Delta-Notch signaling is crucial for development of nearly every tissue in metazoans. Signals received by the Notch receptor influence transcription of select target genes that ultimately restrict the developmental fate of the signal receiving cell with respect to its neighbors. The Notch pathway also functions in contexts of abnormal proliferation and differentiation, e.g. cancer and inflammation. Therefore, understanding the regulation of signaling through the Notch receptor protein at the cellular and molecular level is of great significance. In this dissertation, I investigated three ways in which Notch signaling is regulated, namely (1) proteolysis of the Delta ligand; (2) endocytosis of the Delta ligand; and (3) proteolysis of the Notch receptor.. The Delta protein has three functions. First, Delta is a ligand for Notch when bound to it from an adjacent cell. Second, Delta is an inhibitor of Notch when coexpressed with it in the same cell. Third, Delta is hypothesized to be a receptor and, upon binding to Notch, signals to nucleus. Delta undergoes proteolysis by ADAM proteases and there are two contradictory models for the role of Delta cleavage: (1) cleavage disables Delta function; and (2) cleavage activates Delta function. Overall, the results presented in this dissertation strengthen the first model and weaken the second one. Consistent with the first model, we showed that preventing Delta cleavage strengthens its ligand function. As well, when co-expressed in the same with Notch, Delta cleavage is upregulated therefore disabling Delta function as inhibitor of Notch. In contrast to the second model, we showed that Delta proteolysis does not follow a previously established pattern of cleavages typical of cell surface proteins that are activated by proteolysis. Delta also undergoes endocytosis. Two general models have emerged that are again contradictory: (1) endocytosis downregulates cell surface expression of Delta and therefore diminishes its ability to bind Notch; (2) endocytosis of Delta invokes activation of Notch signaling. Overall, our results strengthen the first model and weaken the second one. In support of the first model, we first demonstrated that Notch activation shows a linear relationship to the amount of Delta ligand present on the cell surface and that subsequent inhibition of cell surface expression of Delta leads to its loss of function. In contrast to the second model, we showed that endocytosis of Delta is not required to activate Notch. We also resolved that earlier evidence in support for this model stemmed from misinterpretations of the properties of a Delta mutant protein. Proteolysis of Notch activates the signaling cascade. Binding of Delta to Notch was previously regarded as a requisite regulatory step to invoke receptor proteolysis. We identified the ability of Kuzbanian and TACE, ADAM proteases that cleave Notch in response to Delta stimulation, to activate Notch in a ligand-independent manner. Altogether, our results demonstrate that proteolysis and endocytosis of Delta are independent mechanisms that act to downregulate Delta function and are therefore an important means of attenuating the Notch signal. Alternatively, we find a novel means of enhancing Notch signals in specific contexts, namely through ligand-independent Notch activation by the ADAMs Kuzbanian and TACE. With respect to the latter observation, Kuzbanian and TACE expression is known to be elevated in several human diseases, and thus predicts that engagement of Notch signaling is a contributing factor in these pathologies.

TRAIL (TNF-related apoptosis inducing ligand) became known for its ability to selectively eliminate cancer cells. This ligand is a member of the TNF (tumor necrosis factor) ligands family and triggers extrinsic apoptotic pathway by binding of its death receptor 4 or 5 (DR4/5), and subsequent formation of death-inducing signalling complex (DISC). This signalling complex is required for successful transmission of apoptotic signal and activation of proximal caspases. However, regulation of the initial steps leading to activation of caspases is still not fully understood. Endocytosis of a TRAIL- DR4/5-DISC complex can be one of modulators of the initiation of extrinsic apoptotic pathway. Recent studies show controversial data documenting that endocytosis of TRAIL receptosomes can in cell type specific manner either positively or negatively influence TRAIL-induced apoptotic signalling. In this study, we focus on the analysis of a role of endocytosis and acidification of endosomal compartments during TRAIL-induced apoptosis in human colorectal cancer cell lines. Our results support the view that both clathrin-dependent endocytosis of TRAIL receptosome and endosomal acidification positively affect activation of caspases during the early stages of TRAIL-induced apoptosis. Inhibition of endocytosis or endosomal...

RALF é um peptídeo hormonal de aproximadamente 5kDa presente em diferentes espécies do reino vegetal regulando negativamente a expansão celular. AtRALF1 é uma isoforma específica de raiz das 37 presentes em Arabidopsis thaliana que regula negativamente o crescimento de raízes seguido de uma mobilização de Ca+2 intracelular e inibição na secreção de prótons (H+). Neste trabalho foi caraterizado a localização e tráfego intracelular do peptídeo AtRALF1. / RALF is a 5kDa peptide hormone ubiquitous in different species of the plant kingdom that regulates cell expansion. AtRALF1 is a root-specific isoform of 37 present in Arabidopsis thaliana that negatively regulates root growth by intracellular calcium mobilization and inhibition of proton secretion (H+). In this work was studied the localization and intracellular trafficking of the AtRALF1 peptide.

Evidências epidemiológicas indicam que a presença de microalbuminúria prediz maior freqüência de eventos cardiovasculares e mortalidade em hipertensos essenciais. A microalbuminúria pode ser decorrente do aumento da permeabilidade glomerular e/ou da diminuição da reabsorção desta macromolécula no túbulo proximal. Todavia não é sabido se os mecanismos que regulam a reabsorção de albumina em túbulo proximal renal encontram-se alterados na hipertensão essencial. Este trabalho teve como objetivo investigar as bases moleculares da microalbuminúria associada à hipertensão arterial essencial, focando na reabsorção tubular de albumina. Para tanto, avaliamos a evolução temporal da excreção urinária de albumina em ratos espontaneamente hipertensos (SHR) com 6 semanas de idade (pressão arterial sistólica, PAS, = 105 ± 4 mmHg), 14 semanas de idade (PAS = 180 ± 2 mmHg) e 21 semanas de idade (PAS = 202 ± 2 mmHg). Ratos normotensos Wistar da mesma idade serviram de controle. Observou-se que a excreção urinária diária de albumina aumentou progressivamente com o aumento da pressão arterial em SHR (10,5 ± 1,9; 92 ± 7,0 e 154 ± 27 g/dia, em SHR com PAS média igual a 105, 180 e 202 mmHg respectivamente). Este aumento progressivo não foi observado em ratos normotensos com idade correspondente, indicando que este fenômeno é decorrente do aumento da pressão arterial e não pode ser atribuído ao aumento da idade dos animais durante o período estudado. A análise das proteínas urinárias por eletroforese em gel de poliacrilamida (SDS-PAGE) mostrou que SHR excretam proteínas do tamanho da albumina ou menores (< 70kDa), padrão típico de proteinúria tubular. Adicionalmente, verificou-se que os níveis de expressão dos receptores endocíticos megalina e cubilina, bem como do canal para cloreto ClC-5 diminuem progressivamente no córtex renal de SHR com o aumento da pressão arterial. Observou-se também uma diminuição significativa na expressão de uma outra macromolécula importante no processo de endocitose mediada por receptor em túbulo proximal renal, a v-H+-ATPase. Entretanto, a diminuição da expressão protéica da subunidade B2 desta ATPase foi estatisticamente significante apenas em SHR com 21 semanas comparado aos com 6 semanas de idade. Não foram encontradas alterações no padrão de expressão de componentes estruturais da barreira glomerular como a nefrina e podocina. Em suma, o nosso estudo demonstra que o aumento da excreção urinária de proteínas, especialmente de albumina, está associado com uma menor expressão de componentes essenciais do aparelho endocítico do túbulo proximal renal. É tentador especular que a disfunção da via endocítica no túbulo proximal renal possa ser o principal mecanismo subjacente ao desenvolvimento de microalbuminúria na hipertensão / Epidemiological evidences indicate that the presence of microalbuminuria predicts a higher frequency of cardiovascular events and mortality in essential hypertensive patients. Microalbuminuria may arise from increased glomerular permeability and/or reduced proximal tubular reabsorption of albumin. However, it remains to be determined whether the mechanisms that regulate the renal proximal tubular reabsorption of albumin are altered in essential hypertension. The purpose of this work was to investigate the molecular basis of microalbuminuria in essential hypertension, focusing on the renal tubular reabsorption of albumin. To this end, we evaluated the temporal evolution of urinary albumin excretion in spontaneously hypertensive rats (SHR) at 6 weeks of age (systolic arterial pressure, SAP, = 105 ± 4 mmHg), 14 weeks of age (SAP = 180 ± 2 mmHg) and 21 weeks of age (SAP = 202 ± 2 mmHg). Age-matched normotensive Wistar rats were used as controls. It was observed that the daily urinary excretion of albumin progressively increased with blood pressure in SHR from 6 to 21 weeks of age (10.5 ± 1.9, 92 ± 7.0 and 154 ± 27 g in SHR with 105, 180 and 202 mmHg of average SAP, respectively). This progressive increase in microalbuminuria has not been observed in age-matched normotensive Wistar rats, indicating that this phenomenon cannot be attributed to age progression over the studied period. SDS-PAGE analysis of urinary proteins showed that microalbuminuric SHR virtually excreted proteins of the size of albumin or smaller (< 70kDa), typical of tubular proteinuria. Additionally, it was verified that the protein expression levels of the endocytic receptors megalin and cubilin as well as of the chloride channel ClC-5 progressively decreased in the renal cortex of SHR from 6 to 21 weeks of age. Moreover, it was observed reduction of expression of another macromolecule that plays an important role in the process of receptor mediated endocytosis in the renal proximal tubule, the v-H+- ATPase, was reduced. However, reduced cortical expression of the B2 subunit of the v- H+-ATPase, was only statistically significant in 21-wk-old vs. 6-wk-old SHR. Expression levels of structural components of the glomerular barrier such as nephrin and podocin were unchanged. To sum up, our study demonstrates that the increase in urinary protein excretion, especially of albumin, is associated with lower expression of key components of the apical endocytic apparatus in the renal proximal tubule. It is tempting to speculate that dysfunction of the apical endocytic pathway in the renal proximal tubule may be the major mechanism underlying development of microalbuminuria in essential hypertension

A pulchellina é uma glicoproteína heterodimérica com duas cadeias, pertencente à família das proteínas inativadoras de ribossomos (RIPs) do tipo 2. A cadeia A é enzimaticamente ativa e é capaz de remover uma adenina da porção 28S do rRNA; a cadeia B é uma lectina que se liga a resíduos de D-Galactose terminais, presentes na membrana. Das 4 isoformas da pulchellina (PI, PII, PIII, PIV), PII é a mais tóxica in vivo, sendo a atividade catalítica da cadeia A similar para todas as isoformas. A interação da cadeia B com os glicoreceptores de membrana e seu conseguinte processo de endocitose é crucial para que cadeia A tóxica entre na célula e torne-se disponível para atuar no seu sítio ribossomal. Assim, visando explorar e encontrar potenciais diferenças no mecanismo de ligação à célula e de endocitose das isoformas, foram realizados experimentos usando microscopia confocal com as toxinas marcadas com Alexa flúor&reg; em células HeLa e MV3. As imagens obtidas mostraram que PII localiza-se na região perinuclear das células enquanto PIV predomina na região cortical. Esses resultados sugeriram que as isoformas apresentam distintos mecanismos de entrada e transporte nas células. Para esclarecer tal questão, a ação da pulchellina em células HeLa tratadas com diversas drogas que atuam em diferentes rotas endocíticas e de translocação, foi monitorada. Os resultados de inibição de síntese proteica mostraram que as células sofrem proteção contra a pulchellina na presença de brefeldina A, indicando que a pulchellina necessita ser transportada via Golgi para executar sua função. Inibidores de glicosilação como tunicamicina, swainsonine e inibidores de síntese proteica, como a puromicina e cicloheximidina sensibilizaram as células à PII e PIV, mas em diferentes taxas. Por outro lado, a puromicina e a cicloheximidina não afetaram a taxa de endocitose das isoformas, o que indica que a pulchellina na ausência dos inibidores compete pelo transporte ou processamento de glicoproteínas recém-sintetizadas. Experimentos de ligação e captação da pulchellina mostraram que PII apresenta 30% menos afinidade pela superfície de células HeLa que PIV, além de apresentar menor taxa endocítica. Esses dados corroboram estudos de FCS (espectroscopia de correlação e fluorescência) que identificaram que a difusão de PIV em células HeLa é maior que de PII. Nos experimentos realizados com inibidores de dinamina, ambas isoformas tiveram as suas taxas de endocitose aumentadas, indicando um efeito compensatório para via endocítica independente de dinamina. Em células incubadas com PDMP e neuraminidase, PIV mostrou uma associação às células reduzida, enquanto PII não se alterou, indicando que PIV pode necessitar de esfingolipídeos e glicocomplexos contendo ácido siálico para ligar e se internalizar nas células testadas. Para investigar essa diferença na interação foram realizados ensaios in vitro de DSC (Calorimetria Diferencial de Varredura) e SPR (ressonância plasmônica de superfície) com as isoformas isoladas. Esses ensaios mostraram que PIV e PII apresentam interações distintas com o gangliosídeo GM1, sendo que a PIV interage mais hidrofobicamente e com uma maior taxa de associação com GM1 que a PII. / Pulchellin is a heterodimeric toxin found in Abrus pulchellus seeds. It is a type 2 ribosome inactivating protein, which consists of a toxic A-chain linked to a sugar binding B-chain. The B-chain mediates its binding to the galactose residues on the cellular membrane in a process that is then followed by an endocytic uptake. Once the A-chain reaches the cytosol it inhibits protein synthesis leading to cell death. In order to explore pulchellin isoforms II and IV (PII and PIV) cell entry and transport mechanisms, experiments monitoring toxin labelled with Alexafuor&reg; in MV3 and HeLa cells were performed using confocal microscopy. We have investigated the pulchellin action in pre-treated HeLa cells with several drugs, targeting different endocytic and translocation routes. Confocal images showed PII tends to be localized in cells cortical region and PIV tend to be localized in cell\'s perinuclear region, suggesting that isoforms have different cell entry and transport mechanisms. The protein synthesis inhibition results showed that brefeldin A protects cells against the toxic effect of pulchellin, which indicates the pulchellin needs to be transported to Golgi to perform its toxic effect. When HeLa cells were incubated with protein synthesis inhibitors, such as puromycin and cycloheximidine and glycosilation inhibitors such as tunicamycin, swainsonine, they were sensitized to pulchellin, but to different extent for PII and PIV. Binding and uptake experiments showed that PII exhibits 30% less affinity than PIV on HeLa cells surface, PII also has lower endocytic rate than PIV in the cells. These data corroborate with FCS (Fluorescence Correlation Spectroscopy) results, which identified that PIV diffuses faster than PII into the celIs. Dynamine inhibitors increased endocytosis rates in both isoforms, indicating that pulchellin is upregulating the dynamine-independent endocytosis, possibly pulchellin is being internalized into the cells by alternative endocytic routes. When HeLa cells were incubated with PDMP and neuraminidase, PIV showed a reduced cell association compared with PII and control, indicating that PIV may require glycocomplexes and sphingolipids containing sialic acid to enter into the cells. DSC (Differential Scanning Calorimetry) and SPR (Surface Plasmon Ressonance) experiments using biomimetic membranes were performed using GM1 ganglioside to check this interaction. The results showed PIV and PII interact with GM1. This results also evidence PIV interact more hidrophobically and with a higher association rate on GM1 than PII.

The Notch signalling pathway plays an essential role in cell-fate decisions and morphogenesis, and is frequently ectopically activated in human cancers. The signal is initiated through DSL ligand-dependent Notch proteolysis, which releases its intracellular domain. However, over-expression of the E3 ubiquitin ligase Deltex can bypass the requirement for Notch ligands and ectopically activate Notch by directing it into the endocytic pathway. It has been shown that Deltex induced endocytic-Notch signalling, but not DSL-induced signalling, requires both HOPS and AP-3 complex components that mediate trafficking to lysosomes and related organelles. I showed through a combination of the analysis of the dxsm mutant allele and by expression of mutant forms of Deltex in cell culture, that the C-terminal region of Deltex is important for Notch signalling, but is not required for Notch endocytosis. This suggests that the C-terminal region contains an interaction site that may direct endocytosed Notch to the correct endocytic compartment. A key question however was whether this endocytic pathway is utilised for full Notch signaling in normal development alongside the canonical activation mechanism. I therefore investigated the expression of Drosophila midline single-minded (sim), which is a Notch signal reporter gene, and embryonic neurogenesis which is repressed by Notch signalling. I found that deltex, HOPS and AP-3 mutants displayed gaps in sim expression and also a neurogenic phenotype similar to Notch loss-of-function, and consistent with a role for ligand-independent Notch activation in normal development. I found that the penetrance of these phenotypes increased when flies were cultured at higher temperature. These results suggest that Dx/HOPS/AP-3-dependent Notch activation provides a developmental robustness to the Notch signalling network. It was also found that AP-3 and HOPS components have an additional role in regulating cell survival, which is partially separable in time using a temperature shift assay. I also revealed a requirement of maternal Notch for cell survival in early embryonic development, which may be related to the HOPS-dependent function. Comparison of deltex, AP-3 and HOPS mutant phenotypes suggested that there may be functional redundancy of deltex with components that regulate Notch endocytosis, and of AP-3 with proteins that mediate subsequent trafficking to the late endosome/lysosome. Finally I characterised the molecular lesions of an allele of the HOPS component light and the AP-3 component ruby and identified lesions which were consistent with the loss-of-function of these genes.

Erythropietin (Epo), acting through its receptor (EpoR), is an essential hemotopietic growth factor that regulates the proliferation, differentiation, and survival of erythroid progenitor cells. Perturbations of Epo/EpoR function cause myeloproliferative disease, such as erythrocytosis, or myelodeficient disease, such as anemia. Therefore, defining the mechanisms by which Epo/EpoR control proliferation and differentiation of erythroid cell lineages attracts interest. Ubiquitin-dependent internalization and degradation is a common regulatory mechanisms affecting signaling from a variety of receptors. Although EpoR has been found to be ubiquitinated, the function of EpoR ubiquitination in the regulation of Epo signaling remains unclear. Therefore, the primary goal of this study was to define the role of EpoR ubiquitination in regulating Epo signaling activities and erythroid cell growth. We showed that EpoR was ubiquitinated in response to ligand stimulation, and that loss of EpoR ubiquitination reduced signaling activity and biological responses to low dosages of Epo. We also identified two EpoR lysines that were the primary targets for ubiquitination, and showed that either ubiquitination site supported the enhanced activities of wild-type-EpoR. Ubiquitination of EpoR was also associated with a change in the endocytic pathway mediating internalization of EpoR. Specifically, constitutive internalization of non-ubiquitinated EpoR was found to depend on dynamin activity, while internalization of ubiquitinated EpoR was dynamin-independent but could be inhibited by disrupting lipid raft microdomains in the plasma membrane. Interestingly, inhibiting internalization of ubiquitinated EpoR (by disrupting lipid rafts) specifically reduced signaling from ubiquitinated receptors without affecting signaling from non-ubiquitinated receptors. Conversely, reducing internalization of non-ubiquitinated EpoR (by inhibiting dynamin) reduced its signaling activity without affecting signaling from ubiquitinated receptors. This strong correlation between EpoR internalization and signaling activity suggests a novel regulatory mechanism in which internalization of EpoR facilitates its signaling activity. In this regard, Epo-induced ubiquitination of EpoR promotes more efficient internalization of ligand-activated receptor and may contribute to enhanced responsiveness to low concentrations of Epo.

Fear and anxiety disorders are potentially crippling conditions that often stem from past experience of trauma and chronic stress. One clear feature of these disorders is the failure to use proximate spatial and contextual information presented in the environment to regulate reflexive physiological threat responses. The central nervous system networks that govern spatial navigation and contextual learning and memory are a series of complex circuitries in which the hippocampus is integrally involved. Deficits in hippocampal function have been linked to severe anterograde and mild retrograde amnesia of semantic and episodic memory, and specific deficits in contextual processing. These deficits manifest as failure to distinguish between the details of contexts that help predict for danger or safety and can thus lead to the overexpression of threat responses that compose the behavioral symptoms of fear disorders. The dentate gyrus (DG) is a subdivision of the hippocampus that serves as the first filter of excitatory flow through the hippocampus. The DG is hypothesized to function in “pattern separation” or the dissociation of similar inputs into dissimilar outputs. Failure in this domain leads to generalization between contexts, a common feature of pathology. Pituitary adenylate cyclase activating polypeptide (PACAP) and the PAC1 receptor are associated with multiple behavioral disorders such as post-traumatic stress disorder, schizophrenia, and bipolar disorder. Mutations in the PAC1 receptor gene are associated with hypervigilance, and modified amygdalar and hippocampal activity. These results are mirrored by rodent studies where central PACAP infusion causes anxiety-like behavior, pain hypersensitivity, anorexia, and reinstatement of drug-seeking. PAC1 receptor transcript is found in high abundance in granule cells of the dentate gyrus and potentiation of DG synapses is impaired in PAC1 knockout mice. PACAP is known to have effects of long-duration, such as those in injury repair, growth, and development, but it also can affect ion channel physiology to control neuronal excitability through several parallel intracellular signaling cascades including those dependent on adenylyl cyclase, phospholipase C, and extracellular signal regulated kinase. Accumulated evidence suggests that recruitment of extracellular signal regulated kinase can be through either adenylyl cyclase-, phospholipase C-, or a receptor endocytosis-dependent mechanism. The experiments described in this dissertation address the role of PACAP in the DG in regulating expression of fear behavior, the effects of PACAP on the excitability of DG granule cells, and the signaling pathways and ion channels responsible for these effects. We found that PACAP infused into the DG amplifies expression of fear to a context but does not affect fear acquisition. Electrophysiology studies demonstrate that treating DG neurons with PACAP increases their excitability, and that parallel signaling mechanisms recruit extracellular signal regulated kinase to drive this excitability. Furthermore, these effects on excitability are attenuated by blocking a persistent inward sodium current. This work represents novel regulation of the DG and its impacts on behavior and identifies a current that likely participates in modulating granule cell excitability in multiple domains. In aggregate, this research traces the path from ligand, to receptor and intracellular signaling, to neurophysiology in order to propose a comprehensive description of behavioral regulation by these processes.

Mutations in the CLN3 gene cause Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), a form of Batten disease that is grouped within the broad class of lysosomal storage diseases. JNCL displays a primary central nervous system phenotype characterized by rapid onset blindness, wide spread brain atrophy and reversal of learned abilities with death occurring 10-20 years after symptom onset. The mechanisms underlying these phenotypes are not known. CLN3 encodes CLN3, a protein with no known molecular function. CLN3 is expressed at very low levels natively in most cells, and is highly hydrophobic. Similar to other lysosomal storage diseases, it is difficult to ascertain the primary versus the secondary defects when the protein functions along the endosomal-lysosomal pathway. In JNCL one common finding among several labs, in various cellular systems, is a fluid-phase endocytotic defect. I took this commonality as a key to CLN3 function, and pursued cell biological pathways required for fluid-phase endocytosis. Fluid-phase endocytosis is regulated by cycling of the small GTPase Cdc42 and I discovered increased Cdc42-GTP in CLN3-null mouse brain endothelial cells. In mouse brain endothelial cells enhanced Cdc42-GTP increased Cdc42 dependent signaling, filopodial formation, and retarded cell migration. I also found reduced plasma membrane association of ARHGAP21, a known negative regulator of Cdc42. My data supports a model where loss of CLN3 reduces ARHGAP21 plasma membrane recruitment, and causes aberrant Cdc42 activation. Thus irregular Cdc42 activation underlies the commonly reported fluid-phase endocytic defects in JNCL. Therapeutic development for JNCL has been hampered in part from the varying phenotypes ascribed to CLN3 deficiency. My discovery that the fluid-phase endocytic defects result from Cdc42 pathway aberrations, which in turn contributed to multiple downstream phenotypes, opened the door to novel JNCL therapeutics. Here I present work showing that a Cdc42 inhibitor corrects the Cdc42 dependent defects in vitro and multiple defects in a JNCL mouse model.

Neurotrophins are a family of growth factors necessary for the development and maintenance of the nervous system. They produce their effects through receptor mediated signaling mechanisms that are highly regulated by sophisticated intracellular transport networks. The impairment of intracellular trafficking of neurotrophins in motor neurons has been identified as one possible factor in the development of motor neuron diseases, but remains inadequately studied. Aided by advances in imaging technology and the development of more powerful and sensitive detection tools for in-vitro studies, the dynamics of intracellular transport of neurotrophins are beginning to be unraveled. However, a primary limiting factor in the study of neurotrophin-transport dynamics in motor neurons has been the lack of alternative and easily available in-vitro systems able to substitute the often difficult and costly primary motor neuron cultures. The aim of this project was to develop a suitable motor neuron model using the NSC-34 cell line for the study of receptor mediated trafficking events through endosomal transport pathways. Successful evaluation and characterization of NSC-34 cells for motor neuron specific markers would result in the investigation of the p75 neurotrophin receptor (p75NTR) trafficking pathways in the presence of exogenous neurotrophins, with a variety of confocal imaging techniques. Chapter 3 describes the optimisation of NSC-34 cell culture conditions through media modification and the development of a suitable growth substrate matrix, which significantly improved cell adhesion, differentiation and the ability to culture the cells for extended time periods in serum free conditions. Quantitative measurements of cell proliferation, culture viability, cell-body size and neurite length are described to highlight the increased value of the cell line for long-term culture and experiments examining a broad range of issues relevant to motor neurons. In Chapter 4, multiple experimental approaches were used to extensively screen the NSC-34 cell line for the presence of motor neuron-specific markers, neurotrophin receptors and proteins involved in regulation of endosomal transport. This characterization established the presence of a developing motor neuron-like neurotrophin receptor profile (p75NTR, TrkB and TrkC), a genetic marker of developing motor neurons, cholinergic markers, proteins regulating transport within the endosomal pathway, and additional proteins previously shown to directly interact with neurotrophin receptors, including sortilin, and the lipid raft associated ganglioside GT1b. Furthermore, evidence is provided that NSC-34 cells undergo apoptosis in response to exogenous nerve growth factor (NGF) or neurotrophin-3 (NT-3), but not brain derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4). In addition characterization of mouse specific p75NTR antibodies is presented to establish their suitability for internalization studies without altering the binding of exogenous neurotrophins to the receptor. Subsequent confocal microscopy examination focusing on p75NTR trafficking in Chapter 5 revealed that internalization and intracellular transport of this receptor is regulated by exogenous neurotrophins at the cell surface where ligand binding and internalization occur, and in endosomal compartments where the bulk of receptors and ligands are targeted to their specific destinations. Evidence is provided showing that p75NTR internalization is altered in the presence of NGF, NT-3, or NT-4, but not BDNF, and the receptor is diverted into non-clathrin mediated endosomal pathways in response to NGF but not BDNF. Immunofluorescence confocal microscopy suggests that p75NTR recycles to the plasma membrane in a Rab4 GTPase dependent manner in the absence of neurotrophins. Addition of neurotrophins diverted p75NTR from the recycling Rab4 positive pathway, into EEA-1 positive sorting endosomes in the presence of NGF or NT-3, or lysosomal degradation in the presence of BDNF or NT-4. This study clearly demonstrates the suitability of the NSC-34 cell line as an alternate in-vitro system for the study of motor neuron biology, particularly the study of neurotrophin receptor trafficking. Taken together the results represented in this study suggest for the first time, that the fate of the p75NTR receptor depends on which neurotrophin is bound. These findings have important implications for understanding the dynamic mechanisms of action of p75NTR in normal neuronal function, and may also offer further insight into the potential role of neurotrophins in the treatment of neurodegenerative diseases.