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.

Orientador: Prof. Dr. Marcelo Augusto Christoffolete / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2014. / Desde o século XV, animais fazem parte da rotina na área da pesquisa, principalmente para estudos de doenças, e hoje em dia o modelo animal mais utilizado para estes estudos é o camundongo, tendo uma participação em mais de 90% das pesquisas em todo o mundo, sendo considerado como uma primeira via para definir funções de genes em mamíferos. Os camundongos são considerados os principais modelos nas técnicas de transgenia animal, porém estas técnicas ainda estão em desenvolvimento, uma vez que as metodologias hoje utilizadas para a geração de animais transgênicos ainda se encontram com uma taxa de sucesso considerada baixa e são dispendiosas, necessitando de muitas etapas. Uma das dificuldades é o contato com a membrana do óvulo devido a zona pelúcida, que é considerada uma barreira física. Vetores virais estão em evidência nas técnicas de transgenia animal, sendo o lentivírus o mais utilizado. Portanto, o objetivo deste projeto é estabelecer um protocolo para a integração de DNA exógeno em óvulos por infecção lentiviral, anteriormente a fertilização in vitro juntamente com a técnica de dissecção parcial da zona pelúcida. Como vetor foi utilizado um lentivírus com GFP em sua construção. Para ocorrer a fertilização in vitro, foram feitas coletas de óvulos em camundongos fêmeas da linhagem C57BL/6, tratadas com injeções hormonais, e coletas de espermatozoides em machos desta mesma linhagem. Os óvulos obtidos foram divididos em grupos controle e com dissecção parcial da zona pelúcida, e estes foram subdivididos em grupos com e sem infecção lentiviral. Entre os grupos houve variação de 20% a 56,25% de embriões em estágio de duas células, e em alguns grupos foi possível alcançar o estágio de blastocisto eclodido. Porém não foi possível visualizar a emissão de fluorescência para confirmar a infecção lentiviral. Em conclusão as metodologias utilizadas tanto para a fertilização in vitro como para a dissecção parcial da zona pelúcida foram de sucesso. Porém a integração do DNA exógeno mostrou resultados não conclusivos, necessitando de estudos futuros. / Since the XV century, animals are used routinely in research, mainly for diseases studies, and nowadays the most used animal model is the mouse, which one has more than 90% of participation in researches around the world and it is considered the first track to define gene function in mammals. Mouse is the main model in transgenic techniques, however the methods available to generate transgenic animals still have a considerable low rate, and also it is expensive, requiring many degrees. An ordinary issue is the contact with the membrane of oocyte due zona pellucida that is considered a physical barrier. In transgenic animals technique, it is in evidence the utilization of viral vectors, and the most used are the lentiviruses. Therefore, the objective of this project is to establish a protocol for the integration of exogenous DNA by lentiviral infection into oocytes, before the in vitro fertilization, using the technique of partial dissection of the zona pellucida. It was used as a vector a lentivirus with GFP in your construction. For in vitro fertilization, were collected oocytes from C57Bl/6 mice, treated with hormones, and sperm from males of the same strain. The obtained oocytes were divided in control group and partial dissection of the zona pellucida group, and then subdivided in groups with and without lentiviral infection. Between the groups, was achieved 20% to 56,25% of two cells stage embryo, and hatched blastocysts stage were obtained at some groups. Therefore it was not possible to visualize florescence emission to confirm the lentiviral infection. In conclusion we have a practicable protocol for in vitro fertilization and partial dissection of the zona pellucida, reaching blastocysts stages in two groups. However the integration of exogenous DNA results were inconclusive, requiring further studies.

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...

Alzheimer’s disease (AD) is histopathologically characterized by neurodegeneration, the formation of intracellular neurofibrillary tangles and extracellular Aβ deposits that derive from proteolytic processing of the amyloid precursor protein (APP). As rodents do not normally develop Aβ pathology, various transgenic animal models of AD were designed to overexpress human APP with mutations favouring its amyloidogenic processing. However, these mouse models display tremendous differences in the spatial and temporal appearance of Aβ deposits, synaptic dysfunction, neurodegeneration and the manifestation of learning deficits which may be caused by age-related and brain region- specific differences in APP transgene levels. Consequentially, a comparative temporal and regional analysis of the pathological effects of Aβ in mouse brains is difficult complicating the validation of therapeutic AD treatment strategies in different mouse models. To date, no antibodies are available that properly discriminate endogenous rodent and transgenic human APP in brains of APP-transgenic animals. Here, we developed and characterized rat monoclonal antibodies by immunohistochemistry and Western blot that detect human but not murine APP in brains of three APP-transgenic mouse and one APP-transgenic rat model. We observed remarkable differences in expression levels and brain region-specific expression of human APP among the investigated transgenic mouse lines. This may explain the differences between APP-transgenic models mentioned above. Furthermore, we provide compelling evidence that our new antibodies specifically detect endogenous human APP in immunocytochemistry, FACS and immunoprecipitation. Hence, we propose these antibodies as standard tool for monitoring expression of endogenous or transfected APP in human cells and APP expression in transgenic animals.

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.