Responses of soil respiration and barley growth to modified supply of oxygen in the soil

Selostus: Maan hengityksen ja ohran kasvun reagointi hapensaannin muutoksiin maassa

Neuropeptide Y at the cellular level – Studies on PreproNPY L7P Polymorphism and the Mitochondrial Form of NPY

Neuropeptide Y (NPY) is a widely expressed neurotransmitter in the central and peripheral nervous systems. Thymidine 1128 to cytocine substitution in the signal sequence of the preproNPY results in a single amino acid change where leucine is changed to proline. This L7P change leads to a conformational change of the signal sequence which can have an effect on the intracellular processing of NPY. The L7P polymorphism was originally associated with higher total and LDL cholesterol levels in obese subjects. It has also been associated with several other physiological and pathophysiological responses such as atherosclerosis and T2 diabetes. However, the changes on the cellular level due to the preproNPY signal sequence L7P polymorphism were not known. The aims of the current thesis were to study the effects of the [p.L7]+[p.L7] and the [p.L7]+[p.P7] genotypes in primary cultured and genotyped human umbilical vein endothelial cells (HUVEC), in neuroblastoma (SK-N-BE(2)) cells and in fibroblast (CHO-K1) cells. Also, the putative effects of the L7P polymorphism on proliferation, apoptosis and LDL and nitric oxide metabolism were investigated. In the course of the studies a fragment of NPY targeted to mitochondria was found. With the putative mitochondrial NPY fragment the aim was to study the translational preferences and the mobility of the protein. The intracellular distribution of NPY between the [p.L7]+[p.L7] and the [p.L7]+[p.P7] genotypes was found to be different. NPY immunoreactivity was prominent in the [p.L7]+[p.P7] cells while the proNPY immunoreactivity was prominent in the [p.L7]+[p.L7] genotype cells. In the proliferation experiments there was a difference in the [p.L7]+[p.L7] genotype cells between early and late passage (aged) cells; the proliferation was raised in the aged cells. NPY increased the growth of the cells with the [p.L7]+[p.P7] genotype. Apoptosis did not seem to differ between the genotypes, but in the aged cells with the [p.L7]+[p.L7] genotype, LDL uptake was found to be elevated. Furthermore, the genotype seemed to have a strong effect on the nitric oxide metabolism. The results indicated that the mobility of NPY protein inside the cells was increased within the P7 containing constructs. The existence of the mitochondria targeted NPY fragment was verified, and translational preferences were proved to be due to the origin of the cells. Cell of neuronal origin preferred the translation of mature NPY (NPY1-36) when compared to the non neuronal cells that translated both, NPY and the mitochondrial fragment of NPY. The mobility of the mitochondrial fragment was found to be minimal. The functionality of the mitochondrial NPY fragment remains to be investigated. L7P polymorphism in the preproNPY causes a series of intracellular changes. These changes may contribute to the state of cellular senescence, vascular tone and lead to endothelial dysfunction and even to increased susceptibility to diseases, like atherosclerosis and T2 diabetes.

Cyanobacteria from the Baltic Sea and Finnish lakes as an energy source and modulators of bioenergetic pathways

Cyanobacteria are a diverse group of oxygenic photosynthetic bacteria that inhabit in a wide range of environments. They are versatile and multifaceted organisms with great possibilities for different biotechnological applications. For example, cyanobacteria produce molecular hydrogen (H2), which is one of the most important alternatives for clean and sustainable energy. Apart from being beneficial, cyanobacteria also possess harmful characteristics and may become a source of threat to human health and other living organisms, as they are able to form surface blooms that are producing a variety of toxic or bioactive compounds. The University of Helsinki Culture Collection (UHCC) maintains around 1,000 cyanobacterial strains representing a large number of genera and species isolated from the Baltic Sea and Finnish lakes. The culture collection covers different life forms such as unicellular and filamentous, N2-fixing and non-N2-fixing strains, and planktonic and benthic cyanobacteria. In this thesis, the UHCC has been screened to identify potential strains for sustainable biohydrogen production and also for strains that produce compounds modifying the bioenergetic pathways of other cyanobacteria or terrestrial plants. Among the 400 cyanobacterial strains screened so far, ten were identified as high H2-producing strains. The enzyme systems involved in H2 metabolism of cyanobacteria were analyzed using the Southern hybridization approach. This revealed the presence of the enzyme nitrogenase in all strains tested, while none of them are likely to have contained alternative nitrogenases. All the strains tested, except for two Calothrix strains, XSPORK 36C and XSPORK 11A, were suggested to contain both uptake and bidirectional hydrogenases. Moreover, 55 methanol extracts of various cyanobacterial strains were screened to identify potent bioactive compounds affecting the photosynthetic apparatus of the model cyanobacterium, Synechocystis PCC 6803. The extract from Nostoc XPORK 14A was the only one that modified the photosynthetic machinery and dark respiration. The compound responsible for this effect was identified, purified, and named M22. M22 demonstrated a dual-action mechanism: production of reactive oxygen species (ROS) under illumination and an unknown mechanism that also prevailed in the dark. During summer, the Baltic Sea is occupied by toxic blooms of Nodularia spumigena (hereafter referred to as N. spumigena), which produces a hepatotoxin called nodularin. Long-term exposure of the terrestrial plant spinach to nodularin was studied. Such treatment resulted in inhibition of growth and chlorosis of the leaves. Moreover, the activity and amount of mitochondrial electron transfer complexes increased in the leaves exposed to nodularin-containing extract, indicating upregulation of respiratory reactions, whereas no marked changes were detected in the structure or function of the photosynthetic machinery. Nodularin-exposed plants suffered from oxidative stress, evidenced by oxidative modifications of various proteins. Plants initiated strategies to combat the stress by increasing the levels of alpha-tocopherol, mitochondrial alternative oxidase (AOX), and mitochondrial ascorbate peroxidase (mAPX).

Mitochondrial disease in Southwestern Finland. Population-based molecular genetic and clinical studies

Mitochondria are present in all eukaryotic cells. They enable these cells utilize oxygen in the production of adenosine triphosphate in the oxidative phosphorylation system, the mitochondrial respiratory chain. The concept ‘mitochondrial disease’ conventionally refers to disorders of the respiratory chain that lead to oxidative phosphorylation defect. Mitochondrial disease in humans can present at any age, and practically in any organ system. Mitochondrial disease can be inherited in maternal, autosomal dominant, autosomal recessive, or X-chromosomal fashion. One of the most common molecular etiologies of mitochondrial disease in population is the m.3243A>G mutation in the MT-TL1 gene, encoding mitochondrial tRNALeu(UUR). Clinical evaluation of patients with m.3243A>G has revealed various typical clinical features, such as stroke-like episodes, diabetes mellitus and sensorineural hearing loss. The prevalence and clinical characteristics of mitochondrial disease in population are not well known. This thesis consists of a series of studies, in which the prevalence and characteristics of mitochondrial disease in the adult population of Southwestern Finland were assessed. Mitochondrial haplogroup Uk was associated with increased risk of occipital ischemic stroke among young women. Large-scale mitochondrial DNA deletions and mutations of the POLG1 gene were the most common molecular etiologies of progressive external ophthalmoplegia. Around 1% of diabetes mellitus emerging between the ages 18 – 45 years was associated with the m.3243A>G mutation. Moreover, among these young diabetic patients, mitochondrial haplogroup U was associated with maternal family history of diabetes. These studies demonstrate the usefulness of carefully planned molecular epidemiological investigations in the study of mitochondrial disorders.