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Canine hip dysplasia (CHD) is a complex developmental orthopedic disorder particularly common in large size breeds. CHD is characterized by the development of a loose and incongruent hip joint. Affected dogs often suffer from secondary osteoarthritis. Radiographic examination reveals flattening of the femoral head and joint widening. An analogous disorder exists in humans. CHD is inherited quantitatively with suggested involvement of the genes of major effect. Genetic studies utilizing dense SNP arrays have revealed few candidate loci and genes for CHD, including the intronic deletion variant of fibrillin 2 (FBN2) gene in Labrador Retriever breed. FBN2 is a promising candidate gene having a functional role in bone and joint development. Therefore, the objective of this study was to investigate the role of FBN2 in the development of CHD in the Finnish dog population and in additional breeds. The specific aims included establishment of well-phenotyped cohorts of samples from four high-risk breeds, namely Labrador Retriever, Golden Retriever, German Shepherd dog and Bernese Mountain Dog and the assessment of the presence and prevalence of the FBN2 deletion in the Finnish dog population affected by CHD. The study cohorts established here will be later utilized also in genome-wide association studies to identify additional CHD loci. Altogether 220 dogs, out of which 53 were Labrador Retrievers, 41 Golden Retrievers, 79 German Shepherd dogs and 47 Bernese Mountain Dogs, were included in the case-control study. For the German Shepherd dogs, the hip status of each dog was further confirmed of by screening the radiographs available from the Finnish Kennel Club archive. All dogs were genotyped by fragment analysis for the FBN2 deletion. The results revealed the presence of the deletion in all four breeds with highest prevalence in the Retriever breeds, in which the deletion haplotype was more common than the wild type. In our study, all CHD-affected dogs in the Labrador Retriever breed had at least one copy of the deletion allele. However, since the deletion allele was common also in the unaffected Labrador Retrievers, no statistically significant allelic association with the deletion and CHD was detected in statistical analysis. In the three other breeds, no association in statistical analysis was found either. Thus, the previously reported positive allelic association between the intronic deletion in the FBN2 gene and CHD was not replicated. Larger genome-wide studies are warranted to identify the major effect CHD loci.

Background: Despite the well-established link between diabetes and dementia risk, the impact of prediabetes and diabetes on the prodromal dementia phase remains controversial. In this study, we investigated whether prediabetes and diabetes increase the risk of cognitive impairment–no dementia (CIND) and accelerate its progression to dementia, as well as the possible underlying mechanisms. Methods: In the Swedish National Study on Aging and Care-Kungsholmen (SNAC-K), one cohort of cognitively-intact individuals (n=1,837) and one cohort of individuals with CIND (n=671) aged ≥60 years were followed for up to 15 years. At baseline and each follow-up (every 3 or 6 years), a neuropsychological test battery was administered, and the domains of episodic memory, processing speed, executive function, visuospatial abilities, and language were derived. CIND was defined as having no dementia and cognitive performance ≤1.5 SDs below age group-specific means in at least one cognitive domain. Dementia was diagnosed according to DSM-IV criteria. Diabetes (controlled and poorly-controlled) was diagnosed by physicians through medical assessment, clinical records, and glycated hemoglobin (HbA1c) ≥6.5%. Prediabetes was identified as HbA1c 5.7-6.4% in diabetes-free participants. Clinicians diagnosed heart disease and collected blood samples used to measure C-reactive protein (CRP). Data were analyzed with Cox regression models adjusted for possible confounders. Results: At baseline, in the cognitively-intact cohort, 133 (7%) participants had diabetes and 615 (34%) had prediabetes. During follow-up (mean 9.2 ± 3.0 years [range=2.2-15.5 years]), 544 (30%) individuals in the cognitively-intact cohort developed CIND. Poorly-controlled diabetes (HbA1c ≥7.5%) was associated with 2-times higher risk of CIND (HR 2.0, 95% CI:1.11-3.48) than diabetes-free participants. In the CIND cohort, 84 (13%) had diabetes and 238 (36%) prediabetes. During follow-up (mean 7.7 ± 4.0 years [range=0.2-15.2 years]), 132 (20%) individuals progressed to dementia. Poorly-controlled diabetes was associated with 3-times higher risk of dementia progression (HR 3.3, 95% CI: 1.29-8.33). Furthermore, comorbid heart disease and diabetes was associated with 2.5-times higher risk of progression to dementia (HR 2.5, 95% CI: 1.17-5.47), particularly if the diabetes was poorly-controlled (HR 5.8, 95% CI: 1.72-19.3). Similarly, having elevated CRP levels and diabetes was associated with increased risk of progression to dementia (HR 4.1, 95% CI: 1.15-14.2), especially in participants with poorly-controlled diabetes (HR 13.6, 95% CI: 1.89-98). No associations between prediabetes and CIND were detected in either cohort. Conclusions: Diabetes, especially if poorly-controlled, increases the risk of cognitive impairment and accelerates its progression to dementia. The diabetes-associated progression from CIND to dementia is further exacerbated by the presence of heart disease and elevated levels of systemic inflammation.

Non-native species can have complex effects on the abundance of native species potentially altering the functioning of ecosystems negatively. Invasive species can outcompete local species competing for resources, ultimately causing the extinction of local species. Inter- and intraspecific competition can be especially vigorous for limited resources. Invasive species have been thought to be a leading cause in native species extinction, and their effects on native species can be especially pronounced during reproductive crucial life-history stages, such as nest-building. Based on previous information about invasive species and their effects on ecosystems, and previous studies conducted related to invasive species, I conducted an experiment at the Tvärminne zoological station in Hanko, southern Finland during May and June of 2021. I conducted a laboratory experiment in which the test species used were the invasive fish species round goby, that has increased its range across the Baltic Sea rapidly, and the native fish species sand goby. The purpose was to see, if there was any effect the invasive species has on the nesting success and motivation of the native species. Methods included five different treatments in aquariums. The results did not differ statistically between different treatments, length was close to statistical significance. However, these results do not demonstrate, that the round goby has no effect on the nest building motivation of sand gobies. Some factors of the experimental setup might have been faulty, and future studies with a larger sample are needed to examine the effects of competition on native species’ abundance.

The main motivator of this thesis was to discover the importance of Chaoborus in a eutrophic and dystrophic lake and evaluate suitable restoration methods to enhance the state of the study lake. The role of Chaoborus in the study lake, Lake Jouttenus, was studied with sampling of both the water column and the sediment, echo-surveys, and diet analyses of fish. The sampling was planned as comprehensive, and the sampling stations were distributed across the whole lake area to examine the density and distribution of Chaoborus. The deeper areas of the lake were emphasized more in sampling because Chaoborus tend to favor those areas. The Chaoborus density was calculated with stratified sampling, which gives a more precise mean density estimate than simple random sampling. In addition, fish were caught on four (4) study occasions to find out if they had included Chaoborus in their diets. The mean density of Chaoborus in Lake Jouttenus was a little lower than expected, only 271 individuals/m2 in areas ≥2 m depth. The highest density of larvae was found from mid-depths between 6.0-7.9 m in the sediment. Only the deepest areas (≥8 m depth) had limnetic Chaoborus and more limnetic than benthic larvae. The mean length of larvae was 8.4 mm in the sediment and 9.0 mm in the water column. The length distributions appeared to be unimodal. Echo-surveys confirmed that the larvae occurred in the water column only in the deepest area in the north of the lake where the hypolimnion had a low oxygen concentration below 4 m depth. The diet analyses showed that roach and perch had eaten Chaoborus but the number of Chaoborus was high only in the diet of roach in early July. In comparison with other studied lakes, the density of Chaoborus was the lowest in Lake Jouttenus. In addition, the mean length of benthic Chaoborus in Lake Jouttenus was lower than in the other studied humic lakes. The distribution of limnetic Chaoborus appeared as restricted to the deepest areas in the lake and elsewhere the larvae occurred only in the sediment. The slow growth of Chaoborus and their low mean density could be explained by the lack of an efficient refuge and probably also the lack of resources. The larvae were unable to reach the epilimnion and their prey safely at daytime and/or the amount and quality of food items for the larvae were poor. The darkness caused by humic substances and low oxygen concentration in the hypolimnion created a refuge for the limnetic larvae only in the deepest area of Lake Jouttenus. Roach and perch ate Chaoborus occasionally. However, mass removal of fish is not recommended as it would decrease the predation pressure by fish on Chaoborus and increase the risk of Chaoborus population growth at the deepest areas and enable their range to extend. Instead of mass removal of fish all methods that aim in reducing the humic substances in the water especially at the lake catchment area might enhance the state of the lake. The clarification of water would diminish the living conditions for Chaoborus in long-term and help with controlling the other troubling factors such as Gonyostomum semen blooms in the study lake.

Background and objectives: Since early adolescence, the bedtimes and wake-up times begin to delay gradually until the early adulthood. This so-called shift to eveningness reaches its maximum at around the age of 20, and it usually occurs earlier in girls than boys. Eveningness has been previously associated with depression, anxiety, sleep problems, somatic symptoms, and other health-related issues in adolescents and adults. The aim of this study is to examine the associations between adolescents’ chronotype and their physical and mental well-being. Methods: This study examined how the self-reported chronotype was associated with self-reported problems related to adolescents’ physical and mental well-being. The chronotypes were divided into 5 types: Definitive Morning-types, Moderate Morning-types, Intermediate-types, Moderate Evening-types, and Definitive Evening-types. The participants were 7th, 8th and 9th graders, and the sample consisted of 6522 students from 83 schools in Finland. Some of the data was gathered at three time points, some at two time points, and some at one time point during the academic year. The associations between chronotype and well-being variables were studied cross-sectionally and some of them also longitudinally. Results: The main findings were that eveningness was associated with difficulty concentrating in lessons, susceptibility to give up easily on difficult tasks, school burnout symptoms, feelings of nervousness and anxiety, excessive worrying, difficulty relaxing, irritability, restlessness, difficulty falling asleep, waking up at night, daytime tiredness, and low mood as compared to morningness. Eveningness was also associated with neck and shoulder pain, lower back pain, and headache, as well as pain in the head and lower back due to the use of digital devices. Eveningness was associated with decreased concentration in lessons and increased susceptibility to give up on difficult tasks across time. On the other hand, feeling lonely and not being accepted as part of the group were associated with morningness. Conclusions: In conclusion, the physical and mental health problems were emphasized among Evening-type adolescents, as compared to Morning-type adolescents. Since adolescents shift toward eveningness, the need for thorough management of sleep and circadian problems should be highlighted, in order to intervene and improve the mental and physical well-being of adolescents both at school and at home.

The motivation of this study was to find new treatment options for the rare cancer pseudomyxoma peritonei (PMP). PMP is a slowly progressing mucinous adenocarcinoma that originates from the appendix and disseminates into the peritoneum where the cancer cells secrete large amounts of MUC2, the main component of intestinal mucus, into the peritoneum. The disulfide isomerase AGR2 helps MUC2 with forming the correct intramolecular disulfide bonds prior secretion, and is essential to MUC2 protein production. The mucus build-up into the peritoneum causes stress on vital organs, and eventually death. Therefore, inhibiting MUC2 production in PMP cancer cells might slow down the disease progression significantly. MAPK/ERK and cAMP/PKA signaling pathways stimulate MUC2 production, and activating mutations in KRAS and GNAS of these pathways are common in PMP. The aim of this study was to elucidate how MUC2 and AGR2 affect each other’s expression levels, and how the MAPK/ERK pathway and the cAMP/PKA pathway targeting substances caffeine, theophylline, cromolyn, fudosteine, octreotide, and lanreotide, affect MUC2 expression in vitro. This study was conducted on human colorectal adenocarcinoma cell lines LS174T, LoVo, and HT29 that all produce large amounts of MUC2. In addition, LS174T and LoVo cell lines carry activating heterozygous mutations in their KRAS genes. MUC2 and AGR2 expression levels were measured on mRNA level with real-time quantitative reverse transcriptase polymerase chain reaction. The effects of MUC2 on AGR2 expression, and vice versa, were tested by silencing each at a time with the appropriate siRNA. MUC2 siRNA suppressed both MUC2 and AGR2 mRNA levels down to 50 % in LS174T cells and down to 40 % in LoVo cells in respect to their control groups. AGR2 siRNA suppressed AGR2 mRNA levels down to 50 % in LS174T cells and even down to 30 % in LoVo cells in respect to their control groups, while there were no statistically significant changes in MUC2 mRNA levels. Caffeine and theophylline inhibit phosphodiesterase of the cAMP/PKA signaling pathway, and in consequence, the hydrolysis of the secondary messenger cAMP, prolonging the activation of the pathway. Caffeine stimulated MUC2 production in LS174T and LoVo cells. In addition, AGR2 mRNA levels increased in LoVo cells, in which the fold change in MUC2 mRNA levels was much greater. Theophylline, a compound found in tea, and used for the treatment of asthma, did not affect MUC2 production. PMP cancer cells express protein S100P. Cromolyn is a pharmaceutical substance used for the treatment of asthma, and it inhibits S100P, and thereby S100P/RAGE signaling -dependent activation of MAPK/ERK pathway. Fudosteine, on the other hand, is a mucoactive pharmaceutical substance that lowers the production of MUC5AC, the main component of lung mucus. Since activating GNAS mutation stimulates the production of both MUC2 and MUC5AC in HT29 cells, the expression of both must be at least partially controlled by same mechanisms. In addition, Somatostatin analogues octreotide and lanreotide inhibit ERK1/2 of the MAPK/ERK pathway, as well as protein kinase A of the cAMP/PKA pathway, and hence cAMP production. However, none of these four tested pharmaceutical substances were able to inhibit MUC2 production in the cell lines used this study in vitro.

Kainate receptors (KARs) act as prominent regulators of neuronal excitability, network activity as well as neurotransmitter release in the developing brain. In the neonatal hippocampus the GluK1 subunit containing KARs take part in regulating the activity of the CA3 interneurons and hence the maintenance of early synchronous network oscillations, which are thought to be vital for developing connections. In the interneurons of the CA3 subfield this regulatory activity is likely performed through a noncanonical, G-protein mediated inhibition of a Ca2+ sensitive medium-duration afterhyperpolarizing current (ImAHP). As in various central neurons the ImAHP has been shown to be regulated by voltage-gated calcium channels (VGCCs) and as the activity of the voltage-gated calcium channels has been in turn shown to be modulated by G-protein coupled signaling of GluK1 KARs, we went on to investigate whether a direct link between GluK1 KARs and VGCCs could be detected in the CA3 stratum radiatum interneurons of neonatal hippocampus. Here we show that the pharmacological inhibition of GluK1 KARs does not affect the amplitude of Ca2+ influx through VGCCs in the CA3 stratum radiatum interneurons of acute hippocampal slices from neonatal mice. As G-protein mediated signaling has been shown to induce alterations in the voltage-dependence of the VGCC-mediated currents, we similarly investigated the effects of GluK1 inhibition on the current-voltage relationship of Ca2+ currents in CA3 interneurons during the first postnatal week as well as during the second postnatal week, since GluK1 subunit is known to undergo developmental changes in its expression during this time. No significant effect was however detected in either of the age groups. Although in our experiments the GluK1-KAR inhibition seemed to induce no statistically significant changes in the Ca2+ current amplitudes or in the voltage-dependence of VGCC-mediated currents in the CA3 interneurons, further, more specific studies should be encouraged to investigate the phenomenon in specific interneuron subtypes and in distinct calcium channel families.

Epileptic patients experience spontaneous recurrent seizures and interictal epileptiform discharges that lead to brain injuries, triggering neuroinflammation and waste product accumulation. Due to the detrimental effect of waste products on brain homeostasis, their removal from the central nervous system is (CNS) is crucial. Meningeal lymphatic vessels (mLVs) located in dura matter contribute to CNS clearance by the drainage of metabolites, waste products, and immune cells from subarachnoid space into cervical lymph nodes. Therefore, because of its role in brain homeostasis, the study of mLVs in different neurological conditions and diseases, including TLE, has gotten increased attention in the last decade. In this study, we sought to understand mLVs role in neuroinflammation and changes in rapid eye movement (REM) sleep stage during epilepsy. For this purpose, we induced mLVs ablation followed by kainic acid (KA) epilepsy model in mice. Shortly, animals were inoculated with AAV-VEGFR3-1-4 to induce mLVs ablation and subsequently challenged with KA to induce status epilepticus. Simultaneously, a control group of animals were injected with a sham AAV and later injection of KA. Afterward, spontaneous EEG activity was registered continuously, and data analysed to compare durations of REM sleep. Also, immunohistochemistry of brain samples was performed to investigate neuroinflammatory changes between experimental groups. Ex-vivo analyses of Iba1 and GFAP expression in brain tissue did not show statistically significant changes in neuroinflammation between experimental groups. However, we observed a trend towards lower expression of inflammatory markers in mLVs ablated animals. The analysis of REM sleep duration shows a progressive reduction of this sleep stage in both groups during the first recording period with a subsequent stabilization during the second one. Our data also indicate that mLVs ablated animals present prolonged REM sleep duration compared to the control group. Although this data contradicts our initial hypothesis it is consistent with the well-established negative correlation between neuroinflammation and REM sleep duration. Future studies should consider a deeper analysis of the glial cell profile for a better understanding of the effect of mLVs dysfunction on epileptic pathology. Moreover, the impact of mLVs ablation on REM sleep duration should be characterized in healthy animals.

Colorectal cancer (CRC), which refers to the cancer of the colon and the rectum currently ranks as the second leading cause of cancer related death worldwide and as the third most common form of cancer in both males and females. The latest reports show that approximately 10% of all new cancer cases globally are diagnosed as CRC annually. Initiation of sporadic CRC is commonly caused by somatic mutations causing the loss of function of the tumor suppressor gene APC. This leads to aberrant activation of the canonical Wnt signalling pathway. The ApcMin/+ mice model the progression of CRC as they carry a constitutive heterozygous nonsense mutation in Apc allele and develop intestinal adenomas. TCF/LEF transcription factor family members are best known as the main downstream effectors of canonical Wnt signalling. In the presence of nuclear β-catenin, TCF/LEF proteins bind to it through their β-catenin-binding domain and activate the transcription of Wnt target genes. The TCF7 gene encodes several isoforms of TCF1 protein, which are traditionally divided into long and short isoforms, transcribed from different promoters. Previously, it has been shown that Tcf7 deletion (Tcf7-/-) in ApcMin/+ mice increases the formation of adenomas. The aim of my study is to better understand the role of Tcf7 and its isoforms in CRC tumorigenesis. To study the Tcf7 deletion in intestinal adenoma development, ApcMin/+; Tcf7mut/mut; Villin CreERT2 mouse strain was established. The expression of the full-length isoforms (p45) is constitutively prevented in the Tcf7mut/mut mice. Moreover, tamoxifen administration to these mice led to the deletion of all isoforms in the intestinal epithelium. The number of intestinal tumors, their sizes and the survival of the Tcf7 deleted ApcMin/+ mice were analyzed and compared to ApcMin/+ mice. Intestinal tissues of the mice were collected after euthanasia. The tissue samples were preserved in paraffin, and later cut into sections for IHC, stained and imaged. The deletion of Tcf7 was confirmed at the RNA level by qPCR, and at the protein level by immunohistochemistry (IHC). IHC and single-cell RNA sequencing was used to further analyze the effect of Tcf7 deletion in mouse intestinal adenomas. The deletion of all Tcf7 isoforms or only the p45 isoforms in ApcMin/+ mice increased robustly the numbers of intestinal tumors. IHC analysis of the intestinal adenomas showed that the deletion of p45 isoforms was sufficient to cause a dramatic decrease in total Tcf1 expression in the adenoma cells. These results were supported by the qPCR results. In summary, our results lead us to believe that the deletion of p45 isoforms causes an acceleration of tumorigenesis in the adenoma model. Without the Apc mutation, the mice did not develop intestinal adenomas. Interestingly, the expression of the Wnt-target gene Prox1 in intestinal adenomas was decreased when Tcf7 was deleted. We next aim to optimize our protocol for single cell dissociation of adenomas and re-run the single-cell RNA sequencing analysis for further analysis of the mechanisms behind the increased tumorigenesis.

Climate change poses an ever-increasing threat on biodiversity as the global mean temperature rises causing changes in weather patterns. Species will have to adapt to the circumstances or follow their climatic niches across space to avoid decline and extinction. Many species are already threatened by extinction due to climate change. Understanding how species are reacting to rising temperatures can help us preserve biodiversity. Genetic adaptation is a long process and takes several generations to occur. A more immediate means to cope with variation is adjusting through phenotypic plasticity, which can help species cope with environmental changes in the short-term. Plasticity can help individuals maintain fitness in different environments and with fluctuating environmental conditions. Flowering phenology is a plastic trait which can have a large impact on reproductive success. Flowering is an important part of a plant’s life cycle as it can produce offspring with new combinations of genes. In this thesis I examine how temperature affects the flowering phenology of Hypericum species and how this thermal plasticity affects fitness. Populations of Hypericum perforatum, H. maculatum and H. montanum from different parts of their distribution across Europe were studied in greenhouse experiments. The plants were grown in four different temperature treatments (16/6°C, 20/10°C, 24/14°C, 28/20°C) and the timing of first flowering was monitored. Seed mass and flower count were recorded and used as measures of fitness. In general, the plants flowered later in the colder temperature treatments. The results differed between species: in H. maculatum the leading-edge populations were less plastic while in H. perforatum differences between areas were negligible. More plastic accessions produced more flowers due to earlier flowering. There was no effect on seed mass. The possible effects of plasticity on overall fitness highlight the need for detailed information on plasticity for predicting species response to climate change.