Voltage-dependent anion-selective channels (VDAC), or mitochondrial porins,regulate the flow of metabolites across the mitochondrial outer membrane. They presumably span the membrane as β-barrels, but the residues forming the individual β-strands are unknown. This information is essential for understanding the structure and function of the protein. Using Neurospora VDAC as a template, published data were reassessed to delineate a unified model for porin structure Bay and Court 2002, which was subsequently refined in collaboration with Greg Runke Runke et al. 2006. The focus of this work was the development and analysis of systems for maintaining high levels of folded porin for the acquisition of high resolution data needed for model testing. The conformation of hexahistidinyl-tagged Neurospora porin in detergent was probed by fluorescence, near-UV circular dichroism and ultraviolet absorption spectroscopy. Derivatives of tryptophan and tyrosine were also examined by fluorescence spectroscopy and UV absorbance spectroscopy to model the interactions between the detergents and the amino acid side chains in the protein. Detergent-specific levels of β-strand and tyrosine exposure were observed. In all cases, the two tryptophan residues reside in weakly asymmetric, hydrophobic environments, suggesting transient tertiary interactions. Porin solubilized in these detergents forms functional channels in liposomes and membrane insertion is accompanied by increased levels of β-strand and loss of protease sensitivity. These data were used to develop mixed detergent folding systems. A mixture of SDS and dodecyl-β-D-maltopyranoside (DDM)supports a β-strand rich conformation at high protein concentrations. The tertiary contacts and protease resistance of the SDS/DDM solubilized porin are very similar to those of the protein following reconstitution into liposomes. Finally, the role of sterols in porin folding was examined, as the addition of sterols to detergent-solubilized VDAC is required for channel formation in artificial membranes. Sterols do not alter the secondary structure of VDAC, and subtle alterations to tertiary interactions were detected, suggesting that sterols do not promote an insertion-competent structure, but rather facilitate insertion into artificial bilayers. In summary, this analysis of the folded states of detergent-solubilized porin has revealed a system that maintains high concentrations of mitochondrial porin in a state that is very promising for structural studies.

Voltage-dependent anion-selective channels (VDAC), or mitochondrial porins,regulate the flow of metabolites across the mitochondrial outer membrane. They presumably span the membrane as β-barrels, but the residues forming the individual β-strands are unknown. This information is essential for understanding the structure and function of the protein. Using Neurospora VDAC as a template, published data were reassessed to delineate a unified model for porin structure Bay and Court 2002, which was subsequently refined in collaboration with Greg Runke Runke et al. 2006. The focus of this work was the development and analysis of systems for maintaining high levels of folded porin for the acquisition of high resolution data needed for model testing. The conformation of hexahistidinyl-tagged Neurospora porin in detergent was probed by fluorescence, near-UV circular dichroism and ultraviolet absorption spectroscopy. Derivatives of tryptophan and tyrosine were also examined by fluorescence spectroscopy and UV absorbance spectroscopy to model the interactions between the detergents and the amino acid side chains in the protein. Detergent-specific levels of β-strand and tyrosine exposure were observed. In all cases, the two tryptophan residues reside in weakly asymmetric, hydrophobic environments, suggesting transient tertiary interactions. Porin solubilized in these detergents forms functional channels in liposomes and membrane insertion is accompanied by increased levels of β-strand and loss of protease sensitivity. These data were used to develop mixed detergent folding systems. A mixture of SDS and dodecyl-β-D-maltopyranoside (DDM)supports a β-strand rich conformation at high protein concentrations. The tertiary contacts and protease resistance of the SDS/DDM solubilized porin are very similar to those of the protein following reconstitution into liposomes. Finally, the role of sterols in porin folding was examined, as the addition of sterols to detergent-solubilized VDAC is required for channel formation in artificial membranes. Sterols do not alter the secondary structure of VDAC, and subtle alterations to tertiary interactions were detected, suggesting that sterols do not promote an insertion-competent structure, but rather facilitate insertion into artificial bilayers. In summary, this analysis of the folded states of detergent-solubilized porin has revealed a system that maintains high concentrations of mitochondrial porin in a state that is very promising for structural studies.

Les analogues de nucléosides (AN), sont des agents importants dans le traitement d'hémopathies malignes et de certaines tumeurs solides. Le catabolisme rapide, la résistance cellulaire au traitement et le grand volume de distribution dans le corps limitent potentiellement l'efficacité thérapeutique des AN. Notre objectif est de concevoir un vecteur permettant un ciblage de ces molécules vers les tissus cancéreux, assurant son internalisation cellulaire et sa protection dans les milieux biologiques. Trois types de vecteurs de taille micronique, submicronique et moléculaire ont été élaborés et caractérisés : microparticules polymériques à base de poly(ε-caprolactone) (PCL), liposomes multilamellaires et complexes d'inclusion de la prodrogue de la cytarabine (Ara-C), qui est active sur certaines lignées cellulaires résistantes à l'Ara-C, l'AraC-SATE (bis(tbutyl-S-acyl-2-thioethyl)-cytidine monophosphate).Les microparticules ont été préparées par la méthode de " double émulsion - évaporation de solvant " en utilisant comme surfactants des copolymères amphiphiles composés de blocs biodégradables de PCL et de blocs bio-éliminables de polyéthylène glycol (PEG). Une série de copolymères mPEG-PCL avec des blocs PCL de différents poids moléculaires a été synthétisée et l'effet de la longueur de chaînes de PCL sur les caractéristiques physico-chimiques des particules a été étudié. Une efficacité d'encapsulation satisfaisante a pu être obtenue, qui a été dix fois plus importante que celle des microparticules à base de PCL seul. Les liposomes ont été préparés par la méthode d'injection d'éthanol. Une étude de formulation a été réalisée afin de sélectionner la formule permettant d'obtenir la meilleure efficacité d'encapsulation. Le test d'internalisation cellulaire et du comportement aérodynamique des liposomes nébulisés ont montré la pertinence des liposomes élaborés pour le ciblage des cellules pulmonaires qui pourrait être d'un grand intérêt pour le traitement des métastases au niveau des poumons.L'encapsulation moléculaire de l'AraC-SATE dans l'hydroxyporpyl-β-cyclodextrine a été réalisée pour augmenter la solubilité apparente de la prodrogue. L'évaluation des complexes sur des cultures de cellules leucémiques murines a montré une activité cytotoxique comparable à celle de la prodrogue indiquant que l'inclusion moléculaire ne modifie pas l'activité biologique de la prodrogue.

Existeixen molts fàrmacs amb un gran potencial en estudis in vitro que no resulten efectius en última instància degut a una pobre absorció o a una insuficiente distribució en el cos humà. Per aquest motiu és necessari tenir mètodes que puguin predir la interacció de fàrmacs amb les membranes cel·lulars a les primeres etapes de desenvolupament del compost, per estalviar temps i diners.Durant el transcurs de la meva tesi vaig estudiar tres tipus de membranes artificials que intentaven imitar l'estructura de les membranes cel·lulars i modelar diferents paràmetres biològics.La primera d'elles era una fase estacionària de cromatografia de líquids anomenada IAM, basada en cadenes de fosfatidilcolina enllaçades a la sílice. Vaig caracteritzar l'acidesa de la seva sílice (és a dir, la presència de silanols lliures), i les propietats de la columna mitjançant diversos models cromatogràfics (LSER, LSER global i model del paràmetre de polaritat). A més, també vaig caracteritzar tota una sèrie de columnes comercials C18 per comparar els resultats amb la columna IAM. Per últim, vaig establir un mètode de predicció de la retenció cromatogràfica de nous compostos a partir del model del paràmetre de polaritat.Un cop caracteritzats els sistemes cromatogràfics vaig buscar similituds amb sistemes biològics mitjançant un paràmetre de distància anomenat "d" que va ser proposat pel nostre grup d'investigació. A continuació vaig comprovar com els sistemes que havíem considerat similars segons el paràmetre de distància presentaven bones correlacions, mentre que aquells que no podien ser considerats semblants per tenir una "d" massa gran presentaven correlacions molt pobres. No només la columna IAM va presentar similituds amb sistemes biològics, sinó també altres columnes C18, com la XTerra RP18 i la XTerra MSC18.La segona membrana artificial estudiada era una membrana líquida d'hexadecà impregnada en un filtre, que separava dos compartiments aquosos. Aquesta tècnica, coneguda com a PAMPA, permet obtenir la permeabilitat passiva dels compostos i predir la seva absorció oral. El meu estudi va consistir en modificar aquesta tècnica afegint albúmina humana al compartiment donador, on estava el fàrmac, de manera que si la proteïna interaccionava fortament amb el composta quedava poca fracció lliure de fàrmac, i per tant, es reduïa bruscament la seva permeabilitat a través de la membrana. La diferència de permeabilitat en presència i absència de proteïna ens permetia calcular la constant de dissociació proteïna-fàrmac, un paràmetre de gran interès a la indústria farmacèutica.La última membrana estudiada va ser un liposoma, en concret de fosfatidilcolina, el mateix constituent de les membranes cel·lulars i de la columna IAM. Vaig estudiar la interacció d'aquests liposomes amb una sèrie de flavonoides presents en aliments com el raïm i beneficiosos per la salut humana degut a les seves propietats antioxidants. Per avaluar aquesta interacció vaig realitzar estudis calorimètrics amb una tècnica anomenada calorimetria d'escaneig diferencial. Quan els liposomes són sotmesos a uns cicles de temperatura d'escalfament/refredament presenten una transició de fase a una temperatura determinada, entre fase gel i fase cristall-líquid. Si es produeix interacció entre un determinat compost i el liposoma, els paràmetres d'aquest pic de transició canvien. Els liposomes, com a model de membrana, eren capaços de donar informació de la interacció produïda: si era més aviat hidrofòbica o polar, si el compost s'introduïa més o menys dins la membrana, etc. / In early drug discovery, artificial membrane models are very important in compound optimization, since some drugs that are potent in vitro assays fail to retain activity in vivo assays. During my doctoral thesis, I have studied three different artificial membranes, which intended to simulate the structure of cell membranes and to model biologic parameters.First of them was a stationary phase for HPLC, an IAM column, based on phosphatidylcholine chains bonded to the silica. I have characterized its silica acidity and their properties by means of several chromatographic models (LSER, global LSER and polarity parameter model). I also characterized several commercial C18 columns in order to compare the results with those of IAM column. Finally, one prediction of retention model was established. One distance parameter "d" was proposed in our group to find similarities between chromatographic and biological systems, by comparing their LSER coefficients. I calculated several "d" distances between different systems, and after that, I corroborated that good correlations could be obtained when distance was smaller than 0.25, while poor correlations were obtained when distance was larger than 0.25. Finally, I concluded that not only IAM column was able to model biological parameters, but also other C18 columns, such as XTerra columns.The second membrane was a liquid membrane of hexadecane coated on a filter, which separates two water compartments (PAMPA assay). This technique is commonly used to determine drug passive permeability and oral absorption. When I added human serum albumin to the donor compartment, this permeability changed, depending on protein binding. The difference on permeability in presence and absence of protein allows calculating the dissociation constant drug-protein (very important parameter for pharmaceutical industry).The last membrane model was a liposome. The interaction between phosphatidylcholine liposomes and flavonoids (natural compounds widely appreciated for their putative health-promoting effects) was studied with the differential scanning calorimetric technique. Liposomes show a main phase transition at one specific temperature and the parameters of this transition change if there is some interaction between liposomes and some compound. Important information can be obtained: strength of interaction, kind of interaction (polar or hydrophobic), etc.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 136-153).

Thèse (M.Sc.)--Université Laval, 2008. / Bibliogr.: f. [106]-126. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

Σκοπός της παρούσας μελέτης είναι η εκτίμηση της συσχέτισης ανάμεσα στην κυτταροτοξικότητα και την μείωση της ακεραιότητας λιποσωμικών μεμβρανών που προκαλούνται από έκδοχα. Εάν υπάρχει, μπορεί να προταθεί η χρήση των λιποσωμάτων ως in vitro τεχνική για την εκτίμηση της κυτταρο-τοξικότητας εκδόχων. Μελετήθηκε η κυτταροτοξικότητα σε 4 κυτταρικές σειρές ( A549, PC3, MDA-MB, MCF-7 ) και η επίδραση στην ακεραιότητα λιποσωμάτων των παρακάτω εκδόχων που χρησιμοποιούνται σε σκευάσματα τοπικής χορήγησης: Labrafac Hydro, Labrafac CC, Transcutol, Cremofor, DL- Lactic acid και Capmul σε συγκεντρώσεις 1% και 10% (v/v) Για την μέτρηση της κυτταροτοξικότητας (στις 6 και 24 ώρες επώασης) χρησιμοποιήθηκε η μέθοδος του MTT (Thiazolyl Blue Tetrazolium Bromide) ενώ για την εκτίμηση της μεμβρανικής ακεραιότητας λιποσωμάτων [χρησιμοποιήθηκαν MLV (πολυστοιβαδιακά) και SUV (μικρά μονοστοιβαδιακά) λιποσώματα, με διάφορες λιπιδικές συστάσεις [PC (φωσφατιδυλοχολίνη), HPC (υδρογονωμένη φωσφατιδυλοχολίνη) και DSPC( διστεαρουλο φωσφατιδυλοχολίνη) με ή χωρίς Chol (χοληστερόλη)] μετρήθηκε η διαφυγή της εγκλωβισμένης στα λιποσώματα καλσεΐνης. Τα πειραματικά αποτελέσματα, έδειξαν ότι η επιβίωση-πολλαπλασιασμός των κυττάρων επηρεάζεται λιγότερο ή περισσότερο από τα έκδοχα: Labrafac Hydro (1% και 10%), Transcutol (10%), Cremofor (1% και 10%), Capmul (1% και 10%) και Lactic acid (1% και 10%) ενώ πολύ λιγότερο από τα έκδοχα Labrafac CC(1% και 10%) καθώς και από το Transcutol 1%. Σε ότι αναφορά την ακεραιότητα των λιποσωμάτων ( έως 24 ώρες επώασης) τα έκδοχα Cremofor, Labrafac, Capmul, Lactic acid και Labrafac hydro επηρεάζουν όλες τις λιπιδικές συστάσεις και τύπους λιποσωμάτων που μελετήθηκαν, ενώ τα έκδοχα Transcutol και Labrafac CC επηρεάζουν πολύ λιγότερο τις πιο ρευστές λιπιδικές μεμβράνες, και σχεδόν καθόλου τις σκληρές. Φαίνεται ότι είναι πιθανή η συσχέτιση των δύο σειρών αποτελεσμάτων. / The aim of this thesis is to study if a correlation may exist between the cytotoxicity of excipients and their effect on the integrity of liposomal membranes. If such a correlation exists perhaps the effect on liposome integrity may be used as an in vitro system to predict excipient cytotoxicity. Methods: The cell toxicity in four cell lines ( A549, PC3, MDA-MB, MCF-7 ) and the influence on liposome integrity of several commonly used excipients was studied. The following excipients which are used in topical formulations were studied: Labrafac Hydro, Labrafac CC, Transcutol, Cremofor, DL- Lactic acid and Capmul at concentrations of 1% and 10% (v/v). For the measurement of cell toxicity (6 and 24 hours of incubation) the MTT-assay (Thiazolyl Blue Tetrazolium Bromide) was used, whereas for the evaluation of the liposome membrane integrity MLV (MultiLaminar Vesicles) and SUV (Small Unilaminar Vesicles) liposomes with different lipid compositions [PC (phosphatidyl choline), HPC (hydrogenated phospatidyl choline) and DSPC (distearoylphosphatidylcholine) with or without Chol (cholesterol)] were prepared and the escape of the calcein encapsulated in the liposomes was measured at different time points during their incubation in presence of the excipients. Results: The experimental results showed that the cell survival-proliferation is influenced more or less by the following excipients (in increasing effect order): Labrafac Hydro (1% and 10%), Transcutol (10%), Cremofor (1% and 10%), Capmul (1% and 10%) and Lactic acid (1% and 10%) while the effect of Labrafac CC(1% and 10%) as well as Transcutol 1%, is minimal. As far as the integrity of the liposomes (up to 24 hours of incubation) is concerned, the excipients Cremofor, Labrafac, Capmul, Lactic acid and Labrafac hydro affect all the lipidic compositions and types of liposomes that were studied, whereas the excipients Transcutol and Labrafac CC influenced only minimally the more liquid membranes and had no effect on the more rigid lipid compositions. It seems that a correlation between the two series of results is possible.

<p>Despite aggressive surgical resection, cytotoxic chemotherapy, and external beam radiotherapy, most cases of glioblastoma (GBM) remain recalcitrant. These outcomes necessitate novel developmental therapeutics that spare normal tissue. Immunotherapy is a promising novel adjuvant treatment that can harness the cytotoxic capacity of the immune system against tumor-associated antigens with exquisite specificity. To circumvent the challenges associated with the advancement of adoptive cellular immunotherapy, we developed a novel treatment platform, which leverages the use of commercially available and clinically translatable nanoparticles (NPs) that can be combined with tumor derived RNA to peripherally activate T cells against GBM antigens. Although cancer vaccines have suffered from weak immunogenicity, we have advanced a NP vaccine formulation that can reshape a host’s immune profile through combinatorial delivery of RNAs encoding for tumor antigens and RNAs encoding for immunomodulatory molecules to mediate long-lived T cell persistence. </p><p>We sought to assess if vaccination with amplified tumor derived RNA encapsulated in lipophilic NPs could be assembled to transfect antigen presenting cells (APCs) in vivo and induce therapeutic anti-tumor immunity in pre-clinical murine tumor models. We hypothesized that RNA encapsulated nanoliposomes would localize to reticuloendothelial organs such as the spleen and liver, transfect APCs therein and induce peripheral antigen specific T cell immunity against GBM. Since activated T cells can cross the blood brain barrier and exert their effector functions against GBM antigens, peripheral transfection of APCs by RNA-NPs represents an attractive vaccination approach for priming endogenous immunity against refractory brain tumors.</p><p>We screened several translatable NP formulations for their ability to transfect dendritic cells (DCs) in vitro with GFP mRNA. We demonstrated that the NP DOTAP was the most promising translatable formulation compared to alternative cationic liposomal preparations and linear polyethylenimine NPs with and without DC targeting mannose receptors. RNA-NP vaccines formulated in DOTAP were shown to induce in vivo gene expression and preserve RNA stability over time. We determined that intravenous (IV) injection of RNA-NPs was requisite for inducing functional antigen specific immunity, which was superior to standard peptide vaccines formulated in complete Freund’s adjuvant (CFA). IV administered RNA-NPs localized to splenic and hepatic white blood cells (WBCs); these cells expanded antigen specific T cells when transferred to naïve immunocompetent mice. RNA-NPs induced increased percentages of B7 co-stimulatory molecules, but also elicited compensatory PD-L1 expression. We enhanced the immunogenicity and anti-tumor efficacy of RNA-NP vaccines by combining RNA-NPs with immune checkpoint blockade against PD-L1. We also enhanced the immunogenicity and efficacy of this platform by simply combining mRNAs encoding for immunomodulatory cytokines (i.e. GM-CSF). Finally, we demonstrated that RNA-NP vaccines mediate anti-tumor efficacy against intracranial and subcutaneous melanomas and engender therapeutic anti-tumor efficacy in a cellular immunotherapy model against a radiation/temozolomide resistant invasive murine high-grade glioma.</p><p>GBM remains invariably associated with poor patient outcomes thus necessitating development of more targeted therapeutics. Clinically translatable RNA-NPs form stable complexes making them amenable to overnight shipping. They induce potent immune responses when administered systemically and mediate robust anti-tumor efficacy that can be enhanced through co-delivery of immunomodulatory RNAs. </p><p>This technology can simultaneously bypass the complexity of cellular therapeutics while cutting down the time to generation of personalized vaccines. Since RNA-NP vaccines can be made within days from a tumor biopsy, providing near immediate immune induction against GBM, these formulations can provide a more feasible and effective therapy with a wide range of applicability for all malignancies that can be targeted using RNA obtained from surgical resection of solid tumors.</p> / Dissertation