Successful human adenovirus (HAdV) replication relies on multiple protein-protein interactions between viral and host proteins. HAdV type 5 (HAdV-5) pVI is a multifaceted protein necessary for viral endosomal escape, activation of viral protease, as well as nuclear shuttling of certain viral proteins. Preliminary mass spectrometry experiments indicated that pVI can bind cellular importins and histone chaperones, of which many are considered novel pVI targets. Here, the binding of the pVI protein to cellular importins was validated, and preliminary studies were done to characterize whether HAdV-5 infection changes importin levels in the infected cells. The validation studies were inconclusive, but it was observed that the accumulation of the importin proteins was not altered in during HAdV-5 infection. In addition, the role of NAP1L1 and NAP1L4, two ubiquitously expressed histone chaperones, was examined during HAdV-5 infection and their effect on HAdV-5 genome structure. Here, it is shown that NAP1L1 knockdown affected viral mRNA and protein as well as hindered viral histone-like protein pVII deposition onto viral DNA during the late stage of infection. In contrast, the NAP1L4 protein was shown to co-localize to viral replication centers (VRCs), and its elimination promoted the pVII protein deposition on virus DNA. These results suggest that NAP1L1 is involved in viral transcription and chromatin assembly, whereas NAP1L4 has anti-viral properties during the assembly process.

<p>AMR as a cause of graft rejection has been long recognized and the presence of pre formed antibodies against donor HLA is a risk factor for increased graft rejection. FlowXM is the current clinical gold standard for detecting harmful DSA in the recipients and a positive FlowXM is considered a strong contraindication to transplantation. However, newer techniques such as SAB provide with a highly sensitive and specific method for DSA detection that is unattainable by FlowXM. But due to the intrinsic limitations associated with SAB assays, the clinical relevance of DSA detected on SAB has been highly disputable. Therefore, the overall aim of this study was to investigate the utility of SAB in predicting harmful DSA levels, by establishing a fluorescence range on SAB that correlated to positive FlowXM. This was done by retrospectively testing the highest serum dilutions on FlowPRA SAB that produced positive B or T cell FlowXM from 15 variably sensitized patients. Thus, a very narrow MFI range on SAB was established, for B and T cells separately, that correlated to positive FlowXM. On B cells this correlate ranged from 2780-7772 MFI (Mean MFI =5641), whereas T cell range was 1089-6731 (Mean MFI= 3226). In order to test these ranges for prediction of positive FlowXM, B and T cell FlowXM tests were carried out using various serum/cell combinations. DSA MFI of >3000 on SAB resulted in a significantly higher T cell positive FlowXM (p</p> / Master of Science (MSc)

<p>Neck braces and head supports used today do not allow wearers to rotate their head while maintaining the support they need. For people with chronic muscle diseases, such as ALS, DHS, Myasthenia Gravis, and Muscular Dystrophy, this inconvenience greatly affects their quality of life in that it hinders their abilities to perform activities of daily living, such as nonverbal communication and knowing their surroundings outside without having to move their entire body. There is a need for a head support device that allows individuals with chronic muscle diseases to rotate their heads, so they may better perform daily activities of living and thus live a more fulfilling life. </p> <p>The purpose of this study was to assess the basic stance of safety of a dynamic head support device that allows individuals with chronic muscle diseases to rotate their heads left and right. The assessment includes an experimental procedure that will conclude whether this device can withstand the load equivalent to an average adult’s head in a stationary position and a dynamic movement.</p> <p>This research proposed a procedure that has a testing apparatus that will place a predetermined load onto the dynamic head support device to stabilize it and then continuously add weight that was checked incrementally. This load was placed on the device while it was centered and static first. The next step in this procedure is to assess whether or not the load on the device can be carried while dynamically moving on the race of the radial sliding track of the device. The data recorded from this experiment will provide the necessary information to determine whether the basic safety requirement of load capacity for a medical device such as the one in this proposed research is met. </p>

Made available in DSpace on 2014-06-11T19:23:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-30Bitstream added on 2014-06-13T19:49:49Z : No. of bitstreams: 1 giovanazzi_rsd_me_botfm.pdf: 1564797 bytes, checksum: e843599819de69ecaa7e684f186d409c (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação Amaral Carvalho / As tentativas de interferir no processo de cicatrização das feridas remotam à antiguidade. Desde então, um grande número de produtos têm surgido no mercado, criando um trincado quebra-cabeça na escolha do melhor curativo. A proposta deste estudo foi avaliar a contribuição do uso de curativos bioativos no tratamento de feridas de origem multifatorial em 14 pacientes do Hospital Amaral Carvalho de Jaú. Estes pacientes foram divididos em três grupos, conforme situação diagnóstica: Grupo I, pacientes oncológicos portadores de feridas de etiologia Oncológica, Grupo II, pacientes oncológicos portadores de feridas de etiologia não Oncológica e Grupo III, pacientes não oncológicos (em remissão) portadores de feridas de origem multifatoriais. Estes pacientes receberam tratamento com curativos bioativos denominados Biogel, Biofibrin e o GelMix. O Biogel é um gel de plaquetas de uso tópico obtido a partir de hemocomponente (CP - Concentrado de Plaquetas) rico em PDGF (Fator de crescimento derivado de plaqueta) e VEGF (Fator de crescimento endotelial). Biofibrin também é um hemocomponente obtido a partir de plasma fresco congelado (PFC) e trombina humana, que quando incorporado à gel dermatológico, facilita o debridamento de feridas. O GelMix consiste na associação do CP e PFC com adição de trombina humana e gluconato de cálcio. Os produtos têm aprovação do CONEP para utilização. No grupo I os pacientes receberam somente Biofibrin por se tratar de ferida de etiologia neoplásica. Apresentaram significativa melhora do odor, controle da dor e do sangramento. O Grupo II utilizou Biofibrin, Biogel e GelMix, sendo considerado eficaz em 86% dos pacientes tratados. Foi observado que estes pacientes tinham um menor grau de co-morbidades e controle da atividade neoplásica. Nos pacientes do Grupo III observou-se que em feridas por isquemia ou deiscência cirúrgica... / The attempts to interfere in the cicatrisation process of wounds remount to the antiquity. Since then, a great amount of products arrived in the commerce, creating an intricate jigsaw for the choice of the better dressing. The proposal of this study was to evaluate the contribution of bioactive dressings in the healing of multiple origins wounds in 14 patients of the Hospital Amaral Carvalho from Jaú. The patients were divided in 3 groups: Group I, oncologic patients with oncologic wounds; Group II, oncologic patients with not oncologic wounds and Group III, oncologic patients in remission with multiple origins wounds. All the patients received treatment with bioactive dressings named Biogel, Biofibrin and GelMix. Biogel is a platelet gel of topical use, obtained from platelets concentrate (CP) which is rich of Platelets Derived Growth Factor (PDGF)) and of Endothelial Growth Factor (VEGF). Biofibrin is also a blood component obtained from Fresh Cooled Plasma (PFC) and human thrombin, with an unbridling facilitation action when mixed in dermatologic gel. GelMix consists in the association of CP with PFC with the addition of human thrombin and calcium gluconate. The products have the CONEP approval to their utilization in humans subjects. Group I received only Biofibrin because of the neoplasic origin of the wound. The patients showed markedly improvement of smelling, pain control and bleeding. Group II received Biofibrin, Biogel and GelMix, which was effective in 86% of the patients. Was observed that this group of patients had a less important degree of comorbidities and controlled neoplasic activity. Group III patients showed that the isquemic or chirurgical disclosed wounds the dressing was effective in 75% of the cases. In the radioterapeuticaly-originated wounds, the dressing was effective only in 50% of the cases. Patients treated in multiprofessional basis obtained better... (Complete abstract click electronic access below)

Resumo: As tentativas de interferir no processo de cicatrização das feridas remotam à antiguidade. Desde então, um grande número de produtos têm surgido no mercado, criando um trincado quebra-cabeça na escolha do melhor curativo. A proposta deste estudo foi avaliar a contribuição do uso de curativos bioativos no tratamento de feridas de origem multifatorial em 14 pacientes do Hospital Amaral Carvalho de Jaú. Estes pacientes foram divididos em três grupos, conforme situação diagnóstica: Grupo I, pacientes oncológicos portadores de feridas de etiologia Oncológica, Grupo II, pacientes oncológicos portadores de feridas de etiologia não Oncológica e Grupo III, pacientes não oncológicos (em remissão) portadores de feridas de origem multifatoriais. Estes pacientes receberam tratamento com curativos bioativos denominados Biogel, Biofibrin e o GelMix. O Biogel é um gel de plaquetas de uso tópico obtido a partir de hemocomponente (CP - Concentrado de Plaquetas) rico em PDGF (Fator de crescimento derivado de plaqueta) e VEGF (Fator de crescimento endotelial). Biofibrin também é um hemocomponente obtido a partir de plasma fresco congelado (PFC) e trombina humana, que quando incorporado à gel dermatológico, facilita o debridamento de feridas. O GelMix consiste na associação do CP e PFC com adição de trombina humana e gluconato de cálcio. Os produtos têm aprovação do CONEP para utilização. No grupo I os pacientes receberam somente Biofibrin por se tratar de ferida de etiologia neoplásica. Apresentaram significativa melhora do odor, controle da dor e do sangramento. O Grupo II utilizou Biofibrin, Biogel e GelMix, sendo considerado eficaz em 86% dos pacientes tratados. Foi observado que estes pacientes tinham um menor grau de co-morbidades e controle da atividade neoplásica. Nos pacientes do Grupo III observou-se que em feridas por isquemia ou deiscência cirúrgica... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The attempts to interfere in the cicatrisation process of wounds remount to the antiquity. Since then, a great amount of products arrived in the commerce, creating an intricate jigsaw for the choice of the better dressing. The proposal of this study was to evaluate the contribution of bioactive dressings in the healing of multiple origins wounds in 14 patients of the Hospital Amaral Carvalho from Jaú. The patients were divided in 3 groups: Group I, oncologic patients with oncologic wounds; Group II, oncologic patients with not oncologic wounds and Group III, oncologic patients in remission with multiple origins wounds. All the patients received treatment with bioactive dressings named Biogel, Biofibrin and GelMix. Biogel is a platelet gel of topical use, obtained from platelets concentrate (CP) which is rich of Platelets Derived Growth Factor (PDGF)) and of Endothelial Growth Factor (VEGF). Biofibrin is also a blood component obtained from Fresh Cooled Plasma (PFC) and human thrombin, with an unbridling facilitation action when mixed in dermatologic gel. GelMix consists in the association of CP with PFC with the addition of human thrombin and calcium gluconate. The products have the CONEP approval to their utilization in humans subjects. Group I received only Biofibrin because of the neoplasic origin of the wound. The patients showed markedly improvement of smelling, pain control and bleeding. Group II received Biofibrin, Biogel and GelMix, which was effective in 86% of the patients. Was observed that this group of patients had a less important degree of comorbidities and controlled neoplasic activity. Group III patients showed that the isquemic or chirurgical disclosed wounds the dressing was effective in 75% of the cases. In the radioterapeuticaly-originated wounds, the dressing was effective only in 50% of the cases. Patients treated in multiprofessional basis obtained better... (Complete abstract click electronic access below) / Orientador: Rosana Rossi Ferreira / Coorientador: Brígida Aparecida Rosa dos Reis / Banca: Ana Gabriela Salvio / Banca: Sonia Regina Perez Evangelista Dantas / Mestre

Coronary artery disease is the leading cause of death worldwide and results from progression of atherosclerosis, which is triggered in part by elevated plasma concentrations of LDL cholesterol. Genome-wide association studies have identified many loci that are associated with circulating lipid levels and bioinformatics tools have been implemented to prioritize positional candidate genes. This project aims to better understand the genetics underlying the regulation of plasma LDL levels and their effect of atherosclerosis using a zebrafish (Danio rerio) model system. A multiplex line with the genes abcg5, abcg8, myrf, col4a3bpa, col4a3bpb, st3gal3, ywhaqa and ywhaqb targeted by CRISPR/Cas9 technique was established using zebrafish with fluorescently labeled macrophages (Tg[mpeg1:mCherry]) and neutrophils (Tg[mpo:EGFP]). Monodansylpentane cadaverase was used to visualize lipids droplets, together with macrophages and neutrophils, in 384 overfed larvae, allowing the visualization and quantification of vascular atherogenic traits at 10 days post-fertilization. Euthanized larvae were homogenized for the quantification of triglycerides, total cholesterol, LDL, HDL, glucose and protein levels. DNA was extracted and larvae were paired-end sequenced for the CRISPR-targeted sites. Linear regression analysis to compare the wild-type larvae against homozygous mutants and additive models for orthologous genes were performed. The lower accumulation of lipids and the lower co-localization of macrophages and neutrophils in the vasculature suggested that the larvae with mutations in the gene abcg5, abcg8, col4a3bpb, and ywhaqb resulted in larvae more protected against atherosclerotic phenotype. The study suggested that loss of function of the targeted genes was associated with atherogenic traits, helping to understand the pathophysiology of atherosclerosis.

Advancements in the field of modified messenger RNA (mRNA) has led to new ventures in the pharmaceutical industry. However, new drug products demand new analytical methods to ensure the efficacy and purity of the drug. Capillary gel electrophoresis (CGE) with UV detection shows great potential for separation of mRNA samples due to the equal mass-to-charge ratio of mRNA and the flexible parameters of the CGE methods. This thesis investigates the optimal parameters of the capillary electrophoresis method, sample treatment procedure and sieving medium composition for enhanced shortmers separation of mRNA by CGE analysis. An RNA ladder with 100-1000 nucleotides and EPO mRNA with 900 nucleotides were used as model compounds. The effect of capillary dimensions and separation temperature on the resolution of the RNA peaks was established through comparative experiments. Sample treatment processes were evaluated to achieve an optimal conformation of the mRNA for CGE analysis. By heating the mRNA sample for 15 min at 80°C all multimers were seemingly eradicated. Moreover, it was found that addition of 4 M of urea to mRNA sample before heating resulted in improved peak shape. A sieving medium consisting of a mix of the two polymers polyvinylpyrrolidine (PVP) and hydroxyethyl cellulose (HEC) proved to have beneficial qualities for separation. The addition of sucrose as viscosity modifier in the sieving medium surprisingly further enhanced the resolution. Moreover, during the project a heavy wash was established which drastically improved repeatability of the analyses through more efficient regeneration of the capillary. ISSN:

The small intestine mucosa is known to be able to adapt itself to several kinds of both physiological and pathological conditions. The adaptive patterns of the structure of the enterocytes, particularly their apical surface (brush border), were studied in three models: (1) in rats, subjected to antrectomy or antral exclusion, combined with gastroduodenostomy and gastrojejunostomy; (2) in rats with alloxan dia­betes; (3) in children with coeliac disease; a) in its active phase; b) after long-term treatment with gluten-free diets; c) after long-term challenge with dietary gluten following treatment; d) after short-term elimination of dietary gluten. Gut mucosa from fasting or fed, normal or sham-operated rats, fasting cats, and short-statured children with no signs of gastrointestinal disease served as controls. - The specimens were prepared for light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Quantitation of structural variables was achieved by means of LM and TEM morphometrical procedures. Differentiation of the rat enterocytes from the base to the crest of the villi was structurally reflected by doubling of their apical cell area, an increase in cell height, and a decrease of both nuclear and mitochondrial volume densities. In mature normal rat enterocytes, high-power SEM showed regularly arranged, nude microvilli in thir apical surfaces, whereas in cat and man the apical surfaces were covered by a thick glycocalyx. - Fasting for 24 hours decreased the total length of the rat small intestine and the height of the enterocytes. Antrectomy and antral exclusion with gastrojejunostomy produced an increase of the apical surfaces of the enterocytes of the seif-emptying duodenal blind loop, whereas no changes occurred after antrectomy with gastroduodeno­stomy. In the jejunum, the apical surface area was reduced both after antrectomy and antral exclusion. In the diabetic rats a slight decrease of the apical surface area, together with an elongation of both the vil­li and the crypts, was observed in the jejunum, whereas no structural changes occurred in the duodenal mucosa. Both in active coeliac disease and after long-term challenge with dietary gluten, SEM analyses showed uniformly destructed villi. The api­cal surfaces of the enterocytes were frequently convex and irregular in size and delineation (the surface of the normal enterocytes was polygo­nal and flat). Ultrastructurally, the apical surfaces were severely damaged with a distortion of the glycocalyx and with marked irregularity of the microvilli. - After gluten elimination, the surface ultrastructu­re of the enterocytes in the coeliac gut mucosa generally showed a rapid, clear-cut restoration despite a remaining severe atrophy of the villi. Successful dietary treatment (after about one year of gluten-free diet) restored the small intestine mucosa to normal as assessed both by LM and low-power SEM. In contrast, high-power SEM often disclosed per­sisting lesions of the enterocytes. Another provocation with gluten for up to 9 days in clinically healed coeliac mucosa did not significantly alter the surface ultrastructure of the enterocytes. / <p>S. 1-52: sammanfattning, s. 53-138: 5 uppsatser</p> / digitalisering@umu

Rodent-borne hantaviruses (family Bunyaviridae) cause two distinct human infections; hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). HFRS is a common viral zoonosis, characterized by fever, renal dysfunction and hemostatic imbalance. Four HFRS-associated hantaviruses have been described: Hantaan virus and Seoul virus mainly found in Asia, Dobrava virus, encountered in the Balkan region and Puumala virus (PUU), causing mild HFRS (nephropathia epidemica; NE) in Europe. HPS, recently discovered in the Americas, involves adult respiratory distress syndrome with a high mortality rate and is caused by Sin Nombre virus. Hantaviruses are enveloped and carry a RNA genome which encodes a polymerase, two glycoproteins and a nucleocapsid protein. The latter elicits a strong humoral immune response in infected patients. The clinical diagnosis of hantavirus infections has until recently relied on serological confirmation by immunofluorescense assay (IFA) and enzyme-linked immunosorbent assay (ELISA) using cell culture derived viral antigens. Due to the hazardous nature of hantaviruses and variable virus yield in cell culture we aimed at using recombinant hantavirus proteins for serological purposes. We expressed PUU N in E. coli (PUU rN) and found that high levels of IgM to this protein could be detected at onset of NE. This indicated that it was useful as the sole antigen for serodiagnosis. Our finding was confirmed by comparing IFA and PUU rN ELISA using 618 sera collected at the regional diagnostic laboratory. Full-length PUU rN is difficult to purify due to aggregation to E. coli remnants. We therefore located the important domain for the humoral immune response by utilizing truncated PUU rN proteins to its amino-terminal region (amino acid 7-94). Amino acid 1-117 of N of the five major human hantavirus pathogens were produced in E. coli. Serological assays based on them could detect IgM and IgG serum responses in 380 HFRS and HPS patients from Sweden, Finland, Slovenia, China, Korea and the USA with high sensitivity. In an epidemiological investigation of hantavirus serum responses in European Russia we unexpectedly found antibody responses to the hantaviruses found in east Asia and the Balkan region in 1.5 %, speaking in favour for the presence of such virus in this region. The degree of cross reactivity within the hantavirus genus was adressed by following the serum responses in NE patients. We found an increase of cross reactivity during the maturation of the immune response from onset of disease up to three years by comparing the IgG reactivity towards the hantavirus aminoterminal rN proteins. The first human isolate of the causative agent of NE in Scandinavia was recovered in cell culture from phytohemagglutinin stimulated leukocytes. Serological analysis revealed that this virus belongs to the PUU hantavirus serotype, distinct from the rodent prototype PUU Sotkamo. The human PUU Umeå is unique but genetically similar to rodent isolates from northern Sweden. / digitalisering@umu.se

Radioimmunotherapy (RIT) and radioimmunolocalisation (RIL) are developing and promising technologies to diagnose and treat tumours by use of radiolabelled antibodies targeting tumour specific antigens. The major reason why RIL and RIT not are efficient enough, is the comparatively low accumulation of radiolabelled antibodies in the tumours. Irrespective of the antigen - antibody system used, the maximal tumour uptake in humans is often limited to below 0.1 % of the total injected dose, with significant radionuclide remaining in the blood pool and extravascular fluid. In the present thesis, the following putative improvement techniques for radioimmunotargeting have been evaluated in an experimental model using HeLa cell-xenografted nude mice: 1) Repetitive, simultaneous targeting of different antigens, 2) Removal of non-targeting antibodies using secondary antiidiotypic antibodies, 3) Preinjection of unlabelled antibody to remove shedded antigen and 4) Use of fractionated antibody administration. By use of multiple injections of mixtures of two different 131I-labelled monoclonal antibodies targeting placental alkaline phosphatase (H7) and cytokeratin 8 (TS1), respectively, a significant tumour growth inhibition compared to controls, was obtained. In the treated group, a negligible increase in tumour volume was seen compared to the control group, in which a 20-fold increase was observed. Quantitative determinations of volume densities of viable tumour cells, necrotic cells and connective tissue demonstrated no significant differences in the relative proportions between the groups, indicating that the irradiation caused decelerated growth. Using hybridoma technology, monoclonal antiidiotypic antibodies were generated against both TS1 and H7. The in vitro and in vivo effects of these antibodies, aH7 and aTSl, were investigated. Both these antiidiotypes were found to generate stable complexes with the radiolabelled idiotypic antibody, as revealed by gel-electrophoresis and autoradiography. Using biosensor technology (BIAcore, Pharmacia) the interactions were followed in real time and the association rate-, dissociation rate-, and affinity constants between the reactants were determined. In vivo, the antiidiotypes promoted a rapid dose dependent clearance of the 125I-labelled idiotypes with a decrease in total body radioactivity and concomitant dramatic increase in non-protein bound 125I excreted in the urine. The syngeneic monoclonal antiidiotypic antibody αTSl, was furthermore evaluated as a secondary clearing antibody at radioimmunolocalisation. Injection of αTSl in a molar ratio of 0.5-0.75:1 to TS1, 24 hours after the 125I-labelled TS1 improved the tumour to normal tissue ratio 2-3 fold. This was due to a decreased level of total body radioactivity as well as a slight decrease in tumour-radioactivity. A model describing the kinetics of the involved components, i.e. the antigen, the idiotype and the antiidiotype was presented. It is concluded that high affinity monoclonal antiidiotypes can be used as tools to regulate the levels of idiotypic antibodies in vivo. This strategy, combined with preinjection of non­labelled idiotypic antibodies, caused accumulated doses of 3 Gy to the tumour and 0.9 Gy to non tumour tissues as calculated for 125I-labelled antibodies (80 MBq/mg) by MIRD formalism based on repetitive quantitative radioimmunoscintigraphies. By approaching the maximal tolerated whole body radiation dose for mice (i.e. 6 Gy), it can be estimated that doses up to 20 Gy are possible to obtain following one single injection of labelled antibody. It was furthermore demonstrated that a single bolus injection of antibody is to be preferred, compared to exactly the same dose divided into three or ten fractions. Thus, not only the dose of radioactivity, but also the amount of antibody should be considered for fractionated RIT. In summary, the thesis demonstrates that several techniques can be used to improve radioimmunolocalisation and to approach the proposed 70 Gy required to sterilise tumours at radioimmunotherapy. / digitalisering@umu.se