A macroprolactinemia é condição freqüente na hiperprolactinemia e em geral, sem impacto clínico. Os dados sobre a atividade biológica da macroprolactina (bbPRL) são controversos e baseados em bioensaio heterólogo com células de rato Nb2. A atividade biológica da bbPRL é observada in vitro e não in vivo, provavelmente porque seu alto peso molecular evita sua passagem pelos capilares. A bioatividade da bbPRL talvez varie de acordo com a especificidade do receptor de prolactina (PRLR). Avaliamos a bioatividade da bbPRL de indivíduos macroprolactinêmicos (Grupo I, n = 18) e da PRL monomérica (mPRL) de pacientes hiperprolactinêmicos sem bbPRL (Grupo II, n = 5) em Nb2 e em células Ba/F-LLP, transfectadas com o PRLR humano. Enquanto ambos ensaios apresentam resultados similares para a atividade de mPRL, nossos resultados indicam que a atividade da bbPRL é presente em ensaio heterólogo e não em ensaio homólogo. O ensaio Ba/F-LLP é sensível e apresenta melhor correlação com a atividade in vivo da bbPRL / Macroprolactinemia is a frequent finding in hyperprolactinemic individuals, usually without clinical impact. Data on biological activity of macroprolactin (bbPRL) is mostly based on a heterologous bioassay (Nb2 cell). Biological activity of bbPRL observed in vitro but not in vivo maybe due to its high molecular weight preventing its passage through capillary barrier. Alternatively, bbPRL bioactivity may differ depending on the PRL receptor species specificity. BbPRL from macroprolactinemic individuals and monomeric PRL (mPRL) from hyperprolactinemic patients without macroprolactinemia were tested in two bioassays: Nb2 and in Ba/F-LLP, which expresses human prolactin receptor. While both bioassays achieve similar results considering mPRL activity, our results indicate that bbPRL displays activity in a heterologous but not in a homologous bioassay, consistently with the apparent absence of bbPRL bioactivity in vivo

Orientadores: Fernanda Klein Marcondes, Tatiana de Sousa da Cunha / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T11:33:32Z (GMT). No. of bitstreams: 1 Briet_LarissadaSilva_M.pdf: 864520 bytes, checksum: ce24e35f8a1a6a6501fb9a3ae6af77f4 (MD5) Previous issue date: 2011 / Resumo: O modelo de natação associado à sobrecarga de peso é um modelo experimental de treinamento físico, mas o desconhecimento da intensidade de esforço que esta sobrecarga representa tem dificultado a padronização de protocolos de condicionamento físico para animais de laboratório. Além disso, pouco se sabe a respeito das respostas musculares e o estresse que pode estar associado à realização deste tipo de treinamento. O objetivo deste estudo foi avaliar, em ratos, os efeitos do treinamento físico diário de alta intensidade, por saltos em água com sobrecarga, na evolução temporal da concentração sangüínea de lactato; na concentração plasmática de corticosterona; na atividade sérica da creatina quinase (CK); na área de secção transversa da fibra muscular (ASTFM) do músculo sóleo e extensor longo dos dedos (EDL); e na expressão das isoformas da cadeia pesada de miosina (MHC) do músculo sóleo. Ratos Wistar machos foram aleatoriamente divididos em dois grupos: treinado e não treinado, e cada grupo foi composto por cinco subgrupos, que correspondiam a cada semana de treinamento. Os animais treinados foram submetidos a 5 semanas de treinamento que consistiu em 4 séries, 10 repetições, 30 segundos de repouso entre as séries, sobrecarga de 50-70% do peso corporal (PC), 5 dias/semana. No final de cada semana, a concentração sanguínea de lactato foi determinada antes, imediatamente após, 20, 40 e 60 minutos após o exercício. O treinamento aumentou a concentração sanguínea de lactato, em relação ao repouso, nas cinco semanas (semana 1=7.2±0.4 vs 2.2±0.3; semana 2=8.1±0.5 vs 2.1±0.1; semana 3=7.9±0.6 vs 2.0±0.2; semana 4=8.2±0.3 vs 1.9±0.1; semana 5=7.7±0.5 vs 1.5±0.1 mmol/L). Os animais treinados apresentaram aumento da concentração plasmática da corticosterona (semana 1=11.4±3.2 vs 3.2±2.1; semana 3=13.0±3.5 vs 2.1±0.9; semana 5=22.6±4.8 vs 6.4±3.2 ng/mL) e diminuição da atividade sérica da CK (semana 1=3191±310 vs 4187±414; semana 3=2828±247 vs 3680±643; semana 5=3330±225 vs 4254±602 U/L) em todas as semanas em relação aos animais não treinados. O treinamento diminui o PC (semana 3=297±7 vs 371±9; semana 5=365±13 vs 401±16 g), a razão peso muscular/PC dos músculos sóleo (semana 3=0.35±0.02 vs 0.57±0.03; semana 5=0.50±0.03 vs 0.61±0.04 g/100g) e EDL (semana 3=0.43±0.02 vs 0.66±0.03; semana 5=0.53±0.02 vs 0.67±0.05 g/100g), e a ASTFM do músculo sóleo (semana 3=2362±144 vs 3031±132; semana 5=2385±104 vs 2918±128 ?m2) a partir da terceira semana em comparação com animais não treinados. Os animais treinados apresentaram diminuição da MHCI (90.3±1.8 vs 99.2±0.80%) e aumento da MHCII (9.8±1.8 vs 0.8±0.8%) no músculo sóleo na quinta semana em comparação com animais não treinados. Os resultados obtidos mostraram que o protocolo de treinamento por saltos em água com sobrecarga empregado é predominantemente anaeróbio, e que a realização deste treinamento, diariamente, sem respeitar o intervalo de recuperação entre as sessões, não promove hipertrofia muscular e constitui-se num estímulo estressor para os animais. Porém, apesar destes efeitos, o treinamento promoveu adaptações no que diz respeito às respostas de lesão muscular (CK) e ao delineamento dos tipos de fibras musculares (MHC), permitindo maior capacidade do músculo em suportar o aumento da carga durante as sessões de treinamento / Abstract: The swimming model associated with weight lift is an experimental model of physical training, but the lack of knowledge about the intensity of this effort difficults the standardization of fitness protocols for laboratory animals. Furthermore, little is known about muscular responses and stress that may be associated with it. The aim of this study was to evaluate, in rats, the effect of jump training into water, carrying an overload, performed daily, on time-course of blood lactate concentration; plasma corticosterone concentration; serum creatine kinase (CK) activity; cross-sectional area of muscle fiber (CSAMF) of soleus and extensor digitorum longus (EDL) muscles; and expression of myosin heavy chain (MHC) isoforms on soleus muscle. Male Wistar rats were randomly divided into two groups: trained and untrained, with five subgroups each one, corresponding to each week of training protocol. Trained animals were submitted for 5 weeks of training that consisted in 4 sets, 10 repetitions, 30 seconds of rest between the sets, 50-70% body weight-load, 5 days/week. At the end of each week, blood lactate concentration was determined before, immediately after, 20, 40 and 60 minutes after exercise training. Physical training increased blood lactate concentration as compared with resting period, in all five weeks (week 1=7.2±0.4 vs 2.2±0.3; week 2=8.1±0.5 vs 2.1±0.1; week 3=7.9±0.6 vs 2.0±0.2; week 4=8.2±0.3 vs 1.9±0.1; week 5=7.7±0.5 vs 1.5±0.1 mmol/L). Corticosterone levels were increased in trained group (week 1=11.4±3.2 vs 3.2±2.1; week 3=13.0±3.5 vs 2.1±0.9; week 5=22.6±4.8 vs 6.4±3.2 ng/mL), and CK activity was decreased (week 1=3191±310 vs 4187±414; week 3=2828±247 vs 3680±643; week 5=3330±225 vs 4254±602 U/L) in all weeks as compared with untrained animals. Physical training decreased body weight (week 3=297±7 vs 371±9; week 5=365±13 vs 401±16 g), muscle weight/body weight ratio of soleus (week 3=0.35±0.02 vs 0.57±0.03; week 5=0.50±0.03 vs 0.61±0.04 g/100g) and EDL muscles (week 3=0.43±0.02 vs 0.66±0.03; week 5=0.53±0.02 vs 0.67±0.05 g/100g), and CSAMF of soleus muscle (week 3=2362±144 vs 3031±132; week 5=2385±104 vs 2918±128 ?m2) on the third and fifth weeks in comparison with untrained groups. Trained animals presented lower MHCI (90.3±1.8 vs 99.2±0.80%) and higher MHCII content (9.8±1.8 vs 0.8± 0.8%) in soleus muscle in the fifth week in comparison to untrained ones. Data showed that the jump training into water, carrying an overload is predominantly anaerobic, and when it is performed daily, without recovery intervals between sessions, there is no muscle hypertrophy and it constitutes a stressor stimulus for animals. However, despite these effects, training protocol promoted adaptations regarding muscle damage (CK) and also muscle fiber type transitions (MHC), increasing muscle ability to support overload increases during training sessions / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Among the peptidyl prolyl isomerases, the Cyclophilin family of proteins has been linked to various cellular activities such as regulation of homeostasis, mitochondrial permeability, and cell death. Their functionality spans throughout the cell and throughout all cell types as different isoforms. Previous studies done on Cyclophilin A revealed an interesting contact ensemble when bound to a substrate. Because of the similarity of CypA to its homologues, it is believed that they too will exhibit the same contact dynamics. We have defined the dynamics of cyclophilin isoforms through Molecular Dynamics simulations and determined their contact dynamics, characterizing their contact ensembles, and their relative dynamical conservation to each other.

P53 est un gène suppresseur de tumeur ubiquitaire qui empêche la prolifération de cellules malignes chez l’humain. En réponse à des dommages à l’ADN ou à des stress cellulaires, p53 entraine l’arrêt du cycle cellulaire et initie la réparation des lésions du génome. Si ces réparations échouent, p53 déclenche alors la mort de la cellule endommagée par apoptose. De plus, p53 présente une forte homologie avec deux autres gènes suppresseurs de tumeur : p63 et p73. Ces trois protéines forment une famille de facteurs de transcription qui protège l’organisme contre le développement de tumeurs. Ce système de défense est enrichi par les multiples isoformes de p53, p63 et p73 dont les rôles sont encore mal décrits. La neutralisation de la fonction de suppression de tumeur de p53, p63 et p73 est un mécanisme clef du développement tumoral auquel participent les mutants hotspots de p53 ainsi que certaines isoformes de p53, p63 et p73 par un effet dominant-négatif. Toutefois, de nombreuses zones d’ombre limitent notre compréhension de ce phénomène. Tout d’abord, l’identification des membres de la famille de p53 impliqués dans l’effet dominant-négatif reste incomplète. Ensuite, les mécanismes responsables de l’effet dominant-négatif sont débattus, suite notamment à l’émergence d’une nouvelle hypothèse impliquant un mécanisme de type prion. Enfin, l’effet dominant-négatif de la famille de p53 pourrait également être mis en cause dans d’autres types de pathologies comme les syndromes développementaux associés à des mutations de p63. Au cours de cette thèse, j’ai étudié l’impact fonctionnel des mutations hotspots de p53 ainsi que celui des principales isoformes de la famille de p53 sur l’activité transcriptionnelle des isoformes actives de p53, p63 et p73. En utilisant comme modèle d’étude un eucaryote simple, la levure S. cerevisiae, nous avons pu démontrer que l’effet dominant-négatif des mutants et isoformes de la famille de p53 repose sur la formation d’hétéro-tétramères entre formes actives et inactives de ces protéines et n’implique pas de mécanisme de type prion. De plus nos travaux ont montré que certains mutants de p53 interfèrent avec les isoformes actives de p63 et p73 par un mécanisme partiellement basé sur la tétramérisation. En outre, nos résultats préliminaires suggèrent que les mutants de p63 impliqués dans les syndromes développementaux EEC, ADULT et NSCL1 exercent également un effet dominant-négatif similaire à celui des mutants de p53. L’identification des mécanismes de l’effet dominant-négatif observé au sein de la famille de p53 permet d’envisager de nouvelles cibles thérapeutiques tant dans les cancers que dans certaines maladies rares du développement humain. / P53 is a ubiquitous tumor suppressor gene that prevents damaged cells from proliferating. Following DNA damage or cellular stress, p53 induces a cell cycle arrest and initiates an attempt to repair the lesions. If the repair fails, p53 triggers the apoptosis of the cell. p53 shares a high homology with two other tumor suppressor genes: p63 and p73. Together they form a family of transcription factors, which are actively protecting the organism from tumor development. This defense network is enriched by multiple N-terminal and C-terminal isoforms of p53, p63 and p73. The loss of p53, p63 and p73 tumor suppression function is a key step of cancer progression. Mutants of p53 and isoforms of p53, p63 and p73 often exhibit a dominant-negative behavior resulting in the loss of p53 tumor suppression activity. However, the extent of the dominant-negative effect within p53 family remains unclear. The mechanisms behind the dominant-negative effect are also debated due to the recent emergence of a prion-like hypothesis. Finally, the dominant-negative effect of p53 family members could be involved in other pathologies such as p63-related developmental syndromes During this PhD, I studied the functional consequences of hotspot mutations of p53 and of the main isoforms of the p53 family on the transcriptional activity of p53, p63 and p73. Using the naïve eukaryotic model S. cerevisiae we have demonstrated that the dominant-negative effect of mutants and isoforms of the p53 family relies on the formation of hetero-tetramers between functional and non-functional members of the family but not on a prion-like mechanism. In addition, certain p53 mutants are able to interfere with p63 and p73 isoforms though a mechanism that is only partially based on tetramerization. Of note, we obtained preliminary results suggesting that mutants of p63, which are involved in EEC, ADULT and NSCL1 developmental syndromes, behave like dominant-negative hotspot mutants of p53. The identification of the mechanisms of the dominant-negative effect occurring within p53 family could lead to new therapeutic targets both in cancer and in rare developmental syndromes.1 EEC : ectrodactyly, ectodermal dysplasia and cleft lip/palate syndrome, ADULT : acro-dermato-ungual-lacrimal-tooth syndrome, NSCL : non-syndromic cleft lip

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Communication between bone cells is important for the maintenance of bone mass. Although osteocytes are deeply embedded within the mineralized matrix, they are essential for the regulation of osteoblast and osteoclast functions. However, the intracellular proteins that control the morphology and function of osteocytes, and their ability to communicate with other bone cells are still unknown. Kalirin is a novel multi-domain GTP exchange factor (GEF) protein that activates the RhoGTPases. Recently, we found that 14 week old female Kalirin knockout (Kal-KO) mice exhibit a 45% decrease in trabecular bone density and have significantly lower cortical area, perimeter, thickness and polar cross-sectional moment of inertia (-12.6%, -7.2%, -7.6% and -21.9%, respectively) than WT mice. Kalirin was found to be expressed in osteoclasts and osteoblasts but its expression and function in osteocytes is currently unclear. We examined the role of Kalirin on the morphology and function of osteocytes. Primary osteocytes were isolated by sequential collagenase digestions from long bones (femurs and tibias) of 10-week old WT and Kal-KO mice. Immunofluorescent staining revealed Kalirin was localized to the perinuclear region of primary osteocytes and MLO-Y4 cells, and was detected along the cytoplasmic processes of primary osteocytes. We also examined primary osteocytes isolated from the long bones of Kal-KO and WT mice for changes in the length and number of cytoplasmic processes. Kal-KO osteocytes were found to express significantly fewer cytoplasmic processes per cell (3.3±0.21) than WT osteocytes (4.7±0.3). In addition, the cytoplasmic processes of Kal-KO osteocytes were shorter (79.5±4.6 µm) than those observed for WT osteocytes (85.4±3.6 µm) (p <0.01). Quantitative PCR revealed the expression of mRNA for the three major Kalirin isoforms (Kal-7, Kal-9, Kal-12) in primary osteocytes and in MLO-Y4 cells. Moreover, the mRNA levels of osteoprotegerin (OPG) and SOST, which are important for controlling osteoclast differentiation and Wnt signaling leading to bone formation, respectively, were reduced in Kal-KO osteocytes. Next, the role of Kalirin in osteocyte morphology and function was further examined. Treatment of MLO-Y4 cells for 5 days with nerve growth factor, which is known to activate Kalirin in neurons, or over-expression of the Ser-Thr kinase domain of Kal-12, promoted cytoplasmic process elongation and upregulated phosphorylated ERK and RhoA levels. Together, these results suggest that Kalirin controls osteocyte morphology and function in part by regulating cytoskeletal remodeling and the activity of ERK and RhoA. Furthermore, Kalirin may control the bone remodeling cycle by regulating osteocyte signaling to osteoclasts and osteoblasts.

This dissertation is divided into three chapters, each discussing the study of different regulatory molecules involved in sensory neurogenesis occurring in the trigeminal placode. Chapter one is a spatiotemporal description of Notch pathway genes in chick opV placode by stage-specific expression analysis, showing expression of many Notch pathway genes and effectors in the opV placode. Notch pathway gene expression is primarily confined to the ectoderm with highest expression of these genes at the beginning stages of peak neuronal differentiation. This information preceded studies of the functional roles that Notch signaling has in the opV placode and how it may affect the transcription factor, Pax3. Chapter two is a study of the transcription factor Pax3 and its role in opV placode development and sensory neuron differentiation. Pax3 is known to activate or repress gene transcription, and its activity may be dependent on the splice variant or isoform present. We show through RT-PCR that alternative splice forms of Pax3 are present at stages of chick development corresponding to cellular competence, cellular differentiation and ingression, and cellular aggregation. We have named these splice forms, Pax3V1 and Pax3V2. Using quantitative RT-PCR we show that Pax3V2 is consistently expressed at lower levels compared to Pax3 during cellular competence and differentiation. In order to determine the function of the three splice forms, we misexpressed them in the opV placode and analyzed the effect on neurogenesis. We looked at markers for neuronal differentiation of targeted cells after in ovo electroporation of Pax3, Pax3V1, and Pax3V2, which showed a significant difference between the control and each construct, but not between the groups of constructs. To enhance the process of neurogenesis we exposed the electroporated embryos to DAPT, a Notch signaling inhibitor that enhances sensory neurogenesis. Using this method we found that misexpression of Pax3 and Pax3V1 resulted in cells failing to differentiate, while Pax3V2 misexpression more closely resembles the neuronal differentiation seen in controls. These results show that the Pax3V2 isoform allows for neuronal differentiation of opV placodal cells after misexpression, while the Pax3 isoform and the Pax3V1 isoform block neuronal differentiation. Chapter three is a study of the necessity of Wnt signaling originating from the neural tube to induce Pax3 expression in the opV placode. A double knockout of Wnt1 and Wnt3a was produced to determine the necessity of these genes in opV placode development. Pax3 expression in the opV placode at E8.5 and E9.5 was markedly reduced in the double mutants when compared to wild type mice. This study shows that Wnt1 and Wnt3a genes are necessary for normal Pax3 expression, but that other signals may contribute to its induction.

No description available.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is known as the master regulator of mitochondrial biogenesis. PGC-1α holds this role by acting as a transcriptional coactivator for an array of transcription factors and nuclear hormone receptors, such as NRF-1/2 and ERRα/γ, whose downstream targets function in mitochondrial biogenesis and oxidative phosphorylation. PGC-1α is regulated both at the transcriptional and post-translational level in several signaling pathways, including p38 MAPK and AMPK. This regulation affects which transcription factor binding events can occur in a given tissue, and thus affects regulation of PGC-1α target genes. PGC-1α is downregulated in many neurodegenerative disorders as well as in muscular dystrophies, diabetes, and aging. Therefore, PGC-1α is prized as a potential therapeutic target to create novel treatments for these various diseases.However, details governing the spatio-temporal regulation of PGC-1α are not completely understood, and overexpression of PGC-1α throughout the body or even in certain tissues or subsets of cells have had detrimental effects in animal and cell models. Therefore, it is necessary to gain knowledge of how to modulate PGC-1α in a tissue-specific manner utilizing these different levels of regulation in order to develop novel therapies. In order to further understand all the functions that have been attributed to PGC-1α, the PGC-1α isoforms need to be accounted for and understood in human tissues. Several murine isoforms have been published, as well as several human brain and muscle isoforms. However, most of these isoforms have only been validated as mature transcripts, and it is not known whether they produce functional protein. Our lab has identified the isoform b transcript in human brain tissue via 5’ RACE and have developed an isoform b specific antibody. This project aimed to characterize the isoform b transcripts and also to validate and optimize this antibody for immunoblotting conditions for detection of further PGC-1α-b isoform protein variants in human tissues. Preliminary studies in our lab have shown that in postmortem frontal cortex from age-matched PD and healthy patients, isoform a transcript levels were 10-15 times more abundant than that of isoform b. These differences in regulation could be partially attributed to the isoform b promoter region being heavily methylated, as shown in this thesis through bisulphite cloning and sequencing as well as 454 bisulphite sequence analysis. The high degree of methylation, correlated with the low level of isoform b transcript in brain and it is not known whether this transcript would be translated into protein in this tissue. In order to probe for isoform b protein expression using human cell lines and tissues, however, it was necessary to create a recombinant protein in order to have a positive control with which to optimize our novel antibody. In our previous 5’ RACE studies, an alternatively spliced PGC-1α-b transcript was found which coded for an early stop codon. This truncated isoform was called PGC-1α-b-3T1, and mature transcript was found in both human skeletal muscle and brain. For this project, PGC-1a-b-3T1 was cloned from human skeletal muscle into a bacterial expression vector to create a recombinant GST fusion protein. This protein was used to validate and optimize our PGC-1α-b specific antibody as well as to determine sensitivity and specificity. The purified recombinant protein contained 3 bands of lower molecular weight that were detected via western blot with both GST and the PGC-1α-b specific antibody. These bands were trypsin cleaved and subjected to mass spectrometry analysis, which verified that all bands detected by the PGC-1a-b specific antibody contained the epitope sequence, and thus binding was specific. This protein was then used to determine western blotting conditions and sensitivity, which is 10 ng using a 1:100 dilution of the antibody. This antibody was then used to probe SH-SY5Y WCL, a human neuroblastoma cell line. Peptide competition assay confirmed 5 PGC-1α-b specific proteins in these lysates. The sizes of these proteins matched to several murine PGC-1α-b isoforms as well as putative PGC-1α-b versions of PGC-1a-a isoforms. These findings provided the putative identities of several endogenous functional human PGC-1α-b isoforms. Mammalian overexpression vectors of these isoforms are still in development. By using this antibody and these expression vectors to further characterize these isoforms, including determining tissue specificity, more knowledge of PGC-1α will be gained. This information could then be used to develop novel, tissue specific treatments for pharmacological intervention of diseases characterized by PGC-1α misregulation.

Adipocytokines released by the adipose tissue play an important role in the regulation of immune and inflammatory responses. In obesity their production is dysregulated, which is one of the major factors contributing to the onset of a chronic low-grade systemic inflammation representing a risk factor for the progression of other diseases, such as atherosclerosis or type-2 diabetes. The main goal of this thesis was to analyze the secretion of selected adipocytokines (adiponectin, IL6 and MCP1) by in-vitro differentiated adipocytes, isolated from the adipose tissue prior to and after a dietary intervention, and this under basal conditions and during stimulated lipolysis. In case of adiponectin, the secretion of its isoforms was analyzed too. The concentration of adiponectin, IL6 and MCP1 was determined by the ELISA method, the Western Blot method was used to determine the distribution of the adiponectin isoforms. The thesis also concentrates on the gene expression of ATF3, ATF4 and HSPA5, factors engaged in the ER stress in the course of the differentiation of adipocytes. The changes in the gene expression were measured by the quantitative Real Time PCR method. At the same time the development of the endoplasmic reticulum (ER) in the course of adipogenesis was monitored by indirect...