The mixed-signal and analog design on a pre-diffused array is a challenging task, given that the digital array is a linear matrix arrangement of minimum-length transistors. To surmount this drawback a specific discipline for designing analog circuits over such array is required. An important novel technique proposed is the use of TAT (Trapezoidal Associations of Transistors) composite transistors on the semi-custom Sea-Of-Transistors (SOT) array. The analysis and advantages of TAT arrangement are extensively analyzed and demonstrated, with simulation and measurement comparisons to equivalent single transistors. Basic analog cells were also designed as well in full-custom and TAT versions in 1.0mm and 0.5mm digital CMOS technologies. Most of the circuits were prototyped in full-custom and TAT-based on pre-diffused SOT arrays. An innovative demonstration of the TAT technique is shown with the design and implementation of a mixed-signal analog system, i. e., a fully differential 2nd order Sigma-Delta Analog-to-Digital (A/D) modulator, fabricated in both full-custom and SOT array methodologies in 0.5mm CMOS technology from MOSIS foundry. Three test-chips were designed and fabricated in 0.5mm. Two of them are IC chips containing the full-custom and SOT array versions of a 2nd-Order Sigma-Delta A/D modulator. The third IC contains a transistors-structure (TAT and single) and analog cells placed side-by-side, block components (Comparator and Folded-cascode OTA) of the Sigma-Delta modulator.

Made available in DSpace on 2014-12-05T18:41:21Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) victor_silva_ioc_dout_2009.pdf: 22194333 bytes, checksum: 32cf0b37f0ab6f7e74531335d04fecbe (MD5) Previous issue date: 2014-11-18 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Protozoários parasitos aparecem como co-patógenos em infecções pelo vírus da imunodeficiência humana (HIV)-1, resultando em um aumento mútuo na replicação viral e parasitária, e facilitando a progressão clínica de ambas as doenças. Os mecanismos pelos quais o HIV-1 induz um aumento na replicação do protozoário são desconhecidos. Neste trabalho, nós investigamos o papel do HIV-1 e da proteína trans-ativadora (Tat) do HIV-1 no aumento da replicação parasitária em macrófagos humanos primários co-infectados ou não com HIV-1 e Leishmania amazonensis ou com HIV-1 e Blastocrithidia culicis. Em alguns experimentos, macrófagos foram infectados somente com L. amazonensis ou B. culicis e expostos à proteína Tat recombinante do HIV-1. As replicações dos protozoários e do HIV-1 foram analisadas por índice endocítico ou ensaio imunoadsorvente ligado a enzima (ELISA) para p24, respectivamente. A infecção pelo HIV-1 dobrou a replicação da Leishmania em macrófagos, e soro contra o Tat do HIV-1 reduziu significativamente a replicação exacerbada do protozoário, indicando uma importante função desta proteína, a qual é liberada pelas células infectadas com HIV-1, neste processo. Corroborando estes resultados, a exposição de macrófagos infectados somente por Leishmania ao Tat recombinante (100 ng/mL) mimetizou a infecção pelo HIV-1. A multiplicação do protozoário diminuiu quando células infectadas por Leishmania foram tratadas com Tat na presença de anticorpos neutralizantes contra o Fator de Crescimento e Transformação (TGF)-b1, demonstrando a participação desta citocina no aumento da replicação da L. amazonensis em macrófagos. O tratamento com Tat induziu a expressão da enzima Ciclo-oxigenase (COX)-2 e a secreção de Prostaglandina E2 (PGE2), e o bloqueio da produção de PGE2 aboliu o aumento da replicação da Leishmania induzida por Tat Adição exógena de PGE2 estimulou o crescimento da Leishmania em macrófagos, e a neutralização imune de TGF-b1 abrandou este efeito. Analisados em conjunto, nós concluímos que Tat estimula a replicação da Leishmania via indução da síntese de PGE2 e conseqüentemente secreção de TGF-b1. Para avaliar se a infecção pelo HIV-1 desativa a atividade microbicida do macrófago, células infectadas com HIV-1 foram co-infectadas com um protozoário não patogênico (Blastocrithidia culicis), e nós observamos que a infecção pelo HIV-1 favoreceu a sobrevivência deste tripanossomatídeo. Por microscopia eletrônica, nós verificamos que tanto o HIV-1 quanto a B. culicis co-habitavam um mesmo macrófago, e que formas em divisão do protozoário podiam ser observadas no interior de macrófagos. De forma similar aos encontrados nos experimentos com Leishmania, o Tat ou o TGF-b1 dobraram o crescimento do protozoário em macrófagos infectados somente por Blastocrithidia. Em conclusão, nós identificamos, pela primeira vez, uma molécula do HIV-1 que promove a multiplicação de um protozoário patogênico (Leishmania), e permite a sobrevivência/crescimento em macrófagos humanos primários de um protozoário habitualmente não patogênico. Uma vez que a neutralização imune do Tat tem sido estudada como uma estratégia de vacinação contra o HIV-1, nossos resultados sugerem que a neutralização desta proteína também pode ser salutar no combate ao protozoário em casos de co-infecção / Protozoan parasites appear as human immunodeficiency virus (HIV)-1 co-pathogens, resulting in a mutuall enhancement of viral and parasite replication, and facilitating the clinical progression of both diseases. The mechanisms by which HIV-1 induces up-modulation of protozoan replication are unknown. In this work, we investigated the role of HIV-1 and HIV-1 transcriptional transactivator (Tat) protein in the enhancement of parasite replication in primary human macrophages co-infected or not with HIV-1. Human macrophages were co-infected with HIV-1 and either with Leishmania amazonensis or Blastocrithidia culicis. In selected assays, macrophages were infected only with L. amazonensis or B. culicis and exposed to recombinant HIV-1 Tat protein. Protozoan and HIV-1 replication were analyzed by endocytic index or p24 Enzyme Linked Immuno Sorbent Assay (ELISA), respectively. HIV-1 infection doubled the Leishmania replication in macrophages, and Tat antiserum significantly reduced the exacerbated parasite replication, pointing to a direct role of Tat protein released from HIV-1-infected cells in this process. Corroborating this finding, exposure of Leishmania-infected macrophages to recombinant Tat (100 ng/mL) mimicked HIV-1 infection. Protozoan replication diminished when Leishmania-infected cells were treated with Tat in the presence of neutralizing anti-Transforming Growth Factor (TGF)-b1 antibodies, showing a participation of this cytokine in the augmentation of L. amazonensis multiplication in macrophages. Interestingly, Tat induced the expression of the enzyme Cyclooxygenase (COX-2) and Prostaglandin E2 (PGE2) secretion, and blockage of PGE2 production abrogated the increased Leishmania replication induced by Tat. Exogenous addition of PGE2 elicited Leishmania replication in macrophages, and immunoneutralization of TGF-b1 blunted this effect. Taken together, we deciphered that Tat stimulates Leishmania replication via induction of PGE2 synthesis and consequently TGF-b1 secretion. To evaluate whether HIV-1 infection deactivates the microbicidal activity of macrophages, HIV- 1-infected cells were co-infected with a non-pathogenic protozoan (Blastocrithidia culicis), and we found that HIV-1 infection favors the survival of this trypanosomatid. By electron microscopy, we verify that both HIV-1 and B. culicis co-habited the same macrophage, and that dividing forms of the protozoan can be observed inside the macrophage. Similarly, Tat or TGF-b1 doubled the protozoan growth in Blastocrithidia-infected macrophages. In conclusion, we identified, for the first time, an HIV-1 molecule that promotes multiplication of a pathogenic protozoan (Leishmania) and permit survival of an otherwise non-pathogenic protozoan in primary human macrophages. Because immunoneutralization of HIV-1 Tat has been studied as a vaccination strategy against HIV-1, our results suggest that neutralization of this protein could be salutary in the combat against the protozoan in the cases of co-infection

The mixed-signal and analog design on a pre-diffused array is a challenging task, given that the digital array is a linear matrix arrangement of minimum-length transistors. To surmount this drawback a specific discipline for designing analog circuits over such array is required. An important novel technique proposed is the use of TAT (Trapezoidal Associations of Transistors) composite transistors on the semi-custom Sea-Of-Transistors (SOT) array. The analysis and advantages of TAT arrangement are extensively analyzed and demonstrated, with simulation and measurement comparisons to equivalent single transistors. Basic analog cells were also designed as well in full-custom and TAT versions in 1.0mm and 0.5mm digital CMOS technologies. Most of the circuits were prototyped in full-custom and TAT-based on pre-diffused SOT arrays. An innovative demonstration of the TAT technique is shown with the design and implementation of a mixed-signal analog system, i. e., a fully differential 2nd order Sigma-Delta Analog-to-Digital (A/D) modulator, fabricated in both full-custom and SOT array methodologies in 0.5mm CMOS technology from MOSIS foundry. Three test-chips were designed and fabricated in 0.5mm. Two of them are IC chips containing the full-custom and SOT array versions of a 2nd-Order Sigma-Delta A/D modulator. The third IC contains a transistors-structure (TAT and single) and analog cells placed side-by-side, block components (Comparator and Folded-cascode OTA) of the Sigma-Delta modulator.

Nanoparticles are used more and more extensively in today's society, especially in the industry sector. Humans get exposed to nanoparticles daily but the effect is a topic that has not been fully explored yet and its effect on humans is still unknown.The purpose of this project was to investigate whether the size of nanoparticles is a factor that influences their effect on humans, mainly the effect on blood activation. In order to study this, nanopaticles of polystyrene with three different sizes (75, 120 and 260 nm) were selected and incubated in a human whole blood model, the Chandler loop. The samples from the Chandler loop experiments were analysed with three different ELISA's: C3a, terminal complement complex (TCC, sC5b-9) and thrombin-antithrombincomplexes (TAT).The results in this study indicate that the smallest nanoparticle has a higher potential for activating the coagulation system than the larger ones. The complement system did not seem to be significantly activated from the nanoparticles. More experiments needs to be done in order to get a better statistic value but just as it is the results look promising and there is a tendency for a higher activation of the coagulation system with the 75 nm nanoparticles.

Ce travail de thèse a comporté deux parties distinctes : l'une porte sur l'étude du complexe TAR/Tat/cycline T1 impliqué dans la transactivation de la transcription de l'ARN du virus HIV-1, l'autre concerne différentes facettes de la régulation de l'épissage de l'ARN du virus HIV-1. L'interaction de la protéine virale Tat avec l'élément TAR présent à l'extrémité 5' des ARN du virus HIV-1 d'une part, et la cycline T1, composant du complexe p-TEFb responsable de l'hyperphosphorylation de l'ARN polymérase II d'autre part, est primordial pour obtenir des ARN viraux de pleine longueur. Dans l'objectif de réaliser une étude structurale du complexe TAR/Tat/cycline T1, l'ARN TAR et un fragment de la cycline T1 ont été produits en grandes quantités. De nombreuses tentatives de complexation des trois partenaires (TAR, Tat et cycline T1) ont été effectuées, mais la qualité des cristaux n'était pas suffisante pour une étude radiocristallographique du complexe. L'épissage est une étape majeure du cycle de multiplication du virus HIV-1. Son ARN comporte 5 sites donneurs et 8 sites accepteurs dont l'utilisation combinée permet la production des 9 ORF virales. Les variations de l'épissage alternatif de l'ARN du virus HIV-1 en fonction de l'expression de protéines virales Tat et Nef ont été étudiées. Nous avons par ailleurs étudié l'effet des protéines SR sur l'utilisation des sites accepteurs A2 et A3. L'étude fine de l'élément régulateur ESEt du site A3 a révélé l'implication de la protéine SR 9G8 dans le schéma complexe de régulation de ce site. / This work of thesis contained two different parts : the one concerns the study of the TAR/Tat/cycline T1 complex involved in the transactivation of the transcription of the HIV-1 RNA, the other one concerns various facets of the regulation of the splicing of the HIV-1 RNA. The interaction of the viral protein Tat with the TAR element present in the 5 ' extremity of the HIV-1 RNA on one hand, and the cycline T1, composing of the complex p-TEFb responsible for the hyperphosphorylation of the RNA polymerase II on the other hand, is essential to obtain viral RNA of full length. In the objective to realize a structural study of the complex TAR/Tat/cycline T1, TAR RNA and a fragment of the cycline T1 were produced in appropriate quantities. Numerous attempts of complexation of three partners (TAR, Tat and cycline T1) were made, but the quality of crystals was not sufficient for a radiocristallographic study of the complex. Splicing is a major stage of the cycle of reproduction of the virus HIV-1. His RNA contains 5 donor splice sites and 8 acceptor splice sites whose combined use allows the production 9 viral ORF. The variations of the alternative épissage of HIV-1 RNA according to the expression of viral proteins Tat and Rev were studied. We besides studied the effect of proteins SR on the use of acceptor splice sites A2 and A3. The fine study of the regulating element ESEt of the site A3 revealed the involvement of the SR protein 9G8 in the complex regulation of this site.

The social diversity of the South African population holds considerable challenge for psychologists, especially in respect of differences in language, culture and socio-economic context. The implications of the diverse nature of expectations and needs of unique individuals, clients as well as professionals, in particular in psychological assessment, are of concern. Projected storytelling in assessment is widely recognised as valuable, especially when working with children and adolescents. The technique has its pitfalls, including the way the stories produced may be influenced by leading questions, applying different methods of interpretation, and administering the instrument in cross-cultural assessment situations. Psychologists presenting projection plates to adolescent clients in South Africa frequently obtain little more than one-liners from standard procedures, raising doubts about viability and reliability of the technique. Prompting and probing need to be enhanced without compromising the projective value of responses or the uniqueness of clients. Feuerstein pioneered mediated intervention for learners with cognitive barriers, and the dynamic assessment of culturally different children. In this study, a dynamic assessment technique of questioning (DATQ) was used to actualise projection potential in mono- and cross-cultural assessment situations. The aim of the study was two-fold: to investigate the influence of a DATQ with projection plates during the psychological assessment of adolescents, and to investigate the influence of culture on such assessment in mono- and cross-cultural situations. A qualitative, multiple case study of ten participants representing five language groups in South Africa was undertaken within a predominantly postmodern epistemology. The tension between assessment from the positivist and post-modern paradigms was acknowledged through applying different perspectives during different stages of the research. A test-training-continuation-of-test situation was created for the administration of seven projection pictures, after which two discussion protocols were used. Data-analysis and interpretation took place in four phases by way of projection analysis (using the Bellak TAT Analysis Blank and Haworth’s analysis of defences), structural analysis (with categories such as word-count, response pattern, formulation, number of statements, prompts, hesitations, repetitions), analysis of the participant’s experience of the Murray-method versus the dynamic assessment technique of questioning, and analysis of possible cross-cultural influences on the assessment (utilizing the Scoring Sheet for the Psychocultural Scoring System (SSPSS) and triangulating the results with the projection analysis and the thematic analysis of the conversation about culture). Findings were derived from intra-comparison (per participant) and inter-comparison (per phase of the assessment) of the analyses. The main conclusions of the study point towards participants’ projective responses increasing and deepening in the direction of self-understanding and wholesome problem solution as well as being structurally enhanced, their emotional experience of the assessment situation being positive, culturally associated values being expressed and cultural barriers to interaction being lessened in both mono- and cross-cultural assessment. Whilst projection isn’t an exclusively context-bound phenomenon and generally occurs irrespective of cultural specificity, it was found that supporting clients through non-directive prompting to voice their associations apparently didn’t interfere with the unconscious content being solicited, irrespective of the mono-/cross-cultural nature of the assessment. / Thesis (PhD)--University of Pretoria, 2011. / Educational Psychology / unrestricted

In South Africa, where a large portion of the population lives in townships, more often than not, the therapist and client do not share a similar cultural context. Cultural knowledge is therefore pertinent to generating a complex and thorough interpretation of any psychological assessments. This study aims to explore possible cultural narratives evoked in the responses to the Thematic Apperception Test so that cross-cultural use of the test would be more effective in the Mamelodi township. The research is done from a narrative point of view, where lived experience is understood by organising it into structured narratives or stories that repeat throughout a person’s life. The pictures of the TAT were viewed as a context that could elicit such life narratives from respondents. TAT stories from five adolescent residents in Mamelodi were thematically analysed as a method of identifying common stories that could reflect the cultural narratives that young persons in Mamelodi draw from to make sense of their world. The results indicated common narratives concerning the following: the experience of violence and danger, the experience of close relationships, dealing with challenges, and the role that clothes play. These findings, and possible findings from similar future research, might aid psychologists towards a better understanding of the TAT in the township context. Copyright / Dissertation (MA)--University of Pretoria, 2013. / Psychology / unrestricted

The mechanisms underlying cannabinoid impairment of fear memory is not clear. This study investigated the effects of the synthetic cannabinoid HU210 and the endocannabinoid hydrolysis inhibitor JZL 195 on fear memory following contextual fear conditioning (CFC; an animal model of fear). The long-term depression (LTD)-blocking peptide Tat-GluR2 was utilized to investigate whether the expression of cannabinoid-induced LTD (CB-LTD) is required for the cannabinoid impairment of acquisition and consolidation of contextual fear memory. HU210 reduced freezing throughout the test phase of the acquisition protocol, which was not affected by pre-administration of Tat-GluR2. High and moderate doses of HU210 reduced freezing during the first and last half, respectively, of the test phase of the consolidation protocol, which was prevented by pre-treatment with Tat-GluR2. HU210 did not affect freezing during the test phase of the retrieval protocol. Thus, these results suggest that HU210 impairs acquisition and consolidation, but not retrieval of contextual fear memory, and that in vivo CB-LTD expression is required for HU210 impairment of the consolidation, but not acquisition, of contextual fear memory. We also observed that HU210 and JZL 195 do not facilitate the acquisition of contextual fear memory extinction.

Cell penetrating peptides have been the focus of drug delivery research for 15 years due to their apparent ability to deliver cargoes inside cells more readily than many other carriers. Using two of the most commonly studied peptides (tat47-57 and R9), the present study differs from previous work by deliberately choosing to observe uptake with lower peptide concentrations closer to potential therapeutic doses, and by implementing raster image correlation spectroscopy (RICS) on a commercial microscope to quantify uptake in parallel to other techniques such as fluorescence correlation spectroscopy (FCS), confocal microscopy, and mass spectroscopy.An initial study using mass spectrometry and ExPASy (Expert Protein Analysis System) revealed that the peptides are stable for at least one hour in PBS. Based on this initial information and other experimental conditions, the study took two main directions with regards to the peptide: the membrane interaction and accumulation in the cell.The peptides interaction with the cell membrane revealed that neither tat-TAMRA nor R9-TAMRA disrupts the membrane of cells: incorporation of FM2-10 in the membrane was not modified in K562 cells whilst it was in presence of the control lytic peptides GALA and mellitin. Based on this information confocal microscopy was utilised to assess the localisation on the cell membrane. Peptide binding to the membrane appeared to be heterogeneous in distribution at 1µM bulk concentration.Accumulation in cells of the peptides was observed incubated at 37°C, confocal microscopy showed punctuated distribution with intracellular aggregations of fluorescence measuring between 2.5-3.5µm in diameter. Co-staining with a nuclear dye revealed these aggregations to be focused around the nucleus of the cell. Initial FCS experiments indicated a concentration dependent accumulation of the peptide in the cells and a decrease of the intracellular diffusion coefficients at high concentration possibly corresponding with molecular crowding. Interestingly the anomalous diffusion model did not statistically improve the results.RICS was implemented to study the kinetics of entry of TAMRA labelled cell penetrating peptides in both Caco-2 and HeLa cells lines at concentrations between 500nM and 2µM. Concentrations above 1µM exhibited higher final intracellular concentrations, yet the measured diffusion coefficients were similarly independent of extracellular concentration. Both peptides appeared to enter the cell quickly with a fast initial uptake over the first 10 minutes, reaching a concentration maxima after 30 minutes.Overall, the study reveals that many published studies may be incorrect as they may only be reporting the presence of a fluorescent dye inside the cell not the peptide. The fast binding of the peptide to the membrane is likely to cause false positive results when traditionally studying internalisation kinetics such as using flow cytometry and confocal microscopy. Correlation spectroscopy techniques such as FCS, provide useful information on internalisation of the peptides, but the single spot measurement is limited when providing information on the entire cell. RICS is a progression of correlation spectroscopy and provides a more representative picture of the cell.

The current study investigated the relationship between clients' object relations functioning and the working alliance. The Social Cognition and Object Relations Scale (SCORS; Westen, 1991), an object relations scoring system for the TAT, was used to assess object relations functioning. Forty-eight therapy clients at a university-based training clinic were administered the TAT, Adult Attachment Scale (AAS), Symptom Checklist 90-Revised (SCL-90-R; Derogatis, 1977), and the short form of the Marlowe-Crowne Social Desirability Scale (MCSD; Crowne & Marlowe, 1960). Following the initial assessment of client characteristics shortly after intake, clients and their therapists rated the working alliance 3 sessions later. Results indicated that the SCORS was significantly correlated with client and therapist ratings of the working alliance. The current study also assessed the predictive validity of the SCORS by examining how its various scales are related to aspects of the working alliance and the other measures used in this study. The findings suggest that the relationship between object relations functioning, the working alliance, symptom severity, and attachment disturbance depends on the aspect of object relations that is being assessed.