3,768 research outputs found
Graduate Recital: Randal Jones, piano
No full textMr. Jones is a student of Mary Norris.Sonata in A Minor, K. 310, Mozart -- Variations Sérieuses, Op. 54, Mendelssohn -- Images, Bk. II, Debussy -- Sonata para Piano, Ginaster
Master Recital: Randal E. Jones, piano
No full textMr. Jones is a student of Mary NorrisPrelude and Fugue in B flat Minor, Bk. 1, Bach -- Sonata in C Minor, Op. 111, Beethoven -- Scherzo in B Minor, Op. 20, Chopin -- Mazurkas, Op. 7 No. 2, Chopin -- Mazurkas, Op. 24 No 1, Chopin -- Mazurkas, Op. 68 No. 2, Chopin -- Alborado del Gracioso, from "Miroirs", Rave
The process of re-designing the geometry curriculum: the case of the Mathematical Association in England in the early twentieth century
No full textThis paper examines a key period of change in geometry teaching in England. Our focus is the character and nature of the recommendations of the 1902 geometry report of the UK Mathematical Association. We analyse historical documents of the Mathematical Association using a theoretical framework informed by work in the sociology of education. Our analysis shows that the character and recommendations of the Mathematical Association report were influenced by various factors including: that Mathematical Association members at the time still respected the traditional Euclidean approach to geometry as a basis for school geometry; that the academic and ‘power’ resources available to the Mathematical Association at the time were not sufficient to enable a complete change from the traditional approach; that a lack of consensus between the various members of the Mathematical Association prevented a more radical proposal; and that the general climate in schools at that time was not prepared for far-reaching changes to the teaching of geometry. These findings accord with other research on educational reform which indicates that curriculum change processes are invariably complex and often subject to much politicking
The process of re-designing the geometry curriculum: the case of the Mathematical Association in England in the early 20th Century
No full textThis paper examines a key period of change in geometry teaching in England. Our focus is the character and nature of the recommendations of the geometry report of the UK Mathematical Association in 1902. We analyse historical documents of the Mathematical Association using a theoretical framework developed from Cooper’s model. Our analysis shows that the character and recommendations of the Mathematical Association report was influenced by various factors including: that the Mathematical Association members still respected the traditional Euclidean approach to geometry as a basis for school geometry; that the academic and power resources available to the Mathematical Association at the time were not sufficient for a complete change from the traditional approach; that conflicts between the various members of the Mathematical Association prevented a complete consensus; and that the climate outside the teaching committee of the Mathematical Association was not ready for radical reform at that time
In vitro and in vivo characterization of the cytomegalovirus and polyomavirus BK specific immune response
No full textDuring my PhD thesis several aspects of the interaction of Cytomegalovirus
and Polyomavirus BK with the host’s immune system were examined (see list
of publications). The overall aim was to compare immune response in healthy
individuals and kidney transplant recipients with or without viral replication.
In healthy individuals, Polyomaviruses BK and JC infect 80% and 60%,
respectively. For CMV seroprevalences may reach up to 80%. Intermittent
virus shedding in urine is observed for BKV in 7%, JCV in 19% and CMV in
0%. However, no virus replication in plasma was detected. Posttransplant,
mainly due to prolonged immune suppression the amount and function of
CMV- and BKV-specific T-cells is impaired. Calcineurin inhibitors lead to a
direct reduction of INFγ production of virus-specific T-cells, whereas antiproliferative
immunosuppressives reduce the expansion capacity in a dosedependent
manner. This may be a major reason for uncontrolled virus
replication.
The humoral response reflects the amount of recent antigen exposure and
does not directly indicate protection from virus replication. Virus-specific
cellular immune responses would probably allow to assessing the risk of
future replication.
Overall the importance of CMV and BKV specific T-cells posttransplant in
controlling virus replication was examined. For both viruses we could
calculate a protective threshold of virus-specific T-cells. CMV-pp65 specific
CD4 T-cells above 0.03% were significantly associated with no CMV
replication during the next eight weeks. Additionally, below this cut-off, CMV
seropositive recipients developed more often GCV-resistant CMV replication.
During BKV replication, patients with more than 69 BKV-LT specific T-cells
per 1 Mio PBMCs were significantly more often showing decreasing BKV
loads in plasma. As virus-specific T-cells seem to be crutial in reducing virus replication, and
reduction of immune suppression harbours the risk of acute rejection, a
booster vaccine could be a new therapeutic option. A booster vaccine could
probably elevate the amount of virus-specific T-cells above a critical threshold
of protection from disease, despite effects of immune suppression.
We tried to identify immunodominant regions with the CMV pp65 and BKV LT
proteins. We used a combined approach of computer prediction algorithms
and experimental verification. Epitope mapping of BKV LT with computer
prediction revealed several clusters, which could be immunodominant and
also potentially be processed and recognized in various HLA backgrounds.
The identified cluster regions were synthesised as 15 and 25mers. Expansion
and re-stimulation with predicted epitopes could so far confirm the HLA A and
B-specific prediction of single 9mers covered by the larger 15 and 25mer
sequences. However, other HLA types need to be tested for direct stimulation
and expansion potential of the predicted epitopes. Additionally, tetramer
staining should be performed for verification.
Based on this research, we will be able to improve current immune monitoring
and probably a high-specific peptide-based vaccine against BKV LT could be
developed and be used to increase the amount of BKV-LT specific T-cells.
Another potentially therapeutic agent could be the blockade of PD1 ligand.
PD1 expression in chronic virus infection lead to impaired CD8+ T-cell
function. CMV-specific CD4 T-cells treated with an inhibitory antibody against
PD1 ligand, and thereby activating CD4+ T-cells, lead to a increase of the
expansion capacity. We have shown, that the anti-PD1 ligand antibody
increases various cytokines. This could be also tested for BK virus.
Measurement of virus-specific T-cells may replace serological assays in the
future, due to a better correlation to effective antiviral control, which can be
used as monitoring tool during infection and post-vaccination
Characterizing determinants of BK Polyomavirus-specific immune response
No full textBK polyomavirus (BKPyV) is one of now 13 human polyomavirus (HPyV) species detected in humans. BKPyV is only known to infect humans and seroprevalence rates of more than 90% have been reported in adult populations around the world. Following primary infection, BKPyV persists in the renourinary tract without causing any disease as evidenced by urinary shedding in 5% - 10% of healthy immunocompetent blood donors.
In immunocompromised persons, however, BKPyV can cause significant diseases whereby uncontrolled high-level replication may lead to organ invasive pathologies in kidneys, bladder, lungs, vasculature, and the central nervous system. The most consistently found diseases are BKPyV-associated hemorrhagic cystitis (BKPyVHC) in 5%-20% allogeneic hematopoietic stem cells transplant patients, and BKPyV-associated nephropathy (BKPyVAN) in 1%-15% of kidney transplant patients. BKPyVHC is highly symptomatic with pain, anemic bleeding, and increased mortality. BKPyVAN is asymptomatic except for progressive renal failure and premature return to dialysis. Both entities are characterized by high-level viral replication i.e. with urine BKPyV loads of 8-10 log10 Geq/mL, plasma BKPyV loads often above 4 log10 Geq/mL, and an allogeneic constellation between the virus-infected host cell and the available T-cell effectors. Despite these similarities, the clinical manifestations are strikingly different suggesting relevant, but experimentally undefined differences in pathogenesis. Thus, BKPyVHC typically occurs within 4 weeks after allogeneic HSCT and is confined to the bladder, and typically without kidney involvement. By contrast, BKPyVAN is diagnosed around 3-6 months after kidney transplantation and confined to the kidney allograft without causing cystitis. Although high-level BKPyV replication should be formally amenable to antiviral drug treatment, no effective and BKPyV-specific antiviral therapy is currently available. Therefore, a better understanding of the immune alteration in both diseases has been deemed essential to identify patients at risk and to develop prophylactic, preemptive and therapeutic strategies.
The currently recommended strategy for BKPyVAN is to screen kidney transplant patients for BKPyV replication and to promptly reduce immunosuppressive therapy in those with significant replication to facilitate mounting of BKPyV-specific T cell responses and thereby preventing progression to disease. This manoeuver has been linked to expanding BKPyV-specific T cell responses in the peripheral blood of kidney transplant patients. However, this approach may place patients at risk for acute rejection episodes that predispose equally well to premature kidney transplant failure. Although the clinical feasibility of reducing immunosuppression and curtailing BKPyV replication has been shown to be effective in prospective cohort studies for many, but not all of kidney transplant patients, this approach has not been possible in allogeneic HSCT patients because of concurrent or imminent graft-versus host disease. Thus, there are significant gaps in the current understanding of the BKPyV– host interaction in the normal host and in the allogeneic setting, which need to be investigated for a more effective and safer management of these significant viral complications.
In this thesis, the interaction of BKPyV and the immune response has been approached from two different angles. In the first project, potential mechanisms of BKPyV immune evasion were studied. Here, we focused on a small accessory protein called agnoprotein encoded as a leader protein in the late viral early region (LVGR). Although HPyV genomes overall show a very similar genome organization, agnoproteins are only found in the genomes of BKPyV and JCPyV that have a kidney tropisms, but not in any of the other 11 presumably non-renotropic HPyVs. We hypothesized that agnoprotein could play a role in immune evasion by downregulating HLA expression. The effects of agnoprotein were studied on HLA class I and II expression in vitro by flow cytometry following transfection of primary human renal tubular epithelial cells, which are the viral target of BKPyV-associated nephropathy. In addition, transfected human UTA-6 cells were studied as well as UTA-6 cells bearing a tetracycline-regulated agnoprotein. As control, the effects were compared with the ICP47 protein of Herpes simplex virus-1, which has been previously reported to effectively down-regulate HLA class I. Although both viral proteins share some similarities at the protein level, our results showed that BKPyV agnoprotein did not down-regulate HLA class I or class II molecules. Also, there was not inhibitory effect on the increase of HLA-class I or class-II surface expression following exposure to interferon-. By contrast, ICP47 reduced HLA class I surface expression, but not class II. We also evaluated effects of agnoprotein on virus epitope-specific T-cell killing by 51Chromium release assay, however no interference could be observed. We concluded that agnoprotein did not contribute to these types of HLA-dependent immune evasion processes. However, further investigations are needed to understand if agnoprotein could contribute to viral immune escape by other mechanisms.
In the second project, we aimed at better characterizing BKPyV-specific CD8 T cell immunity targeting epitopes encoded in the early viral gene region (EVGR). Selected coding sequences of the BKPyV EVGR were submitted to two web-based computer algorithms (SYFPEITHI, IEDB) in order to predict immunodominant 9mer epitopes presented by 14 frequent HLA-class I molecules. For an experimental confirmation, 97 different 9mer epitopes were chemically synthesized and tested in 42 healthy individuals. A total of 39 epitopes could be confirmed by interferon- ELISpot assay in at least 30% of healthy individuals. Interestingly, most of the 9mer epitopes appeared to cluster in short amino acid stretches, and some 9mer could be presented by more than one HLA class I allele as expected for immunodominant domains. HLA-specific presentation was demonstrated by 9mer- MHC-I streptamers for 21/39 (54%) epitopes. The 9mer dependent T-cell killing by 51Chromium release assay and the CD107a surface detection indicated that the 9mer epitopes could be recognized by cytotoxic T-cells. Moving to a clinically relevant situation, 13 9mer epitopes could be validated in 19 kidney transplant patients protected from, or recovering from, BKPyV viremia. The results suggest that, pending further corroboration in larger patient populations, novel 9mer epitopes can be identified, which are associated with CD8 T cell control of BKPyV replication. Thus the identified immunodominant 9mer T-cell epitopes could be further developed for clinical assays to better predict the risk and the recovery of BKPyV diseases, help guiding immunosuppression reduction, and to develop specific adoptive T-cell therapy or vaccine responses to prevent or treat BKPyV-associated disease
Desenvolvimento e aplicação de um sistema celular repórter para herpes simplex virus e padronização de uma PCR quantitativa para poliomavírus BK
No full textDissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2014.Pacientes imunodeprimidos podem apresentar infecções virais com evolução rápida, sintomatologias atípicas graves e muitas vezes fatais, sendo fundamental um diagnóstico precoce para estabelecimento do tratamento efetivo, redução da toxicidade e da resistência aos antivirais. HSV-1, HSV-2 e poliomavírus BK são vírus de importância clínica para imunodeprimidos e podem levar a rejeição de órgãos em transplantados. Assim, o objetivo deste trabalho foi desenvolver um sistema celular repórter, utilizando a proteína fluorescente GFP, para HSV-1 e 2 e implantar uma qPCR utilizando amostras clínicas de pacientes transplantados renais para detecção de poliomavírus BK. O sistema celular repórter foi construído através da transfecção de células Vero com o vetor pZsGreen1-1 ligado ao promotor ICP10 (F3R3 e F4R3) da RR1 do HSV-2. A regulação da expressão da GFP via ICP10 é dependente da infecção viral e acontece por meio da proteína viral transativadora VP16 e de fatores celulares Oct-1 e HCF-1. A efetividade do sistema foi avaliada por infecção viral e pela aplicação de antivirais (Aciclovir, ácido gálico, convalotoxina e extrato de Uncaria sp.) e candidatos antivirais inativos (Extrato de Passiflora edulis e derivados de cardenolídeos). O sistema repórter F4R3 ZsGreen1-1 expressou GFP em função da infecção por HSV-1 e 2, a qual foi detectada por microscopia de fluorescência e/ou citometria de fluxo. Em análise por citometria de fluxo, a fluorescência do sistema repórter correlacionou-se diretamente com os títulos virais (MOI de 4,0 x10-3 a 3,3 x10-4, ou seja, 1 partícula viral a cada 250 a 3000 células), o sistema manteve a capacidade de expressão da GFP na presença de agentes sem propriedade antiviral e não expressou fluorescência quando tratado com antivirais. O sistema F4R3 ZsGreen1-1 mostrou-se um sistema funcional com possíveis aplicações para diagnóstico clínico, para elaboração de testes de resistência aos antivirais e para a pesquisa de novos medicamentos. A qPCR para poliomavírus BK foi implantada utilizando amostras de DNA cedidas pelo HEMOSC com iniciadores dirigidos para o antígeno T viral. O limite de detecção foi de 18 cópias genômicas/ reação com quantificações variando entre 9,8 x 105 a 6,7 x 107 cópias genômicas/ mL. A qPCR foi efetiva para análises de amostras clínicas e apresentou limite de detecção suficiente para avaliação de risco de nefropatia em transplantados renais.Abstract : Immunosuppressed patients can present viral infections with fast evolution, severe atypical symptomatologies and often fatal, being essential the early diagnosis for the establishment of effective treatments, reduction of toxicity and development of resistance to antiviral. HSV-1, HSV-2 and polyomavirus BK are virus of clinical importance for immunosuppresed and can lead to the rejection of transplanted organs. Therefore, the aim of this work was to develop a reporter cellular system, using the fluorescent protein GFP, for HSV-1 and 2, and deploy a qPCR using clinical samples from patients submitted to renal transplant, for the detection of polyomavirus BK. The reporter cellular system was constructed through the transfection of Vero cells with the vector pZsGreen1-1 connected to the promoter ICP10 (F3R3 and F4R3) of the RR1 of the HSV-2. The regulation of the expression of GFP via ICP10 is dependent of the viral infection and happens through the viral transactivating protein VP16, and the cellular factors Oct-1 and HCF-1. The effectivity of the system was evaluated by viral infection and through the application of antiviral (Acyclovir, gallic acid, convalotoxina and extract of Uncaria sp.) and inactive antiviral candidate (Extract of Passiflora edulis and derivatives cardenolide). The reporter system F4R3 ZsGreen1-1 expressed GFP as a function of the infection for HSV-1 and 2, which was detected by fluorescence microscopy and/or flow cytometry. In flow cytometry, the fluorescence of the reporter system was directly correlated with virus titers (MOI 4,0 x10-3 to 3,3 x10-4, that is, 1 viral particle to each 250 to 3000 cells), the system maintained the ability to GFP expression in the presence of agents without antiviral property and no expressed fluorescence when treated with antivirals. The system F4R3 ZsGreen1-1 revealed a functional system with possible applications for clinical diagnosis, elaboration of tests of resistance to the antiviral and for new drugs research. The qPCR to polyomavirus BK was deployed using DNA samples provided by HEMOSC with primers directed to the viral antigen T. The limit of detection was 18 genome copies /reaction with quantification ranging between 9.8 x 105 to 6.7 x 107 genome copies /mL. The qPCR was effective for the analyses of clinical samples and presented enough sensitivity for risk evaluation of nephropathy in renal transplant
JC and BK polyomavirus-like particles as targets of innate and adaptive humoral immunity
No full textJC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) were identified as the first of now more than 12 human polyomaviruses (HPyVs). The average JCPyV and BKPyV seroprevalence rates in adults are 70% and 90%, respectively. After asymptomatic infection both viruses persist in the renourinary tract. In fact, asymptomatic viruria is detectable in one-third of general population. However, in immunocompromised patients, JCPyV and BKPyV replication may progress to significant diseases. Hence, JCPyV can cause progressive multifocal leukoencephalopathy (PML) in patients with HIV-AIDS, malignancies or autoimmune diseases under immunosuppressive treatment. BKPyV can be a cause of polyomavirus-associated nephropathy (PyVAN) in kidney transplant recipients or hemorrhagic cystitis (PyVHC) after allogeneic hematopoietic stem cell transplantation. Due to more frequent application of immunosuppression, the risk of developing these diseases has increased in the last few decades. The risk of PML development is estimated to be 100-fold higher for JCPyV-seropositive patients in comparison to JCPyV-seronegatives. Most cases of PyVAN and PyVHC have been tested positive for BKPyV at the moment of disease diagnosis. Unfortunately, there is no specific antiviral therapy against any of these HPyV diseases. Thus, current strategies to avert PyVAN or PyVHC aim at identifying patients with BKPyV viremia and reducing immunosuppression. Similar strategies for PML have not been effective, since JCPyV viremia is usually not detected prior to or at the diagnosis of disease. The fate of BKPyV and JCPyV virus-like particles (VLPs) was examined in an animal model corresponding to primary viremia in non-immune host. Radioactively labeled VLPs were used to assess blood decay, organ, and hepatocellular distribution of ligand, and non-labeled VLPs to examine cellular uptake by immunohisto- and cytochemistry. Rapid distribution of both BKPyV and JCPyV VLPs to the liver was observed, with lesser uptake in kidney and spleen. Liver uptake was predominantly observed in LSECs. Blood half-life and tissue distribution of both wild-type JCPyV VLPs and two mutant JCPyV VLPs (L55F and S269F), lacking sialic acid binding affinity, were similar, indicating involvement of non-sialic acid receptors in cellular uptake. We concluded that LSECs very effectively cleared a large fraction of blood-borne BKPyV and JCPyV VLPs, indicating a central role of these cells in early removal of polyomavirus from the circulation. Moreover, we observed that a subpopulation of endothelial cells in kidney, the main organ of polyomavirus persistence, showed selective and rapid uptake of VLPs, suggesting a role in viremic organ tropism (Simon-Santamaria et al., p. 54). Giving the increasing clinical need to reliably determine JCPyV and BKPyV IgG levels in patients at risk, we first reviewed and optimized serological tools for JCPyV and BKPyV IgG detection including virus-like particle (VLP)-based ELISA. We demonstrated that although no statistically significant differences in intraassay and interassay variability were revealed for JCPyV serology of 400-fold diluted sera from healthy donors, qualitative differences were seen in the identification of the individual JCPyV serostatus. The cause of discordance for approximately 10% of sera resulted from a low IgG activity close to the cutoff of the assay. Therefore we standardized the ELISA using reference serum for normalization. Moreover, we developed a preadsorption assay with cutoff of 35% reduction of the JCPyV IgG activity after preincubation with JCPyV VLPs. Importantly, we excluded BKPyV antibody cross-reactivity by testing JCPyV IgG positive sera in preadsorption assay using BKPyV VLPs. In conclusion, we showed that VLP-based ELISA with normalization can serve as a reliable tool for JCPyV IgG serology. Additionally, the preadsorption assay can help with unequivocal determination of JCPyV serostatus for samples with low IgG levels. (Kardas et al., p. 72). We also normalized this VLP-based ELISA for BKPyV IgG detection and showed that for seroepidemiology studies, normalized JCPyV and BKPyV IgG ELISA at 1:200 serum dilution provides optimal sensitivity and specificity with the lowest false-positive and false-negative rate. However, for individual risk assessment, 100-, 200-, and 400-fold dilutions combined with preadsorption for low-reactive sera might be the most appropriate (Kardas et al., p. 82). This improved ELISA was used to investigate JCPyV and BKPyV specific antibody levels in several clinical studies: (1) one case of PML patient where positive JCPyV IgG status was compatible with other PML-indicating symptoms (Kurmann et al., p. 90); (2) one case of PyVAN caused by JCPyV rather than BKPyV, as confirmed by JCPyV IgG/IgM positive and BKPyV IgG/IgM negative results (Lautenschlager et al., 99); (3) one case of PyVHC patient after allogeneic hematopoietic stem cell transplantation where increasing BKPyV IgG activities were in line with progression of BKPyV viremia (Koskenvuo et al., p. 106). Further, by serological testing of 122 immunocompetent and 63 immunocompromised patients we demonstrated that the BKPyV IgG level is age-dependent, with the highest values between 20 and 30 years (Schmidt et al., p. 119). In another study we compared serological outcomes of ELISA utilizing two different antigens in terms of prognostic value in prostate cancer development. To accomplish this we utilized improved ELISA for BKPyV IgG activity to both BKPyV VLPs and BKPyV LTag. Testing of 226 patients undergoing radical prostatectomy for primary prostate cancer revealed that BKPyV VP1 serostatus, in contrast to BKPyV LTag, has no prognostic value in prostate cancer progression (Keller et al., p. 125). In conclusion, we provided a new input into knowledge about tropism and clearance of polyomaviruses from blood. Moreover, we established a reliable and sensitive VLP-based assay for specific detection of JCPyV and BKPyV IgG and IgM. Serostatus based on ELISA results was compatible with other symptoms of BKPyV- and JCPyV-related diseases
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Managing the twin pressures in Welsh Local Authorities during the austerity era
No full textHow have Welsh Local Authorities managed the twin pressures of the reductions in central government funding and the continuous increase in demand for their services during the era of austerity
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