Induced defence responses of cereals to pathogen attack

Selostus: Viljojen puolustusreaktiot taudinaiheuttajia vastaan

Macroalgal Defenses Against Herbivory: Causes and Consequences of Intraspecific Variation

In marine benthic communities, herbivores consume a considerable proportion of primary producer biomass and, thus, generate selection for the evolution of resistance traits. According to the theory of plant defenses, resistance traits are costly to produce and, consequently, inducible resistance traits are adaptive in conditions of variable herbivory, while in conditions of constant/strong herbivory constitutive resistance traits are selected for. The evolution of resistance plasticity may be constrained by the costs of resistance or lack of genetic variation in resistance. Furthermore, resource allocation to induced resistance may be affected by higher trophic levels preying on herbivores. I studied the resistance to herbivory of a foundation species, the brown alga Fucus vesiculosus. By using factorial field experiments, I explored the effects of herbivores and fish predators on growth and resistance of the alga in two seasons. I explored genetic variation in and allocation costs of resistance traits as well as their chemical basis and their effects on herbivore performance. Using a field experiment I tested if induced resistance spreads via water-borne cues from one individual to another in relevant ecological conditions. I found that in the northern Baltic Sea F. vesiculosus communities, strength of three trophic interactions strongly vary among seasons. The highly synchronized summer reproduction of herbivores promoted their escape from the top-down control of fish predators in autumn. This resulted into large grazing losses in algal stands. In spring, herbivore densities were low and regulated by fish, which, thus,enhanced algal growth. The resistance of algae to herbivory increased with an increase in constitutive phlorotannin content. Furthermore, individuals adopted induced resistance when grazed and when exposed to water-borne cues originating from grazing of conspecific algae both in the laboratory and in field conditions. Induced resistance was adopted to a lesser extent in the presence of fish predators. The results in this thesis indicate that inducible resistance in F. vesiculosus is an adaptation to varying herbivory in the northern Baltic Sea. The costs of resistance and strong seasonality of herbivory have likely contributed to the evolution of this defense strategy. My findings also show that fish predators have positive cascading effects on F. vesiculosus which arise via reduced herbivory but possibly also through reduced resource allocation to resistance. I further found evidence that the spread of resistance via water-borne cues also occurs in ecologically realistic conditions in natural marine sublittoral. Thus, water-borne induction may enable macroalgae to cope with the strong grazing pressure characteristic of marine benthic communities. The results presented here show that seasonality can have pronounced effects on the biotic interactions in marine benthic communities and thereafter influence the evolution of resistance traits in primary producers.

Alpha2-Adrenoceptor-mediated vascular responses induced by dexmedetomidine

Alpha2-Adrenoceptors are cell-surface G protein coupled receptors that mediate many of the effects of the catecholamines noradrenaline and adrenaline. The three human α2-adrenoceptor subtypes are widely expressed in different tissues and organs, and they mediate many different physiological and pharmacological effects in the central and peripheral nervous system and as postsynaptic receptors in target organs. Previous studies have demonstrated that α2-adrenoceptors mediate both vascular constriction and dilatation in humans. Large inter-individual variation has been observed in the vascular responses to α2-adrenoceptor activation in clinical studies. All three receptor subtypes are potential drug targets. It was therefore considered important to further elucidate the details of adrenergic vascular regulation and its genetic variation, since such knowledge may help to improve the development of future cardiovascular drugs and intensive care therapies. Dexmedetomidine is the most selective and potent α2-adrenoceptor agonist currently available for clinical use. When given systemically, dexmedetomidine induces nearly complete sympatholysis already at low concentrations, and postsynaptic effects, such vasoconstriction, can be observed with increasing concentrations. Thus, local infusions of small doses of dexmedetomidine into dorsal hand veins and the application of pharmacological sympathectomy with brachial plexus block provide a means to assess drug-induced peripheral vascular responses without interference from systemic pharmacological effects and autonomic nervous system regulation. Dexmedetomidine was observed to have biphasic effects on haemodynamics, with an initial decrease in blood pressure at low concentrations followed by substantial increases in blood pressure and coronary vascular resistance at high concentrations. Plasma concentrations of dexmedetomidine that significantly exceeded the recommended therapeutic level did not reduce myocardial blood flow below the level that is observed with the usual therapeutic concentrations and did not induce any evident myocardial ischaemia in healthy subjects. Further, it was demonstrated that dexmedetomidine also had significant vasodilatory effects through activation of endothelial nitric oxide synthesis, and thus when the endothelial component of the blood vessel response to dexmedetomidine was inhibited, peripheral vasoconstriction was augmented. Hand vein constriction responses to α2-adrenoceptor activation by dexmedetomidine were only weakly associated with the constriction responses to α1-adrenoceptor activation, pointing to independent cellular regulation by these two adrenoceptor classes. Substantial inter-individual variation was noted in the venous constriction elicited by activation of α2-adrenoceptors by dexmedetomidine. In two study populations from two different continents, a single nucleotide polymorphism in the PRKCB gene was found to be associated with the dorsal hand vein constriction response to dexmedetomidine, suggesting that protein kinase C beta may have an important role in the vascular α2-adrenoceptor signalling pathways activated by dexmedetomidine.

Interleukin-4 induced leukocyte differentiation

Monocytes, macrophages and dendritic cells (DCs) are important mediators of innate immune system, whereas T lymphocytes are the effector cells of adaptive immune responses. DCs play a crucial role in bridging innate and adaptive immunity. Naïve CD4+ Th progenitors (Thp) differentiate to functionally distinct effector T cell subsets including Th1, Th2 and Th17 cells, which while being responsible for specific immune functions have also been implicated in pathological responses, such as autoimmunity, asthma and allergy. The main objective of this thesis is to dissect the signalling networks involved in the IL-4 induced differentiation of two important leukocyte subtypes, Th2 cells and DCs. Gene expression profiling lead to identification of over 200 genes which are differentially expressed during cytokine induced differentiation of human monocytes to DCs or macrophages and which are likely to be essential for the proper biological functions of these cell types. Transcriptome analysis demonstrated the dynamic regulation of gene expression by IL-12 and IL-4 during the initiation of Th cell differentiation, which was partly counteracted by an immunosuppressive cytokine, TGFβ, present in the culture media. Results from RNAi mediated gene knockdown experiments and global gene expression analysis elucidated that SATB1 regulates multiple genes important for Th cell polarization or function as well as may compete with GATA3 for the reciprocal regulation of IL-5 transcription. In conclusion, the results obtained have extended our system-level understanding of the immune cell differentiation processes and provide an excellent basis for the further functional studies which could lead to development of improved therapeutic approaches for a range of immunological conditions.

Viral and cellular modulation of virus-induced apoptosis in experimental infection models

The aim of this study was to investigate herpes simplex virus type 1 (HSV-1)- and measles virus (MV)-induced cell death. HSV-1 with deletion in genes encoding infected cell protein (ICP)4 and protein kinase Us3 (d120) induced apoptosis and cathepsin activation in epithelial (HEp-2) and monocytic (U937) cells. Inhibition of cathepsin activity decreased the amount of d120-induced apoptosis indicating that d120-induced apoptosis could be cathepsin-mediated. Also, HSV-1 infection increased caspase activation suggesting that d120-induced apoptosis is probably caspase-mediated. Cystatin treatment decreased the activity of cathepsins and the replication of HSV-1 indicating that cathepsins contribute to HSV-1 infection. Interestingly, d120 induced also necroptosis in monocytic cells. This is the first report on necroptosis in HSV-1- infected cells. MV induced apoptosis in uninfected bystander T lymphocytes, probably via interaction of MV-infected monocytes with uninfected lymphocytes. The expression of death receptor Fas was clearly increased on the surface of lymphocytes. The number of apoptotic cells and the activation of cathepsins and caspases were increased in MVinfected U937 cells suggesting that MV-induced apoptosis could be cathepsin- and caspase-mediated. Cystatin treatment inhibited cathepsin activities but not MV-induced apoptosis. Besides HSV-1-induced apoptosis, innate immune responses were studied in HSV-1-infection. HSV-1 viruses with either ICP4 and Us3, or Us3 deletion only, increased the expression of Toll-like receptor (TLR)3 and stimulated its downstream pathways leading to increased expression of type I interferon gene and to functional interferons. These findings suggest that besides controlling apoptosis, HSV-1 ICP4 and Us3 genes are involved in the control of TLR3 response in infected cell.

Regulation of lymphocyte response in vitro and in vivo

CD4+ T helper (Th) cells have an important role in the defence against diverse pathogens. Th cells can differentiate into several functionally distinct subtypes including Th1 and Th2 cells. Th1 cells are important for eradicating intracellular pathogens, whereas Th2 cells pro¬tect our body against extracellular parasites. However if uncontrolled, Th cells can mediate immunopathology such as asthma or allergies, but inappropriate Th response can also lead to autoimmune diseases such as multiple sclerosis or type 1 diabetes. Deeper knowledge of the regulation of the lymphocyte response both in vitro and in vivo is important for un¬derstanding the pathogenesis of immune-mediated diseases and microbe-host interactions. In the work presented in this thesis, the first goal was to elucidate the role of novel factors, PIM kinases and c-FLIP in the regulation of human Th cell differentiation. The oncogenic serine-threonine kinases of the PIM family were shown to be preferentially expressed in Th1 cells and in addition, by using RNA interference, they were also shown to be positive regulators of Th1 differentiation. The PIM depletion experiments suggest that PIM kinases promote the expression of the hallmark cytokine of Th1 cells, IFNγ, and influence the IL12/STAT4 pathway during the early Th1 cell differentiation. In addition to cytokine and T cell receptor (TCR) induced pathways, caspase activity has been shown to regulate Th cell proliferation. In the work presented in this thesis, the two isoforms of the caspase regulator protein, c-FLIP, were shown to be differentially ex¬pressed in Th1 and Th2 cells. Both of the isoforms were up-regulated in response to TCR activation, but the expression of the short isoform was selectively induced by IL4, the Th2 inducing cytokine. Furthermore, the c-FLIP isoforms had distinct and opposite roles during the early differentiation of Th1 and Th2 cells. The knockdown of the long isoform of c-FLIP led to the induction of Th1 marker genes, such as IFNγ and TBET, whereas the depletion of c-FLIP short down-regulated Th2 marker genes IL-4 and GATA3. The third goal was to elucidate the gene expression profiles characterizing the T- and B-lymphocyte responses in vivo during experimental infection by intracellular bacte¬rium Chlamydia pneumoniae. Previously, it has been shown that CD8+ and CD4+ T cells are important for the protection against Chlamydia pneumoniae. In this study, the analysis revealed up-regulation of interferon induced genes during recurrent infection underlining the importance of IFNγ secreted by Th1 and CD8+ T cells in the protection against this pathogen. Taken together, in this study novel regulators of Th cell differ¬entiation were discovered and in addition the gene expression profiles of lymphocytes induced by Chlamydia pneumoniae infection were characterized.