Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Parkinson's disease is a neurodegenerative disease characterized by the buildup of aggregated and spread of misfolded alpha-synuclein. How the misfolded alpha-synuclein contributing to the toxicity and death of neuronal cells has been the focal point of research. The spread of alpha-synuclein has been attributed to many mechanisms, one of which is via cell-derived vesicles called exosomes. This project aims to examine the controllability of exosome release. SH-SY5Y, MCF-7 and CHO-K1 cells were transfected with dopamine receptor 3-green fluorescent protein, G-protein receptor 143 or green fluorescent protein and treated with either dopamine or L-DOPA. Medium was harvested and subjected to ultracentrifugation and a silver stain and western blot were performed. There was no significant difference in the total protein in the exosome fraction lanes between the treatment groups or within them. Another aim was to test the specificity of exosomes. Exosomes isolated from SH-SY5Y or MCF-7 were labeled with Exo-Red dye and introduced to wells containing SH-SY5Y, MCF-7 and CHO-K1 cells at room temperature and -4C. At room temperature, exosomes were observed intercellular in all of the cell lines, however, they did not deliver their content. At -4C exosome uptake was halted and they remained on the surface of the cells. Exo-Red labeled SH-SY5Y exosomes were treated with proteinase K and were introduced to CHO-K1 cells at -4C and room temperature. CHO-K1 did not take up exosomes, suggesting exosomes contain one or more necessary proteins needed to interact with the cellular membrane to initiate internalization. CHO-K1 cells were treated with versene to examine the involvement of integrin proteins. Exo-Red labeled SH-SY5Y exosomes were trapped on the surface of CHO-K1 after versene treatment. Lastly, Exo-Red labeled SH-SY5Y exosomes were biotinylated and magnetically captured then introduced to SH-SY5Y and MCF-7 cells and a silver stain and a biotinylated blot were performed. MCF-7 bound more Exo-Red labeled SH-SY5Y exosomes.

No description available.

The Leishmania spp. are kinetoplastid protozoan parasites that cause a spectrum of highly prevalent and neglected tropical diseases known as leishmaniasis. The parasites must undergo two life forms during their life cycle: the extracellular promastigote life stage within the sand fly vector, and the intracellular amastigote life stage after internalization of host phagocytic cells. In the extracellular life stage, Leishmania promastigotes reside and develop to their infectious metacyclic form solely in the gut lumen of the sand fly, a process known as metacyclogenesis. During this process, other organisms that co-inhabit the sand fly gut, collectively known as the microbiome, influence parasite development. Based on the hypothesis that vector gut microbiota influence the development of parasite virulence, we sequenced midgut microbiomes of the sand fly Lutzomyia longipalpis with or without L. infantum infection. Sucrose fed sand flies contained a highly diverse, stable midgut microbiome. Blood feeding caused a decrease in bacterial richness, which eventually recovered. However, bacterial richness progressively decreased in L. infantum-infected sand flies. Furthermore, parasites altered the relative abundance of several bacterial phylogenies, including Pseudomonas and Serratia. Importantly, antibiotic-mediated perturbation of the midgut microbiome rendered sand flies unable to support parasite growth and consequent development to infectious metacyclic forms, and revealing the level of microbial diversity may induce flies resistant to infection. Together, these data suggest the sand fly midgut microbiome is a critical factor for Leishmania growth and differentiation prior to disease transmission. During the intracellular amastigote life form, macrophages are the primary cell type to phagocytize parasites. The effect of secreted factors such as exosomes from Leishmania-infected human cells and their effect on the immune response has not been extensively investigated. In this thesis, we characterized the proteome of primary human donor monocyte-derived macrophage (MDM) exosomes during L. infantum infection compared to donor-matched uninfected controls, and determined their impact on naïve MDMs measured by cytokine gene expression and resistance to subsequent parasite infection. Proteomic comparisons of infected and uninfected MDM exosomes were made using stable isotopic dimethyl labeling LC-MS/MS technology. A total of 484 human proteins were identified between four donors. Proteins significantly less abundant in exosomes derived from infected MDMs were matrix metalloprotease 9, galectin-3 binding protein, and several Annexins and histone proteins. Proteins more abundant included galectin-1, galectin-9, and serotransferrin and transferrin receptor 1. Interestingly, class I and class II MHC protein chains were differentially abundant in our samples. Furthermore, we observed several Leishmania spp. proteins in exosomes from infected MDMs as well. Naïve MDMs pretreated with exosomes from infected or uninfected MDM for 4 hours were not more resistant to L. infantum infection nor displayed increased gene expression of the pro-inflammatory cytokines IL-1α, IL-1β, IL-6, IL-8 or TNF-α. To date, the work presented in this thesis is the first to comprehensively identify the proteome in primary human MDM exosomes during Leishmania spp. infection, and to determine the impact of these exosomes on the immune response of other naïve human MDMs.

Malignant mesothelioma (MM) is a locally invasive and highly aggressive cancer arising on the mesothelial surface of organ cavities (mainly pleural) as a direct result of asbestos exposure. The latency period of MM is long (20-50yrs) after initial asbestos exposure, and the prognostic outcomes are dismal with median life expectancy of 6-12 months post-diagnosis. There are no useful biomarkers for early MM diagnosis, no successful therapeutic interventions. These vast voids of knowledge led to our hypotheses that secreted vesicles, termed exosomes, play an important role in MM development and tumorigenic properties. Exosomes are nano-sized particles secreted from all cell types and carry biologically active cargo in the form of proteins, RNA, and lipids that can potently act as intercellular messengers in both healthy settings and disease states. We are the first to have conducted studies implicating the roles of exosomes in MM pathogenesis. Firstly, we analyzed the proteomic signature of exosomes from asbestos exposure models, in vitro and in vivo. Our in vitro data demonstrated that asbestos exposed lung epithelial cells and macrophages secrete exosomes with differentially abundant proteins compared to non-exposed controls and some of these proteins are relevant to asbestos exposure toxicology and MM development. Additionally, the exosomes from asbestos exposed cells significantly modulated the gene expression of target mesothelial cells in a way that reflected epithelial to mesenchymal transition and other tumorigenic properties. The in vivo mouse studies illustrated that mouse serum exosomes house differentially abundant proteins after asbestos exposure and this is measurable at an organism wide scale. Secondly, we assayed the miRNA composition of MM tumor exosomes compared to healthy mesothelial cell exosomes and found signature differences in miRNA abundances, particularly that MM tumor cells had significantly higher amounts of tumor suppressor miRNA, particularly miR-16-5p, in their exosomes. This led to the hypothesis that MM tumor cells preferentially secrete tumor suppressor miRNAs via exosomes to rid themselves of the anti-tumor effects. We employed exosomes secretion inhibitors and exosome force-feeding to demonstrate that MM cells do in fact secrete miR-16-5p (along with other tumor suppressor miRNAs) through exosomes and that this property can be targeted as a potentially novel therapeutic advance. Furthermore, we identified a mechanism of miR-16-5p loading into exosomes by the RNA binding protein HuR, and this mechanism is interestingly regulated by miR-16-5p itself in a negative feedback loop. Our studies thus far provide intriguing evidence on the role of exosomes in asbestos exposure and MM biology. We demonstrated the potential for exosomes as protein biomarkers in asbestos exposure and conduits of tumorigenic information to mesothelial cells. In addition, we incriminate exosomes as vehicles of tumor suppressor removal from MM tumor cells and we can target this as a potential n MM therapy.

Prostate cancer is the most common cancer occurring in the men in USA and Europe. According to CDC, incidence of Prostate cancer in African American men in the year 2008 was 234.6 cases per 100,000 compared to 150 cases per 100,000 in Caucasian men, reasons for this disparity remain unclear. Castration resistant prostate cancer is an advanced form of prostate cancer with poor survival rates. 10-20% of prostate cancer patients develop metastatic castration resistant prostate cancer (CRPC) within approximately 5 years of follow-up. Androgen deprivation therapy which is at the center of metastatic prostate cancer is often impeded by development of CRPC. Previous studies have demonstrated that prostatic androgen concentration ranging between 10-25 percent in the treated patients versus the untreated could still continue AR signaling. Previous in vitro studies have demonstrated higher tumor homing potential in normal adipose derived mesenchymal stem cells (ADMSC) from African American patient compared to ADMSC derived from a Caucasian patient when grown in prostate cancer cell condition media. This study attempts to exploit this tumortropicity of ADMSC for selective delivery of alpha keto reductases in the metastasized prostate cancer cells to hydrolyse DHT and other androgens into weaker androgens. Enriched ADMSC were plated in a 6 well plate and were co-transfected with transfected with AKRC14 and GFP. Gene expression was confirmed by PCR and WB. ADMSCs are capable of expressing AKR1C14 on transfection with plasmid. Stem cells expressing AKR1C4 open the avenues for furthering therapeutic strategies in metastatic CRPC by hydrolyzing the androgens. / 1 / Manish Ranjan

Cells can mediate intercellular communication by the secretion and uptake of microvesicles, nano-sized membranous particles that carry signaling molecules, antigens, lipids, mRNA and miRNA between cells. The biological function of these vesicles is dependent upon their composition and cellular origin which is regulated by mechanisms that are not well understood. Based on their molecular content, microvesicles may play a role in immune regulation, cancer progression, the spread of infectious agents and numerous other important normal and pathogenic processes. The proteomic content of microvesicles from diverse sources has been intensely studied. In contrast, little is known about their glycomic content. The glycosylation pattern of a protein or lipid plays a key role in determining its functional properties in several ways. Glycans can determine the trafficking of a protein to particular regions of the cell as well as the protein’s half life. In addition, the glycan-dervied oligomerization of glycolipids and glycoproteins is a known mechanism for the activation of receptors and recognition of ligands on the surface of the cell. Glycomic analysis may thus provide valuable insights into microvesicle function. I utilized lectin microarray technology to compare the glycosylation patterns of microvesicles derived from a variety of biological sources. When compared to cellular membranes, microvesicles were enriched in high mannose, polylactosamine, α2-6 sialic acid, and complex N-linked glycans but exclude terminal blood group A and B antigens. The polylactosamine signature in microvesicles from different cell lines derives from distinct glycoprotein cohorts. After treatment of Sk-Mel-5 cells with lactose to inhibit lectin-glycan interactions, secretion of microvesicle resident proteins was severely reduced. Taken together, this work provides evidence for a role of glycosylation in microvesicle-directed protein sorting. / text

Myelin ist eine mehrschichtige Membran, die die Axone in peripheren (PNS) und Zentrale Nervensystem (ZNS) umhüllt. Die Bildung und Anordnung dieser Struktur ist ein mehrstufiger Prozess, der durch eine Vielzahl extrazellulärer Faktoren reguliert wird. Im ZNS wird Myelin von Oligodendrozyten gebildet. Während der Entwicklung differenzieren die Vorläufer dieser Zellen zu reifen Oligodendrozyten aus. Nachdem sie das geeignete Signal aus ihrer Umgebung erhalten haben, beginnen die Oligodendrozyten die Axone mit Myelinmembranen einzuhüllen.  Allerdings sind die Signale, die diesen Prozess initiieren unbekannt. Mit dieser Arbeit zeigen wir, dass Oligodendrozyten kleine Mikrovesikel - so genannte Exosomen - in den extrazellulären Raum freisetzen, welche die terminale Differenzierung von Oligodendrozyten und die anschließende Myelinbildung verhindern. Es konnte gezeigt werden, dass diese inhibitorische Wirkung durch die Aktivität der RhoA-ROCK-Signalkaskade vermittelt wird. Bemerkenswerterweise war die Exosomenfreisetzumg durch Oligodendrozyten signifikant reduziert, wenn die Zellen mit konditioniertem Medium von Neuronen inkubiert wurden. Unsere Ergebnisse legen nahe, dass Exosomen, die von Oligodendrozyten produziert werden,  Zellen in einem pre-myelinisierten Stadium halten, während die Sekretion von Exosomen in Gegenwart neuronaler Signale reduziert wird und autoinhibitorische Signale aufgehoben werden. Somit können Neuronen die Bildung und Freisetzung von Exosomen regulieren, welche von Oligodendrozyten freigesetzt werden, um die Biogenese und Assemblierung der Myelinmembran zu koordinieren.  Im zweiten Teil der Arbeit wurde die Frage, wie die Kompaktierung des Myelins vermittelt wird, erörtert. Während bekannt ist, dass MBP die Interaktion zwischen Myelinmembranen von cytoplasmatischer Seite aus organisiert, ist der zugrundeliegende molekulare Mechanismus der Interaktion zwischen den äußeren Membranen nach wie vor unklar. Im Allgemeinen erfordert die Interaktion zwischen zwei gegenüberliegenden Membranen die Expression von Adhäsionsmolekülen und die Entfernung von repulsiven Komponenten. Daher untersuchten wir die Rolle des Proteolipid-Proteins (PLP), als mutmaßliches Adhäsionsmolekül, und die Glykocalix, als repulsive Struktur während der Myelinkompaktierung im ZNS. Wir analysierten die Adhäsion von aufgereinigten Myelinpartikeln mit den primären Oligodendrozyten, um die Wechselwirkung zwischen den Myelinschichten zu imitieren. Mit diesem System haben wir gezeigt, dass PLP die Adhäsionsfähigkeit der Myelinmembran erhöht. Mittels Single Particle Force-Spektroskopie fanden wir außerdem heraus, dass PLP die physikalische Stabilität von Myelin verbessert. Zusätzlich beobachteten wir eine signifikante Reduzierung in der Glykokalix während der Oligodendrozytenreifung, die mit einer Zunahme in ihrer Oberflächenaffinität gegenüber den Myelinpartikeln korreliert. Weitere Analysen zeigten, dass die negative Ladung der Zuckeranteile, hauptsächlich der Sialinsäure, für die Verringerung der Myelinadhäsion verantwortlich ist. Daher schlagen wir vor, dass die Adhäsionseigenschaften von PLP zusammen mit der Reduzierung der Glykokalyx, die Adhäsion der Myelinmembran und die  Kompaktierung im ZNS organisieren.

In this project an overview of how synthetic lipid nanoparticles and exosomes can be used for targeted drug delivery is compiled. The goal is to identify aspects that can be in favor for targeted drug delivery and the development of products at Cytiva. The most important fields for Cytiva to understand is the methods and the challenges of cell culturing for production of exosomes, productions of lipid nanoparticles, purification of exosomes, analysis of both exosomes and lipid nanoparticles, and how exosomes and lipid nanoparticles are used as tools for drug delivery. To understand these aspects a description focusing on structural components, specific delivery and cargo loading is also included in the report. Many different components and methods have been found in the different fields mentioned, and the ones that we believe are the most relevant for Cytiva are presented and discussed in the report. We conclude that both exosomes and lipid nanoparticle are suitable options as drug delivery vehicles, especially for their ability to be modified for targeted delivery, encapsulate therapeutic compounds and cross biological barriers. Exosomes are also biostable and possess low immunogenicity. For production the methods identified with highest potential are Hollow-Fiber Bioreactor for cell culturing in production of exosomes and Microemulsion and High-Pressure Homogenization for lipid nanoparticles. Purification is required for exosomes and the most prominent method is Size-Exclusion Chromatography, because of its scalability. After production and purification it is important to be able to detect the vesicles and the most developed and used methods are Nanoparticle Tracking Analysis and Flow Cytometry, beacuse they can use labeling techniques and single vesicle analysis.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lucia Tomášková Supervisor: prof. PharmDr. Tomáš Šimůnek, Ph.D. Title of diploma thesis: Study of exosomes as a drug delivery system in the treatment of glioblastoma Central nervous system disorders are among the most serious diseases affecting humans. They affect not only the patient's life, but also his/her surroundings. Therefore, their therapy, whether at the level of complete cure or alleviation of accompanying symptoms, is a challenge for scientific research. In our research, we focused on glioblastoma multiforme, a brain cancer not yet treatable. The main drawback in therapy is overcoming the blood-brain barrier. Exosomes, such as the body's natural nano-vesicles, have been shown to be a suitable system for delivering drugs to brain tissue. Our research has shown that by a suitable method we are able to obtain sufficient quality exosomes from macrophage and fill them very efficiently with antitumor agents paclitaxel, doxorubicin and temozolomide, while the delivered substances show higher efficacy and fewer side effects than the free form.