7 Abstract Neuroblastoma is the most common extracranial solid tumor of childhood. Despite advances in cancer diagnosis and therapy, the treatment of some forms of neuroblastoma is still complicated. One of the major complications of the chemotherapy is a developed drug resistance. This master thesis deals with the effect of cytostatics on protein and gene expression of selected proteins, which may contribute to chemoresistance of the human neuroblastoma cell line UKF-NB-4. The sensitive line UKF-NB-4 and the resistant line UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI were exposed to cisplatin, doxorubicin, ellipticine for 24, 48 and 72 hours. The Western blot analysis showed that cytostatic agents cisplatin, doxorubicin or ellipticine added to the sensitive neuroblastoma cell line UKF-NB-4 in amounts which are added to resistant neuroblastoma cell lines in order to maintain resistance induced expression of p53 and reduced expression of retinoblastoma protein pRb after 72 hours of cultivation. Differences in the expression of RAS protein, cytochrome P450 1A1, 3A4 and cytochrome b5 has not been shown. Changes in the expression of the studied proteins in resistant lines UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI cultured with and without cytostatic agents were not detected by the Western blot analysis....

Devido ao largo espectro de manifestações na leishmaniose visceral canina (LVC), o exame clínico não é capaz de confirmar o diagnóstico, já que muitos dos sinais clínicos não são exclusivos da LVC, tornando-se imprescindível o diagnóstico laboratorial para confirmação da infecção por L. (L.) infantum chagasi. A sorologia tem sido a primeira escolha para o diagnóstico da leishmaniose canina; porém, a diversidade de técnicas empregadas e de antígenos tem levado a resultados conflitantes. O emprego de formas promastigotas e amastigotas do parasito tem boa concordância no sorodiagnóstico de LVC pela Reação de Imunofluorescência Indireta (RIFI), porém o teste com amastigotas mostrou-se mais sensível sem perda da especificidade. Sendo assim, o objetivo deste estudo foi avaliar o desempenho do ELISA no sorodiagnóstico da LVC empregando antígeno total bruto de formas amastigotas provenientes de cultura axênica (ELISA-AXE) e amastigotas purificadas de tecido de animal experimental cronicamente infectado (ELISA-AMA) comparando com o desempenho do ELISA feito com antígeno total bruto de formas promastigotas (ELISA-PRO) de L. (L.) infantum chagasi. Foram avaliados 115 soros de cães parasitologicamente positivos para a pesquisa de DNA de Leishmania por PCR, domiciliados a pelo menos 2 anos em municípios com alta transmissão de leishmaniose visceral, classificados em sintomáticos (n=67) e assintomáticos (n=48) de acordo com sinais clínicos e exames laboratoriais. Como controle, foram utilizados 81 soros de cães parasitologicamente negativos oriundos do município sem transmissão comprovada de leishmaniose visceral e 13 soros de cães com outras enfermidades. Os resultados não mostraram diferença significante em sensibilidade, especificidade, valores preditivos e acurácia entre os testes ELISA-AXE, ELISA-AMA e ELISA-PRO, com forte correlação e concordância entre os três antígenos testados. O ensaio de Western Blot mostrou reatividade para proteínas de diferentes pesos moleculares, entre 14 a 178kDa dependendo da fonte de antígeno. Os resultados mostraram desempenho semelhantes nos testes de ELISA com amastigotas axênicas, amastigotas purificadas e promastigotas como fonte de antígeno para sorologia da leishmaniose canina. A ampla e diversificada reatividade no Western Blot com poucas bandas altamente especificas sugere o emprego de quimeras de antígenos de diferentes pesos moleculares oriundos tanto de formas amastigotas como de formas promastigotas do parasito no diagnóstico da LVC / Due to the wide spectrum of manifestations in canine visceral leishmaniasis (CVL), clinical exam is not able to confirm the diagnosis, since many of the signs and symptoms are not exclusive of CVL, making it imperative that the laboratory diagnosis for infection confirmation by L. (L.) infantum chagasi. Since the direct search involves invasive material collection, time-consuming and expensive; the serology has been the first choice for diagnosis of canine leishmaniasis. However, the diversity of techniques employed and antigens have led to conflicting results in both diagnostic routine and applied research. Since the use of promastigotes and amastigotes of L. (L.) infantum chagasi have good agreement on serum diagnosis of CVL by reaction of Indirect Immunofluorescence (RIFI), despite the amastigote test proved more sensitive without loss of specificity. The main objective of this study was to evaluate and compare the performance of ELISA in the CVL using crude total antigen of axenic amastigote and promastigote culture, and purified amastigote from experimental chronically infected animal tissue of L. (L.) infantum chagasi. One hundred and fifty-five sera from dogs residing on areas with high disease incidence were evaluated by PCR and diagnosed positive for Leishmania, these were used as positive control on the present study. The animals were examined clinically and in accordance with the characteristic clinical signs of visceral leishmaniasis and laboratory tests, then were classified into 2 groups: symptomatic (n=67) and asymptomatic (n=48) animals. As negative control, eighty-one sera were used from dogs parasitologically negative by PCR in real time from the municipality without proven transmission of visceral leishmaniasis. The results showed no significant difference in sensitivity, specificity, predictive values and accuracy between ELISA-AXE, ELISA-AMA and ELISA-PRO, with strong correlation and concordance between the three antigens tested. The Western Blot assay showed reactivity for proteins of different molecular weights ranging from 14 to 178kDa depending on the antigen source. The results showed similar performance in the ELISA tests with axenic amastigotes, purified amastigotes and promastigotes as source of antigen for serology of canine leishmaniasis. The broad and diverse reactivity in Western Blot with few highly specific bands suggests the use of antigen chimeras of different molecular weights derived from both amastigote and promastigote forms of the parasite in the diagnosis of LVC

Philosophiae Doctor - PhD / Obesity is a medical condition in which an energy imbalance leads to excessive accumulation of body fat. Obesity leads to a reduction in life expectancy through its association with chronic diseases of lifestyle. The prevalence of obesity is rapidly increasing throughout the world. It is now accepted that most cases of obesity result from an interaction between genetic and environmental factors. This rapid increase in obesity generally leads to an increase in morbidity and mortality from chronic diseases such as cardiovascular disease, type 2 diabetes, osteoarthritis and cancer of which obesity is a risk factor. There is a lack of information in molecular research to explain how obesity predisposes individuals to these diseases. Proteomics is a molecular tool and a set of techniques used to identify changes at protein level from a diseased state. This study aims to identify differentially expressed proteins in serum of obese rats fed different isocaloric diets using proteomics.

Hypoxia Inducible Factor-1α in Renal Cell Carcinoma Departments of Surgical and Perioperative Sciences, Urology and Andrology; Radiation Sciences, Oncology; Medical Biosciences, Pathology; and Medical Biosciences, Clinical Chemistry, Umeå University, Umeå, Sweden Background: Renal cell carcinoma (RCC) accounts for approximately 2-3% of all human cancers. A distinguished feature of RCC is vascularisation and among the three dominating RCC types conventional RCC (cRCC) generally is more vascularised than papillary RCC (pRCC) and chromophobe RCC (chRCC). Angiogenesis is a critical step in tumour progression controlled by a balance involving molecules that have positive and negative regulatory activity. A balance distorted by metabolic stress such as hypoxia, acidosis, and inflammation. Hypoxia-Inducible Factor 1α (HIF-1α) is a key transcription factor in angiogenesis and tumour progression, targeting more than a 100 genes involved in vascular growth and regulation, iron metabolism and erythropoesis, collagen matrix formation, regulation of extracellular pH, glucose uptake and metabolism, proliferation, apoptosis, differentiation, and cell viability. Methods: Tumour tissue and corresponding kidney cortex from nephrectomised RCC patients was used in order to characterize HIF-1α expression and one of its target genes, Glucose Transporter 1 (GLUT-1). All tumour samples were thoroughly described regarding tumour type, TNM stage, nuclear grade, tumour size, vein invasion, and patient survival. Utilizing RT-PCR, Westen Blot and Tissue micro array (TMA) we studied HIF-1α mRNA and protein expression as well as GLUT-1 protein expression, correlating them to each other and clinicopathological parameters. Results: Using Western Blot, HIF-1α protein expression differed significantly between the different RCC types and kidney cortex. In cRCC, high expression of HIF-1α was an independent prognostic factor for favourable prognosis. TMA is a useful method to analyze HIF-1α protein expression in RCC. HIF-1α levels were significantly lower in locally aggressive cRCC and patients with high levels of HIF-1 tended to have a better prognosis. GLUT-1 levels were higher in cRCC than in other RCC types and for cRCC a correlation to HIF-1α was seen. HIF-1α mRNA levels were significantly lower in cRCC compared to other RCC types and kidney cortex. An inverse correlation between HIF-1α protein expression and mRNA levels was observed. Summary: These results demonstrate a discrepancy between RCC types, highlighting the need to separately evaluate biological events in different RCC types. Overexpression of HIF-1α protein is not necessarily all bad and translational regulation appears more critical than anticipated. Further studies are encouraged to clarify angiogenic pathways in RCC.

Clostridium chauvoei is a gram-positive, spore-forming anaerobic bacterium. It is the pathogenic agent of blackleg, a disease causing serious toxemia and high mortality in cattle, sheep and many other domestic and wild animals. It is considered the most important Clostridium producing economic losses in livestock. Typically, animals infected with blackleg die rapidly without any signs of illness. Animals quickly die within 12 to 48 hours after contracting the disease. Therefore, the control of this disease is done by commercial vaccines consisting of whole formolized cultures. Immunity against C. chauvoei is associated with whole cell, including its somatic and flagellar antigens while in other clostridial diseases, protective immunity is obtained by the use of vaccines containing toxoids. Moreover, it is essential to obtain new information about the somatic antigens of C. chauvoei. Proteomics is the study of the proteome, the protein complement of the genome. The proteome has been defined as the entire complement of proteins expressed by a cell, organism, or tissue type, and accordingly, proteomics is the study of this complement expressed at a given time or under certain environmental conditions. 2-DE with Immobilized pH Gradients (IPGs) combined with protein identification by Mass Spectrometry (MS) is currently the workhorse for proteomics. Much of information about immunogenic component can be derived from proteomics coupled to Western blotting, namely immunoproteomics. Our study constitutes the first immunoproteomic analysis of C. chauvoei to identify candidate immunogenic antigens for development of new vaccines. Analyses were performed by Western blot and dot blot techniques against the whole cell extract proteins of C. chauvoei separated by 2-DE. Firstly, the growth conditions of two different strains, C. chauvoei ATCC 11957 and C. chauvoei 20 were optimized. After mice immunization studies with experimental vaccines prepared, sera were obtained for evaluation of the immunoglobulin G antibody level by ELISA. After high level of antibody response determination, 1-DE, 2-DE and immunoblot studies were performed for the characterization of immunogenic proteins. In the study, a total of 460 protein spots could be detected on the 2-DE gels by the help of Delta2D image analysis software and 30 of them were reacted with polyclonal antibodies against inactivated whole cells of C. chauvoei. Among these 30 spots, and 8 of them could be characterized by MALDI-TOF MS analyses. Of these 8 spots revealed four different gene products (distinct ORFs). Ornithine decarboxylase, methionine adenosyltransferase, glucose-6-phosphate isomerase, and flagellin protein FliB (C) are the characterized proteins. Glucose-6-phosphate isomerase has been identified as an immunogenic protein for a pathogenic microbe and in C. chauvoei for the first time. Methionine adenosyltransferase and ornithine decarboxylation were identified as immunogenic for C. chauvoei for the first time. The last defined protein is the flagellin protein FliB(C) which is known to be major immunogenic protein of C. chauvoei.

Despite very extensive studies related to molecular processes underlying memory formation, still little known about the potential differences in the brain biochemistry between &ldquo / good&rdquo / and &ldquo / poor&rdquo / learners belonging to a random population of young animals. In the present study, an attempt was taken to correlate the individual variation in short- and long-term spatial memory in three different lines of young, healthy rats: inbred Wistar (W), outcrossed Wistar/Spraque Dawley (W/S) and pigmented Long-Evans rats, with hippocampal levels of selected enzymes known as &ldquo / memory molecules&rdquo / including neuronal (n), endothelial (e) and inducible (i) NOS, CaMKII&alpha / , PKA and ChAT. Additionally, in order to indirectly estimate the activity of CaMKII&alpha / and PKA, hippocampal levels of their phosphorylated forms (pCaMKII&alpha / and pPKA) were assessed. Rats were classified as &ldquo / good&rdquo / and &ldquo / poor&rdquo / learners on the basis of their performance in a partially baited 12-arm radial maze. The hippocampal protein levels were measured using Western Blot technique. In addition to individual variation in animals&rsquo / learning capacity, strain-depended differences have also been observed. Deficient performance recorded in inbred W rats compared to outcrossed W/S rats, and &ldquo / poor&rdquo / learners from both rat groups had predominantly related to the higher frequency of reference memory errors. The results of biochemical assays showed strain-depended differences in the NOS expression. The overall NOS levels were significantly higher in outcrossed W/S rats compared to inbred W rats. In both rat lines, the rate of learning positively correlated with hippocampal levels of nNOS and negatively correlated with iNOS levels. Hippocampal eNOS levels correlated negatively with animals&rsquo / performance but only in the W rats. These results suggested that all 3 NOS isoforms are implemented in the learning process playing, however, different roles in neural signaling. Experiments carried out on Long-Evans rats did not reveal a significant difference in the basal hippocampal levels of the CaMKII&alpha / , however, the level of the pCaMKII&alpha / , was significantly higher in &ldquo / good&rdquo / learners. Also, hippocampal levels of both PKA and pPKA, as well as that of ChAT were significantly higher in &ldquo / good&rdquo / as compared to &ldquo / poor&rdquo / learners. Taken together, the latter findings indicate that low hippocampal expression of PKA and ChAT as well as low CaMKII&alpha / or PKA activation may cause learning deficits in random population of young rats, and thus, these enzymes can be considered target molecules when looking for cognitive enhancers to treat memory deficits in young subjects.

Le canal calcique de type L, Cav1.2, joue un rôle clé dans le couplage excitation-contraction des myocytes ventriculaires. Il a été montré que la sous-unité Cavα1 était sujette à l’épissage alternatif et que ce phénomène pouvait mener à une protéine tronquée en C-terminal au niveau de l’exon 45 (Liao, Yong et al. 2005). D’autres groupes ont étudié différentes délétions au niveau de l’extrémité C-terminale (De Jongh, Warner et al. 1991; Gao, Cuadra et al. 2001). Les courants mesurés dans la configuration cellule entière, était significativement plus grands que le canal « pleine longueur ». Nous avons décidé de tester certaines de ces délétions (ΔC2030, ΔC1935, ΔC1856, ΔC1733, ΔC1700) en présence ou en absence de la sous-unité auxiliaire Cavβ3, susceptible d’interagir avec l’extrémité C-terminale de la sous-unité Cavα1 par l’intermédiaire de son domaine SH3 (Lao, Kobrinsky et al. 2008). Les résultats obtenus dans les ovocytes de Xénope ont mis en évidence que les sous-unités Cavα1.2 tronquées montraient des courants globaux plus élevés que le canal « pleine longueur » en présence de la sous-unité auxiliaire Cavβ3 et que les sous-unités Cavα1.2 tronquées donnaient des courants en absence de la sous-unité Cavβ3 contrairement à la sous-unité Cavα1.2 « pleine longueur ». Afin de vérifier si l’augmentation des courants macroscopiques était le résultat d’une augmentation du nombre de sous-unités Cavα1.2 à la membrane, nous avons choisi de quantifier la fluorescence spécifiquement due à cette sous-unité en utilisant la méthode de cytométrie de flux (FACS : « Fluorescence Activated Cell Sorting »). L’épitope HA a été inséré dans une région extracellulaire de la sous-unité Cavα1 du canal calcique Cav1.2 et un anticorps anti-HA couplé au FITC (« Fluorescein IsoThioCyanate ») a été utilisé pour observer la fluorescence. Nos résultats confirment que la sous-unité Cavα1-HA du canal calcique Cav1.2, s’exprime à la membrane plasmique en présence de la sous-unité auxiliaire Cavβ3, et qu’en absence de celle-ci, ne s’exprime que peu ou pas à la membrane. Les mêmes résultats ont été obtenus pour les trois délétions testées dans les mêmes conditions soit Cavα1.2-HA ΔC1935, Cavα1.2-HA ΔC1856 et Cavα1.2-HA ΔC1733. Ensemble, ces résultats suggèrent que l’augmentation des courants macroscopiques observés après une délétion partielle du C-terminal n’est pas causée par une augmentation du nombre de protéines Cavα1.2 à la membrane. / The L-type calcium channel, Cav1.2, plays an important role in the excitation-contraction coupling of the ventricular myocytes. It has been shown that the alternative splicing of Cavα1.2 subunit could lead to a truncated protein in the C-terminus at exon 45 (Liao, Yong et al. 2005). Many groups have studied deletions in the C-terminus (De Jongh, Warner et al. 1991; Gao, Cuadra et al. 2001). The currents, measured in the whole cell configuration, were significantly higher with the full-length channel. We chose to test some of these deletions (ΔC2030, ΔC1935, ΔC1856, ΔC1733, ΔC1700) in the presence or absence of the Cavβ3 auxiliary subunit which is likely to interact with the C-terminus of the Cavα1.2 subunit through its SH3 domain (Lao, Kobrinsky et al. 2008). The truncated Cavα1.2 subunit, expressed in Xenopus Oocytes, showed macroscopic currents that were greater than those of the full length channel in presence of the Cavβ3 subunit. In addition, the truncated Cavα1.2 subunits displayed currents in the absence of the Cavβ3 subunit in contrast with the Cavα1.2 full length subunit. To investigate whether the larger macroscopic currents resulted in an increase in the number of Cavα1.2 subunits at the plasma membrane, we chose the FACS (« Fluorescence Activated Cell Sorting ») method. An HA-tag was inserted in an extracellular region of the Cavα1.2 subunit and a FITC (« Fluorescein IsoThioCyanate ») coupled anti-HA antibody was used to measure fluorescence. Our results showed that the Cavα1.2-HA subunit of L- type channel is expressed at the plasma membrane in the presence of the Cavβ3 subunit whereas the Cavα1.2-HA subunit is slightly or not expressed at the plasma membrane in its absence. The same results were obtained for the three C-terminal deletions tested under the same conditions (CaVα1.2-HA ΔC1935, CaVα1.2-HA ΔC1856 and CaVα1.2-HA ΔC1733). Taken together, these results suggest that the increased macroscopic currents observed after a partial deletion of the C-terminus is not caused by an increased number of Cavα1.2 proteins expressed at the plasma membrane. Keywords:

El NFAT5 es un factor de transcripción de la familia Rel, la cual incluye a los NFATc y los NF-[kappa]B. Hasta ahora, el NFAT5 había sido caracterizado principalmente como un factor de respuesta a estrés osmótico ya que regula la expresión de genes osmoprotectores y citoquinas en respuesta a hipertonicidad. En este trabajo hemos identificado y caracterizado una nueva función del NFAT5, la regulación de genes (TNF[alfa], IL6, iNOS) activados por la vía de los receptores de tipo Toll (TLR), importantes para la respuesta inmunitaria innata y adaptativa a una amplia variedad de estímulos patogénicos. Nuestros resultados muestran que el NFAT5 es un regulador importante en la ruta de los TLR, jugando un papel en la actividad de varios miembros de esta familia de receptores, tal y como lo hacen los factores de transcripción NF[kappa]B e IRF5. / NFAT5 is a transcription factor of the Rel family, which includes NFATc and NF-[kappa]B. NFAT5 has been mainly characterized as a hypertonicity responsive factor, since it regulates the expression of osmoprotective genes and cytokines in response to osmotic stress. In this work we have identified and characterized a novel role for NFAT5, the regulation of genes (TNF[alfa], IL6, iNOS) activated by the Toll-like receptor pathway (TLR), important for innate and adaptive immunity responses to a wide variety of pathogenic molecules. Our results show that NFAT5 is an important transcription factor of the TLR pathway, playing a role in the activity of several members of this receptor family, like other transcription factors such as NF-[kappa]B and IRF5.

Devido ao largo espectro de manifestações na leishmaniose visceral canina (LVC), o exame clínico não é capaz de confirmar o diagnóstico, já que muitos dos sinais clínicos não são exclusivos da LVC, tornando-se imprescindível o diagnóstico laboratorial para confirmação da infecção por L. (L.) infantum chagasi. A sorologia tem sido a primeira escolha para o diagnóstico da leishmaniose canina; porém, a diversidade de técnicas empregadas e de antígenos tem levado a resultados conflitantes. O emprego de formas promastigotas e amastigotas do parasito tem boa concordância no sorodiagnóstico de LVC pela Reação de Imunofluorescência Indireta (RIFI), porém o teste com amastigotas mostrou-se mais sensível sem perda da especificidade. Sendo assim, o objetivo deste estudo foi avaliar o desempenho do ELISA no sorodiagnóstico da LVC empregando antígeno total bruto de formas amastigotas provenientes de cultura axênica (ELISA-AXE) e amastigotas purificadas de tecido de animal experimental cronicamente infectado (ELISA-AMA) comparando com o desempenho do ELISA feito com antígeno total bruto de formas promastigotas (ELISA-PRO) de L. (L.) infantum chagasi. Foram avaliados 115 soros de cães parasitologicamente positivos para a pesquisa de DNA de Leishmania por PCR, domiciliados a pelo menos 2 anos em municípios com alta transmissão de leishmaniose visceral, classificados em sintomáticos (n=67) e assintomáticos (n=48) de acordo com sinais clínicos e exames laboratoriais. Como controle, foram utilizados 81 soros de cães parasitologicamente negativos oriundos do município sem transmissão comprovada de leishmaniose visceral e 13 soros de cães com outras enfermidades. Os resultados não mostraram diferença significante em sensibilidade, especificidade, valores preditivos e acurácia entre os testes ELISA-AXE, ELISA-AMA e ELISA-PRO, com forte correlação e concordância entre os três antígenos testados. O ensaio de Western Blot mostrou reatividade para proteínas de diferentes pesos moleculares, entre 14 a 178kDa dependendo da fonte de antígeno. Os resultados mostraram desempenho semelhantes nos testes de ELISA com amastigotas axênicas, amastigotas purificadas e promastigotas como fonte de antígeno para sorologia da leishmaniose canina. A ampla e diversificada reatividade no Western Blot com poucas bandas altamente especificas sugere o emprego de quimeras de antígenos de diferentes pesos moleculares oriundos tanto de formas amastigotas como de formas promastigotas do parasito no diagnóstico da LVC / Due to the wide spectrum of manifestations in canine visceral leishmaniasis (CVL), clinical exam is not able to confirm the diagnosis, since many of the signs and symptoms are not exclusive of CVL, making it imperative that the laboratory diagnosis for infection confirmation by L. (L.) infantum chagasi. Since the direct search involves invasive material collection, time-consuming and expensive; the serology has been the first choice for diagnosis of canine leishmaniasis. However, the diversity of techniques employed and antigens have led to conflicting results in both diagnostic routine and applied research. Since the use of promastigotes and amastigotes of L. (L.) infantum chagasi have good agreement on serum diagnosis of CVL by reaction of Indirect Immunofluorescence (RIFI), despite the amastigote test proved more sensitive without loss of specificity. The main objective of this study was to evaluate and compare the performance of ELISA in the CVL using crude total antigen of axenic amastigote and promastigote culture, and purified amastigote from experimental chronically infected animal tissue of L. (L.) infantum chagasi. One hundred and fifty-five sera from dogs residing on areas with high disease incidence were evaluated by PCR and diagnosed positive for Leishmania, these were used as positive control on the present study. The animals were examined clinically and in accordance with the characteristic clinical signs of visceral leishmaniasis and laboratory tests, then were classified into 2 groups: symptomatic (n=67) and asymptomatic (n=48) animals. As negative control, eighty-one sera were used from dogs parasitologically negative by PCR in real time from the municipality without proven transmission of visceral leishmaniasis. The results showed no significant difference in sensitivity, specificity, predictive values and accuracy between ELISA-AXE, ELISA-AMA and ELISA-PRO, with strong correlation and concordance between the three antigens tested. The Western Blot assay showed reactivity for proteins of different molecular weights ranging from 14 to 178kDa depending on the antigen source. The results showed similar performance in the ELISA tests with axenic amastigotes, purified amastigotes and promastigotes as source of antigen for serology of canine leishmaniasis. The broad and diverse reactivity in Western Blot with few highly specific bands suggests the use of antigen chimeras of different molecular weights derived from both amastigote and promastigote forms of the parasite in the diagnosis of LVC

No description available.