Background:Lower back pain is one of the most common health-related complaints in the adult population. Thirty percent of Americans 65 years and older reported symptoms of lower back pain in 2004. With an aging population, the proportion of people over the age of 65 is expected to reach 20% by the year 2030. Because of this increase in older adults, lumbar spinal stenosis (LSS) associated with arthritic changes will also likely increase. In older adults, lower back pain is most often caused by degenerative lumbar spinal stenosis. Stenosis is the narrowing ofthe spinal canal, causing pressure on the nerve roots and is frequently treated surgically. Lumbar spinal stenosis is one of the most common reasons for back surgery in patients 65 years and older 2. However, risks associated with surgery increase with age 3-5 and older patients may choose non-surgical treatment for their lower back pain, including injection treatment. Injection treatment, usually consisting of anti-inflammatory medications and analgesics, has improved since the mid-1990's when fluoroscopic guidance was developed. Information about injection treatment for lower back pain is limited, especially in the older population. An extensive review of published literature regarding injection treatment revealed a paucity of information about older adults diagnosed with lumbar spinal stenosis. In this study, three aims were designed to gain more information about the effectiveness of injection treatment in older patients with lumbar spinal stenosis. In the first (retrospective) study, information about receipt of second injections and time between injections was collected to examine injection usage. In the second and third (prospective) studies, information about pain relief and functional return following injection treatment was collected to examine the effectiveness of injection treatment in patients age 60 and older diagnosed with lumbar spinal stenosis. To our knowledge, such results have not been repolted for this population in the literature. Objective:Injection treatment is a commonly used non-surgical procedure to alleviate lower back pain in older adults. However, older patients do not have enough information about how long pain relief will last after treatment or the amount of pain relief and functional return they will experience. These studies focused on three topics: 1) usage of injection treatment; 2) effectiveness of injection treatment on pain relief; 3) effectiveness of injection treatment on functional return. In addition, the variations of the effectiveness were examined by selected patient attributes. Methods:In a retrospective study, medical records of patients aged 60 years or older from a high volume dedicated spine center at the University of Massachusetts Memorial Hospital were retrospectively reviewed. This study included those diagnosed with degenerative LSS, who had not received an injection for lower back pain within six months, and whom were treated between June I, 2006 and May 31, 2007. In two prospective studies, patients scheduled for lumbar injection treatment between January 1 and June 30, 2008 were selected from the University of Massachusetts Memorial Hospital Spine Center. Selection criteria included patients age 60 and over, diagnosed with degenerative lumbar spinal stenosis and no previous lumbar injection within 6 months or lumbar surgery within 2 years. The Pain sub-score of the SF-36 questionnaire was used to measure pain at baseline and at one and three months post injection. The Physical Component Score (PCS) of the SF-36 questionnaire and the Oswestry Disability Index (ODI) were used to measure function at baseline and at one and three months post injection. Variations in longitudinal changes in scores by patient characteristics were analyzed in both unadjusted (univariate) analyses using one-way analysis of variance (ANOVA), and adjusted (multiple regression) analyses using linear mixed effects models. Results: In the retrospective cohort, the mean age of the cohort was 68, 64% were female, 59% were married, with a mean Body Mass index of 32 kg/m2. Of 92 eligible patients, 57% returned for a second injection within six months of the first. The mean number of months between injections was 4.8 for all patients, ranging from 1 to 22 months. When patient characteristics were examined, the only variable that showed a statistically significant difference was age. Patients aged 70 years and older were found to be 67% less likely to return for a second injection when compared to patients age 60-69 (OR=0.33 (0.12 - 0.94)p In the prospective cohort, information was collected on 62 patients. Mean Pain scores improved significantly from baseline to one month (14.1 points), and from baseline to three months (8.3 points). Post injection changes in Pain scores varied by Body Mass Index (BMI) and baseline emotional health. Based on a linear mixed effects model analysis, higher baseline emotional health, as measured by the SF-36 Mental Component Score (MCS>50), was associated with greater reduction in pain over three months when compared to lower emotional health (MCS Conclusion: Patients over age 70 do not return for repeat injection as frequently as patients age 60-69. In addition, each year a patient ages over age 60, they are 10% less likely to return for a repeat injection. Lower back pain in older adults with LSS is clinically significantly alleviated after injection treatment. In addition, injection treatment for LSS is associated with return of lost function needed for daily living activities in older adults. Pain relief and functional return varies by patient personal and clinical characteristics. Higher emotional health was associated with more pain relief and more functional return experienced over three months following injection treatment. Additional information is needed about why older patients do not return for second injections at the same rate as younger patients and how emotional health affects response to injection treatment in older adults.

Carbo- and heterocyclic compounds are of great interest to chemists. Intramolecular cyclization strategies of donor-acceptor (D-A) cyclopropanes and alkylidene malonate monoamides have excellent potential for synthesis as they offer easy access to structurally-diverse compounds. The work described in this thesis accesses the scope of the In(OTf)3-catalyzed cyclization reaction of cyclopropanes and alkylidene malonate monoamides. In(OTf)3-catalyzed reactions of alkenyl and heteroaryl cyclopropyl ketones were examined in the synthesis of functionalized cyclohexenone-based derivatives (Chapter 2). Subsequent efforts to utilize a tandem cyclopropane ring-opening/Friedel-Crafts alkylation sequence of methyl 1-(1H-indolecarbonyl)-1-cyclopropanecarboxylates to prepare functionalized hydropyrido[1,2-a]indole-6(7H)-ones is discussed in Chapter 3. The extension of this tandem protocol towards the total synthesis of (±)-deethyleburnamonine is the subject of Chapter 6. Intramolecular Friedel-Crafts alkylation of N-indolyl alkylidene malonate monoamides was also examined. An In(OTf)3-catalyzed cyclization of substituted methyl 2-(1H-indole-1-carbonyl) acrylates afforded a series of 1H-pyrrolo[1,2-a]indole-3(2H)-ones (Chapter 4), whereas substrates with the indole 2-position blocked provided access to substituted 4H-pyrrolo[3,2,1-ij]quinolin-4-ones (Chapter 5).

The steroid/thyroid hormone receptor superfamily is a large class of ligand-dependent transcription factors that plays a critical role in regulating the expression of genes involved in a broad range of physiological functions, including development, homeostasis, and reproduction. In the absence of cognate hormone, several receptors are able to repress transcription below the basal level via the recruitment of the nuclear receptor corepressors SMRT and NCoR. Upon hormone binding by the receptor, the corepressor complex is dissociated and a coactivator complex is subsequently recruited. This thesis details the mechanisms by which receptor-associated coactivator 3 (RAC3) interacts with nuclear receptors, particularly the vitamin D, estrogen, and retinoid receptors, and modulates their transcriptional activity. It was discovered that these receptors interact with different α-helical LXXLL motifs of RAC3 in vitro. Mutation of specific motifs differentially impairs the ability of RAC3 to enhance transcription by the receptors in vivo. In addition, the intrinsic transcriptional activation function of RAC3 was also characterized. Here, a single LXXLL motif, NR box v, was found to be essential to activation by serving as a binding surface for the general transcriptional integrator CBP/p300. Finally, the cofactor binding pocket of retinoid receptors was characterized. It was demonstrated that, to a large extent, the coactivator pocket of RARα overlaps with the corepressor pocket, with the exception of helix 12, which is required for coactivator, but not corepressor binding. Recruitment of RAC3 or SMRT also correlates directly with the ability of RARα to activate or repress transcription, respectively. Intriguingly, it was discovered that the AF-2 domain of RXRα inhibited cofactor binding to RXRα heterodimers, for deletion of this domain dramatically enhanced RAC3 and SMRT binding. In addition, it was demonstrated that the RXRα cofactor binding pocket contributed minimally to recruitment of cofactors. Conversely, the AF-2 domain of the partnering monomer and its cofactor pocket were required for these interactions. These findings suggest that the partner of RXRα is the primary docking point for cofactors at RXRα heterodimeric complexes. Taken together, this work contributes significantly to the field of nuclear receptor function in detailing the mechanisms by which the coactivator RAC3 is recruited to nuclear receptors and regulates their transcriptional activity.

Activation of tissue-specific genes is a tightly controlled process that normally involves the combined action of several transcription factors and transcriptional co-regulators. The bone-specific osteoca1cin gene (OC) has been used as a prototype to study both tissue-specific and hormonal responsiveness. In this study we have examined the role of Runx2, VDR and C/EBP factors in the regulation of OC gene transcription. Contributions of the Runx and VDRE motifs to OC promoter activity were addressed by introducing point mutations within the context of the rat (-1.1 kb) osteocalcin promoter fused to a CAT-reporter gene. The functional significance of these mutations was assayed following transient transfection and after genomic integration in ROS 17/2.8 osteoblastic cell lines. Furthermore, we tested the effect of these mutations on the chromatin organization of the OC promoter. Our data show that all three Runx sites are required for maximal activation of the OC promoter and that the distal sites contribute significantly to the basal activity. Strikingly, mutation of the three Runx sites abrogates responsiveness of the OC promoter to vitamin D; this loss is also observed when only the Runx sites flanking the VDRE are mutated. Chromatin changes that result in the appearance of DNase I hypersensitive sites during activation of the OC gene are well documented. Mutation of the three Runx sites results in altered chromatin structure as reflected by absence of DNase I hypersensitive sites at the vitamin D response element and over the proximal, tissue-specific basal promoter. These data are consistent with the critical role of Runx2 in osteoblast maturation and bone development. Mutation of the VDRE resulted in a complete loss of vitamin D responsiveness; however, this mutant promoter exhibited increased basal activity. The two DNase I hypersensitive sites characteristic of the transcriptionally active OC gene in osteoblastics cells were not altered upon mutation of the VDRE element, although restriction enzyme accessibility in the proximal promoter region was decreased. We also found an increased level of histone H3 acetylation at the VDRE mutant promoter in comparison to the endogenous gene. Thus binding of VDR to OC promoter is required to achieve a normal transcriptional regulation and chromatin structure of the OC gene. Although Runx2 is considered a master gene for bone development and osteoblast differentiation, it is noteworthy that osteoblast-specific transcription of the rat OC promoter occurs even in the absence of Runx sites. Therefore, other transcription factor(s) should be able to drive OC expression. We characterized a C/EBP enhancer element in the proximal promoter of the rat osteoca1cin gene that resides in close proximity to a Runx element, essential for tissue-specific activation. We find that C/EBPβ or δ and Runx2 factors interact together in a synergistic manner to enhance OC transcription in cell culture systems. Mutational analysis demonstrated that this synergism is mediated through the C/EBP responsive element in the OC promoter and requires a direct interaction between Runx2 and C/EBPβ or δ. Taken together, our findings strongly support a mechanism in which combinatorial interaction of Runx2, VDR, C/EBPβ or δ and probably other transcription factors are needed for regulating OC expression. In this process Runx factors not only act as simple transcriptional trans activators but also by facilitating modifications in promoter architecture and maintaining an active conformation of the target gene promoter.

(FOR FIGURES REFER THE MAIN PDF FILE) Supramoleculr chemistry, aptly termed by Lehn as the study of molecular sociology, is the chemistry of the intermolecular bond, focusing on the structures and functions of “supermolecules” –chemical system formed by the association between two or more molecular components. While interrelated, this discipline forges beyond the domain of traditional molecular chemistry, which seeks to master the manipulation of the covalent bond between atoms and uncover the principle that governs the structures and properties of molecular species. Supramolecular chemistry assayas to blend the comprehensive resources of molecular chemistry with designed control of the intermolecular interactions to engineers supramolecular with features as well defined as those of the constituent molecular themselves. Not surprisingly, it has been stated that supramoleculars are to molecules and the intermolecular bond what molecules are to atoms and the covalent bond. In the realm of molecular crystals, the focus of supramolecular chemistry and indeed, the scope of the present thesis coverings with that of a rather recent, but rapidly emerging scientific discipline, namely crystal engineering. Coined nearly four decades ago in connection with photodimerization reaction in crystalline cinnamic acids, the term” crystal engineering” has since then broadened its expanse considerably and is, at present, most appropriately defined as“the understanding of intermolecular interactions in the context of crystal packing and the utilization of such understanding in the design of new solids with desired physical and chemical properties”. It would be befitting to remark that it is very pursuit (and more often than not, the elusive target) of being able to make functional solids by design that has allowed crystal engineering to evolve from an object of mere Scientific curiosity to a subject of tremendous utilization value. No proof for this assertion might be greater than that which lies in the fervent efforts put forth by pharmaceutical companies in understanding and controlling drug polymorphism, especially in the wake of the contemporary legal implications attendant with observing such a phenomenon. Polymorphism in molecular crystals results from the possibility of at least two different arrangements of the molecular of a given compound in the solid state and has therefore often been regarded as the” dark side” of crystal engineering. On one hand, polymorphism presents itself as an important probe in the study of structure-property relationship and allows elucidation of the varied macroscopic properties of the same molecule self-assembled in different crystalline environments. On the other hand, the phenomenon poses an implicit complication when predicating the product of a crystallization process forms the goal of a crystal engineering project. This is particularly true in case of crystal structure prediction (CSP) from the molecular structure of a given compound, where the experimentally obtained polymorphic modification may be a kinetic form and therefore, need not correspond to the one ranked lowest in energy from the computational studies. Indeed, this dichotomy between a thermodynamically and a kinetically controlled crystallization process reflects the underlying uncertainty associated with judging the outcome of a crystallization event. In this concept of a supramolecular synthon has been postulated to assimilate both thermodynamic and kinetic alternative, and therefore provide a working model for heuristic crystal design. By analogy with corey’s definition of a molecular synthon, a supramolecular synhon has been described” a structural unit within a supramolecule which can be formed and/or assembled by known or conceivable synthetic operations involving intermolecular interactions”. Being entirely probabilistic in nature, the robutness and thus, the transferability of a particular synthon to a designed crystal is assessed from a systermatic evolution of its recurrence in crystal structures of representative molecules. The Cambridge Structural Database (CSD), which announced the inclusion of the 500000th crystal structures in its archives last year, provides an invaluable cache of experimentally determined structures and the foundation for crystal design in this regard. The practically of the supramolecular synthon approach, now almost synthymous with crystal engineering, has been demonstrated not only in the successful design of a number of functional solids, but also in its possible application in CSP as a knowledge-based alternative. Irrespective of the approach, a basic paradigm can however be constructed from any crystal engineering strategy, viz. construct the molecular building blocks and assemble these, with a prior knowledge of the possible non-covalent interactions, in a manner that leads to the desired crystal structure. This premise will form the central theme of the present thesis, entitled “From molecular to supramolecular: Probing solid state self –assemblies of conformatonally locked polycyclitos and structural siblings”. The dissertation will deal with the nuances of the self-assemblies of four classes of structurally related crystalline polycyclie compounds, all fashioned from a prototypical rigid trans-decalin backbone derived from commonly available aromatic precursors like naphthalene and anthracene. The thesis will be presented in four chapters, each based on one of the four functional make-ups present in the molecular under study. • Chapter 1.Relating intramolecular O-H…Ohydrogen bondigs to conformational locking: Design and self-assemblies of crystalline polyclitols. • Chapter 2.Preferences of supramolecular assemblies towards competing inter- and intramolecular O-H…O hydrogen bonds: A case study in crystalline acyldervaeives of conformarionally locked polyclitols. •Chapter 3.Synthesis of novel polyhydroxylated flustrates: Probing fluorine interactions in a conformatonally constructed environment. • Chapter 4. Strength vs.accessiblity: Universe the patterns of self-recognition in designer conformationally locked aminoacohols. A brief overview of each chapter is presented below. The first chapter of the thesis investigates the supramolecular chemistry of an O-H…O Hydrogen Bond formed between hydroxyl groups that have been constrained to occupy spatiality invariant position in the crystal structure of a polycyclitol (a portmanteau word derived from polycyclic cyclitol). Having been constructed on a grid trans-decalin carbocyclic backbone, the polycyclitols under study 1-6 are conformatonally locked and destined to exhibit an axial rich disposition of the hydroxyl groups, so that the OH functionalities in 1,3-relationship are automatically brought into a favorable geometry for the formation of intramolecular O-H…O hydrogen bonds. Working within this paradigm, which was formulated both logically and on the basis of the observed H-bonding patterns in the crystal structures of several conformationally locked polyols, we were able to demonstrate that intramolecular H-bonding between 1,3-syndisxial OH groups can be used as a tool to preordain the position of the intermolecular O-H…O-bond donors and accepts in the specially crafted polycyclitols 1-3. this observation not only simplified a qualitative visualization of the various packing patterns in 1-3, but also allowed us to propose, based on previously reported CSD analysis, the packing motifs mostlikely to converge with the experimental results. Despite its qualitative nature, the O-H…O hydrogen bonding patters, proposed for 1-3 were found to conform well with those observed experimentally for the tetrols 1 and 3, and even for the two polymorphic modifications of the hexol 2[Figure 1] The determination role played by intramolecular O-H…O bonding in the supramolecular assembly of 2, a novel bicycle C2h symmetric hexol having an all axial disposition of the six hydroxyl functionalities, prompted us to study the crystal packing of the three diastereomeric perhydro-2,3,4q,6,6,8a-naphthalenehexols 4-6. the end-to-end co-operative intramolecular O-H…O-H hydrogen bonding chain on both faces of the molecule, as observed in case of 2, through an axial-equatorial. Figure 1. (left) one of the packing modes proposed for the hexol 2. Note that the H-bonding pattern involves all donor/acceptor oxygen and incorporates infinite chains of O-H…O bonds of O-H….O bonds; (right) Molecular packing observed experimentally in the polymorph of the hexol 2 Transposition of one or more of the peripheral yhdroyl groups. With increased freedom now allowed to the OH groups in the choice of their H-bonding partners, as a compared to 2 crystal packing in the polycyclitols 4-6 evolved from the simplistic model of hydrogen bonding proposed and observed for 2,to ivoke more complex patterns of self assembly mediated through O-H…O-bonds In the second chapter, the crystal structures of four conformationally locked esters, namely tetraaccetate 7/tetrabenzoate 8 of hexol 2 and the diacetate9/dibenzoate 10 of tetrol1,have been analyzed in order to examine the preference of their supramolecular assemblies towards competing inter and intramolecular O-H…O hydrogen bonds. To this end, all the four esters under study were specially crafted on a trans-decalin backbone with the objective of relegating the O-H…O H-bond donors( in form of the 30 OH groups) to the molecular interior and having the peripheral H-bond accepters (in form of the 20 acyl groups) in 1,3-syndiaxial relationship. It was anticipated that this common design element would allow the supramolecular assembly of the easters to evolve along two possible pathway, namely one which employs intermoleculars O-H…O H-bonds (pathway 1) and the other that sacrifises those for intramolecular O-H…O H-bonds and settles for a crystal packing dictated by weak intermolecular interactions alone (pathway 2) A pure sample of 7 crystallized along pathway 1 in two enantiotropic modifications, one obtained at room temperature (form) and the other at 20 C0 (form) [Figure 2]. Behaving much like a temperature guided molecular switch, the tetraacetate 7 could be shifted reversibly between the forms response to changes in the ambient temperature. Thus, the form converted at -4 OC to the denser form, which displayed an unusual kinetic stability till 67 OC and transformed back to the form beyond this temperature. Subsequently, the close similarity between the self-assemble of the two dimonrphs of 7 and the diastereomer 11 was exploited in order to stimulated 7 to fallow the pathway 2 through preferential inhibition of pathway 1[Figure 3]. Interstingly, the nucleation inhibition 11 was obtained serendipitously a route 7 via an apparent breakdownof furst-platter rule. Unlike the tetraceatate 7, crystal packing in the tetrabenzoate 8 preferred to fallow exclusively pathway 2. The individualistic nature of the self-assemblies of 7 and 8 found to be in contrast commonalities noted in the mode of molecular assembly in 9 and 10 both of which conformed to a combination of pathway 1 and 2. A rationale for the preferred crystallization pathway of the four estes 7-10 as well as probable mechanism for the observed reversible transformation between the forms the tetracetate 7 will be put forth in this chapter. Figure 2. (Model for pathway 1) Molecular packing in the forms of the tetraacetate 7. The non-interacting hydrogen atoms have been omitted for clarity. Figure 3. (Model for pathway 2) The nucleation inhibitor 11 and form of the tetraacetate 7. The non-interacting hydrogen atoms have been omitted in the molecular packing diagram for clarity. In light of the wide ranging application of organofluorine compounds and the ambiguity that resides over the disposition of fluorine as a H-bond accepter, the third chapter utilizes three specially designed fluorinated polycyclitols 12-14 investigate the role of covalently bonded flurine in crystal structures of lesser studied aliphatic fluorous substracts and probe its capacity to engage itself in C(sp3)-F…H-X(sp3)(X=O and/or C) H-bounding, in presence of its isostere, the hydrozyl group. Conformatonality locked with well defined spatial disposition of functional groups, all the fluorinated polycyclitols 12-14 bear a fluorohydrin moiety, embedded in a rigid trans-decalin framework. In 12 and 14, it was conceived that the presence of a hydroxyl donor in a favorable 1, 3-syndiaxial relationship to a fluoro group on one side and a hydroxyl group on the other would allow an unambiguous comparison between the two isoteric functionalities (C-OH and C-F) to serve as acceptors for intramolecular hydrogen bonds (O-H…O and purported O-H…F respectively) The difluorodiol 13 was sought to serve as a control to assess the change in the C-F…H-X interactions (if any) which might be observed upon incorporating the peripheral secondary hydroxyl groups in 14. The result presented in this chapter will revel, in particular, that C(sp3) –F…H-C(sp3) hydrogen bonds, though weak and lesser investigated, can indeed be observed and supramolecular recognition motifs, involving such interactions, can be conserved even in crystal structures laden with stronger O-H…O hydrogen bonds [Figure 4}. Figure 4. (Left) Molecular packing in the difluorodiol 13, showing how four intermolecular C-H…F hydrogen bonds forms a part of a R22 H-bonding motif (encircled). This centrosymmentic supramolecular recognition unit is observed even in the molecular packing in the difluorohexol 14 (right). Non-interacting H atoms have been omitted in both diagrams for the sake of clarity. The forth chapter details an in-depth study carried out on the self-assembly of a conformationally locked aminoalchohol 15, in which the amino protons serve as mere spectators, the molecular packing in the crystal being realized through the co-operativity between O-H…N H-bonds and weak π-π stacking interaction (Figure 5b). The crystal structure of 15 was quite intriguing on three sailent grounds (a) previous studies on the supramolecular assemblies in the aminols have shown that both amino and hydroxyl protons participate in H-bonding in the crystal structures of such compounds; (b) the fact that the hydrogen atoms of the NH2 group Figure 5. (Left) Laplacian distribution map in the planes defined by (a) the double bonds, (c) O-H…N-H-bond, and (d) π-π stacking interactions in the aminoalclhol 15. Contours havse been drawn at logarithmic intervals in ▼2 ρb, eÅ-5. Solid lines indicate positive contours and dotted lines negative contours. (b) Molecular packing in 15. Non-interacting H atoms have been omitted for the sake of clarity.remain as mere bystanders in anomalous if one were to abide by the Etter’s rule; (c) the rather well-difined π-π stacking interactions in crystal structure of the aminoalcohol occurs between isolated olefinic bonds-a rarely encountered form of non-covalent interaction. Charge destiny analysis was carried out on the aminoalcoholf 15 not only to catheterize the non-covalent interactions existing in the supramolecular assembly in terms of topological features of electrol destiny at their bond critical points, but also to confirm the non-involvement of the amino H-atoms in any form of either intra- or intermoalecular hydrogen bonds beyond the criteria of mere geometry (Figure a,c,d). The maverick nature of the self-assembly of 15 was elucidated as resulting from the preference of the molecules to assemble with O-H…N H-bonds. This automatically relegated the hydrogen atoms of the tertiary amine to the interior of the conformationally locked cabocycclic scaffold, thereby making them far less accessible than the peripheral C=C bonds.

The Hedgehog (Hh) family of proteins are secreted growth factors that play an essential role in the embryonic development of all organisms and the main components in the pathway are conserved from insects to humans. These proteins affect patterning and morphogenesis of multiple tissues. Therefore, mutations in the Hh pathway can result in a wide range of developmental defects and oncogenic diseases. Because the main components in the pathway are conserved from insects to humans, Drosophilahas been shown to provide a genetically tractable system to gain insight into the processes that Hh is involved in. In this study, the roles of Hh cholesterol modification and endocytosis during gradient fonnation are explored in the Drosophila larval wing imaginal disc. To exclude the possibility of looking at a redistribution of preexisting Hh instead of Hh movement, a spatially and temporally regulated system has been developed to induce Hh expression. Functional Hh-GFP with and without the cholesterol-modification was expressed in a wild-type or shi-tslendocytosis mutant background. The Gal80 system was used to temporally express (pulse) the Hh-GFP transgenes to look at the rate of Hh gradient formation over time and determine whether this process was affected by cholesterol modification and/or endocytosis. Hh with and without cholesterol were both largely detected in punctate structures and the spreading of the different forms of Hh was quantified by measuring distances of these particles from the expressing cells. Hh without cholesterol showed a greater range of distribution, but a lower percentage of particles near the source. Loss of endocytosis blocked formation of intracellular Hh particles, but did not dramatically alter its movement to target cells. Staining for Hh, its receptor Ptc and cortical actin revealed that these punctate structures could be classified into four types of Hh containing particles: cytoplasmic with and without Ptc, and cell surface with and without Ptc. Cholesterol is specifically required for the formation of cytoplasmic particles lacking Ptc. While previous studies have shown discrepancies in the localization of Hh following a block in endocytosis, Hh with and without cholesterol is detected at both apical and basolateral surfaces, but not at basal surfaces. In the absence of cholesterol and endocytosis, Hh particles can be observed in the extracellular space. Through three-dimensional reconstruction and quantitative analysis, this study concludes that the cholesterol modification is required to restrict Hh movement. In addition, the cholesterol modification promotes Ptc-independent internalization. This study also observes that Dynamin-dependent endocytosis is necessary for internalization but does not play an essential role in Hh distribution. The data in this thesis supports the model in which Hh movement occurs via planar diffusion.

Newcastle disease virus (NDV) belongs to the Paramyxoviridae, a family of enveloped RNA viruses that includes many important human and animal pathogens. Although many aspects of the paramyxovirus life cycle are known in detail, our understanding of the mechanisms regulating paramyxovirus assembly and release are poorly understood. For many enveloped RNA viruses, it has recently become apparent that both viral and host cellular determinants coordinate the proper and efficient assembly of infectious progeny virions. Utilizing NDV as a model system to explore viral and cellular determinants of paramyxovirus assembly, we have shown that host cell membrane lipid raft domains serve as platforms of NDV assembly and release. This conclusion was supported by several key experimental results, including the exclusive incorporation of host cell membrane raftassociated molecules into virions, the association of structural components of the NDV particle with membrane lipid raft domains in infected cells and the strong correlation between the kinetics of viral protein dissociation from membrane lipid raft domains and incorporation into virions. Moreover, perturbation of infected cell membrane raft domains during virus assembly resulted in the disordered assembly of abnormal virions with reduced infectivity. These results further established membrane raft domains as sites of virus assembly and showed the integrity of these domains to be critical for the proper assembly of infectious virions. Although specific viral protein-protein interactions are thought to occur during paramyxovirus assembly, our understanding of how these interactions are coordinated is incomplete. While exploring the mechanisms underlying the disordered assembly of non-infectious virions in membrane raft-perturbed cells, we determined that the integrity of membrane raft domains was critical in the formation and virion incorporation of a complex consisting of the NDV attachment (HN) and fusion (F) proteins. The reduced virus-to-cell membrane fusion capacity of particles released from membrane raft-perturbed cells was attributed to an absence of the HN – F glycoprotein-containing complex within the virion envelope. This result also correlated with a reduction of these glycoprotein complexes in membrane lipid raft fractions of membrane raft-perturbed cells. Specifically, it was determined that the formation of newly synthesized HN and F polypeptides into the glycoprotein complex destined for virion incorporation was dependent on membrane lipid raft integrity. Finally, a novel virion complex between the ribonucleoprotein (RNP) structure and the HN attachment protein was identified and characterized. Unlike the glycoprotein complex, the detection of the RNP – HN protein-containing complex was not affected by membrane raft perturbation during virus assembly in the cell. The biological importance of this novel complex for the proper assembly of an infectious progeny virion is currently under investigation. The results presented in this thesis outline the role of host cell membrane lipid raft domains in the assembly and release processes of a model paramyxovirus. Furthermore, the present work extends our understanding of how these particular host cell domains mechanistically facilitate the ordered assembly and release of an enveloped RNA virus.

Systems biology approaches have allowed for comprehensive understanding of complicated biological processes. Here, we’ve developed a global phenotypic profiling method by improving upon transposon mutagenesis methods for identifying genes required for bacterial growth in various conditions. By using the massively parallel power of Illumina sequencing, we precisely redefined the genes required for the growth of Mycobacterium Tuberculosis (Mtb) in vitro. This adapted technique provided more informative data with both increased dynamic range and resolution. As a result, we quantitatively assessed the fitness of individual mutants, as well as identified sub-genic essentiality. Mtb is well adapted to its nutrient-limiting intracellular niche. One important and novel adaptation is its ability to consume cholesterol for both energy and carbon. A combination of this genome-wide phenotypic analysis and global metabolite profiling was used to define the dedicated cholesterol catabolic pathway, as well as important transcriptional and metabolic adaptations required for the consumption of this carbon source. We identified the methylcitrate cycle (MCC) and an unexpected gluconeogenic route as essential pathways. Furthermore, we found that the cholesterol-dependent transcriptional induction of these metabolic enzymes was also essential for growth on this substrate, a function mediated by the Rv1129c regulatory protein. Using a combination of genetic and chemical methods to inhibit these pathways, we show that cholesterol represents a significant source of carbon during intracellular growth in macrophages. Finally, we have begun to define the mechanism by which lipids, such as cholesterol, are imported into the cell by investigating the assembly of the ABC-like lipid transporter, Mce1. The subunits of this system are localized to the cell wall and data is provided to support a novel mechanism for Mce-dependent import of lipids, such as cholesterol. In sum, this global analysis of host cholesterol utilization during infection provides insight into each step of this complicated process; import into the bacterial cell, the degradation of the molecule into primary metabolites, and the transformation of these metabolites into carbon and energy.

O estuário de Santos abriga o maior porto da América Latina e um dos maiores complexos industriais do Brasil. Na década de 1970 foi considerado um dos maiores exemplos de degradação ambiental em ambientes costeiros, e apesar da melhora na qualidade ambiental observada nos últimos anos algumas áreas deste estuário apresentam sedimentos ainda altamente contaminados com compostos tóxicos e genotóxicos, especialmente os Hidrocarbonetos Policíclicos Aromáticos (HPA). Operações de dragagem para manutenção das atividades portuárias são necessárias e consequentemente durante este processo pode ocorrer a ressuspensão de contaminantes presentes nesses sedimentos, tornando-os disponíveis novamente na coluna d\'água. Desta forma é interessante ter uma estratégia eficiente e aplicável no monitoramento da qualidade dessas águas. O Blue rayon (BR) é um adsorvente seletivo para compostos policíclicos com três ou mais anéis fundidos em sua molécula e tem sido utilizado com sucesso no monitoramento de HPA e atividade mutagênica em ambientes aquáticos. Este trabalho teve como objetivo padronizar o uso da técnica de Blue rayon in situ ancorado, onde as fibras são fixas a uma distância padronizada dos sedimentos, associada ao teste de Salmonella/microssoma em microssuspensão a ser aplicada no monitoramento das águas desse estuário. Foram selecionados três pontos de amostragem com diferentes características e sedimentos com variados níveis de contaminação. As duas campanhas iniciais foram realizadas com o objetivo de avaliar a eficiência do BR e de escolher a linhagem de Salmonella mais sensível aos compostos mutagênicos possivelmente presentes nos extratos obtidos bem como verificar a presença de HPA. As cinco campanhas posteriores foram realizadas para verificar o comportamento e a eficiência da estratégia previamente definida no monitoramento das águas desse estuário utilizando experimentos de dose resposta. Os resultados obtidos nas duas primeiras campanhas mostraram que as águas do ponto 1, que contém os sedimentos mais contaminados e recebe os efluentes da COSIPA, apresentam maior contaminação por HPA e atividade genotóxica mais elevada que o ponto 2. Também foi possível determinar que a linhagem de Salmonella YG1041 foi a mais sensível para detectar atividade mutagênica tanto no ponto 1 quanto no ponto 2. Nas cinco campanhas subsequentes as maiores potências mutagênicas foram também observadas no ponto 1 com valores máximos de 5.100 e 36.000 revertentes/g de BR para a linhagem YG1041 na ausência e na presença de ativação metabólica (S9) respectivamente. Nos pontos 2 e 3 as potências observadas foram similares entre si, com valores em torno de 1.000 revertentes/g de BR. Com base nos resultados obtidos nas duas etapas deste trabalho pode-se concluir que a o Blue rayon utilizado in situ a uma distância padronizada dos sedimento associado ao teste de Salmonella/microssoma em microssuspensão com a linhagem YG1041 na presença e na ausência de ativação metabólica (S9) é uma ferramenta que pode ser aplicada no monitoramento da qualidade do estuário de Santos. / Santos estuary is the major Latin American harbor and one of the largest industrial complex of Brazil. In the 1970s, it was considered one of the major examples of coastal degradation. Due to enforcement actions the quality of the environment has improved in the last years in relation to the chemical parameters and toxicity but unfortunately the sediment is still contaminated with toxic and genotoxic compounds especially Polycyclic Aromatic Hydrocarbons (PAHs). For the maintenance of the port activities sediment dredging is needed and during this process contaminants resuspension can occur, leading to the contamination of the water column. Therefore it is interesting to have an efficient strategy that can be used for the water quality monitoring of this area. The Blue rayon is a selective adsorbent to polycyclic compounds containing three or more fused rings and has been successfully used in the monitoring of PAHs and mutagenic activity in aquatic environments. The objective of this work was to evaluate a modified blue rayon hanging technique, denominated in this work \"Blue rayon anchored technique\", where the fibers are placed at a fixed distance from the sediment, associated with the Salmonella/microsome microsuspension assay in order to monitor the water quality of Santos estuary. Three sites with different sources and sediment contamination levels were selected in this study. Two initial samplings were performed in order to evaluate the efficiency of the BR modified technique and find the most sensitive strain of Salmonella typhimurium to the mutagens possibly present in the those waters as well as verify the presence of PAHs. Five subsequent samplings were performed to verify the performance and efficiency of the defined strategy in the estuary water quality monitoring using dose response experiments. In the two initial samplings, the water from the site containing the most contaminated sediment and under influence of COSIPA (site 1) presented higher mutagenic activity and higher levels of PAHs. It was also possible to verify the highest sensitivity of the YG1041 strain in the detection of the mutagenic activity in both sites analyzed. In the subsequent five samplings, higher mutagenic potencies were again observed for site 1, reaching 5,100 and 36,000 revertants per gram of BR for the YG1 041 strain in the absence and presence of S9 respectively. Sites 2 and: presented similar potencies, around 1000 revertants per gram of BR. Based on the obtained results we can conclude that the Blue rayon anchored technique associated with the Salmonella/microsome microsuspension assay with the YG1041 strain in the presence and absence of S9 is an suitable tool to monito Santos estuarine waters.

Compromised calcium homeostasis in the central nervous system (CNS) is implicated as a major contributor in the pathology of neurodegeneration. Dysregulation of Ca2+ homeostasis initiates downstream Ca2+–dependent events that lead to apoptotic and/or necrotic cell death. Increases in the intracellular free calcium concentration ([Ca2+]i) may be the result of Ca2+ influx from the extracellular environment or Ca2+ release from intracellular Ca2+ stores such as the endoplasmic reticulum (ER). Influx from the extracellular environment is controlled predominantly by voltage gated calcium channels (VGCC), such as L–type calcium channels (LTCC) and ionotropic glutamate receptors, such as the N–methyl–D–aspartate (NMDA) receptors. Ca2+ release from the ER occurs through the inositol–1,4,5–triphosphate receptors (IP3Rs) or ryanodine receptors (RyRs) via IP3–induced or Ca2+–induced mechanisms. Mitigation of Ca2+ overload through these Ca2+ channels offers an opportunity for pharmacological interventions that may protect against neuronal death. In the present study the ability of a novel series of polycyclic compounds, both the pentacycloundecylamines and triquinylamines, to regulate calcium influx through LTCC was evaluated in PC12 cells using calcium imaging with Fura–2/AM in a fluorescence microplate reader. We were also able for the first time to determine IC50 values for these compounds as LTCC blockers. In addition, selected compounds were evaluated for their ability to offer protection in apoptosis–identifying assays such as the lactate dehydrogenase release assay (LDH–assay), trypan blue staining assay and immunohistochemistry utilizing the Annexin V–FITC stain for apoptosis. We were also able to obtain single crystal structures for the tricyclo[6.3.0.02,6]undecane–4,9–dien–3,11–dione (9) and tricyclo[6.3.0.02,6]undecane–3,11–dione (10) scaffolds as well as a derivative, N–(3–methoxybenzyl)–3,11–azatricyclo[6.3.0.02,6]undecane (14f). We also evaluated the possibility that the polycyclic compounds might be able to modulate Ca2+ flux through intracellular Ca2+ channels. Computational methods were utilized to accurately predicted IC50 values and develop a QSAR model with marginal error. The linear regression model delivered r2 = 0.83, which indicated a favorable correlation between the predicted and experimental IC50 values. This model could thus serve as valuable predictor for future structural design and optimization efforts. Data obtained from the crystallographic analysis confirmed the NMR–data based structural assignments done for these compounds in previous studies. Obtaining structural information gave valuable insight into the differences in size and geometric constrains, which are key features for the LTCC activity of these compounds. vii In conclusion, we found that all of the compounds evaluated were able to attenuate Ca2+ influx through the LTCC, with some compounds having IC50 values comparable with known LTCC blockers such as nimodipine. Representative compounds were evaluated for their ability to afford protection against apoptosis induced by 200 ?M H2O2. With the exception of compound 14c (the most potent LTCC blocker in the series, IC50 = 0.398 ?M), most compounds were able to afford protection at two or more concentrations evaluated. Compound 14c displayed inherent toxicity at the highest concentrations evaluated (100 ?M). We concluded that compounds representing both types of structures (pentacycloudecylamines and triquinylamines) have the ability to attenuate excessive Ca2+ influx through the LTCC. In general the aza–pentacycloundecylamines (8a–c) were the most potent LTCC blocker which also had the ability to offer protection in the cell viability assays. However, NGP1–01 (7a) had the most favorable pharmacological profile overall with good activity as an LTCC blocker (IC50 = 86 ?M) and the ability to significantly attenuate cell death in the cell viability assays, exhibiting no toxicity. In addition to their ability to modulate Ca2+ influx from the extracellular environment, these compounds also displayed the ability to modulate Ca2+ flux through intracellular Ca2+ channels. The mechanisms by which they act on intracellular Ca2+ channels still remains unclear, but from this preliminary study it would appear that these compounds are able to partially inhibiting Ca2+–ATPase activity whilst possibly simultaneously inhibiting the IP3R. In the absence of extracellular Ca2+ these compounds showed the ability in inhibit voltage–induced Ca2+ release (VICaR), possibly by modulating the gating charge of the voltage sensor being the dihydropyridine receptors. In future studies it might be worthwhile to do an expanded QSAR study and evaluate the aza–pentacycloundecylamines. To clarify the mechanisms by which the polycyclic compounds interact with intracellular Ca2+ channels we should examine the direct interaction with the individual Ca2+ channels independently. The polycyclic compounds evaluated in this study demonstrate potential as multifunctional drugs due to their ability to broadly regulate calcium homeostasis through multiple pathways of Ca2+ entry. This may prove to be more effective in diseases where perturbed Ca2+ homeostasis have devastating effects eventually leading to excitotoxicity and cell death. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.