Dissertations / Theses on the topic 'Cell uptake'

Cell-penetrating peptides (CPPs) have emerged as a group of remarkable delivery vectors for various hydrophilic macromolecules, otherwise excluded from cells due to the protective plasma membrane. Unbiased conclusions regarding e.g. uptake mechanism, intracellular distribution and cargo delivery efficacy is complicated by the use of different methodological parameters by different laboratories. The first paper in this thesis introduced unifying protocols enabling comparison of results from different research groups. One of these methods, HPLC, was used in paper II to investigate CPP uptake and degradation in yeasts. Both parameters varied depending on peptide and yeast species; however pVEC emerged as a promising delivery vector in yeast since it internalized into both species tested without concomitant degradation. Protein mimicry was another investigated phenomenon and in paper III a 22-mer peptide from the p14Arf protein (Arf (1-22)) was found to be sufficient for retaining its function as a tumor suppressor. This peptide comprised a combination of apoptogenic property and CPP in one unity, thus providing opportunity to conjugate cytotoxic agents boosting the tumoricidal activity. Surprisingly, a partially inverted control peptide to Arf (1-22), called M918, was found to be an extraordinary CPP. In paper IV, it was shown to be superior to well-established CPPs in delivery of both peptide nucleic acids and proteins. Albeit the promising results these two peptides displayed, their utility in vivo, as with all peptides, is hampered by rapid degradation. With the aim of improving their stability, Arf (1-22) and M918 were synthesized with D-amino acids in the reverse order, a modification called retro-inverso (RI) isomerization. Their cell-penetrating ability was retained, but the treated cells displayed unexpected morphological alterations indicative of apoptosis. The presented results demonstrate the versatility of CPPs, functioning as vectors in both yeast and mammalian cells and as protein mimicking peptides with biological activity. Their potential as drug delivery agents is obvious; however, peptide degradation is an issue that requires further improvements before clinical success is in reach.<br>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 5: In press.

Insulin maintains glucose homeostasis through its binding of the insulin receptor and activation of the insulin signalling cascade in insulin sensitive tissues. Skeletal muscle is a major endocrine organ, and is responsible for the majority of post-prandial glucose disposal. The maintenance of glucose homeostasis is a delicate balance and impairments in glucose disposal can have significant physiological effects, resulting in the onset of metabolic diseases such as diabetes mellitus. Insulin stimulated glucose uptake involves a number of signalling proteins to enable uptake to occur. In order to understand the complexities associated with the insulin signalling cascade, cell culture models have provided a controlled and easily manipulated environment in which to investigate insulin stimulated glucose uptake in skeletal muscle. While the majority of these experiments have been conducted in conventional monolayer cultures, the growing field of three-dimensional tissue engineering provides an alternative environment in which skeletal muscle cells can be grown to investigate their physiological function. The purpose of this thesis was to investigate the use of different cell culture models for investigating the effects of acute and chronic insulin exposure on skeletal muscle. Initial investigations aimed to establish glucose uptake in tissue engineering skeletal muscle constructs using tritium labelled (H3) 2-deoxy-d-glucose. Monolayer cultures were used to developed base line conditions. In these cultures, concentrations greater than 0.5 μCi for 15 minutes of insulin stimulation suggested an initial assay window for investigating insulin stimulated glucose uptake. However, the duration of insulin stimulation was not effective in measuring uptake in tissue engineered skeletal muscle constructs based upon western blot experiments of Akt phosphorylation, therefore insulin stimulation in skeletal muscle tissue engineered constructs was increased to 30 minutes. Glucose uptake is mediated via specific glucose transporter protein, GLUT1 and GLUT4. Therefore, the transcriptional profile of these transporters was elucidated in monolayer culture and tissue engineered skeletal muscle constructs. Time course experiments showed an increase in GLUT4 transcription in tissue engineered and monolayer culture systems which is associated with an increase in the transcription of skeletal muscle development and myogenic genes. In two dimensional culture, skeletal muscle cells were exposed to insulin during differentiation and in post-mitotic skeletal muscle myotubes to investigating the potential effects upon metabolic genes and proteins involved in insulin signalling. Chronic exposure to insulin during skeletal muscle differentiation reduced insulin signalling and resulted in an increase in basal glucose uptake and ablated insulin stimulated glucose uptake. In contrast, post-mitotic skeletal muscle myotubes did not shown similar changes and were not as responsive to acute insulin exposure. Therefore future experiments exposed skeletal muscle to insulin during differentiation. Using the previous findings as a basis for experimentation, the effects of chronic and acute insulin exposure upon three dimensional skeletal muscle constructs were investigated. Fibrin and collagen constructs were grown for a total period of 14 days. Constructs were exposed to insulin during differentiation and acutely stimulated for 30 minutes at day 14. Although there was a mean increase in Akt protein phosphorylation in both types of tissue-engineered constructs, these changes were not significant following acute insulin stimulation. In addition, glucose uptake in fibrin skeletal muscle constructs increased as a result of acute insulin stimulation however was not significantly difference to unstimulated constructs. The work presented in this thesis provides initial experimental data of the use of different skeletal muscle cell culture models for investigating insulin signalling and glucose uptake. Further research should further characterise these in vitro models for investigating skeletal muscle metabolism.

Cell penetrating peptides have been the focus of drug delivery research for 15 years due to their apparent ability to deliver cargoes inside cells more readily than many other carriers. Using two of the most commonly studied peptides (tat47-57 and R9), the present study differs from previous work by deliberately choosing to observe uptake with lower peptide concentrations closer to potential therapeutic doses, and by implementing raster image correlation spectroscopy (RICS) on a commercial microscope to quantify uptake in parallel to other techniques such as fluorescence correlation spectroscopy (FCS), confocal microscopy, and mass spectroscopy.An initial study using mass spectrometry and ExPASy (Expert Protein Analysis System) revealed that the peptides are stable for at least one hour in PBS. Based on this initial information and other experimental conditions, the study took two main directions with regards to the peptide: the membrane interaction and accumulation in the cell.The peptides interaction with the cell membrane revealed that neither tat-TAMRA nor R9-TAMRA disrupts the membrane of cells: incorporation of FM2-10 in the membrane was not modified in K562 cells whilst it was in presence of the control lytic peptides GALA and mellitin. Based on this information confocal microscopy was utilised to assess the localisation on the cell membrane. Peptide binding to the membrane appeared to be heterogeneous in distribution at 1µM bulk concentration.Accumulation in cells of the peptides was observed incubated at 37°C, confocal microscopy showed punctuated distribution with intracellular aggregations of fluorescence measuring between 2.5-3.5µm in diameter. Co-staining with a nuclear dye revealed these aggregations to be focused around the nucleus of the cell. Initial FCS experiments indicated a concentration dependent accumulation of the peptide in the cells and a decrease of the intracellular diffusion coefficients at high concentration possibly corresponding with molecular crowding. Interestingly the anomalous diffusion model did not statistically improve the results.RICS was implemented to study the kinetics of entry of TAMRA labelled cell penetrating peptides in both Caco-2 and HeLa cells lines at concentrations between 500nM and 2µM. Concentrations above 1µM exhibited higher final intracellular concentrations, yet the measured diffusion coefficients were similarly independent of extracellular concentration. Both peptides appeared to enter the cell quickly with a fast initial uptake over the first 10 minutes, reaching a concentration maxima after 30 minutes.Overall, the study reveals that many published studies may be incorrect as they may only be reporting the presence of a fluorescent dye inside the cell not the peptide. The fast binding of the peptide to the membrane is likely to cause false positive results when traditionally studying internalisation kinetics such as using flow cytometry and confocal microscopy. Correlation spectroscopy techniques such as FCS, provide useful information on internalisation of the peptides, but the single spot measurement is limited when providing information on the entire cell. RICS is a progression of correlation spectroscopy and provides a more representative picture of the cell.

This thesis explored interactions between monocytes and IgG sensitised RBCs as well as synthetic particles for clinical and biomedical application. A monocyte monolayer assay was developed for Red Cell Reference Laboratory in Australia to predict transfusion outcomes, and mechanism and immune modulation associated with this assay and biomedical potential of polystyrene particles were explored using genetic and biological studies. The results obtained will contribute to improved transfusion safety and patient management and contribute knowledge in the fields of biomedical research using nanomaterials.

Regulation of biological processes through the use of genetic elements is a central part of biological research and also holds great promise for future therapeutic applications. Oligonucleotides comprise a class of versatile biomolecules capable of modulating gene regulation. Gene therapy, the concept of introducing genetic elements in order to treat disease, presents a promising therapeutic strategy based on such macromolecular agents. Applications involving charged macromolecules such as nucleic acids require the development of the active pharmaceutical ingredient as well as efficient means of intracellular delivery. Cell-penetrating peptides are a promising class of drug delivery vehicles, capable of translocation across the cell membrane together with molecules otherwise unable to permeate cells, which has gained significant attention. In order to increase the effectiveness of cell-penetrating peptide-mediated delivery, further understanding of the mechanisms of uptake is needed in addition to improved design to make the cell-penetrating peptides more stable and, in some cases, targeted. This thesis encompasses four scientific studies aimed at investigating cell-penetrating peptide and oligonucleotide designs amenable to therapeutic applications as well as elucidating the mechanisms underlying uptake of cell-penetrating peptide:oligonucleotide nanoparticles. It also includes an example of a therapeutic application of cell-penetrating peptide-mediated delivery of oligonucleotides. Paper I presents a study evaluating a range of chemically modified anti-miRNAs for use in the design of therapeutic oligonucleotides. All varieties of oligonucleotides used in the study target miRNA-21 and are evaluated using a dual luciferase reporter system. Paper II introduces a novel cell-penetrating peptide, PepFect15, aiming at combining the desirable properties of improved peptide stability and efficient cellular uptake with a propensity for endosomal escape, to produce a delivery vector well suited for delivery of oligonucleotides. The performance of this new cell-penetrating peptide was evaluated based on its delivery capabilities pertaining to splice-correcting oligonucleotides and anti-miRNAs. Paper III investigates the involvement of scavenger receptor class A in the uptake of various cell-penetrating peptides together with their oligonucleotide cargo. Finally, paper IV aims at using cell-penetrating peptide-mediated delivery to improve the efficiency of telomerase inhibition by antisense oligonucleotides targeting the telomerase enzyme ribonucleotide component.<br><p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Failure of the immune system to differentiate between apoptotic tumour cells and cells rendered apoptotic as part of homeostasis prevents a successful response being mounted against tumours. Apoptotic cells are cleared rapidly by professional phagocytes thus preventing the release of potentially inflammatory or immunogenic material into the surrounding environment. In addition, macrophages release immuno-suppressive cytokines such as tumour growth factor β (TGF-β) and interleukin-10 (IL-10) that dampen the initiation of cytotoxic T cell (CTL) immunity and render T cells tolerant. It was hypothesised that manipulation of this clearance pathway, which is normally employed to prevent autoimmunity, may alleviate immuno-suppression of apoptotic tumour cells and promote the expansion of a tumour antigen-specific T cell response. Milk fat globule – epidermal growth factor 8 (MFG-E8) is a glycoprotein secreted by activated macrophages and immature dendritic cells. It facilitates the uptake of apoptotic cells by acting as a bridging molecule between integrins on the phagocyte and phosphatidylserine (PS) on the apoptotic cell surface. Here, two recombinant dominant-negative MFG-E8 proteins were generated: one which is shown to inhibit PS-dependent apoptotic cell uptake by macrophages by over 40% (DN-MFG-E8), and a second which re-directed apoptotic cells through Fcγ receptors and conferred enhanced phagocytosis by both macrophages and immature dendritic cells (DN-MFG-E8-Fc) in a dose-dependent manner. Cross-presentation of cell-associated antigen by bone marrow-derived dendritic cells (BMDCs) was determined by CD8+ T cell proliferation assays in vitro and in vivo. Loading BMDCs with apoptotic cells via a PS-independent pathway or through Fcγ receptors (FcγRs) reduced their ability to induce CD8+ T cell proliferation in vivo, suggesting the blockade of a mechanism which is intrinsic for DC maturation or migration. Similarly, the balance between activating and inhibitory FcγRs proved essential for effective DC maturation. Apoptotic cells treated with DN-MFG-E8-Fc protein resulted in upregulation of costimulatory molecules, CD86 and CD70, when BMDCs were deficient for the inhibitory FcγR, FcγRIIb. Additionally, the immunosuppressive effect of apoptotic cells on antibody production proved dependent on the exposure of PS; whereby both DN-MFG-E8 and DN-MFG-E8-Fc proteins were shown to alleviate this suppression.

Recent progress in synthetic chemistry has enabled the preparation of new highly-defined polymers that exhibit changes in their structure in response to environmental changes. These responsive nanomaterials may be desirable as carriers of drugs to deliver at the cellular and sub-cellular level. However, the endocytic pathways used by these nanoparticles to access cells must be defined. Carboxylated polystyrene beads (C-PB) of 50 and 100 nm size were chosen as ‘model’ nanomedicines and their route of uptake into cells characterised and compared to thermoresponsive PLGA-b-(PEGMA-co-PPGMA) and PLA-b-(DEGMA-co-OEGMA) block copolymers of 50-150 nm (‘candidate’ drug delivery systems) uptake. A number of protocols were optimised for endocytosis inhibition studies. Results reported that the inhibition of clathrin mediated endocytosis (CME) with chlorpromazine (CPZ) was cell- and time-dependent. After the maximal effect of the inhibitor, the endocytosis of human transferrin (Htf), a marker of CME, recovered up to uninhibited levels in 3T3 and HCT116 cells. Furthermore, high passage number and ageing of cells showed a resistance towards the inhibition of the uptake of Htf with CPZ. Both PLGA-b-(PEGMA-co-PPGMA) and PLA-co-(DEGMA-co-OEGMA) thermoresponsive block copolymers presented colloidal instability and aggregation that impeded further endocytic pathway internalization experiments. However, the results reported in this thesis question some of the interpretation in the literature of the susceptibility of cells to CPZ in the internalization of nanomaterials. New experimental settings for CPZ inhibition studies should be considered and protocols optimised in order to avoid incorrect and potentially misleading outcomes.

Genes are the major regulators of biological processes in every living thing. Problems with gene regulation can cause serious problems for the organism; for example, most cancers have some kind of genetic component. Regulation of biological processes using oligonucleotides can potentially be a therapy for any ailment, not just cancer. The problem so far has been that the targets for oligonucleotide-based therapies all reside on the inside of cells, because the cellular plasma membrane is normally impermeable to large and charged molecules (such as oligonucleotides) a delivery method is needed. Cell-penetrating peptides are a class of carrier molecules that are able to induce the cellular membrane into taking them and their cargo molecules into the cells. Understanding how and why cell-penetrating peptides work is one of the first and most important steps towards improving them to the point where they become useful as carriers for oligonucleotide-based therapies. This thesis is comprised of four scientific papers that are steps toward finding an uptake mechanism for cell-penetrating peptides that have been non-covalently complexed with oligonucleotides. In Paper I, we show that the scavenger receptors are responsible for uptake of the cell-penetrating peptide PepFect14 in complex with a short single-stranded oligonucleotide. Paper II expands upon this first finding and shows that the same receptors are key players in the uptake of several other cell-penetrating peptides that have been complexed with either, long double-stranded plasmid DNA or short double-stranded RNA. Paper III improves the luciferase-based assay for short oligonucleotide delivery by increasing the throughput 4-fold and reducing the cost by 95 %. The fourth manuscript uses the assay developed in paper III to investigate the effects on cell-penetrating peptide-mediated delivery by each of the constituents of a 264-member library of ligands for G-protein coupled receptors. We identify three ligands that dose-dependently increase the luciferase expression compared to control cells. These three ligands are one positive-, one negative allosteric modulator of metabotropic glutamate receptor 5 and one antagonist of histamine receptor 3.<br><p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Earlier work showed transfer of cytoplasm between co-cultured cancer cells and fibroblasts. Cancer cell changed upon uptake of fibroblast contents, with altered morphology and increased cell volume, internal cell complexity and cell migration velocity. The biomechanical basis for transfer was revealed as hydrodynamic, and was termed 'cell-projection pumping'. An important role for cell-projection pumping in driving cancer cell diversity and disease progression was proposed. Assays used in earlier studies averaged the response of many thousands of cancer cells. This may have obscured important effects only detectable at the single cell level. The current thesis used methods for single cell analysis of the effects of cell-projection pumping from fibroblasts to cancer cells. A novel Cartesian Plot analysis method was developed that related transfer of fluorescent contents between co-cultured cancer cells and fibroblasts, to fluorescence in control cells cultured alone. This identified five separate cell sub-populations in co-cultures, and gave a numeric score for transfer to each cell. Results confirmed the morphological effects of transfer earlier seen. The method was extended to study the effect of cell-projection pumping on global DNA methylation, and it was established that global DNA methylation of cancer cells was unaffected. A single-cell tracking method was developed for analysis of time-lapse recordings of co-cultures which confirmed the morphological effects and increased cancer cell migration earlier seen. Data also revealed for the first time, increased cancer cell division subsequent to uptake of fibroblast contents. Importantly, division of cancer cells abrogated the effect of cell-projection pumping, re-setting daughter cells to their native state. This was consistent with absence of changes in DNA methylation, and also supported the possible exploitation of inhibitors for cell-projection pumping as a novel anti-cancer treatment.

Mycoplasma genitalium és un patogen humà que es transmet sexualment i causant d’uretritis, cervicitis i inflamació pelviana. Conté el genoma més petit de qualsevol microorganisme capaç d’autoreplicar-se, amb només 580 kb i 500 gens que codifiquen per proteïnes. L’interès en aquest patogen ha anat augmentat durant els darrers anys degut a la seva cada cop major prevalença i l’aparició de soques resistents a múltiples antibiòtics. En aquesta tesi doctoral, analitzem en detall la divisió cel·lular d’aquest microorganisme, així com la seva resposta a l’absència de metalls. Pel que fa a la divisió, M. genitalium conté una versió molt reduïda de l’operó de divisió cel·lular amb només quatre gens: mraZ, mraW, un gen que codifica per una proteïna de funció desconeguda i ftsZ. Els dos primers gens estan molt conservats en el món bacterià, però no hi ha gaire informació sobre el seu rol en la divisió cel·lular. En aquest treball, caracteritzem els mutants de mraZ i mraW i demostrem els defectes de creixement associats a la seva pèrdua. També establim les dinàmiques de FtsZ en aquest microorganisme i observem que hi ha una relació entre la maquinària de motilitat i FtsZ a M. genitalium. Al segon capítol, descrivim la resposta transcripcional d’aquest patogen a la manca de metalls. Caracteritzem un membre de la família de reguladors transcripcionals Ferric Uptake Regulator (Fur) i també detallem els gens que es troben al seu reguló: un gen que codifica per a una lipoproteïna rica en histidines (hrl) i un transportador de metalls de tipus ABC (MG_304, MG_303, MG_302). Definim experimentalment l’operador de Fur: una seqüència palindròmica conservada que es troba a la regió upstream d’aquests gens. A més, detallem una àmplia resposta transcripcional a l’absència de metalls induïda pel quelant 2,2’-Bipyridyl, una resposta que també es produeix al mutant de fur, el que significa que existeixen vies reguladores Fur-independents relacionades amb l’homeòstasi de metalls. Finalment, mostrem a través de ICP-MS que en el mutant defectiu de fur hi ha un increment important de níquel, el que suggereix que aquest regulador pot tenir un rol important en l’adquisició de níquel. En resum, en aquesta tesi doctoral caracteritzem dos vies reguladores importants de M. genitalium associades amb dos processos essencials: la divisió cel·lular i l’adquisició de metalls.<br>Mycoplasma genitalium es un patógeno humano que se transmite sexualmente y es agente causante de uretritis, cervicitis e inflamación pélvica. Contiene el genoma más pequeño de todos los microorganismos capaces de autoreplicarse, con únicamente 580 kb y 500 genes que codifican para proteínas. El interés en este patógeno ha aumentado recientemente debido a su cada vez más elevada prevalencia y a la aparición de cepas multiresistentes a antibióticos. En esta tesis doctora, analizamos en detalle la división celular de este microorganismo, así como su respuesta a la ausencia de metales. Por lo que respecta a la división, M. genitalium contiene una versión muy reducida del operón de división celular con sólo cuatro genes: mraZ, mraW, un gen que codifica para una proteína de función desconocida y ftsZ. Sobre los dos primeros genes hay poca información disponible sobre su rol en la división, a pesar de que están ampliamente conservados en el mundo bacteriano. En este trabajo, caracterizamos los mutantes de mraZ y mraW y demostramos los defectos en el crecimiento asociados a su pérdida. También establecemos las dinámicas de FtsZ en este microorganismo y observamos que hay una relación entre la maquinaria de motilidad y FtsZ en M. genitalium. En el segundo capítulo, describimos la respuesta transcripcional de este patógeno cuando se enfrenta a la ausencia de metales. Caracterizamos un miembro de la familia de factores de transcripción Ferric Uptake Regulator (Fur) y detallamos los genes que están en su regulón: un gen que codifica para una lipoproteína rica en histidinas (hrl) y un transportador de metales de tipo ABC (MG_304, MG_303, MG_302). También definimos experimentalmente el operador de Fur: una secuencia palindrómica conservada que se encuentra en la región upstream de estos genes. Además, se detalla una amplia respuesta transcripcional a la ausencia de metales inducida por el quelante 2,2'-Bipyridyl, una respuesta que también se produce en el mutante defectivo de fur, con lo cual se evidencia la existencia de vías regulatorias Fur-independientes implicadas en la homeostasis de metales. Finalmente, mostramos a través de ICP-MS que en el mutante de fur hay un incremento importante de níquel, lo que sugiere que este regulador podría tener un rol importante en la adquisición de este metal. En resumen, en esta tesis doctoral caracterizamos dos vías reguladoras importantes de M. genitalium asociadas con dos procesos celular esenciales: la división y la adquisición de metales.<br>Mycoplasma genitalium is a sexually transmitted human pathogen that causes urethritis, cervicitis and pelvic inflammation. It has the smallest genome of any microorganism capable of self-replication, with only 580 kb and barely 500 protein-coding genes. The interest in this pathogen is rising in recent years due to its increasing prevalence and the appearance of multi-drug resistant strains. In this work, we analyze the cell division of this microorganism as well as its response to a metal depletion stress. Regarding the former, M. genitalium encodes a reduced version of the division and cell wall operon that consists of only four genes: mraZ, mraW, a gene coding for an hypothetical protein and ftsZ. The first two genes are widely conserved among bacteria, but there is little to none information about their role in cell division. In this work, we characterize the mraZ and mraW mutants and we demonstrate the growth defects associated with their deletion. We were also able to establish the FtsZ dynamics in this microorganism and we observed that there is a close relation between the cell motility machinery and FtsZ in M. genitalium. In the second chapter, we assess the transcriptional response of this pathogen to metal starvation. We characterize a member of the Ferric Uptake Regulator (Fur) family of transcription factors in this microorganism and we report the genes in its regulon: a gene coding for a histidine-rich lipoprotein (hrl) and an ABC metal transporter (MG_304-MG_303-MG_302). We are able to describe the Fur operator: a conserved palindromic sequence found in the upstream region of these genes. In addition, we also detail a vast transcriptional response to metal depletion induced by the chelator 2,2'-Bipyridyl even in the mutant that lacks the regulator, demonstrating the existence of Fur-independent regulatory pathways. Finally, we reveal an increased nickel uptake in the fur mutant by ICP-MS, suggesting an important role of this regulator in nickel acquisition. Overall, in this work we are able to characterize two important regulatory networks of the genome-reduced M. genitalium associated with two essential processes: cell division and metal uptake.<br>Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina

Gene therapy holds the promise of revolutionizing the way we treat diseases. By using recombinant DNA and oligonucleotides (ONs), gene functions can be restored, altered or silenced according to the therapeutic need. However, gene therapy approaches require the delivery of large and charged nucleic acid-based molecules to their intracellular targets across the plasma membrane, which is inherently impermeable to such molecules. In this thesis, two chemically modified cell-penetrating peptides (CPPs) that have superior delivery properties for several nucleic acid-based therapeutics are developed. These CPPs can spontaneously form nanoparticles upon non-covalent complexation with the nucleic acid cargo, and the formed nanoparticles mediate efficient cellular transfection. In paper I, we show that an N-terminally stearic acid-modified version of transportan-10 (PF3) can efficiently transfect different cell types with plasmid DNA and mediates efficient gene delivery in-vivo when administrated intra muscularly (i.m.) or intradermaly (i.d.). In paper II, a new peptide with ornithine modification, PF14, is shown to efficiently deliver splice-switching oligonucleotides (SSOs) in different cell models including mdx mouse myotubes; a cell culture model of Duchenne’s muscular dystrophy (DMD). Additionally, we describe a method for incorporating the PF14-SSO nanoparticles into a solid formulation that is active and stable even when stored at elevated temperatures for several weeks. In paper III, we demonstrate the involvement of class-A scavenger receptor subtypes (SCARA3 &amp; SCARA5) in the uptake of PF14-SSO nanoparticles, which possess negative surface charge, and suggest for the first time that some CPP-based systems function through scavenger receptors. In paper IV, the ability of PF14 to deliver siRNA to different cell lines is shown and their stability in simulated gastric acidic conditions is highlighted. Taken together, these results demonstrate that certain chemical modifications can drastically enhance the activity and stability of CPPs for delivering nucleic acids after spontaneous nanoparticle formation upon non-covalent complexation. Moreover, we show that CPP-based nanoparticles can be formulated into convenient and stable solid formulations that can be suitable for several therapeutic applications. Importantly, the involvement of scavenger receptors in the uptake of such nanoparticles is presented, which could yield novel possibilities to understand and improve the transfection by CPPs and other gene therapy nanoparticles.<br>At the time of doctoral defence the following paper was unpublished and had a status as follows: Paper nr 4: Submitted

A series of 3'-linked conjugates between an oligodeoxyribonucleotide 13mer and cholic, folic, lipoic and pantothenic acids, cholesterol, vitamin E and a viral fusion peptide have been prepared in an attempt to enhance the cellular uptake properties of the nucleic acid. It was envisaged that the 4 biological acids may improve the cellular uptake of the oligonucleotide through natural receptor-mediated uptake mechanisms, whilst cholesterol, vitamin E and the fusion peptide may enhance interaction of the conjugate with cellular membranes. In the course of preparing conjugates involving cholic, folic, lipoic and panthothenic acids, a methodology was developed that allowed for simple and effective conjugation between moieties bearing a carboxylic acid group and an oligonucleotide sequence, achieving linkage through disulfide and thioether bonds. All of the 3'-linked conjugates were characterised by reverse phase HPLC and electrospray mass spectrometry, and were found to have increased stability towards nucleases present in serum. The solid phase synthesis of a small library of peptides composed of the cationic amino acids lysine, ornithine, histidine and arginine, the hydrophobic amino acid tryptophan, and alanine as a spacer, and their subsequent coupling through an N-terminal cysteine residue to a 5'-maleimide-modified 8mer was also accomplished. The preparation of 49 peptide-oligonucleotide conjugates was achieved in good yield and purity with a simple gel filtration employed as the only purification step. Melt analysis of the hybridisation properties of the constructs indicated that increasing the number of cationic residues within the peptide segment led to elevated <I>T</I><SUB>m</SUB> values on binding with a 16mer target. The binding affinities of the peptide segments were found to be stronger when bound to single-stranded rather than duplex DNA, and dependent on salt concentration and pH. Thermodynamic analysis revealed enhanced free energies of binding for cationic peptide-oligonucleotides compared to the unmodified 8mer.

An outstanding challenge in modern medicine is the safe and efficient delivery of drugs. One approach to improve drug delivery yield and increase specificity towards diseased cells, is to employ a drug carrier to facilitate transport. Promising steps towards developing such a carrier have been taken by the nascent field of nanomedicine: nanometer-sized particles designed to evade premature excretion, non-specific absorption, and the body’s immune response, can reduce undesired drug loss, while also increasing specific drug uptake into diseased cells through targeting surface modifications. However, progress is limited by incomplete knowledge of the ‘ideal’ nanoparticle design as well as a lack of appropriate high resolution construction methods for its implementation. DNA origami, a modular, nanometer-precise assembly method that would enable the rapid testing of particle properties as well as massively parallel fabrication, could provide an avenue to address these needs. In this thesis, I employed the DNA origami method to investigate how nanoscale shape and ligand functionalization affect nanoparticle uptake into cultured endothelial cells. In the first part, I evaluated the uptake yield of a series of eight shapes that ranged from 7.5 nm to 400 nm in their individual dimensions. The best performing shape of that study, a 15 × 100 nm DNA origami nanocylinder, was internalized 18-fold better than a dsDNA control of the same molecular weight. In a follow up study, I decorated this nanocylinder with integrin-targeting cyclic RGD peptides. This surface functionalization increased cellular uptake another 13-fold. In addition, uptake yield and the ratio of internalized versus surface-bound particles depended on the number of ligands present on the nanoparticle surface. This work represents a significant first step towards attaining the ability to design and implement an 'ideal' nanoparticle drug carrier. In the future, the DNA origami method can be used as a platform technology to further expand our understanding of transport properties of drug carriers and achieve safer and more efficient drug delivery.

Thesis (M.S.)--University of California, San Diego, 2009.<br>Title from first page of PDF file (viewed June 22, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 52-62).

Mitochondrial Ca2+ uptake regulates cellular energetic by triggering ATP synthesis. At the same time, Ca2+mit acts as key controller of both cell metabolism and fate. Indeed, a decrease in ATP production elicits autophagy induction, while Ca2+ overload causes organelle dysfunction and release of caspase cofactors. Several pathological conditions, including tumor formation and progression, are directly related to mitochondrial dysfunctions, and reprogramming of mitochondrial metabolism is now considered as an emerging hallmark of cancer. Indeed, even in the presence of oxygen, cancer cells limit their energy supply largely to glycolysis, leading to the so called “aerobic glycolysis” phenotype. Of note, the dependence on glycolytic fueling is further potentiated by hypoxia, a condition that characterizes most tumor microenvironments. Indeed, in response to oxygen deprivation, the hypoxia-indicible-factor-1α (HIF-1α) is stabilized and, consequently, transcription of glucose transporters and glycolysis-related enzymes is induced. In addition, several hereditary tumors have been associated with mutations in key mitochondrial enzymes showing that, in specific settings, an altered mitochondrial metabolism represents a primary trigger for cancer progression. Consistent with these observations, among the most aggressive human breast tumors, triple-negative-breast-cancers (TNBC), a clinically heterogeneous category of breast tumors that lack expression of estrogen, progesterone and HER2 receptors, show profound metabolic alterations with impaired mitochondrial oxidative. Our preliminary observations suggested that, in contrast to pre-malignant cells, TNBC metastatic cells accumulate high [Ca2+] into the mitochondrial matrix upon agonist stimulation. These elevate Ca2+mit transients might be essential for metastatic progression, while sensitize premalignant cells to apoptotic stimuli. Accordingly, we hypothesized alternative roles of mitochondrial Ca2+, distinct from mitochondrial energy production and control of cell death, possibly involved in the metastatic progression of these highly malignant breast tumors. The molecular characterization of the Mitochondrial Calcium Uniporter (MCU), the highly selective channel responsible for Ca2+mit entry into the matrix, opened the chance to modulate mitochondrial function by modifying MCU channel’s activity. We demonstrated that both pharmacological and genetic inhibition of MCU causes a significant decline in TNBC metastatic cell motility and invasiveness. Thus, we investigated how Ca2+mit uptake contributes to mitochondrial Reactive-Oxygen-Species (mROS) production, thus playing a crucial role in the regulation of ROS-dependent cascades. Indeed, ROS molecules are considered fundamental molecular effectors for cancer progression, by eliciting metabolic adaptations and in vivo metastasis formation. Coherently, we observed that antioxidant treatments reduce significantly MDA-MB-231 cell migration. Moreover, in the absence of MCU, production of mROS is significantly reduced, suggesting that mROS might play a crucial role in cell malignancy regulation by Ca2+mit uptake. In addition, MCU silencing inhibits hypoxia-inducible factor 1α (HIF-1α) transcription, thus impairing the expression of HIF-target genes involved in metastasis progression, possibly in a ROS-dependent manner. Of note, rescue of HIF-1α expression restores migration of MCU-silenced TNBC cells. Our results highlight a crucial role of MCU in the control of TNBC metastatic potential and indicate that mitochondrial Ca2+ uptake could represent a novel therapeutic target for clinical intervention.<br>Il flusso di Ca2+ mitocondriale (Ca2+mit) è il principale regolatore della bioenergetica cellulare, grazie al fatto che stimola la produzione di ATP. Allo stesso tempo, il Ca2+mit gioca un ruolo chiave nella regolazione sia del metabolismo sia della morte cellulare. Infatti, una diminuita produzione di ATP porta all’attivazione del processo autofagico (per compensare la mancanza di nutrienti) mentre un accumulo elevato di Ca2+ nel mitocondrio porta inevitabilmente ad una disfunzione dell’organello, seguita dall’attivazione caspasica e quindi dalla morte cellulare . Molte patologie, tra cui la formazione e crescita tumorale, sono direttamente correlabili con le disfunzioni mitocondriali. Inoltre, il reprogramming del metabolismo mitocondriale è ora considerato un aspetto chiave nella patogenesi del cancro. Infatti, anche in presenza di ossigeno, le cellule tumorali limitano gran parte del loro supporto energetico alla glicolisi, raggiungendo lo stato energetico denominato “glicolisi aerobica”. Vale la pena evidenziare che la dipendenza energetica da un regime glicolitico può venir ulteriormente rafforzata in ipossia, condizione che contraddistingue la maggior parte dei microambienti tumorali. Infatti, in risposta alla carenza di ossigeno, il fattore trascrizionale HIF-1α viene stabilizzato e, di conseguenza, viene indotta la trascrizione di proteine di trasporto del glucosio e di enzimi coinvolti direttamente nel metabolismo glicolitico. Inoltre, molti tumori ereditari sono stati associati a mutazioni a carico di enzimi mitocondriali, dimostrando in questo modo che, in particolari condizioni, un’alterazione del metabolismo mitocondriale può esser considerata fattore eziologico per lo sviluppo del tumore. A supporto di queste considerazioni, il tumore mammario metastatico triplo negativo, una delle più aggressive ed eterogenee classi di tumori caratterizzata dalla mancata espressione dei recettori estrogenici, progestinici ed HER2, mostra una profonda alterazione metabolica con ridotta attività ossidativa mitocondriale. I nostri risultati preliminari suggeriscono che, al contrario delle cellule pre-maligne, le metastatiche triplo negative riescono ad accumulare, dopo stimolazione, alte [Ca2+] all’interno della matrice mitocondriale. Transienti di Ca2+mit così elevati possono risultare addirittura essenziali per la progressione metastatica, mentre in cellule pre-maligne sensibilizzano alla morte cellulare. Di conseguenza, abbiamo ipotizzato ruoli alternativi per il Ca2+mit, possibilmente coinvolti nello sviluppo metastatico, che fossero diversi da quelli di produzione energetica mitocondriale e di controllo dell’apoptosi. La caratterizzazione molecolare dell’Uniporto Mitocondriale del Calcio (dall’inglese MCU), canale selettivo per il Ca2+ che ne regola l’ingresso all’interno della matrice, ha aperto la possibilità di modulare la funzione mitocondriale modificando l’attività del canale MCU. Così, si è deciso di investigare come l’ingresso di Ca2+mit contribuisca alla produzione mitocondriale di specie radicaliche dell’ossigeno (ROS), giocando in questo modo un ruolo cruciale nella regolazione di tutti i meccanismi di segnale ROS-dipendenti: i ROS sono considerati dei critici effettori molecolari della progressione tumorale, promuovendo l’adattamento metabolico e la formazione di metastasi in vivo. Di conseguenza, si è osservato che trattamenti antiossidanti riducono significativamente la migrazione delle cellule tumorali triple negative MDA-MB-231. Inoltre, è stato verificato che, in assenza di MCU, la produzione di ROS è significativamente ridotta, suggerendo così che i ROS mitocondriali giocano un ruolo cruciale nella regolazione della malignità tumorale mediata dal Ca2+mit. In aggiunta, è stato dimostrato che il silenziamento di MCU inibisce significativamente la trascrizione del fattore di trascrizione HIF-1α, riducendo di conseguenza l’espressione dei geni da lui regolati, soprattutto di quelli coinvolti nella progressione metastatica e possibilmente con un meccanismo ROS-dipendente. Da notare, a tal proposito, che la ri-espressione di HIF-1α ripristina completamente la capacità di migrazione nelle cellule in cui MCU è silenziato. In conclusione, i nostri risultati mettono in luce un ruolo cruciale di MCU nel controllo del potenziale metastatico del tumore mammario triplo negativo ed indicano che il flusso mitocondriale di Ca2+ può rappresentare un nuovo bersaglio terapeutico per un innovativo approccio clinico.

Atherosclerosis is a disease characterized by cholesterol-rich plaques within the intima of medium and large arteries. Cholesterol deposition is thought to occur by infiltration of low-density lipoprotein (LDL) into lesions followed by uptake into macrophages, generating lipid-loaded “foam cells.” Foam cells can also be generated in vitro by treatment of macrophages with LDL or oxidized LDL (oxLDL). The purpose of the current investigation was to determine the contribution of selective cholesteryl ester (CE) uptake versus whole-particle uptake during LDL-induced foam cell formation in cultured macrophages. Murine bone marrow-derived macrophages (BMMs) exhibited significant cholesterol accumulation when treated with LDL as indicated by quantification of cellular cholesterol and visualization of Oil Red-O-stained neutral lipid droplets. Uptake of LDL cholesterol was determined by measuring uptake of 3H and 125I into BMMs during treatment with [3H]CE/125I-LDL. [3H]CE uptake was linearly related to the LDL concentration at the concentrations used and was much larger than 125I uptake, indicating that the majority of LDL-cholesterol was acquired by nonsaturable, selective CE uptake. This pathway was demonstrated to be independent of whole-particle uptake by showing that inhibition of actin polymerization blocked LDL particle uptake but not selective CE uptake. Analysis by thin-layer chromatography (TLC) indicated that following uptake, [3H]CE was rapidly hydrolyzed into [3H]cholesterol by cells and largely effluxed into the culture medium. In contrast to LDL, studies of [3H]CE/125I-oxLDL uptake demonstrated that CE was acquired from oxLDL by whole-particle uptake with little or no selective CE uptake. Using a series of ten different [3H]CE/125I-oxLDLs oxidized for 0-24 hours, selective [3H]CE uptake was shown to be progressively impaired by LDL oxidation, while 125I-LDL particle uptake was increased as expected. Interestingly, the impairment of selective CE uptake occurred very early in LDL oxidation and this minimally oxidized LDL induced significantly less cholesterol accumulation in BMMs compared to native LDL. Together, these results demonstrate that selective CE uptake is the primary mode of cholesterol uptake from LDL but not oxidized LDL, a finding that has important implications for cholesterol metabolism in atherosclerotic lesions. Future studies seek to identify the molecular components that participate in the macrophage selective CE uptake mechanism.

Glucose and regulation of cell cycle in S. cerevisiae: analysis of mutants impaired in sugar uptake mechanisms Requisito fondamentale per la sopravvivenza di microrganismi a vita libera come il lievito S. cerevisiae è la capacità di regolare il proprio metabolismo e la progressione del ciclo cellulare in modo tale che la crescita sia rapida in presenza di abbondanti nutrienti e si arresti all’esaurirsi degli stessi. Perché questo sia possibile, nutrienti come il glucosio devono generare segnali che vengano recepiti ed elaborati dal complesso macchinario che governa il ciclo cellulare. S. cerevisiae possiede almeno tre meccanismi per rilevare variazioni dei livelli di glucosio nel mezzo di coltura: il pathway di Rgt2/Snf3, che controlla l’espressione dei trasportatori degli zuccheri esosi; il pathway cAMP/PKA, che regolando l’attività della protein-kinasi A promuove l’espressione di geni coinvolti nel metabolismo fermentativo e nella crescita cellulare e inibisce la trascrizione di geni coinvolti nella risposta agli stress; il glucose main repression pathway, che reprime l’espressione di geni coinvolti nella respirazione cellulare, nella gluconeogenesi e nell’utilizzo di fonti di carbonio alternative al glucosio. L’assunzione di glucosio nel citoplasma dall’ambiente esterno avviene attraverso i trasportatori codificati dalla famiglia di geni HXT (HeXose Transporter), che comprende almeno 20 membri: HXT1-17, RGT2, SNF3 e GAL2. Snf3 e Rgt2 sono incapaci di trasportare lo zucchero, ma agiscono piuttosto da sensori del livello di glucosio extracellulare: in particolare, Snf3 rileva basse concentrazioni dello zucchero inducendo l’espressione dei trasportatori ad alta affinità (codificati dai geni HXT2-HXT4), mentre Rgt2 rivela alte concentrazioni di glucosio promuovendo l’espressione dei trasportatori a bassa affinità (HXT1). Nessuno dei trasportatori è essenziale e solo la delezione di tutti i geni HXT (o almeno di quelli compresi tra 1-7 in alcuni background) rende la cellula di lievito incapace di crescere in presenza di glucosio come unica fonte di carbonio. L’espressione dei vari trasportatori è regolata a livello trascrizionale attraverso un complesso network che coinvolge tutti e tre pathway deputati al sensing del glucosio: come risultato, S. Cerevisiae è in grado di mantenere sempre un alto flusso glicolitico esprimendo il set di trasportatori più adatto alla quantità di glucosio disponibile. Le connessioni tra i pathway deputati al sensing del glucosio e gli elementi di regolazione del ciclo cellulare non sono completamente definite, anche perché risulta spesso difficile scindere il duplice ruolo dello zucchero come nutriente e come molecola segnale. Obbiettivo del presente progetto di ricerca è chiarire gli effetti di alterazioni nei meccanismi di sensing e (in modo particolare) di trasporto del glucosio sulla coordinazione tra crescita e divisione cellulare. In una prima fase dello studio sono stati presi in esame alcuni mutanti con delezioni nei geni HXT1-7, codificanti per i principali trasportatori degli zuccheri esosi: i dati presenti in letteratura certificano infatti come queste mutazioni siano sufficienti ad abolire sostanzialmente l’assunzione (uptake) cellulare del glucosio impedendo la crescita dei mutanti su tale fonte di carbonio. I parametri di crescita (tempo di duplicazione, indice di gemmazione, contenuto proteico e di DNA) di ciascuno dei ceppi sono stati determinati in due condizioni sperimentali: i) crescita esponenziale bilanciata in terreno csm/YNB addizionato di 2% etanolo o di una miscela 2% Etanolo + 2% glucosio, da cui è emerso come il glucosio possa esercitare un effetto sulle dimensioni celulari anche nei mutanti hxt (indipendente quindi dal suo ruolo come nutriente). Infatti, analogamente alle cellule wild-type, anche i ceppi con i trasportatori deleti mostrano dimensioni cellulari e contenuto proteico maggiore quando fatti crescere in glucosio+Etanolo, sebbene (diversamente dal ceppo wild type) la loro velocità di crescita sia simile a quella registrata in terreno con solo etanolo; ii) crescita durante shift-up nutrizionale etanolo => glucosio. All’aggiunta dello zucchero le cellule wild-type vanno incontro ad fase iniziale di adattamento (evidenziato dalla diminuzione transiente (10-15%) dell’indice di gemmazione), necessaria per reimpostare profilo trascrizionale, velocità di crescita e dimensioni cellulari e per il successivo passaggio ad un metabolismo energetico di tipo fermentativo. A differenza del ceppo wild type, dopo l’aggiunta di glucosio le cellule hxt(1-7) manifestano una drammatica e prolungata riduzione nell’indice di gemmazione e un forte rallentamento (arresto) nella progressione del ciclo cellulare. In seguito le cellule riprendono a dividersi con una velocità sostanzialmente identica a quella precedente lo shift, mentre volume cellulare e contenuto proteico medio aumentano sensibilmente: l’effetto del glucosio sulle dimensioni cellulari dei mutanti hxt(1-7) è tuttavia transiente e si esaurisce nell’arco di due/tre round di divisioni, quando le cellule tornano ad assumere le dimensioni tipiche della crescita su etanolo. I dati finora riportati sembrano quindi suggerire che, almeno inizialmente, gli effetti del glucosio sulle dimensioni cellulari dipendano dal sensing dello zucchero e non dal suo metabolismo. Tuttavia, sebbene il ceppo hxt(1-7) non sia in grado di crescere su glucosio come unica fonte, rimane comunque dotato di una capacità residua di trasporto dello zucchero, che sebbene insufficiente a sostenere il metabolismo glicolitico, potrebbe comunque assumere un’importanza decisiva per la regolazione delle dimensioni cellulari. Nel tentativo di scindere ancora più nettamente il duplice ruolo del glucosio come nutriente e come molecola segnale, in una successiva fase di studi sono stati utilizzati mutanti con delezioni in tutti i geni per i trasportatori del glucosio (hxt(1-17)), in cui ogni residuo trasporto dello zucchero risulta abolito. In aggiunta, si sono presi in esame una serie di mutanti con una capacità di uptake del glucosio progressivamente ridotta: nel dettaglio, la lista comprende (oltre ovviamente al ceppo wild type di riferimento): i) hxt(1-17)gal2, in cui il trasporto del glucosio è completamente abolito; ii) il ceppo hxt(1-17)) snf3, in cui l’inattivazione del sensore SNF3 rispristina una trascurabile capacità di trasporto del glucosio, insufficiente comunque a garantire la crescita in terreno liquido contenente glucosio come unica fonte: l’assunzione dello zucchero in questo caso sembrerebbe avvenire attraverso un trasportatore non ancora caratterizzato, la cui trascrizione risulta derepressa in assenza di SNF3; iii) il ceppo (hxt(1-17) gal2 HXT1, che esprime in modo costitutivo come unico trasportatore HXT1, un carrier a bassa affinità; iv) ) il ceppo (hxt(1-17) gal2 HXT7, che esprime in modo costitutivo come unico trasportatore HXT7, un carrier ad alta affinità; v) il ceppo snf3 rgt2, in cui l’uptake del glucosio è ridotto a causa dell’inattivazione del principale pathway che regola l’espressione dei maggiori trasportatori; vi) il triplo deleto hxk2 hxk1 glk1, che è in grado di trasportare glucosio nel citoplasma ma non è in grado di metabolizzarlo a causa dell’assenza di tutte e tre le chinasi che catalizzano il primo passaggio della glicolisi. I ceppi sopra elencati sono stati sottoposti alle analisi descritte in precedenza. Nel caso dei ceppi capaci di metabolizzare il glucosio, il tasso di crescita e le dimensioni cellulari su tale fonte sono generalmente correlate all’efficienza del sistema di trasporto dello zucchero nei vari mutanti: sembra esistere una relazione sostanzialmente lineare tra velocià di consumo del glucosio/velocità di crescita/dimensioni cellulari. Unica eccezione pare essere il ceppo snf3 rgt2, che manifesta dimensioni cellulari notevolmente più ridotte rispetto a quanto atteso sulla base del suo tasso di crescita: un risultato che sembrerebbe suggerire un ruolo diretto del pathway Snf3/Rgt3 nei meccanismi che regolano le dimensioni cellulari in risposta ai nutrienti. Diversamente da quanto emerso in precedenza, la crescita dei mutanti hxt(1-17) risulta fortemente inibita in terreni contenenti miscele di etanolo (o altra fonte non fermentabile) e glucosio, anche quando la concentrazione dello zucchero è a livelli sub-ottimali (0.05% anziché 2%). L’aggiunta di glucosio a cellule hxt(1-17) in crescita su etanolo (shift-up nutrizionale) determina l’arresto permanente del ciclo cellulare in G1 (cellule vitali, non gemmate con contenuto di DNA presintetico). Sembra quindi che la semplice presenza di glucosio nell’ambiente extracellulare - ma non il trasporto dello zucchero nel citoplasma - sia sufficiente ad impedire l’utilizzo di fonti di carbonio alternative presenti nel medium: ciò spiegherebbe la mancata crescita in terreni misti glucosio+etanolo da parte di cellule hxt(1-17), incapaci di effettuare l’uptake dello zucchero. Se tale ipotesi fosse corretta, inattivando contemporaneamente tutti i pathway deputati al sensing del glucosio dovrebbe essere possibile ripristinare la crescita di cellule hxt(1-17) in terreni contenenti miscele di glucosio ed etanolo. Al momento, si è appurato che l’inattivazione del ramo del cAMP/PKA pathway passante attraverso Gpr1/Gpa2 non è sufficiente a correggere il difetto di crescita del ceppo hxt(1-17)gal2 in fonte mista glucosio/etanolo. Al contrario, la semplice inattivazione di SNF3 (ma non di RGT2)sembra sostanzialmente azzerare l’effetto citostatico del glucosio sulla crescita del ceppo hxt(1-17) gal2 snf3. L’interpretazione di tale risultato è ovviamente complicata dal fatto che la delezione di SNF3 ripristina parzialmente il trasporto del gluccosio in un ceppo privo di tutti i trasportatori, sebbene, vale la pena ricordare, su scala estremamente ridotta e comunque insufficiente a sostenere la crescita, come confermato attraverso misurazioni dirette della velocità di consumo delo zucchero nel ceppo hxt(1-17)) snf3. Tuttavia, diversi dati in letteratura suggeriscono come il pathway Snf3/Rgt2 partecipi in qualche misura ai meccanismi della glucose repression, in particolare attrverso Mig2, un repressore trascrizionale che in presenza di glucosio collabora con Mig1 nel reprimere la trascrizione di geni richiesti per l’utilizo di fonti di carbonio alternative. La delezione di MIG2 non è tuttavia sufficiente a ripristinare la crescita su etanolo/glucosio del ceppo hxt(1-17)gal2: ulteriori indagini sono dunque necessarie per chiarire quale sia il ruolo giocato da SNF3 nell’intero processo. In aggiunta, il comportamento manifestato dal ceppo hxk2 hxk1 glk1 durante shift-up nutrizionale da etanolo a glucosio sembra ulteriormente confermare come in lievito lo zucchero sia in grado di regolare le dimensioni cellulari indipendentemente dal proprio metabolismo, almeno in una fase iniziale: le cellule hxk2 hxk1 glk1 in crescita su etanolo rispondono all’aggiunta di glucosio aumentando considerevolmente il proprio volume, in misura paragonabile a quanto si registra nel ceppo wild type; tuttavia, contrariamente al ceppo wild type, nel mutante hxk2 hxk1 glk1 l’aumento delle dimensioni celluari si accompagna ad un progressivo rallentamento della velocità di crescita, fino ad un totale arresto del ciclo di divisione cellulare che sopraggiunge a circa 12 ore dallo shift. Dopo una fase di lag piuttosto prolungata ed estremamente variabile, in cui le cellule, pur non dividendosi, si mantengono gemmate, si assiste alla rispresa del ciclo di divisione cellulare: le cellule tornano a dividersi lentamente utilizzando l’etanolo residuo nel terreno e nell’arco di due/tre generazioni assumono nuovamente le tipiche dimensioni ridotte associate alla crescita su fonte di carbonio non fermentabile. Ad ulteriore conferma di come l’effetto del glucosio sia solo temporaneo, dimensioni e contenuto proteico di cellule hxk2 hxk1 glk1 in crescita bilanciata su etanolo o su fonte mista etanolo/glucosio sono sostanzialmente identiche. Nonostante il sorprendente effetto citostativo dello zucchero, l’aumento delle dimensioni cellulari in risposta all’aggiunta di glucosio si manifesta anche nel ceppo privo di tutti i trasportatori (hxt(1-17) gal2)., sebbene in misura meno eclatante rispetto al triplo mutante hxk2 hxk1 glk1. Nell'insieme, tali risultati sembrano confermare come il glucosio sia in grado di modulare le dimensioni della cellula di lievito in maniera (almeno in parte) indipendente dal proprio ruolo come nutriente, funzionando in buona sostanza come un "ormone". Per chiarire le basi molecolari di tale fenomeno è necessario chiarire le connessioni tra i pathway deputati al sensing del glucosio e gli elementi di regolazione del ciclo di divisione cellulare in S. cerevisiae. A tal fine, si è ultimata la costruzione di una serie di mutanti esprimenti versioni “taggate” (HA-tag) di alcuni dei principali regolatori coinvolti nella transizione G1/S (nello specifico Cln3, Cln2, Far1, Sic1 e Clb5), così da facilitare l’analisi dei loro livelli di espressione e di localizzazione subcellulare. Gli studi in questo senso sono tuttavia in una fase ancora troppo preliminare per poter trarre conclusioni definitive. Da utlimo, si è cercato di valutare il contributo relativo di sensing, trasporto e metabolismo del glucosio alla regolazione trascrizionale del gene SUC2, uno dei marcatori più comunemente utilizzati per valutare il fenomeno della glucose repression in S. cerevisiae. SUC2 codifica per l’invertasi, un enzima chiave per l’utilizzo del disaccaride saccarosio e la sua espressione risulta completamente bloccata in presenza di alti livelli di glucosio mentre viene indotta da raffinosio o da bassi livelli di glucosio. I risultati ottenuti con i vari mutanti hanno evidenziato come in presenza di abbondante glucosio il livello basale di attività invertasica sia generalmente proporzionale alla velocità del flusso glicolitico, che dipende in larga misura dalla capacità di trasporto dello zucchero: nei ceppi aventi un sistema di uptake per il glucosio ad efficienza ridotta l’invertasi risulta parzialmente o addirittura competamente derepressa, come nel caso del mutante privo di tutti i trasportatori. Il ceppo snf3 rgt2 sfugge invece a questa regola, in quanto l’attività invertasica risulta sì parzialmente derepressa in presenza di alte concentrazioni di glucosio, ma non più inducibile da bassi livelli di glucosio o raffinosio. In aggiunta, l’inattivazione di SNF3 e di RGT2 abolisce completamente l’induzione dell’attività invertasica nel ceppo hxt(1-17)gal2 in presenza di glucosio. Nell’insieme, i dati appena descritti sembrano suggerire per il pathway Snf3/Rgt2 un ruolo decisamente più rilevante nella regolazione di SUC2 rispetto a quanto gli viene comunemente attribuito. Esperimenti futuri permetteranno di chiarire meglio la questione.

The hydrophobic plasma membrane constitutes an indispensable barrier for cells in living animals. Albeit being pivotal for the maintenance of cells, the inability to cross the plasma membrane is still one of the major obstacles to overcome in order to progress current drug development. A group of substances, with restricted access to the interior of cells, which has shown great promise for future clinical use is oligonucleotides that are exploited to interfere with gene expression. Short interfering RNAs that are utilized to confer gene silencing and splice correcting oligonucleotides, applied for the manipulation of splicing patterns, are two classes of oligonucleotides that have been explored in this thesis. Cell-penetrating peptides (CPPs) are a class of peptides that has gained increasing focus in last years. This ensues as a result of their remarkable ability to convey various, otherwise impermeable, macromolecules across the plasma membrane of cells in a relatively non-toxic fashion. This thesis aims at further characterizing well-established, and newly designed, CPPs in terms of toxicity, delivery efficacy, and internalization mechanism. Our results demonstrate that different CPPs display different toxic profiles and that cargo conjugation alters the toxicity and uptake levels. Furthermore, we confirm the involvement of endocytosis in translocation of CPPs, and in particular the importance of macropinocytosis. All tested peptides facilitate the delivery of splice correcting oligonucleotides with varying efficacy, the newly designed CPP, M918, being the most potent. Finally we conclude that by promoting endosomolysis, by exploring new CPPs with improved endosomolytic properties, the biological response increases significantly. In conclusion, we believe that these results will facilitate the development of new CPPs with improved delivery properties that could be used for transportation of oligonucleotides in clinical settings.

In view of calmodulin's widespread role in calcium-regulated processes, together with the requirement of Ca('2+) for chromaffin cell secretion, a possible role for calmodulin in translating the Ca('2+) signal for secretion is suggested.<br>Isolated bovine adrenal medullary cells were maintained in monolayer culture and their morphological and functional characteristics examined. The cultured chromaffin cell was found capable of accumulating exogenous amines via a high affinity uptake(,1)-like process which closely simulated the neuronal process in many respects but demonstrated neither stereo- nor structural selectivity. The accumulated amines appeared to be uniformly distributed within the chromaffin cell as they were released in parallel with and in equal proportion to the endogenous chromaffin cell catecholamines. This finding proved invaluable in designing a rapid reproducible method for assaying for catecholamine release from the chromaffin cells in culture.<br>Catecholamine secretion from the chromaffin cells in response to stimulation with either acetylcholine or a depolarizing concentration of K('+) is a Ca('2+)-dependent event. The stimulation-induced amine release was reduced in the presence of trifluoperazine, an antipsychotic agent and potent calmodulin antagonist. However, trifluoperazine blocked catecholamine release from the cultured chromaffin cells at a step distal from Ca('2+) entry, thus providing indirect evidence for a role for calmodulin in the secretory process. More direct evidence for a role for calmodulin in the process of secretion was provided with the use of antibodies raised against calmodulin. In this regard, erythrocyte ghost-mediated microinjection of calmodulin antibodies into viable cell cultures inhibited stimulus-evoked catecholamine secretion from the cultured cells without affecting amine accumulation into these cells. These findings support the hypothesis that calmodulin is important in translating the calcium signal for chromaffin cell secretion. On the other hand, calmodulin appears not to be involved in the catecholamine uptake process in these cells.

Les cellules dendritiques (DCs) patrouillent les tissus périphériques à la recherche de dangers potentiels en se déplaçant à travers les tissus et en incorporant de grande quantité de matériel extracellulaire. Cet événement précoce de la réponse immunitaire adaptative est susceptible de déterminer l'amplitude et la qualité de l'activation des lymphocytes T et B. De ce fait, les DCs pourraient avoir besoin d'orchestrer leur motilité et leur fonction de capture des antigènes afin d'initier un réponse immunitaire efficace et adaptée. Afin d'étudier les mécanismes responsables de l'optimisation de l'échantillonnage des tissus par les DCs, nous avons suivi leur migration et leur capacité à capturer des antigènes dans des chambres micro-fluidiques contenant des canaux étroits qui permettent de reproduire l'espace confiné des tissus périphériques. De manière surprenante, nous avons découvert que la migration des DCs et leur aptitude à accumuler des antigènes sont des fonctions antagonistes et dépendent de l'activité du moteur moléculaire Myosine II. Nous avons observé que les DCs se déplacent en alternant des phases rapides au cours desquelles la Myosine II est distribuée de manière asymétrique à l'arrière des cellules, et des phases plus lentes pendant lesquelles la Myosine II est enrichie à l'avant. Les enrichissements transitoires de Myosine II à l'avant des DCs dépendent de l'association de la Myosine II avec la chaîne invariante associée au CMH-II (Ii). Ces évenements favorisent l'absorption d'antigènes et leur transport dans les compartiments endolysosomaux. Des expériences menées avec une pince optique nous ont permis de montrer que l'activité de la Myosine II à l'avant des cellules génère des forces mécaniques qui induisent le transport des vésicules vers l'intérieur de la cellule, probablement en modulant le flux rétrograde d'actine. Ainsi, au cours de cette thèse, nous avons montré que la Myosine II était nécessaire à la fois pour la migration cellulaire et la capture d'antigènes, établissant un mécanisme moléculaire qui permet de coordonner ces deux processus dans le temps et l'espace. Nous proposons que l'alternance de phases de haute mobilité et de phases d'arrêt associées à la capture d'antigènes confère aux DCs une stratégie de recherche intermittente qui leur permettrait d'optimiser la surveillance des tissus périphériques<br>Dendritic cells (DCs) patrol peripheral tissues in search for potential dangers by actively crawling and internalizing extracellular materiel. This initial event of an adaptive immune response is likely to determine the magnitude and quality of T cell and B cell immunity. Therefore, DCs might need to tightly orchestrate their migration and their antigen uptake function in order to mount an efficient and adapted immune response. To investigate the mechanisms responsible for the optimization of tissues sampling by DCs, we monitored their migration and their ability to capture antigens in micro-fluidic chambers containing narrow channels that mimic the confined space of peripheral tissues. Surprisingly, we found that cell migration and antigen accumulation in endolysosomes are antagonistic, both relying on the activity of the motor protein Myosin II. We observed that DCs alternate between phases of fast motility during which Myosin II is asymmetrically distributed at the cell rear, and phases of slow motility during which Myosin is enriched at the cell front. Transient Myosin II enrichments at the leading edge depends on its association with the MHC-II associated Invariant Chain (Ii). These events promote antigen uptake and arrival in endolysosomal compartments. Using optical tweezers, we further showed that Myosin II activity at the leading edge generates mechanical forces that drive vesicles transport toward the cell body probably through the modulation of F-actin retrograde flow. Thus, during my PhD, we have shown that Myosin II is required for both migration and antigen capture, providing a molecular mechanism to couple these two processes and allow their coordination in time and space. We propose that alternation between phases of fast motility and phases of low motility associated with efficient antigen capture imposes an intermittent search behavior on DCs, which might be optimal for environment patrolling

The increased need for macromolecular therapeutics, such as proteins and nucleotides, to reach intracellular targets asks for more effective delivery vectors and a higher level of understanding of their mechanism of action. Cell Penetrating Peptides (CPPs) have been shown to deliver a range of macromolecules into cells either through direct plasma membrane translocation or by endocytosis. All known endocytic pathways involve cellcortex remodelling, a process shown to be regulated by reorganisation of the actin cytoskeleton. Links between actin remodelling and CPP uptake has been shown but more information is required to determine the extent of this association and how it could influence further research into improving the delivery capacity of these entities. This project, by using the CPP octaarginine (R8) investigated how actin disorganisation influences the cellular entry of this peptide when attached to a model fluorophore Alexa 488 or Enhanced Green Fluorescent Protein (EGFP). A confocal microscopy technique was initially developed, allowing for high-resolution and spatial characterization of the actin cytoskeleton at different cell depths. Analysis using this developed method was used to highlight that serum starvation has a strong influence on the capacity of R8 to cause membrane blebs and possibly macropinocytosis. Using a range of direct or indirect actin inhibitors this work also highlighted how they can rapidly cause dramatic cellular deformities beyond the level of actin or more subtly affect actin organisation. Further confocal studies revealed that choice of cell line significantly affects the effect of actin disruption on CPP entry and that this is highly dependent on the nature of the probe. This was exemplified by results showing inhibition of EGFP-R8 uptake in HeLa cells treated with cytochalasin D, ! ! ii! latrunculin B and jasplakinolide but a dramatic increase in uptake in A431 cells when they were treated with these drugs. The regulation of actin dynamics involves various kinases including Rho–associated kinases ROCKs, and Src family kinases. The ROCK inhibitor Y27632 induced the formation of actin needles running perpendicular to the plasma membrane of A431 cells and increased EGFP-R8 internalisation. By contrast, Src inhibitor PP2 had little effect on both the actin cytoskeleton and EGFP-R8 uptake but completely abrogated the effects of Cyt D on cellular uptake. This demonstrates for the first time that pretreatment of actin with one inhibitor can negate the endocytic effects of another actin inhibitor working on a different target. Overall this study highlights the importance of analysing actin in detail to identify how CPPs and possibly other drug delivery vectors and formulations interact with cells to gain entry. Under defined experimental conditions R8 can modify the actin cytoskeleton and requires a functional or dysfunctional actin network to allow for maximal cellular entry.

<p>Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that include Creutzfeldt-Jakob disease, bovine spongiform encephalopathy and sheep scrapie. TSE disease pathology and mechanisms within the central nervous system (CNS) of an infected host largely remains unclear. At the cellular level, the uptake of protease resistant prion protein (PrP-res), which strongly correlates with infectivity and is a valid marker for TSE infection, is one of the earliest events that must occur during TSE infection. Given the difficulty of clearly distinguishing input PrP-res from either PrP-res or protease-sensitive PrP (PrP-sen) made by the cell, the uptake of PrP-res from an infectious inoculum into the host cell remains a poorly understood process. Through the development of a novel assay to exclusively detect input PrP-res we hypothesized that the acute infection of cells by PrP-res is mediated through general processes such as endocytosis, whereas internalization, retention, and propagation of PrP-res are dictated by specific characteristics of both the host cell and PrP-res. Using PrP-res tagged with a unique epitope to the mouse monoclonal antibody 3F4, we developed a detection system to specifically follow the acute cellular uptake of PrP-res. Mouse neural and fibroblast cells were exposed to three different mouse scrapie strains and PrP-res from the inoculum monitored. For all strains, PrP-res uptake was rapid and independent of both cellular prion protein expression and cell type. However, only 30%-40% of the cells were able to internalize PrP-res and PrP-res aggregate size influenced PrP-res uptake. Furthermore, infectious brain homogenate PrP-res was taken up more efficiently then PrP-res in either microsome or partially purified preparations. Our results suggest that PrP-res aggregate size, the PrP-res microenvironment, and/or host cell-specific factors can all influence whether or not a cell takes up PrP-res following exposure to TSE infectivity.</p>

Mitochondria are cellular organelles that play a key role in several physiological processes, including cell proliferation, differentiation, cell death and the regulation of cellular calcium (Ca2+) homeostasis. Increases in mitochondrial Ca2+ activate several dehydrogenases and carriers, inducing enhance in the respiratory rate, H+ extrusion, and ATP production necessary for the correct energy state of the cell. The mitochondrial Ca2+ uptake and release mechanisms are based on the utilization of gated channels for Ca2+ uptake and exchangers for release that are dependent upon the negative mitochondrial membrane potential, which represents the driving force for Ca2+ accumulation in the mitochondrial matrix. In this thesis, the attention was focused on two mechanisms in particular, the mitochondrial Ca2+ influx system by the activity of Mitochondrial Calcium Uniporter (MCU) complex, and the high-conductance channel mitochondrial Permeability Transition Pore (mPTP), responsible for a state of non-selective permeability of the inner mitochondrial membrane (IMM); its opening in non-physiological conditions leads to Ca2+ release from mitochondria and triggers cell death mechanisms. Thus the maintenance of the mitochondrial Ca2+ homeostasis is essential for a proper balance between cell life or death. In particular it will be discussed the possible involvement of MCU in the cell cycle, as the Ca2+ accumulation by MCU is important for the regulation of cell life and energy production. It will be shown that MCU is mainly expressed in specific phases of the cell cycle and this expression positive correlates with the mitochondrial membrane potential. MCU overexpression instead does not alter cell cycle phases. It will also described the role of the c subunit of Fo ATP synthase in mitochondrial permeability transition (MPT) and it will be demonstrated to be a critical component of the mPTP complex. Finally it will be discussed the role of mPTP in mitochondrial Ca2+ efflux and it will be shown that it is a dispensable element for mitochondrial Ca2+ efflux in non-pathological conditions.

Dictyostelium discoideum is a soil dwelling amoeba which has been widely used as a model organism to study cellular processes such as signal transduction, chemotaxis, endocytosis and exocytosis. The process of phagocytosis in Dicytostelium is largely comparable to that of neutrophils and macrophages in the mammalian system. Neutrophils and macrophages are cells of the innate immune system and they engulf infectious bacteria through phagocytosis. Dictyostelium cells uptake yeast and bacteria for their nutrition through phagocytosis, which is an actin dependent mechanism and is a target of multiple signaling inputs. Recent studies have uncovered different proteins involved in the signaling of particle and further studies are required to decipher the intricate mechanism leading to the F-actin rearrangement. Two of the proteins have previously known to be involved in the pathways regulating the F- actin rearrangement name PP2A phosphatase and LKB1 kinase The main objective of this project was to determine how these proteins are affecting the two actin driven particle uptake processes, phagocytosis and fluid uptake. We showed that ablation of PsrA gene which codes the regulatory subunit of PP2A resulted in a defective phagocytosis, whereas the fluid uptake was normal. We also showed for the first time that there was an increase in the phosphorylation of some of the PKB substrate proteins in wild type cell. Cells lacking PsrA gene displayed an aberrant phosphorylation of PKB substrate protein when compared to the wild type cells further confirming the involvement of PKB substrate in phagocytosis. Further, we looked at the effects of LKB1 kinase on phagocytosis by using a LKB1 knockdown construct introduced into wild type cells. The knock down of LKB1 resulted in a higher rate of phagocytosis while introduction of a LKB1 over expressing construct severally decreased the rate of phagocytosis indicating an inhibitory effect of LKB1. Furthermore there was an increase in the PKB substrate protein but a different pattern compared to the psrA- cells. We also carried out adhesion assays on LKB1 knockdown cells and the results showed a higher substrate adhesion as compared to the wild type cells, while psrA- cells had no adhesion defect.

This research focused on investigating and comparing the cytotoxicity and cellular uptake of RRR-alpha-tocopheryl polyethylene glycol succinate (TPGS, with that of alpha-tocopheryl succinate (α-TS). Both TPGS and α-TS are water-soluble forms of vitamin E with important clinical applications. Cytotoxicity assays with RAW 264.7 and LNCaP cells incubated overnight with TPGS or α-TS at concentrations ≥ 12.4 μM suggest that α-TS is more cytotoxic than TPGS. Macrophages were found to be more sensitive than LNCaP cells when treated with similar concentrations of α-TS. For both cell lines, most of the TPGS or α-TS taken up remained esterified after 24 hours. Our results suggest that cell death was due to TPGS and/or α-TS and not alpha-tocopherol. A para-hydroxyanilide of α-TS (p-HATS) that could be used to distinguish between cellular TPGS and α-TS was studied. It was found that p-HATS can be detected electrochemically and that it is hydrolyzed to α-TOH.

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 1993.<br>Includes bibliographical references (leaves 96-101).<br>by Elizabeth Anne Gift.<br>M.S.

As our understanding of the platelet’s systemic role continues to expand beyond hemostasis and thrombosis, interrogation of the platelet’s ability to affect diverse biological processes is required. Studies of the platelet’s non-traditional roles have focused on developing our understanding of the platelet’s relation to specific disease phenotypes as well as elucidation of platelet characteristics, content, and function. The generic content, traditional function and heterogeneity of platelets have long been accepted; more ambiguous and controversial has been how these factors are interrelated. Investigation of platelet content revealed the presence of biologically functional RNA in anucleated platelets, the correlation of platelet RNA to distinct phenotypes, and the ability of platelets to transfer RNA to other vascular cells; however how these processes occur is unclear. To further interrogate platelet RNA processes, we utilized sorting and RNA sequencing to develop platelet subpopulation transcriptome profiles. We found that platelet heterogeneity extends to the platelet transcriptome: distinct RNA profiles exist dependent on platelet size. We hypothesized that this RNA heterogeneity is the result of RNA transfer between platelets and vascular cells. Using in vitro and in vivo modeling, we were able to show the novel ability of platelets to take up RNA from vascular cells, correlating to the unique functional profile associated with small platelet transcriptomes. These findings reveal a role for platelet RNA transfer in platelet RNA heterogeneity, with potential correlation to platelet functional diversity previously proposed. The ability of the platelet to bidirectionally transfer RNA within circulation has implications for vascular health and beyond.

As our understanding of the platelet’s systemic role continues to expand beyond hemostasis and thrombosis, interrogation of the platelet’s ability to affect diverse biological processes is required. Studies of the platelet’s non-traditional roles have focused on developing our understanding of the platelet’s relation to specific disease phenotypes as well as elucidation of platelet characteristics, content, and function. The generic content, traditional function and heterogeneity of platelets have long been accepted; more ambiguous and controversial has been how these factors are interrelated. Investigation of platelet content revealed the presence of biologically functional RNA in anucleated platelets, the correlation of platelet RNA to distinct phenotypes, and the ability of platelets to transfer RNA to other vascular cells; however how these processes occur is unclear. To further interrogate platelet RNA processes, we utilized sorting and RNA sequencing to develop platelet subpopulation transcriptome profiles. We found that platelet heterogeneity extends to the platelet transcriptome: distinct RNA profiles exist dependent on platelet size. We hypothesized that this RNA heterogeneity is the result of RNA transfer between platelets and vascular cells. Using in vitro and in vivo modeling, we were able to show the novel ability of platelets to take up RNA from vascular cells, correlating to the unique functional profile associated with small platelet transcriptomes. These findings reveal a role for platelet RNA transfer in platelet RNA heterogeneity, with potential correlation to platelet functional diversity previously proposed. The ability of the platelet to bidirectionally transfer RNA within circulation has implications for vascular health and beyond.

Heterogeneous reactions between mineral dust aerosols and gas phase volatile organic compounds have the potential to impact important atmospheric chemical processes. However, little is known about the uptake and reactivity of volatile organic compounds on particulates found in the environment. A Knudsen cell was designed and constructed for providing precise measurement of reaction probabilities within these systems. The instrument was validated through a series of experiments. After validating the Knudsen cell against several key benchmarks, the instrument was used to measure the uptake coefficient for ethanol on particulate silicon dioxide. The uptake coefficient of ethanol on silicon dioxide, a common compound in mineral dust aerosols, was determined to be 7 x 10-7. Therefore, uptake of ethanol on silicon dioxide would be competitive with the loss of other volatile organic compounds on silicon dioxide, which show similar rates of uptake. The Knudsen cell was validated and measured the uptake of ethanol on silicon dioxide, and future work with the Knudsen cell will study the uptake of chemical warfare agent simulants on metal oxides.<br>Master of Science

Freshwater (FW) fish inhabit hypotonic environments that can vary markedly both spatially and temporally with respect to ambient salt levels and pH. Despite the chemical variability of FW, fish maintain ionic homeostasis (ionic regulation) and pH homeostasis (acid-base regulation) by manipulating ion transport mechanisms within ion-transporting cells (ionocytes) localised to the gills of adults and the skin of larvae. Ionocytes are mitochondrion-rich (MR) cells that, depending on subtype, express specific ion transporters that facilitate the movement of salts and acid-base equivalents across the gills or skin. In zebrafish (Danio rerio), one of the most well-studied ionocytes is termed the H+-ATPase-rich (HR) cell, which is presumed to be a significant site of transepithelial Na+ uptake/acid-secretion. Proteins that have been found in fish zebrafish HR cells include the Na+/H+ exchanger (NHE3), carbonic anhydrases (CA17a and CA15a), proton ATPase (H+-ATPase) and the ammonia channel, ammonia conducting rhesus C glycoprotein b (Rhcgb), which are all thought to function in Na+ uptake acid–base regulation. Ionic and acid-base regulation are achieved both by adjustments to the activity level of these ion transport proteins, but also by regulating the numbers of specific ionocyte subtypes (e.g. HR cells) during acclimation to environments differing in ionic composition or pH. In previous studies, the quantitative assessment of mRNA levels for genes involved in ionic and acid-base regulation relied on measurements using homogenates derived from whole body (larvae) or gill (adult). Such studies cannot distinguish whether any differences in gene expression arise from adjustments of ionocyte subtype numbers, or transcriptional regulation within individual ionocytes. Surprisingly, there are no data on ionocyte-specific gene expression responses in zebrafish exposed to varying environments including acidic or Na+-deficient water. To rectify this gap in the current knowledge, this thesis utilized the florescence activated cell sorting (FACS) approach to separate the HR cells from other cellular sub-populations. The technique was used to measure the gene expression of several HR cell specific transporters and enzymes in isolated HR cells from zebrafish larvae exposed to low pH (pH 4.0) or low Na+ (5 μM) conditions. The data suggest that treatment of larvae at 4 days post fertilization with acidic water caused an increase in h+atpase, ca17a, ca15a, nhe3b and rhcgb mRNA levels in sorted HR cells. These observations suggest the existence of multiple mechanisms of acid secretion in HR cells of larval zebrafish in acidic water; one in which acid secretion via NHE3b is linked to ammonia excretion via Rhcgb, and another facilitated by H+-ATPase. Furthermore, these results provide molecular evidence to support roles for the CA isoforms in acid-base regulation in HR cells. On the other hand, the low Na+ treatment data suggest that nhe3b and rhcgb are the dominant genes maintaining Na+homeostasis. In summary, the results of this thesis demonstrate that acclimation to low pH or low low Na+ environmental conditions is facilitated by HR cell proliferation and HR cell-specific transcriptional control.

Final article is available at "wileyonlinelibrary.com" Takako Okamoto,Masaki Mandai,Noriomi Matsumura,Ken Yamaguchi,Hiroshi Kondoh,Yasuaki Amano,Tsukasa Baba,Junzo Hamanishi,Kaoru Abiko,Kenzo Kosaka,Susan K. Murphy,Seiichi Mori,Ikuo Konishi "Hepatocyte nuclear factor-1β (HNF-1β) promotes glucose uptake and glycolytic activity in ovarian clear cell carcinoma" Molecular Carcinogenesis 54:35–49 (2013)<br>Kyoto University (京都大学)<br>0048<br>新制・論文博士<br>博士(医学)<br>乙第12804号<br>論医博第2076号<br>新制||医||1001(附属図書館)<br>80848<br>京都大学大学院医学研究科医学専攻<br>(主査)教授 野田 亮, 教授 武藤 学, 教授 稲垣 暢也<br>学位規則第4条第2項該当