Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Dissertation (Ph.D.)--University of Toledo, 2008. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title in Ohio LINK ETD Center record : Na/K-ATPase signaling : from bench to bedside. Title from title page of PDF document. Bibliography: p. 99-120.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 155-167). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Konstanz, Univ., Diss., 2005.

Thesis (M.S.)--Medical University of Ohio, 2006. / "In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Sciences." Major advisor: Zijian Xie. Includes abstract. Document formatted into pages: iv, 50 p. Title from title page of PDF document. Bibliography: pages 40-49.

Made available in DSpace on 2016-06-02T19:32:11Z (GMT). No. of bitstreams: 1 DissGHDP.pdf: 1446855 bytes, checksum: b44d0b337ad1c73932cf0a253c2bdad6 (MD5) Previous issue date: 2004-04-30 / Universidade Federal de Sao Carlos / Copper is an essential trace-element for all live organisms however, when its concentration is high in the aquatic environment, copper is potentially toxic. Most physiological changes caused by copper in fish has been analyzed after acute exposure (96h). The aim of this study was investigate the physiological responses of Prochilodus scrofa to stress, during acute copper exposition (24, 48, 72 and 96 hours) and recovery period after animal transference to clean water, and the time necessary to recovery after animals transference to clean water. The changes associated to fish transference to experimental systems were also evaluated. Juveniles Prochilodus scrofa were acclimated during 30 days and then fish were sampled to take blood for pH, hematocrit, plasma cortisol glucose and ions (Na+, Cl- e K+) analyses and specific Na+/K+-ATPase activity of gills. The remaining fish were divided in two groups: control (C) and exposed to copper (CU) and transferred to glass aquariums where was kept for 4 days. CU groups were exposed to copper (96h-CL50 = 29 µgCu L-1). Sampling was done 1, 2, 3 and 4 days of copper exposure. Surviving fish were then transferred to aquariums with flowing water without copper and sampling was done 1, 2, 7, 15 and 30 days of recovery. Blood sampling was taken for the same analyses above cited. Plasma cortisol concentration increased 300 a 400% in control and copper exposed groups related to acclimated fish, plasma glucose increased 800% only in the copper exposed groups during the exposure period and returned to normal levels in the 2nd day of recovery. In general, the changes in plasma ion (Na+, Cl- e K+) concentrations and pH were not significant in relation to those of acclimated fish however, significant changes were found between the values of control and copper exposed groups during copper exposure and recovery period. The activity of Na+/K+-ATPase of gills was lower in the CU group during the copper exposure period and on the 1st day of recovery. The hematocrit increased during metal exposure but on the 1st day of recovery reached the normal values. Although changes were found on the analyzed parameters, on the 7th days after the transference to clean water all parameters evidenced a tendency to recovery the physiological changes reaching the values of acclimated fish. The present study showed that the stress responses to copper exposure is complex and P. scrofa presents high susceptibility to handling evidencing the importance of comparative analyses involving acclimated and/or unstressed fish together with the controls and exposed fish to a given stressor. / O cobre é um elemento-traço essencial a todos os organismos, entretanto quando em alta concentração no meio aquático pode ser potencialmente tóxico. As alterações fisiológicas causadas pelo cobre em peixes têm sido analisadas, em geral, após exposições agudas (96h). O presente estudo teve como objetivo avaliar as alterações fisiológicas associadas ao estresse, durante o período de exposição aguda ao cobre e o período de recuperação em água sem a presença de cobre. As alterações provocadas pelo estresse associado à transferência dos animais para os sistemas experimentais também foram avaliadas. Prochilodus scrofa jovens foram aclimatados durante 30 dias e posteriormente animais foram amostrados e o sangue coletado para análise de pH e hematócrito sangüíneos, cortisol, glicose e íons (Na+, Cl- e K+) plasmáticos e determinação da atividade da enzima Na+/K+-ATPase das brânquias. Os animais remanescentes foram divididos em dois grupos controle (C) e exposto ao cobre (CU) e transferidos para aquários de vidro onde permaneceram durante 4 dias. O grupo CU foi exposto ao cobre (CL50-96h = 29 µgCu L-1). As amostragens foram efetuadas após 1, 2, 3 e 4 dias de exposição ao cobre. Os animais sobreviventes foram então transferidos para aquários com água corrente sem cobre e amostragens foram efetuadas após 1, 2, 7, 15 e 30 dias de recuperação. Amostras de sangue foram coletadas para as mesmas análises citadas acima. A concentração de cortisol plasmático aumentou 300 a 400% nos grupos controle e expostos ao cobre em relação aos animais aclimatados e a glicose plasmática aumentou 800% somente nos animais expostos ao cobre durante o período de exposição e retornaram aos níveis dos animais aclimatados no 2o dia de recuperação. As alterações nas concentrações dos íons plasmáticos (Na+, Cl- e K+) e pH, em geral, não foram significantes em relação aos animais aclimatados, mas alterações significativas foram observadas entre os grupos controle e exposto ao cobre durante a exposição ao cobre e recuperação. A atividade da enzima Na+/K+-ATPase nas brânquias foi menor no grupo CU durante a exposição ao cobre e 1o dia de recuperação. O hematócrito aumentou significativamente durante a exposição ao metal, mas após o primeiro dia de recuperação retornou aos valores normais. Embora tenha ocorrido alteração nos parâmetros analisados, após o sétimo dia de transferência para água sem cobre houve uma tendência de recuperação em todos os aspectos fisiológicos. Os resultados do presente estudo mostraram que a resposta ao estresse por exposição ao cobre é complexa e que P. scrofa apresenta a alta susceptibilidade à manipulação evidenciando a importância de estudos que envolvem comparações com animais considerados aclimatados ou não estressados e animais de grupos controle e expostos a um dado estressor.

Cytoplasmic dynein is a multisubunit complex involved in retrograde transport of cellular components along microtubules. The heavy chains (HC) are very large catalytic subunits which possess microtubule binding ability. The intermediate chains (IC) are responsible for targeting dynein to its appropriate cargo by interacting with the dynactin complex. The light intermediate chains (LIC) are previously unexplored subunits that have been proposed to modulate dynein activity by regulating the motor or the IC-dynactin interaction. The light chains (LC) are a newly identified class of subunit which are also thought to have regulatory functions. In the first part of this work, I analyzed the relationship between the four SDS-PAGE gel bands that comprise the light intermediate chains. 1- and 2-D electrophoresis before and after alkaline phosphatase treatment revealed that the four bands are derived from two different polypeptides, each of which is phosphorylated. Peptide microsequencing of these subunits yielded sequences that indicated similarity between them. cDNA cloning of the rat LICs revealed the presence of a conserved P-loop sequence and a very high degree of homology between the two different rat LICs and among LICs from different species. The second series of experiments was designed to analyze the association of pericentrin with cytoplasmic dynein. First, various dynein and dynactin subunits were co-associate with pericentrin in these experiments. Co-precipitation from 35S labeled cell extracts revealed a direct interaction between LIC and pericentrin. Comparison of pericentrin binding by LICl and LIC2 showed that only LICl was able to bind. Further investigation of the relationship between LICl and LIC2 demonstrated that each LIC will self-associate, but they will not form heterooligomers. Additionally, using co-overexpression and immunoprecipitation of LICl, LIC2, and HC, I have shown that binding of the two LICs to HC is mutually exclusive. Finally, I investigated the relationships between dynein HC, IC, and LIC by examining the interactions among the subunits. IC and LIC were both found to bind to the HC, but not to each other. Despite the lack of interaction between IC and LIC, they are, in fact, present in the same dynein complexes and they have partially overlapping binding sites within the N-terminal sequence of the HC. The HC dimerization site was determined to extend through a large portion of the N-terminus, and it includes both the IC and LIC binding sites, although these subunits are not required for dimerization. Together these studies implicate the light intermediate chains in dynein targeting. Targeting of dynein to its cargo has been thought to be performed by the dynactin complex, and for one particular cargo, the kinetochore, there is considerable evidence to support this model. The results presented here suggest that the light intermediate chains appear to function in a separate, non-dynactin-based targeting mechanism.

Cells as dynamic, interactive, and self contained units of life have a need for molecular motors that can create physical forces to move cargoes within the cell. Cytoplasmic dynein 1 is one such molecular motor that has many functions in the cell. The number and variety of functions that involve cytoplasmic dynein 1 suggest that there are a number of different binding sites on dynein for different proteins. Cytoplasmic dynein 1 is a multiprotein complex made up of six different subunit families. The many different combinations of subunits that could be used to make up a cytoplasmic dynein 1 holocomplex provides the variety of different binding sites for cargoes that can be individually regulated. The following chapters flush out how light intermediate chain 1 (LIC1), a subunit of cytoplasmic dynein 1, is involved with multiple dynein functions involving the binding of different cargoes to the cytoplasmic dynein 1 holocomplex, and how the binding of these cargoes can be regulated. First, LIC1 is found to be involved in the spindle assembly checkpoint. LIC1 appears to facilitate the removal of Mad1-Mad2, a complex important in producing a wait anaphase signal, from kinetochores. Second, the involvement of LIC1 in the spindle assembly checkpoint requires the phosphorylation of LIC1 at a putative Cdk1 phosphorylation site. This site is located in a domain of LIC1 that binds various proteins suggesting that this phosphorylation could also regulate these interactions. Third, LIC1 is involved in the centrosomal assembly of pericentrin, an important centrosomal protein. From the data presented herein, LIC1 is shaping up as a multifunctional cargo binder for cytoplasmic dynein 1 that requires regulation of its various cargoes.

Chau, Kai Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 136-159). / Abstract also in Chinese. / Chapter I --- Abstract --- p.i / Chapter II --- Acknowledgements --- p.vi / Chapter III --- Table of Contents --- p.vii / Chapter IV --- List of Figures --- p.xv / Chapter Chapter 1: --- Introduction --- p.1 / Chapter Chapter 2: --- Literature review --- p.7 / Chapter 2.1 --- Na+-K+ ATPase --- p.7 / Chapter 2.1.1 --- Introduction / Chapter 2.1.2 --- Structure of Na+-K+ ATPase --- p.9 / Chapter 2.1.1.2 --- Na+-K+ ATPase a subunit --- p.9 / Chapter 2.1.1.3 --- Na+-K+ ATPase β subunit --- p.11 / Chapter 2.1.1.4 --- Composition of the a subunit and β subunit --- p.12 / Chapter 2.1.1.5 --- Isomers of Na+-K+ ATPase --- p.13 / Chapter 2.1.1.6 --- Mechanism of ion exchange --- p.15 / Chapter 2.2 --- Aquaporins --- p.17 / Chapter 2.2.1 --- Introduction --- p.17 / Chapter 2.2.2 --- Structure of AQP-1 --- p.18 / Chapter 2.2.3 --- Distribution and function of AQP-1 --- p.19 / Chapter 2.3 --- Hormone --- p.22 / Chapter 2.3.1 --- Prolactin --- p.22 / Chapter 2.3.1.1 --- Structure of prolactin --- p.22 / Chapter 2.3.1.2. --- Functions of prolactin --- p.24 / Chapter 2.3.2 --- Growth hormone --- p.27 / Chapter 2.3.2.1 --- Structure --- p.27 / Chapter 2.3.2.2 --- Function of growth hormone --- p.28 / Chapter 2.3.3 --- Cortisol --- p.30 / Chapter 2.3.3.1 --- Structure --- p.30 / Chapter 2.3.3.2 --- Functions of cortisol --- p.31 / Chapter 2.4 --- Sparus sarba --- p.34 / Chapter 2.5 --- Urinary bladder of fish --- p.36 / Chapter Chapter 3: --- Effect of salinity on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder of silver sea bream Sparus sarba --- p.38 / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.2 --- Chronic effect of salinity on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.40 / Chapter 3.2.1 --- Materials and Methods --- p.40 / Chapter 3.2.1.1 --- Fish --- p.40 / Chapter 3.2.1.2 --- Tissue sampling --- p.41 / Chapter 3.2.1.3 --- Protein extraction and quantification --- p.41 / Chapter 3.2.1.4 --- Na+-K+ ATPase ATPase activity --- p.42 / Chapter 3.2.1.5 --- RNA extraction and first strand cDNA synthesis --- p.43 / Chapter 3.2.1.6 --- Validation of semi-quantitative RT-PCR --- p.45 / Chapter 3.2.1.7 --- Semi-quantification of expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.47 / Chapter 3.2.1.8 --- Statistical analysis --- p.47 / Chapter 3.2.2 --- Results --- p.48 / Chapter 3.2.2.1 --- Na+-K+ ATPase activity --- p.48 / Chapter 3.2.2.2 --- Relative expression of Na+-K+ ATPase and aquaporin-1 in urinary bladder --- p.48 / Chapter 3.2.3 --- Discussion --- p.54 / Chapter 3.2.3.1 --- Chronic effect of salinity on Na+-K+ ATPase in urinary bladder --- p.54 / Chapter 3.2.3.2 --- Chronic effect of salinity on AQP-1 expression in urinary bladder --- p.59 / Chapter 3.3 --- Effect of abrupt transfer on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.61 / Chapter 3.3.1. --- Materials and Methods --- p.61 / Chapter 3.3.1.1 --- Fish --- p.61 / Chapter 3.3.1.2 --- Tissue sampling --- p.62 / Chapter 3.3.1.3 --- "RNA extraction, first strand cDNA synthesis and RT-PCR" --- p.62 / Chapter 3.3.1.4 --- Statistical analysis --- p.63 / Chapter 3.3.2 --- Results --- p.64 / Chapter 3.3.2.1 --- Effect of abrupt hypo-osmotic transfer on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.64 / Chapter 3.3.2.2 --- Effect of abrupt hyper-osmotic transfer on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.65 / Chapter 3.3.3 --- Discussion --- p.73 / Chapter 3.4 --- Effect of in vitro salinity on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.78 / Chapter 3.4.1 --- Materials and Methods --- p.78 / Chapter 3.4.1.1 --- Fish --- p.78 / Chapter 3.4.1.2 --- Tissue sampling --- p.78 / Chapter 3.4.1.3 --- Preparation of culture medium --- p.79 / Chapter 3.4.1.4 --- "RNA extraction, first strand cDNA synthesis and RT-PCR" --- p.79 / Chapter 3.4.1.5 --- Statistical analysis --- p.80 / Chapter 3.4.2 --- Results --- p.81 / Chapter 3.4.3 --- Discussion --- p.85 / Chapter 3.5 --- Conclusion --- p.86 / Chapter Chapter 4: --- Effect of hormones on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder of silver sea bream Sparus sarba --- p.88 / Chapter 4.1 --- Introduction --- p.88 / Chapter 4.2 --- In vivo effect of hormones on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder of silver sea bream Sparus sarba --- p.91 / Chapter 4.2.1 --- Material and method --- p.91 / Chapter 4.2.1.1 --- Fish --- p.91 / Chapter 4.2.1.2 --- Tissue sampling --- p.92 / Chapter 4.2.1.3 --- "RNA extraction, first strand cDNA synthesis and RT-PCR" --- p.92 / Chapter 4.2.1.4 --- Statistical analysis --- p.92 / Chapter 4.2.2 --- Results / Chapter 4.2.2.1 --- Hormonal effect on mRNA expression of Na+-K+ ATPase and AQP-1 in urinary bladder of sea water adapted fish --- p.93 / Chapter 4.2.2.2 --- Hormonal effect on mRNA expression of Na+-K+ ATPase and AQP-1 in urinary bladder of brackish water adapted fish --- p.97 / Chapter 4.2.3 --- Discussion --- p.101 / Chapter 4.2.3.1 --- Effect of prolactin on mRNA expression of Na+-K+ ATPase and AQP-1 in urinary bladder --- p.101 / Chapter 4.2.3.2 --- Effect of growth hormone on mRNA expression of Na+-K+ ATPase and AQP-1 in urinary bladder --- p.104 / Chapter 4.2.3.3 --- Effect of cortisol on mRNA expression of Na+-K+ ATPase and AQP-1 in urinary bladder --- p.106 / Chapter 4.3 --- In vitro effect of hormone on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder of silver sea bream Sparus sarba --- p.109 / Chapter 4.3.1 --- Materials and methods --- p.109 / Chapter 4.3.1.1 --- Fish --- p.109 / Chapter 4.3.1.2 --- Tissue sampling --- p.110 / Chapter 4.3.1.3 --- Preparation of culture medium --- p.110 / Chapter 4.3.1.4 --- "RNA extraction, first strand cDNA synthesis and RT-PCR" --- p.111 / Chapter 4.3.1.5 --- Statistical analysis --- p.111 / Chapter 4.3.2 --- Results --- p.112 / Chapter 4.3.2.1 --- Effect of prolactin on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.112 / Chapter 4.3.2.2 --- Effect of growth hormone on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.113 / Chapter 4.3.2.3 --- Effect of cortisol on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.123 / Chapter 4.3.3 --- Discussion --- p.124 / Chapter 4.3.3.1 --- Effect of prolactin on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.124 / Chapter 4.3.3.2 --- Effect of growth hormone on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.125 / Chapter 4.3.3.3 --- Effect of cortisol on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.127 / Chapter 4.4 --- Conclusion --- p.129 / Chapter Chapter 5 --- General Conclusions --- p.131 / References --- p.136

The abnormal regulation of H+ ions, leading to a reversed pH gradient in cancer cells when compared to normal cells, is considered to be one of the most distinctive features of cancer. However, this characteristic has yet to be fully exploited as a therapeutic target in cancer. This project assessed whether targeting pH regulating proteins, which permit cancer cells to survive in the hostile hypoxic and acidic tumour microenvironment, could produce an effective therapeutic response in breast cancer. The pH regulating proteins carbonic anhydrase IX (CAIX), Na+/H+ exchanger 1 (NHE1) and vacuolar (H+)-ATPase (V-ATPase) were the focus of this thesis. Initial experiments were conducted in 2D tissue culture before progressing into 3D, using models that more faithfully re-create the in vivo tumour microenvironment. Expression analysis conducted with MCF-7, MDA-MB-231 and HBL-100 human breast cancer cell lines cultured in 2D, and in 3D as multicellular tumour spheroids, showed that protein and mRNA levels of CAIX were very responsive to lower O2 concentrations. Both MDA-MB-231 and HBL-100 cells displayed large increases in CAIX expression levels in hypoxia, with the HBL-100 cell line exhibiting the largest change in CAIX mRNA (42-fold increase) and protein (78-fold increase) levels in 0.5% O2 conditions. Hypoxia inducible factor 1-α (HIF-1α) controls the expression of CAIX, but the induction of CAIX in hypoxic MCF-7 cells was lower in comparison to the other cell lines, despite the presence of similar levels of HIF-1α. The differences in CAIX expression observed between the cell lines was consistent with a varying activity of factor inhibiting HIF-1 (FIH-1), an oxygen sensor that controls signalling through HIF-1α, as siRNA targeting FIH-1 led to increased levels of CAIX in hypoxic MCF-7 cells. While NHE1 protein levels did increase in hypoxic conditions in MCF-7 cells in 3D, overall, the expression levels of both NHE1 and V-ATPase were not as responsive to changes in O2 concentrations when compared to CAIX across differing O2 conditions in each of the cell lines. Inhibitors targeting CAIX, NHE1 and V-ATPase were all shown to reduce cancer cell number in 2D culture conditions. Differing O2 conditions changed the sensitivity of these cell lines to CAIX inhibition. Cells cultured in 20% O2 conditions were responsive to CAIX inhibition, while acute hypoxic cells cultured in 0.5% O2 displayed an increased resistance to drug treatment. Chronically hypoxic cells, which had spent over 10 weeks in 0.5% O2 before treatment, exhibited a re-sensitisation to CAIX inhibition. 3D invasion assays demonstrated that CAIX inhibition significantly reduced the invasion of cells from MDA-MB-231 spheroids into collagen type 1 in both 20% O2 and 0.5% O2 conditions, while drugs targeting either NHE1 or V-ATPase had no such inhibitory effects. Preliminary clonogenic assays, used to assess radiation sensitivity and performed with MDA-MB-231 spheroids, indicated that inhibitors targeting CAIX and NHE1 led to a significant decrease in colony formation when combined with irradiation, compared to either drug treatment or irradiation alone. Further invasion assays, carried out with primary tissue derived from human patients, showed that drugs targeting CAIX retained their inhibitory effects when tested on heterogeneous cancer material of varying tumour subtypes. CAIX inhibition also exhibited anti-cancer effects in vivo on mouse MDA-MB-231 xenografts, significantly reducing the proliferation and growth of tumours within mice. Together, this work demonstrates that inhibitors targeting the pH regulation mechanisms of cancer cells have potential in the treatment of breast cancer, highlighted by their capacity to affect cancer cell number, cancer cell invasion, and their ability to combine with irradiation. Of the 3 pH regulatory molecules studied, CAIX appears to be the target with the most therapeutic potential.