Thyroid hormones (THs) are essential signaling molecules for the postembryonic development of all vertebrates. THs are capable of initiating a diverse set of developmental programs across multiple tissues. The role of TH in regulating gene expression is well-known, but the initiation of TH signaling is still not fully understood. In amphibians, THs are the sole hormones required for the metamorphosis from tadpole to juvenile froglet. Amphibians are a useful model for studying TH signaling, as they undergo extensive, tissue-specific response programs in response to exogenous TH. The metamorphosis of the American bullfrog, Rana [Lithobates] catesbeiana is temperature sensitive. R. catesbeiana tadpoles do not undergo metamorphosis at cold temperatures (4-5 °C) even in the presence of THs that should otherwise prompt it. However, tadpoles undergo metamorphosis at an accelerated rate when returned to warm temperatures (24-25 °C) forty days after their initial TH exposure. R. catesbeiana tadpoles possess a “molecular memory” of TH exposure which establishes the TH signal at cold temperatures and prompts accelerated metamorphosis after a return to warmer temperatures. The mechanisms of the molecular memory which allow it to uncouple the initiation of TH signaling from the execution of the TH response program are not fully understood. Previous research has established that transcripts encoding transcription factors are a substantial component of the TH-dependent transcriptomic response of cultured tailfin (C-Fin) at cold temperatures. However, not all of these putative transcripts encoding transcription factors required active transcription and translation for their induction, which suggests that the initiation of a TH signal involves mechanisms other than regulating gene expression. Herein, we used quantitative polymerase chain reaction (qPCR) and RNA-Sequencing (RNA-Seq) to investigate the TH-dependent transcriptomic response of the back skin, a tissue that undergoes extensive remodeling during metamorphosis. Cultured back skin (C-Skin) was TH-responsive in warm, cold and temperature shift conditions. Forty-four transcripts underwent significant changes in abundance in response to TH in cold temperatures under which the molecular memory is established. Seven of these transcripts encoded putative transcription factors. Surprisingly, the only TH-responsive transcript significantly changed at 4 °C in both the C-Skin and the previously studied C-Fin was thyroid hormone-induced basic leucine zipper-containing protein (thibz). Thibz has been found to be TH-responsive at cold temperatures in the liver, lung, liver, brain, tailfin and back skin of whole animals, which suggests it may be an important regulator of initiating TH signaling. The lack of overlap in the transcriptomic responses of C-Skin and C-Fin may suggest that even the early initiation of TH signaling has tissue-specificity. Alternately, the molecular memory may include mechanisms that do not require active transcription and translation. Transcripts associated with epigenetic modifications and post-transcriptional changes to mRNA stability were also significantly expressed at 4 °C within the C-Skin. Previous investigation of the putative transcription factors in C-Fin revealed that active transcription and translation was not always required for changes in transcript abundance. Multiple mechanisms may be at play in the TH response at different temperatures. In cold temperatures, TH may modulate mRNA stability to influence transcript abundance as a part of initiating TH signaling without executing metamorphosis. Further research is needed to explore potential alternative mechanisms of establishing the molecular memory and the accelerated metamorphic response. The temperature sensitivity of R. catesbeiana’s TH response is incredibly valuable in investigating mechanisms of early TH signaling during postembryonic vertebrate development. / Graduate

Thyroid hormone (TH) is a critical signalling molecule for all vertebrate organisms, playing an especially crucial role in postembryonic development. Given its importance, many studies have focused on further elucidating the initial TH signal response and its method of transduction. Although the primary mechanism of TH response is genomic signalling, alternative mechanisms of early TH signal transduction have been relatively poorly studied. The North American bullfrog, Rana catesbeiana, is a useful model to study these early responses as tadpole post-embryonic development, or metamorphosis, can be experimentally induced through exposure to TH. The experimental induction of the TH signalling program leads to similar morphological endpoints as seen in natural metamorphosis in the transition of a tadpole to a juvenile froglet, such as regression of the tail. This TH-induced developmental program can also be manipulated through temperature where, as temperatures lower, developmental rate is delayed and at 5°C metamorphosis is completely stalled. Interestingly, when tadpoles exposed to TH at 5°C are introduced to permissive temperatures (24°C), an accelerated developmental program ensues, even when no more endogenous TH signal remains. Previous research has shown that this phenomenon can also be seen on the molecular level where only a select few transcripts have been shown to be responsive to TH at 5°C. However, the characteristic, if not augmented, TH response program is seen on the transcriptomic level when tadpoles are shifted to 24°C. This indicates that there is a molecular memory where the TH signal is induced in cold temperatures but not executed until more permissive temperatures arise. The extent and regulation of the transcriptomic program involved in this TH-induced molecular memory has yet to be understood. Herein we use the broader probing technique of RNA-seq analysis to identify potential components of the molecular memory. Eighty-one gene transcripts were TH-responsive at 5°C in cultured R. catesbeiana tail fin indicating that the molecular memory is more complex than previously thought. A number of these transcripts encoded regulators of transcription. Closer examination of select transcripts including a novel krüppel-like factor family member, klfX, at 5oC indicated that not all of the candidate molecular memory transcripts are regulated through active transcription and active translation is not required. When moved into 24°C an accelerated transcriptomic response occurred even when no additional TH is added, suggesting that a priming event occurs by TH exposure at 5°C allowing an accelerated metamorphosis at permissive temperatures. The molecular memory may be used as a means to isolate the initiating TH signalling response and the regulation of this program to allow further elucidation of early TH signalling in post-embryonic development. / Graduate

Ion channels are fundamental molecules in the nervous system that catalyze the flux of ions across the cell membrane. There are mounting evidences suggesting that the kinetic properties of ion channels undergo activity-dependent changes in various pathophysiological conditions. Here such activity-dependent changes were studied in case of two different ion channels; the rat brain derived voltage-gated Na+ channel, rNav1.2 and the human background leak K+ channel, hTREK1 using the single channel patch-clamp technique. Our results on the voltage-gated Na+ channel (Chapter III) illustrated that sustained membrane depolarization, as seen in pathophysiological conditions like epilepsy, induced a defined non-linear variation in the unitary conductance, activation, inactivation and recovery kinetic properties of the channel. Signal processing tools attributed a pseudo-oscillatory nature to the non-linearity observed in the channel properties. Prolonged membrane depolarization also induced a “molecular memory” phenomenon, characterized by clustering of dwell time events and strong autocorrelation in the dwell time series. The persistence of such molecular memory was found to be dependent on the duration of depolarization. Similar plastic changes were observed in case of the hTREK1 channel in presence of saturating concentrations of agonist, trichloroethanol (TCE) (Chapter IV). TREK1 channel behaves similar to single enzyme molecules with a single binding site for the substrate K+ ion whereas TCE acts as an allosteric activator of the channel. We observed that with increasing concentration of TCE (10 M to 10 mM) the catalytic turnover rate exhibited progressive departure from monoexponential to multi-exponential distribution suggesting the presence of ‘dynamic disorder’ analogous to single enzyme molecules. In addition, we observed the induction of strong correlation in successive waiting times and flux intensities, exemplified by distinct mode switching between high and low flux activity, which implied the induction of memory in single ion channel. Our observation of such molecular memory in two different ion channels in different experimental conditions highlights the importance and generality of the phenomenon which is normally hidden under the ensemble behaviour of ion channels. In the final part of the work (chapter V) we observed strong negative cooperativity and half-of-sites saturation kinetics in the interaction of local anesthetic, lidocaine with hTREK1 channel. We also mapped the specific anesthetic binding site in the c-terminal domain of the channel. Further, single channel analysis and the heterodimer studies enabled us to propose a model for this interaction and provide a plausible paradigm for the inhibitory action of lidocaine on hTREK1.

Au cours de ce travail nous avons étudié la réalisation de couches moléculaires d’accroche terminées amine. Sur l’oxyde de silicium l’aminopropyletriméthoxysilane (APTMS) a été déposé à partir d’une solution, et via une méthode originale par voie sèche qui nous a permis de mettre en évidence les temps caractéristiques de greffage et d’organisation de la couche d’APTMS. Sur l’or, les monocouches d’aminoéthanethiol (AET) et d’aminothiophénol (ATP) ont été réalisées à partir d’une solution. Nous avons ensuite étudié les aspects structuraux et cinétiques du greffage des fullerènes C60 sur de telles couches d’accroche, constituées de terminaisons amines soit sur toute la surface soit en des zones isolées (couches binaires). Les techniques de spectroscopie UV-Visible, IRTF, Raman, et XPS ont permis d’observer le greffage des C60 sur les couches aminées. La spectroscopie Raman en mode exalté (SERS) a mis en lumière que les molécules d’ATP étaient plus inclinées après le greffage à reflux des C60. Les analyses des diverses couches à l’échelle moléculaire ont été menées par microscopie à sondes locales (AFM, STM), et les mesures électriques réalisées sur or à l’aide de la pointe STM ont montré le caractère isolant de la couche d’accroche seule et un gap proche de celui du C60 après greffage des fullerènes. Elles ont également mis en évidence que le C60 était greffé sélectivement sur les zones terminées amines des couches d’accroche binaires. Enfin, une application potentielle des couches de C60 étant les mémoires moléculaires, les propriétés électriques des diverses couches réalisées ont été mesurées à l’aide de contacts électriques évaporés. / In this work we studied the preparation of sticking amine- terminated molecular layers. On silicon dioxide, 3-aminopropyltrimethoxysilane (APTMS) was de- posited from a solution, and using an original dry method that allowed us to determine time constants of APTMS layer grafting and organization. On gold surfaces, monolayers of aminoethanethiol (AET) and aminothiophenol (ATP) molecules were prepared from a solution. Then, we studied structural and kinetic aspects of ullerene C60 grafting on such sticking layers, terminated by amines either all over the surface or on isolated areas (binary layers). UV-visible, FTIR, Raman and XPS spectroscopy techniques enabled to observe that C60 was grafted on the amine-terminated layers. Exalted Raman spec- troscopy (SERS) revealed ATP molecules were more tilted after C60 grafting under reflux. Analyses of all the layers were made at a molecular level by local probe microscopy (AFM, STM), and electrical measurements performed on gold using the STM tip showed the in- sulating nature of the sticking layer whereas a gap close to that of C60 appeared after grafting of fullerenes. They also highlighted that C60 was selectively grafted on amine- terminated zones within binary sticking layers. At last, one of potential applications of C60 layers being molecular memory cells, electrical properties of the various studied layers were measured through evaporated electrical contact pads.