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.

[ES] El cáncer de mama es el cáncer más común en mujeres en todo el mundo y la principal causa de muerte por cáncer en mujeres junto al cáncer de pulmón. Este cáncer tiene muy buen pronóstico en general, con una supervivencia del 80%. Sin embargo, el pronóstico del cáncer de mama triple negativo es mucho peor, al no conocerse ninguna diana farmacológica y tratarse de forma inespecífica. La metformina, fármaco prescrito para la diabetes, ha mostrado algunos buenos resultados preliminares como potencial terapia. Por otro lado, el principal tratamiento dirigido de las pacientes HER2+ es el trastuzumab, que neutraliza al receptor HER2 amplificado; sin embargo, un elevado número de pacientes desarrollan resistencias al tratamiento. Los microRNAs son pequeños RNAs no codificantes capaces de regular la expresión génica epigenéticamente, y pueden ser secretados de la célula en vesículas llamadas exosomas. El objetivo de este trabajo es abordar estas dos problemáticas en cáncer de mama. Son necesarios estudios de los mecanismos de acción o resistencia de estos fármacos a través de la regulación epigenética por microRNAs. Queremos determinar la relación del miR-26a y sus dianas con el efecto de la metformina en cáncer de mama triple negativo y estudiar las diferencias de expresión de microRNAs que generan resistencias a trastuzumab en cáncer de mama HER2+, así como estudiar su modo de transmisión entre células. Se realizaron ensayos celulares tratando con metformina las líneas MDA-MB-231, MDA-MB-468 y MCF-7 así como sobreexpresando o inhibiendo miR-26a y se midieron sus dianas teóricas por qPCR. Para las líneas HER2+ se realizó un Affymetrix Genechip miRNA 4.0 microarray comparando líneas SKBR-3wt y BT-474wt con sus respectivas líneas con resistencia generada a trastuzumab y HCC-1954 como resistente innata. Se estudiaron los microRNAs más relevantes del array en las líneas celulares y en pacientes y se comprobó su presencia en exosomas, así como el efecto de los exosomas en la transmisión de la resistencia. La sobreexpresión de miR-26a resultó en una reducción en la viabilidad celular que se recuperó parcialmente al inhibirla. E2F3, MCL-1, EZH2, MTDH y PTEN fueron regulados negativamente por miR-26a y la proteína PTEN también se redujo tras la sobreexpresión de miR-26a. El tratamiento con metformina redujo la viabilidad de las células de cáncer de mama, aumentó la expresión de miR-26a y condujo a una reducción en la expresión de BCL-2, EZH2 y PTEN. La inhibición de miR-26a previene parte del efecto en viabilidad de la metformina y la reducción de la expresión de PTEN y EZH2. En las líneas HER2+, miR-23b-3p y miR-146a-5p fueron los principales candidatos extraídos del array. miR-23b-3p inhibió PTEN significativamente en la línea BT-474. miR-146a-5p aumentó la resistencia de las células SKBR-3 al trastuzumab y su inhibición redujo la resistencia de las SKBR-3r. El aumento de miR-146a-5p en SKBR-3wt tuvo un efecto en ciclo celular aumentando la fase S y la G2/M, inhibiendo la expresión de CDKN1A y aumentando la de CCNB1. Los exosomas de las SKBR-3 contenían miR-146a-5p, con mayores niveles en los de las resistentes (exoR). Los exoR aumentaron la resistencia a trastuzumab, la transición epitelio-mesenquimal y la migración al co-cultivarse con SKBR-3wt, y la angiogénesis en las HUVEC. Nuestros resultados sugieren que el efecto de la metformina está mediado por una mayor expresión de miR-26a y reducción de sus dianas, PTEN y EHZ2. Por tanto, el uso de metformina en el tratamiento del cáncer de mama constituye una prometedora potencial terapia. En HER2+, miR-23b parece provocar resistencia a trastuzumab vía PTEN y miR-146a a través del ciclo celular. Además, miR-146a se transmite en exosomas, que son capaces de reducir la sensibilidad al trastuzumab de las células sensibles y aumentar la TEM, migración y angiogénesis. / [EN] Breast cancer is the most common cancer in women worldwide and the leading cause of cancer death in women along with lung cancer. This cancer has a very good general prognosis, with a survival of 80%. However, the prognosis for triple negative breast cancer is much worse, as it has no pharmacological target and treats it nonspecifically. Metformin, a prescribed diabetes drug, has shown some good preliminary results as potential therapy. On the other hand, the main targeted treatment for HER2 + patients is trastuzumab, which neutralizes the amplified HER2 receptor, but a large number of patients experienced resistance to treatment. MicroRNAs are small non-coding RNAs that are part of epigenetics and are capable of regulating gene expression, and which can be secreted from the cell into vesicles called exosomes. The objective of this work is to address these two problems in breast cancer, which need to study the mechanism of action or resistance of these drugs, through the epigenetics of microRNAs. We want to determine the relationship of miR-26a and its targets with the effect of metformin in triple negative breast cancer and to study the differences in the expression of microRNAs that process resistance to trastuzumab in HER2 + breast cancer, as well as to study its mode of transmission between cells. Cellular assays were performed treating the MDA-MB-231, MDA-MB-468 and MCF-7 lines with metformin as well as overexpressing or inhibiting miR-26a, and their theoretical targets were measured by qPCR. For the HER2+ cell lines, an Affymetrix Genechip miRNA 4.0 microarray was performed comparing SKBR-3wt and BT-474wt lines with their respective cell lines with generated resistance to trastuzumab and HCC-1954 as innate resistance. The most relevant microRNAs of the array in cell lines and in patients were studied and their presence in exosomes was verified, as well as the effect of exosomes in the transmission of resistance. The overexpression of miR-26a resulted in a reduction in cell viability that was partially recovered by inhibiting it. E2F3, MCL-1, EZH2, MTDH, and PTEN were down-regulated by miR-26a, and the PTEN protein was also reduced after overexpression of miR-26a. Metformin treatment reduced the viability of breast cancer cells, increased miR-26a expression, and led to a reduction in BCL-2, EZH2, and PTEN expression. Inhibition of miR-26a partly prevents the effect of metformin in viability and the reduction of the expression of PTEN and EZH2. In the HER2+ lines, miR-23b-3p and miR-146a-5p were the main candidates extracted from the array. miR-23b-3p was shown to significantly inhibit PTEN in the BT-474 cell line. miR-146a-5p increased resistance of SKBR-3wt cells to trastuzumab and its inhibition reduced resistance of SKBR-3r. The increase of miR-146a-5p in SKBR-3wt had effect on the cell cycle by increasing the S phase and the G2/M, inhibiting the expression of CDKN1A and increasing CCNB1 levels. Exosomes isolated from SKBR-3 cell lines contained miR-146a-5p, with higher levels in exosomes from the resistant cell line (exoR). The exoR were shown to increase trastuzumab resistance, EMT, and migration when co-cultivated with SKBR-3wt, and angiogenesis when in culture with HUVEC. Our results indicate that metformin effectively reduces breast cancer cell viability and suggests that the effects of the drug are mediated by an increase in miR-26a expression and a reduction of its targets, PTEN and EHZ2. Thus, the use of metformin constitutes a promising potential triple negative breast cancer therapy. In HER2+ breast cancer, miR-23b appears to elicit resistance to trastuzumab via PTEN and miR-146a throughout the cell cycle. Furthermore, miR-146a is transmitted in exosomes, which have been shown to reduce the sensitivity to trastuzumab of sensitive cells and increase EMT, migration, and angiogenesis. / [CA] El càncer de mama és el càncer més comú en dones arreu del món i la principal causa de mort per càncer en dones junt amb el càncer de pulmó. Aquest càncer té molt bon pronòstic en general, amb una supervivència del 80%. No obstant això, el pronòstic del càncer de mama triple negatiu és molt pitjor, al no conèixer-se'n cap diana farmacològica i tractar-se de forma inespecífica. La metformina, fàrmac prescrit per a la diabetis, ha mostrat alguns bons resultats preliminars com a potencial teràpia. D'altra banda, el principal tractament dirigit de les pacients HER2+ és el trastuzumab, que neutralitza el receptor HER2 amplificat; tanmateix, un elevat nombre de pacients desenvolupen resistències al tractament. Els microRNAs són xicotets RNAs no codificants capaços de regular l'expressió gènica epigenèticament, i poden ser secretats de la cèl·lula en vesícules anomenades exosomes. L'objectiu d'aquest treball és abordar aquestes dues problemàtiques en càncer de mama. Són necessaris estudis dels mecanismes d'acció o resistència d'aquests fàrmacs a través de la regulació epigenètica per microRNAs. Volem determinar la relació del miR-26a i les seues dianes amb l'efecte de la metformina en càncer de mama triple negatiu i estudiar les diferències d'expressió dels microRNAs que generen resistències al trastuzumab en càncer de mama HER2+, així com estudiar la seua manera de transmissió entre cèl·lules. Es van realitzar assajos cel·lulars tractant amb metformina les línies MDA-MB-231, MDA-MB-468 i MCF-7 així com sobreexpressant o inhibint miR-26a i es van mesurar les seues dianes teòriques per qPCR. Per a les línies HER2+ es va realitzar un Affymetrix Genechip miRNA 4.0 microarray comparant línies SKBR-3wt i BT-474wt amb les seues respectives línies amb resistència generada a trastuzumab i HCC-1954 com resistent innata. Es van estudiar els microRNAs més rellevants de l'array en les línies cel·lulars i en pacients i es va comprovar la seua presència a exosomes, així com l'efecte dels exosomes en la transmissió de la resistència. La sobreexpressió de miR-26a resultà en una reducció de la viabilitat cel·lular que es recuperà parcialment en inhibir-la. E2F3, MCL-1, EZH2, MTDH i PTEN foren regulats negativament per miR-26a i la proteïna PTEN també es va reduir en sobreexpressar miR-26a. El tractament amb metformina va reduir la viabilitat de les cèl·lules de càncer de mama, va augmentar l'expressió de miR-26a i va conduir a una reducció en l'expressió de BCL-2, EZH2 i PTEN. La inhibició de miR-26a prevé part de l'efecte en la viabilitat de la metformina i la reducció de l'expressió de PTEN i EZH2. En les línies HER2+, miR-23b-3p i miR-146a-5p foren els principals candidats extrets de l'array. miR-23b-3p va inhibir PTEN significativament en la línia BT-474. miR-146a-5p va augmentar la resistència de les cèl·lules SKBR-3 al trastuzumab i la seua inhibició va reduir la resistència de les SKBR-3r. L'augment de miR-146a-5p en SKBR-3wt va tindre un efecte en cicle cel·lular augmentant la fase S i la G2/M, inhibint l'expressió de CDKN1A i augmentant la de CCNB1. Els exosomes de les SKBR-3 contenien miR-146a-5p, amb majors nivells en els de les resistents (exoR). Els exoR van augmentar la resistència a trastuzumab, la transició epiteli-mesenquimal i la migració en co-cultivar-los amb SKBR-3wt, i l'angiogènesi de les HUVEC. Els nostres resultats suggereixen que l'efecte de la metformina està intervingut per una major expressió de miR-26a i reducció de les seues dianes, PTEN i EHZ2. Per tant, l'ús de metformina al tractament de el càncer de mama constitueix una prometedora potencial teràpia. En HER2+, miR-23b sembla provocar resistència a trastuzumab mitjançant PTEN i miR-146a a través del cicle cel·lular. A més, miR-146a es transmet en exosomes, que són capaços de reduir la sensibilitat al trastuzumab de les cèl·lules sensibles i augmentar la TEM, migració i angiogènesi. / Cabello Navarro, P. (2020). Epigenetic regulation of resistance to treatments in triple negative and HER2+ breast cancer: miRNAs involved [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/153807 / TESIS

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.