Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primary HIV infection stage. This technique was combined with fluorescence correlation spectroscopy (FCS) which enables online detection of the HIV-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA probe at nanomolar concentration was introduced into the NASBA reaction mixture and hybridizing to a distinct sequence of the amplified RNA molecule. The specific hybridization and extension of this probe during amplification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1–1 nM (threshold for unaided FCS detection), the number of amplified RNA molecules in the further course of reaction could be determined. Evaluation of the hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration that was present at the beginning of amplification. The value of initial HIV-1 RNA number enables discrimination between positive and false-positive samples (caused for instance by carryover contamination)—this possibility of discrimination is an essential necessity for all diagnostic methods using amplification systems (PCR as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination of NASBA with FCS may also be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.

Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy

We have investigated the pH dependence of the dynamics of conformational fluctuations of green fluorescent protein mutants EGFP (F64L/S65T) and GFP-S65T in small ensembles of molecules in solution by using fluorescence correlation spectroscopy (FCS). FCS utilizes time-resolved measurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all processes that affect the fluorescence. Fluorescence excitation of a bulk solution of EGFP decreases to zero at low pH (pKa = 5.8) paralleled by a decrease of the absorption at 488 nm and an increase at 400 nm. Protonation of the hydroxyl group of Tyr-66, which is part of the chromophore, induces these changes. When FCS is used the fluctuations in the protonation state of the chromophore are time resolved. The autocorrelation function of fluorescence emission shows contributions from two chemical relaxation processes as well as diffusional concentration fluctuations. The time constant of the fast, pH-dependent chemical process decreases with pH from 300 μs at pH 7 to 45 μs at pH 5, while the time-average fraction of molecules in a nonfluorescent state increases to 80% in the same range. A second, pH-independent, process with a time constant of 340 μs and an associated fraction of 13% nonfluorescent molecules is observed between pH 8 and 11, possibly representing an internal proton transfer process and associated conformational rearrangements. The FCS data provide direct measures of the dynamics and the equilibrium properties of the protonation processes. Thus FCS is a convenient, intrinsically calibrated method for pH measurements in subfemtoliter volumes with nanomolar concentrations of EGFP.

Epigallocathechin-3 Gallate Selectively Inhibits the PDGF-BB–induced Intracellular Signaling Transduction Pathway in Vascular Smooth Muscle Cells and Inhibits Transformation of sis-transfected NIH 3T3 Fibroblasts and Human Glioblastoma Cells (A172)

Enhanced activity of receptor tyrosine kinases such as the PDGF β-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB–, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44mapk/p42mapk) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1–100 μM) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44mapk/p42mapk was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 μM EGCG resulted in an 80% and a complete inhibition of the PDGF-BB–induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1–100 μM) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-Rβ, phosphatidylinositol 3′-kinase, and phospholipase C-γ1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20–50 μM EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-Rβ and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea.

Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells

Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). FCS revealed that a large fraction of both intranuclear oligo(dT) (43%) and oligo(dA) (77%) moves rapidly with a diffusion coefficient of 4 × 10−7 cm2/s. Interestingly, this rate of intranuclear oligo movement is similar to their diffusion rates measured in aqueous solution. In addition, we detected a large fraction (45%) of the intranuclear oligo(dT), but not oligo(dA), diffusing at slower rates (≤1 × 10−7 cm2/s). The amount of this slower-moving oligo(dT) was greatly reduced if the oligo(dT) was prehybridized in solution with (unlabeled) oligo(dA) prior to introduction to cells, presumably because the oligo(dT) was then unavailable for subsequent hybridization to endogenous poly(A) RNA. The FCS-measured diffusion rate for much of the slower oligo(dT) population approximated the diffusion rate in aqueous solution of oligo(dT) hybridized to a large polyadenylated RNA (1.0 × 10−7 cm2/s). Moreover, this intranuclear movement rate falls within the range of calculated diffusion rates for an average-sized heterogeneous nuclear ribonucleoprotein particle in aqueous solution. A subfraction of oligo(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients obtained from FRAP experiments were in agreement with the FCS data. These results demonstrate that oligos can move about within the nucleus at rates comparable to those in aqueous solution and further suggest that this is true for large ribonucleoprotein complexes as well.

Identification of a viability domain in the granulocyte/macrophage colony-stimulating factor receptor beta-chain involving tyrosine-750.

The granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is a heterodimeric receptor expressed by myeloid lineage cells. In this study we have investigated domains of the GMR beta-chain (GMR beta) involved in maintaining cellular viability. Using a series of nested GMR beta deletion mutants, we demonstrate that there are at least two domains of GMR beta that contribute to viability signals. Deletion of amino acid residues 626-763 causes a viability defect that can be rescued with fetal calf serum (FCS). Deletion of residues 518-626, in contrast, causes a further decrement in viability that can be only partially compensated by the addition of FCS. GMR beta truncated proximal to amino acid 517 will not support long-term growth under any conditions. Site-directed mutagenesis of tyrosine-750 (Y750), which is contained within the distal viability domain, to phenylalanine eliminates all demonstrable tyrosine phosphorylation of GMR beta. Cell lines transfected with mutant GMR beta (Y750-->F) have a viability disadvantage when compared to cell lines containing wild-type GMR that is partially rescued by the addition of FCS. We studied signal transduction in mutant cell lines in an effort to identify pathways that might participate in the viability signal. Although tyrosine phosphorylation of JAK2, SHPTP2, and Vav is intact in Y750-->F mutant cell lines, Shc tyrosine phosphorylation is reduced. This suggests a potential role for Y750 and potentially Shc in a GM-CSF-induced signaling pathway that helps maintain cellular viability.

Bulletin.

Each no. has also a distinctive title.

Proceedings of the National Conference of Bargaining Cooperatives.

Mode of access: Internet.

Information.

Mode of access: Internet.

Establishment and characterization of a new epithelial cell line, KC-1, from koala (Phascolarctos cinereus) conjunctiva

A novel, untransformed koala cell line (KC-1) was established by culturing koala conjunctival tissue in growth medium, which has permitted the study of the cell biology of this unique system. After the establishment of the KC-1 cell line, the cells were characterized by light microscopy, doubling time, and Western blot analysis. Light microscopy revealed that the cells have an epithelial morphology. Doubling times were significantly different (P < 0.015) depending on fetal calf serum (FCS) concentration (16.5 h in 10% FCS and 26.5 h in 2% FCS). Cells constricted while in suspension but were shown to attach to the coverslip (or flask) and flatten rapidly, less than 1 h after seeding. To confirm the epithelial nature of the cells, protein was extracted and Western blot analysis was performed. Subsequent probing with primary and secondary antibodies (monoclonal anticytokeratin clone C-11 IgG1 and anti-mouse IgG) revealed two bands at 45 and 52 kDa (compared against a protein molecular weight marker) that correspond to primary type I keratin and major type II keratin, respectively, expressed in simple epithelial cells. The koala cell line was adapted to grow continuously in Dulbecco modified Eagle medium containing 10% FCS for at least 30 passages. This unique cell line is an ideal toot for further investigation on koala cell biology and cytogenetics and for exploration of the pathophysiological mechanism of eye infections caused by different pathogens in koalas.

Investigation of the role of protein kinase C (PKC) in cytostasis induced by tumour promoting phorbol esters and related compounds

Protein kinase C (PKC) is considered to be the major receptor for tumour promoting phorbol esters such as 12-0- tetradecanoylphorbol-13-acetate (TPA). These agents evoke a plethora of biological effects on cells in culture. The growth of A549 human lung carcinoma cells maintained in medium fortified with 10% foetal calf serum (FCS) is arrested for 6 days by TPA and other biologically active phorbol esters. In the work described in this thesis, the hypothesis was tested that modulation of PKC activity is closely related to events pivotal for cytostasis to occur. The effect of several phorbol esters, of newly synthesized analogues of diacylglycerols (DAG) and of bryostatins (bryos) on cell growth and ability to modulate activity of PKC has been investigated.Determination of the subcellular distribution of PKC following treatment of cells with TPA and partial enzyme purification by non-denaturing poly-acrylamide gel electrophoresis revealed translocation of enzyme activity from cytosoUc to paniculate fraction. Chronic exposure of cells to TPA resulted in a time and concentration dependent degradation of enzyme activity. Synthetic DAG and DAG analogues, unable to arrest the growth of cells at non-toxic concentrations, were neither able to affect subcellular PKC distribution nor compete effectively for phorbol ester binding sites at physiologically relevant concentrations. Bryos 1,2,4 and 5, natural products, possessing antineoplastic activity in mice, elicited transient arrest of A549 cell growth in vitro. They successfully competed for phorbol ester receptors in A549 cells with exquisite affinity and induced a shift in sub-cellular PKC distribution, though not to the same extent as PTA. Enzyme down-regulation resulted from prolonged exposure of cells to nanomolar concentrations of bryos. In vivo studies demonstrated that neither PDBu nor bryo 1 was able to inhibit A549 xenograft growth in athymic mice. The growth of A549 cell populations cultured under conditions of serum-deprivation was inhibited only transiently by biologically active phorbol esters. Fortification of serum-free medium with EGF or fetuin was able to partially restore sensitivity to maintained growth arrest by PTA. PKC translocation to the paniculate cellular fraction and subsequent enzyme down-regulation, induced by TPA, occurred in a manner similar to that observed in serum-supplemented cells. However, total PKC activity and cytosolic phorbol ester binding potential were greatly reduced in the serum-deprived cell population. Western blot analysis using monospecific monoclonal antibodies revealed the presence of PKC-a in both A549 cell populations, with significantly reduced protein levels in serum- deprived cells. PKC-/9 was not detected in either cell population.

Developing a process to create and validate an instrument assessing student attainment of competencies at an intercultural university in Mexico

This study took place at one of the intercultural universities (IUs) of Mexico that serve primarily indigenous students. The IUs are pioneers in higher education despite their numerous challenges (Bertely, 1998; Dietz, 2008; Pineda & Landorf, 2010; Schmelkes, 2009). To overcome educational inequalities among their students (Ahuja, Berumen, Casillas, Crispín, Delgado et al., 2004; Schmelkes, 2009), the IUs have embraced performance-based assessment (PBA; Casillas & Santini, 2006). PBA allows a shared model of power and control related to learning and evaluation (Anderson, 1998). While conducting a review on PBA strategies of the IUs, the researcher did not find a PBA instrument with valid and reliable estimates. The purpose of this study was to develop a process to create a PBA instrument, an analytic general rubric, with acceptable validity and reliability estimates to assess students' attainment of competencies in one of the IU's majors, Intercultural Development Management. The Human Capabilities Approach (HCA) was the theoretical framework and a sequential mixed method (Creswell, 2003; Teddlie & Tashakkori, 2009) was the research design. IU participants created a rubric during two focus groups, and seven Spanish-speaking professors in Mexico and the US piloted using students' research projects. The evidence that demonstrates the attainment of competencies at the IU is a complex set of actual, potential and/or desired performances or achievements, also conceptualized as "functional capabilities" (FCs; Walker, 2008), that can be used to develop a rubric. Results indicate that the rubric's validity and reliability estimates reached acceptable estimates of 80% agreement, surpassing minimum requirements (Newman, Newman, & Newman, 2011). Implications for practice involve the use of PBA within a formative assessment framework, and dynamic inclusion of constituencies. Recommendations for further research include introducing this study's instrument-development process to other IUs, conducting parallel mixed design studies exploring the intersection between HCA and assessment, and conducting a case study exploring assessment in intercultural settings. Education articulated through the HCA empowers students (Unterhalter & Brighouse, 2007; Walker, 2008). This study aimed to contribute to the quality of student learning assessment at the IUs by providing a participatory process to develop a PBA instrument.

Changing the Interface of Family and Consumer Sciences at Florida International University

The need to change the interface of the Family and Consumer Sciences (FCS) Program at Florida International University (FlU) has evolved because of changes in our family structure, culture, resources, educational reform, new knowledge in basic disciplines and applied research.

Determinação de recomendações bioclimáticas para habitação de interesse social de quatro climas do Rio Grande do Norte

Recomendações projetuais baseadas no clima são fontes importantes de diretrizes para os arquitetos. Uma das formas de obtenção dessas recomendações é por meio de normas, como a NBR 15220 (ABNT, 2005), que agrupa as cidades em zonas homogêneas quanto as estratégias sugeridas. O zoneamento bioclimático brasileiro divide o território do Rio Grande do Norte em duas zonas, a ZB 7 e ZB 8, que possuem algumas recomendações distintas, como aberturas grandes e vedações leves para a região litorânea contra aberturas pequenas e vedações pesadas para a região semiárida. Entretanto, outras configurações climáticas estão presentes no RN e não possuem clara recomendação. Este é o caso das regiões serranas e das regiões intermediárias entre o litoral e a região semiárida. Em virtude disso, este estudo visa encontrar recomendações bioclimáticas para habitações de interesse social nessas quatro condições climáticas. Para tanto, utilizou-se ferramenta computacional para realização de simulações das condições térmicas de três tipos de habitações populares - alongada, ramificada e compacta - e das seguintes estratégias: presença ou ausência de ventilação natural e sombreamento, massa térmica leve ou pesada, e Fator de Calor Solar alto ou baixo. Essas simulações foram realizadas para quatro condições climáticas do estado. Foram analisados os desempenhos de cada caso para identificar as estratégias e tipos de habitação recomendados para cada clima. De maneira geral, nos climas deNatal, Mossoró e Caicó viu-se que habitações com FCS baixo e com presença de ventilação natural possuem os melhores desempenhos. No clima de Areia, a ausência de ventilação aliada ao baixo FCS é que produziu os melhores desempenhos. O sombreamento melhora o desempenho nas cidades de clima quente, mas aumenta as horas com desconforto ao frio em Areia. A massa térmica varia de acordo com as demais estratégias, tendo casos em que habitações com massa térmica alta possuem melhores desempenhos que outros casos com massa térmica baixa. Por fim, viu-se que as estratégias para Natal, Caicó e Mossoró são bem semelhantes, com a recomendação de ventilação natural, FCS baixo nas vedações e sombreamento. Para Areia, a recomendação é não ventilar, ter FCS baixo e massa térmica alta.

Determinação de recomendações bioclimáticas para habitação de interesse social de quatro climas do Rio Grande do Norte

Recomendações projetuais baseadas no clima são fontes importantes de diretrizes para os arquitetos. Uma das formas de obtenção dessas recomendações é por meio de normas, como a NBR 15220 (ABNT, 2005), que agrupa as cidades em zonas homogêneas quanto as estratégias sugeridas. O zoneamento bioclimático brasileiro divide o território do Rio Grande do Norte em duas zonas, a ZB 7 e ZB 8, que possuem algumas recomendações distintas, como aberturas grandes e vedações leves para a região litorânea contra aberturas pequenas e vedações pesadas para a região semiárida. Entretanto, outras configurações climáticas estão presentes no RN e não possuem clara recomendação. Este é o caso das regiões serranas e das regiões intermediárias entre o litoral e a região semiárida. Em virtude disso, este estudo visa encontrar recomendações bioclimáticas para habitações de interesse social nessas quatro condições climáticas. Para tanto, utilizou-se ferramenta computacional para realização de simulações das condições térmicas de três tipos de habitações populares - alongada, ramificada e compacta - e das seguintes estratégias: presença ou ausência de ventilação natural e sombreamento, massa térmica leve ou pesada, e Fator de Calor Solar alto ou baixo. Essas simulações foram realizadas para quatro condições climáticas do estado. Foram analisados os desempenhos de cada caso para identificar as estratégias e tipos de habitação recomendados para cada clima. De maneira geral, nos climas deNatal, Mossoró e Caicó viu-se que habitações com FCS baixo e com presença de ventilação natural possuem os melhores desempenhos. No clima de Areia, a ausência de ventilação aliada ao baixo FCS é que produziu os melhores desempenhos. O sombreamento melhora o desempenho nas cidades de clima quente, mas aumenta as horas com desconforto ao frio em Areia. A massa térmica varia de acordo com as demais estratégias, tendo casos em que habitações com massa térmica alta possuem melhores desempenhos que outros casos com massa térmica baixa. Por fim, viu-se que as estratégias para Natal, Caicó e Mossoró são bem semelhantes, com a recomendação de ventilação natural, FCS baixo nas vedações e sombreamento. Para Areia, a recomendação é não ventilar, ter FCS baixo e massa térmica alta.

Stochastic Modeling of Reversible Biochemical Reaction-Diffusion Systems and High-Resolution Shock-Capturing Methods for Fluid Interfaces

Thesis (Ph.D.)--University of Washington, 2016-08