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Molecular Pathology of Acute Intermittent Porphyria
Hrdinka, Matouš
Molecular Pathology of Acute Intermittent Porphyria Author: Matouš Hrdinka Acute intermittent porphyria (AIP) is autosomal dominant disorder caused by the partial deficiency of porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthetic pathway. AIP is manifested by life-threatening acute neurological attacks that can be provoked by various factors such as drugs or alcohol. Objective of this study was to identify and characterize the molecular lesions in Czech and Slovak AIP patients. To identify disease-causing mutations screening was performed by PCR, denaturing gradient gel electrophoresis (DGGE), automated DNA sequencing and restriction fragment lenght polymorphism (RFLP) assays. Total of 35 individuals from 8 families were analyzed to detect asymptomatic carriers. Seven mutations were identified, including 3 novel mutations. Of particular interest, one patient had two mutations R173Q and Q204K, both located in exon 10 on the same allele. To further characterize these mutations, human PBGD was cloned into the pGEX-4T-1 vector and mutations were generated by site-directed mutagenesis. The wild-type and mutated enzyme species were expressed in E. coli as GST fusions and purified by affinity chromatography. Conditions of the PBGD enzyme assay were determined and specific...
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The role of membrane microdomains and transmembrane adaptor proteins PRR7 and SCIMP in the regulation of immunoreceptor signaling
Hrdinka, Matouš ; Drbal, Karel (advisor) ; Černý, Jan (referee) ; Kalina, Tomáš (referee)
Dissertation summary The role of membrane microdomains and transmembrane adaptor proteins PRR7 and SCIMP in the regulation of immunoreceptor signaling Matouš Hrdinka How do the plasma membrane microdomains and transmembrane adaptor proteins (TRAPs) influence the outcome of immunoreceptor signaling? These have been the important questions of molecular immunology. In spite of the years of intensive research, these problems remain incompletely understood. The plasma membrane is a highly dynamic heterogeneous bilayer spontaneously organized into microdomains of various size, composition, and lifetime. The lipid rafts are one example of such microdomains and have been implicated in many biological processes, including immunoreceptor signaling. Because rafts are enriched in many signaling proteins, they are believed to function as platforms for signal initiation and propagation. The TRAPs are important organizers and regulators of immunoreceptor signaling. For example, LAT is indispensable in T cell receptor (TCR) signaling and T cell development, PAG for the regulation of Src family tyrosine kinases (SFKs), and NTAL is a multifunctional negative and positive regulator. The presence of these TRAPs in lipid rafts seems to be crucial for their functions, however, is still a matter of debate. Moreover, other so far...
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Molecular Pathology of Acute Intermittent Porphyria
Hrdinka, Matouš
Molecular Pathology of Acute Intermittent Porphyria Author: Matouš Hrdinka Acute intermittent porphyria (AIP) is autosomal dominant disorder caused by the partial deficiency of porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthetic pathway. AIP is manifested by life-threatening acute neurological attacks that can be provoked by various factors such as drugs or alcohol. Objective of this study was to identify and characterize the molecular lesions in Czech and Slovak AIP patients. To identify disease-causing mutations screening was performed by PCR, denaturing gradient gel electrophoresis (DGGE), automated DNA sequencing and restriction fragment lenght polymorphism (RFLP) assays. Total of 35 individuals from 8 families were analyzed to detect asymptomatic carriers. Seven mutations were identified, including 3 novel mutations. Of particular interest, one patient had two mutations R173Q and Q204K, both located in exon 10 on the same allele. To further characterize these mutations, human PBGD was cloned into the pGEX-4T-1 vector and mutations were generated by site-directed mutagenesis. The wild-type and mutated enzyme species were expressed in E. coli as GST fusions and purified by affinity chromatography. Conditions of the PBGD enzyme assay were determined and specific...
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