Overexpression of the protein kinase CK2 increases the survival and resistance to chemotherapy of acute myeloid leukemia cells

Background: The critical role of protein kinase CK2 in the regulation of cellular apoptosis suggests its may be involvement in tumor cell resistance to both conventional and unconventional therapies. Aim of the study: To study the role of CK2 in acute myeloid leukemia (AML) cell survival and response to chemotherapeutic agents. Methods: Apoptotic pathways were evaluated by AnnexinV/Propidium Iodide staining, Western Blot analysis of Caspase3 and Parp cleavage and Real Time PCR of antiapoptotic gene expression. Results: CK2 α catalytic subunit expression level and activity were increased in AML cells as compared to normal CD34+ hematopoietic cells. CK2 inactivation with the synthetic chemical inhibitors K27, CX-4945 or RNA interference induced AML cell apoptosis of p53 wild-type but not of p53-null cells, suggesting that the apoptosis triggered by CK2 inhibition needs the presence of functional p53. Inhibition of CK2 activity with K27 or CX-4945 was associated to an increased sensitivity of the cytotoxic effects of doxorubicin and daunorubicin. Cells were also nucleofected with siRNA oligos directed against CK2 α catalytic, β regulatory or both subunits. Interestingly RNA interference of CK2 β  reduced cell viability and enhanced apoptosis induced by daunorubicin, indicating a prominent role of this subunit in the resistance to chemotherapy. Based on our in-vitro results we have then examined the expression level of CK2 α and β in 32 newly diagnosed AML patients (excluding acute promyelocitic leukemia) from 2008 to 2011. Preliminary data shows a higher expression of the CK2 α subunit in the two AML subgroups with the highest risk based on cytogenetic characteristics. These laters nowadays represent the strongest prognostic factor predicting chemoresistance. Interestingly we did not find a correlation with expression of the regulatory subunit CK2 β. Conclusions: These data highlight the relevance of CK2 in AML cell survival for the influence of this kinase on the activation of anti-apoptotic pathways implicated in AML cell resistance to chemotherapy. A study to establish wether the CK2 expression at diagnosis represents an independent prognostic factor in the progression free survival and overall survival for these patients is needed

STRUCTURAL AND FUNCTIONAL STUDIES ON GABAA RECEPTOR SUBTYPES: A COMPUTATIONAL PATHWAY FOR DESIGNING NOVEL NON-SEDATIVE MODULATORS

In this stressful era, maintaining the proper balance of neuronal excitation and inhibition remains the central demand of human brain. To harmonize the optimal brain functioning, γ-Amino Butyric Acid type A Receptors (GABAA-Rs) play a vital role by mediating the fast inhibitory neurotransmissions. These GABA-gated chloride ion channels maintain the delicate balance between neuronal excitation and inhibition. The formation of GABAA-R uses repertoire of 19 different subunit subtypes α1-6, β1-3, γ1-3, δ, ε, π, θ and ρ1-3, out of which two α1, two β2 and one γ2 form the most abundant native GABAA-R structure. In the absence of heteropentameric human GABAA-R structure the structural biology remains yet to be fully explored. Manipulation of GABAergic transmission is aimed to provide the benefits in the treatment of a host of neurological and psychiatric disorders. We utilised the existing experimental data and carried out a computational study to obtain the structural details of different GABAA-Rs. This computational pathway sequentially proceeds for : i) obtaining the different GABAA-R states and subtypes; ii) understanding the logic of their existence and correlating structure-function details for each of them; iii) unravelling the complete journey of molecular events that fine tune the state dependent channel transitions in normal conditions including ligand unoccupied closed, open, uncapped receptive states and GABA occupied singly and doubly bound states; iv) understanding the nature of cross-talk between two orthosteric sites and third allosteric BZD-site when we brought it into consideration; v) identifying a set of governing rules/markers forming the structural basis of selective modulation for BZD-site agonists at α1- and α2-GABAA-R subtypes. Accordingly, to fulfil the deliberate demand of clinically efficacious α2-selective non-sedative modulator/s the underlying logic is systematically demarcated under single platform. The crux from the early stage modulatory pathways of subtype selective actions provides newer avenues to guide the designing of novel modulator/s having desired pharmacological endpoints in diseased states. Overall, this channelled study is bound to track the structure-function-novel drug designing, based on the understanding of GABAA-R modulatory pathways

Long Term Contracting in a Changing World

I study the properties of optimal long-term contracts in an environment in which the agent’s type evolves stochastically over time. The model stylizes a buyer-seller relationship but the results apply quite naturally to many contractual situations including regulation and optimal income-taxation. I …first show, through a simple discrete example, that distortions need not vanish over time and need not be monotonic in the shock to the buyer’s valuation. These results are in contrast to those obtained in the literature that assumes a Markov process with a binary state space e.g. Battaglini, 2005. I then show that the study of the dynamics of the optimal mechanism can be significantly simplified by assuming the shocks are independent over time. When the sets of possible types in any two adjacent periods satisfy a certain overlapping condition (which is always satisfied with a continuum of types) and some additional regularity conditions hold, then the optimal mechanism is the same irrespective of whether the shocks are the buyer’s private information or are observed also by the seller. These conditions are satisfied, for example, in the case of an AR(1) process, a Brownian motion, but also when shocks have a multiplicative effect as it is often the case in financial applications. Furthermore, the distortions in the optimal quantities are independent of the distributions of the shocks and, when the buyer’s payoff is additively separable, they are also independent of whether the shocks are transitory or permanent. Finally, I show that assuming the shocks are independent not only does it greatly simplify the analysis, it is actually without loss of generality.asymmetric information, stochastic process, dynamic mechanism design, long-term contracting

Long-Term Contracting in a Changing World

I study the properties of optimal long-term contracts in an environment in which the agents type evolves stochastically over time. The model stylizes a buyer-seller relationship but the results apply quite naturally to many contractual situations including regulation and optimal income-taxation. I …rst show, through a simple example, that distortions need not vanish over time and need not be monotonic in the shock to the buyers valuation. These results are in contrast to those obtained in the literature that assumes a Markov process with a binary state space e.g. Battaglini, 2005. I then show that when the sets of possible types in any two adja- cent periods satisfy a certain overlapping condition (which is always satis…ed with a continuum of types), then the dynamics of the optimal mechanism can be signi…cantly simpli…ed by as- suming the shocks are independent over time. Under certain regularity conditions, the optimal mechanism is then the same irrespective of whether the shocks are the buyers private informa- tion or are observed also by the seller. These conditions are satis…ed, for example, in the case of an AR(1) process, a Brownian motion, but also when shocks have a multiplicative e¤ect as it is often the case in …nancial applications. Furthermore, the distortions in the optimal quantities are independent of the distributions of the shocks and, when the buyers valuation is additively separable, they are also independent of whether the shocks are transitory or permanent. Finally, I show that assuming the shocks are independent not only greatly simpli…es the analysis but is actually without loss of generality with a continuum of types.asymmetric information, stochastic process, dynamic mechanism design, long-term contracting

Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding

Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenicprotein that is the basis for both anthrax vaccines and diagnostic methods. Properly foldedantigenic PA is necessary for these applications. In this study a high level of PA was obtained inrecombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, whichfacilitated its efficient purification by simple washing steps; however, it could not be recognizedby specific antibodies. Refolding conditions were subsequently analyzed in a high-throughputmanner that enabled nearly a hundred different conditions to be tested simultaneously. Therecovery of the ability of PA to be recognized by antibodies was screened by dot blot usinga coefficient that provided a measure of properly refolded protein levels with a high degreeof discrimination. The best refolding conditions resulted in a tenfold increase in the intensityof the dot blot compared to the control. The only refolding additive that consistently yieldedgood results was L-arginine. The statistical analysis identified both cooperative and negativeinteractions between the different refolding additives. The high-throughput approach describedin this study that enabled overproduction, purification and refolding of PA in a simple andstraightforward manner, can be potentially useful for the rapid screening of adequate refoldingconditions for other overexpressed antigenic proteins.Fil: Pavan, María Elisa. Biochemiq; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Pavan, Esteban E.. Politecnico di Milano; ItaliaFil: Cairo, Fabian Martin. Biochemiq; ArgentinaFil: Pettinari, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin

Control of flexible joint robotic manipulator using tuning functions design

The goal of this thesis is to design the controller for a single arm manipulator having a flexible joint for the tracking problem in two different cases. A controller is designed for a deterministic case wherein the plant parameters are assumed to be known while another is designed for an adaptive case where all the plant parameters are assumed to be unknown. In general the tracking problem is; given a smooth reference trajectory, the end effector has to track the reference while maintaining the stability. It is assumed that only the output of the manipulator, which is the link angle, is available for measurement. Also without loss of generality, the fast dynamics, that is the dynamics of the driver side of the system are neglected for the sake of simplicity; In the first case, the design procedure adopted is called observer backstepping. Since the states of the system are unavailable for measurement, an observer is designed that estimates the system states. These estimates are fed to the controller which in turn produces the control input to the system; The second case employs a design procedure called tuning functions design. In this case, since the plant parameters are unknown, the observer designed in case one cannot be used for determining the state estimates. For this purpose, parameter update laws and filters are designed for estimation of plant parameters. The filters employed are k-filters. The k-filters and the parameter update laws are given as input to the controller, which generates the control input to the system; For both cases, the mathematical models are simulated using Matlab/Simulink, and the results are verified