T Lymphocyte Myosin IIA is Required for Maturation of the Immunological Synapse

The role of non-muscle myosin IIA (heavy chain encoded by the non-muscle myosin heavy chain 9 gene, Myh9) in immunological synapse formation is controversial. We have addressed the role of myosin IIA heavy chain protein (MYH9) in mouse T cells responding to MHC-peptide complexes and ICAM-1 in supported planar bilayers - a model for immunological synapse maturation. We found that reduction of MYH9 expression levels using Myh9 siRNA in proliferating mouse CD4(+) AND T cell receptor (TCR) transgenic T cells resulted in increased spreading area, failure to assemble the central and peripheral supramolecular activation clusters (cSMAC and pSMAC), and increased motility. Surprisingly, TCR microcluster speed was reduced marginally, however TCR microclusters dissipated prior to forming a cSMAC. TCR microclusters formed in the Myh9 siRNA-treated T cells showed reduced phosphorylation of the Src family kinase (SFK) activation loop and displayed reduced cytoplasmic calcium ion (Ca(2+)) elevation. In addition, Myh9 siRNA-treated cells displayed reduced phosphorylation of the Cas-L substrate domain - a force-dependent SFK substrate - which was observed in control siRNA-treated cells in foci throughout the immunological synapse except the cSMAC. Cas-L exhibited TCR ligation-dependent induction of phosphorylation. These results provide further evidence that T cell activation is modulated by intrinsic force-generating systems and can be viewed as a mechanically responsive process influenced by MYH9

Stomatin-like Protein 2 Links Mitochondria to T-Cell Receptor Signalosomes at the Immunological Synapse and Enhances T-Cell Activation

T cell activation through the antigen receptor (TCR) requires sustained signalling from microclusters in the peripheral region of the immunological synapse (IS). The bioenergetics of such prolonged signaling have been linked to the redistribution of mitochondria to the IS. Here, we report that stomatin-like protein-2 (SLP-2) plays an important role in this process by bridging polarized mitochondria to these signaling TCR microclusters or signalosomes in the IS in a polymerized actin-dependent manner. In this way, SLP-2 helps to sustain TCR-dependent signalling and enhances T cell activation

Application of theory of possibility and random-fuzzy variables (RFVs) in Kalman filter and Bayes' theorem for Industrial conformity analysis

DOTTORATOLa misurazione ha sempre affascinato gli esseri umani. Con la crescente necessità di misurazione, c'è stato un bisogno crescente di teorie matematiche per esprimere la nostra conoscenza sulla misurazione. La teoria dell'errore è stata la prima teoria che ha tentato di quantificare la certezza sulla misurazione. Più recentemente, la teoria dell'incertezza è stata proposta ed è ampiamente accettata ed è attualmente in uso. Come dice il nome, la teoria dell'incertezza quantifica l'incertezza o la quantità di dubbio che abbiamo sulla misurazione. La teoria matematica che è stata adattata per rappresentare è stata a lungo la teoria della probabilità. Ma, con la crescente complessità degli scenari e quindi dei dati negli scenari, la teoria della probabilità non è più adeguata per trattare tutte le classi di dati. La teoria della possibilità e, più specificamente, random-fuzzy variables (RFVs) costituiscono una scelta interessante per rappresentare l'incertezza poiché le RFV possono rappresentare contributi sia sistematici che casuali all'incertezza in modo matematicamente accurato. Ci sono state molte ricerche corrispondenti all'applicazione della teoria della possibilità e delle RFV in diversi campi. Ma, finora, non ci sono state molte ricerche sul filtro di Kalman, specialmente usando RFV. Allo stesso modo, non ci sono state molte ricerche sull'uso di RFV in un ambiente industriale, specialmente nel teorema di Bayes durante un'analisi di conformità. Questo è ciò su cui si concentra la mia tesi. Questa tesi presenta una panoramica dei concetti esistenti in metrologia e filtri kalman. Quindi, viene fornita una nuova definizione per un filtro di Kalman che utilizza RFV. Quindi il filtro di Kalman definito è stato applicato in alcuni casi di studio. Quindi, è stata proposta un'altra versione leggermente modificata del filtro Kalman basato su RFV che consente di compensare parzialmente l'errore sistematico. Infine, è stata presentata una panoramica dell'analisi di conformità insieme a una panoramica dell'uso del teorema di Bayes in metrologia e per la valutazione della conformità. Infine, vengono forniti gli svantaggi dell'uso cieco del teorema di Bayes insieme a una versione modificata del teorema di Bayes in modo tale da utilizzare gli RFV insieme a una simulazione basata e alla convalida sperimentale dell'idea proposta.Measurement has always fascinated humans. With growing need for measurement, there has been a growing need for mathematical theories to express our knowledge about the measurement. The theory of error was the first theory that attempted to quantify the certainty about the measurement. More recently, the theory of uncertainty has been proposed and is widely accepted and is currently in use. As the name says, the theory of uncertainty quantifies the uncertainty or the amount of doubt we have about the measurement. The mathematical theory that has been adapted to represent has long been the theory of probability. But, with growing complexity of scenarios and thereby the data in the scenarios, the theory of probability is no more adequate to deal with all classes of data. The theory of possibility and more specifically, Random-fuzzy variables form an interesting choice to represent uncertainty since RFVs can represent both systematic and random contributions to uncertainty in a mathematically accurate way. There has been a lot of research corresponding to the application of the theory of possibility and RFVs in different fields. But, so far, there has not been much research in Kalman filter especially using RFVs. Similarly, there hasn’t been much research about the use of RFVs in an industrial setting especially in the Bayes’ theorem during a conformity analysis. This is what my thesis focuses on. This thesis presents an overview of the existing concepts in metrology and kalman filters. Then, a new definition for a Kalman filter using RFVs is provided. Then the defined Kalman filter has been applied in a few case studies. Then, another slightly modified version of the RFV based Kalman filter has been proposed which allows to partially compensate for the systematic error. Finally, an overview of the conformity analysis has been presented along with an overview of the use of Bayes’ theorem in metrology and for conformity assessment. Finally, the drawbacks of using the Bayes’ theorem blindly are given along with a modified version of the Bayes’ theorem such that it uses the RFVs is given along with a simulation based as well as experimental validation of the proposed idea.DIPARTIMENTO DI ENERGIA34FERRERO, ALESSANDROMUSSETTA, MARC

Effect of integration of supplemental nutrition with public health programmes in pregnancy and early childhood on cardiovascular risk in rural Indian adolescents: long term follow-up of Hyderabad nutrition trial.

OBJECTIVE: To determine whether integration of nutritional supplementation with other public health programmes in early life reduces the risk of cardiovascular disease in undernourished populations. DESIGN: Approximately 15 years' follow-up of participants born within an earlier controlled, community trial of nutritional supplementation integrated with other public health programmes. SETTING: 29 villages (15 intervention, 14 control) near Hyderabad city, south India. PARTICIPANTS: 1165 adolescents aged 13-18 years. INTERVENTION: Balanced protein-calorie supplementation (2.51 MJ, 20 g protein) offered daily to pregnant women and preschool children aged under 6 years, coupled with integrated delivery of vertical public health programmes. MAIN OUTCOME MEASURES: Height, adiposity, blood pressures, lipids, insulin resistance (homoeostasis model assessment (HOMA) score), and arterial stiffness (augmentation index). RESULTS: The participants from the intervention villages were 14 mm (95% confidence interval 4 to 23; P=0.007) taller than controls but had similar body composition. The participants from the intervention villages had more favourable measures of insulin resistance and arterial stiffness: 20% (3% to 39%; P=0.02) lower HOMA score and 3.3% (1% to 5.7%; P=0.008) lower augmentation index. No strong evidence existed for differences in blood pressures and serum lipids. CONCLUSIONS: In this undernourished population, integrated delivery of supplemental nutrition with other public health programmes in pregnancy and early childhood was associated with a more favourable profile of cardiovascular disease risk factors in adolescence. This pragmatic study provides the most robust evidence to date on this important hypothesis for which classic trials are unlikely. Improved maternal and child nutrition may have a role in reducing the burden of cardiovascular disease in low income and middle income countries

Characterization of cold shock domain proteins and SUMOylation system from Oryza sativa

In the first part of this study, two novel c&barbelow;old s&barbelow;hock domain p&barbelow;roteins from rice (OsCSP) were cloned and subsequently characterized their roles during stress conditions and development. OsCSP1 and OsCSP2 ( Oryza sativa CSD protein) encode putative proteins consisting of an N-terminal CSD and glycine-rich regions that are interspersed by 4 and 2 CX2CX4HX4C (CCHC) retroviral-like zinc fingers, respectively. Using an in vitro DNA binding assay, I demonstrate that OsCSPs exhibit conserved ssDNA binding activity. In vivo functional complementation in a cold-sensitive bacterial strain, that lacks four cold inducible cold shock domain proteins revealed that OsCSPs function as RNA chaperones, similar to their bacterial and winter wheat counterparts. To understand the functions of these genes in rice, I studied the transcriptional regulation in response to abiotic stress conditions. Under cold stress, OsCSP transcript levels are only transiently and marginally increased and the encoded proteins did not accumulate. These transcript and protein data are in sharp contrast with the observed data for winter wheat and Arabidopsis cold shock domain proteins under cold stress. In these species, both transcripts and protein levels of CSPs are increased upon cold stress. Based on these data, it can be hypothesized that the accumulation of cold shock domain proteins may play an important role in determining the cold acclimation capability of the plants. Expression analysis at the protein and RNA levels during development revealed that OsCSPs are highly expressed in the reproductive and meristematic tissues. These results indicate a potential role for rice cold shock domain proteins in plant growth and reproductive development.;In this study, I also characterized the post-translational modification of plant cold shock domain proteins by SUMOylation. Post-translational modifications can impart rapid changes in protein function. SUMOylation involves the reversible attachment of a small protein called SUMO (small ubiquitin-like modifier) to target proteins. The SUMO protein has a similar three dimensional structure as that of ubiquitin and the process of SUMOylation is very similar to that of ubiquitination. However, unlike ubiquitination, SUMOylation is not implicated in protein degradation. SUMO modification can affect the target protein stability, sub-cellular localization protein-protein interactions. Using a computational approach on rice and Arabidopsis cold shock domain proteins, I identified canonical SUMOylation motifs in both rice CSPs and one of the Arabidopsis CSPs. Using in vitro assays, I demonstrated that both OsCSPs can undergo SUMOylation. Using mutational approaches, I identified an important lysine residue for SUMOylation in Arabidopsis AtCSP1. By employing GFP-tagged wild-type and variant AtCSP1 proteins, I demonstrate that SUMOylation appears to affect AtCSP1 protein localization.;In another study, I characterized the entire SUMO conjugation system in rice. The process of SUMOylation involves a cascade of enzymatic reactions involving activation (E1) enzymes, conjugation (E2) enzymes and ligation (E3) enzymes. I compared the protein sequences of all these genes from rice with those from Arabidopsis, yeast and human. This revealed a high amino acid sequence conservation of individual SUMOylation components from yeast to plants and animals. In Arabidopsis, the SUMOylation system has been implicated in plant development and in mediating abiotic stress responses. To understand the role of the rice SUMOylation system during development, I studied the SUMO conjugate profiles and the expression of individual SUMO component genes in various tissues at different stages of plant development. The highest levels of SUMOylated proteins were observed in panicles and meristematic tissues. Expression studies revealed that SUMO component genes are highly expressed in reproductive tissues like developing seeds and panicles. Together, these data implicate an important role for the rice SUMOylation system in plant growth and reproductive development. To understand the role of SUMOylation system in rice, I studied SUMO conjugate profiles and the transcriptional regulation of individual SUMO components during cold, salt and ABA stress conditions. Rice responds to these stresses by accumulating SUMO conjugated proteins, suggesting that protein SUMOylation helps to mediate plant stress responses. Studies on the transcriptional regulation of individual SUMO pathway genes during these stress conditions revealed that most are transcriptionally down-regulated. However, a particular SUMO E3 ligase gene (OsSIZ2) is transiently up-regulated upon exposure to all three stress conditions. Considering the importance of E3 ligases in improving the efficiency and specificity of the SUMO conjugation reactions, OsSIZ2 may mediate accumulation of SUMO conjugates during these stress conditions. Taken together, these data suggest a role for SUMOylation in rice development and stress responses. (Abstract shortened by UMI.)

A Buddhist Monastery and Temple with a Religious Center in New York City

The Buddhist Temple form has remained unchanged in the last 2,500 years. The thesis will become an exploration of contemporary issues of acculturation and transition of Buddhism in the United States. The thesis will examine architecturally, the individual spirit of contemplation and meditation of a monistic religion. This allows the thesis to transform, architecturally and typologically, a Buddhist Temple and Monastery of the Theravada Sect, with a Religious Center

Self-reactive human CD4 T cell clones form unusual immunological synapses

Recognition of self–peptide-MHC (pMHC) complexes by CD4 T cells plays an important role in the pathogenesis of many autoimmune diseases. We analyzed formation of immunological synapses (IS) in self-reactive T cell clones from patients with multiple sclerosis and type 1 diabetes. All self-reactive T cells contained a large number of phosphorylated T cell receptor (TCR) microclusters, indicative of active TCR signaling. However, they showed little or no visible pMHC accumulation or transport of TCR–pMHC complexes into a central supramolecular activation cluster (cSMAC). In contrast, influenza-specific T cells accumulated large quantities of pMHC complexes in microclusters and a cSMAC, even when presented with 100-fold lower pMHC densities. The self-reactive T cells also maintained a high degree of motility, again in sharp contrast to virus-specific T cells. 2D affinity measurements of three of these self-reactive T cell clones demonstrated a normal off-rate but a slow on-rate of TCR binding to pMHC. These unusual IS features may facilitate escape from negative selection by self-reactive T cells encountering very small amounts of self-antigen in the thymus. However, these same features may enable acquisition of effector functions by self-reactive T cells encountering large amounts of self-antigen in the target organ of the autoimmune disease

Rancang Bangun Aplikasi Berbasis Web Untuk Sistem Deteksi Penyakit Malaria Menggunakan Computer Vision

This study discusses the design and development of a web-based application for malariadisease detection using Computer Vision. The main objective of this research is to developa system that can assist medical personnel in detecting malaria infections more quickly andaccurately by utilizing computer vision technology. The proposed system employs theConvolutional Neural Network (CNN) algorithm to analyze microscopic erythrocyteimages obtained from blood samples. Malaria image data used in this study were collectedfrom various online sources as well as through direct observation in hospitals.The systemdevelopment process begins with the collection of malaria image data, which is thenprocessed through preprocessing stages to enhance data quality and prepare it for modeltraining. Once the data is ready, the CNN model is trained using augmented training datato improve the model's generalization. Model evaluation is conducted using test data tomeasure the accuracy and performance of the model in detecting malaria.Evaluation results indicate that the developed CNN model has high accuracy in detecting malaria infections,with satisfactory precision, recall, and F1-score values. The system is also capable ofgenerating detection reports and visualizations that facilitate medical personnel indiagnosis. This system is expected to support efforts to improve malaria diagnosis inhealthcare facilities, particularly in remote areas with limited access to trained medicalpersonnel. Additionally, the system is anticipated to reduce the workload of medicalpersonnel and increase efficiency in the malaria diagnosis process

IN VITRO CYTOTOXIC STUDY OF GREEN SYNTHESIZED GOLD AND SILVER NANOPARTICLES USING ECLIPTA PROSTRATA (L.) AGAINST HT-29 CELL LINE

Objective: The objective of this study is to synthesize gold and silver nanoparticles using the extracts of Eclipta prostrata.Methods: The nanoparticles synthesize is carried out using the powdered leaves and mixed with distilled water. The filtered extract was then mixedwith aqueous solution of HAuCl4 (1 mM) and AgNo3 (1 mM), and the reaction volume was made up to 100 ml. Then, the characterization of nanoparticleswas carried out using ultraviolet, infrared, scanning electron microscope, and the cytotoxic activity of the nanoparticles were investigated against HT-29 cancer cell lines.Results: From the study, it was found that the plant extract was able to synthesize nanoparticles, and the synthesized nanoparticles were found to betoxic against the cancer cell line HT-29.Conclusion: In the present study, both silver and gold nanoparticles were synthesized using the plant extract of E. prostrata. The synthesizednanoparticles were found to be effective against HT 29 cancer cells. The green synthesized nanoparticles were found to be cost-effective, simpler andenvironmentally safe. As the nanotechnology is an emerging field in medicine, the biological synthesis of nanoparticles helps in the other way. Fromthe present study, the nanoparticles synthesized were thus proved against various studies novely. Hope this paves way for the better development ofnanoparticle production in the large scale amount.Keywords: Nanoparticles, Eclipta prostrata, Scanning electron microscope, HT-29 cell line. §Both authors are equally contribute

Wireless Emitter Location Estimation Based on Linear and Nonlinear Algorithms using TDOA Technique

Low-power devices such as cell phones, and wireless routers are commonly used to control Improvised Explosive Devices (IEDs) and as the communication nodes for the sake of command and control. Quickly locating the source of these signals is ambitious, specifically in a metropolitan environment where buildings and towers may cause intervention. This presents a geolocation system that compounds the attributes of several proven geolocation and error mitigation methods to locate an emitter of interest in an urban environment. The proposed geolocation system uses a Time Difference of Arrival (TDOA) approach to estimate the position of the emitter of interest. Using multiple sensors at known locations, TDOA estimates are achieved by the cross-correlation of the signal received at all the sensors. A Weighted Least Squares (WLS) solution, Linear least Square (LLS) method and maximum likelihood (ML) estimation is used to estimate the emitter's location. If the variance of this location estimate is too high, a sensor is detected and identified as possessing a Non-Line of Sight (NLOS) path from the emitter. This poorly located sensor is then removed from the geolocation system and a new position estimate is computed with the remaining sensor TDOA information. The performance of the TDOA system is determined through modeling and simulations. Test results confirm the feasibility of identifying a NLOS sensor, thereby improving the geolocation system's accurateness in a metropolitan environment