Triptofano (TRP) é metabolizado por duas vias, a via serotonérgica e a via das quinureninas. Na via serotonérgica, TRP é metabolizado a serotonina (5-HT) e, em algumas células, à melatonina (MLT) que pode ser oxidada à N1-acetil-N2-formil-5- metoxiquinuramina (AFMK) e N1-acetil-5-metoxiquinuramina (AMK) por ação de peroxidases. Na via das quinureninas o TRP é diretamente metabolizado à N formilquinurenina (NFK) e em seguida a quinurenina (QUIN). A enzima indolamina 2, 3 dioxigenase (IDO) é uma das responsáveis por esta reação. Dada a importância da IDO na tolerância imunológica e pelo fato desta enzima ser induzível nos propusemos a avaliar a existência de uma regulação cruzada entre esta enzima e a via serotonérgica. Avaliando a interferência de AMK sobre a ação de IDO e a interferência de QUIN sobre a formação de AFMK por peroxidases, observamos uma possível interação entre as vias. AMK é um inibidor competitivo clássico de IDO e o Ki encontrado foi de 0,98 mM. QUIN é um inibidor acompetitivo linear simples da formação de AFMK e o Ki encontrado foi de 0,1 mM. A inibição da formação de AFMK também ocorre para a peroxidase humana (mieloperoxidase, MPO). Além de representarem uma regulação cruzada utilizada in vivo, as inibições encontradas podem ser relevantes para a proposta de novos inibidores de IDO e MPO na terapia imunomodulatória. Dado o nosso interesse pelas enzimas IDO e MPO, avaliamos ainda a localização intracelular destas enzimas em células de peritônio de camundongo, tanto residente como ativada com concanavalina A (Con A). O estímulo com Con A representa uma ativação de linfócitos T mediado por interferon gama (IFN-γ) e foi usado como modelo experimental para avaliar condições de localização em células ativadas. Por imunocitoquímica verificamos que IDO e MPO localizam-se próxima à membrana plasmática sendo que uma leve dispersão apenas de MPO foi observada em células ativadas com Con A. A localização intracelular das duas enzimas é no citoplasma, vesículas e núcleo. Curiosamente, verificamos MPO em células isoladas e também em agrupamentos celulares de duas ou mais células. Por citometria de fluxo identificamos macrófagos, linfócitos B1 e agrupamentos celulares como células que contém MPO. A mobilização de MPO durante a ativação celular, a presença de MPO em linfócitos e a presença de MPO e IDO em núcleos são informações novas que sugerem novas atividades para estas enzimas. / Tryptophan (TRP) is metabolized by two mains pathways, the serotoninergic pathway and the kynurenine pathway. In the serotoninergic pathway, TRP is metabolized to serotonin (5-HT) and, in some cells, to melatonin (MLT). The later can even be oxidized to acetyl-N1-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5 -methoxykynuramine (AMK) by peroxidases. In the kynurenine pathway, TRP is metabolized to N-formylkynurenine (NFK) and to kynurenine (KYN). Indoleamine 2, 3 dioxygenase (IDO) is one of those responsible for this reaction. Since IDO is importat in immune tolerance and the fact that this enzyme is inducible by cytokines we proposed whether there is a cross regulation between this enzyme and the serotoninergic pathway. A possible interaction between MLT and TRP oxidation pathways was shown by the AMK influence on IDO activity and QUIN interference on AFMK formation by peroxidases. AMK was shown to be an IDO classical competitive inhibitor with a Ki of 0.98 mM. QUIN was a peroxidase (horseradish peroxidase, HRP) classical uncompetitive inhibitor and Ki was found to be 0,1 mM. AFMK formation inhibition was also found in human peroxidase (myeloperoxidase, MPO). Beyond the in vivo crosstalk, new IDO and MPO inhibitors in immunomodulatory therapy would be proposed by the compounds shown in this study. Given our interest in IDO and MPO, we also evaluated their intracellular localization in both resident and concanavalin A (Con A) activated mice peritoneum cells. Con A stimulation is a IFN-γ mediated T lymphocytes activation and was our experimental model to evaluate activated cells. In light microscopy we observed IDO and MPO localization near the membrane and MPO only had a dispersed localization in Con A activated cells. Cytoplasm, nucleus and vesicles were the intracellular localization of both enzymes. Interestingly, we found MPO in isolated cells and in cell clusters of two or more cells. MPO was founded on macrophages, B1 cells and cell clusters by flow cytometry. The MPO mobilization during cell activation, the presence of MPO in lymphocytes and the presence of MPO and IDO in nuclei are new informations to suggest new activities for these enzymes.

Peptídeos intracelulares são constantemente produzidos pelo sistema ubiquitina proteassomo e muitos são provavelmente funcionais. Aqui, um nonapeptídio derivado da ciclina-D2, específica da transição G1/S, chamado \"pep5\" mostrou um aumento específico durante a fase S do ciclo celular em células HeLa. O pep5 (50-100 μM) induziu a morte celular em células HeLa e em várias outras células tumorais, mas isso só ocorreu quando o pep5 foi sintetizado acoplado ao peptídeo penetrador de células (pep5-cpp). In vivo, o pep5-cpp reduziu o volume do glioblastoma C6 de ratos Wistar em cerca de 50%. A acetilação reduziu a potência do pep5-cpp, enquanto substituições Leu-Ala aboliram totalmente a atividade deste peptídeo. Os resultados de caracterização inicial do mecanismo de morte celular indizida pelo pep5 incluem ativação de caspases 3/7 e 9, inibição da fosforilação Akt2 e inibição da atividade do proteassomo. Esses dados colaboram com a hipótese da função de peptídeos intracelulares na sinalização. / Intracellular peptides are constantly produced by the ubiquitinproteasome system and many are probably functional. Here, a nonapeptide of G1/S-specific cyclin-D2 named pep5 showed a specific increase during the S phase of HeLa cell cycle. Pep5 (50-100 μM) induced cell death in HeLa and in several other tumor cells, only when it was fused to a cell penetrating peptide (pep5-cpp). In vivo, the pep5-cpp reduced the volume of the rat C6 glioblastoma by almost 50%. Acetylation reduced the potency of the pep5-cpp while Leu-Ala substitutions totally abolished the pep5 activity. Findings from the initial characterization of the cell death mechanism of pep5 include caspase 3/7 and 9 activation, inhibition of Akt2 phosphorylation and inhibition of proteasome activity. These data further support the intracellular function of peptides.

A oligopeptidase neurolina (E.C.3.4.24.16; nln ) foi identificado pela primeira vez em membranas sinápticas de cérebro de ratos como sendo capaz de participar no metabolismo de peptídeos bioativos, como neurotensina e bradicinina. Recentemente, foi sugerido que a ausência de Nln pode melhorar a sensibilidade a insulina. Aqui, nós mostrado que camundongos knockout para Nln (KO) são mais tolrerantes à glicose, sensíveis à insulina e apresentam maior gliconeogênese. Os animais KO apresentou um aumento na expressão de mRNA de vários genes relacionados com a gliconeogênese no fígado. A semiquantificação de peptídeos intracelulares revelou um aumento em peptídeos intracelulares específicos no gastrocnêmio e tecido adiposo epididimal, que estão envolvidos com o aumento da tolerância a glicose e maior sensibilidade à insulina nos animais KO. Esses resultados sugerem fortemente a nova possibilidade de que Nln é uma enzima chave no metabolismo energético e pode ser um novo alvo terapêutico para melhorar a captação de glicose e sensibilidade a insulina. / The oligopeptidase neurolysin (EC 3.4.24.16; Nln) was first identified in rat brain synaptic membranes and shown to ubiquitously participate in the catabolism of bioactive peptides such as neurotensin and bradykinin. Recently, it was suggested that Nln reduction could improve insulin sensitivity. Here, we have shown that Nln knockout mice (KO) have increased glucose tolerance, insulin sensitivity and gluconeogenesis. KO mice have increased liver mRNA for several genes related to gluconeogenesis. Isotopic label semi-quantitative peptidomic analysis suggests increase in specific intracellular peptides in gastrocnemius and epididymal adipose tissue, which likely is involved with the increased glucose tolerance and insulin sensitivity in the KO mice. These results suggest the exciting new possibility that Nln is a key enzyme for energy metabolism and could be a novel therapeutic target to improve glucose uptake and insulin sensitivity.

A urocortina 2 (Ucn2) é um peptídeo que pertence à família dos fatores liberadores de corticotrofina (CRF) e, assim como seu receptor específico CRF2R? (corticotropin releasing factor receptor type 2?), encontram-se expressos no músculo esquelético. Embora tenha sido demonstrado que o tratamento sistêmico com Ucn2 seja capaz de induzir hipertrofia e prevenir a perda de massa muscular, nada se conhece acerca dos mecanismos moleculares através dos quais a Ucn2 desempenha suas ações biológicas. O principal objetivo deste trabalho foi investigar o mecanismo de ação da Ucn2 no músculo esquelético de roedores para o aparecimento da resposta hipertrófica e a possível participação das vias de sinalização Akt/mTOR, Akt/Foxo e ERK1/2 neste efeito anabólico. Para isto foi utilizado o modelo de transfecção in vivo da Ucn2 pelo método da eletroporação em músculos tibialis anterior de camundongos. Nestes músculos foram quantificados: 1) o estado de fosforilação de componentes efetores destas vias; 2) moléculas sinalizadoras da via autofágica; 3) a taxa de síntese proteica in vivo e 4) a expressão de genes relacionados à atrofia muscular (atrogenes). Outra metodologia utilizada para verificar o efeito direto da Ucn2 na musculatura esquelética foi a incubação in vitro de músculos soleus e EDL isolados de roedores com este peptídeo a fim de investigar a taxa de degradação proteica total, bem como a atividade dos sistemas proteolíticos lisossomal¸ ubiquitina-proteassoma e dependente de Ca+2. A superexpressão in vivo da Ucn2 por 14 dias promoveu hipertrofia e preveniu a atrofia em músculos tibialis anterior de camundongos normais e submetidos ao modelo de desnervação motora isquiática.Resumo Este crescimento muscular induzido pela Ucn2 in vivo foi associado a ativação das vias de sinalização AMPc/PKA/CREB, AMPc/Epac, Akt/mTOR/S6, Akt/mTOR/4E-BP1 e ERK1/2/eIF4E com consequente estimulação da síntese proteica. Em concordância, utilizando uma técnica de manipulação genética in vivo, demonstramos que a hipertrofia promovida pela Ucn2 é dependente da estimulação das cascatas de sinalização ativadas por Akt e ERK1/2. Ademais, essa alteração fenotípica promovida pela Ucn2 induziu melhora da resistência à fadiga muscular, sendo este impacto funcional dependentente de ERK1/2, mas não de Akt. Além disso, a superexpressão da Ucn2 induziu \"shifting\" para o tipo de fibra oxidativa (tipo I), sendo esta plasticidade possivelmente mediada por PGC-1?, o que pode ter contribuído pelo menos em parte, para o efeito benéfico promovido pela Ucn2 na função muscular. O efeito antiatrófico da Ucn2 in vivo foi associado à estimulação da via Akt/Foxo1,3 concomitante com a redução da atividade transcricional de Foxo, resultando na diminuição da expressão da E3-ligase atrogin-1 e do gene autofágico LC3. Em paralelo, a Ucn2 in vivo promoveu inibição do fluxo autofágico, inferido pelo acúmulo das proteínas LC3-I, LC3-II e p62 nestes músculos. Corroborando os achados in vivo, os efeitos antiproteolíticos da Ucn2 in vitro parecem ser mediados pelo AMPc e envolvem a supressão da atividade do sistema lisossomal/autofágico em músculos EDL de ratos normais. Portanto, além da participação de efetores dowsntream do AMPc, como PKA e EPAC, diferentes quinases participam dos efeitos biológicos da Ucn2 na musculatura esquelética. Esses resultados são importantes para caracterizar novas estratégias terapêuticas capazes de atuar no combate à atrofia muscular em diversas situações catabólicas. / Urocortin 2 (Ucn2) is a peptide that belongs to corticotrophin releasing factors (CRF) family and, as well as its specific receptor CRF2R? (corticotropin releasing factor receptor type 2?), are expressed in skeletal muscle. Although it has been demonstrated that Ucn2 systemic treatment is able to induce hypertrophy and prevent loss of muscle mass, nothing is known about the molecular mechanisms through which Ucn2 plays its biological actions. The main objective of this work was to investigate the Ucn2 mechanism of action in rodent skeletal muscle for the appearance of the hypertrophic response and the possible participation of Akt/mTOR, Akt/Foxo and ERK1/2 signaling pathways in this anabolic effect. For this, an in vivo transfection model of Ucn2 was used by the electroporation method in tibialis anterior muscles of mice. Were quantified in these muscles: 1) the phosphorylation state of effector components of these pathways; 2) signaling molecules of the autophagic pathway; 3) the rate of protein synthesis in vivo and 4) the expression of genes related to muscle atrophy (atrogenes). Another methodology used to verify the direct effect of Ucn2 in skeletal muscle was the incubation of soleus and EDL muscles isolated from rodents with this peptide in vitro in order to investigate the total rate of protein degradation, as well as the activity of the lysosomal, ubiquitin-proteasome and Ca+2 dependent proteolytic systems. Ucn2 overexpression in vivo for 14 days promoted hypertrophy and prevented atrophy in tibialis anterior muscles of normal mice and submitted to the sciatic motor denervation model. This muscle growth induced by Ucn2 in vivo was associated with the activation ofAbstract cAMP/PKA/CREB, cAMP/Epac, Akt/mTOR/S6, Akt/mTOR/4E-BP1 and ERK1/2/eIF4E signaling pathways with consequent stimulation of protein synthesis. In agreement, using a genetic manipulation technique in vivo, we demonstrated that the hypertrophy promoted by Ucn2 is dependent on the stimulation of the signaling cascades activated by Akt and ERK1/2. In addition, this phenotypic alteration promoted by Ucn2 induced an improvement in muscle fatigue resistance, being this functional impact dependent on ERK1/2, but not on Akt. Moreover, Ucn2 overexpression in vivo induced the shift to type I oxidative fiber, and this plasticity is possibly mediated by PGC-1?, which may have contributed at least in part to the beneficial effect promoted by Ucn2 in muscle function. The anti-atrophic effect of Ucn2 in vivo was associated with the stimulation of Akt/Foxo1,3 pathway concomitant with the reduction of Foxo transcriptional activity, resulting in a decrease in the expression of the atrogin-1 E3-ligase and of the autophagic gene LC3. In parallel, Ucn2 in vivo promoted inhibition of autophagic flow, inferred by the accumulation of LC3-I, LC3-II and p62 proteins in these muscles. Corroborating the in vivo findings, the antiproteolytic effects of Ucn2 in vitro appear to be mediated by cAMP and involve the suppression of lysosomal/autophagic system activity in EDL muscles of normal rats. Thus, in addition to the participation of cAMP dowsntream effectors, such as PKA and EPAC, different kinases participate in the biological effects of Ucn2 on skeletal muscle. These results are important to characterize new therapeutic strategies able to prevent muscular atrophy in several catabolic situations.

Os canais de junções comunicantes (gap junctions, JC) são formados por subunidades chamadas de conexinas (Cx). Estas proteínas têm papel relevante no acoplamento celular, participando da condutância de células nervosas ou gliais e modulando vários processos fisiológicos e fisiopatológicos. O objetivo deste trabalho é avaliar o envolvimento das conexinas na nocicepção aguda, por meio de ensaios comportamentais e estudos de mapeamento da sua expressão em células do gânglio da raiz dorsal de ratos. Para tanto, foi analisado o efeito de carbenoxolone (CBX) e quinina (bloqueadores de JCs), assim como de oligonucleotídeos antisense para conexinas 36 e 43 na indução e manutenção de hiperalgesia induzida por carragenina em ratos. Os resultados mostraram que a carragenina induz uma diminuição do limiar nociceptivo em ratos e que esse efeito hiperalgésico da carragenina foi bloqueado pelo tratamento com carbenoxolone (nas doses 20-50mg) e significantemente inibido por quinina (nas doses 20-50mg), sugerindo uma participação das junções comunicantes (JC) no processo. / Gap junctions channels (GJ) are formed by proteic subunits called connexins (Cx). These proteins have an important role in cellular coupling, participating in the conductance of glial and nerve cells or modulating various physiological and pathophysiological processes. The aim of this study is to evaluate Cx36 and Cx43 involvement in acute nociception through behavioral assays, mapping studies of its expression in rat dorsal root ganglion cells. For this purpose, we analyzed the effect of intrathecal treatment with carbenoxolone (CBX) and quinine (GJs blockers), as well as antisense oligonucleotides for connexins 36 and 43 in the induction and maintenance of carrageenan-induced hyperalgesia in rats. The results show that carrageenan induces a nociceptive threshold decrease in rats. The hyperalgesic effect was blocked by treatment with carbenoxolone (20-50mg doses), Cx43 antisense and inhibited significantly by quinine (at doses 20 -50mg) but no with Cx36 antisense, suggesting an involvement of gap junctions (JC) in the process.

Zinc is the second most abundant transition metal in biological systems. This cation is required for the catalytic activity of hundreds of enzymes which mediate protein synthesis, DNA replication and cell division. Despite the central importance of zinc in cellular homeostasis, the mechanism of zinc uptake, compartmentalization and efflux is unknown. Recently, a family of proteins, called ZIP, has been shown to control zinc uptake. Mutations in one of the genes coding for these proteins (ZIP4) can lead to potentially life-threatening diseases like Acrodermatitis Enteropathica and high levels of ZIP4 have been detected in patients suffering from pancreatic cancer. Therefore our goal is to investigate the mechanism of ZIP4 transport and regulation. It was previously shown that the intracellular loop between transmembrane III and IV (M3M4) of ZIP4 is ubiquitinated in the presence of high intracellular zinc which lead to protein degradation. Our initial hypothesis was that the large intracellular domain of ZIP4 (M3M4) is a sensor which detects the intracellular concentration of zinc and regulates the surface expression of ZIP4. In order to test this hypothesis we expressed and purified the M3M4 domain to examine the ability of M3M4 to bind zinc. Our results have demonstrated that M3M4 binds zinc with a 2:1 zinc:protein stoichiometry with nanomolar affinity. We have also shown that upon binding of zinc, M3M4 undergoes a large conformational change.

Chloroplasts are organelles that convert light energy to chemical energy through photosynthesis. The movement of chloroplasts within the cell for the optimization of light absorption is crucial for plant survival. Cellular motor proteins and cytoskeletal tracks can facilitate transport of organelles. As an ancient superfamily of microtubule-dependent motors, kinesins participate in various cellular activities including cytokinesis, vesicle and organelle movements. Based on phylogenetic relationships and functional analysis, the kinesin superfamily has been subdivided into more than 14 families, most of which can be found in plants. With the ever increasing amount of genomic information, it is important and beneficial to systematically characterize and document kinesins within an organism. As a result of my collaborative work with other members of the Vidali lab, a detailed phylogenetic characterization of the 76 kinesins of the kinesin superfamily in the moss Physcomitrella patens is reported here. We found a remarkable conservation of families and subfamily classes with Arabidopsis, which is important for future comparative analyses of functions. Some of the families are composed of fewer members, while other families are greatly expanded in moss. To improve the comparison between species, and to simplify communication between research groups, we proposed a classification of subfamilies based on our phylogenetic analysis. As part of my efforts in studying chloroplasts motility, I investigated the function of two members of Physcomitrella kinesin family 14 class V proteins, Ppkin14-Va and -Vb. These two proteins are orthologs of the Arabidopsis KAC proteins which mediate actin-based chloroplast movement in Arabidopsis thaliana. In contrast, in the Physcomitrella both actin filaments (AFs) and microtubules (MTs) participate in chloroplast movement. Our results show that Ppkin14-Vs are important for maintaining chloroplast dispersion. They also function during chloroplast light avoidance responses via an AF-dependent, rather than MT-dependent mechanism. Although two Ppkin14- Vs do not act as MT-based motors, our phylogenetic study on moss kinesins provides an important source of information to track other potential kinesins that are predicted to move chloroplasts on MTs.

RNA binding proteins (RBPs) have been found to be frequently involved in neurodegenerative diseases (Ash 2014). Mutations in RBPs cause amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia, frontotemporal dementia (FTD) and myopathies (Ash 2014), and recent studies suggest that aggregation of RBPs are a pathological feature frequently encountered in tauopathies (Vanderweyde 2016). Emerging studies on neurodegenerative diseases are now showing an increasingly important role for tau in regulating the biology of RBPs. In this study, we examine findings that show that reducing the RBP T-cell intracellular antigen 1 (TIA1) in vivo not only protects against neurodegeneration, but also prolongs the survival rate in transgenic P301S tau mice. Furthermore, the reduction of TIA1 decreases the number and size of granules co-localizing with stress granule markers, and inhibits the accumulation of tau oligomers, although at the expense of an increased number of neurofibrillary tangles. However, despite the observed increase in neurofibrillary tangles, this TIA1 reduction still manages to increase neuronal survival, rescue behavioral deficits and prolong lifespan. The in vivo data presented in this study suggests an important role for TIA1 in mediating toxicity and provides evidence that RBPs orchestrate a pathway to tau aggregation and the resulting neurodegeneration.

Osteocytes are widely regarded as mechanosensors, capable of detecting changes in the mechanical environment of the bone tissue and modifying cellular responses accordingly. Indeed, an intact osteocyte network is required for bone changes in response to unloading, and studies have shown that loading/unloading influences osteocyte expression of proteins that modulate bone turnover, such as sclerostin and receptor activator of nuclear factor kappa B ligand (RANKL). However, mechanisms underlying osteocyte mechanotransduction remain unclear. For instance, one of the earliest responses of bone cells to mechanical stimuli is a rise in intracellular, or cytosolic, calcium (Ca2+cyt), but the mechanisms by which osteocytes generate or utilize Ca2+ signals to direct bone adaptation are largely unknown. In this thesis, I explored the mechanisms underlying the sustainment of Ca2+cyt oscillations in osteocytes as well as downstream consequences of these patterns. I discovered that Ca2+cyt oscillations are generated in osteocytes by Ca2+ release from the endoplasmic reticulum and that the predominant expression of T-Type voltage sensitive Ca2+ channels in these cells facilitates this behavior. I also explored the role of the actin cytoskeleton – another prominent feature in osteocytes – and found that actin dynamics are important for the generation of Ca2+cyt signals. Furthermore, I confirmed that Ca2+cyt transients subsequently activate actomyosin contractions in osteocytes by monitoring interactions of osteocytes exposed to Ca2+ agonists on micropillar substrates. With this information, I sought to relate Ca2+cyt signaling and actomyosin contractility in osteocytes to their roles as coordinators of bone adaptation. Ca2+-dependent contractions have been shown to facilitate the release of extracellular vesicles, small membrane-enclosed packages of proteins that cells use for communication, in other cell types. I found that mechanical stimulation increased the production and release of extracellular vesicles in osteocytes, and this was dependent on Ca2+ signaling. These extracellular vesicles contained key bone regulatory proteins and were small enough to plausibly transport through the lacunocanalicular system. Thus, I uncovered a novel mechanotransduction pathway by which osteocytes may coordinate tissue-level adaptation. As an extension of this work, I also characterized these behaviors in new osteocyte cell lines which may better reflect native cell physiology. The work in this thesis anchors Ca2+ signaling as a critical osteocyte response to mechanical loading and adds to the body of work exploring how and why these signals are generated. The results of these studies add new information to the still limited knowledge of this important bone cell and extend Ca2+ signaling research by connecting early mechanosensation events to subsequent protein responses to mechanical loading. Understanding the mechanisms behind the robust Ca2+cyt oscillations in osteocytes and how they relate to their roles as coordinators of bone adaptation may improve our ability to prevent or treat bone degeneration in diseases like osteoporosis where mechanosensitivity is impaired.

The myc family of oncogenic transcription factors, which includes c-myc, N-myc and L-myc, control major cellular processes such as proliferation and differentiation by integrating upstream signals and orchestrating global gene transcription. They do this largely through dimerising with Max, which together bind to enhancer (E)-box elements in DNA. Myc proteins function similarly but differ in potency and tissue distribution. For instance, N-myc is expressed predominantly during development in undifferentiated cells of the nervous system, whereas c-myc is ubiquitously expressed in all proliferating cells. Myc proteins, when deregulated, are major drivers of tumourigenesis. Myc deregulation occurs in up to 70% of all human cancers and is often associated with the most aggressive forms. For example, MYCN, the gene encoding N-myc, is amplified in 20-30% of neuroblastomas, and amplification strongly correlates with advanced stage and poor prognosis. Myc proteins are therefore considered “most wanted” targets for cancer therapy, but have long been considered undruggable mainly due to challenges in nuclear drug delivery and physically targeting myc directly given that it is a largely disordered protein that lacks discernible clefts and pockets for small molecules to inhabit. Furthermore, c-myc is important in normal tissue maintenance so the effect of its inhibition in humans is difficult to predict. However, recent in vivo studies showed that systemic myc inhibition (using the peptide pan myc inhibitor Omomyc) has mild and reversible side effects and induces tumour regression. This has alleviated concerns about the side effects that myc inhibition might have, and reinforced the promise of myc as a powerful drug target. However, the translation of Omomyc into the clinic has been hindered by poor cellular delivery. In fact, no direct myc inhibitor has yet been approved, indicating that novel approaches are needed. Moreover, inhibitors in development tend to inhibit all myc family proteins. An inhibitor that could specifically target N-myc might improve safety through bypassing c-myc inhibition. This could be used for the treatment of N-myc-driven cancers such as MYCN-amplified neuroblastoma. Nanobodies, camelid-derived single-domain antibodies, are a relatively new drug class. Whilst some are already in clinical trials for a wide range of diseases, these are specific for cell-surface or extracellular targets. However, their properties also make them ideal for use as intracellular antibodies or ‘intrabodies’. For example, they are small (just 12-15 kDa) and highly soluble due to naturally occurring hydrophobic to hydrophilic amino acid substitutions. Their small size and convex shape makes them advantageous in capturing structures in intrinsically disordered proteins and allows them to reach hidden epitopes not accessible to conventional antibodies, which could improve biological activity. Importantly, nanobodies retain the high specificities and affinities of conventional antibodies. Their small, single-domain nature also means they can be engineered with ease to modify aspects of their localisation and/or function. For example, they can be coupled to carrier molecules to facilitate cellular entry, and a nuclear localisation signal (NLS) can be added to drive them into the nucleus. Also, it was recently shown that an F-box domain could also be incorporated into nanobodies to recruit degradation machinery to its antigen, which depletes the antigen from cells via the proteasomal degradation pathway. Due to their highly advantageous properties, nanobodies raised against N-myc might overcome the barriers to targeting N-myc, providing potent and specific means of directly inhibiting N-myc therapeutically, which has not yet been achieved. In this thesis, nine unique nanobodies were raised against N-myc. These included three against the basic helix-loop-helix leucine zipper (bHLH-LZ) domain where Max dimerises, and six against the transactivation domain where numerous regulatory and cofactor proteins bind, such as the E3 ubiquitin ligase Skp2. Nanobodies against the transactivation domain were more specific for N-myc and were shown to inhibit its Skp-2-mediated ubiquitylation. This could provide novel means of eradicating tumours based on a study showing that inhibition of ubiquitylation at this domain triggers a transcriptional ‘switch’ that induces a non-canonical target gene Egr1, leading to p53-independent apoptosis. A nanobody against the bHLH-LZ (Nb C2) was shown to bind both N- and c-myc to similar magnitudes. Its affinity for N-myc bHLH-LZ was superior to that of the small molecule myc inhibitor 10058-F4, which prolongs survival in a MYCN-dependent mouse model of high-risk neuroblastoma. Nb C2 spontaneously transduced cell membranes and its coupling to a novel small molecule carrier (SMoC) enhanced its cellular uptake. Furthermore, the addition of a NLS increased its nuclear localisation. Preliminary experiments showed that Nb C2 might slow proliferation and induce apoptosis in cancer cell lines expressing c-myc, suggesting that Nb C2 might also be effective against cancers characterised by deregulated c-myc. Taken together, data generated in this thesis have revealed intriguing findings that provide a basis for the development of these nanobodies for the treatment of N-myc- and c-myc-driven cancers.