Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 4 uppsatser.

The fascinating capability of cellular self-organization during tissue development and repair is a central question in developmental biology and regenerative medicine. Understanding the dynamic morphogenic and regenerative processes of biological tissues will have important implications in biology and medicine. Nevertheless, the elucidation of the cellular self-organization processes is hindered by a lack of effective tools for monitoring the spatiotemporal gene expression distribution and a lack of ability to perturb the self-organization processes in living cells and tissues. Multimodal modularities that allow both single cell perturbation and gene detection are required to enable a new paradigm in the investigation of complex tissue morphogenic processes. To address this critical challenge in the field of developmental and regenerative medicine, we are developing a multimodal gold nanorod-locked nucleic acid (GNR-LNA) composite for single cell gene expression analysis in living cells and tissues at the transcriptional level. Using antisense RNA sequences, we design LNA probes for detecting specific molecular targets in living cells. The LNA probes bind to the GNR spontaneously due to the intrinsic affinity between the GNR and LNA. In close proximity, the fluorescent probes are effectively quenched by the GNR. Therefore, a fluorescent signal is only observed when the specific target thermodynamically displaces the LNA probe from the GNR. Furthermore, the GNR also serves as a transducer for photothermal ablation. Thus, we established a novel modularity for imaging the spatiotemporal gene expression distribution in living cells and tissues. The single cell analysis capability of our techniques enables us to adopt a unique approach to study the tissue regenerative processes during normal development and diseases, and this will have a profound impact on regenerative medicine and disease treatment in future. Moreover, we applied this GNR-LNA probe to explore the endothelial cell mRNA dynamics during capillary morphogenesis. Three different types of cells were identified due to their different roles during endothelial cell capillary-like formation process. Our findings indicated that the endothelial cell behavior is directly related to the Dll4 mRNA expression, and Dll4 expression in ECs determine the cell fate. Our GNR-LNA probe enable us to investigate the correlations between Dll4 mRNA expression and cell behavior during capillary morphogenesis. Experimental results indicated that: (1) When the endothelial cells aggregate, the cells migrate with certain displacement, the Dll4 mRNA expression decreases. (2) When the endothelial cells sprout, the cells migrate with small displacement but the cell shape changes to an ellipse shape, the Dll4 mRNA expression begin to increase. (3) When the endothelial cells elongate and form cell-cell contract with adjacent cells, the Dll4 expression decreased to a certain level and keep stable until the cell activity change to another stage. Furthermore, it has been demonstrated endothelial cells compete for the leader cell position during wound healing, collective cell migration, and tip cell formation during angiogenic process. It has been demonstrated that endothelial cells compete for the tip cell formation through Notch signaling pathway. However, how the mechanical force regulates tip cell formation is still unclear, and if mechanoregulation of tip cell formation through Notch pathway still unknown. Mechanical and chemical regulations of tissue morphogenesis and angiogenesis are being investigated in both in vitro capillary-like network formation assay and in vivo mice retina angiogenesis assay. Here, we investigated the mechanoregulation of mechanotransduction of tissue morphogenesis and angiogenesis using both in vitro endothelial cell tube formation model and in vivo mice retina blood vessel development model. Our results demonstrated that (1) Notch pathway negatively regulates tip cell formation: inhibition of Notch pathway (DAPT) enhances tip cell formation, induces Dll4 and Notch1 activity, activation of Notch pathway (Jag1 peptide) inhibits tip cell formation, suppresses Dll4 and Notch1 activity. (2) Mechanical force negatively regulate tip cell formation: (a) Decrease mechanical force via Rho kinase inhibitor Y-27632, myosin II inhibitor Blebbistatin, or laser ablation, enhances tip cell formation and induces Dll4 activity through mediation of Dll4-Notch1 lateral inhibition, (b) increase mechanical force via traction force inducer Nocodazole and Calyculin A, suppresses tip cell formation and inhibits Dll4 activity through activation of Notch pathway. (3) Mechanical force negatively regulates tip cell formation partially via mediation of Notch pathway. Mechanical force is necessary for tip cell formation and negatively regulate tip/stalk selection via Dll4-Notch1 lateral inhibition. Interruption of mechanical force enhance tip cell formation via suppression of Dll4-Notch1 lateral inhibition, thus resulting the increase of Dll4 expression. Enhance of mechanical force inhibits tip cell formation via activation of Dll4-Notch1 lateral inhibition, thus resulting the decreases of Dll4 expression. All these finding wills have great significance for various biomedical applications, such as tissue engineering, cancer, and drug screening.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Infection with hepatitis A virus (HAV) occurs worldwide and is the most common cause of acute viral hepatitis. The highest prevalence of this infection is seen where low standards of sanitation are adopted. Acquired primarily by the fecaloral route, HAV infection is easily disseminated, either by person-to-person contact or by ingestion of contaminated food or water. The HAV is a extremely steady non-enveloped particle, which comprises a single stranded plus-sense RNA with approximately 7,5 kb. The objective of this study was to develop, based into VP1 gene sequence, a DNA probe from the HAV genome by RTPCR to detect the virus. A 783 bp amplicon of HAV genome (Brazilian standard strain HAF-203), amplified by RT-PCR was labelled by coupling of alkaline phosphatase enzyme from Gene ImagesTM AlkPhos DirectTM System (Amersham). Specificity was determined by dot blot hybridization of RNA from standard strain HAF-203 and DNA of the own amplicon. To asses the sensitivity of detection hybridization was done in serially diluted up to 1 pg of purified viral RNA. Sewage water samples were artificially ontaminated with dilutions up to 10 PFU/mL of the virus. The particles were precipitated with amonium sulfate and the total RNA obtained by treatment with proteinase k and phenol:chloroform. Samples were transferred to a nylon membrane by dot blot and hybridized according to labelling system manufacturer s instructions. Dots were detected in spots containing 1 pg of purified RNA, just like the amplicon. The DNA probe did not hybridize to total DNA prepared from BoHV-1, XL2B DNA and Human total RNA. The probe hybridized to samples containing up to 100 PFU/mL of the virus. This results showed the probe specificity and sensitivity level is sufficient to detect the virus in environmental samples, making this technique able to be used in environmental molecular monitoring of the virus. / Infecção pelo vírus da hepatite A (HAV) acontece mundialmente e é a causa mais comum de hepatites virais agudas. A prevalência mais alta desta infecção é observada onde baixos padrões de serviço de saúde pública são adotados. Adquirida principalmente pela rota fecal-oral, a infecção por HAV é disseminada facilmente, através de contato de pessoa-para-pessoa ou por ingestão de comida ou água contaminada. O HAV é uma partícula não envelopada, extremamente estável que possui RNA fita simples de sentido positivo com aproximadamente 7,5 kb. O objetivo deste estudo foi desenvolver, baseado na seqüência do gene VP1, uma sonda de DNA a partir do genoma de HAV por RT-PCR para detectar o vírus. Um amplicon de 783 bp do genoma de HAV (amostra padrão HAF-203), amplificado por RT-PCR foi marcado pela enzima fosfatase alcalina do sistema Gene ImagesTM AlkPhos DirectTM (Amersham). A especificidade foi determinada por hibridização em dot blot com RNA da amostra padrão brasileira HAF-203 e DNA do próprio amplicon. Para testar a sensibilidade de detecção por hibridização foi realizada diluição seriada até 1 pg de RNA viral purificado. Foram contaminadas artificialmente amostras de água de esgoto com diluições até 10 PFU/mL do vírus. As partículas foram precipitadas com sulfato de amônia e RNA total obtido através de tratamento com proteinase k e fenol:clorofórmio. Amostras foram transferidas para uma membrana de nylon por dot blot e hibridizadas de acordo com as instruções do fabricante do sistema. A sonda detectou amostras nos poços contendo até 1 pg de RNA purificado, da mesma forma que o amplicon. A sonda de DNA não hibridizou com DNA preparado de BoHV-1, XL2B DNA e RNA total Humano. A sonda hibridizou com amostras que contêm até 100 PFU/mL do vírus. Estes resultados mostram que a os níveis de especificidade e sensibilidade da sonda é suficiente para detectar o vírus em amostras ambientais, tornando esta técnica aplicável no monitorando molecular ambiental do vírus.

Orientadores: Olga Maria de Toledo Correa, Paulo Pinto Joazeiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T13:23:14Z (GMT). No. of bitstreams: 1 Garcia_EduardoAnselmo_M.pdf: 5637432 bytes, checksum: 7856ba76c45912e029c4e039ce280e21 (MD5) Previous issue date: 2005 / Resumo: O tecido interpúbico no camundongo é conhecido pelas rápidas adaptações sofridas devido as novas demandas impostas durante a prenhez, parto e pós-parto. O desenvolvimento de um ligamento interpúbico é explicado em parte pelas modificações observadas na hidratação deste tecido, assim como, pela presença de Ácido Hialurônico (AH). O presente estudo focou-se na presença do AH na sínfise púbica, uma vez que pouco se sabe a respeito do seu papel durante as modificações sofridas neste período. A localização e quantificação do AH, por meio de uma sonda específica para AH, foi feita em amostras de sínfises púbicas de camundongos virgens, com 12 a 18 dias de prenhez, no dia do parto (D19) e durante o 3° e 5° dia pós-parto. A análise quantitativa fluorométrica indicou um aumento gradual no AH do tecido interpúbico durante a prenhez, seguido de uma diminuição já presente no dia do parto. A análise histoquímica demonstrou a presença de AH na matriz extracelular deste tecido assim como dentro das células. Baseado nestes resultados, pudemos demonstrar a provável participação do AH em três processos. Quando encontrado na matriz extracelular, o AH contribui para a formação de uma estrutura flexível, de consistência rígida, que vai permitir a passagem do feto pelo canal de parto. Ao passo que, quando encontrado intracelularmente, o AH contribui em dois processos distintos: até o 18° dia de prenhez, o AH participa nos processos de proliferação celular, através de possíveis interações com moléculas e estruturas importantes neste processo. O AH talvez também contribua para a proliferação através da formação de uma zona hidratada ao redor das células facilitando o destacamento das mesmas de seu substrato. Finalmente, após o parto e até o 5° dia pós-parto o AH contribui para a manutenção do fenótipo miofibroblástico destas células ajudando no processo de involução sofrido pelo tecido interpúbico no pós-parto / Abstract: The interpubic tissue in mice is known to rapidly adapt itself to the ali new demands imposed during pregnancy, partum and post-partum. The development of an interpubic ligament may be explained, in part, due to the modifications observed in the hydration of this tissue, as well as, the presence of hyaluronan (HA). The present study focuses on the presence of HA in the pubic symphysis, since still very little is known of its role during the modifications suffered through this period. The localization and quantification of HA using a HA-probe were studied in samples of mice pubic symphyses from virgin, 12th to 18th days of pregnancy, the day of delivery (D19) and during 3 and 5 days post-partum. The quantitative fluorimetric analysis indicated a gradual increase of HA in the interpubic tissue throughout pregnancy, being followed by a decrease already present on the day of the delivery. In addition, the histochemical analysis demonstrated the presence of HA in the extracellular matrix of the tissue as well as within its cells. Based on these results, it can be shown that HA may participate in three processes during pregnancy, partum and post-partum. While found in the extracellular matrix, HA contributes in the formation of a flexible structure, yet of rigid consistency, that allows the passage of the embryo for the birth canal. Whereas, while found intracellular, HA contributes in two distinct processes: until the 18th day of pregnancy, HA participates in the process of cellular proliferation through its possible interaction with mitotic cells. HA may also contribute to cellular proliferation through the formation of a hydrated zone from parturition as well as the 5th day post-partum HA contributes to the maintenance of the myofibroblastic phenotype of these cells, aiding in the process of involution suffered by the interpubic tissue following parturition / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

Autosomal dominant epidermolytic ichthyosis (EI) is a rare disease characterized by intra-epidermal blistering due to mutations in either of two keratin genes, KRT1 and KRT10, expressed by suprabasal keratinocytes. Autosomal recessive congenital ichthyosis (ARCI) is a non-blistering, hyperkeratotic disease caused by mutations in one of the following genes: ABCA12, ALOX12B, ALOXE3, TGM1, CYP4F22, NIPAL4 and SLC27A4, which are all essential for skin barrier homeostasis. ARCI and EI often respond well to treatment with retinoids, but the mechanism of action is unclear. The aim of this thesis was to increase the knowledge of pathogenic pathways in ichthyosis and to find new explanations to the effect of retinoids. In vitro studies of immortalized keratinocytes from EI patients showed an abnormal keratin aggregation after heat stress, that could be partially inhibited by pre-treatment with all-trans retinoic acid (ATRA) or retinoic acid receptor α-agonists. ATRA treatment also reduced the relative expression of mutated vs wildtype KRT10. The clearance of ATRA in human keratinocytes was found to be mediated by CYP26B1. In skin biopsies from ARCI patients, immunofluorescence analysis of 12R-LOX, eLOX-3, TGM1, ichthyin and FATP4 showed altered expression, not only of the mutated protein, but also of the other proteins. These observations are consistent with a feedback regulatory mechanism by which the loss of one protein results in an up-regulation of other proteins. Furthermore, 12R-LOX, eLOX-3 and TGM1 were intimately co-localized in stratum corneum, as were ichthyin and FATP4, suggesting that the proteins are linked to the same metabolic pathway. When treated with a CYP26 inhibitor known to raise the endogenous ATRA level of the skin, two patients with NIPAL4 mutations, initially exhibiting increased co-localization signals for 12R-LOX and eLOX-3, displayed normalized lipoxygenase expressions and showed clinical improvement. In conclusion, mechanisms are proposed by which pathogenic keratin aggregations in EI and epidermal protein deficiencies in ARCI patients may be mitigated by retinoids. Furthermore, the vivid crosstalk between proteins incriminated in ARCI suggests that these enzymes operate along a common metabolic pathway essential for producing barrier lipids in stratum corneum. Any abrogation of this production may cause barrier failure, hence resulting in a compensatory hyperkeratosis characteristic of congenital ichthyosis.

As malformações cardíacas congênitas (MCCs) são as malformações mais comuns ao nascimento, representando uma importante causa de morbidade e mortalidade em recém-nascidos. Nos últimos anos, estudos utilizando testes citogenômicos têm permitido elucidar e compreender melhor as causas das MCCs. O objetivo geral desse estudo foi investigar a presença de CNVs em amostras de tecido obtidas post-mortem de portadores de malformações cardíacas congênitas sindrômicas; e os objetivos específicos consistiram em avaliar a frequência das CNVs, destacando as mais relevantes, comparar a presença de CNVs nos diferentes tecidos e realizar a correlação genótipo-fenótipo. Para isso, foram estudados um total de 52 casos de natimortos e recém-nascidos provenientes do Serviço de Verificação de Óbitos - FMUSP. Amostras de DNA extraídas da pele, diafragma e do coração foram avaliadas utilizando o kit AmpFlSTR® MiniFiler(TM) PCR Amplification (Life Technologies(TM), USA) e a técnica de Multiplex Ligation-dependent Probe Amplification (MLPA) com diferentes kits (MCR-Holland, Holanda). A técnica de FISH foi utilizada para a confirmação dos resultados obtidos em um dos casos estudados. Foram encontradas CNVs relevantes em 21 casos, incluindo trissomia do 18 (10 casos), trissomia do 21 (4 casos), trissomia do 13 (2 casos), trissomia do 16 (1 caso), monossomia do X em mosaico (1 caso), dup 4p16 (1 caso), dup 11q25 (1 caso) e del GATA4 éxon 6 (1 caso). A análise genômica se mostrou eficiente na investigação das bases genômicas e na caracterização das diferentes malformações em amostras post-mortem de portadores de MCC sindrômicas / Congenital heart defects (CHDs) are the most common birth defect and represent an important cause of morbidity and mortality in newborns. In recent years, studies using cytogenomic tests have enabled an improved understanding of the causes of CHD. The general objective of this study was to investigate the presence of CNVs in post-mortem tissue samples from patients with congenital syndromic cardiac malformations; and the specific objectives were to evaluate the frequency of CNVs, highlighting the most relevant ones, to compare the presence of CNVs in the different tissues and to perform the genotype-phenotype correlation. For this, a total of 52 stillbirth and newborn cases from the Death Verification Service (SVO), FMUSP were investigated. DNA samples from skin, diaphragm and heart tissues were evaluated using an AmpFlSTR® MiniFiler(TM) PCR Amplification Kit (Life Technologies(TM), California, USA) and Multiplex Ligation-dependent Probe Amplification (MLPA) with different kits (MCRHolland, Amsterdam, The Netherlands). FISH was used to confirm the results of one of the studied cases. The results showed relevant copy number variations (CNVs) in 21 cases, including trisomy 18 (10 cases), trisomy 21 (4 cases), trisomy 13 (2 cases), trisomy 16 (1 case), mosaic monosomy X (1 case), dup 4p16 (1 case), dup 11q25 (1 case) and del GATA4 exon 6 (1 case). Genomic analysis was found to efficiently identify the genomic basis of, and characterize, various malformations found in postmortem samples from syndromic CHD carriers

As malformações cardíacas congênitas (MCCs) são as malformações mais comuns ao nascimento, representando uma importante causa de morbidade e mortalidade em recém-nascidos. Nos últimos anos, estudos utilizando testes citogenômicos têm permitido elucidar e compreender melhor as causas das MCCs. O objetivo geral desse estudo foi investigar a presença de CNVs em amostras de tecido obtidas post-mortem de portadores de malformações cardíacas congênitas sindrômicas; e os objetivos específicos consistiram em avaliar a frequência das CNVs, destacando as mais relevantes, comparar a presença de CNVs nos diferentes tecidos e realizar a correlação genótipo-fenótipo. Para isso, foram estudados um total de 52 casos de natimortos e recém-nascidos provenientes do Serviço de Verificação de Óbitos - FMUSP. Amostras de DNA extraídas da pele, diafragma e do coração foram avaliadas utilizando o kit AmpFlSTR® MiniFiler(TM) PCR Amplification (Life Technologies(TM), USA) e a técnica de Multiplex Ligation-dependent Probe Amplification (MLPA) com diferentes kits (MCR-Holland, Holanda). A técnica de FISH foi utilizada para a confirmação dos resultados obtidos em um dos casos estudados. Foram encontradas CNVs relevantes em 21 casos, incluindo trissomia do 18 (10 casos), trissomia do 21 (4 casos), trissomia do 13 (2 casos), trissomia do 16 (1 caso), monossomia do X em mosaico (1 caso), dup 4p16 (1 caso), dup 11q25 (1 caso) e del GATA4 éxon 6 (1 caso). A análise genômica se mostrou eficiente na investigação das bases genômicas e na caracterização das diferentes malformações em amostras post-mortem de portadores de MCC sindrômicas / Congenital heart defects (CHDs) are the most common birth defect and represent an important cause of morbidity and mortality in newborns. In recent years, studies using cytogenomic tests have enabled an improved understanding of the causes of CHD. The general objective of this study was to investigate the presence of CNVs in post-mortem tissue samples from patients with congenital syndromic cardiac malformations; and the specific objectives were to evaluate the frequency of CNVs, highlighting the most relevant ones, to compare the presence of CNVs in the different tissues and to perform the genotype-phenotype correlation. For this, a total of 52 stillbirth and newborn cases from the Death Verification Service (SVO), FMUSP were investigated. DNA samples from skin, diaphragm and heart tissues were evaluated using an AmpFlSTR® MiniFiler(TM) PCR Amplification Kit (Life Technologies(TM), California, USA) and Multiplex Ligation-dependent Probe Amplification (MLPA) with different kits (MCRHolland, Amsterdam, The Netherlands). FISH was used to confirm the results of one of the studied cases. The results showed relevant copy number variations (CNVs) in 21 cases, including trisomy 18 (10 cases), trisomy 21 (4 cases), trisomy 13 (2 cases), trisomy 16 (1 case), mosaic monosomy X (1 case), dup 4p16 (1 case), dup 11q25 (1 case) and del GATA4 exon 6 (1 case). Genomic analysis was found to efficiently identify the genomic basis of, and characterize, various malformations found in postmortem samples from syndromic CHD carriers

The thesis entitled “New Supramolecular Ion Sensing Probes and their Application in the Detection of Environmentally Relevant Ions” deals with the design and synthesis of several small molecular probes which can specifically sense environmentally relevant ions of (anion or cation) particularly in aqueous or biological medium. The probes have been designed using four different molecular entities which include anthraquinone, oxidized bis-indolyl system, pyrene and rhodamine. The probes afford naked eye detection of a particular ion in the aqueous medium. This work has been divided into six chapters. Chapter 1. Introduction The first chapter gives a brief idea of ion sensor. It provides the description of various approaches used for designing molecular sensors. The chapter further presents an overview of the four different dyes (anthraquinone, oxidized-bis-indole, pyrene and rhodamine) used for designing probes in this work. The properties of these probes, their advantages and disadvantages to use as a signaling subunit have been discussed. This chapter also describes the use of micellar medium for solubilizing different organic dyes in water. Chapter 2. Colorimetric Probes based on Anthraimidazolediones for Selective Sensing of Fluoride and Cyanide ion via Intramolecular Charge Transfer. The second chapter describes the design and synthesis of four different probes based on anthra [1, 2-d] imidazole-6, 11-dione. The anthraquinone part of each molecule has an acceptor moiety whereas substituted nitrogen linked aromatic unit forms the donor site. Each probe acted as strong colorimetric sensor for fluoride and cyanide ion detection and exhibited intramolecular charge transfer (ICT) band which showed significant red-shifts after addition of either the F¯ or CN¯ ion. One of the probes 2 showed selective colorimetric sensing for both cyanide and fluoride ions. In organic medium 2 showed selective color change with fluoride and cyanide, whereas in aqueous organic medium it showed a selective ratiometric response towards cyanide ion. The effect of anionic charge (on the donor moiety) on ICT has been discussed. Among the various donor moieties, the donor site having negative charges on them was found to disperse greater electron density on them. Figure 1. Molecular structures of the sensors Chapter 3 deals with chemodosimetric detection of cyanide ion in water using various oxidized bis-indole based compounds. Chapter 3A. A Chemodosimetric Probe based on a Conjugated and oxidized Bis¬ indolyl System for Selective Naked Eye Sensing of Cyanide ion in Water. The chapter 3A describes the design and synthesis of a new water-soluble bis-indolyl based probe, 5 which possesses two –COOH groups. This probe specifically reacted with the CN¯ ion in pure water at ambient temperature and produced a remarkable change in color from red to colorless. The mechanism of this process was investigated by NMR (1H, 13C and DEPT-135) spectroscopy, mass spectrometry and kinetic studies. The mechanism investigation showed that the cyanide ion reacts with the probe and removes the conjugation of the bis-indolyl moiety of the probe with that of the 4-substituted aromatic ring which renders the probe colorless. Taken together a plausible mechanism of the reaction was presented which showed to operate via a Michael type adduct formation under ambient conditions of pH and temperature in water. The probe gave a detection limit of 0.38 ppm for detection of cyanide ion in water. Figure 2. Molecular structure of the probe 5. Chapter 3B. Micelle Assisted ppb level Detection of Cyanide ion in Water by Chemodosimetry and Visual detection of the Endogenous Cyanide. The chapter 3B deals with the synthesis of a bis-indole based colorimetric probe 6. The probe showed selective detection of the cyanide ion in water at ppb level and a visible detection of endogenous cyanide from cassava (a major staple food in the developing world) by chemodosimetry. The cyanide ion binds with the probe 6 in a chemodosimetric fashion and follows pseudo first-order kinetics in water under appropriate conditions. It showed a highly sensitive detection of the cyanide ion in water with a detection limit of 0.33 ppm. The use of the micellar medium improved the detection limit drastically and a ppb level detection limit was achieved. The probe also showed the detection of the endogenously bound cyanide in cassava both visually and by spectrophotometer. Figure 3. Molecular structure of the probe 6. Chapter 3C. Ratiometric Cyanide ion probe in Water and for the detection of the Endogenously bound cyanide. Chapter 3C presents the synthesis of two new bis-indolyl (7 and 8) based probes for colorimetric detection of cyanide ion in pure water. Compound 8 showed a ratiometric response with cyanide in water and a visual detection of the endogenously bound cyanide ion in cassava. Using compound 8 the selective detection of the cyanide ion in water was achieved with a detection limit of ~ 17 ppb which is almost 13 times lower than the permitted limit as specified by EPA, United States. 7; R = H 8; R = -(OCH2CH2)3CH3 Figure 4. Molecular structures of the probes 1 and 2. Chapter 4 deals with the colorimetric and ratiometric detection of the Cu2+and Hg2+ions using different small synthetic molecular probes. Chapter 4A. Colorimetric Sensors for Ratiometric Detection of Copper and Mercury ions in Biological media and below ppm level in Water. The chapter 4A deals with the synthesis of two novel colorimetric probes (9, 10) using bispicolyl unit as the binding moiety and anthraimidazolediones and bis-indolyl system as a signaling sub-unit. Using the two sensors, Cu2+ion can be detected below the permitted limit (1.3 ppm) in both drinking water and at physiological pH 7.4. Sensor 9 can detect both Cu2+and Hg2+ in water with very low detection limit. It showed specific binding with Cu2+ at physiological pH 7.4 and in presence of serum albumins. Chemosensor 10 can be used for the specific detection of both Cu2+and Hg2in water as well as for the contamination in microorganisms. Figure 5. Molecular structure of the sensors 9 and 10. Chapter 4B. A New Molecular Probe for the Selective Sensing of Cu2+ and Hg2+ ions in Micellar Media and in Live ells.This chapter describes a synthesis of a novel bispicolyl based sensor 11 which can detect Cu2+ ion specifically in water medium and both Cu2+ and Hg2+ ions selectivelyin Brij-58 micellar medium. In micellar medium both the ions can be detected in the ppb level. Using fluorescence spectroscopy these two metal ions can be discriminated.The probe is also be useful for checking metal ion contamination in cellular samples. Figure 6. Molecular structure of the sensor 11. Chapter 4C. Rhodamine based Sensors for Cu2+ and Hg2+ ions in Water and in Biological media. The chapter 4C presents the synthesis and the sensing properties of the three positional isomers of the pyridine end of the rhodamine-pyridine compounds (12-14). The three isomers only differ in the position of nitrogen of the pyridine moiety. Sensor 12, which contains the pyridine nitrogen at the ortho-position showed selective sensing toward Cu2+ ion in both pure water and in buffered physiological media of pH 7.4. It gave a detection limit of ~13 ppb which is 100 times lesser than the EPA permitted limit. The other two sensors 13 and 14, which possessed the pyridine ends with the nitrogen atom at the meta- and the para- positions respectively showed the selective sensing of Hg2+ ion in water and did not show any interaction with the Cu2+ ion. Probes 2 and 3 showed ‘turn-on’ detection of Hg2+ ion both in the UV-vis and the fluorescence emission spectroscopy. Compound 2 and 3 showed a detection limit of ~ 9 and 4 ppb respectively. The NMR titration showed the change in color was due to the opening of the spirolactam ring of the rhodamine. The sensors can also be used for the detection of Cu2+ and Hg2+ ion in real life water samples and in the live cells. Figure 7. Molecular structure of the sensors 12, 13 and 14. Chapter 5. Ratiometric and ppb level Detection of Toxic Transition Metal ions using a Single Probe in Micellar media. This chapter describes the selective sensing of multiple ions using a single probe 15. The probe incorporates pyrene and pyridine as signaling and interacting moiety respectively. The sensor showed different responses towards different metal ions just by varying the medium of detection. In organic solvent (acetonitrile), the probe showed selective detection of Hg2+ ion. In water the fluorescence quenching was observed with three metal ions, Cu2+, Hg2+ and Ni2+. Further just by varying the surface charge of different micellar media, the probe showed selective interaction with Hg2+ ion in neutral micelles (Brij-58). However, in anionic micellar medium (SDS), the probe showed selective changes with both Cu2+ and Ni2+ in the UV-vis spectroscopy. The discrimination between these two ions was achieved by emission spectroscopy, where it showed selective quenching only with Cu2+. Thus using a single probe all the three metal ions Cu2+, Hg2+ and Ni2+ can be detected and discriminated just by varying the surface charge of the micellar medium. Figure 8. Molecular structure of the sensors 15. Chapter 6. Highly sensitive Rhodamine Based Dual Probes for the Visual detection of F¯ and Hg2+ ions in Water. This chapter deals with the design and synthesis of two new rhodamine based probes (16-17) which act as dual probes for the ppb level selective detection of Hg2+ and F¯ ions in water and at physiological pH 7.4. The two probes were synthesized by coupling tert-butyldiphenylsilyl (TBDPS) protected forms of 4-hydroxybenzaldehyde and 2, 4- dihydroxy benzaldehyde with rhodamine hydrazone. The F¯ ion detection is based on the desilylation of the probe, whereas the spirolactam ring opening leads to the detection of Hg2+ ion. The two probes gave turn-on detection of both Hg2+ and F¯ ion selectively in aqueous medium with the detection limit well below the EPA permitted limits. The probes showed detection of both the ions by dual mode with visibly different color and fluorescence under UV-lamp. The F¯ ion interacts with the silyl bond of probe and the cleavage results into yellow color whereas; the addition of Hg2+ ion to the probe solution opened the spirolactam ring and resulted into appearance of pink color. Figure 9. Molecular structure of the probes 16 and 17. (For structural formula pl see the abstract file)