Exosomes are small secreted vesicles of endocytic origin with a size range of 50-150 nm. They are secreted by many cell types and display multiple biological functions including immune-activation, immune-suppression, antigen presentation, and the shuttling of mRNA and miRNA, as well as other cargo. We have characterised the exosomes secreted from two breast cancer cell lines, MDA-MB-231 and MCF7. Exosomes secreted from both cell lines display typical markers including ALIX, Tsg101, CD9 and CD63, and were capable of inducing apoptosis of the Jurkat T cell line, indicating the potential immune-suppressive function of such tumour-derived exosomes. To further investigate the biological potential of exosomes, we loaded purified exosomes with gene specific siRNAs using electroporation, and observed the targeted inhibition of both a known component of the exosome pathway, Rab27a, and also the arthritis associated gene ERAP1, demonstrating the potential novel use of exosomes as therapeutic gene delivery vectors. We have also shown that exosomes derived from MDA-MB-231 cells and the parental cells have different lipid composition, as analysed by lipidomics study. Nanoparticle tracking analysis (NTA), which allows the rapid detection of size and concentration of nanoparticles within the size range 10 nm-1000 nm was tested for its ability to accurately measure size and concentration of exosomes and microvesicles under different conditions. NTA was capable of detecting apoptotic vesicles induced by Taxol and Curcumin treatment. Immunodepletion was used to determine the percentage of CD9 and CD63 positive vesicles. Our data suggest that NTA is a useful technique for measuring size and concentration of exosomes and microvesicles. We hypothesized that NTA could assist in the screening of agents that interfere or promote exosome release. NTA was therefore used to detect increases in exosomes secretion induced by Tamoxifen and Thimerosal treatment, and to monitor the inhibition of exosome secretion from MDA-MB-231 breast cancer cells expressing inhibitory RNA targeted for Rab27a, a component of the exosome pathway. Increases in exosome release induced by Tamoxifen and Thimerosal was detected by NTA and a significant reduction in the release of exosomes by inhibition of Rab27a expression was also observed. Treatment with the known exosomal pathway inhibitor DMA also reduced exosome release, establishing the principle of NTA as a screening technique. We further compared the siRNA targeted cells for their ability to migrate, invade and form anchorage-independent colonies, which were all significantly reduced. Supplementation with MDA-MB-231 derived exosomes restored the ability to form colonies, suggesting exosomes may contribute to metastatic lesion formation. These data suggest that the exosomal pathway is a valid target to disrupt the behaviour of tumour cells and NTA can be used to monitor its activity.

Background The foamy virus (FV) replication cycle displays several unique features, which set them apart from orthoretroviruses. First, like other B/D type orthoretroviruses, FV capsids preassemble at the centrosome, but more similar to hepadnaviruses, FV budding is strictly dependent on cognate viral glycoprotein coexpression. Second, the unusually broad host range of FV is thought to be due to use of a very common entry receptor present on host cell plasma membranes, because all cell lines tested in vitro so far are permissive. Results In order to take advantage of modern fluorescent microscopy techniques to study FV replication, we have created FV Gag proteins bearing a variety of protein tags and evaluated these for their ability to support various steps of FV replication. Addition of even small N-terminal HA-tags to FV Gag severely impaired FV particle release. For example, release was completely abrogated by an N-terminal autofluorescent protein (AFP) fusion, despite apparently normal intracellular capsid assembly. In contrast, C-terminal Gag-tags had only minor effects on particle assembly, egress and particle morphogenesis. The infectivity of C-terminal capsid-tagged FV vector particles was reduced up to 100-fold in comparison to wild type; however, infectivity was rescued by coexpression of wild type Gag and assembly of mixed particles. Specific dose-dependent binding of fluorescent FV particles to target cells was demonstrated in an Env-dependent manner, but not binding to target cell-extracted- or synthetic- lipids. Screening of target cells of various origins resulted in the identification of two cell lines, a human erythroid precursor- and a zebrafish- cell line, resistant to FV Env-mediated FV- and HIV-vector transduction. Conclusions We have established functional, autofluorescent foamy viral particles as a valuable new tool to study FV - host cell interactions using modern fluorescent imaging techniques. Furthermore, we succeeded for the first time in identifying two cell lines resistant to Prototype Foamy Virus Env-mediated gene transfer. Interestingly, both cell lines still displayed FV Env-dependent attachment of fluorescent retroviral particles, implying a post-binding block potentially due to lack of putative FV entry cofactors. These cell lines might ultimately lead to the identification of the currently unknown ubiquitous cellular entry receptor(s) of FVs.

Un grand nombre d'espèces microbiennes, issues d'environnements divers et présentant une large gamme de métabolismes différents, peuvent produire de l'hydrogène par voie fermentaire. Jusqu'à présent, les études ont principalement porté sur l'utilisation de cultures microbiennes pures, voire génétiquement modifiées, en vue d'optimiser la production de biohydrogène à partir de sucres simples ou peu complexes. Les efforts de recherche portent désormais sur l'utilisation de cultures microbiennes mixtes pour produire du biohydrogène à partir de sources organiques plus complexes issus par exemple du traitement de la biomasse. Toutefois, la présence de voies métaboliques alternatives tout comme l'instabilité du processus biologique constitue des verrous scientifiques et techniques qu'il conviendra de lever pour une application potentielle. Ceci nécessite entre autre une meilleur connaissance des interactions bactériennes et donc métaboliques présentent au sein du consortium.Pour cela, nous avons développé une approche innovante et pluridisciplinaire d'ingénierie écologique qui consiste en la conception, la construction et l'étude d'un consortium microbien synthétique afin d'établir les paramètres régissant les réseaux d'interactions métaboliques avec pour objectif d'optimiser la production d'hydrogène. Dans un premier temps nous avons choisit d'étudier les réseaux d'interactions métaboliques entre deux souches modèles connues comme partie prenante d'un consortium bactérien naturel, une bactérie du genre Clostridie et une du genre Desulfovibrio, la première étant productrice d'hydrogène par fermentation / Numerous microorganisms can produce hydrogen by “dark fermentation”. Isolated from various environments, they present a broad range of different metabolisms. Until now, literature reports have mainly dealt with the use of pure microbial cultures producing biohydrogen from simple sugars, such as glucose and sucrose. More recently, studies on biohydrogen production by mixed cultures from complex organic sources have been developed. Even though biohydrogen productivities and conversion yields can be interesting for industrial purposes, several scientific and technical constraints remain to be addressed. In particular, the presence of alternative metabolic ways of hydrogen consumption generally results in chronic instability of the biological processes. To increase the stability and the efficiency of dark fermentative processes, it is now necessary to acquire a better understanding of the metabolic interaction networks existing between producing and consuming microorganisms.We have developed an innovative and multidisciplinary approach to ecological engineering, which consists of the construction and study of synthetic microbial consortia to establish the metabolic networks existing between microorganisms for further optimization of biohydrogen production. First we have studied the networks of metabolic interactions between two bacterial models known as involved in a natural bacterial consortium: a bacterium from Clostridium genus; Clostridium acetobutylicum and one from Desulfovibrio genus, Desulfovibrio vulgaris Hildenborough. The first one being producing of hydrogen by fermentation of complex sugars and the second

Este estudo teve como objetivo avaliar a expressão hipocampal de proteínas e de RNAm das Cx43 e Cx36 no modelo de epilepsia do lobo temporal (ELT) induzido por pilocarpina. Além disso, os efeitos do bloqueador de canais de junções comunicantes (CJC), carbenoxolona (CBX), foram avaliados por eletrofisiologia durante o período de status epilepticus. Os dados referentes à Cx43 demonstraram redução dos níveis proteicos no período latente (p<0,05) e aumento no período crônico do modelo (p<0,01). Os níveis de RNAm de Cx43 não sofreram alterações. Tanto os níveis proteicos quanto os de RNAm de Cx36 não se alteraram. Os dados eletrofisiológicos mostraram redução da potência na banda de frequência entre 15 e 30 Hz no eletrocortigrama, além de redução da amplitude relativa dos potenciais epileptiformes. Foi observado ainda que o grupo tratado com CBX passou a apresentar períodos flat antecipadamente. Os dados deste estudo sugerem um importante papel dos CJC na ELT induzida por pilocarpina, contribuindo para o conhecimento da regulação destes canais na epilepsia. / In this study, the hippocampal protein and mRNA levels of Cx43 and Cx36 were investigated in the pilocarpine model of temporal lobe epilepsy (TLE). In addition, the effects of a gap junction (GJ) blocker (carbenoxolone-CBX) on pilocarpine-induced status epilepticus (SE) were also evaluated by electrophysiological recordings. Our results on Cx43 showed reduction of protein levels in the latent period (p<0.05) and increase in the chronic period of the model (p<0.01), whereas no changes were observed in the mRNA levels. Both protein and mRNA levels of Cx36 showed no changes. The electrophysiological recordings indicated that CBX promoted a marked reduction of power in the 15-30 Hz electrocorticographic frequency. Decrease in the amplitude of the epileptiform potentials was also seen, in addition to anticipation of occurrence of flat periods in the group treated with CBX. Data obtained from this study suggest an important role for GJ channels in the pilocarpine-induced TLE, contributing to a greater understanding of the regulation of these channels in the epilepsy.

Neste projeto caracterizamos a interação da nucleoproteína viral (N) do Vírus Respiratório Sincicial Humano (HRSV) com as proteínas PRMT5 e WDR77, que constituem o metilosomo celular. Confirmamos que essa interação ocorre através de co-imunoprecipitação em células humanas, e de interação in vitro dessas proteínas purificadas. Demonstramos a co-localização dessas proteínas na célula através de microscopias de imunofluorescência e confocal. Inibindo ou aumentando a expressão de PRMT5 não observamos impacto na replicação viral. Verificamos que ocorre metilação em N tanto em resíduos de argininas como de lisinas, com anticorpos específicos para essas modificações, e por espectrometria de massas, indicando significado funcional. Com essa evidência testamos o efeito de inibidores de metilação e demetilação, em argininas e lisinas. Obtivemos efeito inibitório significativo da replicação do HRSV com um inibidor de metilação de lisina, UNC0646, indicando que a interação N-metilossomo tem potencial como alvo terapêutico contra HRSV. / In this project we had as objective to characterize the interaction observed previously in the laboratory of viral nucleoprotein (N) with PRMT5 and WDR77 proteins that constitute the cell metilosome. We confirmed that this interaction occurs through co-imunoprecipitation in human cells and in vitro interaction of these purified proteins. We also demonstrated the co-localization of these proteins in inclusion bodies, by immunofluorescence and confocal microscopy. Inhibiting or enhancing PRMT5 expression we didnt see effect on viral replication. Our results show that methylation occurs in both arginine and lysine residues, through reactivity with antibodies specific to these modifications, and analysis by mass spectrometry. We tested the effect of arginine and lysine methylation and de-methylation inhibitors in viral replication. We obtained significant inhibitory effect on HRSV replication with a lysine methylation inhibitor, UNC0646, indicating that N-metilossome interaction has the potential to be exploited as a therapeutic target in developing antiviral drugs.

Dorsal root ganglia (DRG) cell preparations are commonly used to study the properties of sensory neurons in relation to nociception. A typical DRG cell preparation contains both neurons and glial cells, and in addition to a conventional supportive role of glial cells, an increasing volume of literature has reported interactions between neurons and accompanying glial cells. A typical mixed DRG cell preparation can be separated into a neuron-enriched cell fraction and a preparation of purified glial cells. Using these purified cell fractions, we can study the relative contributions and interactions between neurons and glial cells in regulating neurite outgrowth and adenylyl cyclase-dependent cell signalling activity in vitro. / From our previous studies, pretreating DRG cell cultures with pertussis toxin (PTx) caused neurite retraction over a period of 2 h following the initial stimulus of removal from incubator. The purpose of the current study was to investigate whether this PIx-dependent response was specific to anyone of the three subpopulations of DRG neurons. Interestingly, no neurite retraction response was observed in enriched DRG cultures, including cultures enriched with isolectin B4 (IB4)-positive neurons or IB4-negative neurons. Addition of glial cells or conditioned medium from glial cells to IB4-negative cultures was necessary to restore the PTx-dependent neurite retraction response, which was then only observed in large diameter proprioceptive neurons. To conclude, glial cells constitutively release factor/s that stimulate neurite retraction in larger diameter neurons, and is counterbalanced by neuroprotective Gilo protein signalling pathway. / From our studies, we have provided evidence of bidirectional interactions between neurons and glial cells, with glial cells regulating neurite outgrowth and neurons regulating adenylyl cyclase activity in glial cells. These findings reveal the properties of glial cells in regulating neurite outgrowth and in producing prostanoid-stimulated responses. Moreover, our fmdings provide foundation to understand complex neuron-glia interactions in vivo which will eventually help to overcome obstacles in promoting neurite regeneration and in controlling pain. / In a parallel study, we proved that hyperalgesic agents such as prostaglandin E2 (PGE2) and the prostacyclin (PGI2) mimetic (cicaprost) stimulate cAMP production in DRG cell culture via EP4 and IP receptors, respectively. These prostanoids were presumed to act only on the neurons in typical mixed cell cultures, but since we had acquired purified glial cell preparation, we tested for involvement of glial cells in measurement of agonist-stimulated cAMP production. Interestingly, a purified glial cell cultures also produced EP4 and IP-dependent responses. The expression of EP4 and IP receptors by DRG glia was further confirmed by the detection of EP4 and IP-like immunoreactivity and mRNA. Moreover, these agonist-stimulated responses were greatest in the glial cell preparation, and surprisingly weakest in the neuron-enriched cell cultures. Furthermore, the presence of neurons significantly inhibited both EP4 and IP receptor-dependent signalling in glial cells, but was without effect on forskolin (agonist-independent) stimulation of adenylyl cyclase. In order to characterize this neuron-glia interaction, we tested the adenylyl cyclase activities in glial cell cultures which were treated with conditioned medium derived from neurons or were separated from physical contact with neurons plated on transwell membrane. These studies further suggest that the neuron-glia interactions were dependent on both soluble factors and cell-cell contact. / Ng, Kai Yu. / Adviser: Helen Wise. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 152-172). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

O Vírus Respiratório Sincicial Humano (HRSV) causa doença respiratória principalmente em recém-nascidos e bebês. A infecção por HRSV exerce forte interferência sobre a localização de actina, fenômeno que propomos estar relacionado à interação de TPM com M e P, levando ao rearranjo dos microtúbulos. Os objetivos gerais do projeto são de analisar interações in vitro, em bactéria e em célula entre TPM com M e P. Os dados obtidos indicam que ocorre a interação in vitro entre M e TPM 3 mas não ocorre entre P e TPM 3. Os estudos realizados em célula indicam a interação de M e P com TPM 3. Com siRNA para TPM 3 os dados não foram conclusivos e para a super expressão de TPM 3 verificamos que ocorre inibição da replicação viral. Testamos uma droga que desacopla TPM 3 dos filamentos de actina e os resultados indicam inibição da replicação viral. Concluímos com esses dados que TPM 3 tem papel fundamental no ciclo replicativo do vírus e que sua interação com M tem potencial para ser explorada como alvo terapêutico no desenvolvimento de antivirais contra HRSV. / Human Respiratory Syncytial Virus (HRSV) causes respiratory disease in newborns and babies. The HRSV infection exerts strong interference on intracellular location of actin, a phenomenon that we propose to be connected to the interaction of TPM with M and P, leading to the rearrangement of microtubules. In this project we propose to analyze interactions in vitro, in bacteria and in cells between TPM and P or M. The obtained data indicate that an in vitro interaction between M and TPM occurs but does not occur between P and TPM. Cell studies indicate an interaction of M and P with TPM. With siRNA fot TPM 3 the data were not conclusive, and for overexpression of TPM 3 an inhibition of virus replication was shown. Also, in cell, we obtained results indicating that the use of a cytoskeletal destabilizing drug is affecting viral replication. These data indicate that Tropomyosin plays a key role in the virus cycle and therefore has the potential to be exploited as a therapeutic target in the development of antiviral drugs against HRSV.

Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. Despite the significant disease and economic burden, currently there are no licensed vaccines or antivirals. The understanding of norovirus biology has been hampered by the inability to cultivate HuNoV in cell culture. To establish a tissue culture system, infectious HuNoVs were purified from clinical stool samples. HuNoV replication was tested in different cell types. The B-cell and intestinal organoids culture systems were validated. In addition, using organoids culture a DNA-based reverse genetic system was shown to recover infectious HuNoV. Due to the challenges associated with cultivating HuNoV, murine norovirus (MNV) was used as a surrogate system to understand the role of eIF4E phosphorylation in norovirus pathogenesis, and VP1-RdRp interaction in regulating viral genome replication. MNV infection results in the phosphorylation of the translation initiation factor eIF4E, re-programming host-cell translation during infection. Inhibiting eIF4E phosphorylation reduces MNV replication in cell culture suggesting a role in viral replication. A mouse model with eIF4E S209A, a phosphor-ablative mutation, was established to understand the role of eIF4E phosphorylation in MNV pathogenesis. In vitro and in vivo characterisations demonstrated that eIF4E phosphorylation may have multiple roles in norovirus-host interactions, but overall has little impact on MNV pathogenesis. The shell domain (SD) of norovirus major capsid protein VP1 interacts with viral RNA-dependent RNA polymerase (RdRp) in a genogroup-specific manner to enhance de novo initiation of RdRp, and to promote negative-strand RNA synthesis. To understand how VP1 regulates norovirus genome replication, chimeric MNVs with genogroup-specific residues mutagenised were characterised in vitro and in vivo. A single amino acid mutation was shown to destabilise viral capsid. SDs with reduced VP1-RdRp interaction showed less capacity to stimulate RdRp, resulting in delayed virus replication. In vivo, the replication of an MNV-3 with homologous mutations was abolished, highlighting the crucial role of this interaction.

Delisea pulchra is a red macroalga that produces furanones, a class of secondary metabolites that inhibit the growth and colonization of a range of micro- and macroorganisms. In bacteria, furanones specifically inhibit acyl homoserine lactone (AHL)- driven quorum sensing, which is known to regulate a variety of colonization and virulence traits. This thesis aims to unveil multiple aspects of the chemically mediated interactions between an alga and its bacterial flora. It was demonstrated that the quorum sensing genetic machinery of bacteria is laterally transferred, making traditional 16S rRNA gene based-diversity techniques poorly suited to identify quorum sensing species. Previous studies had shown that AHL-producing bacteria belonging to the roseobacter clade can be readily isolated from D. pulchra. Because of this, it was decided to use a roseobacter epiphytic isolate from this alga, Ruegeria strain R11, to conduct a series of colonization experiments on furanone free and furanone producing D. pulchra. Furanones were shown to inhibit Ruegeria strain R11's colonization and infection of D. pulchra. In addition, it was demonstrated that Ruegeria strain R11 has temperature-regulated virulence, similar to what is seen for the coral pathogen Vibrio shiloi. Rising ocean temperatures may explain bleached D. pulchra specimens recently observed at Bare Island, Australia. To assess whether quorum sensing is common within the roseobacter clade, cultured isolates from the Roseobacter, Ruegeria and Roseovarius genera were screened for AHL production. Half of the bacteria screened produced the quorum sensing signal molecules, AHLs. These AHLs were identified using an overlay of an AHL reporter strain in conjunction with thin layer chromatography (TLC). The prevalence of quorum sensing within the roseobacter clade, suggests that these species may occupy marine niches where cellular density is high (such as surface associated communities on substratum and marine eukaryotes). Diversity studies in marine microbial communities require appropriate molecular markers. The 16S rRNA gene is the most commonly used marker for molecular microbial ecology studies. However, it has several limitations and shortcomings, to which attention has been drawn here. The rpoB gene is an alternate ???housekeeping??? gene used in molecular microbial ecology. Therefore, the phylogenetic properties of these two genes were compared. At most taxonomic levels the 16S rRNA and rpoB genes offer similar phylogenetic resolution. However, the 16S rRNA gene is unable to resolve relationships between strains at the subspecies level. This lack of resolving power is shown here to be a consequence of intragenomic heterogeneity.

La present Tesi Doctoral es presenta com una agrupació de quatre publicacions que resumeixen el treball realitzat al Departament de Genètica de la Facultat de Biologia de la Universitat de Barcelona. El principal objectiu és la caracterització funcional de les regions estructurals de la isoforma muscular de l’enzim deubiquitinat USP25, inicialment definides amb eines bioinformàtiques. A més a més, es pretén fer un estudi de noves interaccions moleculars tipus proteïna-proteïna per la cerca de nous substrats o reguladors enzimàtics. La cèl•lula eucariota posseeix, entre altres, un sistema senyalitzador intracel•lular basat en una família de pèptids, el representant dels quals és la ubiquitina. Aquest sistema presenta diferents categories funcionals, entre elles, els enzims deubiquitinants, un centenar a l’espècie humana. Aquests enzims hidrolitzen l’enllaç que uneix la ubiquitina als seus precursors o substrats, mantenint així l’homeostasi d’aquest pèptid dins la cèl•lula. Una alteració de la seva funció pot portar diferents conseqüències depenent de la via metabòlica que es vegi afectada, doncs suposa una desregulació estequiomètrica dels substrats ubiquitinants respecte els no ubiquitinats. L’enzim deubiquitinant USP25 es va descriure en el grup d’investigació d’aquesta tesi durant la cerca de nous gens relacionats amb la síndrome de Down. Un cop caracteritzat funcionalment com una proteasa específica d’ubiquitina, els estudis d’expressió van mostrar l’existència de tres isoformes proteiques, una d’elles, USP25m, restringida al teixit muscular i cardíac. Tenint en compte que en el fenotip dels pacients amb síndrome de Down, entre altres trets, hi ha deficiència cardiovascular i atonia muscular, els esforços del grup es van centrar en la descripció i anàlisi d’aquesta isoforma. Els primers estudis van mostrar la seva situació citosòlica, l’expressió correlativa amb la diferenciació de cèl•lules musculars i la relació específica amb diverses proteines del sarcòmer. En el treball realitzat per la present Tesi Doctoral, s’han caracteritzat funcionalment diferents regions reguladores descrites a nivell bioinformàtic, així com també s’ha analitzat la seva implicació fisiológica a la funció d’USP25m. A més a més, mitjançant un estudi de cerca de nous interactors proteics, s’ha trobat una nova molècula que pertany a la mateixa via senyalitzadora i que es relaciona de manera específica amb USP25m, la lligasa d’ubiquititna MKRN1 (makorin 1) Mitjançant l’ús de diferents construccions amb delecions i mutacions puntuals de la proteïna que afecten a les regions d’interès, s’ha arribat a diferents conclusions, entre elles, que USP25m és monoubiquitinat i té la capacitat d’autodeubiquitinar-se. La monoubiquitinació en regula la seva activitat enzimàtica i es proposa un mecanisme de regulació basat en la conjugació alternativa de SUMO (una altra molècula de la família de la ubiquitina) i ubiquitina, en el mateix residu aminoacídic, la lisina 99 (Lys99). Els dominis d’unió a ubiquitina regulen el reconeixement de substrat i afavoreixen la monoubiquitinació. USP25m oligomeritza dins la cèl•lula i es troba present en diferents formacions proteiques d’elevat pes molecular. S’ha comprovat la relació específica amb la nova lligasa MKRN1 i es suggereixen diferents escenaris moleculars on poden trobar-se inclosos els dos pèptids / The main aim of the present PhD work, titled “Structural domains and new protein interactions of the deubiquitinating enzyme USP25”, is the functional characterisation of the structural domains of the muscle isoform of USP25, USP25m, as well as the analysis of new protein‐protein interactions. The ubiquitin‐proteasome pathway is widely known as the preferential system to get ride of old or non‐functional proteins. Recently, it has become more apparent that this is not the only function. Ubiquitin (Ub) and all ubiquitin–like (UbLs) molecules acted as regulatory tags involved in different cellular events as subcellular localization, enzyme activation, DNA repair, etc. The intricate Ub‐signalling networks require a tight regulation of both conjugation and deconjugationprocesses, which are controlled by ubiquitin ligases and deubiquitinating enzymes (DUBs), respectively. USP25 is a DUB described while looking for novel genes involved in Down syndrome phenotype. First studies showed that it encoded three alternative protein isoforms, one of them, muscle specific. This muscle isoform, USP25m, is a cytosolic protein, upregulated during myogenesis that interacts in a specific manner with different sarcomeric proteins. Using an “in silico” approach, we were able to identify different structural domains, among them three ubiquitin binding domains (UBDs), and we aimed to characterise its role on USP25m function. By generating a collection of deletion and punctual mutants of the regions of interest, we conclude that USP25m is monoubiquitinated and that the UBDs modulate this modification. The preferential site for monoubiquitination is lysine 99 (K99), a residue that has been reported to undergo sumoylation (SUMO conjugation, being SUMO an UbL). According to our results, mutation of the K99 residue diminishes the deubiquitinating function, proposing a mechanistic model for USP25m regulation based on alternative conjugation of Ub and SUMO on the same residue, K99. Futhermore, while seeking new protein interactions of USP25m we identified Makorin Ring finger protein 1 (MKRN1), which belongs to an ubiquitin ligase family, as a putative interactor. We were capable of characterise its interaction and propose different cellular scenarios were they could interact. / The main aim of the present PhD work, titled “Structural domains and new protein interactions of the deubiquitinating enzyme USP25”, is the functional characterisation of the structural domains of the muscle isoform of USP25, USP25m, as well as the analysis of new protein‐protein interactions. The ubiquitin‐proteasome pathway is widely known as the preferential system to get ride of old or non‐functional proteins. Recently, it has become more apparent that this is not the only function. Ubiquitin (Ub) and all ubiquitin–like (UbLs) molecules acted as regulatory tags involved in different cellular events as subcellular localization, enzyme activation, DNA repair, etc. The intricate Ub‐signalling networks require a tight regulation of both conjugation and deconjugationprocesses, which are controlled by ubiquitin ligases and deubiquitinating enzymes (DUBs), respectively. USP25 is a DUB described while looking for novel genes involved in Down syndrome phenotype. First studies showed that it encoded three alternative protein isoforms, one of them, muscle specific. This muscle isoform, USP25m, is a cytosolic protein, upregulated during myogenesis that interacts in a specific manner with different sarcomeric proteins. Using an “in silico” approach, we were able to identify different structural domains, among them three ubiquitin binding domains (UBDs), and we aimed to characterise its role on USP25m function. By generating a collection of deletion and punctual mutants of the regions of interest, we conclude that USP25m is monoubiquitinated and that the UBDs modulate this modification. The preferential site for monoubiquitination is lysine 99 (K99), a residue that has been reported to undergo sumoylation (SUMO conjugation, being SUMO an UbL). According to our results, mutation of the K99 residue diminishes the deubiquitinating function, proposing a mechanistic model for USP25m regulation based on alternative conjugation of Ub and SUMO on the same residue, K99. Futhermore, while seeking new protein interactions of USP25m we identified Makorin Ring finger protein 1 (MKRN1), which belongs to an ubiquitin ligase family, as a putative interactor. We were capable of characterise its interaction and propose different cellular scenarios were they could interact.