Diagnóstico laboratorial em cães e gatos

O relatório de estágio curricular do Mestrado Integrado em Medicina Veterinária, aqui apresentado, encontra-se dividido em quatro partes distintas. A primeira corresponde à descrição do local de estágio, a segunda engloba a descrição das atividades desenvolvidas com a compilação da respetiva casuística e a terceira consiste numa revisão de literatura sobre as principais áreas laboratoriais acompanhadas: Bioquímica, Hematologia e Urianálise. Na quarta e última parte é abordado um caso clínico de Leishmaniose Canina, acompanhado durante o período de estágio. Este foi realizado no Laboratório Inno, Serviços Especializados em Veterinária, Lda., localizado em Braga, Portugal e abrangeu a área de Diagnóstico Laboratorial Veterinário. A escolha do tema prendeu-se com o grande interesse pela área da Patologia Clínica, associado à sua enorme importância em Medicina Veterinária. No decorrer do estágio, foram solicitadas 29967 análises: 19437 bioquímicas, 2964 hematológicas e 1031 urianálises. Neste relatório pretende-se destacar a importância destas áreas na formulação do diagnóstico médico-veterinário; Laboratory diagnosis in dogs and cats Abstract: This report of my curricular training integrated in the Veterinary Medicine Master Degree of Évora University is divided in three distinctive sections. The first one covers the description of the place where I develop my work - INNO, a specialized Veterinary Laboratory focused mainly on small animal diagnosis, placed in Braga, Portugal; the second one comprise the casuistic and the third is a systematic review of the main laboratory areas of actuation – Biochemistry, Hematology and Urinalysis. The fourth section describes one case of Canine Leishmaniosis accompanied during the period of training. In this period the lab received 29967 analysis, including the following specific exams by areas: biochemistry (n=19437), hematology (n=2964) and urinalysis (n=1031). The purpose of this report is enunciate and evolve about the main and crucial areas of medical diagnostic in small animal practice.

Silencing the gip gene of Phytophthora cinnamomi by iRNA and studying the subcellular localization of GIP and NPP1 proteins

Ink Disease is considered one of the most important causes of the decline of chestnut orchards. The break in yield of Castanea sativa Mill is caused by two species: Phytophthora cinnamomi and Phytophthora cambivora, being the first one the foremost pathogen of ink disease in Portugal. P. cinnamomi is one of the most aggressive and widespread plant pathogen with nearly 1,000 host species. This oomycete causes enormous economic losses and it is responsible for the decline of many plant species in Europe and worldwide. Up to now no efficient treatments are available to fight these pathogens. Because of the importance of chestnut at economical and ecological levels, especially in Portugal, it becomes essential to explore the molecular mechanisms that determine the interaction between Phytophthora species and host plants through the study of proteins GIP (glucanase inhibitor protein) and NPP1 (necrosis-inducing Phytophthora protein 1) produced by P. cinnamomi during the infection. The technique of RNA interference was used to knockdown the gip gene of P. cinnamomi. Transformants obtained with the silenced gene have been used to infect C. sativa, in order to determine the effect of gene silencing on the plant phenotype. To know more about the function of GIP and NPP1 involved in the mechanism of infection, the ORF’s of gip and npp1 genes have been cloned to the pTOR-eGFP vector for a future observation of P. cinnamomi transformants with fluorescent microscopy and determination of the subcellular localization. Moreover the prediction by bioinformatics tools indicates that both GIP and NPP1 proteins are secreted. The results allow to predict the secretory destination of both GIP and NPP1 proteins and confirm RNAi as a potential alternative biological tool in the control and management of P. cinnamomi. Keywords:

Distinct immune responses of juvenile and adult oysters (Crassostrea gigas) to viral and bacterial infections

Since 2008, massive mortality events of Pacific oysters (Crassostrea gigas) have been reported worldwide and these disease events are often associated with Ostreid herpesvirus type 1 (OsHV-1). Epidemiological field studies have also reported oyster age and other pathogens of the Vibrio genus are contributing factors to this syndrome. We undertook a controlled laboratory experiment to simultaneously investigate survival and immunological response of juvenile and adult C. gigas at different time-points post-infection with OsHV-1, Vibrio tasmaniensis LGP32 and V. aestuarianus. Our data corroborates epidemiological studies that juveniles are more susceptible to OsHV-1, whereas adults are more susceptible to Vibrio. We measured the expression of 102 immune-genes by high-throughput RT-qPCR, which revealed oysters have different transcriptional responses to OsHV-1 and Vibrio. The transcriptional response in the early stages of OsHV-1 infection involved genes related to apoptosis and the interferon-pathway. Transcriptional response to Vibrio infection involved antimicrobial peptides, heat shock proteins and galectins. Interestingly, oysters in the later stages of OsHV-1 infection had a transcriptional response that resembled an antibacterial response, which is suggestive of the oyster's microbiome causing secondary infections (dysbiosis-driven pathology). This study provides molecular evidence that oysters can mount distinct immune response to viral and bacterial pathogens and these responses differ depending on the age of the host.

Structural determination and gynecological tumor diagnosis using antibody chip captured proteins

Purpose: To identify markers for gynecological tumor diagnosis using antibody chip capture. Methods: Marker proteins, including cancer antigen 153 (CA153), CA125, and carcinoembryonic antigen (CEA), were analyzed using antibody chip capture of serum samples. Fifteen agglutinin types that specifically recognized five common glycans (fucose, sialic acid, mannose, N - acetylgalactosamine, and N-acetylglucosamine) were used to detect marker protein glycan levels. The levels of CA153, CA125, and CEA from 49 healthy control samples, 31 breast cancer samples, 24 cervical cancer samples, and 19 ovarian cancer samples were used to measure the glycan levels of these marker proteins. Results: In breast cancer samples, CA153 and CA125 were down-regulated (p < 0.01), while differences in ovarian cancer samples were not statistically significant (p > 0.01). The total accuracy was 85.1 %, with 96.8 % accuracy for breast cancer, 75 % in cervical cancer, and 78.9 % in ovarian cancer. Cross-validation analyses showed that breast cancer had 93.5 % accuracy, cervical cancer was 66.7 %, and ovarian cancer was 68.4 %, leading to 78.4 % total accuracy (58/74). Conclusions: The results indicate that better clinical diagnosis of gynecological tumors can be obtained by monitoring changes in glycan levels of serum proteins and types of proteoglycan changes.

Redox regulation of Ras proteins in Dictyostelium discoideum

Reactive oxygen species are a normal consequence of life in an aerobic environment. However when they deviate from the narrow permissible range in cells, oxidative damage can occur. Dictyostelium discoideum is a model organism ideal for the study of cell signaling events such as those affected by oxidative stress. It was previously shown that Ras signaling in Dictyostelium is affected by genetic inactivation of the antioxidant enzyme Superoxide dismutase C (SodC) and in vitro data suggests that the NKCD motif of Ras is the redox target of superoxide.^ The main objective of this project was to determine the mechanism of superoxide mediated Ras regulation in vivo. To accomplish the main objective, we cloned, and in some cases, mutated different Ras proteins and later determined their activity in wild type and sodC- cells. RasC and RasD showed normal activation in sodC- cells, however RasG and RasS displayed high Ras activity. These last two Ras proteins contain the NKC118D motif inside the nucleotide binding region. A mutation of cysteine 118 to alanine in RasG rendered the protein less active in sodC- than the wild type RasG protein and a mutation alanine118 to cysteine in RasD conferred redox sensitivity to this small GTPase. Additionally, the propensity of RasG to be targeted by superoxide was evident when the environment of wild type cells was manipulated to induce the internal generation of superoxide through changes in the extracellular ion levels mainly magnesium. Lack of magnesium ions increased the intracellular level of superoxide and severely hampered directional cell migration. Chemotaxis of cells expressing RasG was negatively impacted by the absence of magnesium ions; however rasG- cells did not seem to be affected in their ability to perform chemotaxis. The last experiment implies that RasG is an important mediator of cell signaling during oxidative stress, responsible for preventing cells from continuing their developmental program. Our study suggests that the cysteine residue in the NKCD motif is essential for mediating the redox sensitivity of Ras proteins in Dictyostelium and that RasG is an essential mediator of the response to oxidative stress in this organism.^

Transthyretin Proteins Regulate Angiogenesis by Conferring Different Molecular Identities to Endothelial Cells

Familial amyloidotic polyneuropathy (FAP) has a high prevalence in Portugal, and the most common form of hereditary amyloidosis is caused by an amyloidogenic variant of transthyretin (TTR) with a substitution of methionine for valine at position 30 (V30M). Until now, the available efficient therapy is liver transplantation, when performed in an early phase of the onset of the disease symptoms. However, transplanted FAP patients have a significantly higher incidence of early hepatic artery thrombosis compared with non-FAP transplanted patients. Because FAP was described as an independent risk factor for early hepatic artery thrombosis, more studies to understand the underlying mechanisms involved in this outcome are of the utmost importance. Knowing that the liver is the major site for TTR production, we investigated the biological effects of TTR proteins in the vasculature and on angiogenesis. In this study, we identified genes differentially expressed in endothelial cells exposed to the WT or V30M tetramer. We found that endothelial cells may acquire different molecular identities when exposed to these proteins, and consequently TTR could regulate angiogenesis. Moreover, we show that V30M decreases endothelial survival by inducing apoptosis, and it inhibits migration. These findings provide new knowledge that may have critical implications in the prevention of early hepatic artery thrombosis in FAP patients after liver transplantation.

Protein cutoff scanning: a comparative analysis of cutoff dependent and cutoff free methods for prospecting contacts in proteins.

In this study, we carried out a comparative analysis between two classical methodologies to prospect residue contacts in proteins: the traditional cutoff dependent (CD) approach and cutoff free Delaunay tessellation (DT). In addition, two alternative coarse-grained forms to represent residues were tested: using alpha carbon (CA) and side chain geometric center (GC). A database was built, comprising three top classes: all alpha, all beta, and alpha/beta. We found that the cutoff value? at about 7.0 A emerges as an important distance parameter.? Up to 7.0 A, CD and DT properties are unified, which implies that at this distance all contacts are complete and legitimate (not occluded). We also have shown that DT has an intrinsic missing edges problem when mapping the first layer of neighbors. In proteins, it may produce systematic errors affecting mainly the contact network in beta chains with CA. The almost-Delaunay (AD) approach has been proposed to solve this DT problem. We found that even AD may not be an advantageous solution. As a consequence, in the strict range up ? to 7.0 A, the CD approach revealed to be a simpler, more complete, and reliable technique than DT or AD. Finally, we have shown that coarse-grained residue representations may introduce bias in the analysis of neighbors in cutoffs up to ? 6.8 A, with CA favoring alpha proteins and GC favoring beta proteins. This provides an additional argument pointing to ? the value of 7.0 A as an important lower bound cutoff to be used in contact analysis of proteins.

Sequential exhaustive extraction of a Mollisol soil, and characterizations of humic components, including humin, by solid and solution state NMR.

A comprehensive sequential extraction procedure was applied to isolate soil organic components using aqueous solvents at different pH values, base plus urea (base-urea), and finally dimethylsulfoxide (DMSO) plus concentrated H2SO4 (DMSO-acid) for the humin-enriched clay separates. The extracts from base-urea and DMSO-acid would be regarded as 'humin' in the classical definitions. The fractions isolated from aqueous base, base-urea and DMSO-acid were characterized by solid and solution state NMR spectroscopy. The base-urea solvent system isolated ca. 10% (by mass) additional humic substances. The combined base-urea and DMSO-acid solvents isolated ca. 93% of total organic carbon from the humin-enriched fine clay fraction (<2 ?m). Characterization of the humic fractions by solid-state NMR spectroscopy showed that oxidized char materials were concentrated in humic acids isolated at pH 7, and in the base-urea extract. Lignin-derived materials were in considerable abundance in the humic acids isolated at pH 12.6. Only very small amounts of char-derived structures were contained in the fulvic acids and fulvic acids-like material isolated from the base-urea solvent. After extraction with base-urea, the 0.5 m NaOH extract from the humin-enriched clay was predominantly composed of aliphatic hydrocarbon groups, and with lesser amounts of aromatic carbon (probably including some char material), and carbohydrates and peptides. From the combination of solid and solution-state NMR spectroscopy, it is clear that the major components of humin materials, from the DMSO-acid solvent, after the exhaustive extraction sequence, were composed of microbial and plant derived components, mainly long-chain aliphatic species (including fatty acids/ester, waxes, lipids and cuticular material), carbohydrate, peptides/proteins, lignin derivatives, lipoprotein and peptidoglycan (major structural components in bacteria cell walls). Black carbon or char materials were enriched in humic acids isolated at pH 7 and humic acids-like material isolated in the base-urea medium, indicating that urea can liberate char-derived material hydrogen bonded or trapped within the humin matrix.

Occupant health and productivity : an Australian perspective

The issue of whether improved building services such as air quality, provision of daylight, thermal comfort etc, have a positive impact on the health and productivity of building occupants is still an open question. There is significant anecdotal evidence supporting the notion that health and productivity of building occupants can be improved by improving the quality of the indoor environment, but there are actually few published quantitative studies to substantiate this contention. This paper reports on a comprehensive review of the worldwide literature which relates health of building occupants with the different aspects of the indoor environment which are believed to impact of these issues, with a particular focus on studies in Australia, The paper analyses the existing research and identifies the key deficiencies in our existing understanding of this problem. The key focus of this research is office and school buildings, but the scope of the literature surveyed includes all commercial buildings, including industrial buildings. There is a notable absence of detailed studies on this link in Australian buildings, although there are studies on thermal comfort, and a number of studies on indoor air quality in Australia, which do not make the connection to health and productivity. Many international studies have focused on improved lighting, and in particular the provision of daylight in buildings, but again there are few studies in Australia which focus in this area.

Passive solar systems for heating, daylighting and ventilation for rooms without an equator-facing facade

Spaces without northerly orientations have an impact on the ‘energy behaviour’ of a building. This paper outlines possible energy savings and better performance achieved by different zenithal solar passive strategies (skylights, roof monitors and clerestory roof windows) and element arrangements across the roof in zones of cold to temperate climates typical of the central and central-southern Argentina. Analyses were undertaken considering daylighting, thermal and ventilation performances of the different strategies. The results indicate that heating,ventilation and lighting loads in spaces without an equator-facing facade can be significantly reduced by implementing solar passive strategies. In the thermal aspect, the solar saving fraction reached for the different strategies were averaged 43.16% for clerestories, 41.4% for roof monitors and 38.86% for skylights for a glass area of 9% to the floor area. The results also indicate average illuminance levels above 500 lux for the different clerestory and monitor arrangements, uniformity ratios of 0.66–0.82 for the most distributed arrangements and day-lighting factors between 11.78 and 20.30% for clear sky conditions, depending on the strategy. In addition, minimum air changes rates of 4 were reached for the most extreme conditions.

In vitro cultivation of adult human chondrocytes : importance of culture system, oxygen and zonal differences

Articular cartilage exhibits limited intrinsic regenerative capacity and focal tissue defects can lead to the development of osteoarthritis (OA), a painful and debilitating loss of cartilage tissue. In Australia, 1.4 million people are affected by OA and its prevalence is increasing in line with current demographics. As treatment options are limited, new therapeutic approaches are being investigated including biological resurfacing of joints with tissue-engineered cartilage. Despite some progress in the field, major challenges remain to be addressed for large scale clinical success. For example, large numbers of chondrogenic cells are required for cartilage formation, but chondrocytes lose their chondrogenic phenotype (dedifferentiate) during in vitro propagation. Additionally, the zonal organization of articular cartilage is critical for normal cartilage function, but development of zonal structure has been largely neglected in cartilage repair strategies. Therefore, we hypothesised that culture conditions for freshly isolated human articular chondrocytes from non-OA and OA sources can be improved by employing microcarrier cultures and a reduced oxygen environment and that oxygen is a critical factor in the maintenance of the zonal chondrocyte phenotype. Microcarriers have successfully been used to cultivate bovine chondrocytes, and offer a potential alternative for clinical expansion of human chondrocytes. We hypothesised that improved yields can be achieved by propagating human chondrocytes on microcarriers. We found that cells on microcarriers acquired a flattened, polygonal morphology and initially proliferated faster than monolayercultivated cells. However, microcarrier cultivation over four weeks did not improve growth rates or the chondrogenic potential of non-OA and OA human articular chondrocytes over conventional monolayer cultivation. Based on these observations, we aimed to optimise culture conditions by modifying oxygen tension, to more closely reflect the in vivo environment. We found that propagation at 5% oxygen tension (moderate hypoxia) did not improve proliferation or redifferentiation capacity of human osteoarthritic chondrocytes. Moderate hypoxia increased the expression of chondrogenic markers during redifferentiation. However, osteoarthritic chondrocytes cultivated on microcarriers exhibited lower expression levels of chondrogenic surface marker proteins and had at best equivalent redifferentiation capacities compared to monolayer-cultured cells. This suggests that monolayer culture with multiple passaging potentially selects for a subpopulation of cells with higher differentiation capacity, which are otherwise rare in osteoarthritic, aged cartilage. However, fibroblastic proteins were found to be highly expressed in all cultures of human osteoarthritic chondrocytes indicating the presence of a high proportion of dedifferentiated, senescent cells with a chondrocytic phenotype that was not rescued by moderate hypoxia. The different zones of cartilage support chondrocyte subpopulations, which exhibit characteristic protein expression and experience varying oxygen tensions. We, therefore, hypothesised that oxygen tension affects the zonal marker expression of human articular chondrocytes isolated from the different cartilage layers. We found that zonal chondrocytes maintained these phenotypic differences during in vitro cultivation. Low oxygen environments favoured the expression of the zonal marker proteoglycan 4 in superficial cells, most likely through the promotion of chondrogenesis. The putative zonal markers clusterin and cartilage intermediate layer protein were found to be expressed by all subpopulations of human osteoarthritic chondrocytes ex vivo and, thus, may not be reliable predictors of in vitro stratification using these clinically relevant cells. The findings in this thesis underline the importance of considering low oxygen conditions and zonal stratification when creating native-like cartilaginous constructs. We have not yet found the right cues to successfully cultivate clinically-relevant human osteoarthritic chondrocytes in vitro. A more thorough understanding of chondrocyte biology and the processes of chondrogenesis are required to ensure the clinical success of cartilage tissue engineering.

Predictions of road traffic noise on residential balconies using a specular and diffusion model

A combined specular reflection and diffusion model using the radiosity technique was developed to calculate road traffic noise level on residential balconies. The model is capable of numerous geometrical configurations for a single balcony situated in the centre of a street canyon. The geometry of the balcony and the street can be altered with width,length and height. The model was used to calculate for three different geometrical and acoustic absorption characteristics for a balcony. The calculated results are presented in this paper.

Whitefly Transmission and Efficient ssDNA Accumulation of Bean Golden Mosaic Geminivirus Require Functional Coat Protein

Bean golden mosaic geminivirus (BGMV) has a bipartite genome composed of two circular ssDNA components (DNA-A and DNA-B) and is transmitted by the whitefly, Bemisia tabaci. DNA-A encodes the viral replication proteins and the coat protein. To determine the role of BGMV coat protein systemic infection and whitefly transmission, two deletions and a restriction fragment inversion were introduced into the BGMV coat protein gene. All three coat protein mutants produced systemic infections when coinoculated with DNA-B onto Phaseolus vulgaris using electric discharge particle acceleration "particle gun." However, they were not sap transmissible and coat protein was not detected in mutant-infected plants. In addition, none of the mutants were transmitted by whiteflies. With all three mutants, ssDNA accumulation of DNA-A and DNA-B was reduced 25- to 50-fold and 3- to 10-fold, respectively, as compared to that of wild-type DNA. No effect on dsDNA-A accumulation was detected and there was 2- to 5-fold increase in dsDNA-B accumulation. Recombinants between the mutated DNA-A and DNA-B forms were identified when the inoculated coat protein mutant was linearized in the common region.

In vitro and in vivo examination of the cell surface glycoprotein CDCP1

A number of reports have demonstrated the importance of the CUB domaincontaining protein 1 (CDCP1) in facilitating cancer progression in animal models and the potential of this protein as a prognostic marker in several malignancies. CDCP1 facilitates metastasis formation in animal models by negatively regulating anoikis, a type of apoptosis triggered by the loss of attachment signalling from cell-cell contacts or cell-extra cellular matrix (ECM) contacts. Due to the important role CDCP1 plays in cancer progression in model systems, it is considered a potential drug target to prevent the metastatic spread of cancers. CDCP1 is a highly glycosylated 836 amino acid cell surface protein. It has structural features potentially facilitating protein-protein interactions including 14 N-glycosylation sites, three CUB-like domains, 20 cysteine residues likely to be involved in disulfide bond formation and five intracellular tyrosine residues. CDCP1 interacts with a variety of proteins including Src family kinases (SFKs) and protein kinase C ä (PKCä). Efforts to understand the mechanisms regulating these interactions have largely focussed on three CDCP1 tyrosine residues Y734, Y743 and Y762. CDCP1-Y734 is the site where SFKs phosphorylate and bind to CDCP1 and mediate subsequent phosphorylation of CDCP1-Y743 and -Y762 which leads to binding of PKCä at CDCP1-Y762. The resulting trimeric protein complex of SFK•CDCP1•PKCä has been proposed to mediate an anti-apoptotic cell phenotype in vitro, and to promote metastasis in vivo. The effect of mutation of the three tyrosines on interactions of CDCP1 with SFKs and PKCä and the consequences on cell phenotype in vitro and in vivo have not been examined. CDCP1 has a predicted molecular weight of ~90 kDa but is usually detected as a protein which migrates at ~135 kDa by Western blot analysis due to its high degree of glycosylation. A low molecular weight form of CDCP1 (LMWCDCP1) of ~70 kDa has been found in a variety of cancer cell lines. The mechanisms leading to the generation of LMW-CDCP1 in vivo are not well understood but an involvement of proteases in this process has been proposed. Serine proteases including plasmin and trypsin are able to proteolytically process CDCP1. In addition, the recombinant protease domain of the serine protease matriptase is also able to cleave the recombinant extracellular portion of CDCP1. Whether matriptase is able to proteolytically process CDCP1 on the cell surface has not been examined. Importantly, proteolytic processing of CDCP1 by trypsin leads to phosphorylation of its cell surface-retained portion which suggests that this event leads to initiation of an intracellular signalling cascade. This project aimed to further examine the biology of CDCP1 with a main of focus on exploring the roles played by CDCP1 tyrosine residues. To achieve this HeLa cells stably expressing CDCP1 or the CDCP1 tyrosine mutants Y734F, Y743F and Y762F were generated. These cell lines were used to examine: • The roles of the tyrosine residues Y734, Y743 and Y762 in mediating interactions of CDCP1 with binding proteins and to examine the effect of the stable expression on HeLa cell morphology. • The ability of the serine protease matriptase to proteolytically process cell surface CDCP1 and to examine the consequences of this event on HeLa cell phenotype and cell signalling in vitro. • The importance of these residues in processes associated with cancer progression in vitro including adhesion, proliferation and migration. • The role of these residues on metastatic phenotype in vivo and the ability of a function-blocking anti-CDCP1 antibody to inhibit metastasis in the chicken embryo chorioallantoic membrane (CAM) assay. Interestingly, biochemical experiments carried out in this study revealed that mutation of certain CDCP1 tyrosine residues impacts on interactions of this protein with binding proteins. For example, binding of SFKs as well as PKCä to CDCP1 was markedly decreased in HeLa-CDCP1-Y734F cells, and binding of PKCä was also reduced in HeLa-CDCP1-Y762F cells. In contrast, HeLa-CDCP1-Y743F cells did not display altered interactions with CDCP1 binding proteins. Importantly, observed differences in interactions of CDCP1 with binding partners impacted on basal phosphorylation of CDCP1. It was found that HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F displayed strong basal levels of CDCP1 phosphorylation. In contrast, HeLa-CDCP1-Y734F cells did not display CDCP1 phosphorylation but exhibited constitutive phosphorylation of focal adhesion kinase (FAK) at tyrosine 861. Significantly, subsequent investigations to examine this observation suggested that CDCP1-Y734 and FAK-Y861 are competitive substrates for SFK-mediated phosphorylation. It appeared that SFK-mediated phosphorylation of CDCP1- Y734 and FAK-Y861 is an equilibrium which shifts depending on the level of CDCP1 expression in HeLa cells. This suggests that the level of CDCP1 expression may act as a regulatory mechanism allowing cells to switch from a FAK-Y861 mediated pathway to a CDCP1-Y734 mediated pathway. This is the first time that a link between SFKs, CDCP1 and FAK has been demonstrated. One of the most interesting observations from this work was that CDCP1 altered HeLa cell morphology causing an elongated and fibroblastic-like appearance. Importantly, this morphological change depended on CDCP1- Y734. In addition, it was observed that this change in cell morphology was accompanied by increased phosphorylation of SFK-Y416. This suggests that interactions of SFKs with CDCP1-Y734 increases SFK activity since SFKY416 is critical in regulating kinase activity of these proteins. The essential role of SFKs in mediating CDCP1-induced HeLa cell morphological changes was demonstrated using the SFK-selective inhibitor SU6656. This inhibitor caused reversion of HeLa-CDCP1 cell morphology to an epithelial appearance characteristic of HeLa-vector cells. Significantly, in vitro studies revealed that certain CDCP1-mediated cell phenotypes are mediated by cellular pathways dependent on CDCP1 tyrosine residues whereas others are independent of these sites. For example, CDCP1 expression caused a marked increase in HeLa cell motility that was independent of CDCP1 tyrosine residues. In contrast, CDCP1- induced decrease in HeLa cell proliferation was most prominent in HeLa- CDCP1-Y762F cells, potentially indicating a role for this site in regulating proliferation in HeLa cells. Another cellular event which was identified to require phosphorylation of a particular CDCP1 tyrosine residue is adhesion to fibronectin. It was observed that the CDCP1-mediated strong decrease in adhesion to fibronectin is mostly restored in HeLa-CDCP1-Y743F cells. This suggests a possible role for CDCP1-Y743 in causing a CDCP1-mediated decrease in adhesion. Data from in vivo experiments indicated that HeLa-CDCP1-Y734F cells are more metastic than HeLa-CDCP1 cells in vivo. This indicates that interaction of CDCP1 with SFKs and PKCä may not be required for CDCP1-mediated metastasis formation of HeLa cells in vivo. The metastatic phenotype of these cells may be caused by signalling involving FAK since HeLa-CDCP1- Y734F cells are the only CDCP1 expressing cells displaying constitutive phosphorylation of FAK-Y861. HeLa-CDCP1-Y762F cells displayed a very low metastatic ability which suggests that this CDCP1 tyrosine residue is important in mediating a pro-metastatic phenotype in HeLa cells. More detailed exploration of cellular events occurring downstream of CDCP1-Y734 and -Y762 may provide important insights into the mechanisms altering the metastatic ability of CDCP1 expressing HeLa cells. Complementing the in vivo studies, anti-CDCP1 antibodies were employed to assess whether these antibodies are able to inhibit metastasis of CDCP1 and CDCP1 tyrosine mutants expressing HeLa cells. It was found that HeLa- CDCP1-Y734F cells were the only cell line which was markedly reduced in the ability to metastasise. In contrast, the ability of HeLa-CDCP1, HeLa- CDCP1-Y743F and -Y762F cells to metastasise in vivo was not inhibited. These data suggest a possible role of interactions of CDCP1 with SFKs, occurring at CDCP1-Y734, in preventing an anti-metastatic effect of anti- CDCP1 antibodies in vivo. The proposal that SFKs may play a role in regulating anti-metastatic effects of anti-CDCP1 antibodies was supported by another experiment where differences between HeLa-CDCP1 cells and CDCP1 expressing HeLa cells (HeLa-CDCP1-S) from collaborators at the Scripps Research Institute were examined. It was found that HeLa-CDCP1-S cells express different SFKs than CDCP1 expressing HeLa cells generated for this study. This is important since HeLa-CDCP1-S cells can be inhibited in their metastatic ability using anti-CDCP1 antibodies in vivo. Importantly, these data suggest that further examinations of the roles of SFKs in facilitating anti-metastatic effects of anti-CDCP1 antibodies may give insights into how CDCP1 can be blocked to prevent metastasis in vivo. This project also explored the ability of the serine protease matriptase to proteolytically process cell surface localised CDCP1 because it is unknown whether matriptase can cleave cell surface CDCP1 as it has been reported for other proteases such as trypsin and plasmin. Furthermore, the consequences of matriptase-mediated proteolysis on cell phenotype in vitro and cell signalling were examined since recent reports suggested that proteolysis of CDCP1 leads to its phosphorylation and may initiate cell signalling and consequently alter cell phenotype. It was found that matriptase is able to proteolytically process cell surface CDCP1 at low nanomolar concentrations which suggests that cleavage of CDCP1 by matriptase may facilitate the generation of LWM-CDCP1 in vivo. To examine whether matriptase-mediated proteolysis induced cell signalling anti-phospho Erk 1/2 Western blot analysis was performed as this pathway has previously been examined to study signalling in response to proteolytic processing of cell surface proteins. It was found that matriptase-mediated proteolysis in CDCP1 expressing HeLa cells initiated intracellular signalling via Erk 1/2. Interestingly, this increase in phosphorylation of Erk 1/2 was also observed in HeLa-vector cells. This suggested that initiation of cell signalling via Erk 1/2 phosphorylation as a result of matriptase-mediated proteolysis occurs by pathways independent of CDCP1. Subsequent investigations measuring the flux of free calcium ions and by using a protease-activated receptor 2 (PAR2) agonist peptide confirmed this hypothesis. These data suggested that matriptase-mediated proteolysis results in cell signalling via a pathway induced by the activation of PAR2 rather than by CDCP1. This indicates that induction of cell signalling in HeLa cells as a consequence of matriptase-mediated proteolysis occurs via signalling pathways which do not involve phosphorylation of Erk 1/2. Consequently, it appears that future attempts should focus on the examination of cellular pathways other than Erk 1/2 to elucidate cell signalling initiated by matriptase-mediated proteolytic processing of CDCP1. The data presented in this thesis has explored in vitro and in vivo aspects of the biology of CDCP1. The observations summarised above will permit the design of future studies to more precisely determine the role of CDCP1 and its binding partners in processes relevant to cancer progression. This may contribute to further defining CDCP1 as a target for cancer treatment.