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Metabolism carcinogens and drugs by the system of monooxygenases
Moserová, Michaela ; Stiborová, Marie (advisor) ; Entlicher, Gustav (referee) ; Čeřovská, Noemi (referee)
Ellipticine, an alkaloid isolated from Apocynaceae plants, exhibits significant antitumor and HIV activities. Ellipticine is a pro-drug, whose pharmacological and genotoxic effects depend on activation by cytochromes P450 (CYP) and peroxidases (Px) to a reactive species generating DNA adducts. To elucidate contribution of CYPs (and which of them) and Px to ellipticine activation, we used rat and mouse models, mice with deleted gene of NADPH:CYP reductase in the liver, thus absenting this enzyme in the liver (HRNTM ) and a control mouse line (WT), rats treated with ellipticine, and microsomal systems isolated from the liver of mouse lines and from the liver, kidney and lung of rats. The purified enzymes, CYP1A1 and 3A4, reconstituted with NADPH:CYP reductase were also used. The effect of cytochrome b5, a facultative component of the mixed function monooxygenase system, on ellipticine oxidation by CYP1A1 and 3A4 was also investigated. Carcinogenic benzo(a)pyrene (BaP), known to covalently bind to DNA after its activation with CYPs, was investigated for its potential to generate DNA adducts and to induce CYP and NADPH:CYP reductase enzymes in mouse livers. We investigated an influence of each of components of the mixed function oxidases (MFO) system on metabolism of BaP. CYP1A1 is widely accepted to be the...
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Plant virus-based biotechnology
Vaculík, Petr ; Čeřovská, Noemi (advisor) ; Ryšánek, Pavel (referee) ; Petrzik, Karel (referee)
The latest model of tertiary structure of capsid protein of potato virus X (PVX CP) was used as a template to design new insertion sites suitable for the preparation of PVX-based antigen presentation system. Based on this model, seven insertion sites (A-G) located in putative surface loops were tested. As an antigen inserted into these sites was used 17 amino acids long epitope derived from human papillomavirus type 16 E7 oncoprotein (E7 epitope) fused with either 6xHis tag or StrepII tag in both possible orientations (6xHis-E7 and E7-6xHis, StrepII-E7 and E7-StrepII). Prior to plant expression, modified PVX CPs were expressed in Escherichia coli MC1061. The results showed that only PVX CP carrying StrepII-E7 or E7-StrepII in the insertion site A formed virus particles. The results from transient expression experiments with modified PVX CPs in Nicotiana benthamiana showed that only the insertion site A (located between 24th and 25th amino acid in the PVX CP) could tolerate all tested inserts. Importantly, viral particles were detected only in the presence of StrepII tag and their stability was affected by the insert orientation (StrepII-E7 vs. E7-StrepII) as only the viral particles presenting E7-StrepII could be purified. Besides the preparation of PVX-based antigen presentation system, an...
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Acetyl - CoA carboxylase - evolution and inhibition
Chalupská, Dominika ; Čeřovská, Noemi (advisor) ; Plchová, Helena (referee) ; Vlček, Čestmír (referee)
Abstract
Acetyl-CoA
carboxylase
(ACC)
is
a
key
enzyme
of
fatty
acid
metabolism
with
multiple
isozymes
often
expressed
in
different
eukaryotic
cellular
compartments.
In
agriculture,
inhibitors
of
plastid
ACC
are
used
as
efficient
herbicides
against
grass
weed.
However,
grass
weed
populations
resistant
to
aryloxyphenoxypropionate
(APP)
and
cyclohexanedione
(CHD)
herbicides
represent
a
major
problem
for
sustainable
agriculture.
Using
PCR
and
sequencing
it
was
found
out
that
five
amino
acid
substitutions
in
plastid
ACC
were
correlated
with
herbicide
resistance
of
Avena
sterilis
ssp.
ludoviciana
Durieu
populations
from
the
northern
grain-growing
region
of
Australia:
Trp-1,999-Cys,
Trp-2,027- Cys,
Ile-2,041-Asn,
Asp-2,078-Gly
and
Gly-2,096-Ala.
We
showed,
using
a
yeast
gene- replacement
system,
that
these
single-site
mutations
also
confer
herbicide
resistance
to
wheat
plastid
ACCase:
Asp-2,078-Gly
confers
resistance
to
APPs
and
CHDs,
Trp-2,027-Cys
and
Ile-2,041-Asn
confer
resistance
to
APPs,
and
Trp-1,999-Cys
confers
resistance
only
to
fenoxaprop.
These
mutations
are
very
likely
to
confer
resistance
to
any
grass
weed
species
under
selection
imposed
by
the
extensive
agricultural
use
of
the
herbicides.
...
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Production of heterologous proteins in plants - human papillomavirus (HPV 16) derived antigens
Folwarczna, Jitka ; Čeřovská, Noemi (advisor) ; Ryšánek, Pavel (referee) ; Kumar, Jiban (referee)
5 Abstract Even though prophylactic vaccine against human papillomavirus (HPV) is currently licensed, infections by the virus continue to be the major health problem mainly in developing countries. Considerable effort is being devoted to preparation of therapeutic vaccine and to decrease of the production costs of current vaccine. Viral proteins such as the E7 oncoprotein and the L2 capsid protein from HPV type 16 are promising targets for the development of the experimental anti-HPV vaccine. The aim of our work was optimization of expression of mutagenized E7 oncoprotein (E7ggg) fused to the C-terminus of Tobacco mosaic virus (TMV) coat protein (CP) or Potato virus X (PVX) CP in viral vectors derived from these plant viruses. The impact of linkers connecting CP and E7ggg fusion partners on expression and stability of fusion proteins was examined. The fusion proteins were first expressed in Escherichia coli (E. coli) MC1061 to assess the characteristics of the recombinant protein prior to their transient expression in both non-transgenic or transgenic Nicotiana benthamiana (N. benthamiana). We have obtained the high level expression in E. coli, but most of the expressed proteins based on TMV CP remained in insoluble inclusion bodies. To increase the ratio of soluble protein various molecular...
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Metabolism carcinogens and drugs by the system of monooxygenases
Moserová, Michaela ; Stiborová, Marie (advisor) ; Entlicher, Gustav (referee) ; Čeřovská, Noemi (referee)
Ellipticine, an alkaloid isolated from Apocynaceae plants, exhibits significant antitumor and HIV activities. Ellipticine is a pro-drug, whose pharmacological and genotoxic effects depend on activation by cytochromes P450 (CYP) and peroxidases (Px) to a reactive species generating DNA adducts. To elucidate contribution of CYPs (and which of them) and Px to ellipticine activation, we used rat and mouse models, mice with deleted gene of NADPH:CYP reductase in the liver, thus absenting this enzyme in the liver (HRNTM ) and a control mouse line (WT), rats treated with ellipticine, and microsomal systems isolated from the liver of mouse lines and from the liver, kidney and lung of rats. The purified enzymes, CYP1A1 and 3A4, reconstituted with NADPH:CYP reductase were also used. The effect of cytochrome b5, a facultative component of the mixed function monooxygenase system, on ellipticine oxidation by CYP1A1 and 3A4 was also investigated. Carcinogenic benzo(a)pyrene (BaP), known to covalently bind to DNA after its activation with CYPs, was investigated for its potential to generate DNA adducts and to induce CYP and NADPH:CYP reductase enzymes in mouse livers. We investigated an influence of each of components of the mixed function oxidases (MFO) system on metabolism of BaP. CYP1A1 is widely accepted to be the...
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Molecular-biological characterization of Potato virus M and Potato leafroll virus
Vaculík, Petr ; Čeřovská, Noemi (advisor) ; Fousek, Jan (referee)
The main aims of diploma thesis were: 1) The sequence analysis of the Czech isolate of Potato virus M (PVM) VIRUBRA 4/009 and phylogenetic analysis of PVM coat proteins sequences 2) The bacterial expression of recombinant triple gene block protein 1 (TGB1) of PVM derived from the Czech isolate VIRUBRA 4/007 3) The construction of expression cassette of Potato leafroll virus (PLRV) coat protein and its transformation into A. tumefaciens for transgenic PLRV resistant plant formation In theoretical part of the thesis the taxonomic classification, morphology, genomic structure and virus transmission are discussed. Furthermore, the main rules concerning the bacterial expression of recombinant proteins and construction of transgenic plants using A. tumefaciens are described. Methodical part is devoted to description of generally used molecular biological and immunochemical methods. The following results were obtained in the thesis: The complete nucleotide sequences of open reading frames coding for three movement proteins (Triple gene block -TGB), coat protein and NA-binding protein of PVM isolate VIRUBRA 4/009; phylogenetic analysis was performed; the TGB1 protein was expressed in bacterial cells and will be used for polyclonal antibodies raising. Finally, the expression cassette containing the PLRV...
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Characterization of recombinant human serine racemase
Hoffman, Hillary Elizabeth ; Konvalinka, Jan (advisor) ; Křen, Vladimír (referee) ; Čeřovská, Noemi (referee)
6 Brief Abstract The pyridoxal-5'-phosphate-dependent enzyme serine racemase (SR) is responsible for the biosynthesis of D-serine in the mammalian central nervous system. D-serine acts as a neurotransmitter and coagonist, together with L-glutamate, of ionotropic N-methyl-D-aspartate receptors (NMDARs). Excitotoxic D-serine levels have been implicated in neuropathologies including Alzheimer's disease and amyotrophic lateral sclerosis. SR inhibitors offer a novel and potentially highly specific approach for attenuation of NMDAR-mediated glutamate excitotoxicity and for further study of the pathway. Many of the SR inhibitors described to date are small, naturally occurring compounds, and novel structures capable of influencing SR's activity are highly sought after. Moreover, structural information about this enigmatic enzyme is lacking, and suitable animal models need to be identified for inhibitor studies. This thesis presents the first published biochemical comparison of mouse and human SR orthologs, validating, at least in part, the use of mouse models in SR research. Additionally, hydroxamic acids are introduced as a novel class of SR inhibitors. While the experimentally determined structure of a mammalian SR remains elusive, random and site-directed mutagenesis experiments in combination with multiple...
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