O intenso uso de compostos orgânicos em conjunto com o grande avanço industrial culminou em um enorme acúmulo de poluentes orgânicos no meio ambiente. Dentre estes poluentes têm-se destacado a presença de hidrocarbonetos aromáticos altamente tóxicos e resistentes à degradação física, química, fotolítica e biológica. Desta maneira, uma nova forma de combater a presença deste tipo de composto no meio ambiente têm sido estudada: o uso de microorganismos, naturais ou geneticamente modificados, capazes de transformá-los em substâncias inertes, como CO2 e água. Tal metodologia é denominada biorremediação. Dentres estes microorganismos destacam-se bactérias dos gêneros Pseudomonas, Aeromonas, Beijerinckia, dentre outros, que têm sido estudadas para esta finalidade. A enzima clorocatecol 1,2-dioxigenase (Pp 1,2-CCD) é uma das proteínas expressas por bactérias do gênero Pseudomonas putida, sendo responsável pela clivagem de hidrocarbonetos aromáticos através da incorporação de ambos os átomos de uma molécula de oxigênio à estrutura do anel aromático, sendo a proteína escolhida para desenvolvermos o presente trabalho. Mais especificamente, nos interessa estudar como o mecanismo de ação da referida enzima é controlado por moléculas extrínsecas, como fosfolipídios. Tal interesse pela interação entre a enzima e fosfolipídios surgiu recentemente quando da obtenção da primeira estrutura cristalográfica de uma enzima da família da CCD (dioxigenases intradióis). Nesta estrutura foi observado um sítio de ligação por monômero para fosfolipídios, o que fez com que várias questões relativas à influência desses sobre a atividade da enzima fossem levantadas. Nosso objetivo foi fazer uso das técnicas de Dicroísmo Circular (CD), Calorimetria e Ressonância Paramagnética Eletrônica (RPE) para estudar alterações conformacionais da enzima e de sua cinética induzidas por moléculas de fosfolipídio, e assim, obter informações que correlacionem as mudanças estruturais com o mecanismo de atividade enzimática da enzima. Os resultados obtidos através do uso daquelas técnicas em conjunto com protocolos que possibilitam a delipidação da enzima mostraram que a presença do fosfolipídios na estrutura da enzima tem influência sobre a atividade enzimática. Quando retiramos o fosfolipídio/ácido graxo, pudemos visualizar uma pequena mudança na estrutura secundária da enzima, um aumento da entalpia de reação, bem como um aumento na velocidade de reação, enquanto que a afinidade da enzima pelo substrato diminuiu. Pudemos também observar uma maior estabilidade térmica da enzima quando na ausência do fosfolipídio/ácido graxo e não foi observado interação da Pp 1,2-CCD com modelos micelares constituídos por lisofosfolipídios. Um breve estudo realizado sobre o papel da força iônica na atividade e na estabilidade térmica da proteína mostrou que na ausência de NaCl, em pH 8, a enzima se mostrou mais ativa, com uma afinidade pelo substrato maior e neste ambiente com baixa força iônica foi observado uma pequena interação da enzima com modelos micelares carregados negativamente. Assim, pudemos concluir que as moléculas anfipáticas, retiradas com os processos de delipidação, apesar de modificarem muito pouco a estrutura secundária da enzima, ainda assim instauram modificações na sua função de catálise do substrato catecol. Esta informação juntamente com os dados sobre inibição do processo reacional ocasionada pelo produto da reação formam um novo conjunto de dados que pode ser utilizado para se alcançar o objetivo mais geral de se controlar a atividade biológica da Pp 1,2-CCD. / The intensive use of organic compounds in conjunction with the industrial advances led to a huge accumulation of organic pollutants in the environment. Among these pollutants it has been noticed the presence of aromatic hydrocarbons that are highly toxic and resistant to physical, chemical and biological degradation. Thus, a new way to deal with the presence of this compounds in the environment has been studied: the use of microorganisms, natural or genetically modified, that can turn them into inert substances such as CO2 and water. This methodology is called bioremediation. Among those microorganisms, bacteria from the gender Pseudomonas, Aeromonas, Beijerinckia, among others, have been studied for this purpose. The enzyme chlorocatechol 1,2-dioxygenase (Pp 1,2-CCD) is one of the proteins expressed by Pseudomonas putida bacteria, being responsible for the cleavage of aromatic hydrocarbons through the incorporation of both atoms of a molecule of oxygen into the aromatic ring structure, being the protein chosen for investigation in this work. More specifically, we are interested in studying how the mechanism of action of this enzyme is controlled by extrinsic molecules such as phospholipids. The interest in the interaction between the enzyme and phospholipids arose recently when the first crystal structure of an enzyme of the intradiol dioxygenase family was reported. In this structure it was observed a binding site for a phospholipid per monomer, which raised many issues concerning its influence on the activity of the enzyme. Our goal was to use the techniques of Circular Dichroism (CD), calorimetry and Electron Magnetic Resonance (EMR) to study enzyme conformational changes and kinetics alterations induced by phospholipid molecules, thus gathering information on the structure-function correlation. The results obtained through those experimental techniques in conjunction with the use of protocols for protein delipidation showed that the presence of phospholipids/fatty acids in the structure of the enzyme play a role in enzyme activity. Upon removal of the phospholipid/fatty acids, we observed small changes in the secondary structure of the enzyme, an increase of the enthalpy of reactions as well as an increase in the reaction rate, whereas the affinity of the enzyme for the substrate decreased. We also observed a higher thermal stability of the Pp 1,2-CCD in the absence of the phospholipids/fatty acids, but no interaction was observed between the Pp 1,2-CCD and lysophospholipid micelles. A brief study of the function of ionic strength on the activity and thermal stability of the protein showed that in the absence of NaCl, at pH 8, the enzyme is more active, showing a greater affinity for the substrate and a low interaction was observed between Pp 1,2-CCD and negatively charged micelles. This information along with the data on the inhibition capacity of the reaction product are a new set of data that can be used to achieve the more general goal of controlling Pp 1,2-CCD biological activity.

<p>This thesis focuses on peptides derived from the Prion, Doppel and Influenza haemagglutinin proteins in the context of bilayer interactions with model membranes and live cells. The studies involve spectroscopic techniques like fluorescence, fluorescence correlation spectroscopy (FCS), circular and linear dichroism (CD and LD), confocal fluorescence microscopy and NMR.</p><p>The peptides derived from the Prion and Doppel proteins combined with their subsequent nuclear localization-like sequences, makes them resemble cell-penetrating peptides (CPPs). mPrPp(1-28), corresponding to the first 28 amino acids of the mouse PrP, was shown to translocate across cell membranes, concomitantly causing cell toxicity. Its bovine counterpart bPrPp(1-30) was demonstrated to enter live cells, with and without cargo, mainly via macropinocytosis. The mPrPp(23-50) peptide sequence overlaps with mPrPp(1-28) sharing the KKRPKP sequence believed to encompass the driving force behind translocation. mPrPp(23-50) was however found unable to cross over cell membranes and had virtually no perturbing effects on membranes.</p><p>mDplp(1-30), corresponding of the first 30 N-terminal amino acids of the Doppel protein, was demonstrated to be almost as membrane perturbing as melittin. NMR experiments in bicelles implied a transmembrane configuration of its alpha-helix, which was corroborated by LD in vesicle bilayers. The positioning of the induced alpha-helix in transportan was found to be more parallel to the bilayer surface in the same model system.</p><p>Positioning of the native Influenza derived fusion peptide in bilayers showed no pH dependence. The glutamic acid enriched variant however, changed its insertion angle from 70 deg to a magic angle alignment relative the membrane normal upon a pH drop from 7.4 to 5.0. Concomitantly, the alpha-helical content dramatically rose from 18% to 52% in partly anionic membranes, while the native peptide’s helicity increased only from 39% to 44% in the same conditions.</p>

G-quadruplex structures are found in important regions of the eukaryotic genome, such as telomeres and regulatory sequences of genes, and are likely to play important roles in regulation of biological events. The significant structural differences with duplex DNA make quadruplex DNA a very attractive target for anticancer drug design. The purpose of this study is to explore conformational space in a series of heterocyclic cations to discover novel structural motifs that can selectively bind and stabilize specific G-quadruplex arrangements. A variety of biophysical techniques such as thermal melting experiments, biosensor surface plasmon resonance, circular dichroism, fluorescence displacement assay and mass spectrometry were employed to evaluate the affinity of the compounds and their recognition properties. The screening of the molecules allowed the identification of not only selective G-quadruplex ligands but also potential quadruplex groove binders. These results can be useful for the development of new efficient telomerase inhibitors which are endowed with pharmacological activity.

There is a high demand for quadruplex-specific compounds that not only bind preferentially to quadruplex DNA over duplex DNA, but also bind to one quadruplex motif over other motifs. Quadruplex structures are recognized as common occurrences in cancer cells, and if a compound could stabilize this structure, it may serve as an effective anti-cancer treatment with minimal side effects. In this study, cyanine dyes’ interactions with DNA were analyzed with fluorescence titrations, UV-Vis thermal studies, circular dichroism titrations, and surface plasmon resonance (SPR) analysis. With these techniques, binding affinity, DNA stabilization, and conformational shifts were analyzed to determine if cyanine dyes could act as quadruplex-specific binding compounds for possible cancer treatments.

Amyloid-proteins are among the most actively researched biological topics today, because they have been associated with many serious human diseases, such as Alzheimer's disease and type II diabetes. In particular the deposition of protein aggregates on cell membranes has been suspected as the causes of the diseases, although the proof is still elusive. Studying the interactions of amyloid-forming peptides with lipid-bilayer membranes may clarify the pathway of the β-aggregate formation and provide new insights into the amyloid hypothesis of diseases. In this thesis, I investigate how three peptides, penetratin, amylin, and LL-37, interact with lipid membranes by using several techniques well-developed in our lab. In the study of penetratin interacting with lipid membranes, we were able to clarify the energy pathway of amyloid formation mediated by membrane-binding. This provides the sole experimental proof for the Jarrett-Lansbury theory of β- amyloid formation. Our investigation on amylin-membrane interaction clarifies how amylin in different forms damage bilayer membranes. Between penetratin and amylin we have clarified the complicated pattern of interactions between amyloid-forming peptides and lipid bilayers. The third peptide LL-37 studied in my thesis turned out to a pore forming peptide. I found the mistake made by previous investigators in several different laboratories that made them erroneously conclude that LL-37 was not a pore forming peptide. The results of these three peptides show that methods we used are a comprehensive set of tools that can reveal a broad range of peptide properties. Both the formation of amyloid aggregates and formation of membrane pores can be explained by a two-state model proposed by Huang describing peptide-membrane interactions. For LL-37, the second state is a pore in membrane. But for penetratin and amylin the second state is an aggregation in the β form. We found that β-aggregates have low affinity within a lipid bilayer, and therefore exit from the bilayer structure. However, this exit process extracts lipid molecules from the bilayer and incorporates them in the peptide aggregates. We suggest that this is the molecular process of how amylin might damage of the membranes of β-cells.

Cations are essential for ribonucleic acids (RNA), as they neutralize the negatively charged phosphate backbone. Divalent metals play important roles in the folding and function of RNA. The relationship between RNA and divalent cations magnesium (Mg(II)) and iron (Fe(II)) has been investigated. Mg(II) is involved in tertiary interactions of many large RNAs, and necessary for ribozyme activity. The influence of Mg(II) on RNA secondary and tertiary structure is investigated experimentally. Mg(II) binding to A-form RNA is accompanied by changes in CD spectra, indicating that Mg-RNA interactions influence the helical structure of RNA duplexes and helical regions of unfolded RNAs. Quantum mechanics calculations are used to probe the energetics of Mg(II)-chelation with phosphate oxygen atoms of nucleic acids. We identify the specific forces that contribute to stability of Mg(II)-chelation complexes in RNA. Fe(II) can serve as a substitute for Mg(II) in RNA folding and function. Fe(II) was abundant on early earth, it is plausible that RNA folding and function was mediated by Fe(II) instead of, or in combination with, Mg(II) in the anoxic environment of early earth. We have investigated oxidoreductase catalytic activity observed in RNA when in combination with Fe(II). This activity, only observed in the presence of Fe(II) and absence of Mg(II)appears to be a resurrection of ancient RNA capabilities that were extinguished upon the depletion of Fe(II) from the environment during the rise of oxygen after the great oxidation event. Finally, metal-ion based cleavage of RNA is used to identify the binding sites of Mg(II) and Fe(II). We observe that both metals cleave RNA in similar positions, providing further support for Fe(II) as a substitute for Mg(II) in RNA.

Supramolekulinė chemija – tyrimų kryptis, nagrinėjanti struktūras, sudarytas iš riboto ir neriboto skaičiaus molekulių (tektonų), sąveikaujančių tarpusavyje silpnosiomis nekovalentinėmis sąveikomis. Žinoma, kad medžiagų savybės užkoduotos ne tik molekulių struktūroje, bet ir jų tarpusavio išsidėstyme. Savo ruožtu, chirališkumas yra vienas iš faktorių, leidžiančių vienoms molekulėms atpažinti kitas. Pagrindiniai disertacijos tikslai: nustatyti 1) chiralinių, konformaciškai suvaržytų bei labilių junginių, turinčių biciklo[3.3.1]nonano fragmentą, chromoforų prigimties, tarpusavio orientacijos ir atstumo įtaką chiroptinėms savybėms, 2) chiralinių tripakeistų aromatinių, turinčių biciklo[3.3.1]nonano pakaitus, ir kompleksinių paladžiociklinių junginių chirališkumo ir struktūros įtaką formuojant įvairaus lygio tvarkias supramolekulines struktūras. Naudojantis apskritiminio dichroizmo spektroskopijair teoriškai atliktais ab initio skaičiavimais charakterizuotos molekulės, turinčios įvairios elektroninės prigimties chromoforus, bei jose vykstantys elektroniniai šuoliai. Susintetinti tripakeisti aromatiniai junginiai, turintys išorinius biciklo[3.3.1]nonano ir įvairių dydžių aromatinius fragmentus. Ištirta tokių save atpažįstančių chiralinių tripakeistų aromatinių junginių struktūros įtaka supramolekulinei asociacijai tirpale ir ant paviršiaus. Nustatyta, kad susintetinti V formos chiralinis ir raceminis dialkinbiciklo[3.3.1]nonenil- ligandai, turintys koordinuojantį piridino pakaitą... [toliau žr. visą tekstą] / The supramolecular chemistry of assemblies composed of a limited or infinite number of the molecular tectons interacting with each other via noncovalent interactions was investigated with a special emphasize on the chirality of the building blocks. The following objectives were pursued in this work: 1) to determine the electronic structure of both conformationally rigid and labile chiral bicyclo[3.3.1]nonane compounds, the mutual orientation and distance of the chromophores and its impact on chiroptical properties, 2) to study the influence of chirality and structure of palladacycle and trisubstituted compounds, containing external bicyclo[3.3.1]nonanyl- and aromatic fragments of different size on the formation of various supramolecular structures. The chiral bicyclo[3.3.1]nonane compounds with chromophores of different electronic nature were synthesized. The possibilities of exciton interaction and charge transfer phenomena were studied in the obtained molecules. The influence of chirality and structure of trisubstituted compounds containing external bicyclo[3.3.1]nonanyl- and aromatic fragments of different size on supramolecular association in solution and on the surface was investigated. In solution, the trisubstituted compounds exist in the form of nanoparticles with regular supramolecular structure. It was shown that the V-shaped chiral and racemic dialkynbicyclo[3.3.1]nonenyl- ligands having coordinating pyridine moiety, form rhomb-shaped palladacycle. The racemic and... [to full text]

There is currently a great interest in the field of porous organosilica materials because of the high surface areas (> 1000 m²/g) and narrow pore size distributions which are beneficial for applications such as chromatography, chiral catalysis, sensing or selective adsorption. Periodic mesoporous organosilicas (PMOs) represent an interesting class of hybrid silica materials because of the wide variety of bridging organic groups which can be incorporated within the precursors [(OR)3Si-R-Si(OR)3] giving rise to materials with exceptional properties. We have synthesized and characterized various aromatic PMOs composed of supporting structural monomers (phenylene- or biphenylenebridged) and functional stilbene monomers (cis and trans) (1, 2). The effect of the different synthetic procedures and varying amounts of functional stilbene monomer on the properties of the materials was examined. The functional transstilbene component was determined to be well distributed in a phenylene-bridged PMO using P123 as a pore template from TEM techniques with Os staining. The trans-stilbene linkers were completely transformed to aryl aldehydes through ozonolysis with dimethylsulfide workup. Further transformation of the carbonyl functionality to an aryl imine showed a moderate level of success. Enantiomeric forms of a novel, chiral PMO precursor (CM) were synthesized and incorporated into biphenylene-bridged PMOs. Under basic pH conditions templated with C18TMACl, although very low levels of CM are incorporated, enantiomeric forms of chiral, porous materials are obtained as was verified by distinct mirror-image circular dichroism spectra. Powder XRD patterns suggest that a tightly packed asymmetric biphenylene arrangement may be necessary for the optical activity. Preliminary results using these materials as a chiral chromatographic phase are promising. Finally, a thin film morphology of an ethane-bridged PMO incorporating a thiol ligand, (3-mercaptopropyl)trimethoxysilane, was prepared on a fibre optic cable and used as a component in a heavy-metal sensing application. / Thesis (Ph.D, Chemistry) -- Queen's University, 2011-03-11 17:24:48.997

Kidney bean proteins and peptides can be developed to serve as an important ingredient for the formulation of high quality foods or therapeutic products that may positively impact on body function and human health. The main goal of this thesis was to determine the in vitro structural and functional characteristics of major proteins and enzymatic protein hydrolysate of red kidney bean (Phaseolus vulgaris). Selective aammonium sulfate precipitation of the kidney bean proteins yielded 88% globulin and 7% albumin.The globulin and albumin are glycoproteins that contained ~4% and 45% carbohydrate contents, respectively. Physicochemical and functional characteristics of the globulin fraction, such as, gelation concentration, foam stability, emulsion capacity, and emulsion stability were superior to those of albumin. Reducing SDS-PAGE revealed vicilin with molecular weight of ~45 kDa as the major globulin in kidney beans. Circular dichroism spectroscopy of the purified vicilin showed reductions in α-helix, and β-pleated sheet conformations upon addition of NaCl or changes in pH. Likewise, the tertiary structures as observed from the near-UV CD spectra were also changed by shifts in pH conditions and NaCl addition. Far UV-CD showed increased β-sheet content up till 60oC from room temperature, but a steady loss in the tertiary structure as temperature was further increased; however, β-sheet structure was still detectable at 80oC. Differential scanning calorimetry thermograms showed a prominent endothermic peak with denaturation temperature at around 90oC, attributed to thermal denaturation of vicilin. Alcalase hydrolysis of kidney bean globulin produced multifunctional peptides that showed potential antihypertensive properties because of the in vitro inhibition of activities of renin and angiotensin I converting enzyme as well as the antioxidant properties. The <1 and 5-10 kDa peptide fractions exhibited highest (p<0.05) renin inhibition and the ability to scavenge 2, 2-Diphenyl-1-picrylhydrazyl free radical, inhibit peroxidation of linoleic acid and reduce Fe3+ to Fe2+. Based on this study, incorporation of kidney bean globulin as an ingredient may be useful for the manufacture of high quality food products. Likewise, the kidney bean protein hydrolysates, especially the <1 kDa fraction represent a potential source of bioactive peptides for the formulation of functional foods and nutraceuticals.

The supramolecular chemistry of assemblies composed of a limited or infinite number of the molecular tectons interacting with each other via noncovalent interactions was investigated with a special emphasize on the chirality of the building blocks. The following objectives were pursued in this work: 1) to determine the electronic structure of both conformationally rigid and labile chiral bicyclo[3.3.1]nonane compounds, the mutual orientation and distance of the chromophores and its impact on chiroptical properties, 2) to study the influence of chirality and structure of palladacycle and trisubstituted compounds, containing external bicyclo[3.3.1]nonanyl- and aromatic fragments of different size on the formation of various supramolecular structures. The chiral bicyclo[3.3.1]nonane compounds with chromophores of different electronic nature were synthesized. The possibilities of exciton interaction and charge transfer phenomena were studied in the obtained molecules. The influence of chirality and structure of trisubstituted compounds containing external bicyclo[3.3.1]nonanyl- and aromatic fragments of different size on supramolecular association in solution and on the surface was investigated. In solution, the trisubstituted compounds exist in the form of nanoparticles with regular supramolecular structure. It was shown that the V-shaped chiral and racemic dialkynbicyclo[3.3.1]nonenyl- ligands having coordinating pyridine moiety, form rhomb-shaped palladacycle. The racemic and... [to full text] / Supramolekulinė chemija – tyrimų kryptis, nagrinėjanti struktūras, sudarytas iš riboto ir neriboto skaičiaus molekulių (tektonų), sąveikaujančių tarpusavyje silpnosiomis nekovalentinėmis sąveikomis. Žinoma, kad medžiagų savybės užkoduotos ne tik molekulių struktūroje, bet ir jų tarpusavio išsidėstyme. Savo ruožtu, chirališkumas yra vienas iš faktorių, leidžiančių vienoms molekulėms atpažinti kitas. Pagrindiniai disertacijos tikslai: nustatyti 1) chiralinių, konformaciškai suvaržytų bei labilių junginių, turinčių biciklo[3.3.1]nonano fragmentą, chromoforų prigimties, tarpusavio orientacijos ir atstumo įtaką chiroptinėms savybėms, 2) chiralinių tripakeistų aromatinių, turinčių biciklo[3.3.1]nonano pakaitus, ir kompleksinių paladžiociklinių junginių chirališkumo ir struktūros įtaką formuojant įvairaus lygio tvarkias supramolekulines struktūras. Naudojantis apskritiminio dichroizmo spektroskopijair teoriškai atliktais ab initio skaičiavimais charakterizuotos molekulės, turinčios įvairios elektroninės prigimties chromoforus, bei jose vykstantys elektroniniai šuoliai. Susintetinti tripakeisti aromatiniai junginiai, turintys išorinius biciklo[3.3.1]nonano ir įvairių dydžių aromatinius fragmentus. Ištirta tokių save atpažįstančių chiralinių tripakeistų aromatinių junginių struktūros įtaka supramolekulinei asociacijai tirpale ir ant paviršiaus. Nustatyta, kad susintetinti V formos chiralinis ir raceminis dialkinbiciklo[3.3.1]nonenil- ligandai, turintys koordinuojantį piridino pakaitą... [toliau žr. visą tekstą]