<p>The capacity of marine organisms to adapt to natural and anthropogenic stressors is an integral component of ocean health. Harmful algal blooms (HABs), which are one of many growing threats in coastal marine ecosystems, represent a historically present natural stressor that has recently intensified and expanded in geographic distribution partially due to anthropogenic activities. In the Gulf of Mexico, HABs of <italic>Karenia brevis</italic> occur almost annually and produce neurotoxic brevetoxins that have been associated with large-scale mortality events of many marine species, including the common bottlenose dolphin (<italic>Tursiops truncatus</italic>). The factors resulting in large-scale dolphin mortality associated with HABs are not well understood, particularly in regards to the seemingly different impacts of HABs in geographically disjunct dolphin populations. My dissertation investigates a genetic basis for resistance to HABs in bottlenose dolphins in central-west Florida and the Florida Panhandle. I used both genome-wide and candidate gene approaches to analyze genetic variation in dolphins that died putatively due to brevetoxicosis and live dolphins from the same geographic areas that survived HAB events. Using restriction site-associated DNA sequencing, I identified genetic variation that suggested both a common genetic basis for resistance to HABs in bottlenose dolphins across the Gulf coast of Florida and regionally specific resistance. Many candidate genes involved in the immune, nervous, and detoxification systems were found in close genomic proximity to survival-associated polymorphisms throughout the bottlenose dolphin genome. I further investigated two groups of candidate genes, nine voltage-gated sodium channel genes selected because of their putative role in brevetoxin binding and four major histocompatibility complex (MHC) loci selected because of their genomic proximity to a polymorphism exhibiting a strong association with survival. I found little variation in the sodium channel genes and conclude that bottlenose dolphins have not evolved resistance to HABs via mutations in the toxin binding site. The immunologically relevant MHC loci were highly variable and exhibited patterns of genetic differentiation among geographic regions that differed from neutral loci; however, genetic variation at the MHC also could not fully explain variation in survival of bottlenose dolphins exposed to HABs. In my final chapter, I consider the advantages and drawbacks of the genome-wide approach in comparison to a candidate gene approach and, as laid out in my dissertation, I recommend using both complementary approaches in future investigations of adaptation in genome-enabled non-model organisms.</p> / Dissertation

Calmodulin (CaM) is a ubiquitous calcium-binding protein responsible for the binding and activation of a vast number of enzymes and signaling pathways. It contains two lobes that bind two calcium ions each, separated by a flexible central linker. This structural flexibility allows CaM to bind and regulate a large number of diverse protein targets within the cell in response to Ca2+ gradients. Voltage gated calcium channels (CaVs), as main sources of extracellular Ca2+, are crucial for a number of physiological processes, from muscle contraction to neurotransmission and endocrine function. These large transmembrane proteins open in response to membrane depolarization and allow gated entry of Ca2+ ions into the cytoplasm. Their regulation is currently the subject of intense investigation due to its pharmacological and scientific importance. CaM has been previously shown to pre-associate and act as a potent inhibitor of one class of high-voltage activated (HVA) channels called L-type channels via its interaction with their C-terminal cytoplasmic region. This interaction is primarily mediated by a conserved CaM-binding motif called the ‘IQ’ motif (for conserved isoleucine and glutamine residues), although the exact molecular details of its involvement in inactivation are currently unclear. Elucidation of these details was the primary objective of this dissertation. Recently, a novel sequence motif within this channel called ‘NSCaTE’ (N-terminal spatial calcium transforming element) has been described as an important contributor to calcium-dependent inactivation (CDI) of L-type channels. It was presumed to be unique to vertebrates, but we also show its conservation in a distantly related L-type channel homolog of Lymnaea stagnalis (pond snail). The interaction of CaM with a number of peptides representing the different regulatory motifs (IQ and NSCaTE) for both mammalian and snail isoforms was characterized in an attempt to better understand their role in CDI. Biophysical work with peptides as well as electrophysiology recordings with an N-terminal truncation mutant of Lymnaea CaV1 homolog were performed to expand our understanding of how the interplay between these channel elements might occur. In brief, the most striking feature of the interaction concerns the strong evidence for a CaM-mediated bridge between the N- and C-terminal elements of L-type channels. Further investigation of the CaM interaction with both IQ and NSCaTE peptides using Ca2+-deficient CaM mutants reveals a preference of both peptides for the Ca2+-C-lobe of CaM, and a much higher affinity of CaM for the IQ peptide, suggesting that the N-lobe of CaM is the main interaction responsible for the physiological effects of NSCaTE. These results are consistent with our electrophysiology findings that reveal a distinct buffer-sensitive CDI in wild type LCaV1 that can be abolished by the N-terminal truncation spanning the NSCaTE region. In addition to L-type channels, CaM has also been shown to have an indirect role in the regulation of low-voltage activated (LVA) or T-type channels (CaV3.x), via their phosphorylation by CaM-dependent protein kinase II (CaMKII). Using a primary sequence scanning algorithm, a CaM-binding site was predicted in a cytoplasmic region of these channels that was also previously shown to be important in channel gating. Biophysical experiments with synthetic peptides spanning this gating brake region from the three human and the single Lymnaea isoform strongly suggest that there is a novel, bona fide CaM interaction in this channel region, and also hint that this interaction may be a Ca2+-dependent switch of some sort. The results confirm a possible new role for CaM in the direct regulation of these channels, although the exact mechanism remains to be elucidated.

Protein interactions are the basis of synaptic function, and studying these interactions on a molecular level is crucial for understanding basic brain function, as well as mechanisms underlying neurological disorders. In this thesis, kinetic and mechanistic characterization of synaptic protein interactions was performed by using surface plasmon resonance biosensor technology. Fragment library screening against the reverse transcriptase of HIV was included, as it served as an outlook for future drug discovery against ligand-gated ion channels. The protein-protein interaction studies of postsynaptic Ca2+ -binding proteins revealed caldendrin as a novel binding partner of AKAP79. Caldendrin and calmodulin bind and compete at similar binding sites but their interactions display different mechanisms and kinetics. In contrast to calmodulin, caldendrin binds to AKAP79 both in the presence and absence of Ca2+ suggesting distinct in vivo functional properties of caldendrin and calmodulin. Homo-oligomeric β3 GABAA receptors, although not yet identified in vivo, are candidates for a histamine-gated ion channel in the brain. To aid the identification of the receptor, 51 histaminergic ligands were screened and a unique pharmacology was determined. A further requirement for identifying β3 receptors in the brain, is the availability of specific high-affinity ligands. The developed biosensor assay displayed sufficient sensitivity and throughput for screening for such ligands, as well as for being employed for fragment-based drug discovery. AMPA receptors are excitatory ligand-gated ion channels, involved in synaptic plasticity, and modulated by auxiliary proteins. Previous results have indicated that Noelin1, a secreted glycoprotein, interacts with the AMPA receptor. By using biochemical methods, it was shown that Noelin1 interacts directly with the receptor. The kinetics of the interaction were estimated by biosensor analysis, thereby confirming the interaction and suggesting low nanomolar affinity. The results provide a basis for functional characterization of a novel AMPA receptor protein interaction. The results demonstrate how secrets of synaptic protein interactions and function were revealed by using a molecular based approach. Improving the understanding of such interactions is valuable for basic neuroscience. At the same time, the technical advancements that were achieved to study interactions of ligand-gated ion channels by surface plasmon resonance technology, provide an important tool for discovery of novel therapeutics against these important drug targets.

Bilayer embedded proteins are affected by stress. This general affirmation is, in this thesis, embodied by two types of proteins: membrane spandex and voltage-gated sodium channels. In this work, we essentially explore, using methods from physics, the theoretical consequences of ideas drawn from experimental biology. Membrane spandex was postulated to exist and we study the theoretical implications and possible benefits for a cell to have such proteins embedded in its bilayer. There are no specific membrane spandex proteins, rather any protein with a transition involving a large enough area change between two non-conducting states could act as spandex. Bacterial cells have osmovalve channels which open at near-lytic tensions to protect themselves against rupture. Spandex expanding at tensions just below the osmovalves’ opening tension could relieve tension enough as to avoid costly accidental osmovalve opening due to transient bilayer tension excursions. Another possible role for spandex is a tension-damper: spandex could be used to maintain bilayer tension at a fixed level. This would be useful as many bilayer embedded channels are known to be modulated by tension. The Stress/shear experienced in traumatic brain injury cause an immediate (< 2 min) and irreversible TTX-sensitive rise in axonal calcium. In situ, this underlies an untreatable condition, diffuse axonal injury. TTX sensitivity indicates that leaky voltage-gated sodium (Nav) channels mediate the calcium increase. Wang et al. showed that the mammalian adult CNS Nav isoform, Nav1.6, expressed in Xenopus oocytes becomes “leaky” when subjected to bleb-inducing pipette aspiration. This “leaky” condition is caused by a hyperpolarized-shift (left-shift or towards lower potentials, typically 20 mV) of the kinetically coupled processes of activation and inactivation thus effectively degrading a well-confined window conductance into a TTX-sensitive Na leak. We propose experimental protocols to determine whether this left-shift is the result of an all-or-none or graded process and whether persistent Na currents are also left-shifted by trauma. We also use modeling to assess whether left-shifted Nav channel kinetics could lead to Na+ (and hence Ca2+ ) loading of axons and to study saltatory propagation after traumatizing a single node of Ranvier.

The aim of this thesis is to analyse the effects of gated communities in the increasing fragmentation of urban space and in the increasing polarisation among different classes in the Turkish context, more specifically in the capital, Ankara. Since the case study is based on an upper-middle class suburban gated community, first, suburbanisation &ldquo / as a wave of urbanisation&rdquo / is analysed. Then, the debates about the middle class and the transformation that this social stratum has undergone are discussed. Later, the formation of gated communities around the world and in Turkey are analysed within the general framework of the transformation of the urban sphere. Finally, the theoretical arguments are scrutinised by incorporating the findings of the case study carried out in Angora Houses. In this study Angora Houses is concluded to be a gated community which is &ldquo / fortified&rdquo / for the preservation of an upper-middle class lifestyle rather than for security concerns and which reproduces socio-spatial inequalities among Ankaraites rather than standing as only the expression of them.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Synaptic plasticity is thought to be integral to learning and memory. The two most common forms of plasticity are long-term potentiation (LTP) and long-term depression (LTD), both of which can be supported either by presynaptic changes in transmitter release probability (Pr), or by postsynaptic changes in AMPA receptor number. It is generally thought that the induction of LTP and LTD at Schaffer-collateral synapses in the hippocampus depends on the activation of NMDA receptors (GluN). Recent studies, however, have demonstrated that both increases and decreases in Pr can be induced under blockade of postsynaptic GluN receptors, suggesting that the activation of postsynaptic GluN receptors by glutamate is only a strict requirement for postsynaptic plasticity. In this thesis, I therefore re-examined the role of glutamate in presynaptic plasticity. I used single synapse imaging along with electrophysiological and pharmacological techniques to independently manipulate and monitor the levels of glutamatergic signalling during synaptic activity. I discovered that glutamate is inhibitory and unnecessary for the induction of LTP at the presynaptic locus. My findings support a novel model of presynaptic plasticity in which the net activity-dependent changes in Pr at an active presynaptic terminal is jointly determined by two opposing processes that can be simultaneously active: 1) postsynaptic depolarization, which, via the activation of L-type voltage-gated Ca²⁺ channels, increases Pr by driving the synthesis and release of nitric oxide from neuronal dendrites and 2) glutamate release, which through the activation of presynaptic GluN receptors, decreases Pr. Computationally, this model suggests that plasticity functions to reduce prediction-errors that arise during synaptic activity, and, thereby offers a biologically plausible mechanism by which neuronal networks may optimize learning at the level of single synapses.

L’expression anormale du canal sodique Nav1.5 dans le cancer du sein est corrélée au développement métastatique et à une mortalité augmentée. Le canal Nav1.5 est localisé dans les invadopodes des cellules cancéreuses mammaires humaines MDA-MB-231 et augmente leur activité protéolytique par une modulation allostérique de l’échangeur NHE-1 et l’activation de protéases acides. In vivo, dans un modèle de xénogreffe sur souris NMRI nude, l’expression de Nav1.5 potentialise la colonisation des poumons par les cellules cancéreuses mammaires humaines. Cette colonisation métastatique est inhibée par un traitement à la ranolazine, un inhibiteur pharmacologique des canaux Nav1.5. La sous-unité β4, auxiliaire des canaux Nav, voit son expression diminuer au cours de la progression cancéreuse, ce qui est associé in vitro à une augmentation de l’invasivité cellulaire. Cette augmentation d’invasivité semble indépendante du canal Nav1.5 et pourrait être associée à une transition des cellules vers un phénotype amiboïde. En conclusion, l’expression de Nav1.5 et la perte d’expression de β4 semblent jouer des rôles complémentaires dans l’invasivité des cellules cancéreuses. / The abnormal expression of sodium channel Nav1.5 in breast cancer is correlated with metastatic development and an increased mortality. The Nav1.5 channel is located in invadopodia in human breast cancer cells MDA-MB-231, where it increases proteolytic activity by allosteric modulation of exchanger NHE-1 and activation of acidic proteases. In vivo, in a xenograft model in nude NMRI mice, the expression of Nav1.5 potentiates lung colonization by human breast cancer cells. Metastatic colonization is inhibited by treatment with ranolazine, a pharmacological inhibitor of Nav1.5. The β4 subunit, an auxiliary subunit of Nav channels, is expressed at low levels or lost when tumors are more aggressive, and its suppression in vitro increases celI invasiveness. This increase seems to be independent of Nav1.5 and could be associated with the transition of cells to an amoeboid phenotype. In conclusion, Nav1.5 expression and the loss of β4 expression seem to play complementary roles in the invasiveness of cancer cells.

Les cellules cancéreuses mammaires invasives expriment des canaux sodiques NaV1.5 dont l’activité semble être associée au développement métastatique. L’activité de ce canal dans les cellules MDA-MB-231 conduit à une acidification péricellulaire favorable à l’activité des cathepsines à cystéine B et S extracellulaires et à la dégradation de la matrice extracellulaire. Au cours de cette thèse, nous avons montré que l’échangeur NHE1 est le principal régulateur du pH des cellules MDA-MB-231 et que l’activité du canal NaV1.5 augmente l’activité d’efflux de protons par NHE1 vraisemblablement par modulation allostérique. NaV1.5 et NHE1 sont co-localisés dans des radeaux lipidiques et plus particulièrement dans les invadopodes des cellules MDA-MB-231. Les activités de NHE1 et NaV1.5 stimulent l’activité protéolytique des invadopodes. Enfin, l’activité du canal NaV1.5 semble moduler le cytosquelette et la morphologie des cellules cancéreuses MDA-MB-231 pour leur donner un phénotype invasif. En conclusion, NaV1.5 augmente l’activité de NHE1 dans les invadopodes stimulant ainsi l’invasivité des cellules cancéreuses mammaires. / Invasive breast cancer cells express NaV1.5 sodium channels which activity seems to be associated with metastatic progression. The activity of the channel in MDA-MB-231 cells leads to a pericellular acidification favourable for the activity of extracellular cysteine cathepsins B and S and for extracellular matrix degradation. During this thesis, we have shown that NHE1 exchanger is the main pH regulator in MDA-MB-231 cells and that the activity of NaV1.5 channels increases protons efflux activity of NHE1 possibly through allosteric modulation. NaV1.5 and NHE1 are co-localised in lipid rafts and in invadopodia of MDA-MB-231 cells. The activity of NHE1 and NaV1.5 promotes the proteolytic activity of invadopodia. Finally, the activity of NaV1.5 channels seems to modulate cytoskeleton and morphology of MDA-MB-231 cancer cells to promote the acquisition of a proinvasive phenotype. In conclusion NaV1.5 increases NHE1 activity in invadopodia to stimulate breast cancer cells invasiveness.

No description available.