Differential regulation of MRN (Mre11-Rad50-Nbs1) complex subunits and telomerase activity in cancer cells

Several lines of evidence suggest that cancer progression is associated with up-regulation or reactivation of telomerase and the underlying mechanism remains an active area of research. The heterotrimeric MRN complex, consisting of Mre11, Rad50 and Nbs1, which is required for the repair of double-strand breaks, plays a key role in telomere length maintenance. In this study, we show significant differences in the levels of expression of MRN complex subunits among various cancer cells and somatic cells. Notably, siRNA-mediated depletion of any of the subunits of MRN complex led to complete ablation of other subunits of the complex. Treatment of leukemia and prostate cancer cells with etoposide lead to increased expression of MRN complex subunits, with concomitant decrease in the levels of telomerase activity, compared to breast cancer cells. These studies raise the possibility of developing anti-cancer drugs targeting MRN complex subunits to sensitize a subset of cancer cells to radio- and/or chemotherapy. (C) 2010 Elsevier Inc. All rights reserved.

Microscopic expression for frequency and wave vector dependent dielectric constant of a dipolar liquid

A microscopic expression for the frequency and wave vector dependent dielectric constant of a dense dipolar liquid is derived starting from the linear response theory. The new expression properly takes into account the effects of the translational modes in the polarization relaxation. The longitudinal and the transverse components of the dielectric constant show vastly different behavior at the intermediate values of the wave vector k. We find that the microscopic structure of the dense liquid plays an important role at intermediate wave vectors. The continuum model description of the dielectric constant, although appropriate at very small values of wave vector, breaks down completely at the intermediate values of k. Numerical results for the longitudinal and the transverse dielectric constants are obtained by using the direct correlation function from the mean‐spherical approximation for dipolar hard spheres. We show that our results are consistent with all the limiting expressions known for the dielectric function of matter.

Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells

Cancer cells are often associated with secondary chromosomal rearrangements, such as deletions, inversions, and translocations, which could be the consequence of unrepaired/misrepaired DNA double strand breaks (DSBs). Nonhomologous DNA end joining is one of the most common pathways to repair DSBs in higher eukaryotes. By using oligomeric DNA substrates mimicking various endogenous DSBs in a cell-free system, we studied end joining (EJ) in different cancer cell lines. We found that the efficiency of EJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of EJ proteins. Interestingly, cancer cells with lower levels of EJ possessed elevated expression of BCL2 and vice versa. Removal of BCL2 by immunoprecipitation or protein fractionation led to elevated EJ. More importantly, we show that overexpression of BCL2 or the addition of purified BCL2 led to the down-regulation of EJ. Further, we found that BCL2 interacts with KU proteins both in vitro and in vivo. Hence, our results suggest that EJ in cancer cells could be negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute toward increased chromosomal abnormalities in cancer.

Novel rhodanine derivatives induce growth inhibition followed by apoptosis

We have designed and synthesized three novel compounds, 5-isopropylidiene derivatives of 3-dimethyl-2-thio-hydantoin (ITH-1), 3-ethyl-2-thio-2,4-oxazolidinedione (ITO-1), and 5-benzilidene-3-ethyl rhodanine (BTR-1), and have tested their chemotherapeutic properties. Our results showed that all three compounds induced cytotoxicity in a time-and concentration-dependent manner on leukemic cell line, CEM. Among the compounds tested, BTR-1 was 5- to 7-fold more potent than ITH-1 and ITO-1 when compared by trypan blue and MTT assays. IC50 value of BTR-1 was estimated to be <10 mu M. Both cell cycle analysis and tritiated thymidine assays revealed that BTR-1 affects DNA replication by inducing a block at S phase. BTR-1 treatment led to increased level of ROS production and DNA strand breaks suggesting activation of apoptosis for induction of cell death. (C) 2010 Elsevier Ltd. All rights reserved.

A Computationally Efficient PWM Algorithm for Multilevel Inverters

Centred space vector PWM (CSVPWM) technique is popularly used for three level voltage source inverters. The reference voltage vector is synthesized by time-averaging of the three nearest voltage vectors produced by the inverter. Identifying the three voltage vectors, and calculation of the dwelling time for each vector are both computationally intensive. This paper analyses the process of PWM generation in CSVPWM. This analysis breaks up a three-level inverter into six different conceptual two level inverters in different regions of the fundamental cycle. Control of 3-level inverter is viewed as the control of the appropriate 2-level inverter. The analysis leads to a systematic simplification of the computations involved, finally resulting in a computationally efficient PWM algorithm. This algorithm exploits the equivalence between triangle comparison and space vector approaches to PWM generation. This algorithm does not involve any 3-phase/2-phase or 2-phase/3-phase transformation. This also does not involve any transformation from rectangular to polar coordinates, and vice versa. Further no evaluation of trigonometric functions is necessary. This algorithm also provides for the mitigation of DC neutral point unbalance, and is well suited to digital implementation. Simulation and experimental results are presented.

Diakoniatyön paikka ihmisten arjessa

This study examines the diaconia work of the Finnish Evangelical Lutheran Church from the standpoint of clients. The role of diaconia work has grown since the early 1990s recession, and since it established itself as one of the actors along with other social organizations. Previous studies have described the changing role of diaconal work, especially from the standpoint of diaconia workers and co-operators. This research goes back to examine, beyond the activities of the diaconia work of everyday practices, its relations of ruling which are determining practices. The theoretical and methodological framework rises from the thinking of Dorothy E. Smith, the creator of institutional ethnography. Its origins are in feminism, Marxism, phenomenology, etnomethodology, and symbolic interactionism. However, it does not represent any school. Unlike the objectivity-based traditional sociology, institutional ethnography has its starting point in everyday life, and people s subjective experience of it. Everyday life is just a starting point, and is used to examine everyday life s experiences of hidden relations of ruling, linking people and organizations. The level of generalization is just on the relations of ruling. The research task is to examine those meanings of diaconia work which are embedded in its clients experiences. The research task is investigated with two questions: how diaconia work among its clients takes shape and what kinds of relations of ruling exist in diaconia work. The meanings of diaconia work come through an examination of the relations of ruling, which create new forms of diaconal work compared with previous studies. For the study, two kinds of data were collected: a questionnaire and ethnographic fieldwork. The first data set was collected from diaconal workers using the questionnaire. It gives background information of the diaconia work process from the standpoint of the clients. In the ethnographic study there were two phases. The first ethnographic material was collected from one local parish by observing, interviewing clients and diaconal workers and gathering documents. The number of observations was 36 customer appointments, and 29 interviews. The second ethnographic material was included as a part of the analysis, in which ruling relations in people s experiences were collected from the transcribed data. Close reading and narrative analysis are used as analysing methods. The analysis has three phases. First, the experiences are identified with close reading; the following step is to select some of the institutional processes that are shaping those experiences and are relevant for the research. At the third stage, those processes are investigated in order to describe analytically how they determine people s experience. The analysis produces another narrative about diaconia work, which provides tools for examining the diaconal work from a new perspective. Through the analysis it is possible to see diaconia as an exchange ratio, in which the exchange takes place between a client and a diaconia worker, but also more broadly with other actors, such as social workers, shop clerks, or with other parishioners. The exchange ratio is examined from the perspective of power which is embedded in the client s experiences. The analysis reveals that the most important relations of ruling are humiliation and randomness in the exchange ratio of diaconia work; valuating spirituality above the bodily being; and replacing official social work. The results give a map about the relations of ruling of diaconia work which gives tools to look at diaconia work s meanings to the clients. The hidden element of humiliation in the exchange ratio breaks the current picture of diaconia work. The ethos of the holistic encounters and empathic practices are shown to be of another kind when spirituality is preferred to the bodily being. Nevertheless, diaconia appears to be a place for a respectful encounter, especially in situations where the public sector s actors are retreating on liability or clients are in a life crisis. The collapse of the welfare state structures imposes on diaconia work tasks that have not previously belonged to it. At the local level, clients receive partners from diaconia workers in order to advocate them in the welfare system. Actions to influence the wider societal structures are not reached because of lacking resources. An awareness of the oppressive practices of diaconia work and their critical reviewing are the keys to the development of diaconia work, since there are such practices even in holistic and respectful diaconia work. While the research raises new information for the development of diaconia work, it also opens up new aspects for developing other kinds of social work by emphasizing the importance of taking people s experiences seriously. Keywords: diaconia work, institutional ethnography, Dorothy E. Smith, experience, customer, relations of ruling.

A multi-stage model for drop breakage in stirred vessels

Existing models for dmax predict that, in the limit of μd → ∞, dmax increases with 3/4 power of μd. Further, at low values of interfacial tension, dmax becomes independent of σ even at moderate values of μd. However, experiments contradict both the predictions show that dmax dependence on μd is much weaker, and that, even at very low values of σ,dmax does not become independent of it. A model is proposed to explain these results. The model assumes that a drop circulates in a stirred vessel along with the bulk fluid and repeatedly passes through a deformation zone followed by a relaxation zone. In the deformation zone, the turbulent inertial stress tends to deform the drop, while the viscous stress generated in the drop and the interfacial stress resist deformation. The relaxation zone is characterized by absence of turbulent stress and hence the drop tends to relax back to undeformed state. It is shown that a circulating drop, starting with some initial deformation, either reaches a steady state or breaks in one or several cycles. dmax is defined as the maximum size of a drop which, starting with an undeformed initial state for the first cycle, passes through deformation zone infinite number of times without breaking. The model predictions reduce to that of Lagisetty. (1986) for moderate values of μd and σ. The model successfully predicts the reduced dependence of dmax on μd at high values of μd as well as the dependence of dmax on σ at low values of σ. The data available in literature on dmax could be predicted to a greater accuracy by the model in comparison with existing models and correlations.

A multi-stage model for drop breakage in stirred vessels

Existing models for dmax predict that, in the limit of μd → ∞, dmax increases with 3/4 power of μd. Further, at low values of interfacial tension, dmax becomes independent of σ even at moderate values of μd. However, experiments contradict both the predictions show that dmax dependence on μd is much weaker, and that, even at very low values of σ,dmax does not become independent of it. A model is proposed to explain these results. The model assumes that a drop circulates in a stirred vessel along with the bulk fluid and repeatedly passes through a deformation zone followed by a relaxation zone. In the deformation zone, the turbulent inertial stress tends to deform the drop, while the viscous stress generated in the drop and the interfacial stress resist deformation. The relaxation zone is characterized by absence of turbulent stress and hence the drop tends to relax back to undeformed state. It is shown that a circulating drop, starting with some initial deformation, either reaches a steady state or breaks in one or several cycles. dmax is defined as the maximum size of a drop which, starting with an undeformed initial state for the first cycle, passes through deformation zone infinite number of times without breaking. The model predictions reduce to that of Lagisetty. (1986) for moderate values of μd and σ. The model successfully predicts the reduced dependence of dmax on μd at high values of μd as well as the dependence of dmax on σ at low values of σ. The data available in literature on dmax could be predicted to a greater accuracy by the model in comparison with existing models and correlations.

Unraveling the helix of change : An activity-theoretical study of health care change efforts and their consequences

M.A. (Educ.) Anu Kajamaa from the University of Helsinki, Center for Research on Activity, Development and Learning (CRADLE), examines change efforts and their consequences in health care in the public sector. The aim of her academic dissertation is, by providing a new conceptual framework, to widen our understanding of organizational change efforts and their consequences and managerial challenges. Despite the multiple change efforts, the results of health care development projects have not been very promising, and many developmental needs and managerial challenges exist. The study challenges the predominant, well-framed health care change paradigm and calls for an expanded view to explore the underlying issues and multiplicities of change efforts and their consequences. The study asks what kind of expanded conceptual framework is needed to better understand organizational change as transcending currently dominant oppositions in management thinking, specifically in the field of health care. The study includes five explorative case studies of health care change efforts and their consequences in Finland. Theory and practice are tightly interconnected in the study. The methodology of the study integrates the ethnography of organizational change, a narrative approach and cultural-historical activity theory. From the stance of activity theory, historicity, contradictions, locality and employee participation play significant roles in developing health care. The empirical data of the study has mainly been collected in two projects, funded by the Finnish Work Environment Fund. The data was collected in public sector health care organizations during the years 2004-2010. By exploring the oppositions between distinct views on organizational change and the multi-site, multi-level and multi-logic of organizational change, the study develops an expanded, multidimensional activity-theoretical framework on organizational change and management thinking. The findings of the study contribute to activity theory and organization studies, and provide information for health care management and practitioners. The study illuminates that continuous development efforts bridged to one another and anchored to collectively created new activity models can lead to significant improvements and organizational learning in health care. The study presents such expansive learning processes. The ways of conducting change efforts in organizations play a critical role in the creation of collective new practices and tools and in establishing ownership over them. Some of the studied change efforts were discontinuous or encapsulated, not benefiting the larger whole. The study shows that the stagnation and unexpected consequences of change efforts relate to the unconnectedness of the different organizational sites, levels and logics. If not dealt with, the unintended consequences such as obstacles, breaks and conflicts may stem promising change and learning processes.

A new model for the breakage frequency of drops in turbulent stirred dispersions

A model of drop breakage in turbulent stirred dispersions based on interaction of a drop with eddies of a length scale smaller than the drop diameter has been developed. It predicts that, unlike the equal breakage assumed by earlier models, a large drop reduces in size due to stripping of smaller segments off it through unequal breakage. It is only when the drop nears the value of the maximum stable drop diameter that it breaks into equal parts. This new model of drop breakage, coupled with the pattern of interaction of drops with eddies of different sizes existing in the vessel, has been used to evaluate not only the breakage frequency, but also the size distribution of the daughter droplets(which was hitherto assumed). The model has been incorporated in the population balance equation and the resulting cumulative size distributions compared with those availble in the literature.

DNA damage recognition in the normal epithelium of human prostate and seminal vesicles

Prostate cancer is one of the most prevalent cancer types in men. The development of prostate tumors is known to require androgen exposure, and several pathways governing cell growth are deregulated in prostate tumorigenesis. Recent genetic studies have revealed that complex gene fusions and copy - number alterations are frequent in prostate cancer, a unique feature among solid tumors. These chromosomal aberrations are though to arise as a consequence of faulty repair of DNA double strand breaks (DSB). Most repair mechanisms have been studied in detail in cancer cell lines, but how DNA damage is detected and repaired in normal differentiated human cells has not been widely addressed. The events leading to the gene fusions in prostate cancer are under rigorous studies, as they not only shed light on the basic pathobiologic mechanisms but may also produce molecular targets for prostate cancer treatment and prevention. Prostate and seminal vesicles are part of the male reproductive system. They share similar structure and function but differ dramatically in their cancer incidence. Approximately fifty primary seminal vesicle carcinomas have been reported worldwide. Surprisingly, only little is known on why seminal vesicles are resistant to neoplastic changes. As both tissues are androgen dependent, it is a mystery that androgen signaling would only lead to tumors in prostate tissue. In this work, we set up novel ex vivo human tissue culture models of prostate and seminal vesicles, and used them to study how DNA damage is recognized in normal epithelium. One of the major DNA - damage inducible pathways, mediated by the ATM kinase, was robustly activated in all main cell types of both tissues. Interestingly, we discovered that secretory epithelial cells had less histone variant H2A.X and after DNA damage lower levels of H2AX were phosphorylated on serine 139 (γH2AX) than in basal or stromal cells. γH2AX has been considered essential for efficient DSB repair, but as there were no significant differences in the γH2AX levels between the two tissues, it seems more likely that the role of γH2AX is less important in postmitotic cells. We also gained insight into the regulation of p53, an important transcription factor that protects genomic integrity via multiple mechanisms, in human tissues. DSBs did not lead to a pronounced activation of p53, but treatments causing transcriptional stress, on the other hand, were able to launch a notable p53 response in both tissue types. In general, ex vivo culturing of human tissues provided unique means to study differentiated cells in their relevant tissue context, and is suited for testing novel therapeutic drugs before clinical trials. In order to study how prostate and seminal vesicle epithelial cells are able to activate DNA damage induced cell cycle checkpoints, we used primary cultures of prostate and seminal vesicle epithelial cells. To our knowledge, we are the first to report isolation of human primary seminal vesicle cells. Surprisingly, human prostate epithelial cells did not activate cell cycle checkpoints after DSBs in part due to low levels of Wee1A, a kinase regulating CDK activity, while primary seminal vesicle epithelial cells possessed proficient cell cycle checkpoints and expressed high levels of Wee1A. Similarly, seminal vesicle cells showed a distinct activation of the p53 - pathway after DSBs that did not occur in prostate epithelial cells. This indicates that p53 protein function is under different control mechanisms in the two cell types, which together with proficient cell cycle checkpoints may be crucial in protecting seminal vesicles from endogenous and exogenous DNA damaging factors and, as a consequence, from carcinogenesis. These data indicate that two very similar organs of male reproductive system do not respond to DNA damage similarly. The differentiated, non - replicating cells of both tissues were able to recognize DSBs, but under proliferation human prostate epithelial cells had deficient activation of the DNA damage response. This suggests that prostate epithelium is most vulnerable to accumulating genomic aberrations under conditions where it needs to proliferate, for example after inflammatory cellular damage.

Formation of a G-quadruplex at the BCL2 major breakpoint region of the t(14;18) translocation in follicular lymphoma

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3'-UTR of the BCL2 gene and are mainly clustered in the major breakpoint region (MBR). Recently, we found that the BCL2 MBR has a non-B DNA character in genomic DNA. Here, we show that single-stranded DNA modeled from the template strand of the BCL2 MBR, forms secondary structures that migrate faster on native PAGE in the presence of potassium, due to the formation of intramolecular G-quadruplexes. Circular dichroism shows evidence for a parallel orientation for G-quadruplex structures in the template strand of the BCL2 MBR. Mutagenesis and the DMS modification assay confirm the presence of three guanine tetrads in the structure. 1H nuclear magnetic resonance studies further confirm the formation of an intramolecular G-quadruplex and a representative model has been built based on all of the experimental evidence. We also provide data consistent with the possible formation of a G-quadruplex structure at the BCL2 MBR within mammalian cells. In summary, these important features could contribute to the single-stranded character at the BCL2 MBR, thereby contributing to chromosomal fragility.

Efficiency of nonhomologous DNA end joining varies among somatic tissues, despite similarity in mechanism

Failure to repair DNA double-strand breaks (DSBs) can lead to cell death or cancer. Although nonhomologous end joining (NHEJ) has been studied extensively in mammals, little is known about it in primary tissues. Using oligomeric DNA mimicking endogenous DSBs, NHEJ in cell-free extracts of rat tissues were studied. Results show that efficiency of NHEJ is highest in lungs compared to other somatic tissues. DSBs with compatible and blunt ends joined without modifications, while noncompatible ends joined with minimal alterations in lungs and testes. Thymus exhibited elevated joining, followed by brain and spleen, which could be correlated with NHEJ gene expression. However, NHEJ efficiency was poor in terminally differentiated organs like heart, kidney and liver. Strikingly, NHEJ junctions from these tissues also showed extensive deletions and insertions. Hence, for the first time, we show that despite mode of joining being generally comparable, efficiency of NHEJ varies among primary tissues of mammals.

Formation of a G-quadruplex at the BCL2 major breakpoint region of the t(14;18) translocation in follicular lymphoma

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3'-UTR of the BCL2 gene and are mainly clustered in the major breakpoint region (MBR). Recently, we found that the BCL2 MBR has a non-B DNA character in genomic DNA. Here, we show that single-stranded DNA modeled from the template strand of the BCL2 MBR, forms secondary structures that migrate faster on native PAGE in the presence of potassium, due to the formation of intramolecular G-quadruplexes. Circular dichroism shows evidence for a parallel orientation for G-quadruplex structures in the template strand of the BCL2 MBR. Mutagenesis and the DMS modification assay confirm the presence of three guanine tetrads in the structure. 1H nuclear magnetic resonance studies further confirm the formation of an intramolecular G-quadruplex and a representative model has been built based on all of the experimental evidence. We also provide data consistent with the possible formation of a G-quadruplex structure at the BCL2 MBR within mammalian cells. In summary, these important features could contribute to the single-stranded character at the BCL2 MBR, thereby contributing to chromosomal fragility.

Ultrafast underdamped solvation: Agreement between computer simulation and various theories of solvation dynamics

A theoretical analysis of the three currently popular microscopic theories of solvation dynamics, namely, the dynamic mean spherical approximation (DMSA), the molecular hydrodynamic theory (MHT), and the memory function theory (MFT) is carried out. It is shown that in the underdamped limit of momentum relaxation, all three theories lead to nearly identical results when the translational motions of both the solute ion and the solvent molecules are neglected. In this limit, the theoretical prediction is in almost perfect agreement with the computer simulation results of solvation dynamics in the model Stockmayer liquid. However, the situation changes significantly in the presence of the translational motion of the solvent molecules. In this case, DMSA breaks down but the other two theories correctly predict the acceleration of solvation in agreement with the simulation results. We find that the translational motion of a light solute ion can play an important role in its own solvation. None of the existing theories describe this aspect. A generalization of the extended hydrodynamic theory is presented which, for the first time, includes the contribution of solute motion towards its own solvation dynamics. The extended theory gives excellent agreement with the simulations where solute motion is allowed. It is further shown that in the absence of translation, the memory function theory of Fried and Mukamel can be recovered from the hydrodynamic equations if the wave vector dependent dissipative kernel in the hydrodynamic description is replaced by its long wavelength value. We suggest a convenient memory kernel which is superior to the limiting forms used in earlier descriptions. We also present an alternate, quite general, statistical mechanical expression for the time dependent solvation energy of an ion. This expression has remarkable similarity with that for the translational dielectric friction on a moving ion.