MRE11-RAD50-NBS1 is a critical regulator of FANCD2 stability and function during DNA double-strand break repair.

Monoubiquitination of the Fanconi anaemia protein FANCD2 is a key event leading to repair of interstrand cross-links. It was reported earlier that FANCD2 co-localizes with NBS1. However, the functional connection between FANCD2 and MRE11 is poorly understood. In this study, we show that inhibition of MRE11, NBS1 or RAD50 leads to a destabilization of FANCD2. FANCD2 accumulated from mid-S to G2 phase within sites containing single-stranded DNA (ssDNA) intermediates, or at sites of DNA damage, such as those created by restriction endonucleases and laser irradiation. Purified FANCD2, a ring-like particle by electron microscopy, preferentially bound ssDNA over various DNA substrates. Inhibition of MRE11 nuclease activity by Mirin decreased the number of FANCD2 foci formed in vivo. We propose that FANCD2 binds to ssDNA arising from MRE11-processed DNA double-strand breaks. Our data establish MRN as a crucial regulator of FANCD2 stability and function in the DNA damage response.

Different concentrations of Mg++ ions affect nuclear matrix protein distribution during thermal stabilization of isolated nuclei.

The nuclear matrix, a proteinaceous network believed to be a scaffolding structure determining higher-order organization of chromatin, is usually prepared from intact nuclei by a series of extraction steps. In most cell types investigated the nuclear matrix does not spontaneously resist these treatments but must be stabilized before the application of extracting agents. Incubation of isolated nuclei at 37C or 42C in buffers containing Mg++ has been widely employed as stabilizing agent. We have previously demonstrated that heat treatment induces changes in the distribution of three nuclear scaffold proteins in nuclei prepared in the absence of Mg++ ions. We studied whether different concentrations of Mg++ (2.0-5 mM) affect the spatial distribution of nuclear matrix proteins in nuclei isolated from K562 erythroleukemia cells and stabilized by heat at either 37C or 42C. Five proteins were studied, two of which were RNA metabolism-related proteins (a 105-kD component of splicing complexes and an RNP component), one a 126-kD constituent of a class of nuclear bodies, and two were components of the inner matrix network. The localization of proteins was determined by immunofluorescent staining and confocal scanning laser microscope. Mg++ induced significant changes of antigen distribution even at the lowest concentration employed, and these modifications were enhanced in parallel with increase in the concentration of the divalent cation. The different sensitivity to heat stabilization and Mg++ of these nuclear proteins might reflect a different degree of association with the nuclear scaffold and can be closely related to their functional or structural role.

Validation Studies of Thermal-hydraulic Code for Safety Analysis of Nuclear Power Plants

This thesis gives an overview of the validation process for thermal hydraulic system codes and it presents in more detail the assessment and validation of the French code CATHARE for VVER calculations. Three assessment cases are presented: loop seal clearing, core reflooding and flow in a horizontal steam generator. The experience gained during these assessment and validation calculations has been used to analyze the behavior of the horizontal steam generator and the natural circulation in the geometry of the Loviisa nuclear power plant. The cases presented are not exhaustive, but they give a good overview of the work performed by the personnel of Lappeenranta University of Technology (LUT). Large part of the work has been performed in co-operation with the CATHARE-team in Grenoble, France. The design of a Russian type pressurized water reactor, VVER, differs from that of a Western-type PWR. Most of thermal-hydraulic system codes are validated only for the Western-type PWRs. Thus, the codes should be assessed and validated also for VVER design in order to establish any weaknesses in the models. This information is needed before codes can be used for the safety analysis. Theresults of the assessment and validation calculations presented here show that the CATHARE code can be used also for the thermal-hydraulic safety studies for VVER type plants. However, some areas have been indicated which need to be reassessed after further experimental data become available. These areas are mostly connected to the horizontal stem generators, like condensation and phase separation in primary side tubes. The work presented in this thesis covers a large numberof the phenomena included in the CSNI code validation matrices for small and intermediate leaks and for transients. Also some of the phenomena included in the matrix for large break LOCAs are covered. The matrices for code validation for VVER applications should be used when future experimental programs are planned for code validation.

Effect of Noncondensable Gases on Circulation of Primary Coolant in Nuclear Power Plants in Abnormal Situations

The present study focuses on two effects of the presence of a noncondensable gas on the thermal-hydraulic behavior of thecoolant of the primary circuit of a nuclear reactor in the VVER-440 geometry inabnormal situations. First, steam condensation with the presence of air was studied in the horizontal tubes of the steam generator (SG) of the PACTEL test facility. The French thermal-hydraulic CATHARE code was used to study the heat transfer between the primary and secondary side in conditions derived from preliminary experiments performed by VTT using PACTEL. In natural circulation and single-phase vapor conditions, the injection of a volume of air, equivalent to the totalvolume of the primary side of the SG at the entrance of the hot collector, did not stop the heat transfer from the primary to the secondary side. The calculated results indicate that air is located in the second half-length (from the mid-length of the tubes to the cold collector) in all the tubes of the steam generator The hot collector remained full of steam during the transient. Secondly, the potential release of the nitrogen gas dissolved in the water of the accumulators of the emergency core coolant system of the Loviisa nuclear power plant (NPP) was investigated. The author implemented a model of the dissolution and release ofnitrogen gas in the CATHARE code; the model created by the CATHARE developers. In collaboration with VTT, an analytical experiment was performed with some components of PACTEL to determine, in particular, the value of the release time constant of the nitrogen gas in the depressurization conditions representative of the small and intermediate break transients postulated for the Loviisa NPP. Such transients, with simplified operating procedures, were calculated using the modified CATHARE code for various values of the release time constant used in the dissolution and release model. For the small breaks, nitrogen gas is trapped in thecollectors of the SGs in rather large proportions. There, the levels oscillate until the actuation of the low-pressure injection pumps (LPIS) that refill the primary circuit. In the case of the intermediate breaks, most of the nitrogen gas is expelled at the break and almost no nitrogen gas is trapped in the SGs. In comparison with the cases calculated without taking into account the release of nitrogen gas, the start of the LPIS is delayed by between 1 and 1.75 h. Applicability of the obtained results to the real safety conditions must take into accountthe real operating procedures used in the nuclear power plant.

Topological phases in small quantum Hall samples

Topological order has proven a useful concept to describe quantum phase transitions which are not captured by the Ginzburg-Landau type of symmetry-breaking order. However, lacking a local order parameter, topological order is hard to detect. One way to detect it is via direct observation of anyonic properties of excitations which are usually discussed in the thermodynamic limit, but so far has not been realized in macroscopic quantum Hall samples. Here we consider a system of few interacting bosons subjected to the lowest Landau level by a gauge potential, and theoretically investigate vortex excitations in order to identify topological properties of different ground states. Our investigation demonstrates that even in surprisingly small systems anyonic properties are able to characterize the topological order. In addition, focusing on a system in the Laughlin state, we study the robustness of its anyonic behavior in the presence of tunable finite-range interactions acting as a perturbation. A clear signal of a transition to a different state is reflected by the system's anyonic properties.

Further molecular profiling of tumors harboring therapeutic targets within non-small cell lung cancer

Background: Treatment of NSCLC has been revolutionized in recent years with the introduction of several targeted therapies for selected genetically altered subtypes of NSCLC. A better understanding of molecular characteristics of NSCLC, which features common drug targets, may identify new therapeutic options. Methods: Over 6,700 non-small cell lung cancer cases referred to Caris Life Sciences between 2009 and 2014. Diagnoses and history were collected from referring physicians. Specific testing was performed per physician request and included a combination of sequencing (Sanger, NGS or pyrosequencing), protein expression (IHC), gene amplification/rearrangement (CISH or FISH), and/or RNA fragment analysis. Results: Tumors profiles from patients with hormone receptor positive disease (HER2, ER, PR, or AR positive by IHC) (n=629), HER2 mutations (n=8) ALK rearrangements (n=55), ROS1 rearrangement (n=17), cMET amplification or mutation (n=126), and cKIT mutation (n=11) were included in this analysis and compared to the whole cohort. Tumors with ALK rearrangement overexpressed AR in 18% of cases, and 7% presented with concomitant KRAS mutation. Lower rates of PTEN loss, as assessed by IHC, were observed in ALK positive (20%), ROS1 positive (9%) and cKIT mutated tumors (25%) compared to the overall NSCLC population (58%). cMET was overexpressed in 66% of ROS1 translocated and 57% of HER2 mutated tumors. cKIT mutations were found co-existing with APC (20%) and EGFR (20%) mutations. Pathway analysis revealed that hormone receptor positive disease carried more mutations in the ERK pathway (32%) compared to 9% in the mTOR pathway. 25% of patients with HER2 mutations harbored a co-existing mutation in the mTOR pathway. Conclusions: Pathway profiling reveals that NSCLC tumors present more often than reported with several concomitant alterations affecting the ERK or AKT pathway. Additionally, they are also characterized by the expression of potential biological modifiers of the cell cycle like hormonal receptors, representing a rationale for dual inhibition strategies in selected patients. Further refining of the understanding of NSCLC biomarker profile will optimize research for new treatment strategies.

Molecular profiling of non-small cell lung cancer by histologic subtype

Background: A substantial proportion of NSCLC has been shown to harbour specific molecular alterations affecting tumour proliferation and resulting in sensitivity to inhibition of the corresponding activated oncogenic pathway by targeted therapies. Comprehensive tumor profiling can diagnose such alterations and may identify new alterations opening additional treatment options for all distinct NSCLC subtypes. Methods: Over 6,700 non-small cell lung cancer cases referred to Caris Life Sciences between 2009 and 2014 were evaluated; clinical diagnoses and detailed tumor pathology were collected from referring physicians. Specific profiling was performed per physician request and included a combination of sequencing (Sanger, NGS or pyrosequencing), protein expression (IHC), gene amplification/rearrangement (CISH or FISH), and/or RNA fragment analysis within potential cancer-related genes and pathways. Results: Patients were grouped into cohorts according to histological subtype - adenocarcinoma (AD) (n=4,286), squamous cell carcinoma (SCC) (n=1,280), large cell carcinoma (LCC) (n=153) and bronchioalveolar carcinoma (BAC) (n=94). Protein overexpression of cMET (>2+ in >50% cells) was higher in AD (35.9%) compared to other subgroups (12-20%) while RRM1 and TOP2A levels were lower in AD. ALK or ROS1 were rearranged in 5.3% of patients with AD compared to 3.7% of patients with LCC and 1.2% of patients with SCC. EGFR mutations were found at low prevalence in both the LCC (0%) and SCC cohorts (2.8%) compared to 21% in AD. Similar lower rates of BRAF mutations were observed in the LCC and SCC cohorts compared to AD (0%, 1.1% and 5.1%). Pathway analysis showed activating mutations in the ERK pathway in 40% of patients with AD. Only 10-12% of patients with LCC or SCC had activating mutations in the ERK pathway. Conclusions: Despite the limitations of this retrospective series, we report comprehensive profiling of the largest cohort of NSCLC. Tumor profiling reveals that ADs may be more addicted to the ERK pathway than other histological subtypes. Drugs which target cMET may also have most utility in AD. Full analysis by histological subtype and additional correlative data on protein expression, gene copy number and mutations will be presented.

Small scale energy production in Helsinki Metropolitan area in emission trading point of view

Emission trading with greenhouse gases and green certificates are part if the climate policy the main target of which is reduce greenhouse gas emissions. The carbon dioxide and fine particle emissions of energy production in Helsinki Metropolitan area are calculated in this study. The analysis is made mainly by district heating point of view and the changes of the district heating network are assessed. Carbon dioxide emissions would be a bit higher, if the district heating network is expanded, but then the fine particle emissions would be much lower. Carbon dioxide emissions are roughly 10 % higher, if the district heating network is expanded at same rate as it has in past five years in the year 2030. The expansion of district heating network would decrease the fine particle emissions about 40 %. The cost of the expansion is allocated to be reduction cost of the fine particle emissions, which is considerably higher than the traditional reduction methods costs. The possible new nuclear plant would reduce the emissions considerably and the costs of the nuclear plant would be relatively low comparing the other energy production methods.

Nuclear Matrix in Apoptotic Cell Death and Cell Proliferation. The role of Nuclear Mitotic Apparatus (NuMA) Protein

The nucleus is a membrane enclosed organelle containing most of the genetic information of the cell in the form of chromatin. The nucleus, which can be divided into many sub-organelles such as the nucleoli, the Cajal bodies and the nuclear lamina, is the site for several essential cellular functions such as the DNA replication and its regulation and most of the RNA synthesis and processing. The nucleus is often affected in disease: the size and the shape of the nucleus, the chromatin distribution and the size of the nucleoli have remained the basis for the grading of several cancers. The maintenance of the vertebrate body shape depends on the skeleton. Similarly, in a smaller context, the shape of the cell and the nucleus are mainly regulated by the cytoskeletal and nucleoskeletal elements. The nuclear matrix, which by definition is a detergent, DNase and salt resistant proteinaceous nuclear structure, has been suggested to form the nucleoskeleton responsible for the nuclear integrity. Nuclear mitotic apparatus protein, NuMA, a component of the nuclear matrix, is better known for its mitotic spindle organizing function. NuMA is one of the nuclear matrix proteins suggested to participate in the maintenance of the nuclear integrity during interphase but its interphase function has not been solved to date. This thesis study concentrated on the role of NuMA and the nuclear matrix as structural and functional components of the interphase nucleus. The first two studies clarified the essential role of caspase-3 in the disintegration of the nuclear structures during apoptosis. The second study also showed NuMA and chromatin to co-elute from cells in significant amounts and the apoptotic cleavage of NuMA was clarified to have an important role in the dissociation of NuMA from the chromatin. The third study concentrated on the interphase function of NuMA showing NuMA depletion to result in cell cycle arrest and the cytoplasmic relocalization of NuMA interaction partner GAS41. We suggest that the relocalization of the transcription factor GAS41 may mediate the cell cycle arrest. Thus, this study has given new aspects in the interactions of NuMA, chromatin and the nuclear matrix.

Licensing Model Development for Small Modular Reactors (SMRs) - Focusing on the Finnish Regulatory Framework

Recently, Small Modular Reactors (SMRs) have attracted increased public discussion. While large nuclear power plant new build projects are facing challenges, the focus of attention is turning to small modular reactors. One particular project challenge arises in the area of nuclear licensing, which plays a significant role in new build projects affecting their quality as well as costs and schedules. This dissertation - positioned in the field of nuclear engineering but also with a significant section in the field of systems engineering - examines the nuclear licensing processes and their suitability for the characteristics of SMRs. The study investigates the licensing processes in selected countries, as well as other safety critical industry fields. Viewing the licensing processes and their separate licensing steps in terms of SMRs, the study adopts two different analysis theories for review and comparison. The primary data consists of a literature review, semi-structured interviews, and questionnaire responses concerning licensing processes and practices. The result of the study is a recommendation for a new, optimized licensing process for SMRs. The most important SMR-specific feature, in terms of licensing, is the modularity of the design. Here the modularity indicates multi-module SMR designs, which creates new challenges in the licensing process. As this study focuses on Finland, the main features of the new licensing process are adapted to the current Finnish licensing process, aiming to achieve the main benefits with minimal modifications to the current process. The application of the new licensing process is developed using Systems Engineering, Requirements Management, and Project Management practices and tools. Nuclear licensing includes a large amount of data and documentation which needs to be managed in a suitable manner throughout the new build project and then during the whole life cycle of the nuclear power plant. To enable a smooth licensing process and therefore ensure the success of the new build nuclear power plant project, management processes and practices play a significant role. This study contributes to the theoretical understanding of how licensing processes are structured and how they are put into action in practice. The findings clarify the suitability of different licensing processes and their selected licensing steps for SMR licensing. The results combine the most suitable licensing steps into a new licensing process for SMRs. The results are also extended to the concept of licensing management practices and tools.

Patch-clamp detection of macromolecular translocation along nuclear pores

The present paper reviews the application of patch-clamp principles to the detection and measurement of macromolecular translocation along the nuclear pores. We demonstrate that the tight-seal 'gigaseal' between the pipette tip and the nuclear membrane is possible in the presence of fully operational nuclear pores. We show that the ability to form a gigaseal in nucleus-attached configurations does not mean that only the activity of channels from the outer membrane of the nuclear envelope can be detected. Instead, we show that, in the presence of fully operational nuclear pores, it is likely that the large-conductance ion channel activity recorded derives from the nuclear pores. We conclude the technical section with the suggestion that the best way to demonstrate that the nuclear pores are responsible for ion channel activity is by showing with fluorescence microscopy the nuclear translocation of ions and small molecules and the exclusion of the same from the cisterna enclosed by the two membranes of the envelope. Since transcription factors and mRNAs, two major groups of nuclear macromolecules, use nuclear pores to enter and exit the nucleus and play essential roles in the control of gene activity and expression, this review should be useful to cell and molecular biologists interested in understanding how patch-clamp can be used to quantitate the translocation of such macromolecules into and out of the nucleus

Antagonist G-mediated targeting and cytotoxicity of liposomal doxorubicin in NCI-H82 variant small cell lung cancer

The aim of the present study was to characterize the interactions of antagonist G (H-Arg-D-Trp-NmePhe-D-Trp-Leu-Met-NH 2)-targeted sterically stabilized liposomes with the human variant small cell lung cancer (SCLC) H82 cell line and to evaluate the antiproliferative activity of encapsulated doxorubicin against this cell line. Variant SCLC tumors are known to be more resistant to chemotherapy than classic SCLC tumors. The cellular association of antagonist G-targeted (radiolabeled) liposomes was 20-30-fold higher than that of non-targeted liposomes. Our data suggest that a maximum of 12,000 antagonist G-targeted liposomes were internalized/cell during 1-h incubation at 37ºC. Confocal microscopy experiments using pyranine-containing liposomes further confirmed that receptor-mediated endocytosis occurred, specifically in the case of targeted liposomes. In any of the previously mentioned experiments, the binding and endocytosis of non-targeted liposomes have revealed to be negligible. The improved cellular association of antagonist G-targeted liposomes, relative to non-targeted liposomes, resulted in an enhanced nuclear delivery (evaluated by fluorimetry) and cytotoxicity of encapsulated doxorubicin for incubation periods as short as 2 h. For an incubation of 2 h, we report IC50 values for targeted and non-targeted liposomes containing doxorubicin of 5.7 ± 3.7 and higher than 200 µM doxorubicin, respectively. Based on the present data, we may infer that receptors for antagonist G were present in H82 tumor cells and could mediate the internalization of antagonist G-targeted liposomes and the intracellular delivery of their content. Antagonist G covalently coupled to liposomal drugs may be promising for the treatment of this aggressive and highly heterogeneous disease.

Immunohistochemical detection of virus through its nuclear cytopathic effect in idiopathic interstitial pneumonia other than acute exacerbation

Idiopathic interstitial pneumonias include complex diseases that have a strong interaction between genetic makeup and environmental factors. However, in many cases, no infectious agent can be demonstrated, and these clinical diseases rapidly progress to death. Theoretically, idiopathic interstitial pneumonias could be caused by the Epstein-Barr virus, cytomegalovirus, adenovirus, hepatitis C virus, respiratory syncytial virus, and herpesvirus, which may be present in such small amounts or such configuration that routine histopathological analysis or viral culture techniques cannot detect them. To test the hypothesis that immunohistochemistry provides more accurate results than the mere histological demonstration of viral inclusions, this method was applied to 37 open lung biopsies obtained from patients with idiopathic interstitial pneumonias. As a result, immunohistochemistry detected measles virus and cytomegalovirus in diffuse alveolar damage-related histological patterns of acute exacerbation of idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia in 38 and 10% of the cases, respectively. Alveolar epithelium infection by cytomegalovirus was observed in 25% of organizing pneumonia patterns. These findings were coincident with nuclear cytopathic effects but without demonstration of cytomegalovirus inclusions. These data indicate that diffuse alveolar damage-related cytomegalovirus or measles virus infections enhance lung injury, and a direct involvement of these viruses in diffuse alveolar damage-related histological patterns is likely. Immunohistochemistry was more sensitive than the histological demonstration of cytomegalovirus or measles virus inclusions. We concluded that all patients with diffuse alveolar damage-related histological patterns should be investigated for cytomegalovirus and measles virus using sensitive immunohistochemistry in conjunction with routine procedures.

Analysis of PWR-PACTEL Small Break LOCA experiment using TRACE

Currently, the power generation is one of the most significant life aspects for the whole man-kind. Barely one can imagine our life without electricity and thermal energy. Thus, different technologies for producing those types of energy need to be used. Each of those technologies will always have their own advantages and disadvantages. Nevertheless, every technology must satisfy such requirements as efficiency, ecology safety and reliability. In the matter of the power generation with nuclear energy utilization these requirements needs to be highly main-tained, especially since accidents on nuclear power plants may cause very long term deadly consequences. In order to prevent possible disasters related to the accident on a nuclear power plant strong and powerful algorithms were invented in last decades. Such algorithms are able to manage calculations of different physical processes and phenomena of real facilities. How-ever, the results acquired by the computing must be verified with experimental data.

Active nuclear import and cytoplasmic retention of activation-induced deaminase

The enzyme activation-induced deaminase (AID) triggers antibody diversification in B cells by catalyzing deamination and consequently mutation of immunoglobulin genes. To minimize off-target deamination, AID is restrained by several regulatory mechanisms including nuclear exclusion, thought to be mediated exclusively by active nuclear export. Here we identify two other mechanisms involved in controlling AID subcellular localization. AID is unable to passively diffuse into the nucleus, despite its small size, and its nuclear entry requires active import mediated by a conformational nuclear localization signal. We also identify in its C terminus a determinant for AID cytoplasmic retention, which hampers diffusion to the nucleus, competes with nuclear import and is crucial for maintaining the predominantly cytoplasmic localization of AID in steady-state conditions. Blocking nuclear import alters the balance between these processes in favor of cytoplasmic retention, resulting in reduced isotype class switching.