6 (FvELF6) and FvCENTRORADIALIS1 (FvCEN1), a homolog of FvTFL1. Furthermore, a gene encoding TCP transcription factor and a homolog of DORMANCY ASSOCIATED MADS BOX (DAM) were identified as candidate genes in QTL regions controlling AXM fate on LG4 and LG5, respectively. This study shed new light into the genetic and environmental control of AXM and SAM fates providing new means to control the balance between vegetative and generative reproduction under different environmental conditions.
  • Cajanus, Kristiina (Helsingin yliopisto, 2018)
    Rintasyöpä on yksi maailman yleisimmistä syövistä. Diagnostiikan ja hoitokeinojen kehittyminen on merkittävästi parantunut taudin ennustetta. Kuitenkin joka vuosi tuhannet naiset saavat rintasyöpädiagnoosin ja läpikäyvät rintasyöpäleikkauksen toivoen parantumista. Nykyaikainen kirurgia vaatii tehokasta kivunlievitystä, sillä voimakas kipu altistaa elimistön merkittävälle rasitukselle. Elimistön suojaamiseksi leikkauksen aikana ja sen jälkeen potilaalle annostellaan kipulääkkeitä, joista opioidit ovat käytetyin kipulääkeryhmä keskivaikean ja vaikean postoperatiivisen kivun hoidossa. Opioidihoitoon liittyy kuitenkin vakavia haittavaikutuksia, kuten pahoinvointi ja hengityslama, joiden vuoksi kaikki opioidit joudutaan titraamaan analgeettiseen annokseen asteittain. Tämä pitkittää riittävän kivunlievityksen saavuttamiseen kuluvaa aikaa.. Oksikodoni on ollut vuosikausia Suomen käytetyin opioidi postoperatiivisen kivun hoidossa. Tämän väitöskirjan tarkoitus oli tutkia, onko oksikodonille määritettävissä analgeettista pitoisuutta sekä määrittää postoperatiivisen kivun voimakkuuteen ja oksikodonin tarpeeseen vaikuttavia tekijöitä. Näiden tekijöiden tunnistaminen helpottaisi riskipotilaiden tunnistamista sekä voimakkaan postoperatiivisen kivun että suuren oksikodonin tarpeen suhteen. Näin hoitohenkilökunta voisi jo ennalta varautua haastavaan postoperatiivisen kivun lievitykseen. Tutkimuspopulaatio koostui 1000 naisesta, jotka olivat menossa rintaleikkaukseen havaitun rintasyövän vuoksi. Potilaiden tauti affisioi vain toista rintaa, eikä heillä ollut havaittu metastaaseja. Ennen leikkausta potilaat täyttivät kyselyt demografisista tiedoistaan, sairaushistoriastaan, kipuoireistaan sekä masennus- ja ahdistustasostaan. Lisäksi potilaiden kuuma- ja kylmäkivun sieto sekä intensiteetti testattiin. Postoperatiivisesti kaikille potilaille annosteltiin suonensisäistä oksikodonia, kunnes he ilmaisivat olevansa kivuttomia. Postoperatiiviset kipuintensiteetit ja oksikodonin käyttö kirjattiin ensimmäisen 20 postoperatiivisen tunnin ajalta. Oksikodonille ei pystytty määrittämään analgeettista pitoisuutta. Postoperatiiviseen liikekivun voimakkuuteen assosioituvat tekijät olivat nuori ikä, kainaloevakuaatio (vs. vartijaimusolmukebiopsia), korkeampi kylmäkipuintensiteetti ja kylmäkivun sieto. Nämä tekijät selittivät yhteensä 8,5 % postoperatiivisen liikekivun voimakkuudesta. Postoperatiivisen liikekivun intensiteetti taas assosioitui vahvasti postoperatiiviseen oksikodonin kulutukseen, samoin kuin BMI, ikä, kainaloevakuaatio, OPRM1 genotyyppi ja preoperatiivinen kipu rinnassa. Nämä tekijät selittivät jopa 28 % oksikodonin tarpeen kokonaisvariaatiosta. Vain postoperatiivinen liikekivun voimakkuus ja kainaloevakuaatio assosioituivat oksikodonin analgeettiseen plasmakonsentraatioon. Yhdessä ne selittivät 17 % tämän variaatiosta. CYP2D6 genotyyppi ei assosioitunut postoperatiiviseen oksikodonitarpeeseen tai analgeettiseen plasma konsentraatioon, mutta se vaikutti plasman postoperatiivisiin oksimorfoni- ja noroksimorfonikonsentraatioihin. Väitöskirjan tulokset auttavat tunnistamaan rintasyöpäleikkauksen läpikäyneitä potilaita, joilla on korkea riski voimakkaalle postoperatiiviselle kivulle sekä suurelle oksikodonin tarpeelle.