The maintenance of protein homeostasis as a response to changing external conditions is crucial for cellular survival and proper function. Since plants cannot adapt by changing location, their need for a rapid intracellular response is accentuated. The AAA ATPase CDC48 maintains protein homeostasis in conjunction with NPL4 and UFD1 by coupling ATP hydrolysis with mechanical force to extract and unfold ubiquitylated proteins from organelle membranes, chromatin, or protein complexes. Our bioinformatic analysis revealed considerable domain and binding motif differences in A. thaliana NPL4 compared to its orthologs in animals and fungi. Using ITC, MST, and SEC-MALS, we found that NPL4 and UFD1 did not heterodimerize, NPL4 bound to CDC48A in the absence of UFD1, and the complex was not stable in vitro. Additionally, we provided the first medium-high-resolution reconstructions of CDC48A in both an AMP-PNP bound and apo state, using cryo-EM. AMP-PNP bound CDC48A was reconstructed in both a tense (3.3 Å) and relaxed (3.5 Å) conformation with the N domain was positioned above or coplanar with the D1 ring, respectively. Our heterogeneity analysis using CryoDRGN revealed continuous flexibility of the N domains between the two conformations. The apo state was reconstructed as a single conformation at 4.4 Å resolution. A cryo-EM reconstruction of the complex was also obtained at a resolution of ~6 Å, which showed expected cofactor stoichiometry and binding positions. Through our efforts, we have observed differences in the interaction between A. thaliana CDC48A and its cofactors UFD1 and NPL4 that may correspond to functional differences between kingdoms.

No description available.

Single particle electron microscopy (EM) allows us to examine the molecular world and gain insights into protein structures implicated in human disease. Visualizing the 3D architecture of the macromolecules can inform drug design and preventative care. While X-ray crystallography and NMR are able to resolve atomic structures, the methodology is better suited for smaller structures with limited flexibility. Single particle EM allows us analyze larger structures that have inherent flexibility. Protein structures can broadly be categorized as symmetry or asymmetric. There are computational advantages when analyzing symmetrical structures. Specifically, structural information can be extrapolated from fewer vantage points. Thus, symmetrical macromolecules are an advantageous for pioneering new methodologies in single particle EM. Rotavirus double layered particles (DLPs) are large macromolecular complexes that display icosahedral symmetry. Previous studies have led to a high resolution structure of transcriptionally inactive rotavirus frozen in time. However, to more fully understand rotavirus we need to examine the structure under transcriptionally active conditions. To expand our understanding, we first evaluated these viral assemblies using cryo-EM under active and inactive conditions. We found both internal and external structural differences. Based on these findings we sought to further our understanding of these nano-machines by developing a liquid cell environment to evaluate their dynamics over time. Our research not only developed a new methodology to evaluate active particles over time, we also found that the mobility of the DLPs were directly correlated to the level of transcriptional activity. When analyzing asymmetrical and flexible protein complexes previous studies have utilized methodologies to limit the proteins' conformational variability. While this does allow for a higher resolution structure, it limits our understanding to a specific orientation and compromises the biological insights. BRCA1 is an asymmetric protein containing a large flexible region and is important in the prevention of breast cancer. We utilize silicon nitride microchips with integrated wells and decorated with a lipid monolayer to capture and image BRCA1 complexes. This imaging platform minimizes heterogeneity and ensures the sample quality while not biasing confirmation. Thus, allowing for high resolution cryo-EM imaging of flexible native proteins. We were able to examine BRCA1 complexes from cells at both the primary and metastatic sites. Our ability to visualize these proteins in their native form provide insights into the variability of BRCA1 in disease progression. We found that BRCA1 complexes isolated from metastatic cells have additional density in the C-terminal domain. Our data suggests this density it due an interaction with p53. Overall, our methodologies highlight the power of single particle EM for studying protein complexes. Furthermore, our findings emphasize the importance of examining protein complexes in their native state. / PHD / Single particle electron microscopy (EM) allows us to examine the molecular world and gain insights into protein structures implicated in human disease. Visualizing the 3D architecture of macromolecules can inform drug design and preventative care. While X-ray crystallography and NMR are able to resolve atomic structures, the methodology is better suited for smaller structures with limited flexibility. Single particle EM allow us analyze larger structures that have inherent flexibility. Protein structures can broadly be categorized as symmetry or asymmetric. There are computational advantages when analyzing symmetrical structures. Specifically, structural information can be extrapolated from fewer vantage points. Thus, symmetrical macromolecules are an advantageous for pioneering new methodologies in single particle EM. Rotavirus double layered particles (DLPs) are large, highly symmetrical macromolecular complexes that represent an ideal model system for developing technology. Previous studies have led to a high resolution structure of inactive rotavirus DLP frozen in time. However, to more fully understand rotavirus we need to examine the structure under active conditions. To expand our understanding, we first evaluated these viral assemblies using cryo-EM under active and inactive conditions. We found structural differences. Based on these findings we sought to further our understanding of these nano-machines by developing a liquid cell environment to evaluate their dynamics over time. Our new methodology revealed new insights into the mobility of the DLPs. When analyzing asymmetrical and flexible protein complexes previous studies have utilized methodologies to limit the proteins’ movement. While this does allow for a higher resolution structure, it limits our understanding to a specific orientation and compromises the biological insights. BRCA1 is a highly flexible asymmetric protein implicated in the development of breast cancer. We utilize specialized microchips to capture and image BRCA1 complexes. This imaging platform ensures sample quality and allows for high resolution cryoEM imaging of flexible native proteins. We were able to examine BRCA1 complexes from cells at both the primary and metastatic sites. Our ability to visualize these proteins in their native form provide insights into the variability of BRCA1 in disease progression. Our data found that BRCA1 complexes isolated from metastatic cells are structurally different than those at the primary site. Overall, our methodologies highlight the power of single particle EM for studying protein complexes. Furthermore, our findings emphasize the importance of examining protein complexes in their native state.

La pause transcriptionnelle marquée par les ARN polymérases (RNAP) est un mécanisme clé pour réguler l'expression des gènes dans tous les règnes de la vie et est une condition préalable à la terminaison de la transcription. Le facteur de transcription bactérien essentiel NusA stimule à la fois la pause et la terminaison de la transcription, jouant ainsi un rôle central. Ici, je présente des reconstructions par cryo-microscopie électronique (cryo-EM) à une seule particule de NusA lié à des complexes d'élongation en présence et en absence d’ARN en épingle à cheveux dans le canal de sortie de l'ARN. Les structures révèlent quatre interactions entre NusA et RNAP qui suggèrent comment NusA stimule le repliement de l’ARN, la pause et la terminaison de la transcription. Un intermédiaire de translocation asymétrique de l'ARN et de l'ADN convertit le site actif de l'enzyme en un état inactif, fournissant une explication structurelle pour l'inhibition de la catalyse. La comparaison de RNAP à différentes étapes de la mise en pause donne un aperçu de la nature dynamique du processus et du rôle de NusA en tant que facteur de régulation. / Transcriptional pausing by RNA polymerases (RNAPs) is a key mechanism to regulate gene expression in all kingdoms of life and is a prerequisite for transcription termination. The essential bacterial transcription factor NusA stimulates both pausing and termination of transcription, thus playing a central role. Here, I present single-particle electron cryo-microscopy (cryo-EM) reconstructions of NusA bound to paused elongation complexes with and without a pause-enhancing hairpin in the RNA exit channel. The structures reveal four interactions between NusA and RNAP that suggest how NusA stimulates RNA folding, pausing, and termination. An asymmetric translocation intermediate of RNA and DNA converts the active site of the enzyme into an inactive state, providing a structural explanation for the inhibition of catalysis. Comparing RNAP at different stages of pausing provides insights on the dynamic nature of the process and the role of NusA as a regulatory factor.

La régulation de la transcription est un processus dynamique faisant intervenir le recrutement de complexes protéiques impliqués dans le remodelage de la chromatine. Parmi eux, mon travail s’est focalisé sur le complexe NuRD (Nucleosome Remodeling and histone Deacetylation) et sa sous-unité de liaison à l’ADN CpG MBD3. Pour cela une approche de biologie structurale intégrative combinant la préparation biochimique, la caractérisation biophysique et l’étude structurale par cryo-EM et cristallographie aux rayons-X a été mise en place. Les caractérisations biophysiques de MBD3 ont permis de mettre en évidence son interaction avec un ADN non-modifié CpG et des cristaux diffractant jusqu’à 3.9 Å ont été obtenu. De plus la région désordonnée en aval du domaine de liaison a été identifiée et son impact dans la formation de complexe caractérisé. Des cristaux pour les différentes constructions en complexe avec l’ADN ont été obtenus et sont actuellement optimisés. Enfin l’optimisation de la purification et la préparation du complexe, ont permis la visualisation du complexe NuRD et mettent en avant pour la première fois une organisation en domaines du complexe. / Transcription regulation of chromatin is a very dynamic process regulated through the recruitment of chromatin-remodeling complexes. My work focuses on NuRD for Nucleosome remodeling and histones deacetylation complex a 1 MDa multi-subunit protein complex and its subunit MBD3 a CpG-binding protein and more precisely on an integrated biology approach of this molecular assembly and its interaction with DNA. It combines biochemical preparation, biophysical characterization, single particle cryo-eletron microscopy and x-ray crystallography. Biophysical analysis show that MBD domain of MBD3 interacts with unmodified CpG DNA, a crystal diffracting up to 3.9 Å were obtained. Moreover a C-terminal intrinsically disordered region of MBD3 were identified and despite is inherent disorder seems to increase the binding affinity of MBD3 for DNA. Crystals were obtained for both constructs in complex with DNA and are currently optimized.Cryo-EM study of NuRD complex allows us to develop and optimized purification and grids preparation for the visualization of the complex. The present results reveal a domain organization of the complex never identify before.

Une approche de biologie structurale intégrative a été mise à profit pour l'étude de l’organisation structurale du complexe NuRD. Mon travail s'est focalisé essentiellement sur trois sous-unités du complexe: MBD3, RbAp46 et RbAp48. J'ai mis en place les protocoles de production et de purification de ces différentes sous-unités, et les ai caractérisé biophysiquement par diverses méthodes. Nous avons ensuite entrepris des études de liaisons sur des nucléosomes reconstitués au laboratoire. Pour MBD3, l'optimisation du complexe nous a permis d'obtenir des cristaux diffractant jusqu'à 7 A de résolution. Parallèlement, une reconstruction 3D préliminaire à partir de données de cryo-microscopie électronique a pu être obtenue à 25A de résolution. Pour RbAp46/48, nous avons pu montrer que ces protéines formaient un complexe stable avec le nucléosome, pavant la voie pour leur future étude structurale par cryo-microscopie électronique ou cristallographie aux rayons-X. / An integrative structural biology approach has been used to study the structural organization of the NuRD complex.My work focused especially on three subunits of this complex: MBD3, RbAp46 and RbAp48. I set up the preparation of the individual subunits and characterized them by various biophysical methods. We next carried out binding assays with homemade human nucleosomes. For MBD3, optimization of the complex led to crystals diffracting up to 7 Å. In parallel, a preliminary 3-D reconstruction at 25 Å resolution has been solved in cryo-EM. For RbAp46/48, crystal we were able to show that these proteins form stable complexes with the nucleosome, paving the way for future structural analysis by cryo-EM or X-ray crystallography.

Peptídeos antimicrobianos (AMP) são importantes componentes do sistema imune de todos os organismos vivos. No plasma de Acanthoscurria rondoniae isolamos 15 AMP com similaridade com a hemocianina. As sete subunidades da hemocianina foram sequenciadas e sua estrutura tridimensional determinada. A rondonina, processada a partir de uma enzima do plasma em condições ácidas apresentou melhor atividade em pH ácido, sinergismo com gomesina, não citotóxico, não interagiu com membranas artificiais lipídicas, protegeu as células da infecção por vírus humanos de RNA e seu mecanismo de ação em leveduras está associado com material genético. Nossos resultados nos ajudam a entender porque aracnídeos sobreviveram por um longo tempo na escala evolutiva. E como as doenças infecciosas estão entre as principais causas de morte da população humana torna-se vital investir na busca de substâncias naturais ou sintéticas que exibam atividades antimicrobianas específicas e, acima de tudo, que as exerçam através de mecanismos de ação alternativos daqueles dos antibióticos disponíveis. / Antimicrobial peptides (AMP) are important components of the immune system of all living organisms. In the plasma of Acanthoscurria rondoniae we isolated 15 AMP with similarity to hemocyanin. The seven subunits hemocyanin were sequenced and determined its three-dimensional structure. The rondonin, processed from a plasma enzyme under acidic conditions showed best activity at acid pH, synergism with gomesin, non-cytotoxic, does not interacted with lipid artificial membrane, protected the cells from infection by human virus RNA and its mechanism of action in yeast is associated with genetic material. Our results help us understand why arachnids have survived for a long time on the evolutionary scale. And how infectious diseases are among the leading causes of death in human population becomes vital to invest in the search for natural or synthetic substances that exhibit specific antimicrobial activities and, above all, that engage through alternative mechanisms of action of these antibiotics available.

Peptídeos antimicrobianos (AMP) são importantes componentes do sistema imune de todos os organismos vivos. No plasma de Acanthoscurria rondoniae isolamos 15 AMP com similaridade com a hemocianina. As sete subunidades da hemocianina foram sequenciadas e sua estrutura tridimensional determinada. A rondonina, processada a partir de uma enzima do plasma em condições ácidas apresentou melhor atividade em pH ácido, sinergismo com gomesina, não citotóxico, não interagiu com membranas artificiais lipídicas, protegeu as células da infecção por vírus humanos de RNA e seu mecanismo de ação em leveduras está associado com material genético. Nossos resultados nos ajudam a entender porque aracnídeos sobreviveram por um longo tempo na escala evolutiva. E como as doenças infecciosas estão entre as principais causas de morte da população humana torna-se vital investir na busca de substâncias naturais ou sintéticas que exibam atividades antimicrobianas específicas e, acima de tudo, que as exerçam através de mecanismos de ação alternativos daqueles dos antibióticos disponíveis. / Antimicrobial peptides (AMP) are important components of the immune system of all living organisms. In the plasma of Acanthoscurria rondoniae we isolated 15 AMP with similarity to hemocyanin. The seven subunits hemocyanin were sequenced and determined its three-dimensional structure. The rondonin, processed from a plasma enzyme under acidic conditions showed best activity at acid pH, synergism with gomesin, non-cytotoxic, does not interacted with lipid artificial membrane, protected the cells from infection by human virus RNA and its mechanism of action in yeast is associated with genetic material. Our results help us understand why arachnids have survived for a long time on the evolutionary scale. And how infectious diseases are among the leading causes of death in human population becomes vital to invest in the search for natural or synthetic substances that exhibit specific antimicrobial activities and, above all, that engage through alternative mechanisms of action of these antibiotics available.

TRIM21 is a cytosolic ubiquitin ligase and an antibody receptor that providesa last line of defense against invading pathogens. By utilizing the diversity ofantibody repertoire to identify pathogens, TRIM21 serves as a link betweenintrinsic cellular defense and adaptive immunity. A variety of diseases havebeen linked to mutations of the TRIM family, including cancer, inflammatorydiseases, and autoimmune diseases. In this project, TRIM21 was producedand purified from Escherichia coli, (E.coli). Protein characterization wasperformed with SDS-PAGE, size exclusion chromatography and cryo-electronmicroscopy (cryo-EM). Previously TRIM21 has been shown to form a dimerwhen produced in SF9. Results from size exclusion chromatography show thatTRIM21 form a larger complex when expressed in E.coli. Cryo-EM resultsshow that the complex structure is more globular than previously thought.Purified TRIM21 was bound to the antibody IC100. SDS-PAGE and sizeexclusion chromatography results show much lower affinity to antibodies thanexpected. / TRIM21 är en cytosolisk ubiquitinligas- och antikroppsreceptor som ger ensista försvarslinje mot invaderande virus. Genom att använda mångfalden avantikroppsrepertoar för att identifiera patogener, fungerar TRIM21 som enlänk mellan inre cellulärt försvar och adaptiv immunitet. En mängd olikasjukdomar har kopplats till mutationer i TRIM-familjen, inklusive cancer,inflammatoriska sjukdomar och autoimmuna sjukdomar. I detta projekt produceradesoch renades TRIM21 från Escherichia coli, (E.coli). Proteinkarakteriseringutfördes med SDS-PAGE, gelfiltreringskromatografi och kryo-elektronmikroskopi(cryo-EM). Tidigare har TRIM21 visat sig bilda en dimer när den producerasi SF9. Resultat från gelfiltrering visar att TRIM21 bildar ett större komplexnär det uttrycks i E.coli. Cryo-EM-resultat visar att den komplexa strukturenär mer klotformig än man tidigare trott. Renad TRIM21 bands till antikroppenIC100. SDS-PAGE och gel-filtrerings resultat visar mycket lägre affinitet tillantikroppar än förväntat.

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.