Radio Frequency Identification (RFID) uses RF radiation to identify physical objects. With decreasing integrated circuit (IC) cost and size, RFID applications are becoming economically feasible and gaining popularity. Researchers at MIT suggest that RFID tags operating in the 900 MHz band (ultrahigh frequency, UHF) represent the best compromise of cost, read range, and capabilities [1]. Passive RFID tags, which exclude radio transmitters and internal power sources, are popular due to their small size and low cost [1]. This project produced Cal Poly’s first ever on-campus printed, assembled, and operational UHF (902 to 928 MHz) passive RFID tag. Project goals include RFID tag antenna design and simulation using the EMPro electromagnetic (EM) simulation tool [47], establishing the tag fabrication process, and testing, operational verification, and comparisons to commercial tag performance. The tag antenna design goal is to meet or exceed the read range performance of the commercial Sirit tag [23] while minimizing the required tag conductive area. This thesis provides an overview of the UHF passive RFID tag fabrication process. Cal Poly’s Graphic Communication Department Laboratory applied a screen‑printing process to print RFID tag antenna patterns onto plastic (PET) substrates. RFID IC-substrate packages were manually attached to tag antennas with conductive adhesives and functionally verified and compared to commercial tag performance. RFID tag antennas were impedance matched (using EMPro) to the Monza 3 RFID IC to maximize IC to antenna power transfer and RFID tag read range.Tag antenna read range (maximum reader-tag communication distance) was characterized in Cal Poly’s Anechoic Chamber, while RFID tag matching characteristics were measured using the differential probe method [33-41] and compared to simulations. Read range results indicate that one of the designs developed in this thesis outperforms a commercial UHF RFID tag.

Juvenil idiopatisk artrit (JIA) är en autoimmun reumatisk sjukdom som drabbar barn före 16 års ålder. Den vanligaste varianten är oligoartikulär JIA, vilket innebär att den påverkar två till fyra leder med symptom så som svullnad, ömhet, stelhet, rodnad eller nedsatt motorik. Oligoartikulär JIA är även associerad med uveit, vilket är en autoimmun ögonsjukdom som förstör synen. Sjukdomen är i nuläget dåligt förstådd och forskning behövs för att kunna identifiera markörer som kan användas för diagnostik. Enzymkopplad immunadsorberande analys (ELISA) är inom vårdens laborativa arbete en mycket vanlig metod för att undersöka patienters serum och identifiera specifika markörer som tyder på sjukdom. En ELISA kan utföras på olika sätt beroende på om den letar efter antigen eller antikroppar. Syftet med denna studie var att optimera en indirekt ELISA för detektion av autoantikroppar och genomföra en metodjämförelse med en redan väl undersökt kulbaserad autoantikroppsmetod. Studien omfattar 14 serumprov från olika patienter och använder sig av rekombinant framställda protein epitope signature tag (PrEST)-antigener för att identifiera autoantikroppar. PrEST-antigener består av ett humant variabelt proteinsegment och en protein-tag som består av ett albuminbindande protein (ABP) med en N-terminal HIS-tag (His6ABP). För att förhindra ospecifik inbindning till His6ABP-delen av PrEST-antigenerna så framställdes His6ABP, med vilket serum inkuberades innan de undersöktes med ELISA. Då resultatet tolkades binärt kunde stor likhet ses mellan de två olika metoderna för samtliga PrEST-antigener som undersöktes, vilket innebär att metoden kan fortsätta utforskas. Resultatet visade också att His6ABP-delen skulle kunna orsaka korsreaktivitet, vilket dock inte är något negativt för metodens validitet. / Juvenile idiopathic arthritis (JIA) is an autoimmune rheumatic disease that affects children before the age of 16. The most common variant is oligoarticular JIA, which means it affects two to four joints with symptoms such as swelling, tenderness, stiffness, redness, or reduced mobility. Oligoarticular JIA is also associated with uveitis, an autoimmune eye disease which damage eyesight. The disease is currently poorly understood, and research is needed to identify markers that can be used for diagnostics. Enzyme-linked immunosorbent assay (ELISA) is a very common method in healthcare laboratory work to examine patients' serum and identify specific markers that indicate disease. An ELISA method can be performed in different ways depending on whether it is looking for antigen or antibodies. The aim of this study was to optimize an indirect ELISA for detection of autoantibodies and conduct a method comparison with an already well established bead-based autoantibody array method. The study included 14 sera samples from different patients and used recombinantly produced protein epitope signature tag (PrEST) antigens to identify autoantibodies. PrEST antigens consist of a human variable protein segment and a protein tag consisting of albumin binding protein (ABP) with an N-terminal HIS-tag (His6ABP). To prevent non-specific binding to the His6ABP portion of the PrEST antigens, His6ABP were recombinantly produced and used for pre-absorption of serum before being examined by ELISA. When the result was interpreted binary, great similarity could be seen between the two different methods for all PrEST antigens examined, which calls for further exploring of the method. The result also showed that the His6ABP part could possibly cause cross-reactivity, which is interesting but doesn’t affect the validity of the method.

<p>Through a number of biological, technological and computational achievements during the 20th century and the devoted work of hundreds of researchers the sequence of the human and other genomes are now available in public databases. The current challenge is to begin to understand the information encoded by the DNA sequence, to elucidate the functions of the proteins and RNA molecules encoded by the genes as well as how they are regulated. For this purpose new technologies within the area of functional genomics are being developed. Among those are powerful tools for gene expression analysis, such as microarrays, providing means to investigate when and where certain genes are used.</p><p>This thesis describes a method that was developed to enable gene expression analysis, on the transcriptome level, in small tissue samples. It relies on PCR amplification of the 3’-ends of cDNA (denoted 3’-end signature tags). PCR is a powerful technology for amplification of nucleic acids, but has not been used much for transcript profiling since it is generally considered to introduce biases, distorting the original relative transcript levels. The described method addresses this issue by generating uniformly sized representatives of the transcripts/cDNAs prior to amplification. This is achieved through sonication which, unlike restriction enzymes, does not require a specific recognition sequence and fragments each transcript randomly. The method was evaluated using cDNA microarrays, Affymetrix™ oligonucleotide arrays and real-time quantitative PCR. It was shown to perform well, yielding transcript profiles that correlate well to the original, unamplified material, as well as being highly reproducible.</p><p>The developed method was applied to stem cell biology. The variability in gene expression between different populations of cultured neural stem cells (neurospheres) was investigated. It was shown that neurospheres isolated from different animals or passaged to different degrees show large fluctuations in gene expression, while neurospheres isolated and cultured under identical conditions are more similar and suitable for gene expression analysis. A second study showed that withdrawing epidermal growth factor (EGF) from the culture medium when treating the cells with an agent of interest has profound effects on gene expression, something which should be taken into consideration in future neurosphere studies.</p>

L’étude de l’assemblage et de l’acheminement à la membrane plasmique des complexes de signalisation des RCPGs va jouer un rôle crucial dans le développement de nouveaux médicaments ayant moins d’effets secondaires. Des outils permettant l’étude de ces phénomènes existent déjà, mais un outil polyvalent qui permettrait d’étudier plusieurs aspects pourrait grandement faciliter et accélérer ces études. L’étiquette SNAP est un candidat intéressant puisqu’avec cette étiquette il est possible de marquer une seule construction avec une variété de différents substrats. Une construction encodant pour le récepteur β2AR avec une étiquette SNAP à son extrémité C-terminale a été ingénérée. Cette construction est apte à lier son ligand, à être acheminée à la membrane plasmique et à homodimériser. La protéine exprimée a été marquée avec le fluorophore BG-430. De la fluorescence non spécifique a été détectée dans la cellule (même en absence de l’étiquette SNAP sur le récepteur). Un essai BRET a été développé, utilisant la construction HA-β2AR-SNAP en tant qu’accepteur et est fonctionnel. L’étiquette SNAP, comme utilisée ici, ne présente pas un aussi bon candidat qu’attendu, puisque le substrat n’ayant pas réagi demeure coincé dans la cellule. Le facteur activateur de plaquette (PAF) et son récepteur (PAFR) jouent un rôle critique dans plusieurs réponses inflammatoires et le récepteur FP est impliqué dans l’accouchement prématuré. Une meilleure compréhension des complexes de signalisation associés à ces récepteurs pourrait être la première étape dans la compréhension de leur signalisation dans des situations normales ou de maladies. Des expériences de BRET étudiant des interactions de bases entre les récepteurs et leurs partenaires d’interactions connus ont été réalisées. Elles ont permis de déterminer que : les récepteurs PAF et FP homodimérisent, que les récepteurs PAF et FP hétérodimérisent, que les protéines Gβγ interagissent de manière constitutive avec ces récepteurs et qu’aucun signal de BRET n’a été détecté avec la protéine Gα et ce, en présence ou en absence de stimulation par un agoniste (suggérant qu’il est nécessaire d’optimiser le système présentement utilisé). / Studies of the assembly and plasma membrane trafficking of G protein-coupled receptor (GPCR) signalling complexes will play an important role in the development of new drugs with fewer side effects. Tools for this purpose have already been developed, but a more versatile tool which would allow assessment of multiple aspects of GPCR trafficking and protein/protein interaction could greatly facilitate and expedite these studies. The SNAP tag is without doubt an interesting candidate for this task. Using this tag, it is possible to label one receptor construct, with a variety of different substrates. A construct encoding the β2AR receptor was engineered with a C-terminal SNAP tag. This construct, when expressed in HEK 293 cells, was able to bind ligand, to traffic to the plasma membrane and to form homodimers. The expressed protein was then labelled with the BG-430 fluorophore. Non-specific fluorescence was detected in the cell, even in the absence of the SNAP tag. A BRET assay was developed using the HA-β2AR-SNAP as a BRET acceptor. The SNAP tag as used in this initial study was not as good as thought previously because uneacted substrate remains trapped in cells (i.e. the background was too high). Platelet-activating factor (PAF) and its receptor (PAF-R) play a critical role in many inflammatory responses and the receptor for prostaglandin F (FP) plays a role in pre-term labour. A better understanding of signalling complexes associated with these receptors might be the first step in a better understanding of their signalling in health and disease. As an initial step in this direction, we used BRET to study basic interactions between these receptors and known signalling partners. PAF-R and FP receptors homodimerize. A heterodimer between the FP and PAF receptors was also detected. Gβγ subunits interact with these receptors in a constitutive way. In the case of the Gα, no BRET signal was detected with or without agonist stimulation suggesting more work is required to optimize the system.

Several classes of antimetabolites have been developed for the treatment of cancer, including numerous inhibitors of nucleotide biosynthesis. N-(phosphonacetyl)-L-aspartate (PALA) and hydroxyurea (HU) are two antimetabolites that inhibit nucleotide biosynthesis; PALA inhibits de novo pyrimidine synthesis and HU inhibits the conversion of ribonucleotide diphosphates to deoxyribonucleotide diphosphates. Due to the similar mechanisms, it was thought that cancer cells would respond similarly to HU and PALA treatment. However, studies in this dissertation revealed strikingly different responses to either HU or PALA treatment in HCT116 cells. A cytoprotective S-phase arrest was activated upon HU treatment while PALA treatment failed to activate the S-phase checkpoint, resulting in p53-dependent apoptosis. The checkpoint effector kinase, Chk1, was not significantly phosphorylated during PALA treatment due to a failure to recruit ATR, the upstream kinase, to chromatin sites. The post-translational modifications of p53, phosphorylation of serines 46 and 392, suggested that PALA treatment promotes the accumulation of a transcriptionally active p53 while HU does not. ChIP analysis showed that p53 bound to pro-apoptotic promoters, therefore activating p53-dependent apoptosis during PALA treatment. To gain more insight into these differential cellular responses, we developed a tandem-affinity purification (TAP) tagged p53 cell line in which a TAP tag was inserted into the C-terminus of the endogenous p53 genetic locus through homologous recombination. This technology allows purification of p53 with its protein binding partners at endogenous expression levels. The tagged p53 accumulated and bound to promoters in response to DNA damage similar to the untagged p53, suggesting that the TAP tag did not interfere with the normal cellular functions of p53. Using mass spectrometry, we can identify the different p53 protein binding partners in response to PALA or HU treatment. We can also determine the variable pattern of post-translational modifications on different drug-stabilized p53 and determine which modifications are responsible for promoting apoptosis versus cytoprotective arrest. We can then exploit the identified proteins and post-translational modifications in the development of new chemotherapeutic agents.

Enquanto as células humanas sintetizam purinas pela via de novo e pela via de recuperação, protozoários parasitas as sintetizam somente pela via de recuperação. Por essa razão, as enzimas que compõem essa via são importantes alvos para o desenvolvimento de novas drogas antiparasitárias. A enzima APRT converte adenina e &#945-D-5-fosforibosil 1-pirofosfato (PRPP) a AMP na via de recuperação de purinas. Nesse trabalho, a APRT e a APRT-His recombinantes foram caracterizadas por métodos bioquímicos e espectroscópicos. As expressões do gene aprt contidos nos vetares pET29+ (Novagen) e pQE30 (Qiagen) renderam 5 e 10 mg.mL-1 de APRT e APRT-His, respectivamente, na forma solúvel. A APRT permaneceu estável e homogênea in vitro em Tris pH 7,5 contendo 5 mM de MgSO4 e 150 mM de KCl mas a APRT-His mostrou-se instável e insolúvel nesse pH e acima de 0,5 mg.mL-1. O estudo de solubilidade revelou que a APRT-His é parcialmente estabilizada em Tris pH 8,5 contendo 150 mM de KCl devendo ser purificada e mantida nesse tampão durante os ensaios espectroscópicos e a adição de 50 mM de histidina mostrou-se eficiente para a concentração da enzima até 8mg.mL-1. A caracterização bioquímica da APRT e da APRT-His revelou que elas são diméricas nos seus tampões e têm PI igual a 6,45 &#177 0,20 e 7,7 &#177 0,16, respectivamente. Os ensaios de atividade enzimática indicaram que a APRT é duas vezes mais ativa do que a APRT-His. Os espectros de CD da APRT-His foram mais intensos do que os espectros da APRT e mostraram perfil de hélice &#945 . Os resultados da desconvolução revelaram que a APRT-His tem cerca de 10% mais hélice-&#945 do que a APRT. O valor de teor de estrutura secundária da APRT equivale aos valores extraídos dos dados cristalográficos da APRT de L donovani e de L tarentolae. Os espectros de emissão de fluorescência mostraram que a APRT-His e a APRT possuem máximos de emissão em 342 e 332 nm, respectivamente. Além disso, eles indicaram que o PRRP e o AMP suprimem a fluorescência do Trp presente na APRT. A supressão foi relacionada à posição dos ligantes localizados no sítio ativo da enzima e a ausência de supressão nas amostras de APRT-His foi relacionada à presença de Mg2+. Os resultados indicam que a presença dos resíduos de histidina na região N-terminal da APRT-His induziu a modificação estrutural da enzima levando a precipitação contínua. Nesse sentido, a ausência dos resíduos de histidina incorporados à enzima favoreceu a estabilidade da proteína in vitro. / Human cells synthesize purine nucleotide by again and salvage pathways, while parasitic protozoa use only salvage pathways. For this reason, the enzymes that compound the salvage pathway are important targets to development of new antiparasitic drugs. The enzyme adenine phosphoribosyltransferase (APRT) converts adenine and &#945-D-5-phosphoribosyll-pyraphosphate (PRPP) to adenosine monophosphate (AMP) at salvage pathway. In this work, the APRT and APRT-His recombinants had been characterized by biochemical and spectroscopic methods. The expression of the aprt genes from L. tarentolae inserted into pET29+ (Novagen) and pQ30 (Qiagen) vectors yielded 5 and 10 mg.mL-1 of the APRT and APRT-His, on soluble form, respectively. The APRT remained stable and homogeneous in vitro at Tris pH 7.5 containing 5 mM MgSO4 and 150 mM KCl, but APRT-His was instable and insoluble above 0.5 mg.mL-1 at the same pH. The solubility study showed that histidine increased the APRT-His solubility and it is partially stabilized at Tris pH 8.5 containing 150 mM KCl. The addition of the histidine 50 mM was efficient for concentrations up to 8 mg.mL-1.Then, the APRT-His was purified and storage in that buffer for spectroscopic assays. The biochemical characterization of the APRT and APRT-His indicated that a both are dimercs in its buffers, and they have isoelectric points at pH 6.45 &#177 0.20 and 7.7 &#177 0.16, respectively. By enzymatic activity assays, the APRT is twice activer than APRT-His. The CD spectra of the APRT-His were more intense than the APRT spectra. and showed helix &#945 profile. The fluorescence spectra marked a maximum emission fluorescence at 342 nm for the APRT-His and 332 um for the APRT. In addition, the spectra revealed that PRPP and AMP quenched the fluorescence of the tryptophan (Trp) into APRT. The quench was related to position of the ligands inside active site of the enzyme and the absent of fluorescence of the Trp, inside APRT-His, was related to absent of the Mg2+. The results has demonstrated that the presence of the histidine residues at N-terminal region of the APRT-His induced to conformational changes of the enzyme following to continuos precipitation. In the same sense, the absent of histidine residues associated to enzyme favored to stability of the protein in vitro.

Made available in DSpace on 2016-04-27T17:05:59Z (GMT). No. of bitstreams: 1 Renato Pereira de Araujo.pdf: 1711666 bytes, checksum: b5ad19e844d9c137553ad64b03b603c7 (MD5) Previous issue date: 2015-03-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / For the visually impaired, identify which path to follow to reach the desired destination without assistance from healthy people is a challenge. To move in an unknown environment is required to interact with the mechanisms of information. However the visually impaired do not yet have a navigation system in indoor environments with real-time response that allows this this communication. This paper presents a device that allows interaction with the tactile floor to aid navigation and orientation. Through an audio interface, the device provides the visually impaired relevant information on the space around it and the navigation options available. The relevant information is disseminated using labels with radio frequency identification technology, integrated into a microcontroller and an audio playback card. Each label has an electronic product code that allows to identify it uniquely. The label installed in a standard place of tactile floor is linked to an audio file with information about the space and the available routes. The reader integrated development board reads the labels and sends the electronic code to the microcontroller, which in turn identifies the code and assigns this audio file to be played. The device was tested by a volunteer who found all labels positioned on the floor tactile alert, clearly identified its position and reached the desired destination without route errors. Used as a complement to tactile signage, this device provides an interaction between the visually impaired and the existing tactile signage. This project will directly contribute to society, providing a convenient navigation system for people with visual disabilities have greater autonomy / Para os deficientes visuais, identificar qual caminho deve ser seguido para chegar ao destino desejado, sem auxílio de pessoas hígidas, é um desafio. Para se deslocar em um ambiente desconhecido é necessária a interação com os mecanismos de informação. Entretanto os deficientes visuais ainda não contam com um sistema de navegação em ambientes internos com resposta em tempo real que possibilite essa comunicação. Este trabalho apresenta um dispositivo que possibilita a interação com o piso tátil para auxiliar a navegação e a orientação. Por meio de uma interface de áudio, o dispositivo proporciona ao deficiente visual as informações úteis sobre o espaço em seu entorno e as opções de navegação disponíveis. As informações pertinentes são difundidas utilizando etiquetas com tecnologia de identificação por rádio frequência, integradas a um micro controlador e uma placa de reprodução de áudio. Cada etiqueta possui um código de produto eletrônico que permite identificá-la de forma exclusiva. A etiqueta instalada em um local padronizado do piso tátil está atrelada a um arquivo de áudio com informações sobre o espaço e as rotas disponíveis. O leitor integrado a placa de desenvolvimento faz a leitura das etiquetas e envia o código eletrônico ao micro controlador, que por sua vez identifica o código e atribui a este o arquivo de áudio que será reproduzido. O dispositivo foi testado por um voluntário que encontrou todas as etiquetas posicionadas junto ao piso tátil de alerta, identificou claramente sua posição e chegou ao destino desejado sem erros de percurso. Usado como complemento da sinalização tátil, este dispositivo possibilita uma interação entre o deficiente visual e a sinalização tátil existente. Este projeto poderá contribuir diretamente para a sociedade, disponibilizando um sistema de navegação conveniente para que as pessoas com deficiência visual tenham maior autonomia

<p>The aim of this study was to find out how features that have been found to be typical of women’s language, such as hedges, tag questions and a high level of talkativeness etc., are represented in a popular TV series. Five cross-sex conversations from one episode of the sitcom <em>The New Adventures of Old Christine </em>were analyzed, and the results show that many of the features of interest, as for instance tag questions, minimal responses and indirect style, are unexpectedly used more frequently by men in this small investigation. In fact, the only feature that was used more frequently by the female main character was hedges. Several factors affect the results of the study, as for instance the fact that the conversations are fictional. The special characteristics of the speakers also affect the results, as well as the tone and the topic of the chosen conversations. Many of the features of interest were used to a very small extent, which is probably a result of the fact that the language in a sitcom is to be entertaining and rather quick, which leaves little or no room for the features studied.</p>

Les collisions proton-proton à une énergie dans le centre de masse de 7 TeV au LHC produiront des bosons Z en grande quantité dont la mesure de la section efficace de production constitue un test du Modèle Standard. Après avoir décrit le contexte théorique et procédé à une description du détecteur ATLAS et des outils de simulation et d'analyse, une partie de cette thèse est consacrée à l'exploitation des événements cosmiques enregistrés dans la phase de préparation aux collisions afin de valider la simulation des formes de gerbes électromagnétiques dans un contexte proche de celui des collisions. Une deuxième partie de la thèse s'attache à la description des éléments clefs intervenant dans la mesure de la section efficace. Des critères de sélection utilisant des variables d'isolation, alternatifs à la sélection standard, ont été étudiés et ont mené à l'observation des premiers candidats Z->e+e- dans ATLAS. Enfin, les principales sources d'erreurs systématiques intervenant dans la mesure de section efficace ont été étudiées. Il s'agit des incertitudes liées d'une part à l'efficacité de sélection des électrons et d'autre part à l'efficacité géométrique du détecteur.

<p>DNA samples from crime scenes or mass disasters are often limited and degraded which limits the possibility of successful traditional STR analysis. Moreover, there is a need to decrease the turnaround time in criminal investigations. These circumstances require a wider set of assays and technologies to be investigated for potential use in forensic DNA analysis, which has been explored in this thesis work. DNA analysis can also provide a useful tool in forensic pathology investigations. </p><p>In a search for mutations involved in The Sudden Infant death Syndrome (SIDS), the entire mitochondrial genome was sequenced in six SIDS infants and shorter mtDNA regions were analysed in paraffin-embedded tissues from an additional 14 SIDS cases. In this sample material no mutations associated with SIDS were found that could explain the death of these infants. </p><p>To reduce time, cost and effort related to sequencing of the mtDNA HVI/HVII regions in caseworks, a HVI/HVII mtDNA linear array assay was used as a pre-screening for exclusions of suspects or evidence samples. Using this assay, 56% of the samples involved in casework analysis could be excluded before sequencing was undertaken.</p><p>The possibility to use the new array technology was explored in a SNP assay targeting both mtDNA and nuclear SNPs. The system relies on minisequencing in solution prior to hybridisation to tag arrays. Using this system, we demonstrate a rapid, highly multiplexable and flexible array-format for SNP analysis.</p><p>The properties of the Pyrosequencing technology being a fast and user-friendly assay was utilised in a study to investigate the possibility to use this method for limited and degraded samples. Ten STR loci, overlapping with standardised kits, were genotyped in 114 Swedish individuals. We found additional variation and higher resolution of repeats at some of these loci that are not detected using standard fragment analysis.</p>