Exploration of new drug delivery pathways: I. Mechanism of folate uptake in cultured human cells. II. Role of nuclear localization signal peptides in the delivery of oligonucleotide into reconstituted nuclei

The roles of the folate receptor and an anion carrier in the uptake of 5- methyltetrahydrofolate (5-MeH_4folate) were studied in cultured human (KB) cells using radioactive 5-MeH_4folate. Binding of the 5-MeH_4folate was inhibited by folic acid, but not by probenecid, an anion carrier inhibitor. The internalization of 5-MeH_4folate was inhibited by low temperature, folic acid, probenecid and methotrexate. Prolonged incubation of cells in the presence of high concentrations of probenecid appeared to inhibit endocytosis of folatereceptors as well as the anion carrier. The V_(max) and K_M values for the carrier were 8.65 ± 0.55 pmol/min/mg cell protein and 3.74 ± 0.54µM, respectively. The transport of 5-MeH4folate was competitively inhibited by folic acid, probenecid and methotrexate. The carrier dissociation constants for folic acid, probenecid and methotreate were 641 µM, 2.23 mM and 13.8 µM, respectively. Kinetic analysis suggests that 5-MeH_4folate at physiological concentration is transported through an anion carrier with the characteristics of the reduced-folate carrier after 5-MeH_4folate is endocytosed by folate receptors in KB cells. Our data with KB cells suggest that folate receptors and probenecid-sensitive carriers work in tandem to transport 5-MeH_4folate to the cytoplasm of cells, based upon the assumption that 1 mM probenecid does not interfere with the acidification of the vesicle where the folate receptors are endocytosed.

Oligodeoxynucleotides designed to hybridize to specific mRNA sequences (antisense oligonucleotides) or double stranded DNA sequences have been used to inhibit the synthesis of a number of cellular and viral proteins (Crooke, S. T. (1993) FASEB J. 7, 533-539; Carter, G. and Lemoine, N. R. (1993) Br. J. Cacer 67, 869-876; Stein, C. A. and cohen, J. S. (1988) Cancer Res. 48, 2659-2668). However, the distribution of the delivered oligonucleotides in the cell, i.e., in the cytoplasm or in the nucleus has not been clearly defined. We studied the kinetics of oligonucleotide transport into the cell nucleus using reconstituted cell nuclei as a model system. We present evidences here that oligonucleotides can freely diffuse into reconstituted nuclei. Our results are consistent with the reports by Leonetti et al. (Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 2702-2706, April 1991), which were published while we were carrying this research independently. We also investigated whether a synthetic nuclear localization signal (NLS) peptide of SV40 T antigen could be used for the nuclear targeting of oligonucleotides. We synthesized a nuclear localization signal peptide-conjugated oligonucleotide to see if a nuclear localization signal peptide can enhance the uptake of oligonucleotides into reconstituted nuclei of Xenopus. Uptake of the NLS peptide-conjugated oligonucleotide was comparable to the control oligonucleotide at similar concentrations, suggesting that the NLS signal peptide does not significantly enhance the nuclear accumulation of oligonucleotides. This result is probably due to the small size of the oligonucleotide.

Robot navigation in dense crowds : statistical models and experimental studies of human robot cooperation

This thesis explores the problem of mobile robot navigation in dense human crowds. We begin by considering a fundamental impediment to classical motion planning algorithms called the freezing robot problem: once the environment surpasses a certain level of complexity, the planner decides that all forward paths are unsafe, and the robot freezes in place (or performs unnecessary maneuvers) to avoid collisions. Since a feasible path typically exists, this behavior is suboptimal. Existing approaches have focused on reducing predictive uncertainty by employing higher fidelity individual dynamics models or heuristically limiting the individual predictive covariance to prevent overcautious navigation. We demonstrate that both the individual prediction and the individual predictive uncertainty have little to do with this undesirable navigation behavior. Additionally, we provide evidence that dynamic agents are able to navigate in dense crowds by engaging in joint collision avoidance, cooperatively making room to create feasible trajectories. We accordingly develop interacting Gaussian processes, a prediction density that captures cooperative collision avoidance, and a "multiple goal" extension that models the goal driven nature of human decision making. Navigation naturally emerges as a statistic of this distribution.

Most importantly, we empirically validate our models in the Chandler dining hall at Caltech during peak hours, and in the process, carry out the first extensive quantitative study of robot navigation in dense human crowds (collecting data on 488 runs). The multiple goal interacting Gaussian processes algorithm performs comparably with human teleoperators in crowd densities nearing 1 person/m², while a state of the art noncooperative planner exhibits unsafe behavior more than 3 times as often as the multiple goal extension, and twice as often as the basic interacting Gaussian process approach. Furthermore, a reactive planner based on the widely used dynamic window approach proves insufficient for crowd densities above 0.55 people/m². We also show that our noncooperative planner or our reactive planner capture the salient characteristics of nearly any dynamic navigation algorithm. For inclusive validation purposes, we show that either our non-interacting planner or our reactive planner captures the salient characteristics of nearly any existing dynamic navigation algorithm. Based on these experimental results and theoretical observations, we conclude that a cooperation model is critical for safe and efficient robot navigation in dense human crowds.

Finally, we produce a large database of ground truth pedestrian crowd data. We make this ground truth database publicly available for further scientific study of crowd prediction models, learning from demonstration algorithms, and human robot interaction models in general.

Characterization of the neural mechanisms supporting the implementation of cognitive control in human decision making

Humans are particularly adept at modifying their behavior in accordance with changing environmental demands. Through various mechanisms of cognitive control, individuals are able to tailor actions to fit complex short- and long-term goals. The research described in this thesis uses functional magnetic resonance imaging to characterize the neural correlates of cognitive control at two levels of complexity: response inhibition and self-control in intertemporal choice. First, we examined changes in neural response associated with increased experience and skill in response inhibition; successful response inhibition was associated with decreased neural response over time in the right ventrolateral prefrontal cortex, a region widely implicated in cognitive control, providing evidence for increased neural efficiency with learned automaticity. We also examined a more abstract form of cognitive control using intertemporal choice. In two experiments, we identified putative neural substrates for individual differences in temporal discounting, or the tendency to prefer immediate to delayed rewards. Using dynamic causal models, we characterized the neural circuit between ventromedial prefrontal cortex, an area involved in valuation, and dorsolateral prefrontal cortex, a region implicated in self-control in intertemporal and dietary choice, and found that connectivity from dorsolateral prefrontal cortex to ventromedial prefrontal cortex increases at the time of choice, particularly when delayed rewards are chosen. Moreover, estimates of the strength of connectivity predicted out-of-sample individual rates of temporal discounting, suggesting a neurocomputational mechanism for variation in the ability to delay gratification. Next, we interrogated the hypothesis that individual differences in temporal discounting are in part explained by the ability to imagine future reward outcomes. Using a novel paradigm, we imaged neural response during the imagining of primary rewards, and identified negative correlations between activity in regions associated the processing of both real and imagined rewards (lateral orbitofrontal cortex and ventromedial prefrontal cortex, respectively) and the individual temporal discounting parameters estimated in the previous experiment. These data suggest that individuals who are better able to represent reward outcomes neurally are less susceptible to temporal discounting. Together, these findings provide further insight into role of the prefrontal cortex in implementing cognitive control, and propose neurobiological substrates for individual variation.

Information processing and higher psychological functions in the disconnected hemispheres of human commissurotomy patients

Several patients of P. J. Vogel who had undergone cerebral commissurotomy for the control of intractable epilepsy were tested on a variety of tasks to measure aspects of cerebral organization concerned with lateralization in hemispheric function. From tests involving identification of shapes it was inferred that in the absence of the neocortical commissures, the left hemisphere still has access to certain types of information from the ipsilateral field. The major hemisphere can still make crude differentiations between various left-field stimuli, but is unable to specify exact stimulus properties. Most of the time the major hemisphere, having access to some ipsilateral stimuli, dominated the minor hemisphere in control of the body.

Competition for control of the body between the hemispheres is seen most clearly in tests of minor hemisphere language competency, in which it was determined that though the minor hemisphere does possess some minimal ability to express language, the major hemisphere prevented its expression much of the time. The right hemisphere was superior to the left in tests of perceptual visualization, and the two hemispheres appeared to use different strategies in attempting to solve the problems, namely, analysis for the left hemisphere and synthesis for the right hemisphere.

Analysis of the patients' verbal and performance I.Q.'s, as well as observations made throughout testing, suggest that the corpus callosum plays a critical role in activities that involve functions in which the minor hemisphere normally excels, that the motor expression of these functions may normally come through the major hemisphere by way of the corpus callosum.

Lateral specialization is thought to be an evolutionary adaptation which overcame problems of a functional antagonism between the abilities normally associated with the two hemispheres. The tests of perception suggested that this function lateralized into the mute hemisphere because of an active counteraction by language. This latter idea was confirmed by the finding that left-handers, in whom there is likely to be bilateral language centers, are greatly deficient on tests of perception.