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.

On reconstruction theorems in noncommutative Riemannian geometry

We present a novel account of the theory of commutative spectral triples and their two closest noncommutative generalisations, almost-commutative spectral triples and toric noncommutative manifolds, with a focus on reconstruction theorems, viz, abstract, functional-analytic characterisations of global-analytically defined classes of spectral triples. We begin by reinterpreting Connes's reconstruction theorem for commutative spectral triples as a complete noncommutative-geometric characterisation of Dirac-type operators on compact oriented Riemannian manifolds, and in the process clarify folklore concerning stability of properties of spectral triples under suitable perturbation of the Dirac operator. Next, we apply this reinterpretation of the commutative reconstruction theorem to obtain a reconstruction theorem for almost-commutative spectral triples. In particular, we propose a revised, manifestly global-analytic definition of almost-commutative spectral triple, and, as an application of this global-analytic perspective, obtain a general result relating the spectral action on the total space of a finite normal compact oriented Riemannian cover to that on the base space. Throughout, we discuss the relevant refinements of these definitions and results to the case of real commutative and almost-commutative spectral triples. Finally, we outline progess towards a reconstruction theorem for toric noncommutative manifolds.

New directions in sparse sampling and estimation for underdetermined systems

A central objective in signal processing is to infer meaningful information from a set of measurements or data. While most signal models have an overdetermined structure (the number of unknowns less than the number of equations), traditionally very few statistical estimation problems have considered a data model which is underdetermined (number of unknowns more than the number of equations). However, in recent times, an explosion of theoretical and computational methods have been developed primarily to study underdetermined systems by imposing sparsity on the unknown variables. This is motivated by the observation that inspite of the huge volume of data that arises in sensor networks, genomics, imaging, particle physics, web search etc., their information content is often much smaller compared to the number of raw measurements. This has given rise to the possibility of reducing the number of measurements by down sampling the data, which automatically gives rise to underdetermined systems.

In this thesis, we provide new directions for estimation in an underdetermined system, both for a class of parameter estimation problems and also for the problem of sparse recovery in compressive sensing. There are two main contributions of the thesis: design of new sampling and statistical estimation algorithms for array processing, and development of improved guarantees for sparse reconstruction by introducing a statistical framework to the recovery problem.

We consider underdetermined observation models in array processing where the number of unknown sources simultaneously received by the array can be considerably larger than the number of physical sensors. We study new sparse spatial sampling schemes (array geometries) as well as propose new recovery algorithms that can exploit priors on the unknown signals and unambiguously identify all the sources. The proposed sampling structure is generic enough to be extended to multiple dimensions as well as to exploit different kinds of priors in the model such as correlation, higher order moments, etc.

Recognizing the role of correlation priors and suitable sampling schemes for underdetermined estimation in array processing, we introduce a correlation aware framework for recovering sparse support in compressive sensing. We show that it is possible to strictly increase the size of the recoverable sparse support using this framework provided the measurement matrix is suitably designed. The proposed nested and coprime arrays are shown to be appropriate candidates in this regard. We also provide new guarantees for convex and greedy formulations of the support recovery problem and demonstrate that it is possible to strictly improve upon existing guarantees.

This new paradigm of underdetermined estimation that explicitly establishes the fundamental interplay between sampling, statistical priors and the underlying sparsity, leads to exciting future research directions in a variety of application areas, and also gives rise to new questions that can lead to stand-alone theoretical results in their own right.

Signal transduction with hybridization chain reactions

Some of the most exciting developments in the field of nucleic acid engineering include the utilization of synthetic nucleic acid molecular devices as gene regulators, as disease marker detectors, and most recently, as therapeutic agents. The common thread between these technologies is their reliance on the detection of specific nucleic acid input markers to generate some desirable output, such as a change in the copy number of an mRNA (for gene regulation), a change in the emitted light intensity (for some diagnostics), and a change in cell state within an organism (for therapeutics). The research presented in this thesis likewise focuses on engineering molecular tools that detect specific nucleic acid inputs, and respond with useful outputs.

Four contributions to the field of nucleic acid engineering are presented: (1) the construction of a single nucleotide polymorphism (SNP) detector based on the mechanism of hybridization chain reaction (HCR); (2) the utilization of a single-stranded oligonucleotide molecular Scavenger as a means of enhancing HCR selectivity; (3) the implementation of Quenched HCR, a technique that facilitates transduction of a nucleic acid chemical input into an optical (light) output, and (4) the engineering of conditional probes that function as sequence transducers, receiving target signal as input and providing a sequence of choice as output. These programmable molecular systems are conceptually well-suited for performing wash-free, highly selective rapid genotyping and expression profiling in vitro, in situ, and potentially in living cells.

Linear chirp control of infrared signal in a biased semiconductor thin film

Ultrashort light-matter interactions between a linear chirped pulse and a biased semiconductor thin film GaAs are investigated. Using different chirped pulses, the dependence of infrared spectra on chirp rate is demonstrated for a 5 fs pulse. It is found that the infrared spectra can be controlled by the linear chirp of the pulse. Furthermore, the infrared spectral intensity could be enhanced by two orders of magnitude via appropriately choosing values of the linear chirp rates. Our results suggest a possible scheme to control the infrared signal.

Signal transduction, regulation, and developmental logic of EGFR signalling in C. elegans

RTKs-mediated signaling systems and the pathways with which they interact (e.g., those initiated by G protein-mediated signaling) involve a highly cooperative network that sense a large number of cellular inputs and then integrate, amplify, and process this information to orchestrate an appropriate set of cellular responses. The responses include virtually all aspects of cell function, from the most fundamental (proliferation, differentiation) to the most specialized (movement, metabolism, chemosensation). The basic tenets of RTK signaling system seem rather well established. Yet, new pathways and even new molecular players continue to be discovered. Although we believe that many of the essential modules of RTK signaling system are rather well understood, we have relatively little knowledge of the extent of interaction among these modules and their overall quantitative importance.

My research has encompassed the study of both positive and negative signaling by RTKs in C. elegans. I identified the C. elegans S0S-1 gene and showed that it is necessary for multiple RAS-mediated developmental signals. In addition, I demonstrated that there is a SOS-1-independent signaling during RAS-mediated vulval differentiation. By assessing signal outputs from various triple mutants, I have concluded that this SOS-1-independent signaling is not mediated by PTP-2/SHP-2 or the removal of inhibition by GAP-1/ RasGAP and it is not under regulation by SLI-1/Cb1. I speculate that there is either another exchange factor for RASor an as yet unidentified signaling pathway operating during RAS-mediated vulval induction in C. elegans.

In an attempt to uncover the molecular mechanisms of negative regulation of EGFR signaling by SLI-1/Cb1, I and two other colleagues codiscovered that RING finger domain of SLI-1 is partially dispensable for activity. This structure-function analysis shows that there is an ubiquitin protein ligase-independent activity for SLI-1 in regulating EGFR signaling. Further, we identified an inhibitory tyrosine of LET-23/ EGFR requiring sli-1(+)for its effects: removal of this tyrosine closely mimics loss of sli-1 but not loss of other negative regulator function.

By comparative analysis of two RTK pathways with similar signaling mechanisms, I have found that clr-1, a previously identified negative regulator of egl-15 mediated FGFR signaling, is also involved in let-23 EGFR signaling. The success of this approach promises a similar reciprocal test and could potentially extend to the study of other signaling pathways with similar signaling logic.

Finally, by correlating the developmental expression of lin-3 EGF to let-23 EGFR signaling activity, I demonstrated the existence of reciprocal EGF signaling in coordinating the morphogenesis of epithelia. This developmental logic of EGF signaling could provide a basis to understand a universal mechanism for organogenesis.