Thesis (Ph.D. in Physiology & Biophysics) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 125-138). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Aqueous Phase Partitioning has a long history of applications to the analytical characterisation of biomolecules. However process applications have attracted the most interest in biotechnology where it has become widely recognized as a cost-effective technique. The main aim of this work was to explore the proposition that partition in Aqueous Two Phase Systems (ATPS) can be used as an analytical tool to detect protein isoforms and to assess the applicability of the method in clinical assays and for quality control in bioprocessing through examination of several analytical problems. The work also examined the development of automated methods of system preparation and sampling techniques to determine the partition coefficient in ATPS. The study demonstrated that the geometrical form of the phase diagram co-existence curve was of crucial importance since this directly affected the accuracy with which systems of defined Tie Line Length and Mass Ratio could be constructed. The TLL %Bias (accuracy) of a theoretical system range in the PEG1000-(NH4)2SO4 system at shorter TLL (12.2) was in the range +80.6% to -100% while at a longer TLL (53.1) the %Bias (accuracy) was reduced to +0.1% to -1.9%. At the same time the MR %Bias (accuracy) at shorter TLL (12.2) was in the range +59.5% to -21.3% while at the longer TLL (53.1) this was reduced to +2.7% to -2.6%. By contrast in the PEG8000-Dextran500 system the TLL %Bias (accuracy) at shorter TLL (13.1) was in the range +3.7% to -4.12%, while at a longer TLL (31.1) the range was +0.74% to -0.67%. The MR %Bias (accuracy) at the shorter TLL (13.1) was in the range +3.6% to -3% while at the longer TLL (31.1) the range was +1.1% to -1.4%. This illustrated that it is more difficult to work with a high degree of accuracy (e.g. %Bias <5%) close to the critical point in PEG-salt systems than in PEG-dextran systems. Two different approaches were taken to examine analytical phase partitioning. In the first approach the structure of the isoforms of a model protein (ovalbumin) were altered enzymatically. Analytical methods involving Strong Anion-Exchange chromatography were developed and applied to the separation of the ovalbumin isoforms. Removal of the phosphorylated groups (dephosphorylation of ovalbumin) was undertaken using alkaline phosphatase and de-glycosylation was attempted using neuraminidase and Endo-glycosidase F. However, both enzymatic approaches to deglycosylation were unsuccessful. Dephosphorylated isoforms were successfully produced and characterised. After partitioning in ATPS a clear difference was demonstrated between the behaviour of the native and dephosphorylated forms of ovalbumin. The mean % recovery in a PEG-salt ATPS was 99.8% (± 3.59) for the naive protein and 75.6% (± 4.03) for the dephosphorylated form. On the other hand, in a PEG3350-Dextran500 system, where solubility was maintained, a significant difference in the partition coefficient (K) of native and dephosphorylated ovalbumin was found. K for native ovalbumin was 0.85 while the partition coefficient of the dephosphorylated ovalbumin was 0.61. Analysis of covariance (ANCOVA) indicated that the regression coefficients of the respective partition isotherms were significantly different (p value < 0.05). In a second approach to examine analytical phase partitioning, chemical modification of a specific target surface amino acid of another model protein (serum albumin) was used to determine the degree of conjugation of the protein and also to determine its oxidative state. The method examined the reactivity of a free surface thiol to a wide range of labels ( (a) 2-methylsulfonyl-5-phenyl -1,3,4 oxidiazole reagent, (b) N-Ethylmaleimide (NEM) reagent, (c) 5, 5’-dithiobis (2-nitrobenzoate)(DTNB) (Ellman’s reagent), (d) N-pyrenylmaleimide (NPM) reagent, (e) Fluorescein-5-maleimide (F-5-M) Reagent). Only DTNB was found to modify the surface free thiol of serum albumin in a highly specific and quantitative manner. In the course of the development of a partitioning assay for surface free thiols of serum albumin significant oxidative properties were found to be associated with poly(ethylene glycol) PEG solutions and several attempts were made to find an oxidatively safe partitioning system by including antioxidants and by removal of contaminants by freeze drying. PEG3350-Dextran500 was found to provide an oxidatively safe environment for the development of a partitioning assay for the determination of albumin free thiols. A phase partitioning assay system capable of quantitatively resolving protein associated free thiols and low molecular weight thiols from a mixture of the two was developed. Correlation coefficients (R2) for the regression of experimentally determined protein free thiols in the presence of different levels of added LMW free thiol on the known addition of protein ranged from 0.77 to 0.83. The results demonstrated that the assay could quantify and distinguish both types of thiol in a simple two-step procedure.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20999号 / 医博第4345号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 武田 俊一, 教授 高田 穣 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 72-79).

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

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.