Teses / dissertações sobre o tema "Amino acids sensing"

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第21148号
農博第2274号
新制||農||1058(附属図書館)
学位論文||H30||N5122(農学部図書室)
京都大学大学院農学研究科食品生物科学専攻
(主査)教授 金本 龍平, 教授 保川 清, 教授 谷 史人
学位規則第4条第1項該当

Nutrient availability regulates eukaryotic cell growth. This study focuses on two signaling pathways, involved in sensing amino acids and carbon sources, which allow cells to respond appropriately to their presence. The first part of this study shows that Ssy1, a plasma membrane localized sensor in the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathway, can detect 19 common L-amino acids with different potencies and affinities based on the physiochemical structure of amino acids. Substituents around alpha carbon are critical for amino acid sensing by Ssy1. Furthermore, a high concentration of cysteine is toxic to cells. Inactivation of SPS signaling confers resistance to cysteine. The second part focuses on the regulation of Hap4, the regulatory subunit of the Hap2/3/4/5 transcriptional factor complex. Many components of the 25-subunit Mediator complex negatively regulate HAP4 expression. Srb8 undergoes post-translational modification in response to changes of the carbon source. Gal11 and Med3 positively regulate HAP4 expression.

The plant glutamate receptors (GLRs) are homologs of mammalian ionotropic glutamate receptors (iGluRs) and are hypothesized to be potential amino acid sensors in plants. Since their first discovery in 1998, the members of plant GLRs have been implicated in diverse processes such as C/N ratio sensing, root formation, pollen germination and plant-pathogen interaction. However, the exact properties of these channels, such as the spectrum of ligands, ion specificities, and subunit compositions are still not well understood. It is well established that animal iGluRs form homo- or hetero-tetramers in order to form ligand-gated cation channels. The first aspect of this research was to determine if plant GLRs likewise require different subunits to form functional channels. A modified yeast-2-hybrid system approach was initially taken and applied to 14 of the 20 AtGLRs to identify a number of candidate interactors in yeast. Forster resonance energy transfer (FRET), which measures the transfer of energy between interacting molecules, was performed in mammalian cells to confirm interaction between a few of those candidates. Interestingly, despite an abundance of overlapping co-localization between heteromeric combinations, only homomeric interactions were identified between GLRs 1.1 and 3.4 in HEK293 cells. Further, amino acids have been implicated in signaling between plants and microbes, but the mechanisms for amino acid perception in defense responses are far from being understood. Recently it was demonstrated that calcium responses initiated by bacterial and fungal microbe-associated molecular patterns (MAMPs) were diminished in seedlings treated with known agonists and antagonists of mammalian iGluRs, suggesting potential roles of GLRs in pathogen responses. Analysis of publicly available microarray data shows altered gene expression of a sub-fraction of GLRs in response to pathogen infection and bacterial elicitors. Thus, the second goal of my PhD research was aimed at determining whether GLRs are involved in the interaction between plants and pathogens. Gene expression changes of a number of candidate GLRs as well as pathogen growth was examined in response to the plant pathogen Pseudomonas syringae pv. tomato DC3000. Interestingly, single gene and multi-gene deficient plants responded differently with regards to pathogen susceptibility, likely as a result of functional compensation between GLRs.
Ph. D.

The lcrF gene of Yersinia pestis, the etiological agent of plague, encodes a transcription activator responsible for inducing expression of several virulence-related proteins (Yops) in response to temperature. The mechanism of this thermoregulation was investigated. Using a yopE::lacZ reporter fusion, lcrF-mediated thermal regulation was observed in Y. pestis and Escherichia coli. The lcrF gene was sequenced, the 30.8 kDa. LcrF protein identified and purified, and LcrF-dependent yopE-specific DNA binding activity was detected. A sequence similarity search revealed that LcrF exhibits 98% homology to VirF of Yersinia enterocolitica and significant homology to the carboxy termini of other members of the AraC family of transcription activators. During localization studies, a significant proportion of LcrF was found associated with the membrane fraction in E. coli. However, pulse-chase experiments indicated that this result is an artifact of fractionation. lcrF-mediated thermal induction of the yopE::lacZ reporter fusion remains intact in a Shigella flexneri virR mutant. The virR mutation is known to affect thermal induction of Shigellavirulence genes, which are also controlled by an activator in the AraC family. As a first step toward identifying the temperature-sensitive step in the regulation of yop expression, lcrF::lacZ transcriptional fusions were constructed and analyzed in Y. pestis and E. coli. The activity of the fusions was not affected by the native pCD1 virulence plasmid, an intact lcrF gene, or temperature. Thus, induction of lcrF transcription is not essential for temperature-dependent activation of yopE transcription. To confirm these results, attempts were made to identify both the native lcrF message in Y. pestis, and a lcrF-lacZ hybrid message in Y. pestis and E. coli. These attempts were unsuccessful. Examination of LcrF protein production revealed temperature-dependent expression in Y. pestis. Surprisingly, high-level T7 polymerase-directed transcription of the lcrF gene in Escherichia coli also resulted in temperature-dependent production of the LcrF protein. Pulse-chase experiments showed that the LcrF protein was stable at both 26 and 37°C, suggesting that translation rate or message degradation is thermally controlled. Comparison of the amount of LcrF protein produced per unit of message at 26 and 37°C in E. coli indicated that the efficiency of translation of lcrF message increased with temperature. mRNA secondary structure predictions suggest that the lcrF Shine-Dalgarno sequence is sequestered in a stem-loop. A model in which decreased stability of this stem-loop with increasing temperature leads to increased efficiency of translation initiation of lcrF message is presented.

Identifying the protein content in a cell in a fast and reliable manner has become a relevant goal in the field of proteomics. This thesis computationally explores the potential for silicon nitride nanopores to sense and distinguish single miniproteins, which are small domains that promise to facilitate the systematic study of larger proteins. Sensing and identification of these biomolecules using nanopores happens by studying modulations in ionic current during translocation. The approach taken in this work was to study two miniproteins of similar geometry, using a cylindrical-shaped pore. I employed molecular mechanics to compare occupied pore currents computed based on the trajectory of ions. I further used density functional theory along with relative surface accessibility values to compute changes in interaction energies for single amino acids and obtain relative dwell times. While the protein remained inside the nanopore, I found no noticeable differences in the occupied pore currents of the two miniproteins for systems subject to 0.5 and 1.0 V bias voltages. Dwell times were estimated based on the translocation time of a protein that exhibits no interaction with the pore walls. I found that both miniproteins feel an attractive force to the pore wall and estimated their relative dwell times to differ by one order of magnitude. This means even in cases where two miniproteins are indistinguishable by magnitude changes in the ionic current, the dwell time might still be used to identify them. This work was an initial investigation that can be further developed to increase the accuracy of the results and be expanded to assess other miniproteins with the goal to aid future experimental work.

The objective of this work is to better understand themetabolism and physiology ofEscherichiacoli(W3110) in defined medium cultures with thelong-term goal of improving cell yield and recombinant proteinproductivity.

The order of amino acid utilization inE. colibatch cultures was investigated in a medium with16 amino acids and glucose. Ser, Pro, Asp, Gly, Thr, Glu andAla were rapidly consumed and depleted at the end of theexponential phase, while His, Arg, Val, Met, Ile, Leu, Phe, Lysand Tyr were consumed slowly during the following linear growthphase. The uptake order correlated to the maximum specificconsumption rate. Of the rapidly consumed amino acids onlyglyine and threonine improved growth when added individually.Serine was the first amino acid to be consumed, but inhibitedglucose uptake initially, which presumably is related to thefunction of PTS. Valine inhibited cell growth could be releasedby isoleucine. The critical medium concentration of valinetoxicity was 1.5 - 3 µmol L^-1. Valine uptake was associated with exchange ofisoleucine out of the cells.

Glycine significantly increased the cell yield,Y_x/s,and growth rate ofE. coliin batch cultures in a glucose-mineral medium.Maximum effect occurred at pH 6.8, at 6 - 12 mmol L^-1glycine, and below 1.15 g dw L^-1.¹³C NMR technique was employed to identify [1-¹³C], [2-¹³C]and [1,2-¹³C]acetate in the cultures supplied with [2-¹³C]glycine. The NMR data revealed that littledegradation of added glycine occurred, and that serine/glycinebiosynthesis was repressed below 1.15 g dw L^-1, implicating that glycine was a source ofglycine, serine, one-carbon units, and threonine. Above 1.15 gdw L^-1, 53% of the consumed glycine carbon was excretedas acetate. Degradation of glycine was associated with anincreased uptake rate, cleavage by GCV, and degradation of bothglycine- and glucose-derived serine to pyruvate. This switch inmetabolism appears to be regulated by quorum sensing.

A cell density-dependent metabolic switch occurred also inthe central metabolism. A 2 - 3 fold decrease in mostglycolytic and TCA cycle metabolites, but an increase inacetyl-CoA, occurred after the switch. The acetate productionrate decreased throughout the culture with a temporary increaseat the switch point, but the intracellular acetate poolremained relatively constant.

Two mixtures of amino acids were fed together with glucosein fed-batch cultures ofE. coliW3110 pRIT44T2, expressing the recombinantprotein ZZT2. One mixture contained 20 amino acids and theother 5 so-called 'protein amino acids': Ala, Arg, Met, His andPhe. Although the amino aids increased the cell yield anddecreased the proteolysis rate in both cases, ZZT2 productionwas decreased. A decrease of ZZT2 synthesis rate is consideredto be the reason. Further studies of the 5 amino acidsindicated that a few amino acids disturb metabolism.

Carbon mass balances were calculated in glucose limitedfed-batch cultures ofE. coli. In the end, the carbon recovery was ~90% basedon biomass, CO₂and acetate, but ~100% if the all carbon in themedium was included. Outer membrane (OM) constituents,lipopolysaccharide, phospholipids, and carbohydratescontributed to 63% of the extracellular carbon. Little celllysis occurred and the unidentified (~30%) carbon was assumedto constitute complex carbohydrates. A novel cultivationtechnique Temperature-Limited Fed-Batch (TLFB) is developed toprevent OM shedding in high-cell density cultures.

Keywords: Escherichia coli, amino acids, glycine, quorumsensing, metabolic switch, metabolite pools, carbon balance,outer membrane, lipopolysaccharide, batch culture, fed-batchculture

Chaque organisme vivant évolue dans son écosystème, où il doit être capable de détecter et d’intégrer de multiples facteurs environnementaux (température, hygrométrie, composés chimiques organiques ou non…). Ces signaux jouent un rôle important dans la communication entre organismes. Chaque individu doit donc mettre en lien ces stimuli externes avec ses propres signaux internes (nutritionnel, métabolique, hormonal, infection…), et adapter son comportement en conséquence pour assurer sa survie et sa reproduction. Pour ce faire des mécanismes complexes de détection se sont développés, notamment le système chimio-sensoriel, permettant la réception et la discrimination de molécules chimiques externes. En parallèle, des organes internes sont sensibles aux signaux internes afin de détecter des carences et de contrôler l’homéostasie cellulaire et tissulaire. Ces processus sont notamment possibles grâce à des protéines transmembranaires qui sont spécialisées dans la réception et/ou le transport d’autres molécules comme les acides aminés (AAs), indispensables à tous les types cellulaires tels que les cellules du système nerveux.Nos travaux de recherche montrent qu’un transporteur d’AAs appartenant à la famille des Solute carrier 7A (SLC7A) nommé Minidiscs (MND) est exprimé dans le cerveau adulte, au niveau des neurones et des cellules gliales. Cette protéine semble se localiser au niveau de la membrane plasmique et de la membrane du réticulum endoplasmique. MND est exprimé dans les neurones formant une structure particulière du cerveau appelée Corps Pédonculés (CPs) et joue un rôle clef dans la réponse de ces neurones à certains L-AAs (L-Asp, L-Arg, L-Glu, L-Lys, L-Ile, L-Leu et L-Thr). Ce résultat démontre ainsi que des transporteurs de la famille des SLC7As sont impliqués dans le contrôle de l’activité des neurones. De plus, cela suggère que les CPs peuvent détecter directement des L-AAs via MND, faisant de cette structure un centre de détection du statut interne nutritionnel de l’individu. La réponse de ces neurones à la L-Leucine MND dépendante fait intervenir la voie de signalisation passant par TOR, mais n’implique pas la Glutamate Deshydrogenase (GDH). Ainsi, MND pourrait potentiellement moduler les comportements associés à cette structure, tels que la mémoire olfactive, l’agressivité... Cependant, nos études comportementales semblent indiquer que la présence de MND dans tous les neurones des CPs n’est pas requise pour la modulation du comportement d’agressivité territoriale des mâles.Mes données indiquent également que MND est requis pour la réponse des CPs au L-Glutamate, qui est aussi un neurotransmetteur. MND étant décrit comme transporteur de L-AAs non chargés et que le L-Glutamate est, dans sa forme physiologique un AA chargé négativement, des partenaires de MND sont potentiellement impliqués dans cette activation. Cinq types de récepteurs au L-Glutamate sont présents dans les mêmes neurones des CPs exprimant MND : NMDAR1, NMDAR2, KAIR1D, mGluR et GluCl��. Il a été possible de montrer que l’activation des CPs via le récepteur NMDAR1 est dépendante de la présence de MND. Toutefois, cette voie de détection de signaux glutamatergiques ne semble pas intervenir dans la régulation du comportement d’agressivité. Nous montrons également que l’activité des CPs en réponse au glutamate qui est dépendante de NMDAR1 semble différer suivant le contexte social. En effet, une réponse plus intense a été observée chez les mâles isolés socialement par rapport aux mâles groupés. Cette réponse au glutamate impliquant NMDAR1 pourrait dépendre de la détection chronique du 11-cis-Vaccenyl Acétate (cVA), une phéromone mâle, suggérant ainsi que l’environnement social joue un rôle sur l’activité des CPs.Ainsi, pris dans leur ensemble, mes résultats montrent que des transporteurs d’acides aminés de type SLC interviennent dans la capacité des neurones à répondre aux AAs comme la L-Leucine et aux neurotransmetteurs comme le glutamate
Every living organism lives in an ecosystem, where it needs to detect and integrate multiple environmental factors (temperature, humidity, organic or non-organic chemical compounds...). These signals play an important role in communication between organisms. Each individual must link these external stimuli with its own internal signals (nutritional, metabolic, hormonal, infection...), and adapt its behavior to ensure its survival and reproduction. To achieve this, complex detection mechanisms have been developed, including the chemosensory system, allowing the reception and discrimination of external chemical molecules. Internal organs are sensitive to internal signals to detect deficiencies and control cellular and tissue homeostasis. These processes are made possible by transmembrane proteins that specialize in detection and/or transporting other molecules such as amino acids (AAs), essential for all cell types such as neuronal cells.My research focuses on an amino acid transporter belonging to the SLC7A family called Minidiscs (MND) and we showed that MND is expressed in the adult brain, in neurons and glial cells. This protein appears to be localized at the plasma membrane and the endoplasmic reticulum. MND is expressed in neurons forming a particular brain structure called Mushroom Bodies (MBs) and plays a key role in the response of these neurons to several L-amino acids (L-Asp, L-Arg, L-Glu, L-Lys, L-Ile, L-Leu, and L-Thr). This result demonstrates that SLC7A transporters are involved in controlling neuron activity and suggests that MBs can directly detect L-amino acids via MND. That making this structure a center for detecting the individual's internal nutritional status. The response of these neurons to L-Leucine MND-dependent involves a TOR pathway and not a GDH one. Due to its localization within the CPs, MND may potentially modulate behaviors associated with this structure. However, the presence of MND in all MB neurons is not required for modulation of male territorial aggressive behavior.My results also show that MND is required for the MBs' response to L-Glutamate which is also a neurotransmitter. MND is described as a transporter of uncharged L-amino acids and not L-Glutamate which is a negatively charged AA. Five L-Glutamate receptors are expressed in the MB neurons expressing MND: NMDAR1, NMDAR2, KAIR1D, mGluR, and GluCl��. We demonstrated that MB activation via the NMDAR1 receptor is MND dependent. This glutamatergic signaling pathway does not appear to be involved in the regulation of aggressive behavior. However, MB activity in response to glutamate involving NMDAR1 appears to be modulated by the social environment. Thus, the response of the MBs is increased in isolated males compared to grouped males. This glutamate response via NMDAR1 could depend on the chronic detection of 11-cis-Vaccenyl Acetate (cVA), a male pheromone. This suggesting that social environment impacts the MB activity.Thus, my results show that SLC-type amino acid transporters are involved in the ability of neurons to respond to neurotransmitters, such as glutamate and AAs

The thesis research was designed to study the characteristics of two important physiological processes in smooth muscle: Ca2+ transport mediated by the plasmalemmal Ca2+-ATPase and muscarinic receptor-G protein interactions. In resting smooth muscle, several Ca2+ extrusion or sequestration processes offset the passive inward leak of Ca2+. Although biochemical evidence suggests that the plasmalemmal Ca2+ pump plays a key role in this process, the precise role of this enzyme could not be proven until a reliable estimate of the inward Ca2+ leak was measured. Recent studies using dispersed smooth muscle cells from the toad stomach provided an estimate of the basal transmembrane Ca2+ flux rate; thus, we examined the transport capacity of the plasmalemmal Ca2+pump in this tissue. Gastric smooth muscle tissue was disrupted by homogenization and nitrogen cavitation. Membranes enriched 20 fold for plasma membrane markers were obtained using differential centrifugation and purification by flotation on discontinuous sucrose gradients. The membrane vesicles exhibited an ATP-dependent 45Ca uptake that was insensitive to azide or oxalate but sensitive to stimulation by calmodulin or inhibition by orthovanadate and the calmodulin antagonists trifluoperazine (TFP) or calmidazolium (CMZ). 45Ca accumulated in the presence of ATP was rapidly released by Ca2+ ionophore but not by agents that stimulate Ca2+ release from the sarcoplasmic rettculum (caffeine, inositol trisphosphate, GTP). However, both CMZ and TFP evoked a Ca2+ release that was comparable to that observed in the presence of Ca2+ ionophore, suggesting that these compounds have profound effects on membrane Ca2+permeability. 45Ca transport exhibited a high affinity for Ca2+ (KD 0.2 μM) and a high transport capacity, producing a > 12,000-fold gradient for Ca2+and a transmembrane flux rate at least 3-fold greater than that observed in resting smooth muscle cells. As a first step toward understanding the biochemical basis for the diversity of muscarinic cholinergic actions on smooth muscle, we examined the distribution of muscarinic receptor subtypes and coupling to guantne nucleotide-binding (G) proteins in airway and gastric smooth muscle. Receptor subtypes were classified in membranes prepared from bovine trachea and toad stomach based on the relative abilities of the selective antagonists pirenzepine (M1), AF-DX 116 (M2) and 4-DAMP (M3) to displace the binding of nonselective antagonist [3H]QNB (quinuclidinyl benzilate). Based on the binding profiles for these antagonists, it was concluded that both smooth muscle types contain a mixture of M2 and M3 subtypes. In trachea the majority of receptors (86%) were M2, whereas in stomach the majority of receptors (88%) were M3. The displacement of [3H]QNB binding by the agonist oxotremorine indicated a mixed population of high affinity (KD = 4 nM) and low affinity (KD = 2-4 μM) binding sites. The addition of GTPγS abolished all high affinity agonist binding, suggesting that coupling of the receptors to G proteins may confer high affinity. Reaction of membranes with pertussis toxin in the presence of [32P]NAD caused the [32P]-labelling of a ~ 41 kD protein in both gastric and tracheal smooth musc1e. Pretreatment of the membranes with pertussis toxin and NAD completely abolished high affinity agonist binding in gastric smooth muscle, but produced little if any decrease in high affinity agonist binding in trachea. We conclude that, although muscarinic receptor activation leads to the elevation of intracellular Ca2+ and to contraction of both airway and gastric smooth muscle, the dissimilar distributions of receptor subtypes and distinct patterns of coupling to G proteins may indicate that each smooth muscle type uses different receptor-G protein interactions to regulate intracellular signalling pathways.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, June 2018.
Cataloged from PDF version of thesis. "June 2018."
Includes bibliographical references.
In order to coordinate growth with organismal physiology, cells must couple anabolic and catabolic metabolism with a variety of environmental cues. In humans, cell growth is controlled primarily by the mechanistic Target of Rapamycin Complex 1 (mTORC1), which senses a diverse array of environmental inputs and in turn regulates nearly all aspects of growth and metabolism. Consistent with its central role in these processes, deregulation of the mTORC1 pathway is also associated with numerous diseases, including epilepsy, diabetes, and cancer. Although decades of study have revealed how growth factor signaling networks regulate mTORC1, the components that detect and signal nutrient availability to mTORC1 have been elusive until the recent discovery of the leucine and arginine sensors, named Sestrin2 and CASTOR1, respectively. However, the molecular mechanisms through which these components detect their respective amino acids have been elusive. To understand how Sestrin2 senses leucine and signals its presence to mTORC1, we solved the X-ray crystal structure of Sestrin2 in complex with leucine to 2.7 A resolution. This structure revealed how Sestrin2 recognizes leucine through a highly specific binding pocket, and utilizes a unique "lid-latch" mechanism to sense leucine. This structural data also allowed us to identify a highly conserved GATOR2-binding site on the surface of Sestrin2, in close proximity to the leucine-binding pocket, providing a plausible allosteric mechanism for the leucine-dependent dissociation of Sestrin2 from GATOR2. Using a similar approach, we then solved the crystal structure of arginine-bound CASTOR1 to 1.8 A resolution. This structure showed that CASTOR1 forms a homodimeric complex that engages arginine at the interface of two domains. Combining this structural information with various biochemical assays, we found that arginine acts as "molecular glue" that bridges these two domains together to allosterically modulate the adjacent GATOR2 binding site, thereby explaining the molecular basis for arginine-dependent mTORC1 activation. Together, the structural and biochemical characterization of Sestrin2 and CASTOR1 presented here have revealed for the first time the mechanisms of mammalian amino acid sensing in atomic detail. These structures have also paved the way for the development of novel compounds targeting mTORC1 through the Sestrin2 and CASTOR1 amino acid-binding pockets, which may have important clinical implications in the context of cancer, diabetes, neurological disorders, and longevity.
by Robert A. Saxton.
Ph. D.

Energy homeostasis is controlled in the hypothalamus. Hypothalamic tanycytes are a type of glial cell that lie in the wall of the third ventricle, between ventricular CSF and the key nuclei controlling feeding, and are potentially linked to this regulation. Tanycytes have been found to express sweet taste receptors; similar receptors exist for amino acids and could be present in tanycytes. Therefore, the aims of this work were to understand the tanycyte amino acid signalling process, determine the impact of diet on tanycyte function, and link tanycytes to energy homeostasis. Using ratiometric Ca2+ imaging, I showed that tanycytes responded to amino acids; the responses also involved ATP release via pannexin 1 and CalHM1 from tanycyte cell bodies and down their processes towards the parenchyma. As IMP increased the amplitude of tanycyte responses to some amino acids, one of the receptors is Tas1r1/Tas1r3. The Tas1r1 subunit was expressed in mouse tanycytes, and Tas1r1-null mice showed sex-specific altered responses to arginine and lysine. mGluR4 antagonist MAP4 reduced the responses to alanine, and mGluR4 was shown to be present in mouse tanycytes. Tas1r1/Tas1r3 and mGluR4 are therefore two of the receptors responsible for tanycyte amino acid signalling. Fasting and essential amino acid deprivation increased tanycyte sensitivity to alanine. Essential amino acid-deprived diet also reduced the intake of an alanine-enriched drink. While amino acid-imbalanced diets did not have strong effects on mouse metabolism over 24 hours, increasing dietary alanine content reduced feeding and increased metabolism in the light phase. These results demonstrate for the first time that tanycytes can detect amino acids, and that they do so via two different pathways, similarly to taste receptor cells in the tongue. Tanycytes can send ATP signals to inform the hypothalamus about nutrient availability. The data also show that tanycyte amino acid sensitivity is diet-dependent. High alanine food has a satiating effect, which could be partially mediated by tanycytes, although a direct link cannot be confirmed at this stage.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, February 2017.
Cataloged from PDF version of thesis. "February 2017."
Includes bibliographical references.
The mechanistic target of rapamycin complex I (mTORC1) protein kinase functions as a master regulator of growth, and its deregulation is common in human disease, including cancer and diabetes. mTORC1 integrates multiple environmental cues to control anabolic and catabolic processes. A key input is amino acids, which function to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. Necessary for this recruitment are the Rag GTPases and several distinct factors that modulate their nucleotide state in response to amino acid availability. Despite these advances, several key questions remain. The components that mediate mTORC1 inhibition upon amino acid deprivation and the identities of the amino acid sensors upstream of mTORC1 are both unknown. To provide insight into these questions, we undertook an unbiased proteomics approach to discover novel mTORC1 regulators. Here, we describe the identification of GATOR2 as a pentameric complex that positively regulates mTORC1 and functions upstream of or in parallel to GATOR1, a GTPase activating protein complex for the Rags and a negative regulator of the mTORC1 pathway. KICSTOR, a four-membered protein complex, is necessary to localize GATOR1 to the lysosome to enable it to suppress mTORC1 activity. GATOR1 components are mutated in cancer and may identify tumors that respond to clinically approved mTORC1 inhibitors. Furthermore, we describe the identification of Sestrin2 and CASTOR1 as GATOR2-interacting proteins that function as leucine and arginine sensors, respectively, for the mTORCI pathway. Both sensors are required to signal the absence of leucine and arginine to mTORC1, and the amino acid-binding capacity of both sensors is necessary for amino acids to activate mTORC1. Altogether, the identification of these mTORC1 regulators furthers our understanding of the mechanisms by which amino acid availability controls cellular growth.
by Lynne Chantranupong.
Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.
Cataloged from PDF version of thesis. "September 2016."
Includes bibliographical references.
The mTOR complex 1 (mTORC 1) protein kinase is a master growth regulator that responds to multiple cues from the local and systemic environment surrounding the cell. Nutrients and growth factors are both required to activate mTORC 1 and to promote growth. While the mechanisms of growth factor signaling have been reasonably well established, we have only begun to unravel in recent years how amino acids signal to mTORC 1, thanks in large part to the identification of the Rag GTPases, which recruit mTORC 1 to the lysosomal surface for catalytic activation, and of the regulators of Rag function. An ultimate goal is to uncover the biochemical basis of the sensing event that triggers this signaling cascade in the first place: which amino acid(s) are sensed and how? Toward this end, we characterize in detail the amino acids required to fully activate mTORCl signaling in HEK-293T cells and identify SLC38A9 as a Rag-interacting amino acid transporter that may serve as an arginine sensor at the lysosome. Finally, we discuss the many open questions that remain to be studied regarding the mechanisms and logic behind amino acid sensing by the mTORC 1 pathway.
by Shuyu Wang.
Ph. D.

Vibrio fischeri, a symbiotic bioluminescent bacterium, serves as one of the best understood model systems for a mechanism of cell-density dependent bacterial gene regulation known as quorum sensing. During quorum sensing in V. fischeri, an acylated homoserine chemical signal (autoinducer) is synthesized by the bacteria and used to sense their own species in a given environment. As the autoinducer levels rise, complexes form between the autoinducer and the N-terminal domain of a regulatory protein, LuxR. In response to autoinducer binding, LuxR is believed to undergo a conformational change that allows the C-terminal domain to activate transcription of the luminescence or lux operon. To further understand the mechanism of LuxR-dependent transcriptional activation of the lux operon, PCR-based site-directed mutagenesis procedures have been used to generate alanine-substitution mutants in the C-terminal forty-one amino acid residues of LuxR, a region that has been hypothesized to play a critical role in the activation process. An in vivo luminescence assay was first used to test the effects of the mutations on LuxR-dependent activation of the lux operon in recombinant Escherichia coli. Luciferase levels present in cell extracts obtained from these strains were also quantified and found to correlate with the luminescence results. Eight strains encoding altered forms of LuxR exhibited a "dark" phenotype with luminescence output less than 50% and luciferase levels less than 50% of the wildtype control strain. Western immunoblotting analysis with cell extracts from the luminescence and luciferase assays verified that the altered forms of LuxR were expressed at levels approximately equal to wildtype. Therefor, Low luminescence and luciferase levels could be the result of a mutation that either affects the ability of LuxR to recognize and bind its DNA target (the lux box) or to establish associations with RNA polymerase (RNAP) at the lux operon promoter necessary for transcriptional initiation. A third in vivo assay was used to test the ability of the altered forms of LuxR to bind to the lux box (DNA binding assay/repression). All of the LuxR variants exhibiting the "dark" phenotype in the luminescence and luciferase assay were also found to be unable to bind to the lux box in the DNA binding assay. Therefore, it can be concluded that the alanine substitutions made at these positions affect the ability of LuxR to bind to the lux box in the presence and absence of RNA polymerase. Another class of mutants exhibited wildtype phenotypes in the luminescence and luciferase assays but were unable to bind to the lux box in the DNA binding assay. The alanine substitutions made at these amino acid residues may be making contacts with RNAP that are important for maintaining the stability of the DNA binding region of LuxR. Alanine substitutions made at these positions have a defect in DNA binding at the promoter of the lux operon only in the absence of RNAP. None of the alanine substitutions made in the C-terminal forty-one amino acids of LuxR were found to affect activation of transcription of the lux operon without also affecting DNA binding. Taken together, these results support the conclusion that the C-terminal forty-one amino acids of LuxR are important for DNA recognition and binding of the lux box rather than positive control of the process of transcription initiation.
Master of Science

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a primary constituent of chronic, polymicrobial infections in the lungs of individuals with cystic fibrosis (CF). A significant consequence of CF is production of thick mucus along epithelial surfaces. In the lungs, this mucus collects and serves as an excellent growth substrate for a range of bacteria including. CF lung fluids (sputum) also enhance the virulence of P. aeruginosa, as production of a signaling molecule critical for virulence, the Pseudomonas quinolone signal (PQS), is enhanced in the presence of phenylalanine and tyrosine in CF sputum. The goal of this dissertation is to better understand how phenylalanine and tyrosine affect PQS production and ultimately P. aeruginosa virulence. To address this, I use transcriptome profiling to determine that genes for phenylalanine and tyrosine catabolism, PQS biosynthesis, and a transcriptional regulator called PhhR are up-regulated in the presence of phenylalanine and tyrosine. I determine that PhhR regulates genes for aromatic amino acid catabolism but not genes for PQS biosynthesis. The PhhR regulon is further characterized by mapping of PhhR-regulated promoters with primer extension, and evidence for direct regulation is presented. To explain enhanced production of PQS in CF sputum, I favor a model in which flux of a shared metabolic precursor, chorismate, toward PQS biosynthesis is enhanced when phenylalanine and tyrosine are present. I investigate this model by examining the first step in PQS biosynthesis, conversion of chorismate to anthranilate by an anthranilate synthase (AS). P. aeruginosa possesses two AS enzymes encoded by the trpEG and phnAB genes, with the former generating anthranilate specifically for tryptophan biosynthesis while the latter generates anthranilate for PQS biosynthesis. I investigate the evolutionary origins of these two enzymes and generate unmarked deletion mutants to dissect their roles in tryptophan and PQS biosynthesis. The ability of PhnAB to compensate for loss of TrpEG at high cell densities is documented, and a model explaining anthranilate sequestering is developed. Knowledge gained from these studies will be useful in developing novel therapeutic strategies.
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碩士
國立中興大學
化學系
93
Infrared spectroscopy provides a simple but powerful method to detect organic compounds. However, the detection of high polar compounds, such as negatively charged amino acid, is limited due to the high polarity and the negatively charged characteristics. In order to improve the selectivity and the sensitivity to the negatively charged amino acids, in this investigation, high polarity amine compounds was successfully immobilized on the internal reflection element by cross-linking method. The prepared sensing phase shows no obvious variation between pH=3 to 9 in aqueous solution. Besides, the network structure of the prepared sensing phase provides high water stability and the positively charged character of the sensing phase makes it possible to detect negatively charged amino acids selectively. Different kinds of amino acids are detected in different pHs to find the proper detecting condition. The results show that under the alkaline condition, both neutral and negatively charged amino acids can be detected by the amine cross-linked sensing phase; under the neutral or slightly acidic conditions, only negatively charged amino acids can be attracted into the sensing phase. For positively charged amino acids, repulsion will be occurred so that positively charged amino acids will not be detected by the amine cross-linked sensing phase in any pH condition. Furthermore, to get the optimal formula in synthesizing the amine cross-linked sensing phase, factors, such as different porogenic agents, molar ratio of tri-(2-amino ethyl)-amine and cross-linking agent, molar ratio of cross-linking agent and porogenic agent, are investigated. The results indicate that adding porogenic agents, especially aliphatic compounds, will increase the signal of the sample molecules apparently. Besides, the cross-linking agent will facilitate the water stability of the sensing phase but will not contribute in drawing sample molecules. Finally, under the optimal condition, the linear range in detecting glutamic acid is between 5 to 50 micro mole and the limit of detection is ca. 2 micro mole under the pH=6 where provides the best selectivity.

碩士
逢甲大學
化學工程學系
106
We’ve successfully synthesized a series of efficient chemical sensors, including two aniline trimer based colorimetric chemical sensors. Colorimetric dual responsive sensor (CDRS) with thiourea binding site can sense CN- anion and changes in pH. Cation-Amino acid sensor (CAS) with chiral thiourea binding site can sense Ag+, Cu2+, Fe3+ and L- Tryptophan. Upon addition of CN- to CDRS in 30% aqueous DMSO, the colorimetric response of solution of CDRS showed a color change from orange to purple. For CAS, upon addition of Ag+, Cu2+, and Fe3+ to it in 50% aqueous DMSO respectively, the colorimetric response of solution of CAS exhibited a color change from light purple to brown, purple, and fuchsia, respectively. Moreover, upon addition of L-Tryptophan to CAS in 10% aqueous DMSO, it showed blue fluorescence. However, no response was observed by adding other amino acid. The results can be recognized by naked eye. The quantative analysis of CN- by CDRS, and quantative analysis of Ag+, Cu2+, Fe3+ by CAS can be achieved by UV-Vis. Spectrometer. The interaction between receptor and analyte ions was realized by 1H NMR spectroscopic titration. CDRS and CAS have shown appreciable selectivity in the presence of multiple ions. In different pH environments, CDRS and CAS also have differnt color change. The CN- anion in aqueous solution can be recognized by solid test kits of CDRS. The Ag+, Cu2+, Fe3+ cations in aqueous solution can be recognized by solid test kits of CAS. The binding constants, detection limits of different receptor toward various ions were also studied.

碩士
國立臺灣大學
農業化學研究所
91
We sequenced a gene fragment in pBluescriptII KS (+), our lab cloned before, containing N-acyl-D-amino acid amidohydrolase (N-D-AAase) from Variovorax paradoxus Iso1. Sequence analysis revealed ten open reading frames, except N-acyl-D-amino acid amidohydrolase, including intergrase, D-amino acid dehydrogenase, two regulators and three ABC transporters. The putative D-amino acid dehydrogenase (DAAO), encodes 416 amino acids, was through gene cloning, expression, and measure activity to confirm its function. We choused two expression systems-pQE30 and pET21a-to express the DAAO, and its activity is very low in pET21a and higher in pQE30. It seemed to have no expression pattern on SDS PAGE, but we could make sure its function through measuring activity. DAAO and N-D-AAase could be induced by N-acetyl-D-amino acids on transcription level and expressed in the same 2.8knt RNA, it is the sum of these two genes length. Through the data, Western blot of N-D-AAase and zymography of DAAO, they also have similar expression pattern when add different kinds of inducers in minimal medium or LB. These data reveals that the two enzymes may constitute an operon to cooperate metabolize similar substrate and be regulated by the same promoter. There are two regulators localized separately at upstream and downstream of this operon. When cloning the different truncated regulators fragment in pUC119 and measure their effect on the activity of N-D-AAase, we found they maybe play a role on the regulation of this operon. The degradation of quorum sensing factors (QSFs) experiment denied our assumption that N-D-AAase maybe active on them. The crude enzyme of N-D-AAase expression strain- pKKdam3 -couldn’t degrade QSFs but the total lysat of wild type could degrade QSFs at 37℃ very soon. When using N-octanoyl-homoserine lactone as substrate, we found a caprylic acid product increase thorough time goes by from the HPLC data. And we analyzed the homoserine lactone derived the OPT-thiol method under the same condition; HSL increased through time, too. From these results, we make propose that there should be an acyl-homoserine lactone（AHL）acylase in V. paradoxus Iso1 to degrade AHL.

Metal ion contamination in surface and ground water is a major threat as it has a direct implication on the health of terrestrial and aquatic flora and fauna. Lead (Pb2+), mercury (Hg2+), cadmium (Cd2+), nickel (Ni2+), copper (Cu2+) and cobalt (Co2+) are few of these metal ions which are classified under the high risk category. Of these, lead and mercury are of greater concern, as even nanomolar concentrations can be lethal, as they can be bio-accumulated and result in physiological as well as neurological disorders. In Asian countries like India and China, heavy metal pollution is more prevalent, as a consequence of poor governmental policies or ineffective or inadequate measures to combat this problem. In recent times, the monitoring and assessment of water pollution is a critical area of study, as it has a direct implication for its prevention and control. The major techniques used for metal ion detection are atomic absorption spectroscopy (AAS), X-ray fluorescence, ion chromatography, neutron activation, etc. Alternatively, the electrochemical, optical and electrical methods provide a platform for the fabrication of portable devices, which can facilitate the on-site analysis of samples in a rapid and cost-effective manner. This has led to a new field of research called chemical sensors or chemo sensory devices. The main aim of this study is to develop various chemosensory materials and test their response towards metal ion sensing. In this study, electroactive polymers have been synthesized for various sensor applications. The focus has been to design synthesize and test various functionalized electroactive polymers (FEAP) for the development of electrochemical, optical and chemoresistive sensors. Electroactive polymers like polyaniline, polypyrrole, polypyrrole grafted to exfoliated graphite oxide and dipyrromethene conjugated with p-(phenylene vinylene) have been synthesized and evaluated after functionalizing with metal coordinating ligands. These metal coordinating ligands were selected, in order to enhance their metal uptake capacity. Various metal ligands like imidazole, tertiary amine group, iminodiacetic acid, and dipyrromethene incorporated either in the polymer backbone or as a part of the backbone have been chosen for the metal binding. These functionalized electroactive polymers (FEAP) served as active material for metal ion sensing. The present investigation is subdivided into three sections. The first part includes design and chemical synthesis of the functionalized polymers by a series of organic reactions. The synthesis has been followed up by characterization using spectroscopic methods including NMR, FTIR, GCMS and Mass spectrometry. In the second part of the investigation, the synthesized polymer has been characterized for the changes in electronic, electric and optical properties after interaction with the selected metal ions. For this, the FEAP is allowed to interact with various metal ions and the changes in the relevant properties have been measured. This includes the study of changes in the conductivity, electronic properties like absorption or emission of the polymer, changes in the redox properties, etc. The third phase of investigation deals with the fabrication of the devices using the active FEAP. The sensor devices comprised of either films, or electrode modified with FEAP or solution of the FEAP, in combination with an appropriate technique has been used for the sensing. The major objectives are enumerated below 1. Functionalzation of polyaniline with imidazole functional group to get imidazole functionalized polyaniline (IMPANI) and study of the electronic, electrical and optical properties of the same. 2. Preparation of films of IMPANI and study of the change in conductivity of the film upon interaction with various metal ions, namely Cu2+, Co2+ and Ni2+ in their chloride form. 3. Synthesis of amine functionalized aniline monomer and chemical graft polymerization onto exfoliated graphite oxide as a substrate to synthesise the amine funtionalised polyaniline grafted to exfoliated graphite oxide (EGAMPANI). Modification of the carbon paste electrode (CPE) with EGAMPANI and study of the electrode characteristic. 4. Study of the electrode properties of EGAMPANI modified carbon paste electrode. 5. Evaluation of the EGAMPANI modified carbon paste electrode as a multi-elemental voltammetric sensor for Pb2+, Hg2+ and Cd2+ in aqueous system. 6. Functionalization of polypyrrole with iminodiacetic acid and characterization of the polymer to synthesis iminodiacetic acid functionalized polypyrrole (IDA-PPy). 7. Modification of the CPE with IDA-PPy by drop casting method and evaluation of the Pb2+ sensing properties. 8. Study of the effect of other metal ions say Hg2+, Co2+, Ni2+, Zn2+, Cu2+ and Cd2+ on the anodic stripping current of Pb2+ using EGAMPANI modified CPE. 9. Synthesis of dipyrromethene-p-(phenylene vinylene) conjugated polymer for heavy metal ion sensing. 10. Study of the changes in the optical absorption and emission properties of the polymer in THF and evaluation of the change in these optical properties upon interaction with the metal ions as analyte. The salient findings of the research work are highlighted as follows, In the first synthesis, aniline has been functionalized with imidazole group and this monomer has been chemical oxidatively polymerized to obtain imidazole functionalized polyaniline (IMPANI). The synthesized polymer possesses a nano-spherical structure, as confirmed from the morphological characterisation using scanning electron microscopy. The IMPANI has been interacted with a representative metal ion, copper (II) chloride, and the copper complexed polymer (Cu-IMPANI) has been subjected to various studies. The coordination of copper with IMPANI results in an increase of molecular weight of the polymer as a result of aggregation, as observed from dynamic light scattering measurements. Apart from this, a significant finding is the decrease of the pH of the system after copper ion coordination attesting to the generation of a secondary hydrochloride ion during the coordination of the copper to the imidazole side chain. This is further confirmed by an increase in conductivity of the Cu-IMPANI compared to IMPANI, measured using the four-probe technique. The increase of conductivity due to copper coordination is one order of magnitude higher. The films which have been prepared from IMPANI and Cu-IMPANI exhibit different morphology. The Cu-IMPANI film prepared by prior co-ordination of Cu ion with IMPANI powder shows a flaky structure, which is not preferable for the conductivity measurements, as a consequence of discontinuity in the medium. To overcome this problem, IMPANI films were initially prepared and then interacted with copper ions for a desired duration, before measurement of the conductivity. This latter procedure enabled the preparation of smooth films for the development of chemoresistive sensors. In continuation of the initial study highlighted above, IMPANI films of thickness 0.02 ± 0.001 mm have been prepared using IMPANI and PANI in DMPU in the ratio of 7:3 by mass. After exposure of the films with respective metal chlorides, such as Ni2+, Co2+ and Cu2+, a change in conductivity is observed in the concentration range of 10-2 to 1 M of metal chlorides. The sensor response may be arranged in the sequence: Ni2+ > Cu2+ > Co2+ at 1M concentration. On the contrary, films prepared from PANI-EB under identical conditions do not exhibit any appreciable change in conductivity. The optimum exposure time is determined to be 10 min for a maximum change in conductivity, after exposure to the chosen metal ions. In the second system taken up for investigation, a tertiary amine containing polyaniline (AMPANI) has been grafted to exfoliated graphite oxide. The amine containing polyaniline grafted to exfoliated graphite oxide (EGAMPANI) has been characterised for structural, morphological and elemental composition. The grafting percentage has been determined to be 7 % by weight of AMPANI on the EGO surface. The synthesized EGAMPANI (5 weight %) has been used to modify carbon paste electrode (CPE) for electrochemical sensor studies. Based on the differential pulse anodic stripping voltammetric studies, the electrochemical response may be arranged in the following sequence: Pb 2+>Cd 2+>Hg 2+ The minimum detection levels obtained are 5×10-6, 5×10-7, and 1.0×10-7 M for Hg2+, Cd2+ and Pb2+ ions respectively. In the next study, an iminodiacetic acid functionalized polypyrrole (IDA-PPy) has been synthesized and characterised for its elemental and structural properties. This has been further used to modify the CPE by drop casting method and used for the specific detection of Pb2+ in acetate buffer. Various parameters governing the electrode performance such as concentration of depositing solution, pH of depositing solution, deposition potential, deposition time, and scan rate, have been optimized to achieve maximum performance and found to be 20 μl, 4.5, -1.3 V, 11 min, 8 mV s-1 respectively for the chosen parameters. Additionally, the influence of other heavy metal ions on the lead response has been studied and it is observed that Co, Cu and Cd ions are found to be interfering. Further, the response of Cd, Co, Cu, Hg, Ni and Zn on IDA-PPy functionalized electrode has been evaluated. The selectivity of IDA-PPy modified electrode for Pb2+ is observed in the concentration range of 1 × 10-7 M and below. The IDA-PPy modified CPE shows a linear correlation for Pb2+ concentration in the range from 1×10-6 to 5×10-9 M and with a lowest limit of detection (LLOD) of 9.6×10-9 M concentration. The efficacy of the electrode for lead sensing has also been evaluated with an industrial effluent sample obtained from a lead battery manufacturing unit. The fourth synthesis pertained to the development of an optical sensor for Fe2+, and Co2+ ions. For this, dipyrromethene as a metal coordinating ligand in conjugation with p-phenylenevinylene has been synthesized and tested for its structural as well as optical properties. It is observed that the polymer shows three absorptions, namely at 294 nm, 357 nm and a major absorption observed as a broad band ranging from 484 to 670 nm. The emission spectrum of the polymer excited at 357 nm shows a characteristic blue emission with a maximum intensity centered at 425 nm. The emission quenching in the presence of various metal ions have been tested and are found to be quenched in presence of Fe2+ and Co2+ ions. All the other metal ions tested namely, Cr3+, Cu2+, and Zn2+ are not found to exhibit any change in the emission spectra below the concentration of 1 × 10-4 M. The linear correlation of the emission intensity with the concentration of the Co2+ and Fe2+ ions has been determined using Stern-Volmer plot. For Co2+ the Stern-Volmer regime is observed from 1×10-4 to 9×10-4 M concentration and the quenching constant Ksv is determined to be 8.67 ×103 M-1. For Fe2+, the linearity is found to be in the regime of 1×10-5 to 9×10-5 M and the quenching constant Ksv is determined to be 7.90 × 103 M-1. In conclusion, different electroactive polymers functionalized with metal coordinating ligands have been synthesized, characterised and evaluated for metal sensing applications. Techniques like electrochemical, optical and conductivity have been used to characterise the response of these FEAP towards metal sensing. It is can be concluded that the electrochemical sensors are more reliable for sensing especially at very low concentrations of metal ions such as Pb, Cd and other techniques like optical and conductimetric are good for detecting metal ions namely Fe, Co, Ni, Cu. The selectivity towards the metal ions is a function of the metal chelating ligand and the extent of sensitivity is dependent upon the technique employed.

Metal ion contamination in surface and ground water is a major threat as it has a direct implication on the health of terrestrial and aquatic flora and fauna. Lead (Pb2+), mercury (Hg2+), cadmium (Cd2+), nickel (Ni2+), copper (Cu2+) and cobalt (Co2+) are few of these metal ions which are classified under the high risk category. Of these, lead and mercury are of greater concern, as even nanomolar concentrations can be lethal, as they can be bio-accumulated and result in physiological as well as neurological disorders. In Asian countries like India and China, heavy metal pollution is more prevalent, as a consequence of poor governmental policies or ineffective or inadequate measures to combat this problem. In recent times, the monitoring and assessment of water pollution is a critical area of study, as it has a direct implication for its prevention and control. The major techniques used for metal ion detection are atomic absorption spectroscopy (AAS), X-ray fluorescence, ion chromatography, neutron activation, etc. Alternatively, the electrochemical, optical and electrical methods provide a platform for the fabrication of portable devices, which can facilitate the on-site analysis of samples in a rapid and cost-effective manner. This has led to a new field of research called chemical sensors or chemo sensory devices. The main aim of this study is to develop various chemosensory materials and test their response towards metal ion sensing. In this study, electroactive polymers have been synthesized for various sensor applications. The focus has been to design synthesize and test various functionalized electroactive polymers (FEAP) for the development of electrochemical, optical and chemoresistive sensors. Electroactive polymers like polyaniline, polypyrrole, polypyrrole grafted to exfoliated graphite oxide and dipyrromethene conjugated with p-(phenylene vinylene) have been synthesized and evaluated after functionalizing with metal coordinating ligands. These metal coordinating ligands were selected, in order to enhance their metal uptake capacity. Various metal ligands like imidazole, tertiary amine group, iminodiacetic acid, and dipyrromethene incorporated either in the polymer backbone or as a part of the backbone have been chosen for the metal binding. These functionalized electroactive polymers (FEAP) served as active material for metal ion sensing. The present investigation is subdivided into three sections. The first part includes design and chemical synthesis of the functionalized polymers by a series of organic reactions. The synthesis has been followed up by characterization using spectroscopic methods including NMR, FTIR, GCMS and Mass spectrometry. In the second part of the investigation, the synthesized polymer has been characterized for the changes in electronic, electric and optical properties after interaction with the selected metal ions. For this, the FEAP is allowed to interact with various metal ions and the changes in the relevant properties have been measured. This includes the study of changes in the conductivity, electronic properties like absorption or emission of the polymer, changes in the redox properties, etc. The third phase of investigation deals with the fabrication of the devices using the active FEAP. The sensor devices comprised of either films, or electrode modified with FEAP or solution of the FEAP, in combination with an appropriate technique has been used for the sensing. The major objectives are enumerated below 1. Functionalzation of polyaniline with imidazole functional group to get imidazole functionalized polyaniline (IMPANI) and study of the electronic, electrical and optical properties of the same. 2. Preparation of films of IMPANI and study of the change in conductivity of the film upon interaction with various metal ions, namely Cu2+, Co2+ and Ni2+ in their chloride form. 3. Synthesis of amine functionalized aniline monomer and chemical graft polymerization onto exfoliated graphite oxide as a substrate to synthesise the amine funtionalised polyaniline grafted to exfoliated graphite oxide (EGAMPANI). Modification of the carbon paste electrode (CPE) with EGAMPANI and study of the electrode characteristic. 4. Study of the electrode properties of EGAMPANI modified carbon paste electrode. 5. Evaluation of the EGAMPANI modified carbon paste electrode as a multi-elemental voltammetric sensor for Pb2+, Hg2+ and Cd2+ in aqueous system. 6. Functionalization of polypyrrole with iminodiacetic acid and characterization of the polymer to synthesis iminodiacetic acid functionalized polypyrrole (IDA-PPy). 7. Modification of the CPE with IDA-PPy by drop casting method and evaluation of the Pb2+ sensing properties. 8. Study of the effect of other metal ions say Hg2+, Co2+, Ni2+, Zn2+, Cu2+ and Cd2+ on the anodic stripping current of Pb2+ using EGAMPANI modified CPE. 9. Synthesis of dipyrromethene-p-(phenylene vinylene) conjugated polymer for heavy metal ion sensing. 10. Study of the changes in the optical absorption and emission properties of the polymer in THF and evaluation of the change in these optical properties upon interaction with the metal ions as analyte. The salient findings of the research work are highlighted as follows, In the first synthesis, aniline has been functionalized with imidazole group and this monomer has been chemical oxidatively polymerized to obtain imidazole functionalized polyaniline (IMPANI). The synthesized polymer possesses a nano-spherical structure, as confirmed from the morphological characterisation using scanning electron microscopy. The IMPANI has been interacted with a representative metal ion, copper (II) chloride, and the copper complexed polymer (Cu-IMPANI) has been subjected to various studies. The coordination of copper with IMPANI results in an increase of molecular weight of the polymer as a result of aggregation, as observed from dynamic light scattering measurements. Apart from this, a significant finding is the decrease of the pH of the system after copper ion coordination attesting to the generation of a secondary hydrochloride ion during the coordination of the copper to the imidazole side chain. This is further confirmed by an increase in conductivity of the Cu-IMPANI compared to IMPANI, measured using the four-probe technique. The increase of conductivity due to copper coordination is one order of magnitude higher. The films which have been prepared from IMPANI and Cu-IMPANI exhibit different morphology. The Cu-IMPANI film prepared by prior co-ordination of Cu ion with IMPANI powder shows a flaky structure, which is not preferable for the conductivity measurements, as a consequence of discontinuity in the medium. To overcome this problem, IMPANI films were initially prepared and then interacted with copper ions for a desired duration, before measurement of the conductivity. This latter procedure enabled the preparation of smooth films for the development of chemoresistive sensors. In continuation of the initial study highlighted above, IMPANI films of thickness 0.02 ± 0.001 mm have been prepared using IMPANI and PANI in DMPU in the ratio of 7:3 by mass. After exposure of the films with respective metal chlorides, such as Ni2+, Co2+ and Cu2+, a change in conductivity is observed in the concentration range of 10-2 to 1 M of metal chlorides. The sensor response may be arranged in the sequence: Ni2+ > Cu2+ > Co2+ at 1M concentration. On the contrary, films prepared from PANI-EB under identical conditions do not exhibit any appreciable change in conductivity. The optimum exposure time is determined to be 10 min for a maximum change in conductivity, after exposure to the chosen metal ions. In the second system taken up for investigation, a tertiary amine containing polyaniline (AMPANI) has been grafted to exfoliated graphite oxide. The amine containing polyaniline grafted to exfoliated graphite oxide (EGAMPANI) has been characterised for structural, morphological and elemental composition. The grafting percentage has been determined to be 7 % by weight of AMPANI on the EGO surface. The synthesized EGAMPANI (5 weight %) has been used to modify carbon paste electrode (CPE) for electrochemical sensor studies. Based on the differential pulse anodic stripping voltammetric studies, the electrochemical response may be arranged in the following sequence: Pb 2+>Cd 2+>Hg 2+ The minimum detection levels obtained are 5×10-6, 5×10-7, and 1.0×10-7 M for Hg2+, Cd2+ and Pb2+ ions respectively. In the next study, an iminodiacetic acid functionalized polypyrrole (IDA-PPy) has been synthesized and characterised for its elemental and structural properties. This has been further used to modify the CPE by drop casting method and used for the specific detection of Pb2+ in acetate buffer. Various parameters governing the electrode performance such as concentration of depositing solution, pH of depositing solution, deposition potential, deposition time, and scan rate, have been optimized to achieve maximum performance and found to be 20 μl, 4.5, -1.3 V, 11 min, 8 mV s-1 respectively for the chosen parameters. Additionally, the influence of other heavy metal ions on the lead response has been studied and it is observed that Co, Cu and Cd ions are found to be interfering. Further, the response of Cd, Co, Cu, Hg, Ni and Zn on IDA-PPy functionalized electrode has been evaluated. The selectivity of IDA-PPy modified electrode for Pb2+ is observed in the concentration range of 1 × 10-7 M and below. The IDA-PPy modified CPE shows a linear correlation for Pb2+ concentration in the range from 1×10-6 to 5×10-9 M and with a lowest limit of detection (LLOD) of 9.6×10-9 M concentration. The efficacy of the electrode for lead sensing has also been evaluated with an industrial effluent sample obtained from a lead battery manufacturing unit. The fourth synthesis pertained to the development of an optical sensor for Fe2+, and Co2+ ions. For this, dipyrromethene as a metal coordinating ligand in conjugation with p-phenylenevinylene has been synthesized and tested for its structural as well as optical properties. It is observed that the polymer shows three absorptions, namely at 294 nm, 357 nm and a major absorption observed as a broad band ranging from 484 to 670 nm. The emission spectrum of the polymer excited at 357 nm shows a characteristic blue emission with a maximum intensity centered at 425 nm. The emission quenching in the presence of various metal ions have been tested and are found to be quenched in presence of Fe2+ and Co2+ ions. All the other metal ions tested namely, Cr3+, Cu2+, and Zn2+ are not found to exhibit any change in the emission spectra below the concentration of 1 × 10-4 M. The linear correlation of the emission intensity with the concentration of the Co2+ and Fe2+ ions has been determined using Stern-Volmer plot. For Co2+ the Stern-Volmer regime is observed from 1×10-4 to 9×10-4 M concentration and the quenching constant Ksv is determined to be 8.67 ×103 M-1. For Fe2+, the linearity is found to be in the regime of 1×10-5 to 9×10-5 M and the quenching constant Ksv is determined to be 7.90 × 103 M-1. In conclusion, different electroactive polymers functionalized with metal coordinating ligands have been synthesized, characterised and evaluated for metal sensing applications. Techniques like electrochemical, optical and conductivity have been used to characterise the response of these FEAP towards metal sensing. It is can be concluded that the electrochemical sensors are more reliable for sensing especially at very low concentrations of metal ions such as Pb, Cd and other techniques like optical and conductimetric are good for detecting metal ions namely Fe, Co, Ni, Cu. The selectivity towards the metal ions is a function of the metal chelating ligand and the extent of sensitivity is dependent upon the technique employed.