Renal cell carcinoma (RCC) is the commonest urogenital tumor, characterized by increased expression of hypoxia inducible factors (HIFs). During normoxia, HIFα subunits are targeted for proteasomal degradation by the product of the von Hippel Lindau gene (pVHL). In RCC, mutations in the VHL gene allow the HIFα subunits to escape degradation and translocate to the nucleus where they activate transcription of their target genes. Although both HIF1α and HIF2α are upregulated in RCC, it has been suggested that HIF2α plays the dominant role. To further elucidate the function of HIF2α in RCC, we generated a transgenic mouse model that permits temporal stabilization of HIF2α in renal tubular cells. Induction of HIF2α results in the rapid development of renal cysts - a feature observed in RCC. Taken together, these results suggest that HIF2α is a key player in development of RCC and an excellent candidate target for therapy in this disorder.

Renal cell carcinoma (RCC) is the commonest urogenital tumor, characterized by increased expression of hypoxia inducible factors (HIFs). During normoxia, HIFα subunits are targeted for proteasomal degradation by the product of the von Hippel Lindau gene (pVHL). In RCC, mutations in the VHL gene allow the HIFα subunits to escape degradation and translocate to the nucleus where they activate transcription of their target genes. Although both HIF1α and HIF2α are upregulated in RCC, it has been suggested that HIF2α plays the dominant role. To further elucidate the function of HIF2α in RCC, we generated a transgenic mouse model that permits temporal stabilization of HIF2α in renal tubular cells. Induction of HIF2α results in the rapid development of renal cysts - a feature observed in RCC. Taken together, these results suggest that HIF2α is a key player in development of RCC and an excellent candidate target for therapy in this disorder.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Gabriela Kocurová Supervisor: Prof. MUDr. Jaroslav Dršata, CSc. Title of diploma thesis: Investigation of proteolytic enzymes expression in different tissues at the transgenic animal model of Huntington's disease by means of biochemical and immunohistochemical methods Background: Huntington's disease (HD) is a neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) domain in the huntingtin (Htt) protein. Because it is known that mutant Htt and especially its small proteolytic fragments are toxic to neurons (particularly those in the striatum and cortex), it has been suggested that proteolysis of mutant huntingtin (mHtt) might play an important role in HD pathogenesis. Therefore, the aim of the present study was to examine the expression of endogenous and mtHtt and possible participation of the proteolytic enzymes from the group of caspases, matrix metalloproteinases (MMPs), kallikreins (KLKs) and calpains in HD pathology of brain tissue. Methods: In this study we used WT and TgHD minipigs for N-terminal part of the human mtHtt (548aaHTT-145Q, both F2 generation, age 36 months; F3 generation, age 48 months in additional experiment), R6/2 mice were used as...

Atrial natriuretic factor (ANF) is a polypeptide hormone produced in heart atria, stored in atrial secretory granules and released into the circulation in response to various stimuli. Proper sorting of ANF at the level of the trans-Golgi network (TGN) is required for the storage of ANF in these specific granules, and this sorting of hormones has been found to be associated with G-proteins. Specifically, the Go protein alpha-subunit (Gαo) was established to participate in the stretch-secretion coupling of ANF, but may also be involved in the transporting of ANF from the TGN into atrial granules for storage and maturation. Based on knowledge of Gαo involvement in hormone production in other endocrine tissues, protein-protein interactions of Gαo and proANF and their immunochemical co-localization in granules, the direct involvement of these two proteins in atrial granule biogenesis is probable. In this study, mice were created using the Cre/lox recombination system with a conditional Gαo knockout in cardiocytes to study and characterize ANF production, secretion and granule formation. Deletion of this gene was successful following standard breeding protocols. Characterization and validation of cellular and molecular content of the knockout mice through mRNA levels, protein expression, peptide content, electron microscopy, and electrocardiography determined that a significant phenotypic difference was observed in the abundance of atrial granules. However, Gαo knockout mice did not significantly alter the production and secretion of ANF and only partially prevented granule biogenesis, likely due to incomplete Gαo knockout. These studies demonstrate an involvement of Gαo in specific atrial granule formation.

Atrial natriuretic factor (ANF) is a polypeptide hormone produced in heart atria, stored in atrial secretory granules and released into the circulation in response to various stimuli. Proper sorting of ANF at the level of the trans-Golgi network (TGN) is required for the storage of ANF in these specific granules, and this sorting of hormones has been found to be associated with G-proteins. Specifically, the Go protein alpha-subunit (Gαo) was established to participate in the stretch-secretion coupling of ANF, but may also be involved in the transporting of ANF from the TGN into atrial granules for storage and maturation. Based on knowledge of Gαo involvement in hormone production in other endocrine tissues, protein-protein interactions of Gαo and proANF and their immunochemical co-localization in granules, the direct involvement of these two proteins in atrial granule biogenesis is probable. In this study, mice were created using the Cre/lox recombination system with a conditional Gαo knockout in cardiocytes to study and characterize ANF production, secretion and granule formation. Deletion of this gene was successful following standard breeding protocols. Characterization and validation of cellular and molecular content of the knockout mice through mRNA levels, protein expression, peptide content, electron microscopy, and electrocardiography determined that a significant phenotypic difference was observed in the abundance of atrial granules. However, Gαo knockout mice did not significantly alter the production and secretion of ANF and only partially prevented granule biogenesis, likely due to incomplete Gαo knockout. These studies demonstrate an involvement of Gαo in specific atrial granule formation.

Background: Consecutive adult neurogenesis is a well-known phenomenon in the ventricular–subventricular zone of the lateral wall of the lateral ventricles (V–SVZ) and has been controversially discussed in so-called “non-neurogenic” brain areas such as the periventricular regions (PVRs) of the aqueduct and the fourth ventricle. Dopamine is a known modulator of adult neural stem cell (aNSC) proliferation and dopaminergic neurogenesis in the olfactory bulb, though a possible interplay between local dopaminergic neurodegeneration and induction of aNSC proliferation in mid/hindbrain PVRs is currently enigmatic. Objective/Hypothesis: To analyze the influence of chronic–progressive dopaminergic neurodegeneration on both consecutive adult neurogenesis in the PVRs of the V–SVZ and mid/hindbrain aNSCs in two mechanistically different transgenic animal models of Parkinson´s disease (PD). Methods: We used Thy1-m[A30P]h α synuclein mice and Leu9′Ser hypersensitive α4* nAChR mice to assess the influence of midbrain dopaminergic neuronal loss on neurogenic activity in the PVRs of the V–SVZ, the aqueduct and the fourth ventricle. Results: In both animal models, overall proliferative activity in the V–SVZ was not altered, though the proportion of B2/activated B1 cells on all proliferating cells was reduced in the V–SVZ in Leu9′Ser hypersensitive α4* nAChR mice. Putative aNSCs in the mid/hindbrain PVRs are known to be quiescent in vivo in healthy controls, and dopaminergic deficiency did not induce proliferative activity in these regions in both disease models. Conclusions: Our data do not support an activation of endogenous aNSCs in mid/hindbrain PVRs after local dopaminergic neurodegeneration. Spontaneous endogenous regeneration of dopaminergic cell loss through resident aNSCs is therefore unlikely.