Mechanism of damage sensing and cell killing following the inhibition of nucleotide excision repair by fludarabine in quiescent cells

Inhibition of DNA repair by the nucleoside of fludarabine (F-ara-A) induces toxicity in quiescent human cells. The sensing and signaling mechanisms following DNA repair inhibition by F-ara-A are unknown. The central hypothesis of this project was that the mechanistic interaction of a DNA repair initiating agent and a nucleoside analog initiates an apoptotic signal in quiescent cells. The purpose of this research was to identify the sensing and signaling mechanism(s) that respond to DNA repair inhibition by F-ara-A. Lymphocytes were treated with F-ara-A, to accumulate the active triphosphate metabolite and subsequently DNA repair was activated by UV irradiation. Pre-incubation of lymphocytes with 3 μM F-ara-A inhibited DNA repair initiated by 2 J/m2 UV and induced greater than additive apoptosis after 24 h. Blocking the incorporation of F-ara-A nucleotide into repairing DNA using 30 μM aphidicolin considerably lowered the apoptotic response. ^ Wild-type quiescent cells showed a significant loss in viability than did cells lacking functional sensor kinase DNA-PKcs or p53 as measured by colony formation assays. The functional status of ATM did not appear to affect the apoptotic outcome. Immunoprecipitation studies showed an interaction between the catalytic sub-unit of DNA-PK and p53 following DNA repair inhibition. Confocal fluorescence microscopy studies have indicated the localization pattern of p53, DNA-PK and γ-H2AX in the nucleus following DNA damage. Foci formation by γ-H2AX was seen as an early event that is followed by interaction with DNA-PKcs. p53 serine-15 phosphorylation and accumulation were detected 2 h after treatment. Fas/Fas ligand expression increased significantly after repair inhibition and was dependent on the functional status of p53. Blocking the interaction between Fas and Fas ligand by neutralizing antibodies significantly rescued the apoptotic fraction of cells. ^ Collectively, these results suggest that incorporation of the nucleoside analog into repair patches is critical for cytotoxicity and that the DNA damage, while being sensed by DNA-PK, may induce apoptosis by a p53-mediated signaling mechanism. Based on the results, a model is proposed for the sensing of F-ara-A-induced DNA damage that includes γ-H2AX, DNA-PKcs, and p53. Targeting the cellular DNA repair mechanism can be a potential means of producing cytotoxicity in a quiescent population of neoplastic cells. These results also provide mechanistic support for the success of nucleoside analogs with cyclophosphamide or other agents that initiate excision repair processes, in the clinic. ^

Capital Accumulation in the Presence of Informal Credit Contracts: Does the Incentive Mechanism Work Better than Credit Rationing Under Asymmetric Information?

Credit markets with asymmetric information often prefer credit rationing as a profit maximizing device. This paper asks whether the presence of informal credit markets reduces the cost of credit rationing, that is, whether it can alleviate the impact of asymmetric information based on the available information. We used a dynamic general equilibrium model with heterogenous agents to assess this. Using Indian credit market data our study shows that the presence of informal credit market can reduce the cost of credit rationing by separating high risk firms from the low risk firms in the informal market. But even after this improvement, the steady state capital accumulation is still much lower as compared to incentive based market clearing rates. Through self revelation of each firm's type, based on the incentive mechanism, banks can diversify their risk by achieving a separating equilibrium in the loan market. The incentive mechanism helps banks to increase capital accumulation in the long run by charging lower rates and lending relatively higher amount to the less risky firms. Another important finding of this study is that self-revelation leads to very significant welfare improvement, as measured by consumptiuon equivalence.

Mechanism of anti-tumor activity of adenovirus type 5 E1A in ovarian cancer

The adenovirus type 5 E1A gene was originally developed as a gene therapy to inhibit tumorigenicity of HER-2-overexpressing cells by transcriptional downregulation of HER-2. Our goal is to improve the overall efficacy of E1A gene therapy. To achieve this goal, we have conducted two preclinical experiments. ^ First, we hypothesized that Bcl-2 overexpressing ovarian cancer is resistant to E1A gene therapy. This hypothesis is based on that the 19 kDa protein product of the adenoviral E1B gene which is homologous to Bcl-2 inhibits E1A-induced apoptosis. Treating high Bcl-2-xpressing cells with E1A in combination with an antisense oligonucleotide to Bcl-2 (Bcl-2-ASO) resulted in a significant decrease in cell viability due to an increased rate of apoptosis relative to cells treated with E1A alone. In an ovarian cancer xenograft model, mice implanted with low HER-2, high Bcl-2 cells, treated with E1A plus Bcl-2-ASO led to prolonged survival. Bcl-2 thus may serve as a predictive molecular marker enabling us to select patients with ovarian cancer who will benefit significantly from E1A gene therapy. ^ Second, we elucidated the molecular mechanism governing the anti-tumor effect of E1A in ovarian cancer to identify a more potent tumor suppressor gene. We identified PEA-15 (phospho-protein enriched in astrocytes) upregulated in E1A transfected low HER-2-expressing OVCAR-3 ovarian cancer cell, which showed decreased cell proliferation. PEA-15 moved ERK from the nucleus to the cytoplasm and inhibited ERK-dependent transcription and proliferation. Using small interfering RNA to knock down PEA-15 expression in OVCAR-3 cells made to constitutively express E1A resulted in accumulation of phosphoERK in the nucleus, an increase in Elk-1 activity, DNA synthesis, and anchorage-independent growth. PEA-15 also independently suppressed colony formation in some breast and ovarian cancer cell lines in which E1A is known to have anti-tumor activity. We conclude that the anti-tumor activity of E1A depends on PEA-15. ^ In summary, (1) Bcl-2 may serve as a predictive molecular marker of E1A gene therapy, allowing us to select patients and improve efficacy of E1A gene therapy. (2) PEA-15 was identified as a component of the molecular mechanism governing the anti-tumor activity of E1A in ovarian cancer, (3) PEA-15 may be developed as a novel therapeutic gene. ^

Are Shirking and Leisure Substitutable? An Empirical Test of Efficiency Wages Based on Urban Economic Theory

Recent theoretical work has examined the spatial distribution of unemployment using the efficiency wage model as the mechanism by which unemployment arises in the urban economy. This paper extends the standard efficiency wage model in order to allow for behavioral substitution between leisure time at home and effort at work. In equilibrium, residing at a location with a long commute affects the time available for leisure at home and therefore affects the trade-off between effort at work and risk of unemployment. This model implies an empirical relationship between expected commutes and labor market outcomes, which is tested using the Public Use Microdata sample of the 2000 U.S. Decennial Census. The empirical results suggest that efficiency wages operate primarily for blue collar workers, i.e. workers who tend to be in occupations that face higher levels of supervision. For this subset of workers, longer commutes imply higher levels of unemployment and higher wages, which are both consistent with shirking and leisure being substitutable.

The mechanism of action of a novel benzo[c]phenanthridine alkaloid, NK314 and the cellular responses

NK314 is a novel synthetic benzo[c]phenanthridine alkaloid that is currently in clinical trials as an antitumor compound, based on impressive activities in preclinical models. However, its mechanism of action is unknown. The present investigations were directed at determining the mechanism of action of this agent and cellular responses to NK314. My studies demonstrated that NK314 intercalated into DNA, trapped topoisomerase IIα in its cleavage complex intermediate, and inhibited the ability of topoisomerase IIα to relax super-coiled DNA. CEM/VM1 cells, which are resistant to etoposide due to mutations in topoisomerase IIα, were cross-resistant to NK314. However, CEM/C2 cells, which are resistant to camptothecin due to mutations in topoisomerase I, retained sensitivity. This indicates topoisomerase IIα is the target of NK314 in the cells. NK314 caused phosphorylation of the histone variant, H2AX, which is considered a marker of DNA double-strand breaks. DNA double-strand breaks were also evidenced by pulsed-field gel electrophoresis and visualized as chromosomal aberrations after cells were treated with NK314 and arrested in mitosis. Cell cycle checkpoints are activated following DNA damage. NK314 induced significant G2 cell cycle arrest in several cell lines, independent of p53 status, suggesting the existence of a common mechanism of checkpoint activation. The Chk1-Cdc25C-Cdk1 G2 checkpoint pathway was activated in response to NK314, which can be abrogated by the Chk1 inhibitor UCN-01. Cell cycle checkpoint activation may be a defensive mechanism that provides time for DNA repair. DNA double-strand breaks are repaired either through ATM-mediated homologous recombination or DNA-PK-mediated non-homologous end-joining repair pathways. Clonogenic assays demonstrated a significant decrease of colony formation in both ATM deficient and DNA-PK deficient cells compared to ATM repleted and DNA-PK wild type cells respectively, indicating that both ATM and DNA-PK play important roles in the survival of the cells in response to NK314. The DNA-PK specific inhibitor NU7441 also significantly sensitized cells to NK314. In conclusion, the major mechanism of NK314 is to intercalate into DNA, trap and inhibit topoisomerase IIα, an action that leads to the generation of double-strand DNA breaks, which activate ATM and DNA-PK mediated DNA repair pathways and Chk1 mediated G2 checkpoint pathway. ^

Mechanism of methylation gene reactivation in human cancer cells

Epigenetic silencing of tumor suppressor genes by DNA hypermethylation at promoter regions is a common event in carcinogenesis and tumor progression. Abrogation of methylation and reversal of epigenetic silencing is a very potent way in cancer treatment. However, the reactivation mechanisms are poorly understood. In this study, we first developed a cell line model system named YB5, derived from SW48 cancer cell line, which bears one copy of stably integrated EGFP gene on Chromosome 1p31.1 region. The GFP gene expression is transcriptionally silenced due to the hypermethylated promoter CMV. However, the GFP expression can be restored using demethylating agent 5-aza-2' deoxycytidine (DAC), and detected by FACS and fluorescent microscopy. Using this system, we observed the heterogeneous reactivation induced by DAC treatment. After flow sorting, GFP negative cells exhibited similar level of incomplete demethylation compared to GFP positive cells on repetitive LINE1 element, tumor suppressor genes such as P16, CDH13, and RASSF1a, and CMV promoter as well. However, the local chromatin of CMV-GFP locus altered to an open structure marked by high H3 lysine 9 acetylation and low H3 lysine 27 tri-methylation in GFP positive cells, while the GFP negative cells retained mostly the original repressive marks. Thus, we concluded that DAC induced DNA hypomethylation alone does not directly determine the level of re-expression, and the resetting of the local chromatin structure under hypomethylation environment is required for gene reactivation. Besides, a lentivirus vector-based shRNA screening was performed using the YB5 system. Although it is the rare chance that vector lands in the neighboring region of GFP, we found that the exogenous vector DNA inserted into the upstream region of GFP gene locus led to the promoter demethylation and reactivated the silenced GFP gene. Thus, epigenetic state can be affected by changing of the adjacent nucleic acid sequences. Further, this hypermethylation silenced system was utilized for epigenetic drug screening. We have found that DAC combined with carboplatin would enhance the GFP% yield and increase expression of other tumor suppressor genes than DAC alone, and this synergistic effect may be related to DNA repair process. In summary, these studies reveal that reversing of methylation silencing requires coordinated alterations of DNA methylation, chromatin structure, and local microenvironment. ^

The role of workplace absence as an organizational mechanism in predicting employee alcohol and drug disorders

Background. EAP programs for airline pilots in companies with a well developed recovery management program are known to reduce pilot absenteeism following treatment. Given the costs and safety consequences to society, it is important to identify pilots who may be experiencing an AOD disorder to get them into treatment. ^ Hypotheses. This study investigated the predictive power of workplace absenteeism in identifying alcohol or drug disorders (AOD). The first hypothesis was that higher absenteeism in a 12-month period is associated with higher risk that an employee is experiencing AOD. The second hypothesis was that AOD treatment would reduce subsequent absence rates and the costs of replacing pilots on missed flights. ^ Methods. A case control design using eight years (time period) of monthly archival absence data (53,000 pay records) was conducted with a sample of (N = 76) employees having an AOD diagnosis (cases) matched 1:4 with (N = 304) non-diagnosed employees (controls) of the same profession and company (male commercial airline pilots). Cases and controls were matched on the variables age, rank and date of hire. Absence rate was defined as sick time hours used over the sum of the minimum guarantee pay hours annualized using the months the pilot worked for the year. Conditional logistic regression was used to determine if absence predicts employees experiencing an AOD disorder, starting 3 years prior to the cases receiving the AOD diagnosis. A repeated measures ANOVA, t tests and rate ratios (with 95% confidence intervals) were conducted to determine differences between cases and controls in absence usage for 3 years pre and 5 years post treatment. Mean replacement costs were calculated for sick leave usage 3 years pre and 5 years post treatment to estimate the cost of sick leave from the perspective of the company. ^ Results. Sick leave, as measured by absence rate, predicted the risk of being diagnosed with an AOD disorder (OR 1.10, 95% CI = 1.06, 1.15) during the 12 months prior to receiving the diagnosis. Mean absence rates for diagnosed employees increased over the three years before treatment, particularly in the year before treatment, whereas the controls’ did not (three years, x = 6.80 vs. 5.52; two years, x = 7.81 vs. 6.30, and one year, x = 11.00cases vs. 5.51controls. In the first year post treatment compared to the year prior to treatment, rate ratios indicated a significant (60%) post treatment reduction in absence rates (OR = 0.40, CI = 0.28, 0.57). Absence rates for cases remained lower than controls for the first three years after completion of treatment. Upon discharge from the FAA and company’s three year AOD monitoring program, case’s absence rates increased slightly during the fourth year (controls, x = 0.09, SD = 0.14, cases, x = 0.12, SD = 0.21). However, the following year, their mean absence rates were again below those of the controls (controls, x = 0.08, SD = 0.12, cases, x¯ = 0.06, SD = 0.07). Significant reductions in costs associated with replacing pilots calling in sick, were found to be 60% less, between the year of diagnosis for the cases and the first year after returning to work. A reduction in replacement costs continued over the next two years for the treated employees. ^ Conclusions. This research demonstrates the potential for workplace absences as an active organizational surveillance mechanism to assist managers and supervisors in identifying employees who may be experiencing or at risk of experiencing an alcohol/drug disorder. Currently, many workplaces use only performance problems and ignore the employee’s absence record. A referral to an EAP or alcohol/drug evaluation based on the employee’s absence/sick leave record as incorporated into company policy can provide another useful indicator that may also carry less stigma, thus reducing barriers to seeking help. This research also confirms two conclusions heretofore based only on cross-sectional studies: (1) higher absence rates are associated with employees experiencing an AOD disorder; (2) treatment is associated with lower costs for replacing absent pilots. Due to the uniqueness of the employee population studied (commercial airline pilots) and the organizational documentation of absence, the generalizability of this study to other professions and occupations should be considered limited. ^ Transition to Practice. The odds ratios for the relationship between absence rates and an AOD diagnosis are precise; the OR for year of diagnosis indicates the likelihood of being diagnosed increases 10% for every hour change in sick leave taken. In practice, however, a pilot uses approximately 20 hours of sick leave for one trip, because the replacement will have to be paid the guaranteed minimum of 20 hour. Thus, the rate based on hourly changes is precise but not practical. ^ To provide the organization with practical recommendations the yearly mean absence rates were used. A pilot flies on average, 90 hours a month, 1080 annually. Cases used almost twice the mean rate of sick time the year prior to diagnosis (T-1) compared to controls (cases, x = .11, controls, x = .06). Cases are expected to use on average 119 hours annually (total annual hours*mean annual absence rate), while controls will use 60 hours. The cases’ 60 hours could translate to 3 trips of 20 hours each. Management could use a standard of 80 hours or more of sick time claimed in a year as the threshold for unacceptable absence, a 25% increase over the controls (a cost to the company of approximately of $4000). At the 80-hour mark, the Chief Pilot would be able to call the pilot in for a routine check as to the nature of the pilot’s excessive absence. This management action would be based on a company standard, rather than a behavioral or performance issue. Using absence data in this fashion would make it an active surveillance mechanism. ^

OXIDATIVE STRESS BASED STRATEGIES FOR ENHANCING THE EFFICACY OF HISTONE DEACETYLASE INHIBITORS (HDACi)

Histone deacetylase inhibitors (HDACi) are anti-cancer drugs that primarily act upon acetylation of histones, however they also increase levels of intracellular reactive oxygen species (ROS). We hypothesized that agents that cause oxidative stress might enhance the efficacy of HDACi. To test this hypothesis, we treated acute lymphocytic leukemia cells (ALL) with HDACi and adaphostin (ROS generating agent). The combination of two different HDACi (vorinostat or entinostat) with adaphostin synergistically induced apoptosis in ALL. This synergistic effect was blocked when cells were pre-treated with the caspase-9 inhibitor, LEHD. In addition, we showed that loss of the mitochondrial membrane potential is the earliest event observed starting at 12 h. Following this event, we observed increased levels of superoxide at 16 h, and ultimately caspase-3 activation. Pre-treatment with the antioxidant N-acetylcysteine (NAC) blocked ROS generation and reversed the loss of mitochondrial membrane potential for both combinations. Interestingly, DNA fragmentation and caspase-3 activity was only blocked by NAC in cells treated with vorinostat-adaphostin; but not with entinostat-adaphostin. These results suggest that different redox mechanisms are involved in the induction of ROS-mediated apoptosis. To further understand these events, we studied the role of the antioxidants glutathione (GSH) and thioredoxin (Trx). We found that the combination of entinostat-adaphostin induced acetylation of the antioxidant thioredoxin (Trx) and decreased intracellular levels of GSH. However, no effect on Trx activity was observed in either combination. In addition, pre-treatment with GSH ethyl ester, a soluble form of GSH, did not block DNA fragmentation. Together these results suggested that GSH and Trx are not major players in the induction of oxidative stress. Array data examining the expression of genes involved in oxidative stress demonstrated a differential regulation between cells treated with vorinostat-adaphostin and entinostat-adaphostin. Some of the genes differentially expressed between the combinations include aldehyde oxidase 1, glutathione peroxidase-5, -6, peroxiredoxin 6 and myeloperoxidase. Taken together, these experimental results indicate that the synergistic activity of two different HDACi with adaphostin is mediated by distinct redox mechanisms in ALL cells. Understanding the mechanism involved in these combinations will advance scientific knowledge of how the action of HDACi could be augmented in leukemia models. Moreover, this information could be used for the development of effective clinical trials combining HDACi with other anticancer agents.

Analysis of the mechanism of replication inhibition of the tumor suppressor gene p53

p53 plays a role in cell cycle arrest and apoptosis. p53 has also been shown to be involved in DNA replication. To study the effect of p53 on DNA replication, we utilized a SV40 based shuttle vector system. The pZ402 shuttle vector, was constructed with a mutated T-antigen unable to interact with p53 but able to support replication of the shuttle vector. When a transcriptional activation domain p53 mutant was tested for its ability to inhibit DNA replication no inhibition was observed. Competition assays with the DNA binding domain of p53 was also able to block the inhibition of DNA replication by p53 suggesting that p53 can inhibit DNA replication through the transcriptional activation of a target gene. One likely target gene, p21$\sp{\rm cip/waf}$ was tested to determine whether p53 inhibited DNA replication by transcriptionally activating p21$\sp{\rm cip/waf}$. Two independent approaches utilizing p21$\sp{\rm cip/waf}$ null cells or the expression of an anti-sense p21$\sp{\rm cip/waf}$ expression vector were utilized. p53 was able to inhibit pZ402 replication independently of p21$\sp{\rm cip/waf}$. p53 was also able to inhibit DNA replication independent of the p53 target genes Gadd45 and the replication processivity factor PCNA. The inhibition of DNA replication by p53 was also independent of direct DNA binding to a consensus site on the replicating plasmid. p53 mutants can be classified into two categories: conformational and DNA contact mutants. The two types of p53 mutants were tested for their effects on DNA replication. While all conformational mutants were unable to inhibit DNA replication three out of three DNA contact mutants tested were able to inhibit DNA replication. The work here studies the effect wild-type and mutant p53 has on DNA replication and demonstrated a possible mechanism by which wild-type p53 could inhibit DNA replication through the transcriptional activation of a target gene. ^

Mechanism of mRNA stability control mediated by AU -rich element: Characterization of cis-elements and trans-factors

Regulation of cytoplasmic deadenylation, the first step in mRNA turnover, has direct impact on the fate of gene expression. AU-rich elements (AREs) found in the 3′ untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. Based on their sequence features and functional properties, AREs can be divided into three classes. Class I or class III ARE directs synchronous deadenylation, whereas class II ARE directs asynchronous deadenylation with the formation of poly(A)-intermediates. Through systematic mutagenesis study, we found that a cluster of five or six copies of AUUUA motifs forming various degrees of reiteration is the key feature dictating the choice between asynchronous versus synchronous deadenylation. A 20–30 nt AU-rich sequence immediately 5 ′ to this cluster of AUUUA motifs can greatly enhance its destabilizing ability and is an integral part of the AREs. These two features are the defining characteristics of class II AREs. ^ To better understand the decay mechanism of AREs, current methods have several limitations. Taking the advantage of tetracycline-regulated promoter, we developed a new transcriptional pulse strategy, Tet-system. By controlling the time and the amount of Tet addition, a pulse of RNA could be generated. Using this new system, we showed that AREs function in both growth- and density-arrested cells. The new strategy offers for the first time an opportunity to investigate control of mRNA deadenylation and decay kinetics in mammalian cells that exhibit physiologically relevant conditions. ^ As a member of heterogeneous nuclear RNA-binding protein, hnRNP D 0/AUF1 displays specific affinities for ARE sequences in vitro . But its in vivo function in ARE-mediated mRNA decay is unclear. AUF1/hnRNP D0 is composed of at least four isoforms derived by alternative RNA splicing. Each isoform exhibits different affinity for ARE sequence in vitro. Here, we examined in vivo effect of AUF1s/hnRNP D0s on degradation of ARE-containing mRNA. Our results showed that all four isoforms exhibit various RNA stabilizing effects in NIH3T3 cells, which are positively correlated with their binding affinities for ARE sequences. Further experiments indicated that AUF1/hnRNP D0 has a general role in modulating the stability of cytoplasmic mRNAs in mammalian cells. ^

Transport properties of CuGaSe(2)-based thin-film solar cells as a function of absorber composition

The transport properties of thin-film solar cells based on wide-gap CuGaSe(2) absorbers have been investigated as a function of the bulk [Ga]/[Cu] ratio ranging from 1.01 to 1.33. We find that (i) the recombination processes in devices prepared from absorbers with a composition close to stoichiometry ([Ga]/[Cu] = 1.01) are strongly tunnelling assisted resulting in low recombination activation energies (E(a)) of approx. 0.95 eV in the dark and 1.36 eV under illumination. (ii) With an increasing [Ga]/[Cu] ratio, the transport mechanism changes to be dominated by thermally activated Shockley-Read-Hall recombination with similar E(a) values of approx. 1.52-1.57 eV for bulk [Ga]/[Cu] ratios of 1.12-1.33. The dominant recombination processes take place at the interface between CdS buffer and CuGaSe(2) absorber independently from the absorber composition. The increase of E(a) with the [Ga]/[Cu] ratio correlates with the open circuit voltage and explains the better performance of corresponding solar cells.

Grid-based histogram arithmetic for the probabilistic analysis of functions

The selection of predefined analytic grids (partitions of the numeric ranges) to represent input and output functions as histograms has been proposed as a mechanism of approximation in order to control the tradeoff between accuracy and computation times in several áreas ranging from simulation to constraint solving. In particular, the application of interval methods for probabilistic function characterization has been shown to have advantages over other methods based on the simulation of random samples. However, standard interval arithmetic has always been used for the computation steps. In this paper, we introduce an alternative approximate arithmetic aimed at controlling the cost of the interval operations. Its distinctive feature is that grids are taken into account by the operators. We apply the technique in the context of probability density functions in order to improve the accuracy of the probability estimates. Results show that this approach has advantages over existing approaches in some particular situations, although computation times tend to increase significantly when analyzing large functions.

BaTboT: a biologically inspired flapping and morphing bat robot actuated by SMA-based artificial muscles

Bats are animals that posses high maneuvering capabilities. Their wings contain dozens of articulations that allow the animal to perform aggressive maneuvers by means of controlling the wing shape during flight (morphing-wings). There is no other flying creature in nature with this level of wing dexterity and there is biological evidence that the inertial forces produced by the wings have a key role in the attitude movements of the animal. This can inspire the design of highly articulated morphing-wing micro air vehicles (not necessarily bat-like) with a significant wing-to-body mass ratio. This thesis presents the development of a novel bat-like micro air vehicle (BaTboT) inspired by the morphing-wing mechanism of bats. BaTboT’s morphology is alike in proportion compared to its biological counterpart Cynopterus brachyotis, which provides the biological foundations for developing accurate mathematical models and methods that allow for mimicking bat flight. In nature bats can achieve an amazing level of maneuverability by combining flapping and morphing wingstrokes. Attempting to reproduce the biological wing actuation system that provides that kind of motion using an artificial counterpart requires the analysis of alternative actuation technologies more likely muscle fiber arrays instead of standard servomotor actuators. Thus, NiTinol Shape Memory Alloys (SMAs) acting as artificial biceps and triceps muscles are used for mimicking the morphing wing mechanism of the bat flight apparatus. This antagonistic configuration of SMA-muscles response to an electrical heating power signal to operate. This heating power is regulated by a proper controller that allows for accurate and fast SMA actuation. Morphing-wings will enable to change wings geometry with the unique purpose of enhancing aerodynamics performance. During the downstroke phase of the wingbeat motion both wings are fully extended aimed at increasing the area surface to properly generate lift forces. Contrary during the upstroke phase of the wingbeat motion both wings are retracted to minimize the area and thus reducing drag forces. Morphing-wings do not only improve on aerodynamics but also on the inertial forces that are key to maneuver. Thus, a modeling framework is introduced for analyzing how BaTboT should maneuver by means of changing wing morphology. This allows the definition of requirements for achieving forward and turning flight according to the kinematics of the wing modulation. Motivated by the biological fact about the influence of wing inertia on the production of body accelerations, an attitude controller is proposed. The attitude control law incorporates wing inertia information to produce desired roll (φ) and pitch (θ) acceleration commands. This novel flight control approach is aimed at incrementing net body forces (Fnet) that generate propulsion. Mimicking the way how bats take advantage of inertial and aerodynamical forces produced by the wings in order to both increase lift and maneuver is a promising way to design more efficient flapping/morphing wings MAVs. The novel wing modulation strategy and attitude control methodology proposed in this thesis provide a totally new way of controlling flying robots, that eliminates the need of appendices such as flaps and rudders, and would allow performing more efficient maneuvers, especially useful in confined spaces. As a whole, the BaTboT project consists of five major stages of development: - Study and analysis of biological bat flight data reported in specialized literature aimed at defining design and control criteria. - Formulation of mathematical models for: i) wing kinematics, ii) dynamics, iii) aerodynamics, and iv) SMA muscle-like actuation. It is aimed at modeling the effects of modulating wing inertia into the production of net body forces for maneuvering. - Bio-inspired design and fabrication of: i) skeletal structure of wings and body, ii) SMA muscle-like mechanisms, iii) the wing-membrane, and iv) electronics onboard. It is aimed at developing the bat-like platform (BaTboT) that allows for testing the methods proposed. - The flight controller: i) control of SMA-muscles (morphing-wing modulation) and ii) flight control (attitude regulation). It is aimed at formulating the proper control methods that allow for the proper modulation of BaTboT’s wings. - Experiments: it is aimed at quantifying the effects of properly wing modulation into aerodynamics and inertial production for maneuvering. It is also aimed at demonstrating and validating the hypothesis of improving flight efficiency thanks to the novel control methods presented in this thesis. This thesis introduces the challenges and methods to address these stages. Windtunnel experiments will be oriented to discuss and demonstrate how the wings can considerably affect the dynamics/aerodynamics of flight and how to take advantage of wing inertia modulation that the morphing-wings enable to properly change wings’ geometry during flapping. Resumen: Los murciélagos son mamíferos con una alta capacidad de maniobra. Sus alas están conformadas por docenas de articulaciones que permiten al animal maniobrar gracias al cambio geométrico de las alas durante el vuelo. Esta característica es conocida como (alas mórficas). En la naturaleza, no existe ningún especimen volador con semejante grado de dexteridad de vuelo, y se ha demostrado, que las fuerzas inerciales producidas por el batir de las alas juega un papel fundamental en los movimientos que orientan al animal en vuelo. Estas características pueden inspirar el diseño de un micro vehículo aéreo compuesto por alas mórficas con redundantes grados de libertad, y cuya proporción entre la masa de sus alas y el cuerpo del robot sea significativa. Esta tesis doctoral presenta el desarrollo de un novedoso robot aéreo inspirado en el mecanismo de ala mórfica de los murciélagos. El robot, llamado BaTboT, ha sido diseñado con parámetros morfológicos muy similares a los descritos por su símil biológico Cynopterus brachyotis. El estudio biológico de este especimen ha permitido la definición de criterios de diseño y modelos matemáticos que representan el comportamiento del robot, con el objetivo de imitar lo mejor posible la biomecánica de vuelo de los murciélagos. La biomecánica de vuelo está definida por dos tipos de movimiento de las alas: aleteo y cambio de forma. Intentar imitar como los murciélagos cambian la forma de sus alas con un prototipo artificial, requiere el análisis de métodos alternativos de actuación que se asemejen a la biomecánica de los músculos que actúan las alas, y evitar el uso de sistemas convencionales de actuación como servomotores ó motores DC. En este sentido, las aleaciones con memoria de forma, ó por sus siglas en inglés (SMA), las cuales son fibras de NiTinol que se contraen y expanden ante estímulos térmicos, han sido usados en este proyecto como músculos artificiales que actúan como bíceps y tríceps de las alas, proporcionando la funcionalidad de ala mórfica previamente descrita. De esta manera, los músculos de SMA son mecánicamente posicionados en una configuración antagonista que permite la rotación de las articulaciones del robot. Los actuadores son accionados mediante una señal de potencia la cual es regulada por un sistema de control encargado que los músculos de SMA respondan con la precisión y velocidad deseada. Este sistema de control mórfico de las alas permitirá al robot cambiar la forma de las mismas con el único propósito de mejorar el desempeño aerodinámico. Durante la fase de bajada del aleteo, las alas deben estar extendidas para incrementar la producción de fuerzas de sustentación. Al contrario, durante el ciclo de subida del aleteo, las alas deben contraerse para minimizar el área y reducir las fuerzas de fricción aerodinámica. El control de alas mórficas no solo mejora el desempeño aerodinámico, también impacta la generación de fuerzas inerciales las cuales son esenciales para maniobrar durante el vuelo. Con el objetivo de analizar como el cambio de geometría de las alas influye en la definición de maniobras y su efecto en la producción de fuerzas netas, simulaciones y experimentos han sido llevados a cabo para medir cómo distintos patrones de modulación de las alas influyen en la producción de aceleraciones lineales y angulares. Gracias a estas mediciones, se propone un control de vuelo, ó control de actitud, el cual incorpora información inercial de las alas para la definición de referencias de aceleración angular. El objetivo de esta novedosa estrategia de control radica en el incremento de fuerzas netas para la adecuada generación de movimiento (Fnet). Imitar como los murciélagos ajustan sus alas con el propósito de incrementar las fuerzas de sustentación y mejorar la maniobra en vuelo es definitivamente un tópico de mucho interés para el diseño de robots aéros mas eficientes. La propuesta de control de vuelo definida en este trabajo de investigación podría dar paso a una nueva forma de control de vuelo de robots aéreos que no necesitan del uso de partes mecánicas tales como alerones, etc. Este control también permitiría el desarrollo de vehículos con mayor capacidad de maniobra. El desarrollo de esta investigación se centra en cinco etapas: - Estudiar y analizar el vuelo de los murciélagos con el propósito de definir criterios de diseño y control. - Formular modelos matemáticos que describan la: i) cinemática de las alas, ii) dinámica, iii) aerodinámica, y iv) actuación usando SMA. Estos modelos permiten estimar la influencia de modular las alas en la producción de fuerzas netas. - Diseño y fabricación de BaTboT: i) estructura de las alas y el cuerpo, ii) mecanismo de actuación mórfico basado en SMA, iii) membrana de las alas, y iv) electrónica abordo. - Contro de vuelo compuesto por: i) control de la SMA (modulación de las alas) y ii) regulación de maniobra (actitud). - Experimentos: están enfocados en poder cuantificar cuales son los efectos que ejercen distintos perfiles de modulación del ala en el comportamiento aerodinámico e inercial. El objetivo es demostrar y validar la hipótesis planteada al inicio de esta investigación: mejorar eficiencia de vuelo gracias al novedoso control de orientación (actitud) propuesto en este trabajo. A lo largo del desarrollo de cada una de las cinco etapas, se irán presentando los retos, problemáticas y soluciones a abordar. Los experimentos son realizados utilizando un túnel de viento con la instrumentación necesaria para llevar a cabo las mediciones de desempeño respectivas. En los resultados se discutirá y demostrará que la inercia producida por las alas juega un papel considerable en el comportamiento dinámico y aerodinámico del sistema y como poder tomar ventaja de dicha característica para regular patrones de modulación de las alas que conduzcan a mejorar la eficiencia del robot en futuros vuelos.

A collaborative filtering similarity measure based on singularities.

Recommender systems play an important role in reducing the negative impact of informa- tion overload on those websites where users have the possibility of voting for their prefer- ences on items. The most normal technique for dealing with the recommendation mechanism is to use collaborative filtering, in which it is essential to discover the most similar users to whom you desire to make recommendations. The hypothesis of this paper is that the results obtained by applying traditional similarities measures can be improved by taking contextual information, drawn from the entire body of users, and using it to cal- culate the singularity which exists, for each item, in the votes cast by each pair of users that you wish to compare. As such, the greater the measure of singularity result between the votes cast by two given users, the greater the impact this will have on the similarity. The results, tested on the Movielens, Netflix and FilmAffinity databases, corroborate the excellent behaviour of the singularity measure proposed.

Dimensional synthesis of a spherical parallel manipulator based on the evaluation of global performance indexes

In this work, the dimensional synthesis of a spherical Parallel Manipulator (PM) with a -1S kinematic chain is presented. The goal of the synthesis is to find a set of parameters that defines the PM with the best performance in terms of workspace capabilities, dexterity and isotropy. The PM is parametrized in terms of a reference element, and a non-directed search of these parameters is carried out. First, the inverse kinematics and instantaneous kinematics of the mechanism are presented. The latter is found using the screw theory formulation. An algorithm that explores a bounded set of parameters and determines the corresponding value of global indexes is presented. The concepts of a novel global performance index and a compound index are introduced. Simulation results are shown and discussed. The best PMs found in terms of each performance index evaluated are locally analyzed in terms of its workspace and local dexterity. The relationship between the performance of the PM and its parameters is discussed, and a prototype with the best performance in terms of the compound index is presented and analyzed.