Design, synthesis and cellular and molecular pharmacology of 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401): A novel non-cross-resistant anthracycline antibiotic with the intrinsic ability to circumvent P -glycoprotein-mediated drug resistance

We designed and synthesized a novel daunorubicin (DNR) analogue that effectively circumvents P-glycoprotein (P-gp)-mediated drug resistance. The fully protected carbohydrate intermediate 1,2-dibromoacosamine was prepared from acosamine and effectively coupled to daunomycinone in high yield. Deprotection under alkaline conditions yielded 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401). The in vitro cytotoxicity and cellular and molecular pharmacology of WP401 were compared with those of DNR in a panel of wild-type cell lines (KB-3-1, P388S, and HL60S) and their multidrug-resistant (MDR) counterparts (KB-V1, P388/DOX, and HL60/DOX). Fluorescent spectrophotometry, flow cytometry, and confocal laser scanning microscopy were used to measure intracellular accumulation, retention, and subcellular distribution of these agents. All MDR cell lines exhibited reduced DNR uptake that was restored, upon incubation with either verapamil (VER) or cyclosporin A (CSA), to the level found in sensitive cell lines. In contrast, the uptake of WP401 was essentially the same in the absence or presence of VER or CSA in all tested cell lines. The in vitro cytotoxicity of WP401 was similar to that of DNR in the sensitive cell lines but significantly higher in resistant cell lines (resistance index (RI) of 2-6 for WP401 vs 75-85 for DNR). To ascertain whether drug-mediated cytotoxicity and retention were accompanied by DNA strand breaks, DNA single- and double-strand breaks were assessed by alkaline elution. High levels of such breaks were obtained using 0.1-2 $\mu$g/mL of WP401 in both sensitive and resistant cells. In contrast, DNR caused strand breaks only in sensitive cells and not much in resistant cells. We also compared drug-induced DNA fragmentation similar to that induced by DNR. However, in P-gp-positive cells, WP401 induced 2- to 5-fold more DNA fragmentation than DNR. This increased DNA strand breakage by WP401 was correlated with its increased uptake and cytotoxicity in these cell lines. Overall these results indicate that WP401 is more cytotoxic than DNR in MDR cells and that this phenomenon might be related to the reduced basicity of the amino group and increased lipophilicity of WP401. ^

Identification and characterization of cellular genes differentially expressed in a subset of tumors with non -functional WT1

Wilms tumor (WT) or nephroblastoma is a genetically heterogeneous pediatric renal tumor that accounts for 6–7% of all childhood cancers in the U.S. WT1, located at 11p13, is the sole WT gene cloned to date. Additional genomic regions containing genes that play a role in the development of Wilms tumor include 11p15, 7p, 16q, 1p, 17q and 19q. This heterogeneity has made it extremely difficult to develop an understanding of the pathways involved in the development of WT, even in the 5–20% of tumors that show mutations at the WT1 locus. My research addresses this gap in our current comprehension of the development of WT. ^ I have used two complementary approaches to extend the current understanding of molecular changes involved in the development of WT. In order to minimize complexities due to genetic heterogeneity, I confined my analysis to the WT1 pathway by assessing those genetically defined tumors that carry WT1 mutations. WT1 encodes a zinc finger transcription factor, and in vitro studies have identified many genes that are potentially regulated in vivo by WT1. However, there is very little in vivo data that suggests that they are transcriptionally regulated endogenously by WT1. In one approach I assessed the role of WT1 in the in vivo regulation of PDGFA and IGF2, two genes that are strong contenders for endogenous regulation by WT1. Using primary tissue samples, I found no correlation between the level of RNA expression of WT1 with either PDGFA or IGF2, suggesting that WT1 does not play a critical role in their expression in either normal kidney or WT. ^ In a parallel strategy, using differential display analysis I compared global gene expression in a subset of tumors with known homozygous inactivating WT1 mutations (WT1-tumors) to the gene expression in a panel of appropriate control tissues (fetal kidney, normal kidney, rhabdoid tumor and pediatric renal cell carcinoma). Transcripts that are aberrantly expressed in this subset of Wilms tumors are candidates for endogenous transcriptional regulation by WT1 as well as for potentially functioning in the development of WT. By this approach I identified several differentially expressed transcripts. I further characterized two of these transcripts, identifying a candidate WT gene in the process. I then performed a detailed analysis of this WT candidate gene, which maps to 7p. Future studies will shed more light on the role of these differentially expressed genes in WT. ^

Circumvention of cellular defense responses to DNA -directed nucleoside analogues by the protein kinase inhibitor, UCN-01

DNA-directed nucleoside analogues, such as ara-C, fludarabine, and gemcitabine, are antimetabolites effective in the treatment of a variety of cancers. However, resistance to nucleoside analogue-based chemotherapy in treatments is still a major problem in therapy. Therefore, it is essential to develop rationales for optimizing the use of nucleoside analogues in combination with other anticancer drugs or modalities such as radiation. The present study focuses on establishing mechanism-based combination strategy to overcome resistance to nucleoside analogues. ^ I hypothesized that the cytostatic concentrations of nucleoside analogues may cause S-phase arrest by activating an S-phase checkpoint that consists of a series of kinases. This may allow cells to repair damaged DNA over time and spare cytotoxicity. Thus, the ability of cells to enact an S-phase arrest in response to incorporation of potentially lethal amounts of nucleoside analogue may serve as a mechanism of resistance to S-phase-specific agents. As a corollary, the addition of a kinase inhibitor, such as UCN-01, may dysregulate the checkpoint response and abrogate the survival of S-phase-arrested cells by suppression of the survival signaling pathways. Using gemcitabine as a model of S-phase-specific nucleoside analogues in human acute myelogenous leukemia ML-1 cells, I demonstrated that cells arrested in S-phase in response to cytostatic conditions. Proliferation continued after washing the cells into drug-free medium, suggesting S-phase arrest served as a resistance mechanism of cancer cells to spare cytotoxicity of nucleoside analogues. However, nontoxic concentrations of UCN-01 rapidly killed S-phase-arrested cells by apoptosis. Furthermore, the molecular mechanism for UCN-01-induced apoptosis in S-phase-arrested cells was through inhibition of survival pathways associated with these cells. In this regard, suppression of the PI 3-kinase-Akt-Bad survival pathway as well as the NF-κB signaling pathway were associated with induction of apoptosis in S-phase-arrested cells by UCN-01, whereas the Ras-Raf-MEK-ERK pathway appeared not involved. This study has provided the rationales and strategies for optimizing the design of effective combination therapies to overcome resistance to nucleoside analogues. In fact, a clinical trial of the combination of ara-C with UCN-01 to treat relapsed or refractory AML patients has been initiated at U.T.M.D. Anderson Cancer Center. ^

Characterization of cellular and molecular functions of the p21 -activated kinase, Shk1, in the fission yeast, Schizosaccharomyces pombe

The p21-activated kinase, Shk1, is an essential serine/threonine kinase required for normal cell polarity, proper mating response, and hyperosmotic stress response, in the fission yeast, Schizosaccharomyces pombe. This study has established a novel role for Shk1 as a microtubule regulator in fission yeast and, in addition, characterized a potential biological substrate of Shk1. Cells defective in Shk1 function were found to exhibit malformed interphase and mitotic microtubules, are hypersensitive to the microtubule disrupting drug thiabendazole (TBZ), and are cold sensitive for growth. Microtubule disruption by TBZ results in a significant reduction of Shk1 kinase activity, which is restored after cells are released from the drug, thus providing a correlation between Shk1 kinase activity and active microtubule polymerization. Consistent with a role for Shk1 as a microtubule regulator, GFP-Shk1 fusion proteins localize to interphase microtubules and mitotic microtubule spindles. Furthermore, loss of Tea1, a presumptive microtubule regulator in fission yeast, exacerbates the growth and microtubule defects of cells deficient in Shk1 function, and results in illicit Shk1 localization. Moreover, loss of the Cdc2 inhibitory kinase Wee1, which has been implicated as a mediator of the Shk1 pathway, leads to significant microtubule defects. Intriguingly, Wee1 protein levels are markedly reduced both by partial loss of Shk1 function and by treatment with TBZ. These results suggest that Shk1 is required for proper regulation of microtubule dynamics in fission yeast and may interact with Tea1 and Wee1 in this regulatory process. ^ To further understand Shk1 function in fission yeast, a yeast two-hybrid screen for proteins that interact with the Shk1 catalytic domain was performed. This screen led to the identification of a novel protein, Skb10 (for S&barbelow;hk1 k&barbelow;inase b&barbelow;inding protein 10). Coprecipitation experiments demonstrated that Skb10 associates with Shk1 in S. pombe cells. (Abstract shortened by UMI.) ^

Binding of the HIV-1 gp120 -V3 to cellular glycosphingolipids is necessary for CXCR4 recruitment and infection

Infection by human immunodeficiency virus type 1 (HIV-1) is a multi-step process, and detailed analyses of the various events critical for productive infection are necessary to clearly understanding the infection process and identifying novel targets for therapeutic interventions. Evidence from this study reveals binding of the viral envelope protein to host cell glycosphingolipids (GSLs) as a novel event necessary for the orderly progression of the host cell-entry and productive infection by HIV-1. Data obtained from co-immunoprecipitation analyses and confocal microscopy showed that the ability of viral envelope to interact with the co-receptor CXCR4 and productive infection of HIV-1 were inhibited in cells rendered GSL-deficient, while both these activities were restored after reconstitution of the cells with specific GSLs like GM3. Furthermore, evidence was obtained using peptide-inhibitors of HIV-1 infection to show that binding of a specific region within the V3-loop of the envelope protein gp120 to the host cell GSLs is the trigger necessary for the CD4-bound gp120 to recruit the CXCR4 co-receptor. Infection-inhibitory activity of the V3 peptides was compromised in GSL-deficient cells, but could be restored by reconstitution of GSLs. Based on these findings, a revised model for HIV-1 infection is proposed that accounts for the established interactions between the viral envelope and host cell receptors while enumerating the importance of the new findings that fill the gap in the current knowledge of the sequential events for the HIV-1 entry. According to this model, post-CD4 binding of the HIV-1 envelope surface protein gp120 to host cell GSLs, mediated by the gp120-V3 region, enables formation of the gp120-CD4-GSL-CXCR4 immune-complex and productive infection. The identification of cellular GSLs as an additional class of co-factors necessary for HIV-1 infection is important for enhancing the basic knowledge of the HIV-1 entry that can be exploited for developing novel antiviral therapeutic strategies. ^

Major cellular and physiological impacts of ocean acidification on a reef building coral

As atmospheric levels of CO2 increase, reef-building corals are under greater stress from both increased sea surface temperatures and declining sea water pH. To date, most studies have focused on either coral bleaching due to warming oceans or declining calcification due to decreasing oceanic carbonate ion concentrations. Here, through the use of physiology measurements and cDNA microarrays, we show that changes in pH and ocean chemistry consistent with two scenarios put forward by the Intergovernmental Panel on Climate Change (IPCC) drive major changes in gene expression, respiration, photosynthesis and symbiosis of the coral, Acropora millepora, before affects on biomineralisation are apparent at the phenotype level. Under high CO2 conditions corals at the phenotype level lost over half their Symbiodinium populations, and had a decrease in both photosynthesis and respiration. Changes in gene expression were consistent with metabolic suppression, an increase in oxidative stress, apoptosis and symbiont loss. Other expression patterns demonstrate upregulation of membrane transporters, as well as the regulation of genes involved in membrane cytoskeletal interactions and cytoskeletal remodeling. These widespread changes in gene expression emphasize the need to expand future studies of ocean acidification to include a wider spectrum of cellular processes, many of which may occur before impacts on calcification.

The photo-physiological response of a model cnidarian-dinoflagellate symbiosis to CO2-induced acidification at the cellular level

We measured the relationship between CO2-induced seawater acidification, photo-physiological performance and intracellular pH (pHi) in a model cnidarian-dinoflagellate symbiosis - the sea anemone Aiptasia sp. -under ambient (289.94 ± 12.54 µatm), intermediate (687.40 ± 25.10 µatm) and high (1459.92 ± 65.51 µatm) CO2 conditions. These treatments represented current CO2 levels, in addition to CO2 stabilisation scenarios IV and VI provided by the Intergovernmental Panel on Climate Change (IPCC). Anemones were exposed to each treatment for two months and sampled at regular intervals. At each time-point we measured a series of physiological responses: maximum dark-adapted fluorescent yield of PSII (Fv/Fm), gross photosynthetic rate, respiration rate, symbiont population density, and light-adapted pHi of both the dinoflagellate symbiont and isolated host anemone cell. We observed increases in all but one photo-physiological parameter (Pgross:R ratio). At the cellular level, increases in light-adapted symbiont pHi were observed under both intermediate and high CO2 treatments, relative to control conditions (pHi 7.35 and 7.46 versus pHi 7.25, respectively). The response of light-adapted host pHi was more complex, however, with no change observed under the intermediate CO2 treatment, but a 0.3 pH-unit increase under the high CO2 treatment (pHi 7.19 and 7.48, respectively). This difference is likely a result of a disproportionate increase in photosynthesis relative to respiration at the higher CO2 concentration. Our results suggest that, rather than causing cellular acidosis, the addition of CO2 will enhance photosynthetic performance, enabling both the symbiont and host cell to withstand predicted ocean acidification scenarios.

Seawater carbonate chemistry, clearance rates, respiration rates, condition index and cellular turnover (RNA: DNA) of Juvenile King Scallop, Pecten maximus in a laboratory experiment

The decline in ocean water pH and changes in carbonate saturation states through anthropogenically mediated increases in atmospheric CO2 levels may pose a hazard to marine organisms. This may be particularly acute for those species reliant on calcareous structures like shells and exoskeletons. This is of particular concern in the case of valuable commercially exploited species such as the king scallop, Pecten maximus. In this study we investigated the effects on oxygen consumption, clearance rates and cellular turnover in juvenile P. maximus following 3 months laboratory exposure to four pCO2 treatments (290, 380, 750 and 1140 µatm). None of the exposure levels were found to have significant effect on the clearance rates, respiration rates, condition index or cellular turnover (RNA: DNA) of individuals. While it is clear that some life stages of marine bivalves appear susceptible to future levels of ocean acidification, particularly under food limiting conditions, the results from this study suggest that where food is in abundance, bivalves like juvenile P. maximus may display a tolerance to limited changes in seawater chemistry.

Phytoplankton calcification as an effective mechanism to alleviate cellular calcium poisoning

Marine phytoplankton has developed the remarkable ability to tightly regulate the concentration of free calcium ions in the intracellular cytosol at a level of ~ 0.1 µmol /l in the presence of seawater Ca2+ concentrations of 10 mmol/1. The low cytosolic calcium ion concentration is of utmost importance for proper cell signalling function. While the regulatory mechanisms responsible for the tight control of intracellular Ca2+ concentration are not completely understood, phytoplankton taxonomic groups appear to have evolved different strategies, which may affect their ability to cope with changes in seawater Ca2+ concentrations in their environment on geological time scales. For example, the Cretaceous (145 to 66 Ma ago), an era known for the high abundance of coccolithophores and the production of enormous calcium carbonate deposits, exhibited seawater calcium concentrations up to four times present-day levels. We show that calcifying coccolithophore species (Emiliania huxleyi, Gephyrocapsa oceanica and Coccolithus braarudii) are able to maintain their relative fitness (in terms of growth rate and photosynthesis) at simulated Cretaceous seawater calcium concentrations, whereas these rates are severely reduced under these conditions in some non-calcareous phytoplankton species (Chaetoceros sp., Ceratoneis closterium and Heterosigma akashiwo). Most notably, this also applies to a non-calcifying strain of E. huxleyi which displays a calcium-sensitivity similar to the non-calcareous species. We hypothesize that the process of calcification in coccolithophores provides an efficient mechanism to alleviate cellular calcium poisoning and thereby offered a potential key evolutionary advantage, responsible for the proliferation of coccolithophores during times of high seawater calcium concentrations. The exact function of calcification and the reason behind the highly-ornate physical structures of coccoliths remain elusive.

Effects of seawater pCO2 and temperature on shell growth, shell stability, condition and cellular stress of Western Baltic Sea Mytilus edulis (L.) and Arctica islandica (L.)

Acidification of the World's oceans may directly impact reproduction, performance and shell formation of marine calcifying organisms. In addition, since shell production is costly and stress in general draws on an organism's energy budget, shell growth and stability of bivalves should indirectly be affected by environmental stress. The aim of this study was to investigate whether a combination of warming and acidification leads to increased physiological stress (lipofuscin accumulation and mortality) and affects the performance [shell growth, shell breaking force, condition index (Ci)] of young Mytilus edulis and Arctica islandica from the Baltic Sea. We cultured the bivalves in a fully-crossed 2-factorial experimental setup (seawater (sw) pCO2 levels "low", "medium" and "high" for both species, temperature levels 7.5, 10, 16, 20 and 25 °C for M. edulis and 7.5, 10 and 16 °C for A. islandica) for 13 weeks in summer. Mytilus edulis and A. islandica appeared to tolerate wide ranges of sw temperature and pCO2. Lipofuscin accumulation of M. edulis increased with temperature while the Ci decreased, but shell growth of the mussels only sharply decreased while its mortality increased between 20 and 25 °C. In A. islandica, lipofuscin accumulation increased with temperature, whereas the Ci, shell growth and shell breaking force decreased. The pCO2 treatment had only marginal effects on the measured parameters of both bivalve species. Shell growth of both bivalve species was not impaired by under-saturation of the sea water with respect to aragonite and calcite. Furthermore, independently of water temperatures shell breaking force of both species and shell growth of A. islandica remained unaffected by the applied elevated sw pCO2 for several months. Only at the highest temperature (25 °C), growth arrest of M. edulis was recorded at the high sw pCO2 treatment and the Ci of M. edulis was slightly higher at the medium sw pCO2 treatment than at the low and high sw pCO2 treatments. The only effect of elevated sw pCO2 on A. islandica was an increase in lipofuscin accumulation at the high sw pCO2 treatment compared to the medium sw pCO2 treatment. Our results show that, despite this robustness, growth of both M. edulis and A. islandica can be reduced if sw temperatures remain high for several weeks in summer. As large body size constitutes an escape from crab and sea star predation, this can make bivalves presumably more vulnerable to predation with possible negative consequences on population growth. In M. edulis, but not in A. islandica, this effect is amplified by elevated sw pCO2. We follow that combined effects of elevated sw pCO2 and ocean warming might cause shifts in future Western Baltic Sea community structures and ecosystem services; however, only if predators or other interacting species do not suffer as strong from these stressors.

Response threshold models, stochastic learning automata and ant colony optimization-based decentralized self-coordination algorithms for heterogeneous multi-tasks distribution in multi-robot systems

In recent decades, there has been an increasing interest in systems comprised of several autonomous mobile robots, and as a result, there has been a substantial amount of development in the eld of Articial Intelligence, especially in Robotics. There are several studies in the literature by some researchers from the scientic community that focus on the creation of intelligent machines and devices capable to imitate the functions and movements of living beings. Multi-Robot Systems (MRS) can often deal with tasks that are dicult, if not impossible, to be accomplished by a single robot. In the context of MRS, one of the main challenges is the need to control, coordinate and synchronize the operation of multiple robots to perform a specic task. This requires the development of new strategies and methods which allow us to obtain the desired system behavior in a formal and concise way. This PhD thesis aims to study the coordination of multi-robot systems, in particular, addresses the problem of the distribution of heterogeneous multi-tasks. The main interest in these systems is to understand how from simple rules inspired by the division of labor in social insects, a group of robots can perform tasks in an organized and coordinated way. We are mainly interested on truly distributed or decentralized solutions in which the robots themselves, autonomously and in an individual manner, select a particular task so that all tasks are optimally distributed. In general, to perform the multi-tasks distribution among a team of robots, they have to synchronize their actions and exchange information. Under this approach we can speak of multi-tasks selection instead of multi-tasks assignment, which means, that the agents or robots select the tasks instead of being assigned a task by a central controller. The key element in these algorithms is the estimation ix of the stimuli and the adaptive update of the thresholds. This means that each robot performs this estimate locally depending on the load or the number of pending tasks to be performed. In addition, it is very interesting the evaluation of the results in function in each approach, comparing the results obtained by the introducing noise in the number of pending loads, with the purpose of simulate the robot's error in estimating the real number of pending tasks. The main contribution of this thesis can be found in the approach based on self-organization and division of labor in social insects. An experimental scenario for the coordination problem among multiple robots, the robustness of the approaches and the generation of dynamic tasks have been presented and discussed. The particular issues studied are: Threshold models: It presents the experiments conducted to test the response threshold model with the objective to analyze the system performance index, for the problem of the distribution of heterogeneous multitasks in multi-robot systems; also has been introduced additive noise in the number of pending loads and has been generated dynamic tasks over time. Learning automata methods: It describes the experiments to test the learning automata-based probabilistic algorithms. The approach was tested to evaluate the system performance index with additive noise and with dynamic tasks generation for the same problem of the distribution of heterogeneous multi-tasks in multi-robot systems. Ant colony optimization: The goal of the experiments presented is to test the ant colony optimization-based deterministic algorithms, to achieve the distribution of heterogeneous multi-tasks in multi-robot systems. In the experiments performed, the system performance index is evaluated by introducing additive noise and dynamic tasks generation over time.

Uniform Distributed Pushdown Automata Systems.

We consider here uniform distributed pushdown automata systems (UDPAS), namely distributed pushdown automata systems having all components identical pushdown automata. We consider here just a single protocol for activating/deactivating components, namely a component stays active as long as it can perform moves, as well as two ways of accepting the input word: by empty stacks (all components have empty stacks) or by final states (all components are in final states), when the input word is completely read. We mainly investigate the computational power of UDPAS accepting by empty stacks and a few decidability and closure properties of the families of languages they define. Some directions for further work and open problems are also discussed.

Connexin-43 in the osteogenic BM niche regulates its cellular composition and the bidirectional traffic of hematopoietic stem cells and progenitors

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals

Decentralized multi-tasks distribution in heterogeneous robot teams by means of ant colony optimization and learning automata

This paper focuses on the general problem of coordinating multiple robots. More specifically, it addresses the self-election of heterogeneous specialized tasks by autonomous robots. In this paper we focus on a specifically distributed or decentralized approach as we are particularly interested on decentralized solution where the robots themselves autonomously and in an individual manner, are responsible of selecting a particular task so that all the existing tasks are optimally distributed and executed. In this regard, we have established an experimental scenario to solve the corresponding multi-tasks distribution problem and we propose a solution using two different approaches by applying Ant Colony Optimization-based deterministic algorithms as well as Learning Automata-based probabilistic algorithms. We have evaluated the robustness of the algorithm, perturbing the number of pending loads to simulate the robot’s error in estimating the real number of pending tasks and also the dynamic generation of loads through time. The paper ends with a critical discussion of experimental results.

Response threshold models and stochastic learning automata for self-coordination of heterogeneous multi-task distribution in multi-robot systems.

This paper focuses on the general problem of coordinating multiple robots. More specifically, it addresses the self-selection of heterogeneous specialized tasks by autonomous robots. In this paper we focus on a specifically distributed or decentralized approach as we are particularly interested in a decentralized solution where the robots themselves autonomously and in an individual manner, are responsible for selecting a particular task so that all the existing tasks are optimally distributed and executed. In this regard, we have established an experimental scenario to solve the corresponding multi-task distribution problem and we propose a solution using two different approaches by applying Response Threshold Models as well as Learning Automata-based probabilistic algorithms. We have evaluated the robustness of the algorithms, perturbing the number of pending loads to simulate the robot’s error in estimating the real number of pending tasks and also the dynamic generation of loads through time. The paper ends with a critical discussion of experimental results.