Academic literature on the topic 'Anti–σ factor Interactions'

The host–pathogen interactions inMycobacterium tuberculosisinfection are significantly influenced by redox stimuli and alterations in the levels of secreted antigens. The extracytoplasmic function (ECF) σ factor σKgoverns the transcription of the serodominant antigens MPT70 and MPT83. The cellular levels of σKare regulated by the membrane-associated anti-σK(RskA) that localizes σKin an inactive complex. The crystal structure ofM. tuberculosisσKin complex with the cytosolic domain of RskA (RskAcyto) revealed a disulfide bridge in the −35 promoter-interaction region of σK. Biochemical experiments reveal that the redox potential of the disulfide-forming cysteines in σKis consistent with its role as a sensor. The disulfide bond in σKinfluences the stability of the σK–RskAcytocomplex but does not interfere with σK–promoter DNA interactions. It is noted that these disulfide-forming cysteines are conserved across homologues, suggesting that this could be a general mechanism for redox-sensitive transcription regulation.

YlaD, a membrane-anchored anti-sigma (σ) factor of Bacillus subtilis, contains a HX3CXXC motif that functions as a redox-sensing domain and belongs to one of the zinc (Zn)-co-ordinated anti-σ factor families. Despite previously showing that the YlaC transcription is controlled by YlaD, experimental evidence of how the YlaC–YlaD interaction is affected by active cysteines and/or metal ions is lacking. Here, we showed that the Pyla promoter is autoregulated solely by YlaC. Moreover, reduced YlaD contained Zn and iron, while oxidized YlaD did not. Cysteine substitution in YlaD led to changes in its secondary structure; Cys3 had important structural functions in YlaD, and its mutation caused dissociation from YlaC, indicating the essential requirement of a HX3CXXC motif for regulating interactions of YlaC with YlaD. Analyses of the far-UV CD spectrum and metal content revealed that the addition of Mn ions to Zn–YlaD changed its secondary structure and that iron was substituted for manganese (Mn). The ylaC gene expression using βGlu activity from Pyla:gusA was observed at the late-exponential and early-stationary phase, and the ylaC-overexpressing mutant constitutively expressed gene transcripts of clpP and sigH, an important alternative σ factor regulated by ClpXP. Collectively, our data demonstrated that YlaD senses redox changes and elicits increase in Mn ion concentrations and that, in turn, YlaD-mediated transcriptional activity of YlaC regulates sporulation initiation under oxidative stress and Mn-substituted conditions by regulating clpP gene transcripts. This is the first report of the involvement of oxidative stress-responsive B. subtilis extracytoplasmic function σ factors during sporulation via a Mn-dependent redox-sensing molecular switch.

In bacteria, a primary σ-factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ-factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative σ-factors are negatively regulated by anti–σ-factors. In Escherichia coli, Salmonella enterica, and many other γ-proteobacteria, the transcription factor Crl positively regulates the alternative σS-regulon by promoting the association of σS with RNAP without interacting with promoter DNA. The molecular mechanism for Crl activity is unknown. Here, we determined a single-particle cryo-electron microscopy structure of Crl-σS-RNAP in an open promoter complex with a σS-regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of σS (σS2), the structure, along with p-benzoylphenylalanine cross-linking, reveals that Crl interacts with a structural element of the RNAP β′-subunit that we call the β′-clamp-toe (β′CT). Deletion of the β′CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-β′CT interaction. We conclude that Crl activates σS-dependent transcription in part through stabilizing σS-RNAP by tethering σS2 and the β′CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators.

σ factors are transcriptional regulatory proteins that bind to the RNA polymerase and dictate gene expression. The extracytoplasmic function (ECF) σ factors govern the environment dependent regulation of transcription. ECF σ factors have two domains σ2 and σ4 that recognize the -10 and -35 promoter elements. However, unlike the primary σ factor σA, the ECF σ factors lack σ3, a region that helps in the recognition of the extended -10 element and σ1.1, a domain involved in the autoinhibition of σA in the absence of core RNA polymerase. Mycobacterium tuberculosis σC is an ECF σ factor that is essential for the pathogenesis and virulence of M. tuberculosis in the mouse and guinea pig models of infection. However, unlike other ECF σ factors, σC does not appear to have a regulatory anti-σ factor located in the same operon. We also note that M. tuberculosis σC differs from the canonical ECF σ factors as it has an N-terminal domain comprising of 126 amino acids that precedes the σC2 and σC4 domains. In an effort to understand the regulatory mechanism of this protein, the crystal structures of the σC2 and σC4 domains of σC were determined. These promoter recognition domains are structurally similar to the corresponding domains of σA despite the low sequence similarity. Fluorescence experiments using the intrinsic tryptophan residues of σC2 as well as surface plasmon resonance measurements reveal that the σC2 and σC4 domains interact with each other. Mutational analysis suggests that the Pribnow box-binding region of σC2 is involved in this interdomain interaction. Interaction between the promoter recognition domains in M. tuberculosis σC are thus likely to regulate the activity of this protein even in the absence of an anti-σ factor.

AbstractThe bacterial RNA polymerase (RNAP) holoenzyme is a multisubunit core enzyme associated with a σ factor that is required for promoter-specific transcription initiation. Besides a primary σ responsible for most of the gene expression during active growth, bacteria contain alternative σ factors that control adaptive responses. A recurring strategy in the control of σ factor activity is their sequestration by anti-sigma factors that occlude the RNAP binding determinants, reducing their activity. In contrast, the unconventional transcription factor Crl binds specifically to the alternative σ factor σS/RpoS, and favors its association with the core RNAP, thereby increasing its activity. σS is the master regulator of the general stress response that protects many Gram-negative bacteria from several harmful environmental conditions. It is also required for biofilm formation and virulence of Salmonella enterica serovar Typhimurium. In this report, we discuss current knowledge on the regulation and function of Crl in Salmonella and Escherichia coli, two bacterial species in which Crl has been studied. We review recent advances in the structural characterization of the Crl-σS interaction that have led to a better understanding of this unusual mechanism of σ regulation.

ABSTRACT In Salmonella enterica serovar Typhimurium,σ 28 and anti-sigma factor FlgM are regulatory proteins crucial for flagellar biogenesis and motility. In this study, we used S. enterica serovar Typhimurium as an in vivo heterologous system to study σ28 and anti-σ28 interactions in organisms where genetic manipulation poses a significant challenge due to special growth requirements. The chromosomal copy of the S. enterica serovar Typhimurium σ28 structural gene fliA was exchanged with homologs of Aquifex aeolicus (an extreme thermophile) and Chlamydia trachomatis (an obligate intracellular pathogen) by targeted replacement of a tetRA element in the fliA gene location using λ-Red-mediated recombination. The S. enterica serovar Typhimurium hybrid strains showed σ28-dependent gene expression, suggesting that σ28 activities from diverse species are preserved in the heterologous host system. A. aeolicus mutants defective for σ28/FlgM interactions were also isolated in S. enterica serovar Typhimurium. These studies highlight a general strategy for analysis of protein function in species that are otherwise genetically intractable and a straightforward method of chromosome restructuring usingλ -Red-mediated recombination.

The alternative sigma (σ) factor E, RpoE or HrpL, has been reported to be involved in stress- and pathogenicity-related transcription initiation in Escherichia coli and many other Gram-negative bacteria, including Erwinia spp. and Pseudomonas spp. A previous study identified the hrpL/rpoE transcript as one of the significant differentially expressed genes (DEGs) during early E. mallotivora infection in papaya and those data serve as the basis of the current project. Here, the full coding DNA sequence (CDS) of hrpL from E. mallotivora (EmhrpL) was determined to be 549 bp long, and it encoded a 21.3 kDa HrpL protein that possessed two highly conserved sigma-70 (σ70) motifs—σR2 and σR4. Nucleotide sequence alignment revealed the hrpL from E. mallotivora shared high sequence similarity to rpoE/hrpL from E. tracheiphila (83%), E. pyrifoliae (81%), and E. tasmaniensis (80%). Phylogenetics analysis indicated hrpL from E. mallotivora to be monophyletic with rpoEs/hrpLs from Pantoea vagans, E. herbicola, and E. tracheiphila. Structural analysis postulated that the E. mallotivora’s alternative σ factor was non-transmembranic and was an extracytoplasmic function (ECF) protein—characteristics shared by other σ factors in different bacterial species. Notably, the protein–protein interaction (PPI) study through molecular docking suggested the σ factor could be possibly inhibited by an anti-σ. Finally, a knockout of hrpL in E. mallotivora (ΔEmhrpL) resulted in avirulence in four-month-old papaya plants. These findings have revealed that the hrpL is a necessary element in E. mallotivora pathogenicity and also predicted that the gene can be inhibited by an anti-σ.

ABSTRACT FpvR is a presumed cytoplasmic membrane-associated anti-sigma factor that controls the activities of extracytoplasmic function sigma factors PvdS and FpvI responsible for transcription of pyoverdine biosynthetic genes and the ferric pyoverdine receptor gene, fpvA, respectively. Using deletion analysis and an in vivo bacterial two-hybrid system, FpvR interaction with these σ factors was confirmed and shown to involve the cytoplasmic N-terminal 67 amino acid resides of FpvR. FpvR bound specifically to a C-terminal region of FpvI corresponding to region 4 of the σ70 family of sigma factors. FpvR and FpvI mutant proteins compromised for this interaction were generated by random and site-directed PCR mutagenesis and invariably contained secondary structure-altering proline substitution in predicted α-helices within the FpvR N terminus or FpvI region 4. PvdS was shown to bind to the same N-terminal region of FpvR, and FpvR mutations compromising FpvI binding also compromised PvdS binding, although some mutations had a markedly greater impact on PvdS binding. Apparently, these two σ factors bind to FpvR in a substantially similar but not identical fashion. Intriguingly, defects in FpvR binding correlated with a substantial drop in yields of the FpvI and to a lesser extent PvdS σ factors, suggesting that FpvR-bound FpvI and PvdS are stable while free and active sigma factor is prone to turnover.

Extra Cytoplasmic Function (ECF) σ factors are a diverse family of alternative σ factors that allow bacteria to sense and respond to changes in the environment. σV is an ECF σ factor found primarily in low GC Gram-positive bacteria and is required for lysozyme resistance in several opportunistic pathogens. In the absence of lysozyme, σV is inhibited by the anti-σ factor RsiV. In response to lysozyme, RsiV is degraded via the process of Regulated Intramembrane Proteolysis (RIP). RIP is initiated by cleavage of RsiV at site-1 which allows the intramembrane protease RasP to cleave RsiV within the transmembrane domain at site-2 and leads to activation of σV. Previous work suggested that RsiV is cleaved by signal peptidase at site-1. Here we demonstrate in vitro that signal peptidase is sufficient for cleavage of RsiV only in the presence of lysozyme and provide evidence that multiple Bacillus subtilis signal peptidases can cleave RsiV in vitro. This cleavage is dependent upon the concentration of lysozyme consistent with previous work that showed binding to RsiV was required for σV activation. We also show that signal peptidase activity is required for site-1 cleavage of RsiV in vivo. Thus, we demonstrate that signal peptidase is the site-1 protease for RsiV.

Neuropathic pain is a debilitating problem affecting millions of people worldwide. This condition affects the quality of life and bears a substantial economic burden on society. Current drug treatments are often inadequate to treat this type of chronic pain, as they provide limited relief and are often accompanied with many undesired and difficult to manage side effects. It is imperative thus, to find new treatments that target alternative mechanisms from the currently approved drugs.The pathogenesis of neuropathic pain is very complex and involves structural, physiological and pharmacological changes throughout the neural axis (from the site of peripheral nerve injury to the spinal cord/brain). While a neuron-centric view has dominated the literature for decades and the way we approach neuropathic pain treatment, recent work has uncovered extensive neuroimmune interactions as substrates of this condition. Interactions between the immune and nervous systems occur at multiple levels, where different types of immune/glial cells and immune-derived substances are implicated in various stages of the pathogenesis. The four experimental chapters contained in this dissertation are in line with this recent recognition of the importance of neuroimmune mechanisms in neuropathic pain. In the work presented here, we sought to unravel novel neuroinflammatory mechanisms driving the aberrant pain condition in both the spinal cord and the peripheral nerve as well as to investigate the role of a potent anti-inflammatory cytokine, TGF-β1, as a potential modulator of these mechanisms. Several important mechanisms have been observed: A) Modulation of the central and peripheral inflammatory reaction with TGF-β1 significantly delays and attenuates mechanical allodynia and thermal hyperalgesia via immunosuppressive actions on glial cells (Chapter 2) and macrophages (Chapter 3). In the spinal cord TGF-β1 inhibits microglial proliferation, astrocytic activation and release of pro-inflammatory cytokines and exerts neuroprotective effects resulting in a decrease in the production of MCP-1. At the site of the injury, exposure to TGF-β1 reduces the number of macrophages, which release pro-inflammatory mediators, and modulates different populations of T-cells. B) In chapter 4 we demonstrate that sciatic nerve injury-induced neuropathic pain is associated with an increased permeability of the blood spinal cord barrier (BSCB). We observed that the spinal inflammatory reaction triggered by nerve injury is a key player in modulating this BSCB impairment. We identified MCP-1 as an endogenous trigger of BSCB leakage, which could be reversed using anti-inflammatory molecules TGF-β1 and IL-10. C) Selective depletion of microglia with MAC-1-saporin results in impairment of mechanical and thermal hypersensitivities at both acute and chronic stages following nerve injury suggesting an important and persistent contribution of microglia in the pathogenesis of neuropathic pain. These results expand our understanding of the neuro-inflammatory mechanisms of neuropathic pain as well as propose that immunosuppression or blockade of the reciprocal signalling pathways between neuronal and non-neuronal cells offer new opportunities for disease modification and more successful management of pain.
La douleur neuropathique est un problème débilitant affectant des millions de personnes dans le monde. Cette maladie affecte la qualité de vie et apporte un fardeau économique considérable à la société. Les traitements actuels sont souvent inadéquats pour traiter ce type de douleur chronique, car ils ne procurent qu'un soulagement limité et s'accompagnent de nombreux effets secondaires indésirables et difficiles à gérer. Aussi, il est impératif que de nouveaux traitements pour la douleur neuropathique soient trouvés.Le problème de la douleur neuropathique est que la pathogenèse de cet état hypersensible est très complexe et implique des changements structuraux, psychologiques et pharmacologiques à travers l'axe neuronal (en partant du site du nerf périphérique lésé jusqu'à la moelle épinière/cerveau). Alors qu'un point de vue neurocentrique a dominé la littérature tout comme l'approche du traitement de la douleur neuropathique pendant des décennies, des travaux récents ont mis en évidence des interactions neuro-immunitaires étendues afin de caractériser la maladie. Les interactions entre les systèmes immunitaires et nerveux se produisent à différents niveaux, avec divers types de cellules immunitaires/gliales et des molécules dérivées du système immunitaire sont impliqués dans plusieurs étapes de la pathogenèse.Les quatre chapitres expérimentaux contenus dans cette thèse sont en lien avec cet état de fait récent de l'importance des mécanismes neuro-immunitaires dans la douleur neuropathique. Dans le travail présenté ici, nous avons cherché à éclaircir ces nouveaux mécanismes neuro-inflammatoires entrainant cette condition de douleur aberrante à la fois dans la moelle épinière et le nerf périphérique afin d'étudier le rôle d'une cytokine anti-inflammatoire active, TGF-β1, comme modulateur potentiel de ces mécanismes. Plusieurs mécanismes importants ont été observés: A) La modulation de la réaction inflammatoire centrale et périphérique avec TGF-β1 retarde et diminue de manière significative l'allodynie mécanique et l'hyperalgésie thermique via des actions immunosuppressives sur les cellules gliales (chapitre 2) et les macrophages (chapitre 3). Dans la moelle épinière, TGF-β1 inhibe la prolifération microgliale, l'activation des astrocytes et la libération de cytokines pro-inflammatoires et exerce des effets neuro-protecteurs entrainant une diminution de la production de MCP-1. À l'endroit de la lésion, l'exposition à TGF-β1 réduit le nombre de macrophages libérant des médiateurs inflammatoires et module différentes populations de cellule T. B) Dans le chapitre 4, nous démontrons que la douleur neuropathique induite par la lésion du nerf sciatique est associée à une augmentation de la perméabilité de la barrière hématoencéphalique (BHE). Nous avons observé que la réaction inflammatoire de la moelle déclenchée par la lésion du nerf a un rôle clé dans la modulation de la perméabilité de la BHE. Nous avons identifié MCP-1 comme étant un déclencheur endogène de la perméabilité de la BHE, cette dernière pouvant être inversée en utilisant des molécules telles que TGF-β1 et IL-10. C) La diminution sélective des microglies grâce à la MAC-1-saporine résulte en l'affaiblissement de l'hypersensibilité mécanique et thermique à la fois dans les étapes aigues et chronique suivant la lésion du nerf suggérant une contribution importante et persistante des microglies dans la pathogenèse de la douleur neuropathique. Ces résultats étendent notre compréhension des mécanismes neuro-inflammatoire de la douleur neuropathique en plus de suggérer que l'immunosuppression ou le blocage des voies de signalisation réciproques entre les cellules neurales et non-neurales offre de nouvelles opportunités à la modification de la maladie et une meilleure gestion de la douleur.

Des résines dérivées du polystyrene réticule ont été synthétisées. Ces polymères sont fonctionnalisés par des groupements chimiques analogues à ceux de l'adn et des phospholipides. L'affinité biospécifique de ces resines pour des protèines humaines qui admettent pour substrat l'adn et les phospholipides : anticorps anti adn, anti phospholipide, facteurs de transcription de l'adn. Les anticorps qui sont présents dans le sang des patients atteints de lupus érythemateux dissemine (l. E. D) ont montré une affinité spécifique tres élevée pour les dérivés fonctionnels du polystyrene;ces interactions impliquent le fragment fab des immunoglobulines lupiques et des sites distribués de façon statistique à la surface des polymères. Les polymères fonctionnels phosphoryles permettent en tant que phase stationnaire la résolution chromatographique d'extraits cellulaires contenant des facteurs de transcription de l'adn

Adaptation to external environmental conditions is essential for the survival of a bacterial cell. Bacteria have thus evolved multiple mechanisms to sense environmental stimuli and to couple this information into an appropriate cellular response. The cellular response, in its simplest sense involves a change in the cellular content which is achieved by regulating gene expression. Gene expression in bacteria is primarily regulated at the first step, also referred to as transcription initiation. Extensive studies on this mechanism over the past two decades provide a molecular picture of this process. The main enzyme in this process is the DNA dependent RNA polymerase (RNAP). The RNAP enzyme however lacks a specificity factor that dictates which genes are to be selectively expressed. Selectivity is enforced by a dissociable subunit, the sigma (σ) factor. σ factors have specificity determinants that allow recognition of promoter sequences in the DNA. The sequence features on DNA include the –10 elements (Pribnow box), –35 elements, the extended -10 region, the spacing between –10 and –35 elements and the upstream sequence to –35 promoter element. σ factors recognize a few or several of these promoter sequence features thereby providing an efficient mechanism for RNAP recruitment at a specific promoter. σ factors substantially differ in sequence and structural elements. The principal σ factor of the σ70 family has several domains. This includes specific domains that interact with –10 and –35 promoter elements and the sequence between these two promoter elements. The N-terminal domain of σ factors of the σ70 family also encodes regions enabling auto-regulation of this initiation factor. A defining feature that distinguishes σ70 members from other σ factors (σ38 and σ54) is that σ70 does not require ATP for its activity. The number of σ factors in a bacterial cell varies across species. For example Streptococcus pneumonia has one σ factor, Lactococcus lactis has two, Haemophilus influenza has four while Mycobacterium tuberculosis has thirteen σ factors. The exceptions are Streptomyces coelicolor with 65and Sorangium cellulosum with 109 σ factors. The number of σ factors in a bacterial cell is suggested to be correlated with the diverse environmental conditions encountered by the bacterium and the genomic size. The focus of work reported in this thesis is on M. tuberculosis σ factors. There are large variations in the number of σ factors in different mycobacterial species. While M. leprae has two, M. tuberculosis has thirteen σ factors. This variation in the number of σ factors has widely been believed to aid rapid signal transduction of environmental stimuli into changes in gene expression. While other transcription factors enable the recruitment of RNAP to specific promoter sequences, repressors that abrogate transcription and effectors that modulate transcription also dictate transcription levels. The intra cellular levels of a σ factor is often the primary determinant of the expression profile. A specific group of σ factors referred to as Extra Cytoplasmic Function (ECF) σ factors, govern the cellular response to specific environmental stimuli. The ECF family of σ factors has been shown to be regulated by diverse mechanisms. These include transcriptional, translational and post translational control by protein – protein interactions. The focus of this thesis was to understand the regulatory mechanism that involves σ factor interacting proteins. ECF σ factors that are governed by protein – protein interactions are often co-expressed with a regulator protein, the anti-σ factor. In several cases, they are a part of the same operon. This ensures similarity in the expression levels of σ factors and their cognate anti-σ factors. The selective dissociation of an inactive σ factor from a σ/anti-σ complex is also effected by diverse mechanisms. These include structural changes in response to environmental stimuli, conformational changes brought about by the binding of metabolites or by targeted proteolysis of anti-σ factors. The cellular concentration of an activated σ factor is thus often subject to the rate at which it is released from an inactive complex. The notion of specific σ/anti σ factor pairs has recently been challenged with a suggestion that a σ factor could also make non-specific interactions with other anti-σ factors. This finding has implications for the widely accepted model for bacterial gene expression that relies on a cellular estimate of free active σ factors. In this model, referred to as the partitioning of σ factor space model of bacterial transcription, σ factors compete for a limited pool of apo-RNAP and recruit the enzyme to target promoter elements. Two aspects of the mechanism that governs σ factor activity were explored in the course of the studies reported in this thesis. The first is recognition of a particular stress by structurally similar anti-σ factors. This is described in the second chapter of this thesis. The relative sensitivity of redox sensors plays an important role in providing a calibrated response to environmental stimuli and cellular homeostasis. This cellular machinery plays a crucial role in the human pathogen M. tuberculosis as it encounters diverse microenvironments in the host. The redox sensory mechanism in M. tuberculosis is governed by two component and one component systems, alongside ECF σ factors. ECF σ factors that govern the cellular response to redox stimuli are negatively regulated by forming a complex with proteins called zinc associated anti – σ factors (ZAS). ZAS proteins release their cognate σ factor in response to oxidative stress. The relative sensitivity of the ZAS sensors to redox processes dictates the concentration of free ECF σ factors in the cell. However, factors governing the redox threshold of these sensors remain unclear. The molecular characterization of three σ factor/ZAS pairs - σL/RslA, σE/RseA and σH/RshA using a combination of biochemical, biophysical and electrochemical techniques revealed the differences in redox sensitivity in these proteins despite apparent structural similarity. This finding can potentially rationalize the hierarchy in the activation of the cognate ECF σ factors under oxidative stress. Put together, the study described in chapter 2 provides a basis for examining sequence and conformational features that modulate redox sensitivity within the confinement of a conserved structural scaffold. The other aspect that was examined in the course of this study, described in chapter 3 of this thesis, was on crosstalk between σ/anti σ factors. In this study, we evaluated the affinity and specificity for σ/anti-σ factor interaction. We then analyzed the conformational determinants that enforce fidelity in σ/anti-σ interactions. The experimental data that we obtained on interactions between cognate and non-cognate σ/anti-σ pairs provide a template to evaluate tolerance between specific and non-specific interactions. The results from this analysis suggest non-cognate interactions are feasible. These interactions are likely to govern the extent to which different σ factors are activated in response to a particular environmental stimulus. It appears likely that this mechanism could provide a route to bacterial survival, perhaps allowing phenotypic diversity that help circumvent an environmental insult. Another aspect we addressed in the course of this work was the evaluation of specific targeting of a prokaryotic transcription initiation factor. Peptide ligands of a M. tuberculosis σ factor, targeting the RNAP-σ interface were evaluated biochemically and biophysically. A sixteen residue long helical peptide from the core RNAP that could interact with σ4 region and suitably designed control peptides are validated in vitro. Consistent findings from in silico and in vitro observations validated the design strategy that can also be extended across σ factors. This study suggests that designed peptide binders can be explored as chemical tools for studying the regulation of transcription. The fifth chapter of this thesis provides a summary of the findings from the three research themes described in this thesis. This thesis has three appendices. The first two report projects that were discontinued as they were not feasible from the perspective of structural characterization. While Appendix I reports preliminary studies on hemagglutinin-antibody complex, Appendix II describes structural studies on Escherichia coli toxin–anti toxin pairs to evaluate a conformational rationale for protein–protein interactions. Appendix III is a compilation of sequence and structural data that was used for the bioinformatics analysis presented in chapter 3. Put together the studies described in this thesis reveal exquisite adaptation strategies employed by M. tuberculosis to ensure survival under diverse micro-environments in the host.

A distinctive feature of host-pathogen interactions in the case of Mycobacterium tuberculosis is the asymptomatic latent phase of infection. The ability of the bacillus to survive for extended periods of time in the host suggests an adaptive mechanism in M. tuberculosis that can cope with a variety of environmental stresses and other host stimuli. Extensive genomic studies and analysis of knock-out phenotypes revealed elaborate cellular machinery in M. tuberculosis that ensures a rapid cellular response to host stimuli. Prominent amongst these are two-component systems and σ factors that exclusively govern transcription re-engineering in response to environmental stimuli. M. tuberculosis σK is a σ factor that was demonstrated to control the expression of secreted antigenic proteins. The study reported in this thesis was geared to understand the molecular basis for σK activity as well as to explore conditions that would regulate σK activity. Transcription in bacteria is driven by the RNA polymerase enzyme that can associate with multiple σ factors. σ factors confer promoter specificity and thus directly control the expression of genes. The association of different σ factors with the RNA polymerase is essential for the temporal and conditional re-engineering of the expression profile. Environment induced changes in expression rely on a subset of σ factors. This class of σ factors (also referred to as Class IV or Extra-cytoplasmic function (ECF) σ factors) is regulated by a variety of mechanisms. The regulation of an ECF σ factor activity at the transcriptional, translational or posttranslational steps ensures fidelity in the cellular concentration of free, active ECF σ factors. In general, ECF σ factors associate with an inhibitory protein referred to as an anti-σ factor. The release of a free, active σ factor from a σ /anti-σ complex is thus a mechanism that can potentially control the cellular levels of an active σ factor in the cell. M. tuberculosis σK is associated with a membrane bound anti-σK (also referred to as RskA) (Said-Salim et al., Molecular Microbiology 62: 1251-1263: 2006). The extracellular stimulus that is recognized by RskA remains unclear. However, recent studies have suggested the possibility of a regulated proteolytic cascade that can selectively degrade RskA and other membrane associated anti-σ factors. The goal of the study was to understand this regulatory mechanism with a specific focus on the M. tuberculosis σK/RskA complex. The structure of the cytosolic σK/RskA complex and the associated biochemical and biophysical characteristics revealed several features of this /anti-σ complex that were hitherto unclear. In particular, these studies revealed a redox sensitive regulatory mechanism in addition to a regulated proteolytic cascade. These features and an analysis of the M. tuberculosis σK/RskA complex vis-à-vis the other characterized σ/anti σfactor complexes are presented in this thesis. This thesis is organized as follows- Chapter 1 provides an overview of prokaryotic transcription. A brief description of the physiology of M. tuberculosis is presented along with a summary of characterized factors that contribute to the pathogenecity and virulence of this bacillus. The pertinent mechanistic issues of σ/anti-σ factor interactions are placed in the context of environment mediated changes in M. tuberculosis transcription. A summary of studies in this area provides a background of the research leading to this thesis. Chapters 2 and 3 of this thesis describe the structural and mechanistic studies on the σK/RskA complex. The crystal structure of the σK/RskA complex revealed a disulfide bond in domain 4 (σK4). σK4 interacts with the -35 element of the promoter DNA. The disulfide forming cysteines were seen to be conserved in more than 70% of σK homologs, across both gram-positive and gram-negative bacteria. The conservation of the disulfide-forming cysteines led us to further characterize the role of this disulfide in σK/RskA interactions. These were examined by several biochemical and biophysical experiments. The redox potential of these disulfide bond forming cysteine residues were consistent with the proposed role of a sensor. The crystal structure and biochemical studies thus suggest that M. tuberculosis σK is activated under reducing conditions. Chapter 4 of this thesis describes the progress made thus far in the structural and biochemical characterization of an intra-membrane protease, M. tuberculosis Rip1 (Rv2869c). This protein is an essential component of the proteolytic cascade that selectively cleaves RskA. The proteolytic steps that govern the selective degradation of an anti-σ factor were first characterized in the case of E. coli σE (Li, X. et al. Proc. Natl. Acad. Sci. USA, 106:14837-14842, 2009). This cascade is triggered by the concerted action of a secreted protease (also referred to as a site-1 protease) and a trans-membrane protease (also referred to as a site-2 protease). M. tuberculosis Rip1 was demonstrated to be bona-fide site 2 protease that acts on three anti-σ factors viz., RskA, RslA and RsmA (Sklar et al., Molecular Microbiology 77:605-617; 2010). To further characterize the role of Rip1 in the proteolytic cascade, this intra-membrane protease was cloned, expressed and purified for structural, biochemical and biophysical analysis. The preliminary data on this membrane protein is described in this chapter. The conclusions from the studies reported in this thesis and the scope for future work in this area is described in Chapter 5. Put together, the σK/RskA complex revealed facets of σ/anti-σ factor interactions that were hitherto unrecognized. The most prominent amongst these is the finding that an ECF σfactor can respond to multiple environmental stimuli. Furthermore, as seen in the case of the σK/RskA complex, the σ factor can itself serve as a receptor for redox stimuli. Although speculative, a hypothesis that needs further study is whether these features of the σK/RskA complex contribute to the variable efficacy of the M. bovis BCG vaccine. In this context it is worth noting that σK governs the expression of the prominent secreted antigens- MPT70 and MPT83. The studies reported in this thesis thus suggest several avenues for future research to understand mycobacterial diversity, immunogenicity and features of host-pathogen interactions. The appendix section is divided into two subparts- Appendix 1 of the thesis is a review on peptidase V. This is a chapter in The Handbook of Proteolytic enzymes (Elsevier Press, ISBN:9780123822192). Appendix 2 of the thesis includes technical details and an extended materials and methods section.

A distinctive feature of host-pathogen interactions in the case of Mycobacterium tuberculosis is the asymptomatic latent phase of infection. The ability of the bacillus to survive for extended periods of time in the host suggests an adaptive mechanism in M. tuberculosis that can cope with a variety of environmental stresses and other host stimuli. Extensive genomic studies and analysis of knock-out phenotypes revealed elaborate cellular machinery in M. tuberculosis that ensures a rapid cellular response to host stimuli. Prominent amongst these are two-component systems and σ factors that exclusively govern transcription re-engineering in response to environmental stimuli. M. tuberculosis σK is a σ factor that was demonstrated to control the expression of secreted antigenic proteins. The study reported in this thesis was geared to understand the molecular basis for σK activity as well as to explore conditions that would regulate σK activity. Transcription in bacteria is driven by the RNA polymerase enzyme that can associate with multiple σ factors. σ factors confer promoter specificity and thus directly control the expression of genes. The association of different σ factors with the RNA polymerase is essential for the temporal and conditional re-engineering of the expression profile. Environment induced changes in expression rely on a subset of σ factors. This class of σ factors (also referred to as Class IV or Extra-cytoplasmic function (ECF) σ factors) is regulated by a variety of mechanisms. The regulation of an ECF σ factor activity at the transcriptional, translational or posttranslational steps ensures fidelity in the cellular concentration of free, active ECF σ factors. In general, ECF σ factors associate with an inhibitory protein referred to as an anti-σ factor. The release of a free, active σ factor from a σ /anti-σ complex is thus a mechanism that can potentially control the cellular levels of an active σ factor in the cell. M. tuberculosis σK is associated with a membrane bound anti-σK (also referred to as RskA) (Said-Salim et al., Molecular Microbiology 62: 1251-1263: 2006). The extracellular stimulus that is recognized by RskA remains unclear. However, recent studies have suggested the possibility of a regulated proteolytic cascade that can selectively degrade RskA and other membrane associated anti-σ factors. The goal of the study was to understand this regulatory mechanism with a specific focus on the M. tuberculosis σK/RskA complex. The structure of the cytosolic σK/RskA complex and the associated biochemical and biophysical characteristics revealed several features of this /anti-σ complex that were hitherto unclear. In particular, these studies revealed a redox sensitive regulatory mechanism in addition to a regulated proteolytic cascade. These features and an analysis of the M. tuberculosis σK/RskA complex vis-à-vis the other characterized σ/anti σfactor complexes are presented in this thesis. This thesis is organized as follows- Chapter 1 provides an overview of prokaryotic transcription. A brief description of the physiology of M. tuberculosis is presented along with a summary of characterized factors that contribute to the pathogenecity and virulence of this bacillus. The pertinent mechanistic issues of σ/anti-σ factor interactions are placed in the context of environment mediated changes in M. tuberculosis transcription. A summary of studies in this area provides a background of the research leading to this thesis. Chapters 2 and 3 of this thesis describe the structural and mechanistic studies on the σK/RskA complex. The crystal structure of the σK/RskA complex revealed a disulfide bond in domain 4 (σK4). σK4 interacts with the -35 element of the promoter DNA. The disulfide forming cysteines were seen to be conserved in more than 70% of σK homologs, across both gram-positive and gram-negative bacteria. The conservation of the disulfide-forming cysteines led us to further characterize the role of this disulfide in σK/RskA interactions. These were examined by several biochemical and biophysical experiments. The redox potential of these disulfide bond forming cysteine residues were consistent with the proposed role of a sensor. The crystal structure and biochemical studies thus suggest that M. tuberculosis σK is activated under reducing conditions. Chapter 4 of this thesis describes the progress made thus far in the structural and biochemical characterization of an intra-membrane protease, M. tuberculosis Rip1 (Rv2869c). This protein is an essential component of the proteolytic cascade that selectively cleaves RskA. The proteolytic steps that govern the selective degradation of an anti-σ factor were first characterized in the case of E. coli σE (Li, X. et al. Proc. Natl. Acad. Sci. USA, 106:14837-14842, 2009). This cascade is triggered by the concerted action of a secreted protease (also referred to as a site-1 protease) and a trans-membrane protease (also referred to as a site-2 protease). M. tuberculosis Rip1 was demonstrated to be bona-fide site 2 protease that acts on three anti-σ factors viz., RskA, RslA and RsmA (Sklar et al., Molecular Microbiology 77:605-617; 2010). To further characterize the role of Rip1 in the proteolytic cascade, this intra-membrane protease was cloned, expressed and purified for structural, biochemical and biophysical analysis. The preliminary data on this membrane protein is described in this chapter. The conclusions from the studies reported in this thesis and the scope for future work in this area is described in Chapter 5. Put together, the σK/RskA complex revealed facets of σ/anti-σ factor interactions that were hitherto unrecognized. The most prominent amongst these is the finding that an ECF σfactor can respond to multiple environmental stimuli. Furthermore, as seen in the case of the σK/RskA complex, the σ factor can itself serve as a receptor for redox stimuli. Although speculative, a hypothesis that needs further study is whether these features of the σK/RskA complex contribute to the variable efficacy of the M. bovis BCG vaccine. In this context it is worth noting that σK governs the expression of the prominent secreted antigens- MPT70 and MPT83. The studies reported in this thesis thus suggest several avenues for future research to understand mycobacterial diversity, immunogenicity and features of host-pathogen interactions. The appendix section is divided into two subparts- Appendix 1 of the thesis is a review on peptidase V. This is a chapter in The Handbook of Proteolytic enzymes (Elsevier Press, ISBN:9780123822192). Appendix 2 of the thesis includes technical details and an extended materials and methods section.

Factor Quinolinone Inhibitor 1 (FQI1), discovered as a small molecule inhibitor of the transcription factor LSF, causes cell death in many cancer cell lines and inhibits tumor growth in tumor xenografts and an endogenous hepatocellular carcinoma model in mice. Significantly, multiple animal studies have shown minimal to no toxicity after FQI1 treatment, making it a promising potential lead chemotherapeutic for multiple cancer types. In determining how FQI1 causes cancer cell death, it was previously shown that FQI1 treatment, like knockdown of LSF expression by siRNA, produced a mitotic arrest with condensed but unaligned chromosomes, but with no clearly observable transcriptional dysregulation. In this thesis, I establish that introducing FQI1 to cells already in mitosis induces a mitotic arrest in colorectal cancer cells, demonstrating that FQI1 inhibits mitotic processes directly while these processes are occurring. This mitotic arrest is characterized by defects in the mitotic spindle and limited connections of mitotic spindles to the kinetochores, as indicated by a dramatic decrease cold-stable microtubules in mitosis. Additionally, in a dose-dependent manner, FQI1 treatment resulted in supernumerary γ-tubulin-containing mitotic centrosomes and γ-tubulin-deficient aster-like bodies, indicating a defect in centrosome stability. As FQI1 is known to be a specific inhibitor of LSF, with its dose dependence for LSF inhibition directly proportional to its ability to inhibit cell proliferation, these findings suggested the novel hypothesis that LSF regulates mitosis through non-transcriptional mechanisms by interacting with key mitotic proteins required for proper spindle formation and metaphase alignment. By mass spectrometry, multiple proteins were identified that interact with biotinylated LSF in mitosis in a FQI1-sensitive manner, with several related to the formation and stability of the mitotic spindle. Proximity ligation assays validated endogenous LSF interactions with CKAP5, a processive microtubule polymerase that protects kinetochore microtubules from depolymerization, and MISP, a requirement for proper mitotic spindle positioning. However, in this assay these interactions were not demonstrably FQI1-sensitive. In conclusion, FQI1 treatment results in defects in kinetochore-microtubule attachment and centrosome stability, triggering a mitotic arrest. Combined with the target specificity of FQI1, this suggests the hypothesis that LSF is required for proper mitotic spindle formation through its protein interactions in mitosis.
2022-10-16T00:00:00Z

University of Technology Sydney. Faculty of Science.
Tumour necrosis factor (TNF) is potent pro-inflammatory and anti-viral cytokine, acting via two cellular receptors, TNFR1 and TNFR2 that induces apoptosis and inflammation. Poxviruses encode homologues of TNF-receptors (viral TNFRs) that independently interact with both TNF, and simultaneously with cellular TNFRs, to subvert TNF-induced anti-viral apoptosis. The vTNFRs are expressed during poxvirus infection and are considered as bona fide virulence factors. The recently discovery of a “Pre-ligand Assembly Domain (PLAD)” within the N-terminus of the cellular TNFRs is shown to be required for receptor trimerisation and efficient cell death signalling. Whilst it has previously shown that the rabbit-trophic Myxoma (MYX) viral TNFR also contains a PLAD required for viral TNFR:cellular TNFR interactions, little is known about the human-trophic poxvirus TNFRs, nor physical characteristics of the interactions of vTNFRs and cellular TNFRs. To assess the importance of the PLAD domain in TNFR structure, function and viral subversion of TNFRs, this study focused on naturally occurring mutations in the TNFR PLAD domain, that occur in transient periodic fevers (TRAPS) – a clinical syndrome of febrile attacks of inflammation. TRAPS PLAD domain mutations were generated in a TNFR1-YFP in plasmids by site-directed mutagenesis and cloning. WT and TRAPS mutant TNFR1 constructs were transfected into U20S cells and TNFR1 location was determined by confocal microscopy. Neither WT TNFR1 nor TRAPS TNFRs were unable to be detected at the cell surface by both widefield and confocal microscopy despite published data on surface expression of WT TNFR1. WT TNFR1-YFP fusion proteins were found to be expressed within endocytic vesicles known as receptosomes and also as aggregates in a membranous structure resembling Golgi/ER. In addition it was found that TRAPS mutations in particular those affecting critical amino acids such as cysteines in disulphide bonds, display reduced TNFR-induced cell death as determined by flow cytometry. To better understand the biology of the vTNFR association with cellular TNFRs, and with WHO Smallpox committee approval, the human tropic poxviral TNFRs from Variola (Smallpox) (VAR) and Monkeypox (MPV) were synthesised and cloned as CFP/YFP and MycHis expression plasmids. Using multi-colour flow cytometry we have shown that, like the MYXT2 vTNFR, VARG4R and MPVJ2R TNFRs are potent intracellular inhibitors of TNFR1-induced cell death. As each vTNFR was able to inhibit TNFR-induced cell death, an assay was developed by flow cytometry to measure the intracellular abundance of the vTNFRs in the presence of cellular TNFR overexpression. MYXT2 was found to increase in intracellular abundance however for unknown reasons VARG4R and MPVJ2R did not convincingly increase in abundance. A structure for each of the vTNFRs was then attempted to be determined by X-ray crystallography, however bacterial expression of the both the cellular TNFRs and viral TNFRs proteins were unable to be obtained. Lastly to determine the structural orientations and conformations of cellular vTNFR interactions, a method of fluorescence resonance energy transfer (FRET) was established by flow cytometry. Using the generated C-terminal fusion -CFP and -YFP TNFRs, interactions were assessed between each of the cellular and vTNFRs. It was found that in addition to the reduced cell death TRAPS TNFRs when expressed with WT TNFR1, TRAPS mutations also cause reduced FRET possibly due to altered conformations in the receptor. Again mutations affecting more critical structural amino acids were found to have a more dramatic effect. Moreover differences were observed between mutations in distribution of FRET histograms further indicating altered network formations of higher order complexes. Next the FRET method was used to assess interactions between each of the vTNFRs with WT human TNFRs as well as with themselves and other vTNFRs. However no FRET was detectable between each of the molecules despite evidence of MYXT2 associating with human TNFR1 and TNFR2. Thus Comparative homology modelling and automated docking simulations were performed to explain possible orientations of the interactions tested in FRET. These data suggest that the interactions of vTNFRs with cellular TNFRs may possibly occur in a C-N anti-parallel orientation and not the previously predicted PLAD-PLAD interactions. Taken together, these data further our understanding of basic TNFR biology as well as for the first time characterise an entire panel of PLAD TRAPS mutations. It also furthers the characterisation of the very limited evidence of vTNFR subversion of TNFRs for the human trophic viral proteins VARG4R and MPVJ2R. Overall these results show the importance of PLAD interactions to TNFR biology and a possible new avenue in which TNFR signalling may be exploited in the development of new therapeutics.

Control of Rhipicephalus microplus is predominantly mediated by the application of acaricides, but the rapid acquisition of resistance by this species and environmental pollution resulting from discarded acaricides, necessitates the discovery of new control measures. Due to the fact that Rhipicephalus spp. are genetically diverse and often have more than one host, it has been difficult to identify a common protective vaccine candidate able to target all species of this genus. Only one anti-tick antigen, Bm86, has been commercialized to date and is sold as GAVAC® and GAVACPlus® in South America. In an attempt to identify protective antigens, a protein termed subolesin was identified using expression library immunisation. RNAi studies showed that subolesin knockdown causes the degeneration of tick guts, salivary glands, reproductive tissues and embryos. Subolesin additionally mediates tick gene expression, impacts the innate immune response and affects tick infection by Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. The R. microplus EF-1α homolog was identified as a subolesin-interacting protein via yeast two-hybrid and co-affinity purification experiments. RNAi experiments have suggested that EF-1α is another possible anti-tick vaccine candidate since it exhibits a similar phenotype as subolesin upon knockdown. The aim of the present research was to express R. microplus EF-1α in the yeast, Pichia pastoris and to exploit the yeast two-hybrid system in an attempt to identify its protein-binding partners. This will provide insight into understanding the translational machinery of this species and of ixodid ticks. Recombinant EF-1α was expressed as a 24 kDa protein, validated by western blotting. A highly representative cDNA library was produced from R. microplus mixed lifestages mRNA, fractionated and cloned into a two-hybrid prey vector. No definitive hits were obtained during the two-hybrid screen of reporter genes, as E-values attained after tblastx and PSI-BLAST analysis were higher than the required limit of 1 x 10-4.
Dissertation (MSc)--University of Pretoria, 2013.
Biochemistry
unrestricted

Economy and mankind are inextricably interlinked. Just as the economy or the production of material wealth is unimaginable without a man, so human existence and development are impossible without the wealth created in the economy. Shortly, both the goal and the means of achieving and realization of the economy are still the human resources. People have long ago noticed that it was the economy that created livelihoods, and the delays in their production led to the catastrophic events such as hunger, poverty, civil wars, social upheavals, revolutions, moral degeneration, and more. Therefore, the special interest of people in understanding the regulatory framework of the functioning of the economy has existed and exists in all historical epochs [A. Sisvadze. Economic theory. Part One. 2006y. p. 22]. The system of economic disciplines studies economy or economic activities of a society. All of them are based on science, which is currently called economic theory in the post-socialist space (the science of economics, the principles of economics or modern economics), and in most countries of the world - predominantly in the Greek-Latin manner - economics. The title of the present book is also Modern Economics. Economics (economic theory) is the science that studies the efficient use of limited resources to produce and distribute goods and services in order to satisfy as much as possible the unlimited needs and demands of the society. More simply, economics is the science of choice and how society manages its limited resources. Moreover, it should be emphasized that economics (economic theory) studies only the distribution, exchange and consumption of the economic wealth (food, beverages, clothing, housing, machine tools, computers, services, etc.), the production of which is possible and limited. And the wealth that exists indefinitely: no economic relations are formed in the production and distribution of solar energy, air, and the like. This current book is the second complete updated edition of the challenges of the modern global economy in the context of the coronary crisis, taking into account some of the priority directions of the country's development. Its purpose is to help students and interested readers gain a thorough knowledge of economics and show them how this knowledge can be applied pragmatically (professionally) in professional activities or in everyday life. To achieve this goal, this textbook, which consists of two parts and tests, discusses in simple and clear language issues such as: the essence of economics as a science, reasons for origin, purpose, tasks, usefulness and functions; Basic principles, problems and peculiarities of economics in different economic systems; Needs and demand, the essence of economic resources, types and limitations; Interaction, mobility, interchangeability and efficient use of economic resources. The essence and types of wealth; The essence, types and models of the economic system; The interaction of households and firms in the market of resources and products; Market mechanism and its elements - demand, supply and price; Demand and supply elasticity; Production costs and the ways to reduce them; Forms of the market - perfect and incomplete competition markets and their peculiarities; Markets for Production Factors and factor incomes; The essence of macroeconomics, causes and importance of origin; The essence and calculation of key macroeconomic indicators (gross national product, gross domestic product, net national product, national income, etc.); Macroeconomic stability and instability, unemployment, inflation and anti-inflationary policies; State regulation of the economy and economic policy; Monetary and fiscal policy; Income and standard of living; Economic Growth; The Corona Pandemic as a Defect and Effect of Globalization; National Economic Problems and New Opportunities for Development in the conditions of the Coronary Crisis; The Socio-economic problems of moral obsolescence in digital technologies; Education and creativity are the main solution way to overcome the economic crisis caused by the coronavirus; Positive and negative effects of tourism in Georgia; Formation of the middle class as a contributing factor to the development of tourism in Georgia; Corporate culture in Georgian travel companies, etc. The axiomatic truth is that economics is the union of people in constant interaction. Given that the behavior of the economy reflects the behavior of the people who make up the economy, after clarifying the essence of the economy, we move on to the analysis of the four principles of individual decision-making. Furtermore, the book describes how people make independent decisions. The key to making an individual decision is that people have to choose from alternative options, that the value of any action is measured by the value of what must be given or what must be given up to get something, that the rational, smart people make decisions based on the comparison of the marginal costs and marginal returns (benefits), and that people behave accordingly to stimuli. Afterwards, the need for human interaction is then analyzed and substantiated. If a person is isolated, he will have to take care of his own food, clothes, shoes, his own house and so on. In the case of such a closed economy and universalization of labor, firstly, its productivity will be low and, secondly, it will be able to consume only what it produces. It is clear that human productivity will be higher and more profitable as a result of labor specialization and the opportunity to trade with others. Indeed, trade allows each person to specialize, to engage in the activities that are most successful, be it agriculture, sewing or construction, and to buy more diverse goods and services from others at a relatively lower price. The key to such human interactions is that trade is mutually beneficial; That markets are usually the good means of coordination between people and that the government can improve the results of market functioning if the market reveals weakness or the results of market functioning are not fair. Moroever, it also shows how the economy works as a whole. In particular, it is argued that productivity is a key determinant of living standards, that an increase in the money supply is a major source of inflation, and that one of the main impediments to avoiding inflation is the existence of an alternative between inflation and unemployment in the short term, that the inflation decrease causes the temporary decline in unemployement and vice versa. The Understanding creatively of all above mentioned issues, we think, will help the reader to develop market economy-appropriate thinking and rational economic-commercial-financial behaviors, to be more competitive in the domestic and international labor markets, and thus to ensure both their own prosperity and the functioning of the country's economy. How he/she copes with the tasks, it is up to the individual reader to decide. At the same time, we will receive all the smart useful advices with a sense of gratitude and will take it into account in the further work. We also would like to thank the editor and reviewers of the books. Finally, there are many things changing, so it is very important to realize that the XXI century has come: 1. The century of the new economy; 2. Age of Knowledge; 3. Age of Information and economic activities are changing in term of innovations. 1. Why is the 21st century the century of the new economy? Because for this period the economic resources, especially non-productive, non-recoverable ones (oil, natural gas, coal, etc.) are becoming increasingly limited. According to the World Energy Council, there are currently 43 years of gas and oil reserves left in the world (see “New Commersant 2007 # 2, p. 16). Under such conditions, sustainable growth of real gross domestic product (GDP) and maximum satisfaction of uncertain needs should be achieved not through the use of more land, labor and capital (extensification), but through more efficient use of available resources (intensification) or innovative economy. And economics, as it was said, is the science of finding the ways about the more effective usage of the limited resources. At the same time, with the sustainable growth and development of the economy, the present needs must be met in a way that does not deprive future generations of the opportunity to meet their needs; 2. Why is the 21st century the age of knowledge? Because in a modern economy, it is not land (natural resources), labor and capital that is crucial, but knowledge. Modern production, its factors and products are not time-consuming and capital-intensive, but science-intensive, knowledge-intensive. The good example of this is a Japanese enterprise (firm) where the production process is going on but people are almost invisible, also, the result of such production (Japanese product) is a miniature or a sample of how to get the maximum result at the lowest cost; 3. Why is the 21st century the age of information? Because the efficient functioning of the modern economy, the effective organization of the material and personal factors of production largely depend on the right governance decision. The right governance decision requires prompt and accurate information. Gone are the days when the main means of transport was a sailing ship, the main form of data processing was pencil and paper, and the main means of transmitting information was sending letters through a postman on horseback. By the modern transport infrastructure (highways, railways, ships, regular domestic and international flights, oil and gas pipelines, etc.), the movement of goods, services and labor resoucres has been significantly accelerated, while through the modern means of communication (mobile phone, internet, other) the information is spreading rapidly globally, which seems to have "shrunk" the world and made it a single large country. The Authors of the book: Ushangi Samadashvili, Doctor of Economic Sciences, Associate Professor of Ivane Javakhishvili Tbilisi State University - Introduction, Chapters - 1, 2, 3, 4, 5, 6, 9, 10, 11,12, 15,16, 17.1,18 , Tests, Revaz Shengelia, Doctor of Economics, Professor of Georgian Technical University, Chapters_7, 8, 13. 14, 17.2, 17.4; Zhuzhuna Tsiklauri - Doctor of Economics, Professor of Georgian Technical University - Chapters 13.6, 13.7,17.2, 17.3, 18. We also thank the editor and reviewers of the book.

Diabetes mellitus (DM) is considered a risk factor for the development of coronary artery disease (CAD). Metformin, an anti-diabetic drug, has been shown to lower the cardiovascular events in pre-clinical and clinical studies. Many research articles suggests that metformin has a protective effect on CAD beyond its hypoglycemic effects. Patients with diabetes type 2 have an increased risk for cardiovascular disease and commonly use combination therapy consisting of the anti-diabetic drug metformin and a cholesterol-lowering statin. Statins have been found to be a safe and effective approach to reduce serum low density lipoprotein cholesterol (LDL-C) levels, which is the cornerstone for primary and secondary prevention of atherosclerosis. However, regular statin monotherapy in some patients may not be sufficient to achieve a therapeutic LDL-C. It has been reported that statins increased the incidence of new-onset diabetes in a dose dependent manner especially in women, the elderly, or in the presence of a family history of type 2 diabetes (T2D) and Asian ethnicity. The molecular mechanisms contributed to antioxidation, anti-inflammation, and anti-apoptosis. In this chapter, we aimed to investigate whether the combined administration of metformin and atorvastatin could achieve superior protective effects on different disease treatment purpose and to elucidate its molecular mechanisms of the combinations.

The recovery of skin wounds is a complex biological process involving three basic mechanisms: inflammatory phase, re-epithelialization followed by granulation and tissue remodeling. The interactions between inflammatory cells, fibroblasts, and keratinocytes induce microenvironmental changes at the wound site. Tissue remodeling is initiated by matrix-producing proteins and protease enzymes and collagen fibers in the dermis. A saponin extracted from ginseng, known as ginsenoside, has been shown to accelerate neovascularization in burn wounds in mice. It also increases levels of vascular endothelial growth factor and interleukin (IL-β). IL-β accelerate wound healing by promoting accumulation of macrophages at skin wound sites. Saponins are major active constituents of ginseng. They contain many ginsenosides. The purified ginsenosides or the extracts of ginseng root have been reported to have beneficial effects on damaged skin. For instance, red ginseng root extract protected skin from acute UVB-irradiation. Ginsenoside F1, an enzymatically modified derivative of the ginsenoside Rg1, protected HaCaT against UVB-induced apoptosis. Panax ginseng root extract promotes type I collagen synthesis in human dermal fibroblasts (HDF) via the Smad activation pathway and exhibits antioxidant activity against free radicles including diphenyl-p-picrylhydrazyl treatment. In addition, ginsenoside Rb1 promotes healing process of burn wound by enhancing angiogenesis. Among the various ginsenosides, ginsenoside Rb1 has been found to most potent agent for wound healing.

Abstract This study experimentally investigates the statistics of wall-pressure fluctuations and their source inside attached cavitation under different cavity regimes. Experiments were conducted in the divergent section of a convergent-divergent channel at a constant Reynolds number of Re = 7.8 × 105 based on throat height, and different cavitation numbers σ = 1.18, 0.92, 0.82 and 0.78. Four high-frequency unsteady pressure transducers were flushed-mounted in the divergent section downstream the throat where cavitation develops to sample the unsteady pressure signals induced by cavity behaviors. Flow visualization and wall-pressure measurement in high frequency on the order of MHz were employed using a synchronizing sampling technique. Results are presented for sheet/cloud cavitating flows. Specifically, sheet cavitation with both inception shear layer and fully cavitated shear layer and cloud cavitation under re-entrant jet dominated shedding and shock wave dominated shedding are studied. Compared with re-entrant jet, the interactions between shock wave and cavity could induce pressure peaks with high magnitude within cavity, which will collapse the local vapor along its propagating path and reduce local void fraction. Furthermore, statistics analysis shows that within the cavity, wall-pressure fluctuations increase with the distance to cavity leading edge increase in the first half of cavity length, and the moments of the probability density distribution skewness and kurtosis factor decrease, indicating the asymmetry and intermittency of wall-pressure fluctuation signals decrease. In shock wave dominated cavity shedding condition, the skewness and kurtosis factor increase. These results can provide data to improve the accuracy of turbulence modeling in numerical simulation of turbulent cavitating flow.

In order to develop specific immunologic reagents for mapping functionally important sites on FVIII, we have prepared rabbit polyclonal antibodies against synthetic peptides of FVIII derived from regions along the entire FVIII amino acid sequence. To date, a total of 70 peptides have been synthesized and characterized by amino acid and HPLC analysis. The peptides were coupled to keyhole limpet hemocyanin with glutaraldehyde as a linkage reagent and used to immunize rabbits. Antisera were tested by ELISA assay on polystyrene microtiter plates coated with either the peptide immunogen, or purified FVIII. The antisera were also tested for their ability to inhibit FVIII clotting activity and to react with separated FVIII polypeptides on immunoblots.Of the 70 peptides, all reacted with the peptide immunogen, 45 reacted with purified FVIII and 33 reacted with FVIII on immunoblots. Because we had obtained evidence that cleavage of the amino terminal region of the 80 kDa polypeptide may play a role in FVIII activation by thrombin, a series of partially overlapping peptides, 15 residues in length, were synthesized in this area. After affinity purifying these antibodies on columns of FVIII immobilized on agarose, adjusting the antibodies to equal antigen binding titers by dot immunoblotting and testing for inhibition of FVIII activity, only one antibody could strongly inhibit FVIII clotting activity. This inhibition could be blocked by the peptide itself at nanomolar concentrations and no significant inhibition could be shown by antibodies to partially overlapping peptides individually, or in combination. These data suggest that a site important to FVIII function can be localized to a 15 amino acid residue region of the 80 kDa polypeptide of FVIII. In addition, a second inhibitoryantibody was identified which was produced against a peptide in the carboxy terminal region of the 54 kDa thrombin fragment of FVIII and this area is currently being studied in a similar manner. In addition, two monoclonal anti-FVIII synthetic peptide antibodies have been produced which react with purified FVIII on immunoblots. One of these antibodies also functions as an immunoadsorbent when linked to agarose and FVII can be purified in this manner, using the synthetic peptide as eluant. It is evident that antibodies to synthetic peptides of FVIII can be useful probes of FVIII structure, function and interactions as well as being of use in FVIII purification.

Paroxysmal nocturnal hemoglobinuria (PNH)-is frequently complicated by thrombosis. It has been suggested that the abnormal interactions of PNH platelets with complement contribute to thrombosis. Using purified complement proteins, we have previously demonstrated that the platelets from some patients with PNH do not demonstrate elevated activity of C3bBb, the alternative pathway C3 amplification enzyme complex, even though they lack the C3bBb regulatory protein, decay accelerating factor (DAF). As measured by fluorescence flow cytometry, washed platelets from both normal donors and PNH patients released the fluid phase C3bBb regulatory protein, factor H, in response to the deposition of purified complement proteins. Platelet factor H was localized to the alpha granules by immunocytochemical techniques. A quantitative radioimmunoassay demonstrated that normal platelets released 54 ± 6 ng factor H/108 platelets in response to thrombin stimulation. PNH platelets contained less factor H (22 ±7 ng/108 platelets) than normal platelets. Thrombin stimulated platelets from patients with elevated C3bBb activity released less than half of the factor H measured in detergent extracts. However, thrombin stimulated platelets from PNH patients exhibiting normal C3bBb activity released nearly all their factor H. The release of factor H from normal platelets was blocked by treating the platelets with metabolic inhibitors. In the absence of factor H release, the activity of the C3bBb complex increased three-fold. In addition, the number of molecules of 1251-factor B bound per C3b increased from 0.40 to 0.92 when factor H release was blocked. The inhibition of DAF by anti-DAF had no effect on the activity of C3bBb if factor H could be released from the platelets. However, when factor H release was blocked by treatment with metabolic inhibitors, the inhibition of DAF by anti-DAF increased the activity of C3bBb by 40%. Therefore, in the absence of DAF, platelets can regulate complement activation by the alternative pathway via the release of platelet factor H. Since factor H is an alpha granule protein, platelet release in the presence of activated complement may contribute to the occurrence of thrombosis.

Platelet (plt) factor 4 (PF4) is an alpha granule protein which can modulate T lymphocyte function. T cells may help regulate megakaryocytopoiesis. Therefore, we hypothesized that T cell-PF4 interactions might play a role in autoregulating marrow megakaryocyte (MEG) production. To test this idea, we studied MEG colony formation in plasma clot cultures containing human serum derived solely from pit poor normal AB plasma, enriched hematopoietic progenitor cells (HPC), autologous T cells, and exogenous PF4. Highly purified PF4 (single band on SDS gel) was prepared from outdated human pits by a combination of heparin-agarose, Sephacryl G-200, and Sephadex G-50 column chromatography. HPC were prepared by depleting normal light density marrow mononuclear cells of adherent monocytes, and T cells. T cells were further fractionated into helper (Leu 3+) and suppressor (Leu 2+) subtypes by solid phase immunoabsorption ("panning"). MEG colonies were enumerated by indirect immunofluorescence with an anti-human platelet glycoprotein antiserum. HPC(5×105/ml) were co-cultured with Leu 3+, or Leu 2+ T cells at target;T cell ratios of 2:1 (n=3; n=4 respectively) and l:l(n=4; n=4 respectively) in the presence of 2.5 μg/ml PF4. Under these growth conditions, MEG colony formation was unchanged (p>0.5) when compared to colonies formed by HPC in the absence of PF4. When the above experiments were repeated (n=2-3/condition) at a higher PF4 concentration [25 μg/ml], MEG colony formation was markedly (>60%) inhibited. To determine if PF4 directly inhibited MEG or erythroid progenitor cell growth (CFU-Meg; CFU-E) in vitro, HPC were cloned in PF4 (25μg/ml) without added T cells. Mean ± SEM of MEG and CFU-E derived colonies formed without vs. with PF4 was as follows:These results suggest that: 1) PF4 may be a non-T cell dependent, lineage specific inhibitor of CFU-MEG, and 2) PF4 may play a role in autoregulating human megakaryocytopoiesis.

The present paper aims at determining the effect of several design and manufacturing parameters on the frictional response of the underhead contact in a bolted joint comprising a socket-head M6 class 12.9 screw. The rationale of the research moves from the need to understand the frictional behavior of screw joints used in machine tools as a means to join roller monoguides with the moving carriages of three-axis tooling units. The experimentation takes into account different underhead materials (Steel, Cast Iron, Aluminum), different roughness levels (Ra = 0.8, 1.6, 3.2), different surface treatments (No treatment, oxidised, DLC), as well as repeated tightening operations. The experimentation has been run by a specifically developed specimen, consisting of two parts: an instrumented sleeve, equipped with a double array of strain gauges capable of sampling both the axial preload and the underhead torque, and interchangeable underhead washers, with an anti-rotation device, whose material and surface treatment are suitably changed among the different sets. The tightening torque is recorded by a digital torque wrench. The collected data are processed by ANoVa tools, to investigate the significance of each factor, as well as related interactions. The friction coefficients associated with the different operating parameters are a useful tool to support the design task of bolted joints.

Transient growth of energy is known to occur even in stable dynamical systems due to the non-normality of the underlying linear operator. This has been the object of growing attention in the field of hydrodynamic stability, where linearly stable flows may be found to be strongly nonlinearly unstable as a consequence of transient growth. We apply these concepts to the generic case of coupled-mode flutter, which is a mechanism with important applications in the field of fluid-structure interactions. Using numerical and analytical approaches on a simple system with two degrees of freedom and anti-symmetric coupling we show that the energy of such a system may grow by a factor of more than 10, before the threshold of coupled-mode flutter is crossed. This growth is a simple consequence of the non-orthogonality of modes arising from the non-conservative forces. These general results are then applied to three cases in the field of flow-induced vibrations: (a) panel flutter (two-degrees-of-freedom model, as used by Dowell (1995)), (b) follower force (two-degrees-of-freedom model as used by Bamberger (1981)), and (c) fluid-conveying pipes (two-degree-of-freedom model, as used by Benjamin (1961) and Paidoussis (1998)), for different mass ratios. For these three cases we show that the magnitude of transient growth of mechanical energy before the onset of coupled-mode flutter is substantial enough to cause a significant discrepancy between the apparent threshold of instability and the one predicted by linear stability theory.

Thrombospondin (TSP), a multifunctional 450 KD glycoprotein is a secretory product of thrombin stimulated platelets. It is a major component of the platelets alpha granule constituting approximately 3% of total platelet protein. Thrombospondin does not circulate in appreciable concentrations ∽0 100 ng/ml); however, the tissue distribution is broad. In addition to its expression on the membrane of activated platelets, the protein is synthesized by fibroblasts endothelial cells, glial cell smooth muscle cells alveolar pneumocytes mononuclear phagocytes and various tumor cells. TSP is a major constituent of the extracellular matrix and has been demonstrated in the vessel wall, basement membrane and glandular connective tissue. Fibroblasts, smooth muscle cells and endothelial cells in tissue culture incorporate TSP into the extracellular matrix. Matrix TSP is under cell-cycle regulatory control. Mesenchymal cells in the proliferative phase synthesize greater amounts of TSP than non growing cells. Platelet derived growth factor induces smooth muscle cell and glial cell synthesis of TSP. Atheromatous lesions contain increased amounts of TSP compared to normal vessels emphasizing the potential role of TSP in the interaction of proliferating cells with the matrix. TSP binds specifically, saturably, and reversibly to mouse peritoneal macrophages and to cells of the monocyte-like human cell line U937. Binding was time dependent and was optimal in the presence of both Ca++ and Mg++. PMA stimulated U937 cells and activated macrophages bound TSP to an equivalent extent as resting cells. The TSP binding site on the surface of U937 cells and peripheral blood monocytes mediates the adhesive interaction between these cells and thrombin-stimulated platelets. Using a sensitive rosetting assay we found that monocytes were not rosetted by resting platelets while >90% were rosetted by thrombin-stimulated platelets. Monoclonal and polyclonal anti-TSP antibodies markedly inhibited rosetting as did TSP itself. Antifibronectin or non-immune control antibodies did not inhibit rosetting, nor did fibronectin, fibrinogen, the fibronectinadhesion tetrapeptide arg-gly-asp-ser (RGDS), or heparin. The TSP membrane receptor, an 88 KD glycoprotein, formely known as GPIV has been identified in platelets, endothelial cells, monocytes and a variety of tumor cells. TSP may thus serve as a molecular bridge linking activated platelets with monocytes at sites of early vascular injury. Such interactions involving the TSP receptor complex may be of critical importance in the regulation of thrombosis and the initiation of atherosclerosis.

Interaction between cells and between cells and extracellular matrices are critical for a number of biological processes, including organ development, cell differenciation, cell motility, and the inimune' response. These interactions are mediated by a family of adhesion receptors that recognize short sequences such as Arg-Gly-Asp (RGD). These receptors share similar structural properties. They are heterodimers composed of a and B subunits and sometime express common epitopes. This suggests that the structural and functional relationship of these receptors may result from the transcription of related genes or may arise from cell specific post-transcriptional events. Thus, analysis of the biosynthesis and processing of these receptors would provide valuable insights into the molecular mechanism which control their expression at the surface,of different cells. Platelet membrane glycoprotein (GP) IIb-IIIa is a member of this receptor family. This protein is a non covalent heterodimer composed of two distinct polypeptides, Glib which consists of two subunits Ilba and IlbB (Mr = 116 kD, Mr = 25 kD) and GPIIIa (Mr = 100 kD, reduced). GPIIb-IIIa functions at site of platelet aggregation and serves as receptor for RGD containing factors including fibrinogen, fibronectin and von Willebrand factor. We report here on the investigation of the biosynthetic pathways of this RGD receptor in human megakaryocytes. High number of megakaryocytic cells from the megakaryoblastic stage to the polyploid mature megakaryocyte were obtained from liquid culture of cryopreserved leukocyte stem cell concentrates from patients with chronic myelogenous leukemia (CML). After sorting, using a FACS IV and indirect immunofluores-cent labeling with monoclonal antibodies anti-GPIIb-IIIa, 95 % of the cells in culture were of the megakaryocytic lineage. These megakarocytes represented an excellent tool to delineate at the molecular level events associated with the biosynthesis of GPIIb-IIIa.Metabolic labeling and pulse-chase experiments indicated that GPIIb and GPIIIa are synthesized from separate mRNA and that the two subunits of GPIIb derive from a common precursor. This was further confirmed by cell-free translation of megakaryocyte mRNA and the identification of separate cDNA containing sequences coding for the pro-GPIIb and for GPIIIa. These cDNA were isolated from a Xgt11 expression library constructed with purified megakaryocyte RNA, and were used to size the messengers coding for the two polypeptides. A single mRNA species of 3.9 kB was found to encode the pro-GPIIb, whereas two different mRNA species of 2.9 kB and 4. 1 kB were identified with the GPIIIa cDNA.The newly synthesized GPIIIa associates early with the pro-GPIIb in the rough endoplasmic reticulum. Examination of the glycosylation pathways with endoglycosidase H, tunicamycin and monensin indicated that high mannose oligosaccharides are added to the GPIIIa and pro-GPIIb polypeptide backbone. The pro-GPIIb is then processed with conversion of high mannose to the complex type carbohydrate, whereas GPIIIa remains endoH sensitive. Glycosylation of pro-GPIIb-IIIa and processing of oligosaccharides are prerequisite for proteolytic maturation of pro-GPIIb and the expression of the mature complex at the surface of the cell. Thus post-translational processing of GPIIb-IIIa requires an early assembly of the complex. This may have important implications in the maturation of megakaryocyte granules and in the molecular mechanism underlying the Glanzmann thrombastenic disease.

The objectives of the BARD proposal were to determine the mechanisms of nonspecific cytotoxic cells (NCC) that are necessary to provide heightened innate resistance to infection and to identify the antigenic determinants in Streptococcus iniae that are best suited for vaccine development. Our central hypothesis was that anti-bacterial immunity in trout and tilapia can only be acquired by combining "innate" NCC responses with antibody responses to polysaccharide antigens. These Objectives were accomplished by experiments delineated by the following Specific Aims: Specific aim (SA) #1 (USA) "Clone and Identify the Apoptosis Regulatory Genes in NCC"; Specific aim #2 (USA)"Identify Regulatory Factors that Control NCC Responses to S. iniae"; Specific aim #3 (Israel) "Characterize the Biological Properties of the S. iniae Capsular Polysaccharide"; and Specific aim #4 (Israel) "Development of an Acellular Vaccine". Our model of S. iniae pathogenesis encompassed two approaches, identify apoptosis regulatory genes and proteins in tilapia that affected NCC activities (USA group) and determine the participation of S.iniae capsular polysaccharides as potential immunogens for the development of an acellular vaccine (Israel group). We previously established that it was possible to immunize tilapia and trout against experimental S. difficile/iniaeinfections. However these studies indicated that antibody responses in protected fish were short lived (3-4 months). Thus available vaccines were useful for short-term protection only. To address the issues of regulation of pathogenesis and immunogens of S. iniae, we have emphasized the role of the innate immune response regarding activation of NCC and mechanisms of invasiveness. Considerable progress was made toward accomplishing SA #1. We have cloned the cDNA of the following tilapia genes: cellular apoptosis susceptibility (CAS/AF547173»; tumor necrosis factor alpha (TNF / A Y 428948); and nascent polypeptide-associated complex alpha polypeptide (NACA/ A Y168640). Similar attempts were made to sequence the tilapia FasLgene/cDNA, however these experiments were not successful. Aim #2 was to "Identify Regulatory Factors that Control NCC Responses to S. iniae." To accomplish this, a new membrane receptor has been identified that may control innate responses (including apoptosis) of NCC to S. iniae. The receptor is a membrane protein on teleost NCC. This protein (NCC cationic antimicrobial protein-1/ncamp-1/AAQ99138) has been sequenced and the cDNA cloned (A Y324398). In recombinant form, ncamp-l kills S. iniae in vitro. Specific aim 3 ("Characterize the Biological Properties of the S.iniae Capsular Polysaccharide") utilized an in- vitro model using rainbow trout primary skin epithelial cell mono layers. These experiments demonstrated colonization into epithelial cells followed by a rapid decline of viable intracellular bacteria and translocation out of the cell. This pathogenesis model suggested that the bacterium escapes the endosome and translocates through the rainbow trout skin barrier to further invade and infect the host. Specific aim #4 ("Development of an Acellular Vaccine") was not specifically addressed. These studies demonstrated that several different apoptotic regulatory genes/proteins are expressed by tilapia NCC. These are the first studies demonstrating that such factors exist in tilapia. Because tilapia NCC bind to and are activated by S. iniae bacterial DNA, we predict that the apoptotic regulatory activity of S. iniae previously demonstrated by our group may be associated with innate antibacterial responses in tilapia.

Tomato yellow leaf curl geminivirus (TYLCV) is a major pathogen of cultivated tomato, causing up to 100% crop loss in many parts of the world. In Israel, where TYLCV epidemics have been recorded since the 1960' s, this viral disease is well known and has been of economic significance ever since. In recent years, TYLCV outbreaks also occurred in the "New World" - Cuba, The Dominican Republic, and in the USA, in Florida, Georgia and Louisiana. Thus, TYLCV substantially hinders tomato growth throughout the world. Surprisingly, however, little is known about the molecular mechanisms of TYLCV interaction with the host tomato cells. The present proposal, a continuation of the project supported by BARD from 1994, expanded our understanding of the molecular mechanisms by which TYLCV enters the host cell nucleus for replication and transcription and exits it for the subsequent cell-to-cell spread. Our project sought two objectives: I. To study the roles of the viral capsid protein (CP) and host cell factors in TYLCV nuclear import. II. To study the roles of CP and host cell factors in TYLCV nuclear export. Our research toward these goals have produced the following major achievements: . Developed a one-hybrid assay for protein nuclear export and import (#3 in the List of Publications). . Identified a functional nuclear export signal (NES) in the capsid protein (CP) of TYLCV (#3 in the List of Publications). . Discovered homotypic interactions between intact TYLCV CP molecules and analyzed these interactions using deletion mutagenesis of TYLCV CP (#5 in the List of Publications). . Showed developmental and tissue-specific expression of the host factor required for nuclear import of TYLCV CP, tomato karyopherin alpha 1, in transgenic tomato plants (#14 in the List of Publications). . By analogy to nuclear import of TYLCV ,identified an Arabidopsis VIPI protein that participates in nuclear import of Agrobacterium T -complexes via the karyopherin alpha pathway (#4,6, and 8 in the List of Publications). These research findings provided significant insights into (i) the molecular pathway of TYLCV entry into the host cell nucleus, and (ii) the mechanism by which TYLCV is exported from the nucleus for the cell-to-cell spread of infection. Furthermore, the obtained knowledge will help to develop specific strategies to attenuate TYLCV infection, for example, by blocking viral entry into and/or exit out of the host cell nucleus. Also, as much of our findings is relevant to all geminiviruses, new anti- TYLCV approaches developed based on the results of our research will be useful to combat other members of the Geminivirus family. Finally, in addition to the study of TYLCV nuclear import and export, our research contributed to our understanding of general mechanisms for nucleocytoplasmic shuttling of proteins and nucleic acids in plant cells.

The general objective of this study was to develop rationally designed mimetic antagonists (and agonists) of the PK/PBAN Np class with enhanced bio-stability and bioavailability as prototypes for effective and environmentally friendly pest insect management agents. The PK/PBAN family is a multifunctional group of Nps that mediates key functions in insects (sex pheromone biosynthesis, cuticular melanization, myotropic activity, diapause and pupal development) and is, therefore, of high scientific and applied interest. The objectives of the current study were: (i) to identify an antagonist biophores (ii) to develop an arsenal of amphiphilic topically active PK/PBAN antagonists with an array of different time-release profiles based on the previously developed prototype analog; (iii) to develop rationally designed non-peptide SMLs based on the antagonist biophore determined in (i) and evaluate them in cloned receptor microplate binding assays and by pheromonotropic, melanotropic and pupariation in vivo assays. (iv) to clone PK/PBAN receptors (PK/PBAN-Rs) for further understanding of receptor-ligand interactions; (v) to develop microplate binding assays for screening the above SMLs. In the course of the granting period A series of amphiphilic PK/PBAN analogs based on a linear lead antagonist from the previous BARD grant was synthesized that incorporated a diverse array of hydrophobic groups (HR-Suc-A[dF]PRLa). Others were synthesized via the attachment of polyethylene glycol (PEG) polymers. A hydrophobic, biostablePK/PBAN/DH analog DH-2Abf-K prevented the onset of the protective state of diapause in H. zea pupae [EC50=7 pmol/larva] following injection into the preceding larval stage. It effectively induces the crop pest to commit a form of ‘ecological suicide’. Evaluation of a set of amphiphilic PK analogs with a diverse array of hydrophobic groups of the formula HR-Suc-FTPRLa led to the identification of analog T-63 (HR=Decyl) that increased the extent of diapause termination by a factor of 70% when applied topically to newly emerged pupae. Another biostablePK analog PK-Oic-1 featured anti-feedant and aphicidal properties that matched the potency of some commercial aphicides. Native PK showed no significant activity. The aphicidal effects were blocked by a new PEGylated PK antagonist analog PK-dF-PEG4, suggesting that the activity is mediated by a PK/PBAN receptor and therefore indicative of a novel and selective mode-of-action. Using a novel transPro mimetic motif (dihydroimidazole; ‘Jones’) developed in previous BARD-sponsored work, the first antagonist for the diapause hormone (DH), DH-Jo, was developed and shown to block over 50% of H. zea pupal diapause termination activity of native DH. This novel antagonist development strategy may be applicable to other invertebrate and vertebrate hormones that feature a transPro in the active core. The research identifies a critical component of the antagonist biophore for this PK/PBAN receptor subtype, i.e. a trans-oriented Pro. Additional work led to the molecular cloning and functional characterization of the DH receptor from H. zea, allowing for the discovery of three other DH antagonist analogs: Drosophila ETH, a β-AA analog, and a dF analog. The receptor experiments identified an agonist (DH-2Abf-dA) with a maximal response greater than native DH. ‘Deconvolution’ of a rationally-designed nonpeptide heterocyclic combinatorial library with a cyclic bis-guanidino (BG) scaffold led to discovery of several members that elicited activity in a pupariation acceleration assay, and one that also showed activity in an H. zea diapause termination assay, eliciting a maximal response of 90%. Molecular cloning and functional characterization of a CAP2b antidiuretic receptor from the kissing bug (R. prolixus) as well as the first CAP2b and PK receptors from a tick was also achieved. Notably, the PK/PBAN-like receptor from the cattle fever tick is unique among known PK/PBAN and CAP2b receptors in that it can interact with both ligand types, providing further evidence for an evolutionary relationship between these two NP families. In the course of the granting period we also managed to clone the PK/PBAN-R of H. peltigera, to express it and the S. littoralis-R Sf-9 cells and to evaluate their interaction with a variety of PK/PBAN ligands. In addition, three functional microplate assays in a HTS format have been developed: a cell-membrane competitive ligand binding assay; a Ca flux assay and a whole cell cAMP ELISA. The Ca flux assay has been used for receptor characterization due to its extremely high sensitivity. Computer homology studies were carried out to predict both receptor’s SAR and based on this analysis 8 mutants have been generated. The bioavailability of small linear antagonistic peptides has been evaluated and was found to be highly effective as sex pheromone biosynthesis inhibitors. The activity of 11 new amphiphilic analogs has also been evaluated. Unfortunately, due to a problem with the Heliothis moth colony we were unable to select those with pheromonotropic antagonistic activity and further check their bioavailability. Six peptides exhibited some melanotropic antagonistic activity but due to the low inhibitory effect the peptides were not further tested for bioavailability in S. littoralis larvae. Despite the fact that no new antagonistic peptides were discovered in the course of this granting period the results contribute to a better understanding of the interaction of the PK/PBAN family of Nps with their receptors, provided several HT assays for screening of libraries of various origin for presence of PK/PBAN-Ragonists and antagonists and provided important practical information for the further design of new, peptide-based insecticide prototypes aimed at the disruption of key neuroendocrine physiological functions in pest insects.