Este estudo foi elaborado com o objetivo de elucidar os mecanismos de resistência às quinolonas presentes em enterobactérias isoladas de pacientes de duas regiões metropolitanas brasileiras. Foram avaliadas possíveis alterações nas proteínas relacionadas com o sítio de ação das quinolonas, bem como a presença de plasmídeos e integrons que carregam determinantes gênicos que codificam resistência às quinolonas. A associação destes com mecanismos plasmideais de resistência aos antibióticos -lactâmicos também foi analisada. Foram avaliadas 257 enterobactérias resistentes ao ácido nalidíxico isoladas de pacientes hospitalizados e da comunidade no período de 2000 a 2005. Por PCR e seqüenciamento, foram analisadas as mutações presentes nos genes cromossômicos gyrA e parC e as estruturas gênicas associadas com determinantes plasmideais de resistência às quinolonas, Qnr, e aos -lactâmicos de amplo espectro. Foram determinados os perfis plasmideais e a transferabilidade dos plasmídeos que carrearam estes determinantes. A extração e a análise das proteínas de membrana externa foi realizada para avaliar uma possível perda ou diminuição da expressão de porinas, também relacionada com os mecanismos de resistência avaliados. Todas as amostras foram resistentes ao ácido nalidíxico, apresentando concentração inibitória mínima (CIM) superior a 16 g/mL, e em média, 70% apresentaram diminuição de sensibilidade às fluoroquinolonas testadas. De 257 enterobactérias resistentes ao ácido nalidíxico, em seis enterobactérias (2,3%), incluindo 3 E. coli, 2 K. pneumoniae e 1 C. freundii foi encontrado o gene qnrB. Cinco linhagens apresentaram suas seqüências idênticas ao gene qnrB2, e uma linhagem, C. freundii JF79, apresentou uma seqüência idêntica ao gene qnrB8. Em uma linhagem de E. cloacae foi detectado o gene qnrA1 (0,37%). Os genes qnrA e qnrB apresentaram-se localizados em plasmídeos que apresentaram de 55 a 180 kilobases. A análise da estrutura gênica indicou que os genes qnrA1 e qnrB2 estavam associados com um integron classe 1 e localizados entre o elemento ISCR1 e a segunda cópia do segmento conservado 3. Na coleção bacteriana avaliada, as principais mutações observadas foram nos códons 83 e 87 em gyrA, e nos códons 80 e 84 em parC. Todas as enterobactérias que exibiram unicamente mutações no gene gyrA apresentaram CIM 16 µg/mL para o ácido nalidíxico. A diferença encontrada nos valores da CIM de fluoroquinolonas em enterobactérias que apresentaram as mesmas substituições em GyrA e ParC foi explicada pela ausência ou diminuição da expressão de porinas. Em relação à produção de -lactamase de espectro ampliado (ESBL), sua presença foi comprovada em 24 (9,3%) enterobactérias. O seqüenciamento de blaCTX-M identificou 18 determinantes: CTX-M-2 (n=13), incluindo dois novos variantes, CTX-M-8 (n=2) e CTX-M-9 (n=3) mediados por plasmídeos de 48 a 180 kilobases, não conjugativos, em maioria. O gene blaSHV-5 foi detectado em seis enterobactérias. Todos os determinantes do grupo 2 apresentaram-se associados com um elemento ISCR1, enquanto que aqueles do grupo 9 estiveram relacionados com ISEcp1. Concluindo, o principal mecanismo de resistência às quinolonas detectado foi a presença de substituições em GyrA e ParC, apesar de outros mecanismos, como a diminuição da expressão de porinas estarem envolvidos nas enterobactérias avaliadas. A associação da produção de ESBL foi significativa devido ao encontro de uma grande diversidade de genótipos circulando na comunidade, com uma predominância de enterobactérias produtoras de CTX-M. Quanto aos determinantes Qnr descritos neste estudo, que notoriamente foram os primeiros relatos no Brasil, somente dois deles apresentaram-se relacionados com a produção de ESBL. / The aim of this study was to investigate the main mechanisms of quinolone resistance among enterobacterial isolates recovered from hospitalized patients and outpatients in Brazil. The modification of the quinolone targets with changes of DNA gyrase and of topoisomerase IV genes and the presence of determinants codifying plasmid- mediated quinolone and oxymino- cephalosporins resistance were investigated. Two hundred fifty seven non-duplicate nalidixic-acid resistant enterobacterial isolates recovered from January 2000 to May 2005 were analysed. Mutations in the topoisomerases gyrA and parC genes and the genetic structures surrounding Qnr and CTX-M determinants were recognized by PCR and sequencing. Conjugation experiments were performed to determine whether the qnr- and blaCTX-M carrying plasmids were self transferable. Also, decrease in the level of porin expression related to quinolone resistance was assessed. All enterobacterial isolates were resistant to nalidixic-acid, showing minimal inhibitory concentrations (MIC) 16 g/mL and 70% of these isolates showed decreased susceptibility to fluoroquinolone. Six qnrB-positive (2.3%) out of 257 nalidixic-acid resistant enterobacterial isolates, were identified, including 3 Escherichia coli, 2 Klebsiella pneumoniae and 1 Citrobacter freundii. Five isolates had an identical qnrB2 sequence and one isolate, C. freundii 79, possessed the qnrB8. A single Enterobacter cloacae carrying a plasmid encoding qnrA gene was identified (0.37%). All isolates were negative for the qnrS genes. Plasmid-mediated quinolone resistance ranged from 55- to 180-kb in size. Sequence analysis of the genetic structures surrounding of the qnrA and qnrB genes identified an ISCR1 element at the left-hand boundary and a partial copy of the 3-end segment of class 1 integrons. Concerning the changes of DNA gyrase and topoisomerase IV, the most common modification in the enterobacterial isolates analyzed were present at codons 83 and 87 in GyrA and in ParC at codons 80 and 84. All isolates that exhibited mutations in gyrA gene showed nalidixic-acid MIC 16 µg/mL. The finding of different MIC values to fluoroquinolones in enterobacterial isolates with the same GyrA and ParC modification was explained by a decreasing in the level of porin expression. Regarding -lactam resistance mechanisms, twenty four (9.3%) ESBL-producing enterobacterial isolates were detected. Sequencing of the CTX-M-encoding genes identified 18 determinants belonging to CTX-M-2 (n=13), CTX-M-8 (n=2) and CTX-M-9 (n=3) groups. CTX-M-2 group determinants included blaCTX-M-2 and the two novel variants. Plasmids harboring blaCTX-M genes ranged from 48- to 180- kb in size and were not transferable, in their majority. The blaSHV-5 genes were detected in all the 6 blaCTX-M negative isolates. All alleles belonging to the group 2 were associated with ISCR1 element, while all blaCTX-M-9 genes were related to ISEcp1 element. In conclusion, alterations in the targets of quinolones, GyrA and ParC was the main mechanism of quinolone resistance identified in this study, although a decreasing of the porins expression had been identified among nalidixic-acid resistant enterobacterial isolates. In the surveyed area, the prevalence of ESBL producers was important (9.3%), provided that this finding was related to a large diversity of genotypes circulating in the community, mainly CTX-M-producing isolates. This study constituted the first epidemiological survey of QnrA and QnrB determinants among Brazilian isolates. Interestingly, the qnrB2 gene was identified in non ESBL producers isolates and qnrS genes were not found.

This thesis examined molecular changes in the quinolone-resistance determining regions (QRDRs) of the topoisomerase genes, gyrA and parC of Neisseria gonorrhoeae and Neisseria meningitidis and their contribution to fluoroquinolone resistance (FQR). Initially models of FQR emergence were developed from analysis of resistant mutants generated in vitro. The effects of the nature and order of sequential changes in GyrA and ParC on FQR were explored by correlating QRDR changes with ciprofloxacin minimum inhibitory concentration (MIC) determinations. The in vitro models were validated by comparisons of QRDR changes and MICs in two populations of wild-type FQR N. gonorrhoeae over a wide MIC range (0.09 to 24??g/mL), and in a wild type FQR meningococcus. The in vitro activities of three newer quinolones with differential activity on GyrA and ParC were compared with that of ciprofloxacin. Key findings were that the initial QRDR changes always occurred in gyrA and were the predominant influence on phenotypic expression of FQR. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in gonococci and two GyrA and one ParC change those in meningococci. GyrA alterations at Ser-91 in gonococci and Thr???91 in meningococci were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on FQR. ParC substitutions, Ser-87???Arg and Glu-91???Gly in gonococci and Cys- 85???Asp and Glu-91???Lys in meningococci led to the expression of the highest FQR levels. Examination of FQR in wild-type meningococci was necessarily restricted, but analyses using the broader MIC range available in in-vitro-derived FQR meningococci (0.09 to 16??g/mL) revealed the first ParC changes in N. meningitidis. The study also redefined QRDR boundaries and described novel mutations within them. The nature of sequence changes in GyrA and ParC in FQR Neisseria also affected the relative activities of the three newer quinolones. Trovafloxacin was the most active quinolone in vitro but MIC differences with ciprofloxacin were mutation-dependent. Grepafloxacin and moxifloxacin were only slightly more active than ciprofloxacin in the presence of multiple QRDR changes. This thesis provides a comprehensive analysis of the relationship between QRDR alterations and FQR in N. gonorrhoeae and offers insights into the potential for FQR development in N. meningitidis.

Chapter 1: Reaction of benzoaldehyde with wittig agents or isophone to build up conjugate carbon chain, then combined with electron acceptor to furnished the chromophores. The charge-transfer chromophores, which have the first molecular hyperpolarizability

This thesis aimed to investigate the features and properties of the ground states, transient species and photoproducts involved in the photophysical and photochemical processes for four kinds of drug compounds: lomefloxacin (LF), norfloxacin (NF), tiaprofenic acid (TPA), and flurbiprofen (Fp). The investigation used femtosecond transient absorption (fs-TA), nanosecond transient absorption (ns-TA), UV/Vis absorption spectra (UV/Vis), nanosecond transient resonance Raman (ns-TR2) and nanosecond time-resolved resonance Raman spectroscopy (ns-TR3), as well as density functional theory (DFT) calculations. Although many previous investigations have indicated that photo-defluorination or photo-decarboxylation reactions may account for the phototoxicity for these compounds, detailed information on the mechanisms remains unclear. In this thesis, the photo-defluorination reaction of LF was explored in neutral water at pH 7.2. The fs-TA results revealed that the lowest lying excited singlet state species (S1) partially decayed into the ground state through fluorescence emission and partially underwent cleavage of the carbon-fluorine bond at position 8 to generate into a singlet aryl cation. Subsequently, intersystem crossing (ISC) allowing the transformation from singlet cation to triplet carbene was observed. Finally, a cyclization reaction with the N-ethyl chain took place for the triplet carbene to generate the final product. The mechanism underlying NF phototoxicity involves a photo-defluorination reaction in neutral water (pH=7.2). The fs-TA spectra indicated that the S1 underwent efficient ISC to swiftly transform into lowest excited triplet (T1) The ns-TA gained under nitrogen-saturated condition observed a new transient species produced from T1 that was proposed to be a transient species derived from the photo-defluorination reaction involving a SN2Ar* mechanism. The photo-defluorinated product ultimately experienced an ISC process to produce the final product. The photo-decarboxylation mechanism of TPA was studied in a neutral phosphate buffered solution (PBS). The fs-TA data revealed that S1 went through an efficient ISC to rapidly transform into T1 that then undergoes a photo-decarboxylation reaction to produce a triplet biradical species (denoted as TB3). The ns-TA and ns-TR3 results supplied evidence of the protonation process of TB3 that produces the neutral species (denoted as TBP3) that then decayed through ISC to give rise to the singlet TBP species, which underwent further reaction to make the final product (DTPA). The photo-decarboxylation reaction of Fp was explored in pure acetonitrile (MeCN). The second excited singlet (S2) went through internal conversion (IC) to decay to S1. Intriguingly, three different pathways for S1 decay co-exist. One pathway is fluorescence emission and the second is an ISC process. The third pathway is the homolysis of the carbon α bond reaction that proceeds to generate two radical species, one being a carboxyl species and the other being the residual, denoted as FpR that was liable to be oxidized under an oxygen-saturated condition to yield a new radical species with the addition of one oxygen molecule which is denoted as FOR that then experienced intramolecular hydrogen transfer (IHT) and dehydroxylation (DHO) to produce the final product. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

This thesis examined molecular changes in the quinolone-resistance determining regions (QRDRs) of the topoisomerase genes, gyrA and parC of Neisseria gonorrhoeae and Neisseria meningitidis and their contribution to fluoroquinolone resistance (FQR). Initially models of FQR emergence were developed from analysis of resistant mutants generated in vitro. The effects of the nature and order of sequential changes in GyrA and ParC on FQR were explored by correlating QRDR changes with ciprofloxacin minimum inhibitory concentration (MIC) determinations. The in vitro models were validated by comparisons of QRDR changes and MICs in two populations of wild-type FQR N. gonorrhoeae over a wide MIC range (0.09 to 24??g/mL), and in a wild type FQR meningococcus. The in vitro activities of three newer quinolones with differential activity on GyrA and ParC were compared with that of ciprofloxacin. Key findings were that the initial QRDR changes always occurred in gyrA and were the predominant influence on phenotypic expression of FQR. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in gonococci and two GyrA and one ParC change those in meningococci. GyrA alterations at Ser-91 in gonococci and Thr???91 in meningococci were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on FQR. ParC substitutions, Ser-87???Arg and Glu-91???Gly in gonococci and Cys- 85???Asp and Glu-91???Lys in meningococci led to the expression of the highest FQR levels. Examination of FQR in wild-type meningococci was necessarily restricted, but analyses using the broader MIC range available in in-vitro-derived FQR meningococci (0.09 to 16??g/mL) revealed the first ParC changes in N. meningitidis. The study also redefined QRDR boundaries and described novel mutations within them. The nature of sequence changes in GyrA and ParC in FQR Neisseria also affected the relative activities of the three newer quinolones. Trovafloxacin was the most active quinolone in vitro but MIC differences with ciprofloxacin were mutation-dependent. Grepafloxacin and moxifloxacin were only slightly more active than ciprofloxacin in the presence of multiple QRDR changes. This thesis provides a comprehensive analysis of the relationship between QRDR alterations and FQR in N. gonorrhoeae and offers insights into the potential for FQR development in N. meningitidis.

This thesis examined molecular changes in the quinolone-resistance determining regions (QRDRs) of the topoisomerase genes, gyrA and parC of Neisseria gonorrhoeae and Neisseria meningitidis and their contribution to fluoroquinolone resistance (FQR). Initially models of FQR emergence were developed from analysis of resistant mutants generated in vitro. The effects of the nature and order of sequential changes in GyrA and ParC on FQR were explored by correlating QRDR changes with ciprofloxacin minimum inhibitory concentration (MIC) determinations. The in vitro models were validated by comparisons of QRDR changes and MICs in two populations of wild-type FQR N. gonorrhoeae over a wide MIC range (0.09 to 24??g/mL), and in a wild type FQR meningococcus. The in vitro activities of three newer quinolones with differential activity on GyrA and ParC were compared with that of ciprofloxacin. Key findings were that the initial QRDR changes always occurred in gyrA and were the predominant influence on phenotypic expression of FQR. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in gonococci and two GyrA and one ParC change those in meningococci. GyrA alterations at Ser-91 in gonococci and Thr???91 in meningococci were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on FQR. ParC substitutions, Ser-87???Arg and Glu-91???Gly in gonococci and Cys- 85???Asp and Glu-91???Lys in meningococci led to the expression of the highest FQR levels. Examination of FQR in wild-type meningococci was necessarily restricted, but analyses using the broader MIC range available in in-vitro-derived FQR meningococci (0.09 to 16??g/mL) revealed the first ParC changes in N. meningitidis. The study also redefined QRDR boundaries and described novel mutations within them. The nature of sequence changes in GyrA and ParC in FQR Neisseria also affected the relative activities of the three newer quinolones. Trovafloxacin was the most active quinolone in vitro but MIC differences with ciprofloxacin were mutation-dependent. Grepafloxacin and moxifloxacin were only slightly more active than ciprofloxacin in the presence of multiple QRDR changes. This thesis provides a comprehensive analysis of the relationship between QRDR alterations and FQR in N. gonorrhoeae and offers insights into the potential for FQR development in N. meningitidis.

This thesis examined molecular changes in the quinolone-resistance determining regions (QRDRs) of the topoisomerase genes, gyrA and parC of Neisseria gonorrhoeae and Neisseria meningitidis and their contribution to fluoroquinolone resistance (FQR). Initially models of FQR emergence were developed from analysis of resistant mutants generated in vitro. The effects of the nature and order of sequential changes in GyrA and ParC on FQR were explored by correlating QRDR changes with ciprofloxacin minimum inhibitory concentration (MIC) determinations. The in vitro models were validated by comparisons of QRDR changes and MICs in two populations of wild-type FQR N. gonorrhoeae over a wide MIC range (0.09 to 24??g/mL), and in a wild type FQR meningococcus. The in vitro activities of three newer quinolones with differential activity on GyrA and ParC were compared with that of ciprofloxacin. Key findings were that the initial QRDR changes always occurred in gyrA and were the predominant influence on phenotypic expression of FQR. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in gonococci and two GyrA and one ParC change those in meningococci. GyrA alterations at Ser-91 in gonococci and Thr???91 in meningococci were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on FQR. ParC substitutions, Ser-87???Arg and Glu-91???Gly in gonococci and Cys- 85???Asp and Glu-91???Lys in meningococci led to the expression of the highest FQR levels. Examination of FQR in wild-type meningococci was necessarily restricted, but analyses using the broader MIC range available in in-vitro-derived FQR meningococci (0.09 to 16??g/mL) revealed the first ParC changes in N. meningitidis. The study also redefined QRDR boundaries and described novel mutations within them. The nature of sequence changes in GyrA and ParC in FQR Neisseria also affected the relative activities of the three newer quinolones. Trovafloxacin was the most active quinolone in vitro but MIC differences with ciprofloxacin were mutation-dependent. Grepafloxacin and moxifloxacin were only slightly more active than ciprofloxacin in the presence of multiple QRDR changes. This thesis provides a comprehensive analysis of the relationship between QRDR alterations and FQR in N. gonorrhoeae and offers insights into the potential for FQR development in N. meningitidis.

A diferenciação entre processos infecciosos e inflamatórios representa um grande desafio na área de diagnóstico. A complexação do antimicrobiano ciprofloxacino (CIP) com o tecnécio (99mTc-CIP) vem sendo estudada com objetivo de desenvolver um radiofármaco com alta especificidade no diagnóstico de infecções, baseado em seu amplo espectro de atividade antibacteriana. O mecanismo de ação do fármaco consiste na ligação deste com a enzima ADN-girase da bactéria, permitindo sua ligação à bactéria ativa e a obtenção de imagens devido a fótons emitidos pelo 99mTc. Tendo em vista que o local de formação do complexo envolve os mesmos grupos funcionais responsáveis pela ligação do fármaco à enzima, sua eficácia pode ser prejudicada. A diferenciação entre processos infecciosos e inflamatórios representa um grande desafio na área de diagnóstico. A complexação do antimicrobiano ciprofloxacino (CIP) com o tecnécio (99mTc-CIP) vem sendo estudada com objetivo de desenvolver um radiofármaco com alta especificidade no diagnóstico de infecções, baseado em seu amplo espectro de atividade antibacteriana. O mecanismo de ação do fármaco consiste na ligação deste com a enzima ADN-girase da bactéria, permitindo sua ligação à bactéria ativa e a obtenção de imagens devido a fótons emitidos pelo 99mTc. Tendo em vista que o local de formação do complexo envolve os mesmos grupos funcionais responsáveis pela ligação do fármaco à enzima, sua eficácia pode ser prejudicada. O objetivo deste trabalho foi avaliar as condições ideais para ligação do 99mTc a duas quinolonas (CIP e pefloxacino), utilizando diferentes agentes redutores (SnCl2 e FSA), pH da solução e temperatura de incubação. A complexação dos fármacos ao radioisótopo 99mTc foi avaliada através do controle radioquímico, utilizando cromatografia em camada delgada. Posteriormente, testou-se sua atividade antimicrobiana in vitro utilizando diferentes microorganismos. O agente redutor FSA não foi eficiente na formação do complexo, resultando em uma concentração alta de Tc livre (37%). A formulação com Sn aumentou a formação de colóide com aquecimento (100 ºC). A complexação dos antimicrobianos ao Tc impediu sua ligação à ADN-girase. A taxa de ligação variou de 10% (filtração) a 16% (centrigugação). Além disso, verificou-se boa correlação entre a formação de colóide e quantidade de radiação ligada à bactéria em ambos os testes. Esta perda de atividade vem de encontro a alguns estudos clínicos relatados na literatura, que revelam a não diferenciação entre processos infecciosos e processos inflamatórios pelas quinolonas marcadas com 99mTc. / Differentiation between inflammation and infection represents a major challenge in clinical diagnostics. Several studies using ciprofloxacin (CIP), a broad spectrum antimicrobial agent, complexed with technetium (99mTc) have been reported in order to evaluate its capacity to diagnose infections. CIP mechanism of action involves its binding with bacterial DNA-gyrase during the growth phase and the acquisition of images due to 99mTc radioactivity. The fact that the sites of complexation are the same as those involved in bacterial binding it may affect its antimicrobial activity. The objective of the present work was to evaluate the ideal conditions for the complexation of CIP and pefloxacin with 99mTc using stannous chloride and formamidinesulfinic acid (FSA) as reducing agents, different pH and temperatures. The efficiency of complexation was monitored using radiochemical control as well as thin layer chromatography. Furthermore the antimicrobial activity of both complexes was evaluated in vitro. FSA was not adequate as a reducing agent and SnCl2 was better at room temperature than using heat (100 oC). The complexes did not bound well to the bacterias with binding efficiencies ranging from 10 (filtration method) to 16% (centrifugation method). Besides that, a good correlation between colloid formation (impurity) and radioactivity bound to bacterias was found. This decrease in bacterial activity was previously reported in some articles suggesting that quinolones complexed with 99mTc are not suitable to diagnose infections in the presence of inflammation.

Focus of this study was on the development of one of the faster, simpler, cost effective and environmentally friendly sample pre-treatment techniques which employs a supported liquid membrane, in this case a Hollow-fiber supported liquid membrane (HF-SLM) for determination of seven (7) quinolone antibiotics (enrofloxacin, ciprofloxacin, danofloxacin, difloxacin, norfloxacin, nalidixic acid and sarafloxacin) in bovine kidney samples followed by LC-MS/MS analysis. The key parameters of the method were optimized and the method was validated following the 2002/657 EC guidelines. The optimum HF-SLM conditions were therefore; NaH2PO4 as a donor phase at pH 7, 0.1% formic acid at pH 3 as acceptor phase. Triethylamine was the optimized liquid membrane and the stirring time was optimized at 1 hour. Separation of the 7 quinolones including 3 internal standards (enrofloxacin-d5, norfloxacin-d5 and difloxacin-d3) was carried out on a Phenomenex Kinetex 2.6 μm XB-C18, 100 mm x 4.6 mm, 100Å column. Validation parameters such as Correlation coefficients (r2) ranging from 0.9714-0.9975 were obtained, while limit of detection (LOD) ranged between 3-39 ug kg-1 and limit of quantification (LOQ) ranged between 10-130 ug kg-1. The obtained limits at which it can be concluded with an error probability of α = 95% that a sample is non-compliant (CCα) ranged from 28 – 422 ug kg-1 while CCβ; the smallest content of the substance that may be detected, identified or quantified in a sample with an error probability of β = 95%, ranged from 29 – 454 ug kg-1. The method was found to be reproducible with CVs ≤ 23 %. The tested samples from Botswana local abattoirs showed no presence of quinolone antibiotics when the method was applied to real bovine kidney samples. Hollow-fiber supported liquid membrane can therefore be used for extraction of biological samples since it is a “greener technique” which uses less solvent which are less harmful to the environment when disposed as compared to dispersive Solid Phase Extraction (dSPE). / Chemistry / M. Sc. (Chemistry)