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Buněčná signalizace a molekulární komplexy TRH receptoru / Cell signalling and molecular complexes of the TRH receptorDrastichová, Zdeňka January 2012 (has links)
1 Summary The first part of this thesis is preoccupied with the identification of protein alterations in the membrane fraction of HEK293-E2M11 cells after prolonged TRH treatment. The isolated membrane fraction enriched in plasma membranes contained markedly increased the amount of Na,K-ATPase, TRH receptor and G-proteins compared to the postnuclear supernatant. By using 2D electrophoresis and mass spectrometry, the levels of 42 proteins were identified to be altered in samples of PM- enriched fractions from TRH-treated (16 h; 10 μM) cells. Out of these proteins only ezrin and stomatin-like 2 are known to be localized in the plasma membrane. Five proteins (mitofilin, MTHSP75, prohibitin, stomatin like-2, peroxiredoxin III) whose levels were increased after the prolonged TRH treatment represent proteins localized in mitochondria. All of them are important for proper structure and function of mitochondria. The ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax was markedly higher in cells treated with TRH than in control untreated cells. Hence, it can be concluded that prolonged TRH treatment may significantly affect mitochondrial membrane and function of mitochondria. The second part of this thesis deals with the identification of molecular protein complexes of TRH-R and/or Gq/11 protein. The presumed...
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Phosphoinositide Phase Behavior in Complex Lipid Monolayer SystemsKing, Katrice 19 April 2016 (has links)
Although phosphatidylinositol (PI) and phosphoinositides (PIPs) only comprise a small percentage of the inner leaflet of the plasma membrane, they mediate a large variety of signaling events. In previous studies, we have observed the absence of macroscopically discernible domains in mixtures of PI/PC and PI(4,5)P2/PC. The addition of cholesterol to these mixtures results in condensation of the monolayer and hence domain formation. To better mimic the ionic conditions and hydrogen bonding properties of the inner leaflet plasma membrane, we investigated in this study the effect of common inner leaflet plasma membrane lipids like phosphatidylethanolamine (PE), phosphatidylserine (PS) and PI, on phosphoinositide domain behavior in the presence of cholesterol and/or bivalent cations.
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Membrane and Mitochondrial Responses to Cryobiological ConditionsReardon, Anthony J Unknown Date
No description available.
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Caloxins: A Novel Class of Plasma Membrane Ca2+ Pump InhibitorsPande, Jyoti 06 1900 (has links)
Ionized calcium (Ca2+) is a signaling messenger that controls numerous
cellular processes essential for life. The fidelity of Ca2+ signaling depends on the
mechanisms that dynamically regulate its cytosolic concentration and maintain it
at a low level in a resting cell. Plasma Membrane Ca2+ ATPase (PMCA) is a high
affinity Ca2+ extrusion pathway involved in Ca2+ homeostasis and signal
transduction. PMCA are encoded by 4 genes (PMCA1-4), which are expressed in
a tissue dependent manner. The diversity of PMCA isoforms is further increased
by alternative splicing. Changes in PMCA activity occur in heart failure and
hypertension. Specific inhibitors of other ion transporters such as thapsigargin and
digoxin, have made their mark in cell biology, but the currently used inhibitors of
PMCA (vanadate and eosin) are non-specific. Thus, selective inhibitors of PMCA
are needed to discern its role in Ca2+ signaling in physiology and pathophysiology.
We introduced the concept of caloxins - peptides that specifically inhibit the
activity of PMCA by binding to one of its five extracellular domains (exdoms) 1
to 5. The earlier caloxins including 2a1 and 3a1 were obtained by screening a
phage display random 12-amino acid peptide (Ph.D-12) library for binding to
synthetic peptides based on the exdom sequences. However, they all had low
affinity.
The objective of this research was to develop caloxins with high affinity
and PMCA4 isoform selectivity. A two-step screening method was developed to
screen the Ph.D-12 library to first bind to the synthetic exdom of PMCA4, followed by affinity chromatography using PMCA protein purified from human erythrocyte ghosts (mainly PMCA4). This method was used to obtain caloxins 1b1 and 1b2 to bind to the N and C-terminal halves of the exdom 1 of PMCA4, respectively. Both caloxins 1b1 and 1b2 had a 10-fold higher affinity than the prototype caloxin 2a1 and showed slight PMCA4 isoform preference. To engineer inhibitors with greater affinity and PMCA4 isoform selectivity, Ph.D caloxin 1b1 like peptide library was constructed. Most of the peptides expressed in this
library differed from caloxin 1b1 in 0, 1, 2 or 3 amino acid residues at random.
The library was screened to obtain several peptides one of which was caloxin 1c2.
Caloxin 1c2 had 200-fold higher affinity than caloxin 2a1 and was isoform
selective, with greater than 10-fold affinity for PMCA4 than for PMCA isoforms
1, 2 or 3. Thus, caloxin 1c2 is the first high affinity PMCA inhibitor that also is
selective for an individual PMCA isoform.
The second aim of this research was to establish that caloxin 1c2 binds to
PMCA protein in erythrocyte ghosts. Two photoreactive caloxin 1c2-derivatives
containing the photoactivable residue benzoylphenylalanine (Bpa) and a C-terminal
biotin tag were used. Bpa substituted tryptophan at position 3 (3Bpa1c2-biotin) and serine at position 16 (16Bpa1c2-biotin) in caloxin 1c2. Both the derivatives inhibited PMCA activity in the erythrocyte ghosts. The intensity of the biotin label in the photolabeled erythrocyte ghosts was much stronger with 3Bpa1c2-biotin, which was then used in the subsequent experiments. The photolabeled proteins in erythrocyte ghosts were detected as a 250-270 kDa doublet in Western blots using streptavidin and the PMCA specific antibody. The
degree of photolabeling depended on the UV-crosslinking time, and on the
concentrations of 3Bpa1c2-biotin and the ghost protein. The selectivity of the
photolabeling site was confirmed by decreased photolabel incorporation at 250-270 kDa doublet in the presence of excess caloxin 1c2 and the synthetic exdom
1X peptide of PMCA4. The photolabeled erythrocyte ghosts were solubilized and
analyzed by immunoprecipitation with the PMCA specific antibody. The
immunoprecipitate showed a 250-270 kDa doublet in Western blots using
streptavidin. This confirmed that PMCA protein was photolabeled by the
photoreactive derivatives of caloxin 1c2. Thus, caloxin 1c2 inhibits PMCA
activity by binding to the exdom 1X of PMCA4.
My work in M.Sc. initiated the concept of caloxins in the literature. This
research has taken it to the stage where we can obtain caloxins selective for
individual PMCA isoforms. This contrasts with the relative paucity of inhibitors
specific for individual isoforms of other ion pumps. The high affinity isoform
selective caloxin 1c2 and previous caloxins are being used to study PMCA
physiology in our lab and by other researchers. Since caloxins act when added
extracellularly and it is possible to obtain PMCA isoform selective caloxins, it is
anticipated that they will aid in understanding the role of PMCA in signal
transduction and homeostasis in health and disease. / Thesis / Doctor of Philosophy (PhD)
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Identification and Molecular Characterization of dveli, the drosophila ortholog of C. Elegans lin-7MacMullin, Allison A. 06 1900 (has links)
Receptors and signal transduction complexes are assembled in a precise manner at
specific subdomains of the plasma membrane. Recent research has implicated scaffolding
proteins in organizing these receptor and signaling complexes. One well characterized
example is the C. elegans LIN-2/LIN-7 /LIN-1 0 complex. This complex is essential in the
proper localization of LET -23, the EGFR ortholog, to the basolateral membrane surface of
vulval epithelial cells.
The mammalian orthologs of the LIN-2/LIN-7 /LIN-10 complex have been
identified. CASKIVELI!Mintl/Xllalpha function as a tripartite complex in neurons,
presynaptically and postsynaptically. Presynaptically, the multi protein complex aids in
linking cell adhesion to ion influx, synaptic vesicle fusion with the presynaptic membrane.
and subsequent neurotransmitter release. At the post-synaptic membrane, the
CASKIVELI!Mintl/Xllalpha complex is hypothesized to function in the sorting and
proper localization of the NMDA type glutamate receptor, reflecting the function of the C.
elegans orthologs in receptor localization.
We have identified the Drosophila orthologs ofLIN-2/CASK, LIN-7NELI, and
LIN-10/Mintl/Xllalpha, termed CMG, dVELI and dMINT. respectively. These proteins
were found to be highly conserved among species. The Drosophila YELl protein was
initially identified by the McGlade laboratory, University of Toronto, where it was found to
bind phosphorylated Drosophila EGFR (DER). We have mapped the chromosomal
location of dveli, determined RNA transcript distribution and protein localization, and
initiated a P-element mutagenesis screen to generate a dveli mutant. Furthermore, candidate
genes for other proteins known to associate with LIN-7 (PALS) have been identified by
sequence analysis.
dVELI expression begins early in the larval stage. It is concentrated mostly in neuropil areas, sites of synaptic connections. This expression pattern continues into adult
development. Within the larval CNS, dVELI protein is localized to the neuropil areas of the
ventral nerve cord and brain. NMJ staining further localizes dVELI almost exclusively to
the post-synaptic density. This post-synaptic localization resembles that of mammalian
YELls, wherein the complex is thought to aid in glutamate receptor sorting and localization.
The similarity in structure and expression patterns of dVELI to that of its mammalian
orthologs suggests a model in which the Drosophila complex aids in the localization of
receptors to post -synaptic specializations in neurons. / Thesis / Master of Science (MSc)
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Synthesis and evaluation of new PET radioligands for imaging central norepinephrine transporters /Schou, Magnus, January 2006 (has links)
Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 7 uppsatser.
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Apoptosis-driven microenvironmental conditioning by microvesicles in non-Hodgkin lymphomaPatience, Lauren Alexandra January 2017 (has links)
Plasma membrane derived microvesicles (MV) are nanoscale particles released from cells both constitutively and in response to stimuli including stress, apoptosis and oncogenic transformation. Due to their mechanism of biogenesis, the majority of MV expose phosphatidylserine (PS) on their surface and as such can be identified by staining with annexin V (AxV). First observed nearly 40 years ago as coagulant ‘dust’ originating from activated platelets, MV were initially studied for their role in thrombosis. In more recent years it has become apparent that MV release is increased in several diseases including cancer; this, in conjunction with their ability to carry cargo such as proteins and nucleic acid species, strongly implicates them in disease pathology. Given their small size it is considered likely that MV are able to travel to distal sites within the body allowing the widespread dissemination of effects otherwise not achievable by their parent cells. In the context of malignancy, the contribution of MV is especially important in that MV have been demonstrated to have roles in oncogenic transformation, promotion of tumour growth and increasing metastatic potential. Although clearly important in pathogenesis, their small size makes qualitative and quantitative analysis extremely difficult. Furthermore, the study of MV has been greatly hampered by a lack of standardised protocols for their isolation and as such the majority of studies have been in vitro. In line with this, the relevance of observed effects to in vivo systems is often questioned; given the high quantities of MV used in in vitro systems, the question of whether these concentrations bear any relevance in vivo remain to be answered. We hypothesise that the high rates of apoptosis observed in many tumours, most notably in the high grade B cell malignancy, Non-Hodgkin’s lymphoma (NHL), provides an environment whereby MV are continually released into the surrounding milieu allowing for an amplification of effects. As apoptosis has been previously implicated in promoting tumourigenesis we propose that this is extended to include MV released from apoptotic tumour cells (aMV). Given the numerous technical challenges involved in MV research, initial studies involved identifying the limitations of the instruments available for MV analysis. Preliminary experiments identified considerable resolution issues with the older style EPICS XL flow cytometer (Beckman Coulter) and so a newer flow cytometer, The Attune™ (Thermo Fisher), capable of higher resolution was utilised for the remainder of the project. Despite this improvement, flow cytometry was demonstrated to be less effective at quantifying MV than nanoparticle tracking analysis (NTA). As the fluorescent capacity of NTA is still in its infancy, it was used in concert with flow cytometry in order to quantify and phenotype MV as accurately as possible. As there is currently no concensus on an optimal method of MV isolation subsequent studies focused on determining a method of MV isolation that was appropriate for our experimental system. To this end, centrifugation, filtration and antibody coated magnetic bead-based methods were all tested and their limitations identified. In terms of bead-based isolation strategies, the generation of AxV, protein S, gla domain and gas 6 fusion proteins was attempted with the intention to conjugate to magnetic beads and provide a novel means to isolate aMV. Unfortunately this aspect of the project was ultimately abandoned due to time constraints and although commerically available antibody coated beads were tested for their ability to isolate MV, later co-culture experiments demonstrated that the beads had off target effects that were deleterious to cells. As a result, centrifugation and filtration methods were next researched and validated extensively. TEM analysis of MV morphology identified damage likely induced by the high-speed centrifugation of a fragile apoptotic cell population. As such, a protocol combining low speed centrifugation and filtration was designed and validated by several methods including TEM and staining with AxV. The surface levels of parent cell markers (CD19 and CD20) and apoptosis associated proteins were compared in aMV and vMV (MV released from viable tumour cells) and results demonstrated that B cell surface markers were off loaded into MV to a greater extent following apoptosis. Additional phenotypic studies extended previous work from the group demonstrating the presence of apoptotic cell associated molecular patterns (ACAMPs) capable of binding a panel of antibodies to LPS. Flow cytometry results confirmed the presence of ACAMPs on aMV and results from co-culture experiments with CD14 positive and negative cells suggested that unlike recognition of LPS, binding via ACAMPs was not CD14 dependent. The protein and nucleic acid content of MV was also studied and interestingly, results demonstrated significantly increased quantities of DNA and RNA in aMV compared to vMV. Furthermore, aMV were also shown to contain the matrix metalloproteinases, MMP2 and MMP12 alluding to a role for aMV in angiogenesis. The final stage of the project was focused on determining the roles of aMV in the tumour microenvironment and effects relating to cell growth, cell cycle and angiogenesis were studied and compared to vMV. Results showed that both aMV conditioned supernatant and aMV concentrated by the centrifugation were able to significantly increase the growth of the parent cell population. Further studies using DAPI staining to determine the cell cycle status of cells co-cultured with aMV demonstrated an increase in DNA synthesis and cell division upon incubation with aMV. An in vitro angiogenesis assay was designed to determine any pro-angiogenic capabilities of aMV given the earlier results demonstrating the presence of MMPs. These results provided some of the most interesting findings of the project and showed that aMV were able to increase the angiogenic potential of human endothelial cells (HUVECs); an effect that was shown to be greatly reduced following storage at either 4 or - 80°C. These results demonstrated that aMV possess factors capable of manipulating the tumour microenvironment to favour disease progression and that previously described pro-tumour functions of MV are increased as a result of apoptosis. These findings have implications both in terms of extending the previously described hallmarks of cancer and also when designing a course of therapy whereby in some instances the generation of large amounts of apoptosis may in fact serve to promote regeneration of the tumour cell population.
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Hippocampal neurogenesis in the SERT ALA56 mouse model to autismUnknown Date (has links)
The causes of autism spectrum disorder (ASD) are not all known, but it is suspected that the serotonin transporter (SERT) plays an important role for some subjects with ASD. Mutations in the SLC6A4 gene, that encodes SERT, including the Ala56 mutation (Gly56Ala), have been found in some autism patients. This mutation makes the transporter more active and reduces the probability of serotonergic neurotransmission in the brain, which is linked to behavioral changes that are associated with core domain deficits of ASD 1.
Depression also has been linked to decreases in the availability of serotonin (5-hydroxytryptamine; 5-HT) in the central nervous system (CNS), and is associated with reduced hippocampal neurogenesis. Selective serotonin reuptake inhibitors (SSRIs), drugs used to block SERTs, are used to treat depression and/or anxiety by inhibiting SERT to increase synaptic 5-HT levels. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection
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Docosahexaenoic acid differentially modulates plasma membrane targeting and subcellular localization of lipidated proteins in colonocytesSeo, Jeongmin 12 April 2006 (has links)
Correct localization of lipidated cytosolic proteins to the plasma membrane (PM) is mediated by interactions between lipid anchors of proteins and cell membranes. Previously, dietary fish oil and its major n-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA), have been shown to decrease Ras membrane association, concomitantly reducing rat colon tumor incidence and Ras signaling, compared with corn oil and linoleic acid (LA), a highly prevalent vegetable fat and dietary PUFA in the U.S. diet. In order to explore the potential regulatory role of the cellular lipid environment in PM targeting of lipidated proteins, young adult mouse colon (YAMC) cells were treated with 50 µM DHA, LA, or oleic acid (OA) 24 h prior to and 36-48 h after transfection with green fluorescent protein (GFP) fusion constructs of various lipidated cytosolic proteins. Relative expression of each GFP fusion protein at the PM and the Golgi in living cells was quantified using z-serial confocal microscopy and digital image processing. DHA differentially altered the subcellular localization of Ras isoforms and Src-related tyrosine kinases in a reversible manner. DHA significantly decreased the PM localization and increased the endomembrane association of H-Ras,
N-Ras, and Lck, which are targeted to the PM via the exocytic pathway, regardless of their functional state. In contrast, the subcellular distribution of K-Ras and Fyn, of which transport is independent of the vesicular transport pathway, was unaffected by DHA. Moreover, DHA selectively inhibited lipidated cytosolic protein targeting since the PM delivery of transmembrane protein cargo was unaffected, indicating that DHA does not alter the bulk flow of secretory vesicular traffic. Overall, the present study presents compelling evidence that select dietary constituents with membrane lipid-modifying properties can differentially modulate subcellular localization of important lipidated signaling proteins depending on their intracellular trafficking route to the PM.
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Defining the Mechanisms by which Palmitoylation Regulates the Localization and Function of RGS4Dissanayake, Kaveesh 31 December 2010 (has links)
Regulator of G-protein signalling 4 (RGS4) modulates Gq and Gi signalling at the plasma membrane (PM). It has been demonstrated that the addition of palmitate to cysteine residues is an important regulator of RGS protein localization and function. The family of palmitate transferase enzymes shares a conserved Asp-His-His-Cys (DHHC) motif. We set out to establish the DHHC isoform(s) that affect RGS4 activity in HEK201 cells. Confocal microscopy revealed that overexpression of DHHCs 3 and 7 mobilized RGS4 to the Golgi. Knockdown of either DHHC3 or DHHC7 attenuated RGS4 inhibition of Gαq-coupled Ca2+ release and reduced RGS4 PM localization. Consistent with a role in promoting RGS4 lipid bilayer targeting, dominant negative mutants of the five most highly expressed DHHCs in HEK201 cells also diminished RGS4 PM association. Together, these data suggest that members of the mammalian DHHC family regulate RGS4 localization and function, likely through palmitoylation of its target cysteine residues.
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