Finite element 3D reconstruction of the pulmonary acinus imaged by synchrotron X-ray tomography

The alveolated structure of the pulmonary acinus plays a vital role in gas exchange function. Three-dimensional (3D) analysis of the parenchymal region is fundamental to understanding this structure-function relationship, but only a limited number of attempts have been conducted in the past because of technical limitations. In this study, we developed a new image processing methodology based on finite element (FE) analysis for accurate 3D structural reconstruction of the gas exchange regions of the lung. Stereologically well characterized rat lung samples (Pediatr Res 53: 72-80, 2003) were imaged using high-resolution synchrotron radiation-based X-ray tomographic microscopy. A stack of 1,024 images (each slice: 1024 x 1024 pixels) with resolution of 1.4 mum(3) per voxel were generated. For the development of FE algorithm, regions of interest (ROI), containing approximately 7.5 million voxels, were further extracted as a working subunit. 3D FEs were created overlaying the voxel map using a grid-based hexahedral algorithm. A proper threshold value for appropriate segmentation was iteratively determined to match the calculated volume density of tissue to the stereologically determined value (Pediatr Res 53: 72-80, 2003). The resulting 3D FEs are ready to be used for 3D structural analysis as well as for subsequent FE computational analyses like fluid dynamics and skeletonization.

Modeling mechanical response of heterogeneous materials

Heterogeneous materials are ubiquitous in nature and as synthetic materials. These materials provide unique combination of desirable mechanical properties emerging from its heterogeneities at different length scales. Future structural and technological applications will require the development of advanced light weight materials with superior strength and toughness. Cost effective design of the advanced high performance synthetic materials by tailoring their microstructure is the challenge facing the materials design community. Prior knowledge of structure-property relationships for these materials is imperative for optimal design. Thus, understanding such relationships for heterogeneous materials is of primary interest. Furthermore, computational burden is becoming critical concern in several areas of heterogeneous materials design. Therefore, computationally efficient and accurate predictive tools are highly essential. In the present study, we mainly focus on mechanical behavior of soft cellular materials and tough biological material such as mussel byssus thread. Cellular materials exhibit microstructural heterogeneity by interconnected network of same material phase. However, mussel byssus thread comprises of two distinct material phases. A robust numerical framework is developed to investigate the micromechanisms behind the macroscopic response of both of these materials. Using this framework, effect of microstuctural parameters has been addressed on the stress state of cellular specimens during split Hopkinson pressure bar test. A voronoi tessellation based algorithm has been developed to simulate the cellular microstructure. Micromechanisms (microinertia, microbuckling and microbending) governing macroscopic behavior of cellular solids are investigated thoroughly with respect to various microstructural and loading parameters. To understand the origin of high toughness of mussel byssus thread, a Genetic Algorithm (GA) based optimization framework has been developed. It is found that two different material phases (collagens) of mussel byssus thread are optimally distributed along the thread. These applications demonstrate that the presence of heterogeneity in the system demands high computational resources for simulation and modeling. Thus, Higher Dimensional Model Representation (HDMR) based surrogate modeling concept has been proposed to reduce computational complexity. The applicability of such methodology has been demonstrated in failure envelope construction and in multiscale finite element techniques. It is observed that surrogate based model can capture the behavior of complex material systems with sufficient accuracy. The computational algorithms presented in this thesis will further pave the way for accurate prediction of macroscopic deformation behavior of various class of advanced materials from their measurable microstructural features at a reasonable computational cost.

QSAR Studies on Thiazolidines: A Biologically Privileged Scaffold

A large number of drugs and biologically relevant molecules contain heterocyclic systems. Often the presence of hetero atoms or groupings imparts preferential specificities in their biological responses. Amongst the heterocyclic systems, thiazolidine is a biologically important scaffold known to be associated with several biological activities. Some of the prominent biological responses attributed to this skeleton are antiviral, antibacterial, antifungal, antihistaminic, hypoglycemic, anti-inflammatory activities. This diversity in the biological response profiles of thiazolidine has attracted the attention of many researchers to explore this skeleton to its multiple potential against several activities. Many of these synthetic and biological explorations have been subsequently analyzed in detailed quantitative structure-activity relationship (QSAR) studies to correlate the respective structural features and physicochemical properties with the activities to identify the important structural components in deciding their activity behavior. In this, drugs or any biologically active molecules may be viewed as structural frames consisting of strategically positioned functional groups that will interact effectively with the complementary groups/sites of the receptor. With this in focus, the present article reviews the QSAR studies of diverse biological activities of the thiazolidines published during the past decade.

Integration of merged delayed-enhanced magnetic resonance imaging and multidetector computed tomography for the guidance of ventricular tachycardia ablation: a pilot study

BACKGROUND Delayed enhancement (DE) MRI can assess the fibrotic substrate of scar-related VT. MDCT has the advantage of inframillimetric spatial resolution and better 3D reconstructions. We sought to evaluate the feasibility and usefulness of integrating merged MDCT/MRI data in 3D-mapping systems for structure-function assessment and multimodal guidance of VT mapping and ablation. METHODS Nine patients, including 3 ischemic cardiomyopathy (ICM), 3 nonischemic cardiomyopathy (NICM), 2 myocarditis, and 1 redo procedure for idiopathic VT, underwent MRI and MDCT before VT ablation. Merged MRI/MDCT data were integrated in 3D-mapping systems and registered to high-density endocardial and epicardial maps. Low-voltage areas (<1.5 mV) and local abnormal ventricular activities (LAVA) during sinus rhythm were correlated to DE at MRI, and wall-thinning (WT) at MDCT. RESULTS Endocardium and epicardium were mapped with 391 ± 388 and 1098 ± 734 points per map, respectively. Registration of MDCT allowed visualization of coronary arteries during epicardial mapping/ablation. In the idiopathic patient, integration of MRI data identified previously ablated regions. In ICM patients, both DE at MRI and WT at MDCT matched areas of low voltage (overlap 94 ± 6% and 79 ± 5%, respectively). In NICM patients, wall-thinning areas matched areas of low voltage (overlap 63 ± 21%). In patients with myocarditis, subepicardial DE matched areas of epicardial low voltage (overlap 92 ± 12%). A total number of 266 LAVA sites were found in 7/9 patients. All LAVA sites were associated to structural substrate at imaging (90% inside, 100% within 18 mm). CONCLUSION The integration of merged MDCT and DEMRI data is feasible and allows combining substrate assessment with high-spatial resolution to better define structure-function relationship in scar-related VT.

Design, synthesis and pharmacological characterization of analogs of 2-aminoethyl diphenylborinate (2-APB), a known store-operated calcium channel blocker, for inhibition of TRPV6-mediated calcium transport

2-Aminoethyl diphenylborinate (2-APB) is a known modulator of the IP3 receptor, the calcium ATPase SERCA, the calcium release-activated calcium channel Orai and TRP channels. More recently, it was shown that 2-APB is an efficient inhibitor of the epithelial calcium channel TRPV6 which is overexpressed in prostate cancer. We have conducted a structure-activity relationship study of 2-APB congeners to understand their inhibitory mode of action on TRPV6. Whereas modifying the aminoethyl moiety did not significantly change TRPV6 inhibition, substitution of the phenyl rings of 2-APB did. Our data show that the diaryl borinate moiety is required for biological activity and that the substitution pattern of the aryl rings can influence TRPV6 versus SOCE inhibition. We have also discovered that 2-APB is hydrolyzed and transesterified within minutes in solution.

Characterization of Hsp70 binding and nucleotide exchange by the yeast Hsp110 chaperone Sse1.

SSE1 and SSE2 encode the essential yeast members of the Hsp70-related Hsp110 molecular chaperone family. Both mammalian Hsp110 and the Sse proteins functionally interact with cognate cytosolic Hsp70s as nucleotide exchange factors. We demonstrate here that Sse1 forms high-affinity (Kd approximately 10-8 M) heterodimeric complexes with both yeast Ssa and mammalian Hsp70 chaperones and that binding of ATP to Sse1 is required for binding to Hsp70s. Sse1.Hsp70 heterodimerization confers resistance to exogenously added protease, indicative of conformational changes in Sse1 resulting in a more compact structure. The nucleotide binding domains of both Sse1/2 and the Hsp70s dictate interaction specificity and are sufficient for mediating heterodimerization with no discernible contribution from the peptide binding domains. In support of a strongly conserved functional interaction between Hsp110 and Hsp70, Sse1 is shown to associate with and promote nucleotide exchange on human Hsp70. Nucleotide exchange activity by Sse1 is physiologically significant, as deletion of both SSE1 and the Ssa ATPase stimulatory protein YDJ1 is synthetically lethal. The Hsp110 family must therefore be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.

Functionally Optimized Neuritogenic Farinosone C Analogs: SAR-Study and Investigations on Their Mode of Action

Several natural products derived from entomopathogenic fungi have been shown to initiate neuronal differentiation in the rat pheochromocytoma PC12 cell line. After the successful completion of the total synthesis program, the reduction of structural complexity while retaining biological activity was targeted. In this study, farinosone C served as a lead structure and inspired the preparation of small molecules with reduced complexity, of which several were able to induce neurite outgrowth. This allowed for the elaboration of a detailed structure-activity relationship. Investigations on the mode of action utilizing a computational similarity ensemble approach suggested the involvement of the endocannabinoid system as potential target for our analogs and also led to the discovery of four potent new endocannabinoid transport inhibitors.

MECHANISMS OF SPECIES DIFFERENCES IN BRAIN ACETYLCHOLINESTERASE SENSITIVITY TO ORGANOPHOSPHATE INHIBITION

In vitro incubation of acetylcholinesterase from brain tissue of several species with organophosphate compounds indicated that the concentrations required to inhibit 50% of acetylcholinesterase activity (IC(,50)) differed from species to species for the same compound (Murphy, et al., 1968; Andersen, et al., 1972, 1977 and 1978).^ The hypothesis that non-specific binding proteins (Lauwerys and Murphy, 1969a,b) exerts a protective effect on acetylcholinesterase, and thus cause the differences observed in IC(,50) studies was tested by a ('3)H-DFP binding experiment. It was found that differences in the amount of non-specific binding protein cannot explain the observed differences observed in IC(,50) studies.^ An alternative hypothesis, that acetylcholinesterase from different species have different affinities for binding and/or different rates of phosphorylation by organophosphate insecticides was tested by determining the apparent affinity constant (k(,a)) and apparent rate of phosphorylation (k(,p)). Kinetic studies indicated that acetylcholinesterases from different species have different sensitivities to inhibition by organophosphate insecticides, and the differences are due to different affinities for binding and/or different rates of phosphorylation by the same organophosphate compound.^ Studies of the spontaneous reactivation of acetylcholinesterase after inhibition by organophosphate insecticides also indicated that acetylcholinesterases from different species have different rates and extents of spontaneous reactivation. This further substantiates the hypothesis that acetylcholinesterases from different species have different kinetic characteristics with respect to organophosphate insecticides inhibition.^ Eleven paraoxon analogs were synthesized for a quantitative structure-activity relationship study. It was found that the electron-withdrawing power ((sigma)) and hydrophobicity ((PARAGR)) of the substituent are important in determining the anti-cholinesterase activity of paraoxon analogs. Thus, predictions of species differences in acetylcholinesterase sensitivities to paraoxon analogs can be made if the physicochemical parameters ((sigma) and (PARAGR)) of the substituents are known.^ In another approach, i.e. enzyme modeling, the sensitivity of rat brain acetylcholinesterase to organophosphate insecticides was used as the independent variable to predict the sensitivities of acetylcholinesterases from other species to the same compound. Regression equations were derived for each species based on nineteen organophosphate insecticides studied. It was found, that in addition to paraoxon analogs, this method is also applicable to other organophosphate compounds with wide variations in structure. Thus, the sensitivities of acetylcholinesterases from other species can also be predicted from the sensitivity of rat brain acetylcholinesterase. ^

Genetic and functional analyses of cell division proteins FtsZ and FtsA in Escherichia coli

In the current model for bacterial cell division, the FtsZ protein forms a ring that marks the division plane, creating a cytoskeletal framework for the subsequent action of other essential division proteins such as FtsA and ZipA. The putative protein complex ultimately generates the division septum. The essential cell division protein FtsZ is a functional and structural homolog of eukaryotic tubulin, and like tubulin, FtsZ hydrolyzes GTP and self-assembles into protein filaments in a strictly GTP-dependent manner. FtsA shares sequence similarity with members of the ATPase superfamily that include actin, but its actual function remains unknown. To test the division model and elucidate functions of the division proteins, this dissertation primarily focuses on the analysis of FtsZ and FtsA in Escherichia coli. ^ By tagging with green fluorescent protein, we first demonstrated that FtsA also exhibits a ring-like structure at the potential division site. The localization of FtsA was dependent on functional FtsZ, suggesting that FtsA is recruited to the septum by the FtsZ ring. In support of this idea, we showed that FtsA and FtsZ directly interact. Using a novel E. coli in situ assay, we found that the FtsA-FtsZ interaction appears to be species-specific, although an interspecies interaction could occur between FtsA and FtsZ proteins from two closely related organisms. In addition, mutagenesis of FtsA revealed that no single domain is solely responsible for its septal localization or interaction with FtsZ. To explore the function of FtsA, we purified FtsA protein and demonstrated that it has ATPase activity. Furthermore, purified FtsA stimulates disassembly of FtsZ polymers in a sedimentation assay but does not affect GTP hydrolysis of FtsZ. This result suggests that in the cell, FtsA may function similarly in regulating dynamic instability of the FtsZ ring during the cell division process. ^ To elucidate the structure-function relationship of FtsZ, we carried out thorough genetic and functional analyses of the mutagenized FtsZ derivatives. Our results indicate that the conserved N-terminal domain of FtsZ is necessary and sufficient for FtsZ self-assembly and localization. Moreover, we discovered a critical role for an extreme C-terminal domain of FtsZ that consists of only 12 residues. Truncated FtsZ derivatives lacking this domain, though able to polymerize and localize, are defective in ring formation in vivo as well as interaction with FtsA and ZipA. Alanine scanning mutagenesis of this region pinpointed at least five residues necessary for the function of FtsZ. Studies of protein levels and protein-protein interactions suggested that these residues may be involved in regulating protein stability and/or FtsZ-FtsA interactions. Interestingly, two of the point mutants exhibited dominant-negative phenotypes. ^ In summary, results from this thesis work have provided additional support for the division machinery model and will contribute to a better understanding of the coordinate functions of FtsA and FtsZ in the cell division process. ^

Molecular mechanisms of prostacyclin receptor signaling: The intracellular domains in G protein coupling

To better understand the mechanisms of how the human prostacyclin receptor (1P) mediates vasodilation and platelet anti-aggregation through Gs protein coupling, a strategy integrating multiple approaches including high resolution NMR experiments, synthetic peptide, fluorescence spectroscopy, molecular modeling, and recombinant protein was developed and used to characterize the structure/function relationship of important segments and residues of the IP receptor and the α-subunit of the Gs protein (Gαs). The first (iLP1) and third (iLP3) intracellular loops of the IP receptor, as well as the Gαs C-terminal domain, relevant to the Gs-mediated IP receptor signaling, were first identified by observation of the effects of the mini gene-expressed corresponding protein segments in HEK293 cells which co-expressed the receptor and Gαs. Evidence of the IP iLP1 domain interacted with the Gαs C-terminal domain was observed by fluorescence and NMR spectroscopic studies using a constrained synthetic peptide, which mimicked the IP iLP1 domain, and the synthetic peptide, which mimicked Gαs C-terminal domain. The solution structural models and the peptide-peptide interaction of the two synthetic protein segments were determined by high resolution NMR spectroscopy. The important residues in the corresponding domains of the IP receptor and the Gαs predicted by NMR chemical shift mapping were used to guide the identification of their protein-protein interaction in cells. A profile of the residues Arg42 - Ala48 of the IP iLP1 domain and the three residues Glu392 ∼ Leu394 of the Gαs C-terminal domain involved in the IP/Gs protein coupling were confirmed by recombinant proteins. The data revealed an intriguing speculation on the mechanisms of how the signal of the ligand-activated IP receptor is transmitted to the Gs protein in regulating vascular functions and homeostasis, and also provided substantial insights into other prostanoid receptor signaling. ^

Development of HIF-1α/HIF-1β heterodimerization inhibitors using a novel bioluminescence reporter assay system for in vitro high throughput screening and in vivo imaging

Tumor growth often outpaces its vascularization, leading to development of a hypoxic tumor microenvironment. In response, an intracellular hypoxia survival pathway is initiated by heterodimerization of hypoxia-inducible factor (HIF)-1α and HIF-1β, which subsequently upregulates the expression of several hypoxia-inducible genes, promotes cell survival and stimulates angiogenesis in the oxygen-deprived environment. Hypoxic tumor regions are often associated with resistance to various classes of radio- or chemotherapeutic agents. Therefore, development of HIF-1α/β heterodimerization inhibitors may provide a novel approach to anti-cancer therapy. To this end, a novel approach for imaging HIF-1α/β heterodimerization in vitro and in vivo was developed in this study. Using this screening platform, we identified a promising lead candidate and further chemically derivatized the lead candidate to assess the structure-activity relationship (SAR). The most effective first generation drug inhibitors were selected and their pharmacodynamics and anti-tumor efficacy in vivo were verified by bioluminescence imaging (BLI) of HIF-1α/β heterodimerization in the xenograft tumor model. Furthermore, the first generation drug inhibitors, M-TMCP and D-TMCP, demonstrated efficacy as monotherapies, resulting in tumor growth inhibition via disruption of HIF-1 signaling-mediated tumor stromal neoangiogenesis.

Advanced Protein Modeling Method: Benchmarking and Applications in Computer-Aided Drug Discovery

Development of homology modeling methods will remain an area of active research. These methods aim to develop and model increasingly accurate three-dimensional structures of yet uncrystallized therapeutically relevant proteins e.g. Class A G-Protein Coupled Receptors. Incorporating protein flexibility is one way to achieve this goal. Here, I will discuss the enhancement and validation of the ligand-steered modeling, originally developed by Dr. Claudio Cavasotto, via cross modeling of the newly crystallized GPCR structures. This method uses known ligands and known experimental information to optimize relevant protein binding sites by incorporating protein flexibility. The ligand-steered models were able to model, reasonably reproduce binding sites and the co-crystallized native ligand poses of the β2 adrenergic and Adenosine 2A receptors using a single template structure. They also performed better than the choice of template, and crude models in a small scale high-throughput docking experiments and compound selectivity studies. Next, the application of this method to develop high-quality homology models of Cannabinoid Receptor 2, an emerging non-psychotic pain management target, is discussed. These models were validated by their ability to rationalize structure activity relationship data of two, inverse agonist and agonist, series of compounds. The method was also applied to improve the virtual screening performance of the β2 adrenergic crystal structure by optimizing the binding site using β2 specific compounds. These results show the feasibility of optimizing only the pharmacologically relevant protein binding sites and applicability to structure-based drug design projects.

Functional analysis of p53 phosphorylation and a p53 hot spot mutation

The tumor suppressor p53 is a phosphoprotein which functions as a transcriptional activator. By monitoring the transcriptional activity, we studied how p53 functions is regulated in relation to cell growth and contact inhibition. When cells were arrested at G1 phase of the cell cycle by contact inhibition, we found that p53 transactivation function was suppressed. When contact inhibition was overridden by cyclin E overexpression which stimulates cell cycle progression, p53 function was restored. This observation led to the development of a cell density assay to study the regulation of p53 function during cell cycle for the functional significance of p53 phosphorylation. The murine p53 is phosphorylated at serines 7, 9, 12, 18, 37, 312 and 389. To understand the role of p53 phosphorylation, we generated p53 constructs encoding serine-to-alanine or serine-to-glutamate mutations at these codons. The transcriptional activity were measured in cells capable of contact inhibition. In low-density cycling cells, no difference in transcriptional activity was found between wild type p53 and any of the mutants. In contact-inhibited cells, however, only mutations of p53 at serine 389 resulted in altered responses to cell cycle arrest and to cyclin E overexpression. The mutant with serine-to-glutamate substitution at codon 389 retained its function in contact inhibited cells. Cyclin E overexpression in these cells induced p53 phosphorylation at serine 389. Furthermore, we showed that phosphorylation at serine 389 regulates p53 DNA binding activity. Our findings implicate that phosphorylation is an important mechanism for p53 activation.^ p53 is the most frequently mutated gene in human tumors. To study the mechanism of p53 inactivation by mutations, we carried out detailed analysis of a murine p53 mutation with an arginine-to-tryptophane substitution at codon 245. The corresponding human p53 mutation at amino acid 248 is the most frequently mutated codon in tumors. We showed that this mutant is inactive in suppressing focus formation, binding to DNA and transactivation. Structural analysis revealed that this mutant assumes the wild type protein conformation. These findings define a novel class of p53 mutations and help to understand structure-function relationship of p53. ^

Estructura y desarrollo en la enseñanza de Jacques Lacan : Primera parte

El trabajo expone las principales conclusiones de una investigación centrada en el problema de las relaciones estructura-desarrollo en la enseñanza de Jacques Lacan. Después de revisar los problemas que tal relación presenta, se delimitan dos momentos de tal enseñanza en los que se articulan los tiempos lógicos de transformación del niño como sujeto a partir de su posición de objeto. El trabajo aborda el primer momento, la inscripción del sujeto en la estructura.: de lo imaginario a lo simbólico en una teoría del desarrollo estructurado Se subraya el desplazamiento teórico que se opera en la noción de desarrollo cuando Lacan lo aborda a partir de la estructura del inconsciente en sus relaciones con los tres registros imaginario, simbólico y real, contemplando el anudamiento entre el lenguaje, el cuerpo y la pulsión

Estructura y desarrollo en la enseñanza de Jacques Lacan : Primera parte

El trabajo expone las principales conclusiones de una investigación centrada en el problema de las relaciones estructura-desarrollo en la enseñanza de Jacques Lacan. Después de revisar los problemas que tal relación presenta, se delimitan dos momentos de tal enseñanza en los que se articulan los tiempos lógicos de transformación del niño como sujeto a partir de su posición de objeto. El trabajo aborda el primer momento, la inscripción del sujeto en la estructura.: de lo imaginario a lo simbólico en una teoría del desarrollo estructurado Se subraya el desplazamiento teórico que se opera en la noción de desarrollo cuando Lacan lo aborda a partir de la estructura del inconsciente en sus relaciones con los tres registros imaginario, simbólico y real, contemplando el anudamiento entre el lenguaje, el cuerpo y la pulsión