Luminescence-based assay methods in drug discovery

The drug discovery process is facing new challenges in the evaluation process of the lead compounds as the number of new compounds synthesized is increasing. The potentiality of test compounds is most frequently assayed through the binding of the test compound to the target molecule or receptor, or measuring functional secondary effects caused by the test compound in the target model cells, tissues or organism. Modern homogeneous high-throughput-screening (HTS) assays for purified estrogen receptors (ER) utilize various luminescence based detection methods. Fluorescence polarization (FP) is a standard method for ER ligand binding assay. It was used to demonstrate the performance of two-photon excitation of fluorescence (TPFE) vs. the conventional one-photon excitation method. As result, the TPFE method showed improved dynamics and was found to be comparable with the conventional method. It also held potential for efficient miniaturization. Other luminescence based ER assays utilize energy transfer from a long-lifetime luminescent label e.g. lanthanide chelates (Eu, Tb) to a prompt luminescent label, the signal being read in a time-resolved mode. As an alternative to this method, a new single-label (Eu) time-resolved detection method was developed, based on the quenching of the label by a soluble quencher molecule when displaced from the receptor to the solution phase by an unlabeled competing ligand. The new method was paralleled with the standard FP method. It was shown to yield comparable results with the FP method and found to hold a significantly higher signal-tobackground ratio than FP. Cell-based functional assays for determining the extent of cell surface adhesion molecule (CAM) expression combined with microscopy analysis of the target molecules would provide improved information content, compared to an expression level assay alone. In this work, immune response was simulated by exposing endothelial cells to cytokine stimulation and the resulting increase in the level of adhesion molecule expression was analyzed on fixed cells by means of immunocytochemistry utilizing specific long-lifetime luminophore labeled antibodies against chosen adhesion molecules. Results showed that the method was capable of use in amulti-parametric assay for protein expression levels of several CAMs simultaneously, combined with analysis of the cellular localization of the chosen adhesion molecules through time-resolved luminescence microscopy inspection.

Novel CO2 Regulated Proteins in Synechocystis PCC 6803

The large biodiversity of cyanobacteria together with the increasing genomics and proteomics metadata provide novel information for finding new commercially valuable metabolites. With the advent of global warming, there is growing interest in the processes that results in efficient CO2 capture through the use of photosynthetic microorganisms such as cyanobacteria. This requires a detailed knowledge of how cyanobacteria respond to the ambient CO2. My study was aimed at understanding the changes in the protein profile of the model organism, Synechocystis PCC 6803 towards the varying CO₂ level. In order to achieve this goal I have employed modern proteomics tools such as iTRAQ and DIGE, recombinant DNA techniques to construct different mutants in cyanobacteria and biophysical methods to study the photosynthetic properties. The proteomics study revealed several novel proteins, apart from the well characterized proteins involved in carbon concentrating mechanisms (CCMs), that were upregulated upon shift of the cells from high CO₂ concentration (3%) to that in air level (0.039%). The unknown proteins, Slr0006 and flavodiiron proteins (FDPs) Sll0217-Flv4 and Sll0219-Flv2, were selected for further characterization. Although slr0006 was substantially upregulated under C_i limiting conditions, inactivation of the gene did not result in any visual phenotype under various environmental conditions indicating that this protein is not essential for cell survival. However, quantitative proteomics showed the induction of novel plasmid and chromosome encoded proteins in deltaslr0006 under air level CO₂ conditions. The expression of the slr0006 gene was found to be strictly dependent on active photosynthetic electron transfer. Slr0006 contains conserved dsRNA binding domain that belongs to the Sua5/YrdC/YciO protein family. Structural modelling of Slr0006 showed an alpha/beta twisted open-sheet structure and a positively charged cavity, indicating a possible binding site for RNA. The 3D model and the co-localization of Slr0006 with ribosomal subunits suggest that it might play a role in translation or ribosome biogenesis. On the other hand, deletions in the sll0217-sll218- sll0219 operon resulted in enhanced photodamage of PSII and distorted energy transfer from phycobilisome (PBS) to PSII, suggesting a dynamic photoprotection role of the operon. Constructed homology models also suggest efficient electron transfer in heterodimeric Flv2/Flv4, apparently involved in PSII photoprotection. Both Slr0006 and FDPs exhibited several common features, including negative regulation by NdhR and ambiguous cellular localization when subjected to different concentrations of divalent ions. This strong association with the membranes remained undisturbed even in the presence of detergent or high salt. My finding brings ample information on three novel proteins and their functions towards carbon limitation. Nevertheless, many pathways and related proteins remain unexplored. The comprehensive understanding of the acclimation processes in cyanobacteria towards varying environmental CO₂ levels will help to uncover adaptive mechanisms in other organisms, including higher plants.

The Cellular Oxygen Sensor PHD2 in Cancer Growth

Adequate supply of oxygen is essential for the survival of multicellular organisms. However, in several conditions the supply of oxygen can be disturbed and the tissue oxygenation is compromised. This condition is termed hypoxia. Oxygen homeostasis is maintained by the regulation of both the use and delivery of oxygen through complex, sensitive and cell-type specific transcriptional responses to hypoxia. This is mainly achieved by one master regulator, a transcription factor called hypoxiainducible factor 1 (HIF-1). The amount of HIF-1 is under tight oxygen-dependent control by a family of oxygen-dependent prolyl hydroxylase domain proteins (PHDs) that function as the cellular oxygen sensors. Three family members (PHD1-3) are known to regulate HIF of which the PHD2 isoform is thought to be the main regulator of HIF-1. The supply of oxygen can be disturbed in pathophysiological conditions, such as ischemic disorders and cancer. Cancer cells in the hypoxic parts of the tumors exploit the ability of HIF-1 to turn on the mechanisms for their survival, resistance to treatment, and escape from the oxygen- and nutrient-deprived environment. In this study, the expression and regulation of PHD2 were studied in normal and cancerous tissues, and its significance in tumor growth. The results show that the expression of PHD2 is induced in hypoxic cells. It is overexpressed in head and neck squamous cell carcinomas and colon adenocarcinomas. Although PHD2 normally resides in the cytoplasm, nuclear translocation of PHD2 was also seen in a subset of tumor cells. Together with the overexpression, the nuclear localization correlated with the aggressiveness of the tumors. The nuclear localization of PHD2 caused an increase in the anchorage-independent growth of cancer cells. This study provides information on the role of PHD2, the main regulator of HIF expression, in cancer progression. This knowledge may prove to be valuable in targeting the HIF pathway in cancer treatment.

Coarse Global Visual Localization of a Mobile Robot

Localization, which is the ability of a mobile robot to estimate its position within its environment, is a key capability for autonomous operation of any mobile robot. This thesis presents a system for indoor coarse and global localization of a mobile robot based on visual information. The system is based on image matching and uses SIFT features as natural landmarks. Features extracted from training images arestored in a database for use in localization later. During localization an image of the scene is captured using the on-board camera of the robot, features are extracted from the image and the best match is searched from the database. Feature matching is done using the k-d tree algorithm. Experimental results showed that localization accuracy increases with the number of training features used in the training database, while, on the other hand, increasing number of features tended to have a negative impact on the computational time. For some parts of the environment the error rate was relatively high due to a strong correlation of features taken from those places across the environment.

Performance Evaluation of Six-Sectored Configuration in Hexagonal WCDMA (UMTS) Cellular Network Layout

The objective of this master's thesis is to evaluate the optimum performance of sixsectored hexagonal layout of WCDMA (UMTS) network and analyze the performance at the optimum point. The maximum coverage and the maximum capacity are the main concern of service providers and it is always a challenging task for them to achieve economically. Because the optimum configuration of a network corresponds to a configuration which minimizes the number of sites required to provide a target service probability in the planning area which in turn reduces the deployment cost. The optimum performance means the maximum cell area and themaximum cell capacity the network can provide at the maximum antenna height satisfying the target service probability. Hexagon layout has been proven as the best layout for the cell deployment. In this thesis work, two different configurations using six-sectored sites have been considered for the performance comparison. In first configuration, each antenna is directed towards each corner of hexagon, whereas in second configurationeach antenna is directed towards each side of hexagon. The net difference in the configurations is the 30 degree rotation of antenna direction. The only indoor users in a flat and smooth semi-urban environment area have been considered for the simulation purpose where the traffic distribution is 100 Erl/km2 with 12.2 kbps speech service having maximum mobile speed of 3 km/hr. The simulation results indicate that a similar performance can be achieved in both the configurations, that is, a maximum of 947 m cellrange at antenna height of 49.5 m can be achieved when the antennas are directed towards the corner of hexagon, whereas 943.3 m cell range atantenna height of 54 m can be achieved when the antennas are directed towards the side of hexagon. However, from the interference point of view the first configuration provides better results. The simulation results also show that the network is coverage limited in both the uplink and downlink direction at the optimum point.

Mobile robot localization using sonar ranging and wlan intensity maps

 Main goal of this thesis was to implement a localization system which uses sonars and WLAN intensity maps to localize an indoor mobile robot. A probabilistic localization method, Monte Carlo Localization is used in localization. Also the theory behind probabilistic localization is explained. Two main problems in mobile robotics, path tracking and global localization, are solved in this thesis. Implemented system can achieve acceptable performance in path tracking. Global localization using WLAN received signal strength information is shown to provide good results, which can be used to localize the robot accurately, but also some bad results, which are no use when trying to localize the robot to the correct place. Main goal of solving ambiguity in office like environment is achieved in many test cases.

Multi-sensor Simultaneous Localization and Mapping

Simultaneous localization and mapping(SLAM) is a very important problem in mobile robotics. Many solutions have been proposed by different scientists during the last two decades, nevertheless few studies have considered the use of multiple sensors simultane¬ously. The solution is on combining several data sources with the aid of an Extended Kalman Filter (EKF). Two approaches are proposed. The first one is to use the ordinary EKF SLAM algorithm for each data source separately in parallel and then at the end of each step, fuse the results into one solution. Another proposed approach is the use of multiple data sources simultaneously in a single filter. The comparison of the computational com¬plexity of the two methods is also presented. The first method is almost four times faster than the second one.

Role and Function of c-Jun Protein Complex in Cancer Cell Behavior

Transcription factors play a crucial role in the regulation of cell behavior by modulating gene expression profiles. Previous studies have described a dual role for the AP-1 family transcription factor c-Jun in the regulation of cellular fate. In various cell types weak and transient activations of c-Jun N-terminal kinase (JNK) and c-Jun appear to contribute to proliferation and survival, whereas strong and prolonged activation of JNK and c-Jun result in apoptosis. These opposite roles played by c-Jun are cell type specific and the molecular mechanisms defining these antonymous c-Jun-mediated responses remain incompletely understood. c-Jun activity in transformed cells is regulated by signalling cascades downstream of oncoproteins such as Ras and Raf. In addition, the pro-proliferative role and the survival promoting function for c-Jun has been described in various cancer models. Furthermore, c-Jun was described to be overexpressed in different cancer types. However, the molecular mechanisms by which c-Jun exerts these oncogenic functions are not all clearly established. Therefore it is of primary interest to further identify molecular mechanisms and functions for c-Jun in cancer. Regulation of gene expression is tightly dependent on accurate protein-protein interactions. Therefore, co-factors for c-Jun may define the functions for c-Jun in cancer. Identification of protein-protein interactions promoting cancer may provide novel possibilities for cancer treatment. In this study, we show that DNA topoisomerase I (TopoI) is a transcriptional co-factor for c-Jun. Moreover, c-Jun and TopoI together promote expression of epidermal growth factor receptor (EGFR) in cancer cells. We also show that the clinically used TopoI inhibitor topotecan reduces EGFR expression. Importantly, the effect of TopoI on EGFR transcription was shown to depend on c-Jun as Jun-/- cells or cells treated with JNK inhibitor SP600125 are resistant to topotecan treatment both in regulation of EGFR expression and cell proliferation. Moreover, c-Jun regulates the nucleolar localization and the function of the ribonucleic acid (RNA) helicase DDX21, a previously identified member of c-Jun protein complex. In addition, c-Jun stimulates rRNA processing by supporting DDX21 rRNA binding. Finally, this study characterizes a DDX21 dependent expression of cyclin dependent kinase (Cdk) 6, a correlation of DDX21 expression with prostate cancer progression and a substrate binding dependency of DDX21 nucleolar localization in prostate cancer cells. Taken together, the results of this study validate the c-Jun-TopoI interaction and precise the c-Jun-DDX21 interaction. Moreover, these results show the importance for protein-protein interaction in the regulation of their cellular functions in cancer cell behavior. Finally, the results presented here disclose new exciting therapeutic opportunities for cancer treatment.

Conservation Laws in Cellular Automata

Conservation laws in physics are numerical invariants of the dynamics of a system. In cellular automata (CA), a similar concept has already been defined and studied. To each local pattern of cell states a real value is associated, interpreted as the “energy” (or “mass”, or . . . ) of that pattern.The overall “energy” of a configuration is simply the sum of the energy of the local patterns appearing on different positions in the configuration. We have a conservation law for that energy, if the total energy of each configuration remains constant during the evolution of the CA. For a given conservation law, it is desirable to find microscopic explanations for the dynamics of the conserved energy in terms of flows of energy from one region toward another. Often, it happens that the energy values are from non-negative integers, and are interpreted as the number of “particles” distributed on a configuration. In such cases, it is conjectured that one can always provide a microscopic explanation for the conservation laws by prescribing rules for the local movement of the particles. The onedimensional case has already been solved by Fuk´s and Pivato. We extend this to two-dimensional cellular automata with radius-0,5 neighborhood on the square lattice. We then consider conservation laws in which the energy values are chosen from a commutative group or semigroup. In this case, the class of all conservation laws for a CA form a partially ordered hierarchy. We study the structure of this hierarchy and prove some basic facts about it. Although the local properties of this hierarchy (at least in the group-valued case) are tractable, its global properties turn out to be algorithmically inaccessible. In particular, we prove that it is undecidable whether this hierarchy is trivial (i.e., if the CA has any non-trivial conservation law at all) or unbounded. We point out some interconnections between the structure of this hierarchy and the dynamical properties of the CA. We show that positively expansive CA do not have non-trivial conservation laws. We also investigate a curious relationship between conservation laws and invariant Gibbs measures in reversible and surjective CA. Gibbs measures are known to coincide with the equilibrium states of a lattice system defined in terms of a Hamiltonian. For reversible cellular automata, each conserved quantity may play the role of a Hamiltonian, and provides a Gibbs measure (or a set of Gibbs measures, in case of phase multiplicity) that is invariant. Conversely, every invariant Gibbs measure provides a conservation law for the CA. For surjective CA, the former statement also follows (in a slightly different form) from the variational characterization of the Gibbs measures. For one-dimensional surjective CA, we show that each invariant Gibbs measure provides a conservation law. We also prove that surjective CA almost surely preserve the average information content per cell with respect to any probability measure.

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.

Granulation tissue formation -The effect of hydroxyapatite coating of cellulose on cellular differentiation

Haavan jyväiskudoksen muodostuminen – Hydroksiapatiittipinnoi-tetun selluloosasienen vaikutus solujen erilaistumiseen paranemisprosessin aikana Etsittäessä uusia luun bioyhteensopivia täytemateriaaleja selluloosasieni päällystettiin luun koostumusta muistuttavalla runsaasti piitä sisältävällä hydroksiapatiittikerroksella. Vastoin odotuksia hydroksiapatiittipinnoitettu selluloosa ei parantanut luun kasvua, vaan päinvastoin ylläpiti tulehdusta ja sidekudossolujen hakeutumista vamma-alueelle. Ihon alle implantoituna sama sienimateriaali edisti merkittävästi haavan verekkään jyväiskudoksen kasvua. Tämän löydöksen perusteella hydroksiapatiittipinnoitetun selluloosasienen vaikutusta haavan soluihin paranemisprosessin aikana tutkittiin tarkemmin ja havaittiin, että tulehdussolujen lisäksi sieniin kertyi tavallista enemmän sekä hematopoieettisia että mesenkymaalisia kantasoluja. Hematopoieettiset kantasolut sijaitsevat luuytimessä lähellä luun sisäpintaa. Luun hydroksiapatiitista vapautuu kalsiumioneja luun jatkuvan fysiologisen uudismuodostuksen ja hajottamisen yhteydessä. Kantasolut etsiytyvät luuytimeen kalsiumia aistivien reseptorien välityksellä. Koska luun pintakerrosta muistuttavasta hydroksiapatiittipinnoitteesta vapautuu kalsiumia, tämän ajateltiin toimivan selityksenä sille, että hematopoieettiset kantasolut hakeutuvat runsaslukuisesti juuri hydroksiapatiittipinnoitettuihin selluloosasieniin. Tämän hypoteesin mukaisesti hydroksiapatiittipinnoitettujen selluloosapalkkien läheisyydestä löydettiin suuria määriä kalsiumreseptoreja sisältäviä soluja. Jatkotutkimuksissa todettiin lisäksi, että hematopoieettiset kantasolut pystyivät sienissä erilaistumaan hemoglobiinia tuottaviksi soluiksi. Havaittujen punasolulinjan merkkiaineiden perusteella näyttäisikin siltä, että haavan paranemiskudoksessa tapahtuu paranemisen aikana ekstramedullaarista erytropoieesia. Nämä soluja ohjaavat vaikutukset saattavat olla hyödyllisiä vaikeasti paranevien haavojen hoidossa.

On Undecidable Dynamical Properties of Reversible One-Dimensional Cellular Automata

Cellular automata are models for massively parallel computation. A cellular automaton consists of cells which are arranged in some kind of regular lattice and a local update rule which updates the state of each cell according to the states of the cell's neighbors on each step of the computation. This work focuses on reversible one-dimensional cellular automata in which the cells are arranged in a two-way in_nite line and the computation is reversible, that is, the previous states of the cells can be derived from the current ones. In this work it is shown that several properties of reversible one-dimensional cellular automata are algorithmically undecidable, that is, there exists no algorithm that would tell whether a given cellular automaton has the property or not. It is shown that the tiling problem of Wang tiles remains undecidable even in some very restricted special cases. It follows that it is undecidable whether some given states will always appear in computations by the given cellular automaton. It also follows that a weaker form of expansivity, which is a concept of dynamical systems, is an undecidable property for reversible one-dimensional cellular automata. It is shown that several properties of dynamical systems are undecidable for reversible one-dimensional cellular automata. It shown that sensitivity to initial conditions and topological mixing are undecidable properties. Furthermore, non-sensitive and mixing cellular automata are recursively inseparable. It follows that also chaotic behavior is an undecidable property for reversible one-dimensional cellular automata.

The Integrin Tail: A Tale of Cell Motility and Division

Integrin transmembrane receptor functions are regulated by adaptor molecules binding to their alpha and beta subunit intracellular domains, or tails, thus affecting integrin traffic and adhesion during e.g. cell motility. Interestingly, many cellular proteins function in both cell motility and cell division, thus raising the possibility that integrins might be involved in regulating the cell cycle. A thorough understanding of cell division is essential in cell biology and in human malignancies. It is well established that failures to complete cell cycle can give rise to genetically unstable cells with tumorigenic properties. Transformed cells promote the disruption of intercellular adhesions such as tight junctions, and this correlates with the onset of cell motility, invasion and unfavorable prognosis in cancer. In this study, we analyzed integrin regulation, mediated by adaptor binding to the  subunit tail, during cell motility and cell division. We revealed a novel molecular mechanism by which Rab21, through association with the integrin alpha subunits, drives integrin endosomal traffic during mitotic phases. In addition, we found indications for this finding in vivo, as RAB21 gene deletions were mapped in ovarian and prostate cancer samples. Importantly, the multinucleated phenotype of cultured ovarian cancer cells could be reverted by Rab21 overexpression. In this thesis work, we also show how the tight junction protein ZO-1 unexpectedly interacts with the 5 integrin cytoplasmic domain in the lamellipodia to promote cell motility and at the cleavage furrow to support separation of the daughter cells. The alpha5-ZO-1 complex formation was dependent on PKC which regulates ZO-1 phosphorylation and its subcellular localization. In addition, by an in situ detection method, we showed that a subset of metastatic human lung cancers expressed the alpha5beta-ZO-1 complex. Taken together, we were able to identify new molecular pathways that regulate integrin functions in an alpha tail-mediated fashion. These findings firmly suggest that genetic alterations in integrin traffic may lead to progression of tumorigenesis as a result of failed cell division. Also, the interplay of integrins and ZO-1 in forming spatially regulated adhesive structures broadens our view of crosstalk between pathways and distinct adhesive structures that can be involved in cancer cell biology.

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.