Magistrinio darbo metu sukonstruotos mielių plazmidės, turinčios pelių poliomos viruso pagrindinį kapsidės baltymą VP1 su jame įterptais hepatito C viruso apvalkalo baltymų peptidais. Šiomis plazmidėmis buvo transformuotos S. cerevisiae mielės. Transformuotos mielės sintetina chimerinius baltymus: MPyV-VP1-BC-E2 [384-414], MPyV-VP1-HI-E2 [384-414], MPyV-VP1-BC-E1 [310-329], MPyV-VP1-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447], MPyV-VP1-HI-E2 [412-i447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-i447], MPyV-VP1-BC-E2 [412-447]-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447]-HI-E1 [310-329]. Į virusus panašias daleles (VPD) renkasi tik MPyV-VP1-BC-E2 [384-414], MPyV-VP1-HI-E2 [384-414], MPyV-VP1-BC-E1 [310-329] baltymai. Kiti baltymai: MPyV-VP1-BC-E1 [310-329], MPyV-VP1-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447], MPyV-VP1-HI-E2 [412-i447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-i447], MPyV-VP1-BC-E2 [412-447]-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447]-HI-E1 [310-329] nesirinko į VPD. / In the present study, plasmids for expression of major capsid proteins VP1 of murine polyomavirus with inserted sequences from Hepatitis C virus envelope proteins in yeast S. cerevisiae were constructed. The plasmids were used to transform yeast cells. The transformed yeast produced proteins: MPyV-VP1-BC-E2 [384-414], MPyV-VP1-HI-E2 [384-414], MPyV-VP1-BC-E1 [310-329], MPyV-VP1-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447], MPyV-VP1-HI-E2 [412-i447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-i447], MPyV-VP1-BC-E2 [412-447]-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447]-HI-E1 [310-329]. Virus-like particles (VLPs) composed MPyV-VP1-BC-E2 [384-414], MPyV-VP1-HI-E2 [384-414], MPyV-VP1-BC-E1 [310-329]. Other proteins: MPyV-VP1-BC-E1 [310-329], MPyV-VP1-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447], MPyV-VP1-HI-E2 [412-i447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-447], MPyV-VP1-BC-E1 [310-329]-HI-E2 [412-i447], MPyV-VP1-BC-E2 [412-447]-HI-E1 [310-329], MPyV-VP1-BC-E2 [412-i447]-HI-E1 [310-329] did not form VLPs.

Interaction of polyomaviruses with proteasomal system of host cells Abstract: Viral family Polyomaviridae includes besides model organisms - mouse polyomavirus and SV40 virus, also human pathogens, for example, BK virus. Polyomaviruses are small non- enveloped viruses with double-stranded DNA. Understanding of their life cycle is important for their use in gene therapy and immunotherapy as well as for prevention and treatment of complications caused by these viruses. This thesis is focused on early phases of MPyV and SV40 infection studying, mainly on delivery of viral genome to nucleus and role of proteasomal system in this stage of infection. It was found out that inhibition of proteasomes by specific inhibitor leads to increase of early non-structural protein LT expression, which was chosen as marker for viral entry to the nucleus and successful viral expression. Relative localization of proteasomes and VP1 protein of MPyV and SV40 was monitored and it showed 10% colocalization of mentioned structures. Further, it was found out that proteasomal inhibitor MG-132 negatively influences the replication of both viral and cellular DNA. Next aim of this diploma thesis was to prepare antigen - unique part of VP2 protein of BKV - for producing antibody. Expression vector with inserted fragment of unique part of...

K. Babiarová Ph.D. Thesis ABSTRACT Cancer immunotherapy is concerned generally with the activation of cancer immunity specific for tumor antigens (TA) produced by cancer cells. My PhD thesis focused on the development of different types of cancer vaccines expressing various TA and predominantly on the determination of the efficacy of these vaccines. For studying TA-specific cancer cellular immunity in mice immunized with these vaccines, I used mainly the ELISPOT-IFNγ assay. First, DNA, recombinant vaccinia virus (rVACV) and peptide vaccines against WT1 positive tumors were prepared. They consist of a fragment of WT1 protein with motifs predicted to bind to Db murine MHC class I. The administration of peptide vaccines by tattoo delivery in combination with unmethylated CpG motifs and anti-TGFβ monoclonal antibody was the most effective. Next, I was interested in the immunotherapy of chronic myeloid leukemia (CML). Hruskova et al. prepared the mouse polyomavirus-like particles (MPyV-VLP) carrying the junction region of BCR-ABL fusion protein (1). In our laboratory, there were constructed the other types of CML vaccines with the expression of the junction region of BCR-ABL fusion protein, such as DNA or rVACV, too. Prepared vaccines failed to induce effective cancer immune response. It seems that BCR-ABL...

In this study, we focused on interactions of structural proteins of mouse polyomavirus (MPyV) and BK virus (BKV) with the nuclear lamina. Our goal was to examine whether and how can the virus, hence viral structural proteins, interact with the nuclear lamina and how would these interactions affect its properties. We supposed, that the expression of viral proteins would induce disintegration of the structure of nuclear lamina, thus enabling nuclear egress of virions in the late phase of infection. Viral structural proteins were expressed transiently in cells transfected with an expression vector pMPyV LATE. In these cells, VP1 was localized in a likewise manner as it shows in infected cells - mostly in a perinuclear area. Concurrently, defects in staining of nuclear lamina were observed in these cells, similarly to infected cells. Also, another expression vector was used in our experiments, the pMPyV mut3 VP1 encoding for a mutated protein VP1. When transiently expressed in cells, the mutated VP1 protein showed mostly diffuse nuclear localization. However, we observed significant morphological deformations and defective staining of the nuclear lamina. These observations imply an important role of VP1 in mechanical and biochemical properties alterations of the nuclear lamina in transfected and...

Murine polyomavirus is an important member of Polyomaviridae family offering potential applications in gene therapy and immunotherapy. Viral mutant analysis is crucial for study of the virus, however, commonly used methods of its production are laborious and give low yields. This thesis involves development of the new experimental system that can produce intact viral genome from recombinant plasmid in vivo using Cre/loxP-mediated recombination. One loxP site is unavoidably introduced into newly generated viral genome during recombination. Two variants of production plasmids generating wild type viral genome with incorporation of loxP between the poly(A) signal sites of early and late genes or into the intronic region of early genes were prepared. LoxP insertion between the poly(A) signal sites has a dramatic effect on viral gene expression and leads to complete loss of virus infectivity. Conversely, the infectious virus was obtained from the viral genome containing loxP site in the early intronic region. To ensure expression of Cre recombinase I also prepared stably transfected cell lines which can simplify the virus production. This thesis shows that newly designed system gives satisfactory yield of the virus, solves restrictions connected with commonly used methods and can be used for low infectious viral...