Rozprawy doktorskie na temat „Molecules - Biological Interests”

Una de les àrees més prometedores en recerca biomèdica i farmacèutica és el disseny molecular computacional, que intenta establir relacions entre propietats físico-químiques i activitat biològica.
L'èxit d'aquestes tècniques depen críticament de la qualitat de la descripció molecular. En aquest sentit, metodologies basades en potencials d'interacció molecular (MIP) són eines útils per la comparació de compostos que presenten comportaments biològics semblants.
Aquest projecte desenvolupa eines per comparar molècules basades en la caracterització de llurs MIPs. El programa de similaritat molecular MIPsim ha estat desenvolupat i aplicat a diferents problemes biològics.
Aquesta tesi consisteix en quatre estudis científics que mostren l'ús del MIPSim en aliniament molecular, catalisi enzimàtica, en acoratge de molècules dins el lligand i en estudis 3D-QSAR.
One of the most promising areas in biomedical and pharmaceutical research is computer assisted molecular design, which tries to stablish relationships between physicochemical properties and biological activity.
The success of these techniques depends critically on the quality of the molecular description. In this sense, methodologies based on molecular interaction potentials (MIP) are useful tools for the comparison of compounds displaying related biological behaviours.
This project aims to develop tools to compare 'molecules based on the characterization 'of their MIPs. To this end, the molecular similarity program MIPSim has been further developed and applied to different biological problems.
This thesis consists on four scientific studies showing the use of MIPSim for molecular alignment, enzymatic catalysis, ligand-protein docking and 3D-QSAR analyses.

It was observed in the ‘80s that the radiation damage on biological systems strongly depends on processes occurring at the microscopic level, involving the elementary constituents of biological cells. Since then, lot of attention has been paid to study elementary processes of photo- and ion-chemistry of isolated organic molecule of biological interest. This work fits in this framework and aims to study the radiation damage mechanisms induced by different types of radiations on simple halogenated biomolecules used as radiosensitizers in radiotherapy. The research is focused on the photofragmentation of halogenated pyrimidine molecules (5Br-pyrimidine, 2Br-pyrimidine and 2Cl-pyrimidine) in the VUV range and on the 12C4+ ion-impact fragmentation of the 5Br-uracil and its homogeneous and hydrated clusters. Although halogen substituted pyrimidines have similar structure to the pyrimidine molecule, their photodissociation dynamics is quite different. These targets have been chosen with the purpose of investigating the effect of the specific halogen atom and site of halogenation on the fragmentation dynamics. Theoretical and experimental studies have highlighted that the site of halogenation and the type of halogen atom, lead either to the preferential breaking of the pyrimidinic ring or to the release of halogen/hydrogen radicals. The two processes can subsequently trigger different mechanisms of biological damage. To understand the effect of the environment on the fragmentation dynamic of the single molecule, the ion-induced fragmentation of homogenous and hydrated clusters of 5Br-uracil have been studied and compared to similar studies on the isolated molecule. The results show that the “protective effect” of the environment on the single molecule hold in the homogeneous clusters, but not in the hydrated clusters, where several hydrated fragments have been observed. This indicates that the presence of water molecules can inhibit some fragmentation channels and promote the keto-enol tautomerization, which is very important in the mutagenesis of the DNA.

It was observed in the ‘80s that the radiation damage on biological systems strongly depends on processes occurring at the microscopic level, involving the elementary constituents of biological cells. Since then, lot of attention has been paid to study elementary processes of photo- and ion-chemistry of isolated organic molecule of biological interest. This work fits in this framework and aims to study the radiation damage mechanisms induced by different types of radiations on simple halogenated biomolecules used as radiosensitizers in radiotherapy. The research is focused on the photofragmentation of halogenated pyrimidine molecules (5Br-pyrimidine, 2Br-pyrimidine and 2Cl-pyrimidine) in the VUV range and on the 12C4+ ion-impact fragmentation of the 5Br-uracil and its homogeneous and hydrated clusters. Although halogen substituted pyrimidines have similar structure to the pyrimidine molecule, their photodissociation dynamics is quite different. These targets have been chosen with the purpose of investigating the effect of the specific halogen atom and site of halogenation on the fragmentation dynamics. Theoretical and experimental studies have highlighted that the site of halogenation and the type of halogen atom, lead either to the preferential breaking of the pyrimidinic ring or to the release of halogen/hydrogen radicals. The two processes can subsequently trigger different mechanisms of biological damage. To understand the effect of the environment on the fragmentation dynamic of the single molecule, the ion-induced fragmentation of homogenous and hydrated clusters of 5Br-uracil have been studied and compared to similar studies on the isolated molecule. The results show that the “protective effect” of the environment on the single molecule hold in the homogeneous clusters, but not in the hydrated clusters, where several hydrated fragments have been observed. This indicates that the presence of water molecules can inhibit some fragmentation channels and promote the keto-enol tautomerization, which is very important in the mutagenesis of the DNA.

1) Developement of radio-labelled SNPs for the targeted detection and treatment of Her2-positive breast cancer.Aim of this work was to develop a SNP-based system loaded with radioactive/fluorescent probes and functionalized with the half-chain of a monoclonal antibody, Trastuzumab, which specifically recognizes the human epidermal growth factor receptor 2 (Her2), overexpressed in 25-30% of human breast tumours. The silica core was covalently functionalized with FITC, further protected by a 10 nm silica shell and stabilized in saline buffer by means of differently terminated PEGs (SNP). Such nanoparticles were then conjugated with Trastuzumab half-chain (SNP-TZ). Finally, both SNP and SNP-TZ were derivatized with nitrilo-triacetic acid and labelled with 99mTc-Tricarbonyl complex, giving rise to SNP-NTA and SNP-NTA-TZ NPs. The functionalization steps were monitored both by size and z-potential measurements and the impact of each chemical moietiy on the NP behaviour in cells was assessed in prelabeling in vitro experiments comparing SNP, SNP-NTA, SNP-TZ and SNP-NTA-TZ. Targeting specificity of TZ-functionalized or TZ-free SNPs was studied in in vitro, in vivo and ex vivo experiments, both employing fluorescence and radionuclide techniques. Our results suggested that active targeting provided higher efficiency and selectivity in tumor detection compared to passive diffusion, confirming that our synthetic strategy provided stable nanoconjugates and did not affect their binding efficiency to HER2 expressing cells.2) Development of doxorubicin-loaded nonporous SNPs. Nonporous SNPs were chosen as the starting point to produce different drug carriers, bearing the well-known anticancer drug doxorubicin. Different silica nanoformulations containing the well-known anticancer drug doxorubicin were compared: OuterDox NPs, in which doxorubicin was covalently linked on the silica surface, InnerDox NPs, in which the chemotherapeutic was covalently immobilized in the core of the same particles and DoubleDox NPs, containing the drug both externally and internally.The nanoformulations were studied in terms of carrier degradation and payload release in physiological conditions.The in vitro efficiency was also investigated.3)Development of glutathione-sensitive apoferritin NPs for the controlled delivery of luciferin.Although bioluminescence imaging has been successfully used in a variety of applications to obtain information regarding biological processes in vivo, the detection of photon emission is limited by the short half-life of luciferin (less than 30 minutes), its modest cell penetration and inhomogeneous diffusion into different tissues. In this context, we developed a glutathione-sensitive NP for stimuli-responsive release of luciferin within cancer cells. The nanoconjugate bears luciferin by means of a disulfide containing linker (Luc-linker), which, in the presence of a reducing agent, undergoes an intramolecular cyclization reaction that results in the release of free luciferin. The correct luciferin release mechanism was checked in cell-free in vitro bioluminescence tests: an abundant photon production was detected when Luc-linker was preincubated with DTT and then reacted with luciferase, while no light emission was seen without DTT pretreatment. Luc-linker was then attached to apoferritin (HFn) NP surface, exploiting the free thiol groups of cysteine residues, leading to Luc-linker@HFn NPs.After the conjugation, an HPLC method was developed for the quantification of conjugation efficiency and drug loading, requiring a preliminary separation of the linker from the hosting HFn NPs. The Luc-linker@HFn was then tested in vitro to initially elucidate the bioluminescent kinetics and compare the luminous signal to the one of nanoparticle-free luciferin.
1) Developement of radio-labelled SNPs for the targeted detection and treatment of Her2-positive breast cancer.Aim of this work was to develop a SNP-based system loaded with radioactive/fluorescent probes and functionalized with the half-chain of a monoclonal antibody, Trastuzumab, which specifically recognizes the human epidermal growth factor receptor 2 (Her2), overexpressed in 25-30% of human breast tumours. The silica core was covalently functionalized with FITC, further protected by a 10 nm silica shell and stabilized in saline buffer by means of differently terminated PEGs (SNP). Such nanoparticles were then conjugated with Trastuzumab half-chain (SNP-TZ). Finally, both SNP and SNP-TZ were derivatized with nitrilo-triacetic acid and labelled with 99mTc-Tricarbonyl complex, giving rise to SNP-NTA and SNP-NTA-TZ NPs. The functionalization steps were monitored both by size and z-potential measurements and the impact of each chemical moietiy on the NP behaviour in cells was assessed in prelabeling in vitro experiments comparing SNP, SNP-NTA, SNP-TZ and SNP-NTA-TZ. Targeting specificity of TZ-functionalized or TZ-free SNPs was studied in in vitro, in vivo and ex vivo experiments, both employing fluorescence and radionuclide techniques. Our results suggested that active targeting provided higher efficiency and selectivity in tumor detection compared to passive diffusion, confirming that our synthetic strategy provided stable nanoconjugates and did not affect their binding efficiency to HER2 expressing cells.2) Development of doxorubicin-loaded nonporous SNPs. Nonporous SNPs were chosen as the starting point to produce different drug carriers, bearing the well-known anticancer drug doxorubicin. Different silica nanoformulations containing the well-known anticancer drug doxorubicin were compared: OuterDox NPs, in which doxorubicin was covalently linked on the silica surface, InnerDox NPs, in which the chemotherapeutic was covalently immobilized in the core of the same particles and DoubleDox NPs, containing the drug both externally and internally.The nanoformulations were studied in terms of carrier degradation and payload release in physiological conditions.The in vitro efficiency was also investigated.3)Development of glutathione-sensitive apoferritin NPs for the controlled delivery of luciferin.Although bioluminescence imaging has been successfully used in a variety of applications to obtain information regarding biological processes in vivo, the detection of photon emission is limited by the short half-life of luciferin (less than 30 minutes), its modest cell penetration and inhomogeneous diffusion into different tissues. In this context, we developed a glutathione-sensitive NP for stimuli-responsive release of luciferin within cancer cells. The nanoconjugate bears luciferin by means of a disulfide containing linker (Luc-linker), which, in the presence of a reducing agent, undergoes an intramolecular cyclization reaction that results in the release of free luciferin. The correct luciferin release mechanism was checked in cell-free in vitro bioluminescence tests: an abundant photon production was detected when Luc-linker was preincubated with DTT and then reacted with luciferase, while no light emission was seen without DTT pretreatment. Luc-linker was then attached to apoferritin (HFn) NP surface, exploiting the free thiol groups of cysteine residues, leading to Luc-linker@HFn NPs.After the conjugation, an HPLC method was developed for the quantification of conjugation efficiency and drug loading, requiring a preliminary separation of the linker from the hosting HFn NPs. The Luc-linker@HFn was then tested in vitro to initially elucidate the bioluminescent kinetics and compare the luminous signal to the one of nanoparticle-free luciferin.

En médecine, les nanotechnologies permettent le développement de nouvelles techniques de soin comme l’hyperthermie local ou la délivrance ciblée de médicaments. Ces applications impliquent de nouveaux défis scientifiques concernant la conception de nanosystems et les propriétés de leur environnement biologique. Dans cette thèse, nous avons analysé plusieurs aspects de la relaxation thermique de tels systèmes. Nous avons mise en œuvre la fois des simulations de Dynamique Moléculaire et des mesures expérimentales de microscopie d’imagerie en temps de vie de fluorescence. Nous présentons une étude numérique du transfert thermique depuis une nanoparticule en solution aqueuse et montrons qu’attacher un polymère à sa surface permet de réduire la résistance thermique entre la particule et son environnement. Nous avons modélisé des bicouches lipidiques pour calculer leurs propriétés diélectriques et leur viscosité a été étudiée par microscopie de fluorescence. Ces expériences sont réalisées sur des membranes suspendues et des vésicules unilamellaires géantes et démontrent que la viscosité des bicouches lipidiques diminue avec la température et l’application d’une tension transmembranaire induisant un changement de structure
In medicine, nanotechnologies give the opportunity to create new care practices such as local hyperthermia and targeted drug delivery. These applications imply new scientific challenges concerning the design of nanodevices and the properties of their biological environment. In this thesis, we have analysed several aspects of heat relaxation of such systems. We have used both Molecular Dynamics numerical simulations and Fluorescence-lifetime imaging microscopy experiments. We present a study of heat transfer from a solvated nanoparticle and show that attaching a polymer on its surface reduces the thermal resistance between the particle and its aqueous environment. We have modelled lipid bilayers to compute their dielectric properties and their viscosity have been investigated by fluorescence imaging. The experiments conducted on both suspended lipid membrane and giant unilamellar vesicles show that the viscosity decreases when the temperature increases and when a transmembrane voltage is applied to inducing a structural change

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2006.
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As proteínas recombinantes de interesse terapêutico vêm ganhando cada vez mais espaço na indústria farmacêutica e atualmente já movimentam um mercado anual de cerca de 50 a 60 bilhões de dólares em todo o mundo. As células de mamíferos são as hospedeiras de expressão preferencialmente escolhidas no caso de proteínas que requerem um grau sofisticado de processamento pós-traducional, sendo crescente a iniciativa de identificação de novas linhagens de células, especialmente humanas, como sistemas alternativos de expressão às células utilizadas. Nosso grupo de pesquisa tem interesse na produção de antígenos para seleção de anticorpos com potencial neutralizante, especialmente os antígenos de superfície do envelope viral de HIV-1, agente etiológico da pandemia mundial de AIDS, que atualmente apresenta mais de 40 milhões de infectados. O presente trabalho teve por objetivo a avaliação preliminar das células de ducto de glândula submandibular humana (HSG) como sistema de expressão heteróloga alternativo às células de ovário de hamster chinês (CHO-K1). Comparativamente, foi avaliada a eficiência de transfecção, assim como a de expressão transiente do anticorpo quimérico anti-Z-DNA Z22, na forma recombinante de fragmento FvFc pelas duas linhagens celulares. Outro objetivo foi a produção de versões recombinantes das glicoproteínas virais de HIV-1. Os resultados apontaram as células HSG como um bom sistema alternativo para a produção de proteínas heterólogas secretadas, especialmente quando transfectadas por co-precipitação com fosfato de cálcio, sendo ainda necessários alguns ajustes, uma vez que os choques osmóticos com glicerol e DMSO, considerados pontencializadores da transfecção, mostraram-se tóxicos da forma como foram executados. Foram amplificados e clonados em vetor de expressão para células de mamíferos os segmentos gênicos correspondentes a quatro versões recombinantes das glicoproteínas do envelope viral de HIV-1 (gp160, gp140, gp120 e gp41+PS), subtipo C que, de acordo com as nossas análises, utiliza CCR5 como co-receptor. Até o presente momento, não foi possível a detecção das glicoproteínas recombinantes, expressas de forma transiente em células CHO-K1, sendo necessários ajustes, principalmente na etapa de transfecção. _______________________________________________________________________________ ABSTRACT
Recombinant therapeutic proteins have become more and more important in the pharmaceutical industry, and nowadays they are responsible for an injection of about 50 to 60 million dollar a year into the worldwide market. Animal cell cultures are the preferential expression systems for those proteins which require extensive posttranslational modifications. In this view, the identification of alternative expression systems is an issue of increasing concern, specially considering human cell lines. Our research group has been interested in the production of antigens to be used for the selection of neutralizing antibodies, particularly those antigens derived from the envelope surface of HIV-1, the etiologic agent of the pandemic infection of AIDS, which nowadays affects more than 40 million people. This work aimed the preliminary evaluation of the human salivary gland duct cells (HSG) as a heterologous expression system alternative to the Chinese hamster ovary cells (CHO-K1). The transfection efficiency for both cell lines was comparatively evaluated, as well as the transient expression of the anti-Z-DNA Z22 chimeric antibody, as a recombinant FvFc fragment. Another objective was the production of recombinant versions of HIV-1 glycoproteins. Our results pointed out to the HSG cells as a good alternative system for the production of secreted heterologous proteins, specially when transfected by co-precipitation with calcium phosphate. Some adjusts are still needed, considering that the glycerol and DMSO osmotic shocks, generally considered as transfection pontentializers, proved to be toxic in the employed protocol. The genic fragments corresponding to four recombinant versions of the HIV-1 envelope glycoproteins (gp160, gp140 gp120 and gp41+PS), subtype C, were amplified and cloned in a mammal cells expression vector. According to our analysis, this virus subtype uses CCR5 as co-receptor. So far, it was not possible to detect the recombinant glycoproteins expressed in a transient form in the CHO-K1 cells. In order to achieve this objective, some adjustments are still necessary, specially concerning the transfection protocol.

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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico
The total number of prokaryotic cells on Earth has been estimated at 4 to 6x1030 and only about 1% of microorganisms present in the environment can be cultivated by standard techniques of cultivation and plating. Therefore, it is a huge biological and genetic pool that can be exploited, for the identification and characterization of genes with biotechnological potential. Within this perspective, the metagenomics approach was applied in this work. Functional screening methods were performed aiming to identify new genes related to DNA repair and / or oxidative stress resistance, hydrocarbon degradation and hydrolytic activities (lipase, amylase and protease). Metagenomic libraries were built utilizing DNA extracted from soil samples collected in Jo?o C?mara RN. The libraries were analyzed functionally using specific substrate containing solid medium (hydrolytic activity), supplemented with H2O2 (DNA repair and / or resistance to oxidative stress) and liquid medium supplemented with light Arabian oil (activity, degradation of hydrocarbons). After confirmation of activity and exclusion of false-positive results, 49 clones were obtained, being 2 positive for amylase activity, 22 resistant to oxidative stress generated by H2O2 and 25 clones active for hydrocarbons degradation. Analysis of the sequences showed hypothetical proteins, dienelactona hydrolase, DNA polymerase, acetyltransferase, phosphotransferase, methyltransferase, endonucleases, among other proteins. The sequence data obtained matched with the functions tested, highlighting the success of metagenomics approaches combined with functional screening methods, leading to very promising results
O n?mero total de c?lulas procari?ticas na Terra tem sido estimado em 4 a 6x1030 sendo que apenas cerca de 1% dos microrganismos presentes no meio ambiente pode ser cultivado, atrav?s de t?cnicas padr?o de cultivo e plaqueamento, se apresentando, portanto, como um enorme pool biol?gico e gen?tico que pode ser explorado, visando a identifica??o e a caracteriza??o de genes com potencial biotecnol?gico. Dentro desta perspectiva, a abordagem metagen?mica foi aplicada neste trabalho a partir de metodologias de sele??o funcional visando ? identifica??o de novos genes relacionados ao reparo de DNA e/ou resist?ncia a estresse oxidativo, genes relacionados com atividade de degrada??o de hidrocarbonetos, e atividade hidrol?tica (lipase, amilase e protease). Com esse objetivo, uma biblioteca metagen?mica, constru?da a partir de amostras de solo coletadas no munic?pio de Jo?o C?mara RN, foi analisada funcionalmente utilizando meios s?lidos contendo substratos espec?ficos (atividade hidrol?tica), suplementados com H2O2 (reparo de DNA e/ou resist?ncia a estresse oxidativo) e meio liquido suplementado com ?leo ?rabe leve (atividade de degrada??o de hidrocarbonetos). Ap?s confirma??o da atividade e exclus?o de falsos-positivos foram obtidos 49 clones, sendo 2 positivos para atividade amilase, 22 resistentes ao estresse oxidativo gerado por H2O2 e 25 com atividade de degrada??o de hidrocarbonetos, cuja an?lise das seq??ncias revelou,al?m de prote?nas hipot?ticas, dienolactona hidrolase, DNA polimerases, acetiltransferase, fosfotransferase, metiltransferase, endonucleases entre outras, cuja coer?ncia com as fun??es ensaiadas, ressalta o sucesso da abordagem metagen?mica aliada a metodologias funcionais e, os resultados obtidos bastante promissores. PALAVRAS CHAVE: Metagenoma, Biotecnologia

Le but de ce travail était la préparation de dérivés cyclobutaniques du GABA optiquement purs et leur utilisation dans la préparation de γ/α-peptides pouvant adopter une structure tridimensionnelle bien définie. Pour cela, deux stratégies ont été développées. La première consistait en l’utilisation de la β-Cyclodextrine comme hôte supramoléculaire chirale lors de cyclizations photochimiques énantiosélectives. La tentative de cyclisation [2+2] intramoléculaire du N-allyl-N-(4-methoxyphenyl)acrylamide n’a conduit qu’à un δ-lactame issu d’une électrocyclisation 6π. L’électrocyclisation de la 1,3-dihydro-2H-azepin-2-one nous a permis d’obtenir le γ-latame bicyclique précurseur du (+)-cis-3,4CB-GABA avec un excès énantiomérique de 45%. La deuxième stratégie était basée sur une synthèse racémique du N-Boc-cis-3,4CB-GABA suivi d’une séparation des deux énantiomères par CLHP semi-préparative avec une colonne chirale. Les (-) et (+)-cis-3,4CB-GABA optiquement purs ont ainsi été obtenu à l’échelle du gramme. Ces deux énantiomères (-) et (+)-cis-3,4CB-GABA ont ensuite été utilisés pour la préparation de deux séries de peptides mixtes γ/α, diastéréoisomères [(S,S/R) et (R,R/R)] à courtes chaines contenant alternativement le cis-3,4CB-GABA et le D-Alanine. L'analyse des conformations des dipeptides des deux séries par Diffraction des Rayons X, n'a montré aucune interaction intramoléculaire mais plutôt un assemblage de liaisons d'hydrogène intermoléculaires entre les molécules du dipeptide. D'autre part, les études RMN 1D et 2D (en solution) ont montré que le tétrapeptide des séries (S,S/R) pourraient avoir une structure hélicoïdale 12/10, tandis que son analogue diastéréoisomères des séries (R,R/R), a montré, en solution, une nouvelle structure sous forme de ruban 7/9
This work revolves around the synthesis of ennatiomerically pure cyclobutane derivatives of GABA, and their use in the preparation of hybrid γ/α-peptides that could adopt a well-defined three dimensional secondary structure. In this aim we developed two strategies. The first one employed native β-Cyclodextrin as a supramolecular chiral host to achieve enantiodifferentiating photochemical cyclizations. Attempting to perform an intramolecular [2+2] cyclization of N-allyl-N-(4-methoxyphenyl)acrylamide, we only obtained a δ-lactam resulting from a 6π electrocyclization, whereas the electrocyclization of 1,3-Dihydro‑2H‑azepin-2-one allowed access to a 45% enantiomerically enriched bicyclic γ-lactam precursor of (+)-cis-3,4CB-GABA. The second strategy was based on a racemic synthesis of N-Boc-cis-3,4CB-GABA followed by a separation of the two enantiomers using a semi-preparative HPLC fitted with a chiral column. This allowed access to optically pure (-) and (+)-cis-3,4CB-GABA, on a gram scale. Furthermore, the enantiomerically pure (-) and (+)-cis-3,4CB-GABA, were used to synthesize, and fully characterize two series [the (S,S/R) and the (R,R/R)] of short diasteriomeric hybrid γ/α-peptides composed of alternating cis-3,4CB-GABA and D-Alanine. Analysis of the conformational behavior of the dipeptides from both series by X-Ray diffraction on a single crystal, showed no intramolecular interactions but rather an array of intermolecular hydrogen bonding between the dipeptide molecules. On the other hand, a series of 1D and 2D NMR experiments showed that the tetrapeptide of the (S,S/R)-series could attain a 12/10 helical structuration, whereas its diasteriomeric analog of the (R,R/R)-series, displayed evidence of an unprecedented 7/9 folding pattern in solution

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2010.
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A holocelulose é o componente mais abundante da biomassa vegetal e é composto principalmente por celulose, hemicelulose e pectina. O bagaço de cana é o maior resíduo da agroindústria brasileira e é uma fonte de carbono economicamente viável para microrganismos produzirem enzimas holocelulolíticas de aplicação industrial. Os fungos filamentosos são eficientes produtores de xilanases, e suas enzimas têm sido utilizadas em todo o mundo em processos industriais. No presente estudo, uma xilanase (Xyl) do fungo Aspergillus niger crescido sobre bagaço de cana foi purificada e caracterizada visando a sua aplicação industrial. A curva de indução enzimática do fungo indicou alta atividade xilanolítica a partir do segundo dia, mantendo-se constante ao longo de 50 dias. A enzima teve sua maior atividade a 50°C e pH 4,5. A meia-vida aumentou 2,3 vezes quando Xyl foi incubada com tampão acetato de sódio pH 4,5. Estes resultados apontam para a possibilidade de aproveitamento desta xilanase na indústria têxtil, na panificação e em biorefinarias. Diversos íons foram testados, mas nenhum foi capaz de estimular a atividade de Xyl. Dentre os modificadores químicos de aminoácidos, o NBS foi o maior inibidor da atividade de Xyl, sugerindo o envolvimento de L-triptofano na ligação ao substrato ou na catálise. O ?- mercaptoetanol e o L-triptofano foram os maiores ativadores da enzima. Os valores de KM e Vmax encontrados foram de 47,08 mg/mL e 3,02 UI/mL, respectivamente, e há indícios de que Xyl dependa das ramificações da xilana para se ancorar ao substrato. A massa molecular estimada foi de cerca de 33 kDa, e o perfil bidimensional revelou a presença de isoformas ou enzimas múltiplas na amostra. Os resultados da espectrometria de massa sugerem que as xilanases são conservadas entre as espécies do gênero Aspergillus. As imagens de microscopia eletrônica de varredura mostram a degradação do bagaço de cana por enzimas de A. niger. As imagens de microscopia de força atômica sugerem que Xyl pertença à família GH10, mas sua atividade holocelulolítica residual a classificam com GH11. _________________________________________________________________________________ ABSTRACT
Holocelulose is the most abundant component of biomass and it is basically composed of cellulose, hemicellulose and pectin. Sugar cane bagasse is the major waste of brazilian agroindustry and it is a cheap carbon source for microorganisms to produce holocellulolytic enzymes of industrial application. Filamentous fungi are good xylanase producers and their enzymes have been used in industrial processes all over the world. In this study a xylanase (Xyl) produced by the fungus Aspergillus niger over sugar cane bagasse was purified and characterized aiming its biotechnological application. The fungus produces higher amounts of xylanolytic activity from the second day on, and this activity remains relatively constant up to the 50th day. The enzyme presented the best activity at 50°C and pH 4,5. The half-life increased 2,3 times when Xyl was incubated with sodium acetate buffer pH 4,5. The results point out to the application of this enzyme in the textile industry, bakery and biorefineries. None of the tested ions was capable of increasing Xyl activity. NBS was Xyl major inhibitor, suggesting that Ltryptophan is involved in the substrate linkage or catalysis. β- mercaptoethanol and L-tryptophan were the best enzyme activators. The KM and Vmax values were 47,08 mg/mL and 3,02 UI/mL, respectively, and it is possible that Xyl depends on xylan side chains to stabilize over the substrate structure. The estimated molecular mass was about 33 kDa, and the 2Delectrophoresis analysis suggested the existence of multiple forms of xylanases. The mass spectrometry results suggest that the xylanases are conserved among the Aspergillus species. The electron scanning microscopy images show the degradation of sugar cane bagasse by A. niger enzymes. The atomic force microscopy images suggest that Xyl belongs to GH10, but its residual holocelulolytic activity classifies Xyl as a member of GH11 family.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES
Studies on diversity and biology of insects colonizing carcasses exposed to the natural environment have gradually increased and contributed to the development of forensic entomology in Brazil. These surveys provide information that may assist in estimating the postmortem interval (PMI) and in the resolution of other issues related to the legal scope. This study aimed to characterize the fauna of insects associated with exposed pig carcasses in the region of Porto Alegre, Rio Grande do Sul, Brazil, relating the effects of biotic and abiotic factors to the community of these organisms. In addition, alternative methodologies were investigated, based on DNA barcode analysis, to identify species of necrophagous Diptera of forensic interest for the study site. Experiments were conducted in the months of January and September of 2014, relating them to hot and dry and cold and wet seasons, respectively. As experimental model, domestic male pigs of approximately 12 kg were used. Immediately after death, carcasses were placed in metal cages under a modified "Shannon" trap. For sampling adult insects, active collections of winged insects were made and pitfall traps were used for the terrestrial ones. For the collection of immature insects, trays containing sawdust were placed under the carcass. The tray?s contents were later removed and transferred to plastic pots with an organza cover to allow them to cramp and complete their development until emergence. Collections were performed daily, as well as photographic records to characterize the stages of decomposition. Abiotic data such as temperature, relative humidity and rainfall were obtained from the National Institute of Meteorology (INMET). In total, 16.321 insects belonging to orders Diptera (78 species) and Coleoptera (56 species) were collected. Four stages of decomposition were recognized: fresh, bloated, decay and dry. In the hot and dry season with temperature averages of 31.3? C, relative air humidity of 55.3% and total rainfall of 13.1 mm, the decomposition occurred in 10 days, and 2.326 specimens were collected. In the cold and wet season, with temperature averages of 19.3? C, humidity of 78.4% and total precipitation of 161.4 mm, the decomposition lasted 34 days, and 13.995 specimens were collected. Diptera was predominant in both seasons and represented the only order of insects that used the carcass as a resource for the development of their offspring, including those belonging to the families Calliphoridae, Muscidae and Sarcophagidae. Beetle diversity was higher in the cold and wet season, with representatives of Dermestes maculatus (Dermestidae), Euspilotus azureus (Histeridae) and Oxelytrum discicolle (Silphidae). Differing from other similar studies in the hot and dry season, there was an increase in the number of accidental insects (Chrysomelidae), a fact that can be attributed to the high temperatures recorded, evidencing the strong influence of abiotic factors on the diversity of the community of insects that colonized the carcasses. For the molecular characterization, 40 male individuals of seven species of flesh flies of forensic interest were morphologically determined previously. Fragments were obtained from the COI (cytochrome c oxidase subunit I) region of the mitochondrial DNA of approximately 595bp length, which were useful for differentiation and characterization of the different taxa proposed here.Thus, the results obtained here evidenced the importance of regional studies on taxa of forensic importance, mainly due to the biotic and abiotic influences on the local community associated with the carcasses.
Estudos sobre a diversidade e biologia de insetos que colonizam carca?as expostas ao ambiente natural t?m aumentado gradativamente e contribu?do para o fomento da entomologia forense no Brasil. Essas pesquisas trazem informa??es que podem auxiliar na estimativa do intervalo p?s-morte (IPM) e na resolu??o de outras quest?es relacionadas ao ?mbito legal. Esse trabalho teve como objetivo caracterizar a fauna de insetos associados a carca?as de su?nos expostas na regi?o de Porto Alegre, Rio Grande do Sul, Brasil, relacionando os efeitos dos fatores bi?ticos e abi?ticos sobre a comunidade destes organismos. Adicionalmente, foram investigadas metodologias alternativas, a partir da an?lise do DNA barcode, para identifica??o de esp?cies de d?pteros necr?fagos de interesse forense para o local de estudo. Os experimentos foram conduzidos nos meses de janeiro e setembro de 2014, relacionando-os as esta??es quente e seca e fria e ?mida, respectivamente. Como modelo experimental foram utilizados su?nos dom?sticos machos de aproximadamente 12 kg. Imediatamente ap?s a morte as carca?as foram dispostas em gaiolas met?licas sob uma armadilha modificada do tipo ?Shannon?. Para a amostragem dos insetos adultos foram feitas coletas ativas para insetos alados e armadilhas de queda do tipo pitfall para os terrestres. Para a coleta de imaturos foram dispostas bandejas sob a carca?a contendo serragem, a qual tinha seu conte?do removido e transferido para potes pl?sticos com cobertura de organza para permitir que empupassem e completassem seu desenvolvimento at? a emerg?ncia. As coletas foram realizadas diariamente, assim como os registros fotogr?ficos para caracteriza??o dos est?gios de decomposi??o. Dados abi?ticos tais como temperatura, umidade relativa do ar e precipita??o foram obtidos junto ao Instituto Nacional de Meteorologia (INMET). Ao todo foram coletados 16.321 insetos pertencentes ?s ordens Diptera (78 esp?cies) e Coleoptera (56 esp?cies). Quatro est?gios de decomposi??o foram reconhecidos: fresco, gasoso, avan?ado e seco. Na esta??o quente e seca, com m?dias de temperatura de 31,3 ?C, umidade relativa do ar de 55,3% e precipita??o total de 13,1mm, a decomposi??o ocorreu em 10 dias, tendo sido coletados 2.326 esp?cimes. J? na esta??o fria e ?mida, com m?dias de temperatura de 19,3 ?C, umidade de 78,4% e precipita??o total de 161,4mm, a decomposi??o durou 34 dias, tendo sido coletados 13.995 esp?cimes. Diptera foi predominante em ambas as esta??es e representou a ?nica ordem de insetos que utilizou a carca?a como recurso para o desenvolvimento de sua prole, dentre os quais aqueles pertencentes ?s fam?lias Calliphoridae, Muscidae e Sarcophagidae. A diversidade de besouros foi maior na esta??o fria e ?mida, com representatividade de Dermestes maculatus (Dermestidae), Euspilotus azureus (Histeridae) e Oxelytrum discicolle (Silphidae). Diferindo de outros estudos semelhantes ocorridos na esta??o quente e seca, houve maior abund?ncia de insetos acidentais (Chrysomelidae), fato que pode ser atribu?do as altas temperaturas registradas, evidenciando a forte influ?ncia dos fatores abi?ticos sobre a diversidade da comunidade de insetos que colonizaram as carca?as. Para a caracteriza??o molecular foram previamente determinados morfologicamente 40 indiv?duos machos de sete esp?cies de sarcofag?deos de interesse forense. Foram obtidos fragmentos da regi?o COI (citocromo oxidase I) do DNA mitocondrial de aproximadamente 595 pb, os quais se mostraram ?teis para diferencia??o e caracteriza??o dos distintos t?xons aqui propostos. Dessa forma, os resultados aqui obtidos evidenciam a import?ncia de estudos regionais sobre os t?xons de import?ncia forense, sobretudo decorrente das influ?ncias abi?ticas sobre a comunidade local associada ?s carca?as.

Orientador: Everlon Cid Rigobelo
Banca: Diego Cunha Zied
Banca: Davi Rodrigo Rossatto
Banca: José Eduardo Marcondes de Almeida
Banca: Felipe Batistella Filho
Resumo: A representatividade da agricultura brasileira é reconhecida mundialmente, especialmente na produção de grãos como a soja, o milho e o feijão. A produtividade nas lavouras, especialmente na última década, tem apresentado crescimento expressivo, entretanto, um dos aspectos negativos associados a esse processo é o aumento concomitante no uso de defensivos e fertilizantes. Atualmente, o tema se tornou uma preocupação de saúde pública e ambiental dada a toxicidade e recalcitrância desses compostos. Dentre formas alternativas ao uso desses insumos o estudo de micro-organismos é promissor, pois eles constituem um grande patrimônio genético que alberga vias metabólicas de grande interesse para diversas atividades humanas, incluindo a agricultura. Os micro-organismos interagem de diversas maneiras com as plantas beneficiando-as e garantindo recursos para si em troca. Eles podem atuar melhorando o aporte de nutrientes, os mecanismos de defesa e de promoção de crescimento em condições de estresse biótico e abiótico. Assim, o presente estudo teve como objetivos o isolamento de fungos a partir de amostras de solo, com enfoque para fungos do gênero Purpureocillium; sua caracterização molecular e de seu potencial de promoção de crescimento de plantas in vitro; e a seleção de linhagens para serem testadas in planta em soja, milho e feijão. O isolamento foi realizado utilizando meios convencionais e seletivos para a obtenção dos fungos. Os isolados tiveram seu DNA extraído e as regiões ITS (... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The representativeness of Brazilian agriculture is worldwide recognized, especially in the grains production such as soybean, maize and beans. Crops' yield, especially in the last decade, has presented significant growth, however, one of the negative aspects associated with this process is the concomitant increase in the use of pesticides and fertilizers. Currently, this issue has become a public health and environmental concern because of the toxicity and recalcitrance of these compounds. Among alternatives to the use of these inputs the study of microorganisms is promising because they constitute a great genetic patrimony, which houses metabolic pathways of great interest for several human activities, including agriculture. Microorganisms are able to interact in different ways with plants benefiting them and ensuring resources for themselves in return. They can act by improving nutrient supply, defense mechanisms and promoting growth under conditions of biotic and abiotic stress. Thus, the present study aimed the isolation of fungi from soil samples, focusing on the search for fungi of the genus Purpureocillium; their molecular characterization and the assessment of their in vitro plant growth promotion potential; and the selection of strains to be tested in planta in soybean, maize and bean. Fungal isolation was performed using conventional and selective media. The isolates had their DNA extracted and the ITS (Internal Transcribed Spacer) region of the ribosomal DNA and pa... (Complete abstract click electronic access below)
Doutor

El presente proyecto de investigación está enfocado al desarrollo de sensores químicos fluoro-cromogénicos, para la detección y determinación de especies químicas de interés biológico, industrial y medioambiental de forma selectiva y con alta sensibilidad. En forma general, se busca el diseñar nuevos sistemas sensores basados en compuestos (receptores) formados por dos unidades: una unidad coordinante que interacciona con el anión a determinar y una unidad generadora de señal que alerta del reconocimiento molecular efectuado. Durante este estudio se están preparando diversas moléculas receptoras funcionalizandas con grupos modificadores de estructura para evaluar su influencia sobre las capacidades de detección y selectividad como receptores de especies específicas en diferentes condiciones y medios. Las diferentes aproximaciones en prueba implican a su vez el diseño y síntesis molecular, así como el análisis de las diferentes señales ópticas producidas en el reconocimiento, con el fin de diseñar sistemas de alta eficacia y eficiencia, y con posibilidades reales de aplicación.
Santos Figueroa, LE. (2014). New approaches for the development of chromo-fluorogenic sensors for chemical species of biological, industrial and environmental interest [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/43216
TESIS
Premiado

Proteins are large biomolecules that play essential functional and structural roles within cells and that typically act through their interaction with other proteins and biomolecules, forming highly specific functional complexes. Thus, a major biological challenge is to provide structural and energetics details for such interactions. In this context, computational methods can successfully contribute to predict and characterize the mechanistic aspects of protein function, in which conformational flexibility plays a major role. However, an accurate consideration of protein plasticity within computational modeling of protein function at molecular level is still far from trivial, mostly because of both technical and methodological limitations. Thus, the main purpose of this PhD thesis is the assessment, development and application of computational tools for the structural, energetic and dynamic characterization of protein molecules and their interactions. To fulfill these objectives, the first part of the thesis consists in the review and comparison of several existing computational protocols for the characterization of protein-protein interfaces, as well as in the evaluation and discussion of the performance of pyDock, the docking protocol previously developed in our group, as resulted from the last CAPRI (Critical Assessment of PRediction of Interactions) editions. These analyses confirm that current computational protocols aimed to model the phylogenetic, structural and energetic properties of residues within protein-protein interfaces show reasonably good predicting performance and consistency. However, as shown by CAPRI experiment, despite recent methodological advances in docking, dealing with protein plasticity is still a crucial bottle-neck. Based on these premises, the second part of the thesis is focused on understanding the role of protein conformational heterogeneity in protein-protein recognition. Subsequently, a novel protocol to integrate unbound conformational ensembles within a docking framework has been devised and systematically tested. The analysis of conformational heterogeneity in precomputed unbound ensembles reveals that docking encounters are favoured by improving the energetic complementarity of the docking partners rather than the geometrical similarity to the bound state. Moreover, the unbiased use of such ensembles is a successful strategy to incorporate flexibility into a docking approach for low-medium flexible cases, especially those that presumably follow a conformational selection mechanism. Finally the last part of the thesis consists in the application of computational methods to the modeling of protein interactions and the exhaustive exploration of their conformational space within different realistic contexts, thanks to the expertise previously acquired on the theoretical basis of protein interactions. Thus, the main lines of research include the energetic characterization of host-pathogen protein interactions (i.e., host GTPase Rab5 with pathogen phospholipase VipD), the ab-initio modeling of the encounter complex ensembles of redox proteins (i.e. PSI with alternative electron donors, cytochrome c6 and plastocyanin), and finally the description of the structural and dynamic basis of a protein kinase dysfunction under pathological conditions (i.e., MEK1 oncogenic and CFC-related mutants). The results confirmed that computational modeling can complement experimental data to improve the understanding of biological processes involving protein interactions and can help to rationalize and quantify the structural, energetic and dynamic effects of pathological mutations at molecular level.
Las proteínas son grandes biomoléculas que desarrollan funciones esenciales en las células, muy a menudo mediante la formación de complejos altamente específicos con otras proteínas y biomoléculas. Por tanto, uno de los mayores retos científicos en la actualidad es el estudio completo a nivel estructural y energético de todas las interacciones entre proteínas de interés biológico y terapéutico. Sin embargo, la consideración precisa de la plasticidad de las proteínas en los métodos computacionales de modelado molecular no es trivial, debido a limitaciones tanto técnicas como metodológicas. En este contexto, el objetivo principal de esta tesis doctoral ha sido el desarrollo, aplicación y evaluación de herramientas computacionales para la caracterización estructural, energética y dinámica de las proteínas y sus interacciones. Para cumplir con estos objetivos, durante la primera parte de la tesis se ha llevado a cabo la revisión de varios protocolos computacionales para la caracterización de las superficies de interación entre proteínas. Los métodos analizados proporcionan unas predicciones razonablemente consistentes y fiables. También se ha llevado a cabo la evaluación de la eficacia predictiva de nuestro método pyDock en CAPRI, un experimento comunitario de evaluación de métodos de modelado estructural de complejos entre proteínas. En general, a pesar de los avances metodológicos en los protocolos de docking, el modelado eficaz de la plasticidad de las proteínas sigue siendo un reto importante en el campo. En base a los análisis anteriores, se ha llevado a cabo un estudio sistemático sobre la importancia de la heterogeneidad conformacional en el reconocimiento entre proteínas. Los resultados indican que los ensamblados conformacionales generados a partir de proteínas en solución contienen confórmeros con mejor complementariedad energética que la estructura cristalográfica de dichas proteínas y que favorecen su reconocimiento intermolecular. A partir de estos resultados, se ha propuesto un nuevo métodode docking que usa ensamblados conformacionales generados a partir de las proteínas en solución. Esta estrategia resulta particularmente efectiva en casos poco o medianamente flexibles. Finalmente, en la última parte de la tesis se ha llevado a cabo la aplicación de métodos computacionales al modelado de varios casos de interés biomédico. En conclusión, los avances metodológicos en cuanto al modelado de proteínas y sus interacciones, junto a la inclusión eficaz de la flexibilidad conformacional, permiten tener herramientas computacionales cada vez más útiles para complementar los datos experimentales y mejorar la comprensión de procesos biológicos relevantes.

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Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná
Currently, studies of aspects of evolutionary biology in neotropical fishes diversity are still lacking, despite the strong environmental impact caused by human action that has had a negative effect in native species, many of which probably still unknown to science. The knowledge of genetic variability within and between populations is of utmost importance for planning of both fish breeding and conservation of natural populations. In the present study we have analyzed, especially through molecular cytogenetics techniques, 29 specimens of Pseudoplatystoma reticulatum (Paraguay river, state of Mato Grosso do Sul), 14 specimens of Pimelodus britskii (Igua?u river, state of Paran?), 6 specimens of Sorubim lima (Paraguay river, state of Mato Grosso do Sul) and 10 specimens of Steindachneridion parahybae (Para?ba do Sul river, state of S?o Paulo). Results indicate 2n = 56 chromosomes for all species, a distinct karyotype formula composed mainly of biarmed chromosomes. Other particular features were found, regarding the location for Ag+NORs and fluorescent in situ hybridization with 5S and 18S probes. Except P. britskii, which showed NORs in the terminal region of the long arm, all species showed Ag+NORs in the terminal position of the short arm. Another peculiar feature of this fish was the co-localization of one of the 5S and 18S rDNA sites, which could be considered an apomorphy to the others Pimelodidae studied. The present work aims to contribute to the collection of data on the cytogenetics studies of Siluriformes, considering that much of the here presented information are the first characterization of the karyotype macro-struture of analysed fishes.
Considerando-se a diversidade ictiofaun?stica neotropical, estudos sobre aspectos de biologia evolutiva s?o ainda pouco expressivos. Em contraste, o forte impacto ambiental causado pela a??o humana tem refletido negativamente sobre as esp?cies nativas, muitas ainda desconhecidas da ci?ncia. O conhecimento da variabilidade gen?tica intra e inter populacional ? de extrema import?ncia para o planejamento tanto da piscicultura quanto para a conserva??o das popula??es naturais. No presente trabalho foram analisados citogeneticamente, sobretudo aplicando t?cnicas de citogen?tica molecular, 29 exemplares de Pseudoplatystoma reticulatum (rio Paraguai, MS), 14 esp?cimes de Pimelodus britskii (rio Igua?u, PR), 6 indiv?duos de Sorubim lima (rio Paraguai, MS), e 10 exemplares de Steindachneridion parahybae (rio Para?ba do Sul, SP). Os dados obtidos revelaram 2n = 56 cromossomos para todas as esp?cies analisadas, f?rmulas cariot?picas distintas, compostas basicamente de cromossomos de dois bra?os e caracter?sticas pr?prias em rela??o ? localiza??o das RONs atrav?s da impregna??o pelo ?on Ag+ e pela hibrida??o fluorescente in situ com sondas de DNAr 5S e 18S. Com exce??o de P. britskii, que apresentou RONs na regi?o terminal do bra?o longo, todas as outras esp?cies apresentaram marca??es das Ag+RONs em posi??o terminal no bra?o curto. Outra caracter?stica peculiar de P. britskii foi a localiza??o sint?nica de um dos s?tios de DNAr 5S e o 18S em cromossomos subteloc?ntricos, atributo que pode ser considerado uma apomorfia em rela??o a outros pimelod?deos estudados. O presente estudo visa contribuir com o acervo de dados citogen?ticos a respeito dos Siluriformes em estudo, considerandose que muitas das informa??es correntes tratam-se das primeiras caracteriza??es da macroestrutura cariot?pica das esp?cies de peixes analisadas.

A series of water-soluble platinum(II) terpyridine complexes with functionalized alkynyl ligands and a boronic acid-containing polymer, PAAPBA, have been synthesized and characterized. The photophysical and electrochemical properties of all the platinum(II) complexes have been studied. Some of the complexes have been demonstrated to show ground-state aggregation in organic solvents and aqueous solutions at high concentrations, leading to Pt…Pt and/or π–π interactions and hence the emergence of metal-metal-to-ligand charge transfer (MMLCT) transitions in both the UV−visible and emission spectra. The induced self-assembly of [Pt(tpy)(C≡CC6H4−CH2NMe3-4)](OTf)2 by PAAPBA has been explored for the development of glucose sensing protocols and α-glucosidase assay by monitoring the triplet metal-metal-to-ligand charge transfer (3MMLCT) emission in the near-infrared (NIR) region. [Pt(tpy){C≡CC6H4− {NHC(=NH2+)(NH2)}-4}](OTf)2 has been observed to undergo induced aggregation in the presence of citrate, with good selectivity over other mono- and dicarboxylates in the tricarboxylic acid (TCA) cycle. Enzymatic activity of citrate lyase has also been probed by the emission spectral changes of the complex in the NIR region. A series of water-soluble alkynylplatinum(II) terpyridine complexes and water-soluble conjugated polyelectrolytes (CPEs) have been synthesized and characterized. The UV–vis absorption and emission properties of the platinum(II) complexes and CPEs have been investigated in organic solvents and/or aqueous buffer solutions. The electrochemical properties and ground-state aggregation at high concentrations of the platinum(II) complexes have also been examined. Two-component ensembles containing selected platinum(II) complexes and PPE-SO3− have been studied, and Förster resonance energy transfer (FRET) has been demonstrated from the PPE-SO3− donor to the aggregated complexes as acceptors. The ensemble containing PPE-SO3− and [Pt(tpy)(C≡CC6H4CH2NMe3-4)](OTf)2 has been employed for a “proof-of-principle” label-free detection of human serum albumin (HSA) in pH 3 buffer solutions with high selectivity and sensitivity, while another ensemble containing PPE-SO3− and [Pt{tpy(C6H4CH2NMe3-4)-4’}(C≡CC6H5)](OTf)2 has been utilized for selective label-free detection of G-quadruplex structure of the human telomeric DNA in physiological buffer solutions. A series of water-soluble platinum(II) terpyridine complexes with stimuli-responsive alkynyl ligands and a series of water-soluble platinum(II) metallosupramolecular triblock copolymers have been synthesized and characterized. The photophysical and electrochemical properties as well as the ground-state aggregation of the complexes have been investigated. Some of them have been found to show different electronic absorption and emission properties in aqueous solution at different pHs due to aggregation/deaggregation of the complexes. One of the complexes has been employed for live-cell imaging experiments to locate acidic organelles, such as lysosomes, in MDCK cells. The water-soluble platinum(II) metallosupramolecular triblock copolymers have been found to show an increase in 3MMLCT emission intensity in the red-NIR region with temperature, which has been attributed to the formation of spherical polymeric micelles. The platinum(II) triblock copolymer with pH-responsive –CH2NMe2 moieties has been demonstrated as a NIR-emitting dual sensor for pH and temperature through the changes in hydrophilicity and hence the emission properties with pH and temperature simultaneously.
published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy

A genética forense tem grande importância na geração de provas em casos de violência sexual, paternidade criminal, identificação de cadáveres e investigação de evidências de locais de crime. A análise de STRs apresenta grande poder de discriminação, mas é uma metodologia multi-etapas, trabalhosa, cara e em muitos casos a análise genética é prejudicada pela baixa quantidade e qualidade das evidências coletadas. Este estudo teve como objetivo desenvolver e caracterizar uma metodologia de triagem de amostras forenses através da análise de perfis de dissociação em alta resolução (HRM) de regiões do DNA mitocondrial, o qual está presente em maior número de cópias e mais resistente a degradação. Para tanto, foram extraídos DNAs de 68 doadores. Estas amostras foram sequenciadas e analisadas por HRM para sete alvos no DNA mitocondrial. Também foram realizados ensaios para determinar a influência do método de extração, da concentração e nível de degradação do DNA no perfil de HRM obtido para uma amostra. Os resultados demonstraram a capacidade da técnica de excluir indivíduos com sequências diferentes da referência comparativa em cinco regiões amplificadas. Podem ser analisadas em conjunto, amostras de DNA com variação de concentração de até a ordem de 100 vezes e extraídas por diferentes metodologias. Condições de degradação de material genético não prejudicaram a obtenção de perfis de dissociação em alta resolução. A sensibilidade da técnica foi aprimorada com a análise de produtos de amplificação de tamanho reduzido. A fim de otimizar o ensaio foi testada a análise de HRM em reações de PCR duplex. Um dos pares de amplificação forneceu perfis de HRM compatíveis com resultados obtidos de reações com amplificação de apenas um dos alvos. Através da análise conjunta das cinco regiões, esta metodologia visa a identificação de indivíduos não relacionados com as referências comparativas, diminuindo o número de amostras a serem analisadas por STRs, reduzindo gastos e aumentando a eficiência da rotina de laboratórios de genética forense.
The forensic genetics has an important role in the generation of evidence in cases of sexual assault, criminal paternity, identification of corpses and crime scenes investigation. The analysis of STRs has great power of discrimination, but it is a multi-stage methodology, complex, expensive and in many cases the genetic analysis is hampered by the low quantity and quality of evidence collected. This study aimed to develop and characterize a forensic samples screening methodology to examine high resolution melting profiles (HRM) of regions of the mitochondrial DNA, which is present in more copies and more resistant to degradation. Thus, we extracted DNA from 68 donors. These samples were sequenced and analyzed by HRM to seven mitochondrial DNA targets. Tests were also conducted to determine the influence of extraction method, concentration and DNA degradation level of HRM profile obtained for a sample. The results demonstrated the technical ability to exclude individuals with different sequences of comparative reference amplified in five regions. Can be analyzed together samples with varying concentration to the order of 100 times and extracted by different methods. Genetic material degradation conditions did not prevent obtaining high resolution melting profiles. The sensitivity of the technique was improved with the analysis of reduced size amplification products. In order to optimize the assay HRM analysis was tested in duplex PCR reactions. A pair of amplification provided HRM profiles consistent with results from amplification in reactions with only one of the targets. Through the joint analysis of the five regions, this approach aims to identify individuals not related to comparative references, reducing the number of samples to be analyzed by STRs, reducing costs and increasing the efficiency of the routine of forensic genetics laboratories.

博士
國立交通大學
應用化學系碩博士班
104
Small molecules can probe the biological systems thereby understanding of biological processes is possible. Hence, they have been exploited in designing drug molecules against various biological targets. The present thesis deals with the design and synthesis of heterocyclic small molecules of biological interest. The thesis is divided into three chapters for the sake of convenience and better understanding. The first chapter deals with a facile and efficient synthesis of novel oxo, thio and seleno hydantoin fused tetrahydroazepino [4, 5-b]indoles. Naturally occurring iboga class alkaloid inspired seven-member azepino[4,5-b]indole ring was synthesized as a new scaffold through Pictet-Spengler reaction followed by skeletal rearrangement of aziridine ring. To improve the efficiency of the synthetic route, the double bond of the rearranged olefinic product 1-45 was reduced and privileged hydantoin moiety was constructed on the core system through urea formation using variety of isocyanates, isothiocyanates and isoselenocyanates followed by intramolecular cyclization to incorporate elements of diversity. The regeneration of the double bond of intermediate 1-49 afforded hydantoin-fused tetrahydroazepino [4, 5-b]indoles. In the second chapter, an efficient and regioselective synthesis of novel 1,2,3-triazole-fused-1,5,-benzoxazocinones through intramolecular cyclization of substituted ethynyl triazoyl benzoic acids was explored. A crucial precursor 5-iodo-1,2,3-triazole benzoate was obtained from substituted anthranalic acid esters in a single step through CuAAC reaction using CuI/NBS catalytic system. Carbon-carbon triple bond was installed through Sonogashira coupling reaction by various terminal alkynes. Finally, the 1,4,5-substituted ethynyl triazoyl benzoic acids were obtained by AgOTf mediated intramolecular cyclization. The third chapter describes the design and synthesis of new biprivileged molecular scaffolds with diverse structural features. Commercially available, simple heterocyclic building blocks such as 4-fluoro-3-nitrobenzoic acid, 2-chloro-3-nitrobenzoic acid, and indoline were utilized for the synthesis of the novel heterocycles. Pictet–Spengler-type condensation was used as a key step to construct tetracyclic indolo-benzodiazepines and indolo-quinoxalines linked with substituted benzimidazoles. Analysis of single crystals of representative compounds showed that these molecular skeletons have the potential to present various substituents with distinct three-dimensional orientations.

L'obiettivo di questo lavoro di tesi era determinare la struttura tridimensionale di tre proteine umane, Heat shock protein 60 (HSP60), Activation-induced cytidine deaminase (AICDA) e Cholesterol 7-alpha-monooxygenase (CYP7A1), attraverso la tecnica di diffrazione di raggi X. La chaperonina umana HSP60 è una proteina mitocondriale, espressa in maniera costitutiva. La proteina è stata espressa in E. coli e purificata tramite cromatografia di affinità immobilizzata dello ione metallo, utilizzando un tag di sei istidine inserito all'estremità N-terminale della proteina, e tramite cromatografia a esclusione molecolare. Le prove di cristallizzazione non danno risultati positivi, probabilmente per problemi di eterogeneità della proteina. La AICDA umana è una citidina deaminasi, la quale è selettivamente espressa nei linfociti B e svolge un ruolo cruciale nell'ipermutazione somatica e nella ricombinazione per scambio di classe degli anticorpi. L'espressione della proteina è stata tentata in E. coli con differenti vettori ed è stata ottenuta con successo con il plasmide pGEX-4T-1, che permette di esprimere la GST all'estremità N terminale della proteina oggetto di studio; sfortunatamente, il protocollo di purificazione non è stato efficace. La CYP7A1 è un enzima microsomale che catalizza la conversione del colesterolo a 7α-idrossicolesterolo, la prima reazione della sintesi degli acidi biliari, e questa è la reazione limitante la velocità della via metabolica; la proteina è espressa solamente nel fegato. L'espressione della proteina umana CYP7A1 in E. coli è stata difficoltosa e, sebbene siano stati testati differenti vettori, non è stato ottenuto un buon livello di espressione della proteina. Per superare questi problemi, la CYP7A1 di pesce zebra (Danio rerio) è stata espressa in E. coli, ma senza apprezzabili miglioramenti. Un'altra parte della mia tesi ha riguardato la determinazione della struttura tridimensionale della Methionine aminopeptidase 1 umana con due differenti inibitori. La proteina catalizza l'eliminazione della metionina N-terminale dalle proteine nascenti. Inibitori contro questa proteina sono di grande interesse medico grazie al loro potenziale impiego come farmaci antitumorali. Questa parte di tesi è stata svolta presso il laboratorio del Dr. L. Mario Amzel sotto la supervisione della Dr. Sandra B. Gabelli - Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, School of Medicine (Baltimore, USA).
The aim of this thesis work was to determine the three-dimensional structure of three human proteins, Heat shock protein 60 (HSP60), Activation-induced cytidine deaminase (AICDA) and Cholesterol 7-alpha-monooxygenase (CYP7A1), by X-ray diffraction of single crystals. The human chaperonine HSP60 is a mitochondrial protein, expressed in a constitutive manner. The protein was expressed in E. coli and purified by immobilized metal ion affinity chromatography, using a histidine tag fused to the N-terminus of the protein, and by size exclusion chromatography. The crystallization trials do not give positive results, probably due to protein heterogeneity problems. Human AICDA is a cytidine deaminase, which is selectively expressed in B lymphocytes and plays a crucial role in antibody somatic hypermutation and class switch recombination. Protein expression was attempted in E. coli with different vectors and was successfully achieved with the pGEX-4T-1 plasmid, that allows to express GST at the N-terminus of the target protein; unfortunately, the purification protocol was not effective. CYP7A1 is a microsomal enzyme that catalyzes the conversion of cholesterol to 7α-hydroxycholesterol, the first reaction of bile acid synthesis and the rate-limiting step of the metabolic pathway; the protein is expressed only in the liver. The expression of human CYP7A1 in E. coli was troublesome and, although different vectors were tested, a good level of protein expression was not obtained. In order to overcome these problems, zebrafish (Danio rerio) CYP7A1 was expressed in E. coli, but without any substantial improvement. Another part of this thesis work concerned the determination of the three-dimensional structure of the human Methionine aminopeptidase 1 with two different inhibitors. The protein catalyzes the removal of the N-terminal methionine from nascent proteins. Inhibitors against this protein are of great medical interest because of their potential employment as anticancer drugs. This part of the thesis was performed at Dr. L. Mario Amzel's laboratory under the direct supervision of Dr. Sandra B. Gabelli - Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, School of Medicine (Baltimore, USA).

Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes, widely expressed in all kingdoms of life and encoded by eight evolutionarily unrelated gene families: α-, β-, γ-, δ-, ζ-, η-, θ- and ι-CAs. The CAs catalyze the reversible hydration of carbon dioxide into bicarbonate and proton that is physiologically crucial for all living beings because involved in respiration, pH and CO2 homeostasis, transport of CO2/HCO3- and a multitude of biosynthetic reactions. In Homo sapiens, fifteen α-class CA isoforms were identified, which are implicated in a plethora of physiological processes such as electrolytes secretion in many tissues/organs, metabolic reactions (e.g. gluconeogenesis, lipogenesis, ureagenesis), bone resorption, calcification, and carcinogenesis. Thus, an abnormal expression/activity of specific human CAs results in a multitude of human pathological processes that can be targeted with a pharmacological intervention based on CA modulation. Moreover, numerous α-, β-, γ- and η-CAs were identified in many bacteria, protozoa, and fungi that act as human pathogens. In this context, CAs were shown to be crucial for the virulence, growth or acclimatization of the parasites in the hosts. Their inhibition produces growth impairment and defects in the pathogen, being a promising strategy for chemotherapy. The research activity included in this Ph.D. thesis fits in the context of the spreading interest of the scientific community on CAs as drug targets for the treatment of a multitude of disorders. Thus, a set of projects involving drug-design, synthesis, biological evaluation and in silico investigation of the ligand-target interactions of new CA inhibitors (CAIs) were the focus of the three-year Ph.D. cycle. In the first reported project, the concept of tail- (and dual tail) approach was extended up to the three tail approach. This study aimed to increase the selectivity of the benzenesulfonamide scaffold against certain CA isoforms over others, through the introduction of three pendants called “tails” with different lipophilic/hydrophilic nature. Hence, it was possible both to target simultaneously the most variable residues of the inner/outer rim of the hydrophobic and hydrophilic half of the active site and to interact with the peculiar accessory subpockets which differentiate the various isoforms. The synthesized three-tailed inhibitors (TTIs) resulted to be more selective against the glaucoma-associated CA isoforms (hCA II, IV and XII) with respect to the mono-tailed precursors. A massive in silico study of the TTIs-targets interactions was carried out to extend the X-ray crystallography results and the IOP-lowering ability of the three most promising derivatives was evaluated. The second project adopted the multi-targets approach for the treatment of multifactorial diseases, such as inflammation and tumors. Inhibitors of target CA isoforms, to repristinate the correct synovial fluid pH value (against IV, IX and XII), were endowed with an H2S releasing ability, to exploit the gasotransmitter relieving effect in inflammation, developing innovative H2S releaser-CAI hybrids. The stopped-flow kinetic assay pointed out selective inhibition profiles against CAs IX and XII for most synthesized compounds over the off-target CAs I and II. Four compounds were submitted to the Paw pressure and Incapacitance tests to evaluate their ability as anti-inflammatory agents. In the third project, an in silico study was conducted to optimize the CA IV inhibitory properties of a set of benzenesulfonamide derivatives, that produced potent but unselective CA IV inhibition. A peculiar hydrophilic cleft of the CA IV active site (pocket A) was the target for the drug optimization process. The introduction of a pendant with an H-bond acceptor/donor group in a position suitable for the interaction with pocket A was predicted to improve the binding to the target CA and not to off-target isoforms. As a result, the newly reported set of derivatives showed enhanced CA inhibition up to a subnanomolar range. The research of new CAI chemotypes is an important strategy to selectively inhibit specific isoforms over others. In the fourth and fifth projects, the joint use of docking studies, MM-GBSA and molecular dynamic (MD) simulations allowed to evaluate the binding mode of derivatives with CAI scaffolds other than sulfonamides such as hydantoin, saccharin and acesulfame. A zinc binder inhibition mechanism was computed for the hydantoins, which are able to exist in a deprotonated form at the physiological pH. A mechanism based on the anchorage to zinc-bound water molecule was instead proposed and assessed for tertiary sulfonamides, saccharin- and acesulfame derivatives. The sixth project was here reported as representative for a series of in silico investigations carried out to figure out the SAR of several sets of benzenesulfonamide CAIs. The design of N-nitrosulfonamide silver salts was reported to combine the compounds selective inhibition of certain pathogens CAs (over human isozymes) and the antiseptic properties of silver. Indeed, a subset of such compounds showed effective and selective inhibition of the CAs from Trypanosoma cruzi (TcCA) and Leishmania donovani chagasi (LdcCA) compared to hCAs I and II as well as effective chemotherapic action in vitro against several protozoan strains and forms. Another project consisted of the in silico characterization of the binding mode of benzoxaborole derivatives within the α-, β-, and γ-CA active site of the bacterial Vibrio cholerae (VchCA). Primarily the targets homology models were built to proceed therefore with docking studies, MM-GBSA and MD calculations with the boron-based ligands. The computational analysis, for the first time carried out on a γ-class CA, showed that benzoxaborole derivatives adopt both a tetrahedral and trigonal bipyramidal zinc coordination in complex with VchCA (α-CA), a tetrahedral geometry within VchCAβ and two alternative penta-coordinated geometries around the zinc ion in VchCAγ active site. GABAA receptors (GABAARs) are another crucial target for the treatment of several human diseases, such as anxiety disorders, insomnia, epilepsy, restlessness, and aggressive behaviors. Many clinically important drugs were discovered to target GABAARs, interacting at different allosteric binding sites such as benzodiazepine and benzodiazepine site ligands, barbiturates, intravenous and volatile anesthetics, anticonvulsants, ethanol and neuroactive steroids, and performing their pharmacological effects. Nowadays, several natural products such as flavonoids, menthol, magnolol, honokiol, coronaridines and others, were found to modulate GABAARs. All of these compounds exhibit diverse pharmacology mediated by known and unknown binding sites and their potential for clinical application is currently being explored. In this scenario, the identification of the binding site and the investigation of the binding mode of coronaridine congeners, such as (+)-catharanthine, is an actual challenge. The joint use of electrophysiological, radioligand displacement and in silico studies promoted the identification of the (+)-catharanthine binding site at the β(+)α(-), concluding that this ligand acts according to a loreclezole-like mechanism. Finally, during my stage in the organic chemistry laboratory of Prof. Vittorio Pace (University of Vienna) I was involved in a study on the reductive lithiation arene-catalyzed of imines as a new method for the synthesis of amino alcohols.