Polymerization kinetics and polymerization stress in resin composites after accelerated aging as a function of the expiration date

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Analysing the possible influence of transposon TCl4.7 insertion on the function of the genome of Cydia pomonella granulovirus

CpGV-MCp5 is a natural mutant of the Cydia pomonella Granulovirus (Mexican isolate) (CpGV-M) that harbors an insect host transposon termed TCl4.7 in its genome. TCl4.7 is located between the open reading frames Cp15 and Cp16 and separates two homologous regions hr3 and hr4, which have been recently shown to be origins of replication of CpGV-M. The MCp5 has a significant replication disadvantage in the presence of the wild-type CpGV-M. In this study, the possible effects of TCl4.7 transposon insertion on the genome function of its insertion site has been analysed. The role of Cp15 and Cp16 in the context of the virus infection cycle was examined by generating a CpGV-Bacmid (CpBAC) and Cp15 knock-out (CpBACCp15KO) and Cp16 knock-out (CpBACCp16KO) mutants. The mutant CpBACCp15KO was not able to replicate in CM larvae suggesting that Cp15 was essential for virus replication. In contrast, the mutant CpBACCp16KO infected CM larvae and produced viable occlusion bodies (OBs) demonstrating that Cp16 is a non-essential gene for virus in vivo infection of C. pomonella. The temporal transcription of Cp15 and Cp16, as well as of Cp31 (F protein) as a control, was analysed using RT-PCR and quantitative real-time PCR. It suggested a general delay or reduction of gene transcription of MCp5 compared to the parental CpGV-M. Western blot analyses using anti-Cp15 and anti-Cp16 polyclonal antibodies, however, did not show any immuno-reactive response. Thus, a direct influence of TCl4.7 on the expression of Cp15 and Cp16 could not be substantiated. To investigate whether the interruption of hr3 and hr4 palindromes affects the virus replication, two mutant bacmids with a deletion of hr3 and hr4 (CpBAChr3/hr4-KO) and another with an insertion of a Kanamycin resistance cassette between hr3 and hr4 (CpBAChr3-kan-hr4) were generated. Both mutant bacmids replicated and produced infectious virus OBs, which did not significantly differ in their median lethal concentration (LC50) and median survival time (ST50) compared to the parental CpBAC. Interestingly, the mutant CpBAChr3-kan-hr4 was very effectively out-competed by parental CpBAC, when CM larvae were co-infected with known ratios of OBs of CpBAC and the mutant CpBAChr3-kan-hr4. These observations suggested a functional co-operation between hr3 and hr4 which was interrupted by the KanR insertion in CpBAChr3-kan-hr4 and possibly by TCl4.7 transposon insertion in the mutant MCp5. This hypothesis may explain the observed replication disadvantage of the mutants MCp5 and CpBAChr3-kan-hr4 in the presence of the parental viruses CpGV-M and CpBAC, respectively.

Function of liver activation-regulated chemokine/CC chemokine ligand 20 is differently affected by cathepsin B and cathepsin D processing

Chemokine processing by proteases is emerging as an important regulatory mechanism of leukocyte functions and possibly also of cancer progression. We screened a large panel of chemokines for degradation by cathepsins B and D, two proteases involved in tumor progression. Among the few substrates processed by both proteases, we focused on CCL20, the unique chemokine ligand of CCR6 that is expressed on immature dendritic cells and subtypes of memory lymphocytes. Analysis of the cleavage sites demonstrate that cathepsin B specifically cleaves off four C-terminally located amino acids and generates a CCL20(1-66) isoform with full functional activity. By contrast, cathepsin D totally inactivates the chemotactic potency of CCL20 by generating CCL20(1-55), CCL20(1-52), and a 12-aa C-terminal peptide CCL20(59-70). Proteolytic cleavage of CCL20 occurs also with chemokine bound to glycosaminoglycans. In addition, we characterized human melanoma cells as a novel CCL20 source and as cathepsin producers. CCL20 production was up-regulated by IL-1alpha and TNF-alpha in all cell lines tested, and in human metastatic melanoma cells. Whereas cathepsin D is secreted in the extracellular milieu, cathepsin B activity is confined to cytosol and cellular membranes. Our studies suggest that CCL20 processing in the extracellular environment of melanoma cells is exclusively mediated by cathepsin D. Thus, we propose a model where cathepsin D inactivates CCL20 and possibly prevents the establishment of an effective antitumoral immune response in melanomas.

A STUDY ON THE FEASIBILITY OF UNIVERSAL CHIP CONTROL IN MACHINING

A novel solution to the long standing issue of chip entanglement and breakage in metal cutting is presented in this dissertation. Through this work, an attempt is made to achieve universal chip control in machining by using chip guidance and subsequent breakage by backward bending (tensile loading of the chip's rough top surface) to effectively control long continuous chips into small segments. One big limitation of using chip breaker geometries in disposable carbide inserts is that the application range is limited to a narrow band depending on cutting conditions. Even within a recommended operating range, chip breakers do not function effectively as designed due to the inherent variations of the cutting process. Moreover, for a particular process, matching the chip breaker geometry with the right cutting conditions to achieve effective chip control is a very iterative process. The existence of a large variety of proprietary chip breaker designs further exacerbates the problem of easily implementing a robust and comprehensive chip control technique. To address the need for a robust and universal chip control technique, a new method is proposed in this work. By using a single tool top form geometry coupled with a tooling system for inducing chip breaking by backward bending, the proposed method achieves comprehensive chip control over a wide range of cutting conditions. A geometry based model is developed to predict a variable edge inclination angle that guides the chip flow to a predetermined target location. Chip kinematics for the new tool geometry is examined via photographic evidence from experimental cutting trials. Both qualitative and quantitative methods are used to characterize the chip kinematics. Results from the chip characterization studies indicate that the chip flow and final form show a remarkable consistency across multiple levels of workpiece and tool configurations as well as cutting conditions. A new tooling system is then designed to comprehensively break the chip by backward bending. Test results with the new tooling system prove that by utilizing the chip guidance and backward bending mechanism, long continuous chips can be more consistently broken into smaller segments that are generally deemed acceptable or good chips. It is found that the proposed tool can be applied effectively over a wider range of cutting conditions than present chip breakers thus taking possibly the first step towards achieving universal chip control in machining.

Generating Realistic Camera Shake for Virtual Scenes

When depicting both virtual and physical worlds, the viewer's impression of presence in these worlds is strongly linked to camera motion. Plausible and artist-controlled camera movement can substantially increase scene immersion. While physical camera motion exhibits subtle details of position, rotation, and acceleration, these details are often missing for virtual camera motion. In this work, we analyze camera movement using signal theory. Our system allows us to stylize a smooth user-defined virtual base camera motion by enriching it with plausible details. A key component of our system is a database of videos filmed by physical cameras. These videos are analyzed with a camera-motion estimation algorithm (structure-from-motion) and labeled manually with a specific style. By considering spectral properties of location, orientation and acceleration, our solution learns camera motion details. Consequently, an arbitrary virtual base motion, defined in any conventional animation package, can be automatically modified according to a user-selected style. In an animation package the camera motion base path is typically defined by the user via function curves. Another possibility is to obtain the camera path by using a mixed reality camera in motion capturing studio. As shown in our experiments, the resulting shots are still fully artist-controlled, but appear richer and more physically plausible.

Thermal 2-loop master spectral function at finite momentum

When considering NLO corrections to thermal particle production in the “relativistic” regime, in which the invariant mass squared of the produced particle is K2 ~ (πT)2, then the production rate can be expressed as a sum of a few universal “master” spectral functions. Taking the most complicated 2-loop master as an example, a general strategy for obtaining a convergent 2-dimensional integral representation is suggested. The analysis applies both to bosonic and fermionic statistics, and shows that for this master the non-relativistic approximation is only accurate for K2 ~(8πT)2, whereas the zero-momentum approximation works surprisingly well. Once the simpler masters have been similarly resolved, NLO results for quantities such as the right-handed neutrino production rate from a Standard Model plasma or the dilepton production rate from a QCD plasma can be assembled for K2 ~ (πT)2.

GENETIC ANALYSIS OF THE FUNCTION OF THE DROSOPHILA DOUBLESEX-RELATED FACTOR dmrt93B

DMRT (Doublesex and Mab-3 related transcription factor) proteins generally associated with sexual differentiation in many organisms share a common DNA binding domain and are often expressed in reproductive tissues. Aside from doublesex, which is a central factor in the regulation of sex determination, Drosophila possesses three different dmrt genes that are of unknown function. Because the association with sexual differentiation and reproduction is not universal and some DMRT proteins have been found to play other developmental roles we chose to further characterize one of these Drosophila genes. We carried out genetic analysis of dmrt93B, which was previously found to be expressed sex-specifically in the developing somatic gonad and to affect testis morphogenesis in RNAi knockdowns. In order to disrupt this gene, the GAL4 yeast transcriptional activator followed by a polyadenylation signal was inserted after the dmrt93B start codon and introduced into the genome by homologous recombination. Analysis of the knock-in mutation as well as a small deletion removing all dmrt93B sequence demonstrate that loss of function causes partial lethality at the late pupal stage. Surprisingly, these mutations have no significant effect on gonad formation or male fertility. Analysis of GAL4-driven GFP reporter expression indicates that the dmrt93B promoter activity is highly specific to neurons in the suboesophageal and proventricular ganglion in larva and adult of both sexes suggesting a possible role in digestive tract function. Using the Capillary Feeder (CAFÉ) assay to measure daily food intake we find that reduction in this gene’s function leads to an increase in food consumption. These results suggest dmrt93 plays an important role in the formation or maintenance of neurons that affect feeding and support the idea that dmrt genes may not be restricted to roles in sexual differentiation.

Early stages of experimental colon carcinogenesis inhibit mast cell-dependent mucosal immune function of the distal colon

Inhibition of local host immune reactions is one mechanism contributing to tumor progression. To determine if alterations in local immune functioning occur during colon carcinogenesis, a model mucosal immune response, type I hypersensitivity against the intestinal parasite Trichinella spiralis, was first characterized in normal mice and then examined during experimental colon carcinogenesis. Segments of sensitized colon mounted in Ussing chambers and challenged with T. spiralis-derived antigen resulted in a rise in short-circuit current ($\rm\Delta I\sb{sc}$) that was antigen-specific and inhibited by furosemide, implicating epithelial Cl$\sp-$ secretion as the ionic mechanism. The immune-regulated Cl$\sp-$ secretion by colonic epithelial cells required the presence of mast cells with surface IgE. Inhibition of potential anaphylactic mediators with various pharmacological agents in vitro implicated prostaglandins and leukotrienes as the principal mediators of the antigen-induced $\rm\Delta I\sb{sc}$, with 5-hydroxytryptamine also playing a role. Distal colon from immune mice fed an aspirin-containing diet (800 mg/kg powdered diet) ad libitum for 6 wk had a decreased response to antigen, confirming the major role of prostaglandins in generating the colonic I$\sb{\rm sc}$. To determine the effects of early stages of colon carcinogenesis on this mucosal immune response, mice were immunized with T. spiralis 1 day after or 8 wk prior to the first of 6 weekly injections of the procarcinogen 1,2-dimethylhydrazine (DMH). Responsiveness to antigenic challenge was suppressed in the distal colon 4-6 wk after the final injection of DMH. One injection of DMH was not sufficient to inhibit antigen responsiveness. The colonic epithelium remained sensitive to direct stimulation by exogenous Cl$\sp-$ secretagogues. Decreased antigen-induced $\rm\Delta I\sb{sc}$ in the distal colon was not due to systemic immune suppression by DMH, as the proximal colon and jejunum maintained responsiveness to antigen. Also, rejection of a secondary T. spiralis infection from the small intestine was not altered. Tumors eventually developed 25-30 wk after the final injection of DMH only in the distal portions of the colon. These results suggest that early stages of DMH-induced colon carcinogenesis manipulate the microenvironment such that mucosal immune function, as measured by immune-regulated Cl$\sp-$ secretion, is suppressed in the distal colon, but not in other regions of the gut. Future elucidation of the mechanisms by which this localized inhibition of immune-mediated ion transport occurs may provide possible clues to the microenvironmental changes necessary for tumor progression in the distal colon. ^

Bounds for the probability generating functional of a Gibbs point process

We derive explicit lower and upper bounds for the probability generating functional of a stationary locally stable Gibbs point process, which can be applied to summary statistics such as the F function. For pairwise interaction processes we obtain further estimates for the G and K functions, the intensity, and higher-order correlation functions. The proof of the main result is based on Stein's method for Poisson point process approximation.

U7 snRNP-specific Lsm11 protein: dual binding contacts with the 100 kDa zinc finger processing factor (ZFP100) and a ZFP100-independent function in histone RNA 3' end processing

The 3' cleavage generating non-polyadenylated animal histone mRNAs depends on the base pairing between U7 snRNA and a conserved histone pre-mRNA downstream element. This interaction is enhanced by a 100 kDa zinc finger protein (ZFP100) that forms a bridge between an RNA hairpin element upstream of the processing site and the U7 small nuclear ribonucleoprotein (snRNP). The N-terminus of Lsm11, a U7-specific Sm-like protein, was shown to be crucial for histone RNA processing and to bind ZFP100. By further analysing these two functions of Lsm11, we find that Lsm11 and ZFP100 can undergo two interactions, i.e. between the Lsm11 N-terminus and the zinc finger repeats of ZFP100, and between the N-terminus of ZFP100 and the Sm domain of Lsm11, respectively. Both interactions are not specific for the two proteins in vitro, but the second interaction is sufficient for a specific recognition of the U7 snRNP by ZFP100 in cell extracts. Furthermore, clustered point mutations in three phylogenetically conserved regions of the Lsm11 N-terminus impair or abolish histone RNA processing. As these mutations have no effect on the two interactions with ZFP100, these protein regions must play other roles in histone RNA processing, e.g. by contacting the pre-mRNA or additional processing factors.

REGULATION OF BRAIN NORADRENERGIC-RECEPTOR FUNCTION BY ADRENOCORTICOTROPHIN AND ANTIDEPRESSANT DRUGS (ADRENERGIC RECEPTOR, CYCLIC-AMP, ADENYLATE CYCLASE, IMIPRAMINE, STRESS)

Human behavior appears to be regulated in part by noradrenergic transmission since antidepressant drugs modify the number and function of (beta)-adrenergic receptors in the central nervous system. Affective illness is also known to be associated with the endocrine system, particularly the hypothalamic-pituitary-adrenal axis. The aim of the present study was to determine whether hormones, in particular adrencorticotrophin (ACTH) and corticosterone, may influence behavior by regulating brain noradrenergic receptor function.^ Chronic treatment with ACTH accelerated the increase or decrease in rat brain (beta)-adrenergic receptor number induced by a lesion of the dorsal noradrenergic bundle or treatment with the antidepressant imipramine. Chronic administration of ACTH alone had no effect on (beta)-receptor number although it reduced norepinephrine stimulated cyclic AMP accumulation in brain slices. Treatment with imipramine also reduced the cyclic AMP response to norepinephrine but was accompanied by a decrease in (beta)-adrenergic receptor number. Both the imipramine and ACTH treatments reduced the affinity of (beta)-adrenergic receptors for norepinephrine, but only the antidepressant modified the potency of the neurotransmitter to stimulate second messenger production. Neither ACTH nor imipramine treatment altered Gpp(NH)p- or fluoride-stimulated adenylate cyclase, cyclic AMP, cyclic GMP, or cyclic GMP-stimulated cyclic AMP phosphodiesterase, or the activity of the guanine nucleotide binding protein (Gs). These findings suggested that post-receptor components of the cyclic nucleotide generating system are not influenced by the hormone or antidepressant. This conclusion was verified by the finding that neither treatment altered adenosine-stimulated cyclic AMP accumulation in brain tissue.^ A detailed examination of the (alpha)- and (beta)-adrenergic receptor components of norepinephrine-stimulated cyclic AMP production revealed that ACTH, but not imipramine, administration reduced the contribution of the (alpha)-receptor mediated response. Like ACTH treatment, corticosterone diminished the (alpha)-adrenergic component indicating that adrenal steroids probably mediate the neurochemical responses to ACTH administration. The data indicate that adrenal steroids and antidepressants decrease noradrenergic receptor function by selectively modifying the (alpha)- and (beta)-receptor components. The functional similarity in the action of the steroid and antidepressants suggests that adrenal hormones normally contribute to the maintenance of receptor systems which regulate affective behavior in man. ^

A Universal Formula for Continuous Utility

A single formula assigns a continuous utility function to every representable preference relation.

Genetic integration of a nuclear -encoded mitochondrial gene with cardiac function

Cardiovascular disease (CVD) is the leading cause of death in the United States. One manifestation of CVD known to increase mortality is an enlarged, or hypertrophic heart. Hypertrophic cardiomyocytes adapt to increased contractile demand at the genetic level with a re-emergence of the fetal gene program and a downregulation of fatty acid oxidation genes with concomitant increased reliance on glucose-based metabolism. To understand the transcriptional regulatory pathways that implement hypertrophic directives we analyzed the upstream promoter region of the muscle specific isoform of the nuclear-encoded mitochondrial gene, carnitine palmitoyltransferase-1β (CPT-1β) in cultured rat neonatal cardiac myocytes. This enzyme catalyzes the rate-limiting step of fatty acid entry into β-oxidation and is downregulated in cardiac hypertrophy and failure, making it an attractive model for the study of hypertrophic gene regulation and metabolic adaptations. We demonstrate that the muscle-enriched transcription factors GATA-4 and SRF synergistically activate CPT-1β; moreover, DNA binding to cognate sites and intact protein structure are required. This mechanism coordinates upregulation of energy generating processes with activation of the energy consuming contractile promoter for cardiac α-actin. We hypothesized that fatty acid or glucose responsive transcription factors may also regulate CPT-1β. Oleate weakly stimulates CPT-1β activity; in contrast, the glucose responsive Upstream Stimulatory Factors (USF) dramatically depresses the CPT-1β reporter. USF regulates CPT-1β through a novel physical interaction with the cofactor PGC-1 and abrogation of MEF2A/PGC-1 synergistic stimulation. In this way, USF can inversely regulate metabolic gene programs and may play a role in the shift of metabolic substrate preference seen in hypertrophy. Failing hearts have elevated expression of the nuclear hormone receptor COUP-TF. We report that COUP-TF significantly suppresses reporter transcription independent of DNA binding and specific interactions with GATA-4, Nkx2.5 or USF. In summary, CPT-1β transcriptional regulation integrates mitochondrial gene expression with two essential cardiac functions: contraction and metabolic substrate oxidation. ^

Intrinsic oxidative stress in cancer cells: A biochemical basis for therapeutic selectivity using ROS -generating agents

A major goal of chemotherapy is to selectively kill cancer cells while minimizing toxicity to normal cells. Identifying biological differences between cancer and normal cells is essential in designing new strategies to improve therapeutic selectivity. Superoxide dismutases (SOD) are crucial antioxidant enzymes required for the elimination of superoxide (O2·− ), a free radical produced during normal cellular metabolism. Previous studies in our laboratory demonstrated that 2-methoxyestradiol (2-ME), an estradiol derivative, inhibits the function of SOD and selectively kills human leukemia cells without exhibiting significant cytotoxicity in normal lymphocytes. The present work was initiated to examine the biochemical basis for the selective anticancer activity of 2-ME. Investigations using two-parameter flow cytometric analyses and ROS scavengers established that O2·− is a primary and essential mediator of 2-ME-induced apoptosis in cancer cells. In addition, experiments using SOD overexpression vectors and SOD knockout cells found that SOD is a critical target of 2-ME. Importantly, the administration of 2-ME resulted in the selective accumulation of O 2·− and apoptosis in leukemia and ovarian cancer cells. The preferential activity of 2-ME was found to be due to increased intrinsic oxidative stress in these cancer cells versus their normal counterparts. This intrinsic oxidative stress was associated with the upregulation of the antioxidant enzymes SOD and catalase as a mechanism to cope with the increase in ROS. Furthermore, oxygen consumption experiments revealed that normal lymphocytes decrease their respiration rate in response to 2-ME-induced oxidative stress, while human leukemia cells seem to lack this regulatory mechanism. This leads to an uncontrolled production of O2·−, severe accumulation of ROS, and ultimately ROS-mediated apoptosis in leukemia cells treated with 2-ME. The biochemical differences between cancer and normal cells identified here provide a basis for the development of drug combination strategies using 2-ME with other ROS-generating agents to enhance anticancer activity. The effectiveness of such a combination strategy in killing cancer cells was demonstrated by the use of 2-ME with agents/modalities such as ionizing radiation and doxorubicin. Collectively, the data presented here strongly suggests that 2-ME may have important clinical implications for the selective killing of cancer cells. ^

The function of the murine complement *C3a and *C5a anaphylatoxin receptors in endotoxemia, bacteremia and septic shock

The complement system functions as a major effector for both the innate and adaptive immune response. Activation of the complement cascade by either the classical, alternative, or lectin pathway promotes the proteolysis of C3 and C5 thereby generating C3a and C5a. Referred to as anaphylatoxins, the C3a and C5a peptides mediate biological effects upon binding to their respective receptors; C3a binds to the C3a receptor (C3aR) while C5a binds to the C5a receptor (C5aR, CD88). Both C3a and C5a are known for their broad proinflammatory effects. Elevated levels of both peptides have been isolated from patients with a variety of inflammatory diseases such as COPD, asthma, RA, SLE, and sepsis. Recent studies suggest that C5a is a critical component in the acquired neutrophil dysfunction, coagulopathy, and progressive multi-organ dysfunction characteristic of sepsis. The primary hypothesis of this dissertation was that preventing C3a-C3aR and C5a-C5aR mediated pro-inflammatory effects would improve survival in endotoxic, bacteremic and septic shock. To test this hypothesis, the murine C3aR and C5aR genes were disrupted. Following disruption of both the C3aR and C5aR genes, no abnormalities were identified other than the absence of their respective mRNA and protein. In models of both endotoxic and bacteremic shock, C3aR deficient mice suffered increased mortality when compared to their wild type littermates. C3aR deficient mice also had elevated circulating IL-1β levels. Using a model of sepsis, C3aR deficient mice had a higher circulating concentration of IL-6 and decreased peritoneal inflammatory infiltration. While these results were unexpected, they support an emerging role for C3a in immunomodulation. In contrast, following endotoxic or bacteremic shock, C5aR deficient mice experienced increased survival, less hemoconcentration and less thrombocytopenia. It was later determined that C5a mediated histamine release significantly contributes to host morbidity and mortality in bacteremic shock. These studies provide evidence that C5a functions primarily as a proinflammatory molecule in models of endotoxic and bacteremic shock. In the same models, C3a-C3aR interactions suppress the inflammatory response and protect the host. Collectively, these results present in vivo evidence that C3a and C5a have divergent biological functions. ^