Merkel cell polyomavirus-specific CD8 T cells in Merkel cell carcinoma: T cell receptor diversity & novel immune therapies

Thesis (Ph.D.)--University of Washington, 2016-06

Gene codon composition determines differentiation-dependent expression of a viral capsid gene in keratinocytes in vitro and in vivo

By establishing mouse primary keratinocytes (KCs) in culture, we were able, for the first time, to express papillomavirus major capsid (L1) proteins by transient transfection of authentic or codon-modified L1 gene expression plasmids. We demonstrate in vitro and in vivo that gene codon composition is in part responsible for differentiation-dependent expression of L1 protein in KCs. L1 mRNA was present in similar amounts in differentiated and undifferentiated KCs transfected with authentic or codon-modified L1 genes and had a similar half-life, demonstrating that L1 protein production is posttranscriptionally regulated. We demonstrate further that KCs substantially change their tRNA profiles upon differentiation. Aminoacyl-tRNAs from differentiated KCs but not undifferentiated KCs enhanced the translation of authentic L1 mRNA, suggesting that differentiation-associated change to tRNA profiles enhances L1 expression in differentiated KCs. Thus, our data reveal a novel mechanism for regulation of gene expression utilized by a virus to direct viral capsid protein expression to the site of virion assembly in mature KCs. Analysis of two structural proteins of KCs, involucrin and keratin 14, suggests that translation of their mRNAs is also regulated, in association with KC differentiation in vitro, by a similar mechanism

Interaction between conventional dendritic cells and natural killer cells is integral to the activation of effective antiviral immunity

Dendritic cells (DCs) regulate various aspects of innate immunity, including natural killer (NK) cell function. Here we define the mechanisms involved in DC - NK cell interactions during viral infection. NK cells were efficiently activated by murine cytomegalovirus ( MCMV) - infected CD11b(+) DCs. NK cell cytotoxicity required interferon-alpha and interactions between the NKG2D activating receptor and NKG2D ligand, whereas the production of interferon-gamma by NK cells relied mainly on DC-derived interleukin 18. Although Toll-like receptor 9 contributes to antiviral immunity, we found that signaling pathways independent of Toll-like receptor 9 were important in generating immune responses to MCMV, including the production of interferon-alpha and the induction of NK cell cytotoxicity. Notably, adoptive transfer of MCMV-activated CD11b(+) DCs resulted in improved control of MCMV infection, indicating that these cells participate in controlling viral replication in vivo.

Campfire culture and computer culture: Museums of the present

In Australia indigenous peoples have never had a treaty with the dominant cultures; and their on-going marginalisation is some testimony to this. However, they have not languished entirely in a policy free environment: media is one area where some policy advances have been made; but media policy development has experienced a number of problems. It has tended to be monolithic in a situation demanding multi and complex treatments. And funding, as always, never seems sufficient to meet those multi and complex needs. This paper examines a small remote community on the island of Milingimbi off the northern coast of Arnhem Land in Australia's far north. People in East Arnhem Land refer to themselves collectively as Yolngu. This community is not typical of many documented cases of media relations between indigenous and non-indigenous peoples; however, the fact that it tends to overturn much of the conventional scholarship surrounding indigenous peoples and the media, helps shed new light on the inadequacy of not only monolithic media policy, but the inadequacy of media-only approaches to policy. Arguably, the significance of the media in Milingimbi is part of a 'triangulated' relationship between indigenous and dominant cultures. That triangulation also involves appropriate forms of government and education, which coupled with appropriate media appear to offer new ways of seeing self-government alongside relative cultural and economic autonomy.

Human brain slices for epilepsy research:pitfalls, solutions and future challenges

Increasingly, neuroscientists are taking the opportunity to use live human tissue obtained from elective neurosurgical procedures for electrophysiological studies in vitro. Access to this valuable resource permits unique studies into the network dynamics that contribute to the generation of pathological electrical activity in the human epileptic brain. Whilst this approach has provided insights into the mechanistic features of electrophysiological patterns associated with human epilepsy, it is not without technical and methodological challenges. This review outlines the main difficulties associated with working with epileptic human brain slices from the point of collection, through the stages of preparation, storage and recording. Moreover, it outlines the limitations, in terms of the nature of epileptic activity that can be observed in such tissue, in particular, the rarity of spontaneous ictal discharges, we discuss manipulations that can be utilised to induce such activity. In addition to discussing conventional electrophysiological techniques that are routinely employed in epileptic human brain slices, we review how imaging and multielectrode array recordings could provide novel insights into the network dynamics of human epileptogenesis. Acute studies in human brain slices are ultimately limited by the lifetime of the tissue so overcoming this issue provides increased opportunity for information gain. We review the literature with respect to organotypic culture techniques that may hold the key to prolonging the viability of this material. A combination of long-term culture techniques, viral transduction approaches and electrophysiology in human brain slices promotes the possibility of large scale monitoring and manipulation of neuronal activity in epileptic microcircuits.

Administrators' perspectives of culture at a multicampus community college

The purpose of this study was to explore how administrators’ perceived the campus and administrative cultures found on a single campus of a multicampus community college system. A review of the literature revealed that the culture found in higher education institutions contains a high degree of human interactions, has a myriad of cultures, and that individuals play a significant role in the maintenance or the evolution of the cultures present. The study site was Neighborhood Campus which is one campus of a large urban community college system containing a total of eight campuses, Urban College. Kuh’s conventional organizational models served to identify the model on Neighborhood Campus, Levin’s cultural definitions described the campus culture, and cultural definitions from Bergquist and Pawlak formed the framework for the administrative culture. The study was guided by the following research questions: What are the administrators’ perspectives of the campus culture on a community college campus and what are the administrators’ perspectives of the administrative culture on a community college campus? A qualitative case study method was used, data collection included interviews, document and videograph reviews, and observations of administrative meetings. The participants for the interview portion of the study included 10 individuals defined as administrators. ^ The study revealed that administrators’ perspectives of these cultures demonstrated five themes (student-centered, size, location, Hispanics, and family) served as contributors to the campus culture. The administrative culture was supported by six themes (size, team, collaboration, open, Inclusion, and rewards and recognition). ^ The findings revealed three of Kuh’s conventional organizational models (rational, bureaucratic, and collegial models) were seen as being in place at Neighborhood Campus. Levin’s traditional and service cultures were seen in the campus culture with the service culture demonstrating dominance. Using Bergquist and Pawlak’s definitions, components of the collegial, managerial, and developmental cultures appear to be present in the administrative culture with the collegial culture serving as the dominant administrative culture. ^ Through an understanding of these cultures and themes, administrators can provide leadership that is sensitive to these cultures, especially if institutional change is required.^

Effect of diatom supplementation during the nursery rearing of Litopenaeus vannamei (Boone, 1931) in a heterotrophic culture system

Bio-floc shrimp culture systems have been investigated in an attempt to optimize water use and prevent the discharge of effluent into the environment. The importance of microalgae in maintaining water quality and nutrition of the shrimp is well known in conventional systems; however, its maintenance amid bio-flocs and its role in the shrimp performance in this system are still poorly understood. The aim of this study was to evaluate the contribution of diatoms in the performance of Litopenaeus vannamei reared during the nursery phase in intensive system with minimal water exchange. Shrimp (0.31 ± 0.10 g) were reared among diatoms, bio-flocs and the combination of the two forming the mixture medium. The survival of shrimp was high in all treatments (90–97%). However, the shrimp reared among diatoms showed higher weight gain (P\0.05) and feed conversion ratio significantly more efficient, reaching a value of 0.47. The results indicate the importance of diatoms in bio-floc culture systems and points out to future research in an attempt to maintain a constant presence of these microalgae in culture medium without requiring successive inoculations.

Soft tissue sarcoma: biology and therapeutic correlates

Soft tissue sarcomas (STS) comprise a heterogenenous group of greater than 50 malignancies of putative mesenchymal cell origin and as such they may arise in diverse tissue types in various anatomical locations throughout the whole body. Collectively they account for approximately 1% of all human malignancies yet have a spectrum of aggressive behaviours amongst their subtypes. They thus pose a particular challenge to manage and remain an under investigated group of cancers with no generally applicable new therapies in the past 40 years and an overall 5-year survival rate that remains stagnant at around 50%. From September 2000 to July 2006 I undertook a full time post-doctoral level research fellowship at the MD Anderson Cancer Center, Houston, Texas, USA in the department of Surgical Oncology to investigate the biology of soft tissue sarcoma and test novel anti- sarcoma adenovirus-based therapy in the preclinical nude rat model of isolated limb perfusion against human sarcoma xenografts. This work, in collaboration with colleagues as indicated herein, led to a number of publications in the scientific literature furthering our understanding of the malignant phenotype of sarcoma and reported preclinical studies with wild-type p53, in a replication deficient adenovirus vector, and oncolytic adenoviruses administered by isolated limb perfusion. Additional collaborative and pioneering preclinical studies reported the molecular imaging of sarcoma response to systemically delivered therapeutic phage RGD-4c AAVP. Doxorubicin chemotherapy is the single most active broadly applicable anti-sarcoma chemotherapeutic yet only has an approximate 30% overall response rate with additional breakthrough tumour progression and recurrence after initial chemo-responsiveness further problematic features in STS management. Doxorubicin is a substrate for the multi- drug resistance (mdr) gene product p-glycoprotein drug efflux pump and exerts its main mode of action by induction of DNA double-strand breaks during the S-phase of the cell cycle. Two papers in my thesis characterise different aspects of chemoresistance in sarcoma. The first shows that wild-type p53 suppresses Protein Kinase Calpha (PKCα) phosphorylation (and activation) of p-glycoprotein by transcriptional repression of PKCα through a Sp-1 transcription factor binding site in its -244/-234 promoter region. The second paper demonstrates that Rad51 (a central mediator of homologous recombination repair of double strand breaks) has elevated levels in sarcoma and particularly in the S- G2 phase of the cell cycle. Suppression of Rad51 with small interfering RNA in sarcoma cell culture led to doxorubicin chemosensitisation. Reintroduction of wild-type p53 into STS cell lines resulted in decreased Rad51 protein and mRNA expression via transcriptional repression of the Rad51 promoter through increased AP-2 binding. In light of poor response rates to chemotherapy, escape from local control portends a poor prognosis for patients with sarcoma. Two papers in my thesis characterise aspects of sarcoma angiogenesis, invasion and metastasis. Human sarcoma samples were found to have high levels of matrix metalloproteinase-9 (MMP-9) with expression levels that correlated with p53 mutational status. MMP-9 is known to degrade extracellular collagen, contribute to the control of the angiogenic switch necessary in primary tumour progression and facilitate invasion and metastasis. Reconstitution of wild-type p53 function led to decreased levels of MMP-9 protein and mRNA as well as zymography-assessed MMP-9 proteolytic activity and decreased tumour cell invasiveness. Reintroduction of wild-type p53 into human sarcoma xenografts in-vivo decreased tumour growth and MMP-9 protein expression. Wild-type p53 was found to suppress mmp-9 transcription via decreased binding of NF-κB to its -607/-595 mmp-9 promoter element. Studies on the role of the VEGF165 in sarcoma found that sarcoma cells stably transfected with VEGF165 formed more aggressive xenografted tumours with increased vascularity, growth rate, metastasis, and resistance to chemotherapy. Use of the anti-VEGFR2 antibody DC101 enhanced doxorubicin sensitivity at sub-conventional dosing, inhibited tumour growth, decreased development of metastases, and reduced tumour micro-vessel density while increasing the vessel maturation index. These effects were explained primarily through effects on endothelial cells (e.c.s), rather than the tumour cells per se, where DC101 induced e.c. sensitivity to doxorubicin and suppressed e.c. production of MMPs. The p53 tumour suppressor pathway is the most frequently mutated pathway in sarcoma. Recapitulation of wild-type p53 function in sarcoma exerts a number of anti-cancer outcomes such as growth arrest, resensitisation to chemotherapy, suppression of invasion, and attenuation of angiogenesis. Using a modified nude rat-human sarcoma xenograft model for isolated limb perfusion (ILP) delivery of wild-type p53 in a replication deficient adenovirus vector I showed that functionally competent wild-type p53 could be delivered to and detected in human leiomyosarcoma xenografts confirming preclinical feasibility - although not efficacious due to low transgene expression. Viral fibre modification to express the RGD tripeptide motif led to greater viral uptake by sarcoma cells in vitro (transductional targeting) and changing the transgene promoter to a response element active in cells with active telomerase expression restricted the transgene expression to the tumour intracellular environment (transcriptional targeting). Delivery of the fibre-modified, selectively replication proficient oncolytic adenovirus Ad.hTC.GFP/ E1a.RGD by ILP demonstrated a more robust, and tumour-restricted, transgene expression with evidence of anti-sarcoma effect confirmed microscopically. Collaborative studies using the fibre modified phage RGD-4C AAVP confirmed that systemic delivery specifically, efficiently, and repeatedly targets human sarcoma xenografts, binds to αv integrins in tumours, and demonstrates a durable, though heterogeneous, transgene expression of 1-4 weeks. Incorporation of the Herpes Simplex Virus thymidine kinase (HSVtk) transgene into RGD-4C AAVP permitted CT-PET spatial and temporal molecular imaging in vivo of transgene expression and allowed quantification of tumour metabolic activity both before and after interval administration of a systemic cytotoxic with predictable and measurable response to treatment before becoming apparent clinically. These papers further the medical and scientific community’s understanding of the biology of soft tissue sarcoma and report preclinical studies with novel and promising anti- sarcoma therapeutics.

Manure and sorbent fertilisers increase on-going nutrient availability relative to conventional fertilisers

The key to better nutrient efficiency is to simultaneously improve uptake and decrease losses. This study sought to achieve this balance using sorbent additions and manure nutrients (spent poultry litter; SL) compared with results obtained using conventional sources (Conv; urea nitrogen, N; and phosphate–phosphorus; P). Two experiments were conducted. Firstly, a phosphorus pot trial involving two soils (sandy and clay) based on a factorial design (Digitaria eriantha/Pennisetum clandestinum). Subsequently, a factorial N and P field trial was conducted on the clay soil (D. eriantha/Lolium rigidum). In the pot trial, sorbent additions (26.2 g of hydrotalcite [HT] g P− 1) to the Conv treatment deferred P availability (both soils) as did SL in the sandy soil. In this soil, P delivery by the Conv treatments declined rapidly, and began to fall behind the HT and SL treatments. Addition of HT increased post-trial Colwell P. In the field trial low HT-rates (3.75 and 7.5 g of HT g P− 1) plus bentonite, allowed dry matter production and nutrient uptake to match that of Conv treatments, and increased residual mineral-N. The SL treatments performed similarly to (or better than) Conv treatments regarding nutrient uptake. With successive application, HT forms may provide better supply profiles than Conv treatments. Our findings, combined with previous studies, suggest it is possible to use manures and ion-exchangers to match conventional N and P source productivity with lower risk of nutrient losses.

Improvement of the yeast slide culture technique

Este trabalho tem como objetivo melhorar a técnica de cultura em lâmina para ser usada na avaliação da viabilidade de leveduras sob diferentes condições fisiológicas. Inicialmente, foram otimizadas as condições ideais para o cultivo em lâmina de uma estirpe laboratorial (BY4741) e de uma estirpe industrial (NCYC 1214) da levedura Saccharomyces cerevisiae. O melhor protocolo foi obtido utilizando: YEPD agar com uma espessura de cerca de 2 mm; 20 μL de uma suspensão de 1 x 105 células/mL para a estirpe BY4741 ou de 5 x 104 células/mL para a estirpe NCYC 1214; uma câmara de humedecimento com 100 μL de água desionizada e um tempo de incubação de 24 h, a 25 ° C. Com o objetivo de facilitar a contagem das microcolónias, foi adicionado um corante (calcofluor white, CFW) ao meio YEPD agar. Ensaios preliminares, em YEPD líquido, contendo diferentes concentrações de CFW, permitiram verificar que o corante, até 5,0 μg/L, não inibe o crescimento da levedura. Uma concentração de 2,5 μg/L de CFW permitiu a coloração da parede das leveduras, não se observando células com morfologia alterada, sendo esta a concentração de CFW selecionado nos estudos subsequentes. A técnica de cultura em lâmina, com ou sem CFW, foi aplicada para avaliar a viabilidade de células saudáveis (células em fase exponencial de crescimento), células submetidas a stress de etanol [células expostas a 20% (v/v) de etanol, a 25 ºC, durante 2 h] e células envelhecidas (células incubadas em água, a 25 ° C, durante 48 h), da estirpe laboratorial. A percentagem de células viáveis não foi significativamente diferente entre as duas técnicas (com ou sem CFW), após uma incubação de 24 horas. Finalmente, a técnica de cultura de lâmina, contendo CFW, foi comparada com duas técnicas habitualmente usadas na indústria cervejeira: fermentação de curta duração e determinação da percentagem de células gemuladas. Os resultados obtidos através da técnica de cultura de lâmina, desenvolvida, seguem um padrão similar aos obtidos nos ensaios de fermentação de curta duração e aos da determinação da percentagem de células gemuladas. Os resultados obtidos sugerem que a técnica de cultura em lâmina, combinada com CFW, parece ser uma alternativa, fácil, rápida (em 24 h) e reprodutível, relativamente ao método convencional (técnica de plaqueamento), para a avaliação da viabilidade de células de levedura. Deverá ser realizado trabalho adicional a fim de validar o método com estirpes industriais.

Detection of viral diseases in shrimp Litopenaeus vannamei in Iran with emphasis on white spot disease prevention by using marine alga extract

Nowadays, following was expanded shrimp breeding and culture; viral diseases have been main problem which threatened shrimp industry in the country. Therefore, shrimp samples were obtained from different stages of Litopenaeus vannmei life cycle (larval, post larval, juveniles, adults and broodstocks) based on clinical signs in the breeding center and shrimp farming from Bushehr, Khozestan and Sistan and Baluchestan provinces. Viral diseases were detected by PCR (Polymerase Chain Reaction), histopathology and transmission electron microscopy (TEM) methods. Results of the PCR were indicated present white spot virus (WSV) in juveniles, sub adults and adults shrimp with medium intensity from three provinces, but it was not showed in larval and post larval stages. Histopathological sections were indicated hypertrophy and basophilic Cowdry type A formation in nucleus cells of gill, haematopoietic, lymphoid and epithelial's cuticles and intestinal tissues which was associated with small vacuoles increased in B cells of hepatopancreas tissue of infection shrimps. Transmission electronic microscopic studies were demonstrated that the length and diameter virus was detected, respectively, 300 ± 20 nm and 75 ± 5 nm. Considerable, results of the PCR were only displayed IHHNV in juvenile, adult and broodstock shrimps from breeding and farming center of Bushehr province. The main lesion pathology was formed eosinophilic Cowdry type A in nucleus cells of gill, haematopoietic, lymphoid and epithelial's cuticles and intestinal tissues. Whereas penaeid shrimps are lack specific immune system, hence, in the present study was used of marine alga (Lurensia snideria) collected from along costal Persian Gulf of Bushehr province for viral diseases were prevented. Powder alga extract were added with a ratio of 1 % to shrimp diet. Total haemocyte count (THC) and total protein plasma (TPP) were increased after 5 days of oral administration diets. When shrimps were infected by with spot virus experimentally, THC and TPP gradually were increased in both two groups (shrimps fed with diet containing alga extract and without alga extract) after 48h. Nevertheless; THC, TPP and survival of shrimp fed with diet containing alga extract were more than shrimp control in 15 days. So, oral administration Lurensia snideria extract was capable prevention infected L. vannamei via stimulant specific immune system.

Hardpan and maize root distribution under conservation and conventional tillage in Agro-Ecological Zone IIA, Zambia

Hardpans (plough/hoe pans) are commonly believed to restrict plant root growth and crop yields under conventional small-scale agriculture in sub-Saharan Africa. This study questions the notion of widespread hardpans in Zambia and their remedy under conservation tillage. Soil penetration resistance was measured in 8x12 grids, covering 80 cm wide and 60 cm deep profiles in 32 soil pits. Large and fine maize roots were counted in 8x6 grids. Soil samples from mid-rows were analysed for pH, exchangeable H+, exchangeable Al3+, cation exchange capacity, total N and extractable P (Bray 1) at six depths from 0-10 to 50-60 cm. Cultivation-induced hardpans were not detected. Soils under conservation tillage were more compact at 5 cm depth than soils under conventional tillage. No differences in root distributions between conservation and conventional tillage were found. Maize ( Zea mays L. ) roots were largely confined to a relatively small soil volume of about 30 cm x 30 cm x 30 cm. Root growth appeared to be restricted by a combination of low concentrations of N and P. Soil acidity and Al saturation appeared to play a minor role in root distribution. L-shaped taproots in soils under manual tillage reported earlier were not necessarily due to hardpans, but may rather be caused by temporarily dry, impenetrable subsoils early in the rain season. There is no scientific basis for the recommendation given to farmers by agricultural extension workers to “break the hardpan” in fields under manual or animal tillage in the study areas.

EFFECT OF BAP, NAA AND GA3, EITHER ALONE OR IN COMBINATION, ON MERISTEM CULTURE AND PLANTLET ESTABLISHMENT IN SWEET POTATO (CV BRONDAL)

In Zimbabwe, the average sweet potato yield (6 t/ha) is relatively low when compared to Asian counterparts (17 t/ha). These low crop yields have been blamed on weevil infestations and viral infections which account for 60-90% of sweet potato yield losses in Africa. Meristem tip culture, a Centre for Potato Improvement (CIP) initiated tissue culture technique, has been widely used to eradicate viruses from clonally propagated crops and has been noted to be one of the instrumental techniques that helped China to increase sweet potato yields. In an effort to adopt the meristem tip culture technique for the production of virus-free planting material of a local sweet potato (cv Brondal), a study was conducted to evaluate the effect of Benzylamino purine (BAP), 1-Naphthaleneacetic acid (NAA) and Gibberellic acid (GA3) (either alone or in combination) on cultured Brondal meristems. The different hormonal treatments were assessed on the following parameters: plantlet regenerative capacity, multiple plantlet production, shoot height, average leaf number per shoot and average node number per shoot, ten weeks after meristem culture. All treatments containing a combination of BAP (1 mg-L) and GA3 (at either 5 mg-L, 10 mg-L, or 20 mg-L) had a significantly (p<0.01) higher plantlet regenerative capacity of 33-66% when compared to other treatment combinations. Only treatments, 10 mg-L GA3 + 1 mg-L BAP and 20 mg-L GA3 + 1 mg-L BAP were capable of inducing multiple plantlet formation, producing an average of three plantlets/meristem and two plantlets/meristem respectively. Overall, treatment 10 mg-L GA3 + 1 mg-L BAP gave rise to significantly (p<0.01) taller shoots (20 mm) compared to the rest of the treatments used. For average leaf number per shoot, all GA3 treatments (5 mg-L, 10 mg-L, or 20 mg-L) supplemented with 1 mg-L BAP gave significantly (p<0.01) higher numbers of leaves (six leaves/shoot) than the rest of the treatments. Treatments 10 mg-L GA3 + 1 mg-L BAP and 20 mg-L GA3 + 1 mg-L BAP gave rise to the highest number of nodes per shoot, producing an average of three nodes per shoot. In sharp contrast to treatments containing a combination of BAP and GA3, all treatments containing a combination of BAP and NAA performed poorly in all parameters tested for plant regeneration of Brondal sweet potato variety. In conclusion, the best hormonal treatment for culturing Brondal meristems proved to be 10 mg-L GA3 + 1 mg-L BAP.

Sulphated polysaccharides from Ulva clathrata and Cladosiphon okamuranus seaweeds both inhibit viral attachment/entry and cell-cell fusion, in NDV infection

Sulphated polysaccharides (SP) extracted from seaweeds have antiviral properties and are much less cytotoxic than conventional drugs, but little is known about their mode of action. Combination antiviral chemotherapy may offer advantages over single agent therapy, increasing efficiency, potency and delaying the emergence of resistant virus. The paramyxoviridae family includes pathogens causing morbidity and mortality worldwide in humans and animals, such as the Newcastle Disease Virus (NDV) in poultry. This study aims at determining the antiviral activity and mechanism of action in vitro of an ulvan (SP from the green seaweed Ulva clathrata), and of its mixture with a fucoidan (SP from Cladosiphon okamuranus), against La Sota NDV strain. The ulvan antiviral activity was tested using syncytia formation, exhibiting an IC50 of 0.1 μg/mL; ulvan had a better anti cell-cell spread effect than that previously shown for fucoidan, and inhibited cell-cell fusion via a direct effect on the F0 protein, but did not show any virucidal effect. The mixture of ulvan and fucoidan showed a greater anti-spread effect than SPs alone, but ulvan antagonizes the effect of fucoidan on the viral attachment/entry. Both SPs may be promising antivirals against paramyxovirus infection but their mixture has no clear synergistic advantage