Evaluation of methods and applications for behavioural profiling of transgenic mice

During the last 10-15 years interest in mouse behavioural analysis has evolved considerably. The driving force is development in molecular biological techniques that allow manipulation of the mouse genome by changing the expression of genes. Therefore, with some limitations it is possible to study how genes participate in regulation of physiological functions and to create models explaining genetic contribution to various pathological conditions. The first aim of our study was to establish a framework for behavioural phenotyping of genetically modified mice. We established comprehensive battery of tests for the initial screening of mutant mice. These included tests for exploratory and locomotor activity, emotional behaviour, sensory functions, and cognitive performance. Our interest was in the behavioural patterns of common background strains used for genetic manipulations in mice. Additionally we studied the behavioural effect of sex differences, test history, and individual housing. Our findings highlight the importance of careful consideration of genetic background for analysis of mutant mice. It was evident that some backgrounds may mask or modify the behavioural phenotype of mutants and thereby lead to false positive or negative findings. Moreover, there is no universal strain that is equally suitable for all tests, and using different backgrounds allows one to address possible phenotype modifying factors. We discovered that previous experience affected performance in several tasks. The most sensitive traits were the exploratory and emotional behaviour, as well as motor and nociceptive functions. Therefore, it may be essential to repeat some of the tests in naïve animals for assuring the phenotype. Social isolation for a long time period had strong effects on exploratory behaviour, but also on learning and memory. All experiments revealed significant interactions between strain and environmental factors (test history or housing condition) indicating genotype-dependent effects of environmental manipulations. Several mutant line analyses utilize this information. For example, we studied mice overexpressing as well as those lacking extracellular matrix protein heparin-binding growth-associated molecule (HB-GAM), and mice lacking N-syndecan (a receptor for HB-GAM). All mutant mice appeared to be fertile and healthy, without any apparent neurological or sensory defects. The lack of HB-GAM and N-syndecan, however, significantly reduced the learning capacity of the mice. On the other hand, overexpression of HB-GAM resulted in facilitated learning. Moreover, HB-GAM knockout mice displayed higher anxiety-like behaviour, whereas anxiety was reduced in HB-GAM overexpressing mice. Changes in hippocampal plasticity accompanied the behavioural phenotypes. We conclude that HB-GAM and N-syndecan are involved in the modulation of synaptic plasticity in hippocampus and play a role in regulation of anxiety- and learning-related behaviour.

Heparin-binding growth-associated molecule (HB-GAM) in activity-dependent neuronal plasticity in hippocampus

Cell adhesion and extracellular matrix (ECM) molecules play a significant role in neuronal plasticity both during development and in the adult. Plastic changes in which ECM components are implicated may underlie important nervous system functions, such as memory formation and learning. Heparin-binding growthassociated molecule (HB-GAM, also known as pleiotrophin), is an ECM protein involved in neurite outgrowth, axonal guidance and synaptogenesis during perinatal period. In the adult brain HB-GAM expression is restricted to the regions which display pronounced synaptic plasticity (e.g., hippocampal CA3-CA1 areas, cerebral cortex laminae II-IV, olfactory bulb). Expression of HB-GAM is regulated in an activity-dependent manner and is also induced in response to neuronal injury. In this work mutant mice were used to study the in vivo function of HB-GAM and its receptor syndecan-3 in hippocampal synaptic plasticity and in hippocampus-dependent behavioral tasks. Phenotypic analysis of HBGAM null mutants and mice overexpressing HB-GAM revealed that opposite genetic manipulations result in reverse changes in synaptic plasticity as well as behavior in the mutants. Electrophysiological recordings showed that mice lacking HB-GAM have an increased level of long-term potentiation (LTP) in the area CA1 of hippocampus and impaired spatial learning, whereas animals with enhanced level of HB-GAM expression have attenuated LTP, but outperformed their wild-type controls in spatial learning. It was also found that GABA(A) receptor-mediated synaptic transmission is altered in the transgenic mice overexpressing HB-GAM. The results suggest that these animals have accentuated hippocampal GABAergic inhibition, which may contribute to the altered glutamatergic synaptic plasticity. Structural studies of HB-GAM demonstrated that this protein belongs to the thrombospondin type I repeat (TSR) superfamily and contains two β-sheet domains connected by a flexible linker. It was found that didomain structure is necessary for biological activity of HB-GAM and electrophysiological phenotype displayed by the HB-GAM mutants. The individual domains displayed weaker binding to heparan sulfate and failed to promote neurite outgrowth as well as affect hippocampal LTP. Effects of HB-GAM on hippocampal synaptic plasticity are believed to be mediated by one of its (co-)receptor molecules, namely syndecan-3. In support of that, HB-GAM did not attenuate LTP in mice deficient in syndecan-3 as it did in wild-type controls. In addition, syndecan-3 knockout mice displayed electrophysiological and behavioral phenotype similar to that of HB-GAM knockouts (i.e. enhanced LTP and impaired learning in Morris water-maze). Thus HB-GAM and syndecan-3 are important modulators of synaptic plasticity in hippocampus and play a role in regulation of learning-related behavior.

Functional dissection of alternative secretory pathways in the yeast S. cerevisiae

Eukaryotic cells are characterized by having a subset of internal membrane compartments, each one with a specifi c identity, structure and function. Proteins destined to be targeted to the exterior of the cell need to enter and progress through the secretory pathway. Transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi takes place by the selective packaging of proteins into COPII-coated vesicles at the ER membrane. Taking advantage of the extensive genetic tools available for S. cerevisiae we found that Hsp150, a yeast secretory glycoprotein, selectively exited the ER in the absence of any of the three Sec24p family members. Sec24p has been thought to be an essential component of the COPII coat and thus indispensable for exocytic membrane traffic. Next we analyzed the ability of Hsp150 to be secreted in mutants, where post-Golgi transport is temperature sensitive. We found that Hsp150 could be selectively secreted under conditions where the exocyst component Sec15p is defective. Analysis of the secretory vesicles revealed that Hsp150 was packaged into a subset of known secretory vesicles as well as in a novel pool of secretory vesicles at the level of the Golgi. Secretion of Hsp150 in the absence of Sec15p function was dependent of Mso1p, a protein capable of interacting with vesicles intended to fuse with the plasma membrane, with the SNARE machinery and with Sec1p. This work demonstrated that Hsp150 is capable of using alternative secretory pathways in ER-to-Golgi and Golgi-to-plasma membrane traffi c. The sorting signals, used at both stages of the secretory pathway, for secretion of Hsp150 were different, revealing the highly dynamic nature and spatial organization of the secretory pathway. Foreign proteins usually misfold in the yeast ER. We used Hsp150 as a carrier to assist folding and transport of heterologous proteins though the secretory pathway to the culture medium in both S. cerevisiae and P. pastoris. Using this technique we expressed Hsp150Δ-HRP and developed a staining procedure, which allowed the visualization of the organelles of the secretory pathway of S. cerevisiae.

Molecular genetics of tooth agenesis

Congenital missing of teeth, tooth agenesis or hypodontia, is one of the most common developmental anomalies in man. The common forms in which one or a few teeth are absent, may cause occlusal or cosmetic harm, while severe forms which are relatively rare always require clinical attention to support and maintain the dental function. Observation of tooth agenesis is also important for diagnosis of malformation syndromes. Some external factors may cause developmental defects and agenesis in dentition. However, the role of inheritance in the etiology of tooth agenesis is well established by twin and family studies. Studies on familial tooth agenesis as well as mouse null mutants have also identified several genetic factors. However, these explain syndromic or rare dominant forms of tooth agenesis, whereas the genes and defects responsible for the majority of cases of tooth agenesis, especially the common and less severe forms, are largely unknown. In this study it was shown, that a dominant nonsense mutation in PAX9 was responsible for severe tooth agenesis (oligodontia) in a Finnish family. In a study of tooth agenesis associated with Wolf-Hirschhorn syndrome, it was shown that severe tooth agenesis was present if the causative deletion in 4p spanned the MSX1 locus. It was concluded that severe tooth agenesis was caused by haploinsufficiency of these transcription factors. A summary of the phenotypes associated with known defects in MSX1 and PAX9 showed that, despite similarities, they were significantly different, suggesting that the genes, in addition to known interactions, also have independent roles during the development of human dentition. The original aim of this work was to identify gene defects that underlie the common incisor and premolar hypodontia. After excluding several candidate genes, a genome-wide search was conducted in seven Finnish families in which this phenotype was inherited in an autosomal dominant manner. A promising locus for second premolar agenesis was identified in chromosome 18 in one family and this finding was supported by results from other families. The results also implied the existence of other loci both for second premolar agenesis and for incisor agenesis. On the other hand the results did not lend support for comprehensive involvement of the most obvious candidate genes in the etiology of incisor and premolar hypodontia. Rather, they suggest remarkable genetic heterogeneity of tooth agenesis. The available evidence suggests that quantitative defects during tooth development predispose to a failure to overcome a developmental threshold and to agenesis. The results of the study increase the understanding of the etiology and heredity of tooth agenesis. Further studies may lead to identification of novel genes that affect the development of teeth.

FGFR1 regulated gene-expression, cell proliferation and differentiation in the developing midbrain and hindbrain

The neuroectodermal tissue close to the midbrain hindbrain boundary (MHB) is an important secondary organizer in the developing neural tube. This so-called isthmic organizer (IsO) regulates cellular survival, patterning and proliferation in the midbrain (Mb) and rhombomere 1 (R1) of the hindbrain. Signaling molecules of the IsO, such as fibroblast growth factor 8 (FGF8) and WNT1 are expressed in distinct bands of cells around the MHB. It has been previously shown that FGF-receptor 1 (FGFR1) is required for the normal development of this brain region in the mouse embryo. In the present study, we have compared the gene expression profiles of wild-type and Fgfr1 mutant embryos. We show that the loss of Fgfr1 results in the downregulation of several genes expressed close to the MHB and in the disappearance of gene expression gradients in the midbrain and R1. Our microarray screen identified several previously uncharacterized genes which may participate in the development of midbrain R1 region. Our results also show altered neurogenesis in the midbrain and R1 of the Fgfr1 mutants. Interestingly, the neuronal progenitors in midbrain and R1 show different responses to the loss of signaling through FGFR1. As Wnt1 expression at the MHB region requires the FGF signaling pathway, WNT target genes, including Drapc1, were also identified in our screen. The microarray data analysis also suggested that the cells next to the midbrain hindbrain boundary express distinct cell cycle regulators. We showed that the cells close to the border appeared to have unique features. These cells proliferate less rapidly than the surrounding cells. Unlike the cells further away from the boundary, these cells express Fgfr1 but not the other FGF receptors. The slowly proliferating boundary cells are necessary for development of the characteristic isthmic constriction. They may also contribute to compartmentalization of this brain region.

Structure, function and intracellular dynamics of alphavirus replication complexes

Intracellular membrane alterations are hallmarks of positive-sense RNA (+RNA) virus replication. Strong evidence indicates that within these exotic compartments, viral replicase proteins engage in RNA genome replication and transcription. To date, fundamental questions such as the origin of altered membranes, mechanisms of membrane deformation and topological distribution and function of viral components, are still waiting for comprehensive answers. This study addressed some of the above mentioned questions for the membrane alterations induced during Semliki Forest virus (SFV) infection of mammalian cells. With the aid of electron and fluorescence microscopy coupled with radioactive labelling and immuno-cytochemistry techniques, our group and others showed that few hours after infection the four non structural proteins (nsP1-4) and newly synthesized RNAs of SFV colocalized in close proximity of small membrane invaginations, designated as spherules . These 50-70 nm structures were mainly detected in the perinuclear area, at the limiting membrane of modified endosomes and lysosomes, named CPV-I (cytopathic vacuoles type I). More rarely, spherules were also found at the plasma membrane (PM). In the first part of this study I present the first three-dimensional reconstruction of the CPV-I and the spherules, obtained by electron tomography after chemical or cryo-fixation. Different approaches for imaging these macromolecular assemblies to obtain better structure preservation and higher resolution are presented as unpublished data. This study provides insights into spherule organization and distribution of viral components. The results of this and other experiments presented in this thesis will challenge currently accepted models for virus replication complex formation and function. In a revisitation of our previous models, the second part of this work provides the first complete description of the biogenesis of the CPV-I. The results demonstrate that these virus-induced vacuoles, where hundreds of spherules accumulate at late stages during infection, represent the final phase of a journey initiated at the PM, which apparently serves as a platform for spherule formation. From the PM spherules were internalized by an endocytic event that required the activity of the class I PI3K, caveolin-1, cellular cholesterol and functional actin-myosin network. The resulting neutral endocytic carrier vesicle delivered the spherules to the membrane of pre-existing acidic endosomes via multiple fusion events. Microtubule based transport supported the vectorial transfer of these intermediates to the pericentriolar area where further fusions generated the CPV-I. A signal for spherule internalization was identified in one of the replicase proteins, nsP3. Infections of cells with viruses harbouring a deletion in a highly phosphorylated region of nsP3 did not result in the formation of CPV-Is. Instead, thousands of spherules remained at the PM throughout the infection cycle. Finally, the role of the replicase protein nsP2 during viral RNA replication and transcription was investigated. Three enzymatic activities, protease, NTPase and RNA-triphosphatase were studied with the aid of temperature sensitive mutants in vitro and, when possible, in vivo. The results highlighted the interplay of the different nsP2 functions during different steps of RNA replication and sub-genomic promoter regulation, and suggest that the protein could have different activities when participating in the replication complex or as a free enzyme.

Ozone-induced signaling in Arabidopsis thaliana

Tropospheric ozone (O3) is one of the most common air pollutants in industrialized countries, and an increasing problem in rapidly industrialising and developing countries in Asia, Africa and South America. Elevated concentrations of tropospheric O3 can lead to decrease in photosynthesis rate and therefore affect the normal metabolism, growth and seed production. Acute and high O3 episodes can lead to extensive damage leading to dead tissue in plants. Thus, O3 derived growth defects can lead to reduction in crop yield thereby leading to economical losses. Despite the extensive research on this area, many questions remain open on how these processes are controlled. In this study, the stress-induced signaling routes and the components involved were elucidated in more detail starting from visual damage to changes in gene expression, signaling routes and plant hormone interactions that are involved in O3-induced cell death. In order to elucidate O3-induced responses in Arabidopsis, mitogen-activated protein kinase (MAPK) signaling was studied using different hormonal signaling mutants. MAPKs were activated at the beginning of the O3 exposure. The activity of MAPKs, which were identified as AtMPK3 and AtMPK6, reached the maximum at 1 and 2 hours after the start of the exposure, respectively. The activity decreased back to clean air levels at 8 hours after the start of the exposure. Both AtMPK3 and AtMPK6 were translocated to nucleus at the beginning of the O3 exposure where they most likely affect gene expression. Differences were seen between different hormonal signaling mutants. Functional SA signaling was shown to be needed for the full protein levels and activation of AtMPK3. In addition, AtMPK3 and AtMPK6 activation was not dependent on ethylene signaling. Finally, jasmonic acid was also shown to have an impact on AtMPK3 protein levels and AtMPK3 activity. To further study O3-induced cell death, an earlier isolated O3 sensitive Arabidopsis mutant rcd1 was mapped, cloned and further characterized. RCD1 was shown to encode a gene with WWE and ADP-ribosylation domains known to be involved in protein-protein interactions and cell signaling. rcd1 was shown to be involved in many processes including hormonal signaling and regulation of stress-responsive genes. rcd1 is sensitive against O3 and apoplastic superoxide, but tolerant against paraquat that produces superoxide in chloroplast. rcd1 is also partially insensitive to glucose and has alterations in hormone responses. These alterations are seen as ABA insensitivity, reduced jasmonic acid sensitivity and reduced ethylene sensitivity. All these features suggest that RCD1 acts as an integrative node in hormonal signaling and it is involved in the hormonal regulation of several specific stress-responsive genes. Further studies with the rcd1 mutant showed that it exhibits the classical features of programmed cell death, PCD, in response to O3. These include nuclear shrinkage, chromatin condensation, nuclear DNA degradation, cytosol vesiculation and accumulation of phenolic compounds and eventually patches of HR-like lesions. rcd1 was found to produce extensive amount of salicylic acid and jasmonic acid in response to O3. Double mutant studies showed that SA independent and dependent processes were involved in the O3-induced PCD in rcd1 and that increased sensitivity against JA led to increased sensitivity against O3. Furthermore, rcd1 had alterations in MAPK signature that resembled changes that were previously seen in mutants defective in SA and JA signaling. Nitric oxide accumulation and its impact on O3-induced cell death were also studied. Transient accumulation of NO was seen at the beginning of the O3 exposure, and during late time points, NO accumulation coincided with the HR-like lesions. NO was shown to modify defense gene expression, such as, SA and ethylene biosynthetic genes. Furthermore, rcd1 was shown to produce more NO in control conditions. In conclusion, NO was shown to be involved in O3-induced signaling leading to attenuation of SA biosynthesis and other defense related genes.

Molecular details of phage phi6 RNA-dependent RNA synthesis

For most RNA viruses RNA-dependent RNA polymerases (RdRPs) encoded by the virus are responsible for the entire RNA metabolism. Thus, RdRPs are critical components in the viral life cycle. However, it is not fully understood how these important enzymes function during viral replication. Double-stranded RNA (dsRNA) viruses perform the synthesis of their RNA genome within a proteinacous viral particle containing an RdRP as a minor constituent. The phi6 bacteriophage is the best-studied dsRNA virus, providing an excellent background for studies of its RNA synthesis. The purified recombinant phi6 RdRP is highly active in vitro and it possesses both RNA replication and transcription activities. The crystal structure of the phi6 polymerase, solved in complex with a number of ligands, provides a working model for detailed in vitro studies of RNA-dependent RNA polymerization. In this thesis, the primer-independent initiation of the phi6 RdRP was studied in vitro using biochemical and structural methods. A C-terminal, four-amino-acid-long loop protruding into the central cavity of the phi6 RdRP has been suggested to stabilize the incoming nucleotides of the initiation complex formation through stacking interactions. A similar structural element has been found from several other viral RdRPs. In this thesis, this so-called initiation platform loop was subjected to site-directed mutagenesis to address its role in the initiation. It was found that the initiation mode of the mutants is primer-dependent, requiring either an oligonucleotide primer or a back-priming initiation mechanism for the RNA synthesis. The crystal structure of a mutant RdRP with altered initiation platform revealed a set of contacts important for primer-independent initiation. Since phi6 RdRP is structurally and functionally homologous to several viral RdRPs, among them the hepatitis C virus RdRP, these results provide further general insight to understand primer-independent initiation. In this study it is demonstrated that manganese phasing could be used as a practical tool for solving structures of large proteins with a bound manganese ion. The phi6 RdRP was used as a case study to obtain phases for crystallographic analysis. Manganese ions are naturally bound to the phi6 RdRP at the palm domain of the enzyme. In a crystallographic experiment, X-ray diffraction data from a phi6 RdRP crystal were collected at a wavelength of 1.89 Å, which is the K edge of manganese. With this data an automatically built model of the core region of the protein could be obtained. Finally, in this work terminal nucleotidyl transferase (TNTase) activity of the phi6 RdRP was documented in the isolated polymerase as well as in the viral particle. This is the first time that such an activity has been reported in a polymerase of a dsRNA virus. The phi6 RdRP used uridine triphosphates as the sole substrate in a TNTase reaction but could accept several heterologous templates. The RdRP was able to add one or a few non-templated nucleotides to the 3' end of the single- or double-stranded RNA substrate. Based on the results on particle-mediated TNTase activity and previous structural information of the polymerase, a model for termination of the RNA-dependent RNA synthesis is suggested in this thesis.

Functional characterization of MutL homologue mismatch repair proteins and their variants

Mismatch repair (MMR) mechanisms repair DNA damage occurring during replication and recombination. To date, five human MMR genes, MSH2, MHS6, MSH3, MLH1 and PMS2 are known to be involved in the MMR function. Human MMR proteins form 3 different heterodimers: MutSα (MSH2 and MSH6) and MutSβ (MSH2 and MSH3), which are needed for mismatch recognition and binding, and MutLα (MLH1 and PMS2), which is needed for mediating interactions between MutS homologues and other MMR proteins. The other two MutL homologues, MLH3 and PMS1, have been shown to heterodimerize with MLH1. However, the heterodimers MutLγ (MLH1and MLH3) and MutLβ (MLH1 and PMS1) are able to correct mismatches only with low or no efficiency, respectively. A deficient MMR mechanism is associated with the hereditary colorectal cancer syndrome called hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome. HNPCC is the most common hereditary colorectal cancer syndrome and accounts for 2-5% of all colorectal cancer cases. HNPCC-associated mutations have been found in 5 MMR genes: MLH1, MSH2, MSH6, PMS2 and MLH3. Most of the mutations have been found in MLH1 and MSH2 (~90%) and are associated with typical HNPCC, while mutations in MSH6, PMS2 and MLH3 are mainly linked to putative HNPCC families lacking the characteristics of the syndrome. More data of MLH3 mutations are needed to assess the significance of its mutations in HNPCC. In this study, were functionally characterized 51 nontruncating mutations in the MLH1, MLH3 and MSH2 genes to address their pathogenic significance and mechanism of pathogenicity. Of the 36 MLH1 mutations, 22 were deficient in more than one assay, 2 variants were impaired only in one assay, and 12 variants behaved like the wild type protein, whereas all seven MLH3 mutants functioned like the wild type protein in the assays. To further clarify the role and relevance of MLH3 in MMR, we analyzed the subcellular localization of the native MutL homologue proteins. Our immunofluorescence analyses indicated that when all the three MutL homologues are natively expressed in human cells, endogenous MLH1 and PMS2 localize in the nucleus, whereas MLH3 stays in the cytoplasm. The coexpression of MLH3 with MLH1 results in its partial nuclear localization. Only one MSH2 mutation was pathogenic in the in vitro MMR assay. Our study on MLH1 mutations could clearly distinguish nontruncating alterations with severe functional defects from those not or only slightly impaired in protein function. However, our study on MLH3 mutations suggest that MLH3 mutations per se are not sufficient to trigger MMR deficiency and the continuous nuclear localization of MLH1 and PMS2 suggest that MutLα has a major activity in MMR in vivo. Together with our functional assays, this confirms that MutLγ is a less efficient MMR complex than MutLα.

Plant guard cell anion channel SLAC1 regulates stomatal closure

Plants are rooted to their growth place; therefore it is important that they react adequately to changes in environmental conditions. Stomatal pores, which are formed of a pair of guard cells in leaf epidermis, regulate plant gas-exchange. Importantly, guard cells protect the plant from desiccation in drought conditions by reducing the aperture of the stomatal pore. They serve also as the first barrier against the major air pollutant ozone, but the behaviour of guard cells during ozone exposure has not been sufficiently addressed. Aperture of the stomatal pore is regulated by the influx and efflux of osmotically active ions via ion channels and transporters across the guard cell membrane, however the molecular identity of guard cell plasma membrane anion channel has remained unknown. In the frame of this study, guard cell behaviour during ozone exposure was studied using the newly constructed Arabidopsis whole-rosette gas-exchange system. Ozone induced a Rapid Transient Decrease (RTD) in stomatal conductance within 10 min from the start of exposure, which was followed by a recovery in the conductance within the next 40 min. The decrease in stomatal conductance was dependent on the applied ozone concentration. Three minutes of ozone exposure was sufficient to induce RTD and further ozone application during the closure-recovery process had no effect on RTD, demonstrating that the whole process is programmed within the first three minutes. To address the molecular components responsible for RTD, the ozone response was measured in 59 different Arabidopsis mutants involved in guard cell signalling. Four of the tested mutants slac1 (originally rcd3), ost1, abi1-1 and abi2-1 lacked RTD completely. As the ozone sensitive mutant slac1 lacked RTD, the next aim of this study was to identify and characterize SLAC1. SLAC1 was shown to be a central regulator in response to all major factors regulating guard cell aperture: CO2, light/darkness transitions, ozone, relative air humidity, ABA, NO, H2O2, and extracellular Ca2+. It encodes the first guard cell plasma membrane slow type anion channel to be identified at the molecular level. Interestingly, the rapid type anion conductance was intact in slac1 mutant plants. For activation, SLAC1 needs to be phosphorylated. Protein kinase OST1 was shown to phosphorylate several amino acids in the N-terminal tail of SLAC1, Ser120 was one of its main targets, which led to SLAC1 activation. The lack of RTD in type 2C protein phosphatase mutants abi1-1 and abi2-1, suggests that these proteins have a regulatory role in ozoneinduced activation of the slow type anion channel.

Novel Molecular Mechanisms of Arabidopsis Disease Resistance

Plants are capable of recognizing phytopathogens through the perception of pathogen-derived molecules or plant cell-wall degradation products due to the activities of pathogen-secreted enzymes. Such elicitor recognition events trigger an array of inducible defense responses involving signal transduction networks and massive transcriptional re-programming. The outcome of a pathogen infection relies on the balance between different signaling pathways, which are integrated by regulatory proteins. This thesis characterized two key regulatory components: a damage control enzyme, chlorophyllase 1 (AtCHL1), and a transcription factor, WRKY70. Their roles in defense signaling were then investigated. The Erwinia-derived elicitors rapidly activated the expression of AtCLH1 and WRKY70 through different signaling pathways. The expression of the AtCHL1 gene was up-regulated by jasmonic acid (JA) but down-regulated by salicylic acid (SA), whereas WRKY70 was activated by SA and repressed by JA. In order to elucidate the functions of AtCLH1 and WRKY70 in plant defense, stable transgenic lines were produced where these genes were overexpressed or silenced. Additionally, independent knockout lines were also characterized. Bacterial and fungal pathogens were then used to assess the contribution of these genes to the Arabidopsis disease resistance. The transcriptional modulation of AtCLH1 by either the constitutive over-expression or RNAi silencing caused alterations in the chlorophyll-to-chlorophyllide ratio, supporting the claim that chlorophyllase 1 has a role in the chlorophyll degradation pathway. Silencing of this gene led to light-dependent over-accumulation of the reactive oxygen species (ROS) in response to infection by Erwinia carotovora subsp. carotovora SCC1. This was followed by an enhanced induction of SA-dependent defense genes and an increased resistance to this pathogen. Interestingly, little effect on the pathogen-induced SA accumulation at the early infection was observed, suggesting that action of ROS might potentiate SA signaling. In contrast, the pathogen-induced JA production was significantly reduced in the RNAi silenced plants. Moreover, JA signaling and resistance to Alternaria brassicicola were impaired. These observations provide support for the argument that the ROS generated in chloroplasts might have a negative impact on JA signaling. The over-expression of WRKY70 resulted in an enhanced resistance to E. carotovora subsp. carotovora SCC1, Pseudomonas syringae pv. tomato DC3000 and Erysiphe cichoracearum UCSC1, whilst an antisense suppression or an insertional inactivation of WRKY70 led to a compromised resistance to E. carotovora subsp. carotovora SCC1 and to E. cichoracearum UCSC1 but not to P. syringae pv. tomato DC3000. Gene expression analysis revealed that WRKY70 activated many known defense-related genes associated with the SAR response but suppressed a subset of the JA-responsive genes. In particular, I was able to show that both the basal and the induced expression of AtCLH1 was enhanced by the antisense silencing or the insertional inactivation of WRKY70, whereas a reduction in AtCLH1 expression was observed in the WRKY70 over-expressors following an MeJA application or an A. brassicicola infection. Moreover, the SA-induced suppression of AtCLH1 was relieved in wrky70 mutants. These results indicate that WRKY70 down-regulates AtCLH1. An epistasis analysis suggested that WRKY70 functions downstream of the NPR1 in an SA-dependent signaling pathway. When challenged with A. brassicicola, WRKY70 over-expressing plants exhibited a compromised disease resistance while wrky70 mutants had the opposite effect. These results confirmed the WRKY70-mediated inhibitory effects on JA signaling. Furthermore, the WRKY70-controlled suppression of A. brassicicola resistance was mainly through an NPR1-dependent mechanism. Taking all the data together, I suggest that the pathogen-responsive transcription factor WRKY70 is a common component in both SA- and JA-dependent pathways and plays a crucial role in the SA-mediated suppression of JA signaling.

On gravitational fields of stars in f(R) gravity theories

Einstein's general relativity is a classical theory of gravitation: it is a postulate on the coupling between the four-dimensional, continuos spacetime and the matter fields in the universe, and it yields their dynamical evolution. It is believed that general relativity must be replaced by a quantum theory of gravity at least at extremely high energies of the early universe and at regions of strong curvature of spacetime, cf. black holes. Various attempts to quantize gravity, including conceptually new models such as string theory, have suggested that modification to general relativity might show up even at lower energy scales. On the other hand, also the late time acceleration of the expansion of the universe, known as the dark energy problem, might originate from new gravitational physics. Thus, although there has been no direct experimental evidence contradicting general relativity so far - on the contrary, it has passed a variety of observational tests - it is a question worth asking, why should the effective theory of gravity be of the exact form of general relativity? If general relativity is modified, how do the predictions of the theory change? Furthermore, how far can we go with the changes before we are face with contradictions with the experiments? Along with the changes, could there be new phenomena, which we could measure to find hints of the form of the quantum theory of gravity? This thesis is on a class of modified gravity theories called f(R) models, and in particular on the effects of changing the theory of gravity on stellar solutions. It is discussed how experimental constraints from the measurements in the Solar System restrict the form of f(R) theories. Moreover, it is shown that models, which do not differ from general relativity at the weak field scale of the Solar System, can produce very different predictions for dense stars like neutron stars. Due to the nature of f(R) models, the role of independent connection of the spacetime is emphasized throughout the thesis.

Search for R-Parity Violating Decays of Sneutrinos to eμ, μτ, and eτ Pairs in pp̅ Collisions at √s=1.96 TeV

We present a search for tau sneutrino production using the Tevatron ppbar collision data collected with the CDF II detector and corresponding to an integrated luminosity of 1 fb^-1. We focus on the scenarios predicted by the R-parity violating (RPV) supersymmetric models in which tau sneutrinos decay to two charged leptons of different flavor. With the data consistent with the standard model expectations, we set the upper limits on sigma(ppbar -> tau sneutrino)*BR(tau sneutrino ->emu,mutau,etau) and use these results to constrain the RPV couplings as a function of tau sneutrino mass.