Polyethylene glycol-stabilized lipodisks have emerged as a novel type of lipid-based nanoparticles with high potential as both drug carriers and biomimetic membranes. In this thesis we assess both of these applications, and show how the properties of the lipodisks can be further developed and optimized. Initially, we show that the antimicrobial peptides melittin, alamethicin and magainin 2, in spite of their very different physico-chemical properties and suggested modes of action on membranes, all have high affinity to lipodisks. Using melittin as a model peptide, we confirm a maintained antimicrobial effect of disk-formulated peptides. We also show that melittin dissociates slowly from the disks, resulting in extended drug release and prolonged antibacterial effect. Additionally, we present evidence that the peptide is protected against enzymatic degradation when formulated in the disks. Further, we develop a stable HPLC-MS system with immobilized lipodisks as model membranes. The stability of the system is confirmed by drug partitioning analysis using 15 different drug compounds. We also show how the lipodisk column can be supplemented with cyclooxygenase by in situ incorporation of the protein in the lipodisks. The specific binding of the protein to the disks is confirmed using QCM-D. Finally, by changing the polymer length and applying a new preparation protocol, we have optimized the lipodisks for use as drug carriers and biomimetic membranes. Previous lipodisk studies have been conducted on systems containing PEG-lipids with polymer molecular weights of 2000 or 5000 Da. Also, conventional protocols for the preparation of lipodisks typically require a PEG-lipid concentration of 15 mol% or more. Here we show that stable lipodisks can also be produced using PEG-lipids with a 1000 Da molecular weight polymer and that the use of shorter PEG-lipids dramatically improve the amount of lipodisks that can be immobilized on silica surfaces. Moreover, through the development of a method in which lipid mixtures are sonicated at low temperatures, we produce lipodisks containing as little as 2 mol% PEG-lipid. We present data verifying that these disks are superior to disks with higher PEG-lipid content in terms of their ability to incorporate externally added PEG-lipids functionalized with targeting agents.

By using cryo-electron microscopy, we analyzed the morphology and structure of long double-stranded DNA chains condensed upon addition of varying amounts of the tetravalent polycation spermine (polyamine). Experiments have been performed i) with chains diluted in the bulk and ii) with individual chains confined in a virus capsid.Bulk experiments have been done with lambda DNA (48.5 kbp) at low concentration (0.03 mM Ph) and in low salt conditions (10 mM Tris HCl, 1 mM EDTA, pH 7.6). We explored a wide range of spermine concentration, from the onset of precipitation (0.05 mM sp) up to above the resolubilization limit (400 mM sp). Sixteen min after mixing spermine and DNA, samples have been trapped in thin films and vitrified in liquid ethane to keep ionic conditions unchanged, and imaged at low temperature with low doses of electrons (cryoTEM). DNA chains mostly form large aggregates of toroids in which DNA chains are hexagonally packed with interhelical spacings of 2.93, 2.88, and 2.95 nm at 0.05, 1 and 100 mM spermine, respectively, in agreement with previous X-ray data. At higher spermine concentration (200 mM), hexagonal toroids are replaced by cholesteric bundles with a larger interhelical spacing (3.32 nm). We conclude that the shape and the structure of the liquid crystalline sp-DNA condensates are linked to the DNA interhelix spacing and determined by the ionic conditions i.e. by the cohesive energy between DNA strands. Outside of the precipitation domain (400 mM spermine), DNA chains form a soluble network of thin fibers (4-6 nm in diameter) that let us reconsider the state of these DNA chains in excess of spermine. We also designed experiments to visualize condensates formed 6-60 sec after mixing Lambda DNA with 0.05 mM spermine, under identical buffer conditions. Among multiple original shapes (not found after 16 min), the presence of stretched and helical elongated fibers seen only 9sec after addition of spermine let us propose that DNA chains are immediately stretched upon addition of spermine, relax into helical structures and finally form small toroids (containing in some cases less than one Lambda chain) that further grow and aggregate. We also analyzed the dimensions and structural details of the complete collection of toroids, and reveal the existence of geometric constraints that remain to be clarified. Since it was only exceptionally possible to prevent the aggregation of DNA in dilute solution, we used another approach to observe the collapse of single DNA chains. We handled a population of T5 viruses containing a fraction of their initial genome (12-54 kbp long). The Na-DNA chain, initially confined in the small volume of the capsid (80nm in diameter) is collapsed by the addition of spermine. Compared to the first set of experiments, we explored a higher DNA concentration range (0.45 mM Phosphates in the whole sample) and the spermine concentration was varied from 0.05 to 0.5 mM (which corresponds to much lower +/- charge ratios). Experiments are thus performed close to the precipitation line, in the coexistence region, between the region where all chains are in a coil conformation, and the region where all chains are collapsed into toroids. We describe the existence of intermediate states between the coil and the toroidal globule that were not reported yet. In these "hairy toroids", part of the DNA chain is condensed in the toroid and the other part stays uncondensed outside of it. The interhelical spacing was also measured; it is larger in these partly-condensed toroids than in the fully organized toroids formed at higher spermine concentration.These two series of experiments show the interest of cryoEM to analyze the structural polymorphism and local structure of spermine-DNA aggregates. We also demonstrated how the confinement interferes with DNA condensation and the interest to investigate such effects that are important in the biological context.

Nous avons utilisé la méthode de CEMOVIS (Cryo-Electron Microscopy Of Vitreous Sections) pour étudier l’ultrastructure des cellules HT29 (lignée cancéreuse colique humaine) et plus particulièrement l’organisation de la chromatine au sein du noyau. Pour améliorer la méthode, nous avons développé un micromanipulateur qui facilite la collecte des coupes et leur transfert sur la grille. Nous avons également cherché à préparer de nouveaux films métalliques (en remplacement du carbone) permettant une meilleure adhésion des coupes sur le support Au vu des premiers tests réalisés, les films de TiO2 que nous avons fabriqués au laboratoire et caractérisés par microscopie électronique (HR, spectroscopie et cartographie EELS) semblent offrir des perspectives intéressantes que nous attribuons à leur propriétés de conducteur électrique à basse température (ce qui reste à démontrer). Les organites cellulaires (noyaux, réseaux de filaments du cytosquelette, systèmes multilamellaires) ont été identifiés in situ. Les conditions d’imagerie choisies nous ont permis d’obtenir une résolution permettant d’identifier les deux feuillets des bicouches membranaires. Dans le noyau, nous avons observé des motifs striés, distants de 2.7 à 3.5 nm que nous attribuons à la molécule d’ADN enroulée autour du cœur d’histones. Comparées aux images de phases denses de nucléosomes, ces images suggèrent que les nucléosomes (jamais identifiés in situ jusqu’à présent) présentent un ordre très local au sein de la chromatine, que nous discutons à la lumières des modèles polymériques actuels. / The ultrastructure of HT29 cells (human epithelial adenocarcinoma cell line) was studied by CEMOVIS (Cryo-Electron Microscopy of Vitreous Sections) with a special emphasis on chromatin organization in the cell nucleus. We proposed methodological improvements for this technique:- We first developed a grid holding micromanipulator to facilitate both cryosections collect and deposition on carbon-coated TEM grids.- We also developed new metallic thin films (to replace carbon-base supports) to enhance the adhesion of cryosections on their support. The TiO2 thin films that we produced and analysed by electron microscopy (high resolution imaging, EELS and chemical mapping) seem to be an interesting alternative to carbon films for the deposition of cryosections. Their adhesive properties could be due to Titanium high electric conductance at low temperature (although this relation has not been clearly demonstrated yet).In HT 29 cells, we indentified cell organites (nucleus; cytoskeleton filament bundles, multilamellar bodies) in situ. Selected imaging conditions provide for a high enough resolution to visualise the two membrane leaflets. In the cell nucleus, we observed striated patterns separated from 2.7 to 3.5 nm that we assume to be DNA molecule turns wrapped around the histone protein core. Compared with the dense phases formed in vitro by nucleosome core particle in solution, our images suggest that nucleosomes are locally ordered in chromatin. This observation is discussed regarding the chromatin polymeric models.

Nanomedicine is one of the most promising fields in nanotechnology nowadays. The use of nanoparticles as carriers aims to improve efficiency of drugs that possess low solubility in aqueous environment (very hydrophobic molecules) or that have a lot of undesired side effects. In this way, nanoparticles offer both a protection for the molecules and a carrying vehicle. On this ground, cubosomes are nanoparticles capable of storing both hydrophilic and hydrophobic molecules within its structure, in addition, cubosomes have approximately 50% hydrophilic and hydrophobic areas. Therefore, they can carry much more molecules than liposomes for instance. In particular, cubosomes are quite easy to produce due to its base product, lipids (like monoolein (GMO) or phytantriol (PHY)) that self-assembly in water media. In this project, both lipids were chosen to produce the cubosomes from well-established protocols in literature. A model drug, miletofsine (MILT), was chosen to study the interaction of such nanosystem with a guest molecule. GMO cubosomes revealed to have Im3m crystallographic symmetry and lattice parameter 15.3(7) nm, particles presented sizes 300(8) nm and moderate polydispersion 0.160(20). TEM revealed squared particles with sizes ~350 nm, cryo-EM presented particles with internal structure and varied size (from 200 to > 500 nm). From FFT analysis, the calculated lattice parameter remained in the order of ~10 nm compatible with SAXS measurements. MILT loading into cubosomes was possible up to 4% w/w without loss of cubosomes structure. For 5% w/w MILT, the nanoparticles were already loosing their crystalline structure, as evidenced by cryo-EM. TEM analysis reveals that as more MILT is loaded into the cubosomes, their sizes increased. For sample 1.5% w/w MILT cryo-EM presents nanoparticles with organized internal structure and an envelope (hypothesized to be a polymer coating) in its surface. Calculated lattice parameters are in the order of ~10 nm. Myverol (Myv) is a commercial mixture that contains ~60% GMO, in this project it was proposed a bottom up protocol for Myv-based cubosomes. The production of these nanoparticles also revealed, by SAXS, Im3m symmetry and lattice parameter 12.30(12) nm. DLS revealed particle size 280(5) nm and moderate polydispersion 0.115(52). TEM shows square and cubic nanoparticles with sizes ~500 nm. MILT loading into Myv-cubosomes revealed that the drug interacts with the nanoparticle by enlarging their lattice parameter as more MILT is loaded (up to 4% w/w). Curiously, for some MILT concentrations the presence of other unknown cubic structures was evidenced by SAXS. TEM revealed nanoparticles with huge polydispersion, with sizes raging from 200 nm to 2 µm. PHY based cubosomes were successfully reproduced by the chosen protocol, in both water, PBS buffer and 2.25% glycerol medium. SAXS revealed crystallographic structure Pn3m and lattice parameter 6.74(04) nm. DLS measured sizes ~450 nm and moderate polydispersion 0.161(10). NTA measurements were consistent with DLS, revealing a broad size distribution and total particle concentration of ~1016 particles/mL for each sample. TEM revealed square and rounder particles in varied size. Cryo-EM micrographs presented particles with internal structure and varied size confirming moderate polydispersion. The FFT analysis revealed calculated lattice parameters ~6.5 nm, compatible with SAXS data. Samples were submitted to lyophilization and found that after re-hydration they still hold the same characteristics (morphology, size) as the original sample. Extrusion was also performed in order to improve polydispersion and control particle size, again cubosomes held their internal structure after the process, diminishing their sizes and improving monodispersion. MILT was loaded into cubosomes via co-solubilization and addition after the nanoparticles were formed. Up to 5% w/w the cubosomes incorporated MILT without loss of crystallographic structure, but at 10%, 15% and 20% w/w, the drug provoked phase change for Im3m symmetry. At the lower concentrations, MILT enlarged the lattice parameter of cubosomes and it was hypothesized that MILT inserted itself into the bilayer of the nanoparticles. DLS reveales that the drug does not change particle size or polydispersion. TEM revealed square and rounder particles in sizes slightly bigger than DLS. For sample 4% w/w, Cryo-EM presented particles with internal structure and calculated lattice parameter ~7 nm compatible with SAXS measurements for this sample. Co-solubilization and addition after nanoparticle preparation proved out to have the same effect on cubosomes loaded with MILT. All samples were submitted to higher temperatures to investigate phase change, based on phase diagram of the lipid. It was found that for the blank samples at 65 °C the cubosomes suffer phase change for isotropic phase L2, when MILT is loaded into the nanoparticles this phase change does not happen. DLS revealed also that at higher temperatures, particle size does not change, neither polydispersion. Finally, cubosomes proved to be remarkable nanoparticles that hold their physico-chemical characteristics even when submitted to extreme environments (lyophylization, extrusion and higher temperatures.) / Nanomedicina é o campo de estudo mais promissor dentro da nanotecnologia atualmente. O uso de nanopartículas visa melhorar a eficiência de fármacos que possuem baixa solubilidade em meios aquosos (moléculas muito hidrofóbicas) ou que possuem muitos efeitos colaterais indesejados. Neste contexto, as nanopartículas oferecem proteção e veículo para tais moléculas. Para isso, cubossomos são nanopartículas capazes de encapsular tanto as moléculas hidrofóbicas como as hidrofílicas em sua estrutura. Cubossomos também apresentam aproximadamente 50% de áreas hidrofílica e hidrofóbica, sendo capaz de encapsular grandes quantidades de moléculas teóricamente. Particularmente, cubossomos são nanopartículas de fácil produção devido à sua matéria prima serem lipídios (por exemplo, monoleína (GMO) ou fitantriol (PHY)) que se auto associam em meio aquoso. Neste projeto, os dois lipídios citados foram escolhidos para a produção dos cubossomos empregando-se protocolos bem estabelecidos da literatura. Uma fármaco modelo, miltefosina (MILT), foi escolhida para o estudo da interação com as nanopartículas. Cubossomos de monoleína (GMO) revelaram simetria cristalográfica Im3m e parâmetro de rede de 15.3(7) nm, as nanopartículas apresentaram tamanhos em torno de 300(8) nm e PDI 0.160(20). MET revelou partículas quadradas com tamanhos ~350 nm e a crio microscopia mostrou partículas com estrutura interna bem definida e tamanhos variados (200 a 500 nm), os parâmetros de rede calculados se mostraram da ordem de ~10 nm, compatíveis com as medidas de SAXS. O encapsulamento da MILT nos cubossomos foi possível até 4% w/w sem perda de morfologia. Para 5% w/w MILT as nanopartículas já apresentavam perda de cristalinidade na sua estrutura, evidenciado por crio microscopia. Análise por MET revelou que quanto mais MILT era encapsulada nos cubossomos, maiores ficaram as nanopartículas. Com a amostra de 1.5% w/w foi feita a crio microscopia, que revelou cubossomos com estrutura interna bem definida e um envelope (possivelmente formado pelo polímero) na sua superfície. Os parâmetros de rede calculados foram da ordem de ~10 nm também. O myverol (Myv) é uma mistura comercial que contém aproximadamente 60% de GMO, e neste projeto foi proposto um protocolo bottom up para cubossomos feitos de Myv. A produção dessas nanopartículas também revelou, por SAXS, estrutura cristalográfica Im3m e um parâmetro de rede de 12.30(12) nm. DLS apresentou partículas de tamanho 280(5) nm e polidispersão moderada 0.115(52). MET mostrou partículas quadradas e cúbicas com tamanhos de ~500 nm. O encapsulamento da MILT revelou que o fármaco interage com os cubossomos aumentando seu parâmetro de rede, até uma concentração de 4% w/w. Curiosamente, para algumas concentrações de MILT havia presença de outras estruturas evidenciadas por SAXS. MET revelou nanopartículas com muita polidispersão, com tamanhos variando entre 200 nm e 2 µm. Cubossomos de PHY foram reproduzidos com sucesso a partir do protocolo escolhido, em meios aquoso, tampão PBS e 2.25% glicerol. SAXS revelou nanopartículas com simetria cristalográfica Pn3m e parâmetro de rede 6.74(04) nm. Por DLS, o tamanho das partículas foi de ~450 nm e polidispersão moderada 0.161(10). Medidas de NTA foram consistentes com DLS, mostrando uma larga distribuição de tamanhos e concentração de partículas ~1016 partículas/mL. MET revelou cubossomos quadrados e mais arredondados de tamanhos variados. Criomicroscopia apresentou partículas com estrutura interna bem definida, tamanhos variados (confirmando a polidispersão) e parâmetro de rede calculado em ~6.5 nm, compatível com medidas de SAXS. Essas amostras também foram submetidas a liofilização e descobriu-se que mesmo depois da re-hidratação, as partículas ainda mantiveram as mesmas características da amostra original. A extrusão também foi feita com o objetivo de melhorar a polidispersão e controlar o tamanho das partículas, novamente, os cubossomos demonstraram manter sua estrutura interna depois desse processo, diminuindo seus tamanhos e diminuindo a polidispersão dos sistema. MILT foi encapsulada de duas formas: passiva (co-solubilização) e ativa (adição depois que as nanopartículas foram formadas). Com até 5% w/w de MILT incorporada, os cubossomos mantiveram sua estrutura cristalográfica, porém em concentrações de 10%, 15% e 20% w/w, o fármaco provocou transição de fase para simetria Im3m. Em baixas concentrações, MILT aumentou os parâmetros de rede dos cubossomos e a hipótese levantada foi que a droga se insere na bicamada lipídica das nanopartículas. DLS revelou que MILT não altera o tamanho das partículas nem sua polidispersão. MET revelou partículas quadradas e arredondadas com tamanhos maiores que os medidos por DLS. Para a amostra 4% w/w, a crio microscopia foi realizada e as partículas encontradas apresentaram estrutura interna e parâmetro de rede calculado ~7 nm, compatível com medidas de SAXS. Co-solubilização e adição depois do preparo se mostraram equivalentes para o encapsulamento da MILT. Todas as amostras também foram submetidas a um estudo de temperaturas para investigar transições de fase, baseando-se nos diagramas de fase dos lipídios. Foi descoberto que os cubossomos, sem a droga, a 65 °C sofrem transição para a fase isotrópica L2 e quando MILT está incorporada essa transição não acontece. DLS também revelou que as partículas não têm seus tamanhos alterados com o aumento de temperatura. Por fim, cubossomos mostraram ser excepcionais conseguindo manter suas características físico-químicas mesmo quando submetidos a ambientes extremos, como a liofilização, a extrusão e a altas temperaturas.

Nanomedicine is one of the most promising fields in nanotechnology nowadays. The use of nanoparticles as carriers aims to improve efficiency of drugs that possess low solubility in aqueous environment (very hydrophobic molecules) or that have a lot of undesired side effects. In this way, nanoparticles offer both a protection for the molecules and a carrying vehicle. On this ground, cubosomes are nanoparticles capable of storing both hydrophilic and hydrophobic molecules within its structure, in addition, cubosomes have approximately 50% hydrophilic and hydrophobic areas. Therefore, they can carry much more molecules than liposomes for instance. In particular, cubosomes are quite easy to produce due to its base product, lipids (like monoolein (GMO) or phytantriol (PHY)) that self-assembly in water media. In this project, both lipids were chosen to produce the cubosomes from well-established protocols in literature. A model drug, miletofsine (MILT), was chosen to study the interaction of such nanosystem with a guest molecule. GMO cubosomes revealed to have Im3m crystallographic symmetry and lattice parameter 15.3(7) nm, particles presented sizes 300(8) nm and moderate polydispersion 0.160(20). TEM revealed squared particles with sizes ~350 nm, cryo-EM presented particles with internal structure and varied size (from 200 to > 500 nm). From FFT analysis, the calculated lattice parameter remained in the order of ~10 nm compatible with SAXS measurements. MILT loading into cubosomes was possible up to 4% w/w without loss of cubosomes structure. For 5% w/w MILT, the nanoparticles were already loosing their crystalline structure, as evidenced by cryo-EM. TEM analysis reveals that as more MILT is loaded into the cubosomes, their sizes increased. For sample 1.5% w/w MILT cryo-EM presents nanoparticles with organized internal structure and an envelope (hypothesized to be a polymer coating) in its surface. Calculated lattice parameters are in the order of ~10 nm. Myverol (Myv) is a commercial mixture that contains ~60% GMO, in this project it was proposed a bottom up protocol for Myv-based cubosomes. The production of these nanoparticles also revealed, by SAXS, Im3m symmetry and lattice parameter 12.30(12) nm. DLS revealed particle size 280(5) nm and moderate polydispersion 0.115(52). TEM shows square and cubic nanoparticles with sizes ~500 nm. MILT loading into Myv-cubosomes revealed that the drug interacts with the nanoparticle by enlarging their lattice parameter as more MILT is loaded (up to 4% w/w). Curiously, for some MILT concentrations the presence of other unknown cubic structures was evidenced by SAXS. TEM revealed nanoparticles with huge polydispersion, with sizes raging from 200 nm to 2 µm. PHY based cubosomes were successfully reproduced by the chosen protocol, in both water, PBS buffer and 2.25% glycerol medium. SAXS revealed crystallographic structure Pn3m and lattice parameter 6.74(04) nm. DLS measured sizes ~450 nm and moderate polydispersion 0.161(10). NTA measurements were consistent with DLS, revealing a broad size distribution and total particle concentration of ~1016 particles/mL for each sample. TEM revealed square and rounder particles in varied size. Cryo-EM micrographs presented particles with internal structure and varied size confirming moderate polydispersion. The FFT analysis revealed calculated lattice parameters ~6.5 nm, compatible with SAXS data. Samples were submitted to lyophilization and found that after re-hydration they still hold the same characteristics (morphology, size) as the original sample. Extrusion was also performed in order to improve polydispersion and control particle size, again cubosomes held their internal structure after the process, diminishing their sizes and improving monodispersion. MILT was loaded into cubosomes via co-solubilization and addition after the nanoparticles were formed. Up to 5% w/w the cubosomes incorporated MILT without loss of crystallographic structure, but at 10%, 15% and 20% w/w, the drug provoked phase change for Im3m symmetry. At the lower concentrations, MILT enlarged the lattice parameter of cubosomes and it was hypothesized that MILT inserted itself into the bilayer of the nanoparticles. DLS reveales that the drug does not change particle size or polydispersion. TEM revealed square and rounder particles in sizes slightly bigger than DLS. For sample 4% w/w, Cryo-EM presented particles with internal structure and calculated lattice parameter ~7 nm compatible with SAXS measurements for this sample. Co-solubilization and addition after nanoparticle preparation proved out to have the same effect on cubosomes loaded with MILT. All samples were submitted to higher temperatures to investigate phase change, based on phase diagram of the lipid. It was found that for the blank samples at 65 °C the cubosomes suffer phase change for isotropic phase L2, when MILT is loaded into the nanoparticles this phase change does not happen. DLS revealed also that at higher temperatures, particle size does not change, neither polydispersion. Finally, cubosomes proved to be remarkable nanoparticles that hold their physico-chemical characteristics even when submitted to extreme environments (lyophylization, extrusion and higher temperatures.) / Nanomedicina é o campo de estudo mais promissor dentro da nanotecnologia atualmente. O uso de nanopartículas visa melhorar a eficiência de fármacos que possuem baixa solubilidade em meios aquosos (moléculas muito hidrofóbicas) ou que possuem muitos efeitos colaterais indesejados. Neste contexto, as nanopartículas oferecem proteção e veículo para tais moléculas. Para isso, cubossomos são nanopartículas capazes de encapsular tanto as moléculas hidrofóbicas como as hidrofílicas em sua estrutura. Cubossomos também apresentam aproximadamente 50% de áreas hidrofílica e hidrofóbica, sendo capaz de encapsular grandes quantidades de moléculas teóricamente. Particularmente, cubossomos são nanopartículas de fácil produção devido à sua matéria prima serem lipídios (por exemplo, monoleína (GMO) ou fitantriol (PHY)) que se auto associam em meio aquoso. Neste projeto, os dois lipídios citados foram escolhidos para a produção dos cubossomos empregando-se protocolos bem estabelecidos da literatura. Uma fármaco modelo, miltefosina (MILT), foi escolhida para o estudo da interação com as nanopartículas. Cubossomos de monoleína (GMO) revelaram simetria cristalográfica Im3m e parâmetro de rede de 15.3(7) nm, as nanopartículas apresentaram tamanhos em torno de 300(8) nm e PDI 0.160(20). MET revelou partículas quadradas com tamanhos ~350 nm e a crio microscopia mostrou partículas com estrutura interna bem definida e tamanhos variados (200 a 500 nm), os parâmetros de rede calculados se mostraram da ordem de ~10 nm, compatíveis com as medidas de SAXS. O encapsulamento da MILT nos cubossomos foi possível até 4% w/w sem perda de morfologia. Para 5% w/w MILT as nanopartículas já apresentavam perda de cristalinidade na sua estrutura, evidenciado por crio microscopia. Análise por MET revelou que quanto mais MILT era encapsulada nos cubossomos, maiores ficaram as nanopartículas. Com a amostra de 1.5% w/w foi feita a crio microscopia, que revelou cubossomos com estrutura interna bem definida e um envelope (possivelmente formado pelo polímero) na sua superfície. Os parâmetros de rede calculados foram da ordem de ~10 nm também. O myverol (Myv) é uma mistura comercial que contém aproximadamente 60% de GMO, e neste projeto foi proposto um protocolo bottom up para cubossomos feitos de Myv. A produção dessas nanopartículas também revelou, por SAXS, estrutura cristalográfica Im3m e um parâmetro de rede de 12.30(12) nm. DLS apresentou partículas de tamanho 280(5) nm e polidispersão moderada 0.115(52). MET mostrou partículas quadradas e cúbicas com tamanhos de ~500 nm. O encapsulamento da MILT revelou que o fármaco interage com os cubossomos aumentando seu parâmetro de rede, até uma concentração de 4% w/w. Curiosamente, para algumas concentrações de MILT havia presença de outras estruturas evidenciadas por SAXS. MET revelou nanopartículas com muita polidispersão, com tamanhos variando entre 200 nm e 2 µm. Cubossomos de PHY foram reproduzidos com sucesso a partir do protocolo escolhido, em meios aquoso, tampão PBS e 2.25% glicerol. SAXS revelou nanopartículas com simetria cristalográfica Pn3m e parâmetro de rede 6.74(04) nm. Por DLS, o tamanho das partículas foi de ~450 nm e polidispersão moderada 0.161(10). Medidas de NTA foram consistentes com DLS, mostrando uma larga distribuição de tamanhos e concentração de partículas ~1016 partículas/mL. MET revelou cubossomos quadrados e mais arredondados de tamanhos variados. Criomicroscopia apresentou partículas com estrutura interna bem definida, tamanhos variados (confirmando a polidispersão) e parâmetro de rede calculado em ~6.5 nm, compatível com medidas de SAXS. Essas amostras também foram submetidas a liofilização e descobriu-se que mesmo depois da re-hidratação, as partículas ainda mantiveram as mesmas características da amostra original. A extrusão também foi feita com o objetivo de melhorar a polidispersão e controlar o tamanho das partículas, novamente, os cubossomos demonstraram manter sua estrutura interna depois desse processo, diminuindo seus tamanhos e diminuindo a polidispersão dos sistema. MILT foi encapsulada de duas formas: passiva (co-solubilização) e ativa (adição depois que as nanopartículas foram formadas). Com até 5% w/w de MILT incorporada, os cubossomos mantiveram sua estrutura cristalográfica, porém em concentrações de 10%, 15% e 20% w/w, o fármaco provocou transição de fase para simetria Im3m. Em baixas concentrações, MILT aumentou os parâmetros de rede dos cubossomos e a hipótese levantada foi que a droga se insere na bicamada lipídica das nanopartículas. DLS revelou que MILT não altera o tamanho das partículas nem sua polidispersão. MET revelou partículas quadradas e arredondadas com tamanhos maiores que os medidos por DLS. Para a amostra 4% w/w, a crio microscopia foi realizada e as partículas encontradas apresentaram estrutura interna e parâmetro de rede calculado ~7 nm, compatível com medidas de SAXS. Co-solubilização e adição depois do preparo se mostraram equivalentes para o encapsulamento da MILT. Todas as amostras também foram submetidas a um estudo de temperaturas para investigar transições de fase, baseando-se nos diagramas de fase dos lipídios. Foi descoberto que os cubossomos, sem a droga, a 65 °C sofrem transição para a fase isotrópica L2 e quando MILT está incorporada essa transição não acontece. DLS também revelou que as partículas não têm seus tamanhos alterados com o aumento de temperatura. Por fim, cubossomos mostraram ser excepcionais conseguindo manter suas características físico-químicas mesmo quando submetidos a ambientes extremos, como a liofilização, a extrusão e a altas temperaturas.

No description available.

A ausência de trabalhos verificando a melhor concentração de espermatozoides para se congelar o sêmen de cachaços em palhetas de 0,5 mL, gerou a necessidade deste estudo. Foi proposto, por meio deste expererimento, encontrar uma quantidade ideal, ou seja, uma concentração em que se consiga congelar o maior número de espermatozoides por palheta, sem aumentar os danos já causados pelo processo de criopreservação dos espermatozoides de cachaços. Diante disso, foram realizadas 5 coletas de 5 cachaços (n=25), utilizando apenas a fração rica do ejaculado. Estes foram divididos em cinco concentrações espermáticas diferentes, a saber: 100, 200, 300, 600 e 800x106 espermatozoides/mL e congelados em palhetas de 0,5mL. Após o processo de criopreservação, realizado por um sistema automatizado, as plalhetas foram acondicionadas em botijão criogênico. Para as análises, 2 palhetas de cada concentração foram descongeladas em banho maria (37ºC por 30 segundos) e submetido às avaliações das características de motilidade por meio do CASA, citometria de fluxo para a análise da integridade das membranas acrossomal e plasmática, potencial mitocondrial, peroxidação lipídica e fluidez da membrana espermática (capacitação), além da morfologia espermática por microscopia de contraste de interferência diferencial de fase. As variáveis avaliadas foram submetidas à análise de variância e ao teste de média PDDIF, ao nível de 5%. O aumento da concentração espermática, na palheta de 0,5mL, afetou significativamente (p<0,05) a motilidade total e progressiva, velocidade progressiva, velocidade de trajeto, linearidade, retilinearidade, frequência de batimento flagelar e hiperativação. O aumento do número de espermartozoides na palheta não alterou (p>0,05) a porcentagem de células que apresentavam simultaneamente a integridade das membranas plasmática e acrossomal e com potencial mitocondrial (MIAIAP), o aumento não influenciou (p>0,05) as variáveis peroxidação lipídica, capacitação e morfologia espermática. Baseado nos resultados, pode-se sugerir a congelação de até 300x106 espermatozoides/mL, na palheta de 0,5 mL, sem maiores danos à motilidade e integridade das membranas espermáticas. / The lack of studies veryfing the best concentration of sperm to freeze the boar semen in 0.5 ml straws, bring forth necessity for this study. The experiment proposed to find an optimal amount, in other words a concentration that is possible to freeze the higher number of spermatozoa per straw without increasing the damage already caused by the process of cryopreservation of boar spermatozoa. Therefore, there were 5 semen collections of 5 boars (n = 25) using only the rich fraction of the ejaculate. They were divided into five different sperm concentrations, namely 100, 200, 300, 600 and 800x106 sperm/mL and frozen in 0.5 mL straw. After the cryopreservation process, carried out by an automated system, straws were placed in cryogenic cylinders. For the analysis, two straws of each concentration were thawed in water bath (37°C for 30 seconds) and subjected to evaluations of motility characteristics through CASA, flow cytometry to analyze the integrity of the acrosome and plasma membrane, mitochondrial potential, peroxidation and lipid fluidity of sperm membrane (capacitation), and the sperm morphology by phase differential interference contrast microscopy. The variables were subjected to analysis of variance and the average test PDDIF, at 5%. The increased sperm concentration, in the 0.5mL straw, affected significantly (p <0.05) total and progressive motility, progressive velocity, path velocity, linearity, straightness, flagellar beat frequency and hyperactivation. The increase in the spermatozoa number, in 0,05 mL straw, did not change (p> 0.05) the percentage of cells that simultaneously showed the integrity of plasma and acrosomal membranes and with high mitochondrial potential (MIAIHM), the increase did not affect (p> 0.05) the lipid peroxidation variables, training and sperm morphology. Based on the results, it can be suggested to freeze with 300x106 spermatozoa/mL, in 0.5 mL straw, without major damage to the integrity and motility of the sperm membrane.

<div>Viruses are a group of contagious microbes that have compact structures, containing a nucleic acid core and a protein shell. The replication of viruses requires assistance from hosts which can be almost any cellular organism. Viral infections are often associated with diseases and have been a major threat to the human race. To cope with viral diseases, we need to understand viruses, including their structures, life cycle, pathogenesis and interactions with their hosts. The first structure of a human virus was determined by the Rossmann lab in 1985 using X-ray crystallography.</div><div>Thanks to the recent advances in both hardware and software, cryo-electron microscopy (cryo-EM) has emerged as a powerful tool to study virus structures. Cryo-EM allows structural determination for a wide range of specimens to high resolution comparable to what can be achieved by X-ray crystallography. Currently two techniques of cryo-EM are commonly used in structural virology: single particles analysis (SPA) and electron tomography (ET). </div><div>Single particle analysis has been used to determine the structures of viruses complexed with host factors in three studies that are to be discussed with more details in chapters 2-4. </div><div>The structure of B19 parvovirus complexed with Fabs of a neutralizing human antibody was determined to 3.2 Å resolution. This structure showed that amino acids from three neighboring VP2 proteins form a quaternary structure epitope. In addition, the structure of human rhinovirus-C (RV-C) complexed with its cellular receptor, CDHR3, was determined to 3.9 Å resolution. Despite the low occupancy of the receptors, a “powerful” localized 3D classification procedure helped to select viral particles that had more bound receptors. Furthermore, structures were determined to 10 Å resolution of bacteriophage ΦX174 bound to lipopolysaccharide (LPS) bilayers, before and after genome ejection. These structures showed a series of conformational changes that occurred when a phage penetrated the bacterial membranes. These studies are good examples of applying cryo-EM to investigate virus-host interactions.</div><div>However, single particle analysis requires samples to be isolated, homogenous and monodispersed. On the contrary, tomography allows in situ studies and is applicable to samples with more flexibility and more heterogeneity. In the case of ΦX174, the structural changes that are involved in the assembly of the H-tube during infection remains a huge mystery. To provide an environment that is more similar to the surface of a bacterial cell, LPS-containing liposomes were mixed with ΦX174 viruses. It was then observed that the ΦX174 particles bound to these liposomes in a very compact manner which was impossible interpret with single particle analysis. Using cryo-ET, 3D volumes of liposome-ΦX174 complexes were reconstructed and structural details were visualized by sub-tomogram classification and averaging.</div><div>The emergence of cryo-EM has not only made high-resolution structural studies possible but also broadened the scope of samples with which virologists could work. Moreover, studies on flexible and heterogeneous complexes between viruses and host factors are now possible using either single particle analysis or electron tomography. These techniques will help us to understand virus-host relationships and finally, to develop effective anti-viral therapies.</div>

No description available.

Adeno-associated Virus (AAV) Rep proteins are multifunctional proteins that carry out various DNA transactions required for the life cycle of AAV. The Rep proteins have been found to be important for genome replication, gene regulation, site-specific integration and play an essential role in genome packaging. There are two main groups of Rep proteins: large and small Reps; both groups are SF3 helicase family members. During DNA packaging, studies have shown that the small Rep proteins are critical to produce fully packed particles. Using stopped-flow kinetic analysis, we show a significant difference in helicase activity between the small and large Rep proteins that support the notion that the small Rep proteins are the primary motor to package DNA due to more efficient motor activity. That leaves the large Rep proteins to serve a different role during packaging. In previous studies, we have shown that the large Rep proteins have the ability to change their oligomeric state depending on the nature of the DNA substrate. We can observe double octameric rings with single-stranded DNA (ssDNA) and heptameric complex with double-stranded DNA (dsDNA). To understand Rep protein structural plasticity, we solved a 6.96 Å cryo-EM structure of Rep68*/ssDNA complex illustrating that the formation of Rep octamer rings is dominated by interactions between their N-terminal origin-binding domain (OBD) using the same interface utilized to recognize dsDNA specifically. Our analysis of the structural data suggests that the double octameric ring structure is stabilized by ssDNA that bridges octameric rings together. The structure shows that the helicase domains are highly flexible and that ssDNA is present at the center of the ring. In addition, we have solved a preliminary 12 Å model of Rep68*/dsDNA complex showing a heptameric ring encircling a DNA molecule. Our structural and functional data offer insights to the various Rep-DNA scaffolds that can perform diverse functions during the AAV life cycle.