Pancreatic cancer is a devastating disease that leaves patients with a very poor prognosis and few therapeutic options. Many of the treatment options available are the same that have been used for almost 2 decades. There is a dire need for both novel treatments for this disease as well as novel strategies of treatment. This body of work will introduce and provide evidence in support of a novel combination therapy for pancreatic cancer treatment, a novel strategy of modifying currently used chemotherapeutics for pancreatic cancer therapy, and a novel transgenic preclinical mouse model of pancreatic cancer. Sabutoclax, an antagonist of the anti-apoptotic Bcl-2 proteins, and Minocycline, a commonly used antibiotic, show potent synergy when used in combination in both pancreatic cancer cells and in multiple immune-deficient and immune-competent mouse models of pancreatic cancer. Sabutoclax alone is capable of inducing cell cycle arrest and apoptosis in cells and its cytotoxicity is enhanced significantly when combined with Minocycline. This combination results in the loss of Stat3 activation both in vitro and in vivo, which is essential for its toxicity. It also inhibits tumor growth and prolongs survival in the KPC transgenic mouse model of pancreatic cancer. Also presented here are studies that demonstrate efficacy in vivo of modified versions of Gemcitabine and Paclitaxel. These drugs are linked to a peptide that shows specificity for the EphA2 receptor, which is overexpressed on the surface of pancreatic cancer cells and only minimally on normal cells. This peptide results in increased cellular uptake of drug, as it is bypassing its normal mechanism of entry. These normal mechanisms are often dysregulated in cancer, leading to decreased uptake and drug resistance. The use of these modified drugs show significantly increased tumor growth inhibition as compared to the parent drug alone. Finally, we provide data on the characterization of a novel transgenic mouse model of pancreatic cancer. This model, the Pan Met View (PMV) mouse, combines the commonly used KPC transgenic mouse model of pancreatic cancer and a mouse that expresses a Luciferase reporter gene under the control of the cancer-specific promoter, CCN1. Our data shows that double transgenic PMV mice can now be used to follow primary tumor and metastasis development in real time by Bioluminescent imaging (BLI) through disease progression and potentially therapy. This strategy will enhance the use of genetically engineered mouse models (GEMMS) to study cancer initiation and progression with potential to non-invasively monitor therapy. These chapters present novel and exciting data that have the potential to open multiple avenues of translational study and result in significant advances in pancreatic cancer therapy.

Preclinical research using animals often precedes and informs clinical trials; however, most attempts to translate findings from “bench-to-bedside” fail. There is growing concern that an important cause of failed translations is that much of preclinical research is not reproducible, with poor experimental methodology believed to be a major contributor. Four studies were conducted: (1) an assessment of reported study designs of preclinical experiments published in leading cardiovascular journals; (2) an examination of sex bias in preclinical cardiovascular research; (3) a comparison of experimental practices between male and female preclinical cardiovascular researchers; and (4) an analysis of the influence of journal initiatives on preclinical research quality. These studies suggest that (1) methodological shortcomings are prevalent and persistent in preclinical cardiovascular research; (2) women’s involvement in preclinical cardiovascular research is positively associated with considering sex as a biological variable; and (3) journals can exert considerable influence on the quality of published data.

Over the past three decades, nitric oxide (NO) has been recognised as one of the most versatile and important players in many aspects of physiology, including immune responses. More recently, S-nitrosylation, the incorporation of a NO moiety into a protein thiol group, has emerged as a major post-translational modification (PTM) during pathophysiological responses in plants and animals. The main goal of this work was to investigate the role of S-nitrosylation in physiology and innate immunity of animals using the genetic reference system, Drosophila melanogaster. The S-nitrosylated derivative of glutathione (GSH), S-nitrosoglutathione (GSNO), is the main non-protein S-nitrosothiol (SNO) in the cell and extracellular fluids. GSNO can trans-S-nitrosylate other thiols and is considered a reservoir of NO bioactivity. The levels of GSNO and total S-nitrosylation have been shown to be controlled by S-nitrosoglutathione reductase (GSNOR) in yeast, plants and mammals. By employing an overlapping deletion technique to knock-out gsnor, a role for S-nitrosylation in the immune response of D. melanogaster is proposed. Compared to wild type flies, gsnor overlapping deletion flies presented lower expression of antimicrobial peptides in response to infections, and succumbed more rapidly to both Gram-positive bacterial and fungal pathogens. As the Toll pathway mediates responses against these pathogens, key components of this network were tested for their propensity to being S-nitrosylated. Two CLIP-domain serine proteases of the Toll signalling pathway, Persephone (PSH) and Spätzle-Processing Enzyme (SPE), were shown to be S-nitrosylated both in vitro and in vivo and this process seemed to control the quaternary structure of these proteins and interfere with the immune response of D. melanogaster. At least for PSH, S-nitrosylation at C254 has an immune significance as the expression of non-Snitrosylable PSHC254S in gsnor knock-out flies partially recovered the resistance of these animals to infections with the entomopathogenic fungus Beauveria bassiana. These findings might represent a novel mechanism by which NO and S-nitrosylation regulate immunity. Further results presented in this thesis reveal an interplay between reactive oxygen species (ROS) and reactive nitrogen species (RNS) in D. melanogaster physiology and immunity. Similarly to what has been reported in Arabidopsis thaliana, gsnor knock-out flies presented higher tolerance to the herbicide paraquat, an inducer of superoxide (O2 -) production. Moreover, additional mutations in Catalase (Cat), a hydrogen peroxide (H2O2) scavenger enzyme, partially restored the immunodeficiency phenotypes of gsnor knock-out flies. These findings suggest an inter-relation between the levels of ROS and RNS during stress responses of plants and animals. In addition, CRISPR/Cas9 technology was employed to generate gsnor knock-outs in the genome of D. melanogaster. These flies were shown to have no GSNOR activity, presented lower tolerance to pharmacological-induced nitrosative stress and succumbed faster to infections with B. bassiana compared to wild type flies. These results support the role played by GSNOR in regulating NO homeostasis and immunity in D. melanogaster.

C-type natriuretic peptide (CNP) is synthesised and released by the endothelium and plays a vital role in the maintenance of vascular homeostasis (Moyes et al., 2014). However, a similar regulatory role of endogenous CNP in the heart has yet to be elucidated. Therefore, I have used three unique mouse strains with endothelium (Tie2-Cre), cardiomyocyte (αMHC-Cre) and fibroblast (Col1α2-Cre)-restricted deletion of CNP to investigate if the peptide modulates coronary vascular reactivity and cardiac function. Methods: Langendorff isolated hearts were used to investigate the effect of CNP deletion on coronary vascular reactivity in response to the endothelium-dependent vasodilators bradykinin (10nmol) and acetylcholine (0.1-1nmol). Vasodilatation associated with reperfusion was investigated by transient cessation of flow (20-80 seconds). Ischaemia reperfusion (IR) injury (35 minutes ischaemia followed by 60 minutes reperfusion) was also investigated in cell-specific knockout (KO) animals. Isoprenaline (ISO; 20mg/kg/day, 7days)- and pressure overload (abdominal aortic constriction [AAC]; 6 weeks)-induced heart failure were used to study the effect of CNP deletion during cardiac stress, with cardiac function assessed by echocardiography. Cardiac fibrosis and hypertrophy were determined by picro-sirius red and wheat-germ agglutinin fluorescence staining, respectively. A subset of experiments was repeated in mice with global deletion of natriuretic peptide receptor-C (NPR-C) to delineate the signalling pathway triggered by CNP. Real time qPCR was used to determine hypertrophic and fibrotic gene expression in left ventricles isolated from mice subjected to AAC or sham. Neonatal cardiomyocytes were isolated to investigate angiotensin (Ang)II-induced hypertrophy. Results: Coronary endothelial reactivity was reduced in endothelial CNP (ecCNP) KO mice compared to wild type (WT) in response to bradykinin, acetylcholine and reperfusion-induced vasodilatation. These observations were paralleled in NPR-C KO animals. ecCNP KO did not exacerbate IR injury, whilst mice with cardiomyocyte-restricted deletion of CNP (cmCNP KO) and NPR-C KO animals exhibited a larger infarct size compared to WT. cmCNP KO mice also displayed greater cardiac dysfunction and fibrosis after ISO infusion or AAC compared to WT; similar results were observed in fbCNP KO and NPR-C KO animals. Infusion of CNP (0.2mg/kg/day; osmotic mini-pump, s.c.) in WT, but not NPR-C KO, animals rescued the decline in cardiac function. CNP (1μM) administration in isolated cardiomyocyte also blunted Ang II-induced hypertrophy. Pro-hypertrophic and pro-fibrotic gene expression (ANP, β-MHC and MMP-2) was augmented in cmCNP KO and NPR-C KO mice compared to littermate controls following AAC. Conclusions: Endothelial, cardiomyocyte and fibroblast-derived CNP have distinct, complementary roles in the heart, modulating cardiac function by influencing coronary vascular tone and protecting against heart failure and IR injury. These protective effects of CNP are mediated, at least in part, via NPR-C activation. Developing CNP mimetics or selective NPR-C agonists could be a novel therapeutic intervention in cardiovascular disease.

A massificação dos estudos da medicina translacional permite aos pesquisadores que usufruam de fontes de dados das mais diversas áreas. Uma área de suma importância e a bioinformatica, que agrega o alta capacidade de processamento computacional disponível atualmente, com a infindável quantidade de dados gerada por métodos de sequenciamento de ultima geração, para entregar aos pesquisadores uma quantidade rica de dados para serem analisados. Apesar da disponibilidade desses dados, a expertise necessária para analisa-los dificulta que profissionais com pouco conhecimento em bioinformatica, estatística e ciência da computação possam realizar pesquisas e analises com estes dados. Dada esta situação, este trabalho consistiu em criar uma ferramenta que tira proveito da integração de múltiplas bases de dados proporcionada pelo framework IPTrans, permitindo que usuários da área biomédica realizem analises com os dados contidos nessas bases. Com base em outras ferramentas existentes e em um levantamento de requisitos junto a potenciais usuários, foram identificadas as funcionalidades mais importantes e assim foi projetada e implementada a IPTrans Advanced Analysis Tool (IPTrans A2Tool). Esta ferramenta permite que usuários façam analises de expressão diferencial mais comuns como heatmaps, volcano plots, consenso de agrupamentos e blox-plot. Além disso, a ferramenta proporciona um algoritmo de mineração de dados baseado na extração de regras de associação entre dados clínicos e biomoleculares, que permite ao usuário descobrir novas associações entre a expressão dos genes dados clínicos e fenotípicos. Adicionalmente a este trabalho, foi criado também o BioBank Warden, um sistema de controle de dados clínicos e amostras biomoleculares, que foi utilizado como uma das fontes de dados para o IPTrans A2Tool. Este sistema permite que usuários adicionem informações clinicas de pacientes e também das amostras extraídas para a realização de estudos. Uma avaliação preliminar de usabilidade, realizada junto a profissionais da área biomédica, mostrou que as ferramentas possuem potencial para serem utilizadas no contexto da medicina translacional. / The great number of translational medicine studies allows researchers to make benefit of data sources from various fields. An area of great importance is bioinformatics, which combines the high computational processing capabilities found nowadays with the endless amount of data generated by next-generation sequencing methods, to give researchers a rich amount of data to be analyzed. Despite the availability of such data, the expertise required to analyze it makes difficult for professionals with little knowledge in bioinformatics, statistics or computer science, to conduct research and analysis on this data. Given this situation, this work was intended to create a tool that takes advantage of multiple databases integration capabilities provided by IPTrans and that allows users to perform analysis on the data contained in these databases. To accomplish that other tools were studied in order to observe which features our framework should aggregate and thus was created the IPTrans A2Tool (IPTrans Advanced Analysis Tool). This tool allows users to perform differential expression analysis and generate output as heatmaps, volcano plots, consensus clustering and blox-plots. In addition, the tool provides an association rule extraction algorithm between clinical and biomolecular data, allowing the user to discover hidden associations between the expression of analyzed genes and clinical data. As a by-product of this work was also created the BioBank Warden a clinical data and biomolecular samples management system that was used as one of the data sources for IPTrans A2Tool. This system allows users to add patients clinical information and also of samples taken for carrying out studies. In addition, the system provides a strong research group and project permission management that ensures only authorized people to have access to patients data.

A biofilm is a complex community of cells enveloped in a self-produced polymeric matrix. Entry into a biofilm is exquisitely controlled at the level of transcription and in Bacillus subtilis it requires the concerted efforts of several major transcription factors including the repressor SinR and activator DegU. I initially identified that these transcriptional regulators control biofilm formation via parallel pathways. Through investigating the regulation of biofilm formation by SinR and DegU, I discovered that biofilm formation is also regulated at the post-translational level. This was achieved by identifying three key proteins which are needed for biofilm formation. These proteins are PtkA, a bacterial tyrosine kinase; TkmA, the cognate modulator of PtkA; and PtpZ, a bacterial tyrosine phosphatase. By introducing amino acid point mutations within the catalytic domains of PtkA and PtpZ it was identified that the kinase phosphatase activities, respectively, are essential function.In addition, PtkA contains a conserved C-tyrosine cluster that is the site autophosphorylation. Investigation of the role of the C-terminal tyrosine cluster tentatively suggests that this domain acts to block access to the active site of PtkA, thus affecting the ability of PtkA to phosphorylate its targets. Deletion of the gene coding for TkmA demonstrated that this modulator was also required for biofilm formation. It was also demonstrated that TkmA may interact with other protein partners, at least in the absence of PtkA, raising the question of how signal specificity is maintained. Finally, a systematic mutagenesis approach was used with the aim of identifying the target(s) of PtkA and PtpZ during biofilm formation but,despite extensive efforts, it remained elusive. The findings presented in this thesis highlight the complexity of biofilm formation by B. subtilis by revealing an additional level of regulation in the form of protein tyrosine phosphorylation.

The O-linked N-acetylglucosamine post-translational modification (O-GlcNAcylation) is the dynamic and reversible attachment of N-acetylglucosamine to serine and threonine residues of target proteins. It is abundant in metazoa, involving hundreds of proteins linked to a plethora of biological functions with implications in human diseases. The process is catalysed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), that add and remove the sugar moiety, respectively. Ogt gene knock-out is embryonic lethal in a range of animal models, hampering the study of the biological role of O-GlcNAc. O-GlcNAcylation of nuclear and cytoplasmic proteins has been extensively studied, however little is known about the role of O-GlcNAc in mitochondria. A previous report suggested the presence of a mitochondrial OGT isoform (mOGT) in human cell lines in addition to the well-characterised nucleocytoplasmic one (ncOGT). Since this report more than one decade ago, this putative mOGT has not been studied further. Similarly, hundreds of O-GlcNAcylated nucleocytoplasmic proteins have been identified by high-throughput proteomic screens, whereas only a few mitochondrial proteins have been detected. Nevertheless, several studies suggest that altered O-GlcNAc signalling affects mitochondrial function and morphology, with potential clinical implications. The aim of this thesis work was to study and characterise the biological role of mOGT and determine the mitochondrial O-GlcNAc proteome. Firstly, the presence of mOGT in human cell lines and mouse tissues was investigated. Surprisingly, analysis of genomic sequences indicates that this isoform cannot be expressed at protein level in most of the species analysed, except human and some primates. In fact, the putative mOGT cDNA in most of the genomes analysed contains a stop codon that excludes the presence of such isoform. In addition, mOGT was not detected at protein level in a wide range of human cell lines. Knock-down experiments and Western blot analysis of all the predicted OGT isoforms suggested the expression of only a single OGT isoform. In agreement with this, overexpression of ncOGT in HEK 293 suspension cells led to increased O-GlcNAcylation of mitochondrial proteins, suggesting that ncOGT is necessary and sufficient for the generation of the mitochondrial O-GlcNAc proteome. These data point to a model where O-GlcNAc cycling of mitochondrial proteins occurs in the cytosol, followed by their import into mitochondria. Alternatively, ncOGT itself might be transported into mitochondria where it can take part to O-GlcNAc cycling inside the organelle. In parallel, some advance in determining the O-GlcNAc mitochondrial proteome has been undertaken. Different mitochondrial fractionation protocols, combined with O-GlcNAc enrichment methods have been explored in order to map novel glycosylation sites on mitochondrial proteins. A novel technique established in our research group, employing a bacterial OGA orthologue as a bait to trap O-GlcNAcylated proteins, has been applied to crude mitochondrial fractions allowing the identification of several hits, although site mapping has not been yet achieved. The second chapter describes the work that has been done to improve and optimise novel O-GlcNAc inhibitors previously designed in the laboratory, called goblins. The original objective was to make these molecules cell-permeable and possibly target them to mitochondria in order to inhibit mOGT. Several strategies were explored to deliver the compounds into living cells, including the use of transfection reagents and covalent linkage to linear cell-penetrant peptides. The above methods did not achieve cellular uptake, although recently designed cyclic cell-penetrant peptides, linked to fluorescein, were internalised by HeLa cells with immediate diffuse nucleocytoplasmic staining. These molecules will be linked to goblins aiming to use the inhibitors for cell biology studies. A different approach, based on the permeabilisation of Drosophila embryos, enabled the penetration of goblins into the organisms with consequent reduction of global O-GlcNAc levels. This method allowed the use of these novel bisubstrate inhibitors in vivo for the first time, with potential applications in studying the role of O-GlcNAc in Drosophila development and possibly for future therapeutic purposes after further development of the scaffold.

<p>Have you thought of why you get tired or why you get hungry? Something in your body keeps track of time. It is almost like you have a clock that tells you all those things.</p><p>And indeed, in the suparachiasmatic region of our hypothalamus reside cells which each act like an oscillator, and together form a coherent circadian rhythm to help our body keep track of time. In fact, such circadian clocks are not limited to mammals but can be found in many organisms including single-cell, reptiles and birds. The study of such rhythms constitutes a field of biology, chronobiology, and forms the background for my research and this thesis.</p><p>Pioneers of chronobiology, Pittendrigh and Aschoff, studied biological clocks from an input-output view, across a range of organisms by observing and analyzing their overt activity in response to stimulus such as light. Their study was made without recourse to knowledge of the biological underpinnings of the circadian pacemaker. The advent of the new biology has now made it possible to "break open the box" and identify biological feedback systems comprised of gene transcription and protein translation as the core mechanism of a biological clock.</p><p>My research has focused on a simple transcription-translation clock model which nevertheless possesses many of the features of a circadian pacemaker including its entrainability by light. This model consists of two nonlinear coupled and delayed differential equations. Light pulses can reset the phase of this clock, whereas constant light of different intensity can speed it up or slow it down. This latter property is a signature property of circadian clocks and is referred to in chronobiology as "Aschoff's rule". The discussion in this thesis focus on develop a connection and also a understanding of how constant light effect this clock model.</p>

<p>Positron Emission Tomography (PET) has gained an important roll in clinical for diagnosis, staging and prognosis of a range of cancer types. Utilization of PET for monitoring and evaluation of cancer treatment is an attractive but almost new concept. The proper choice of PET-tracer as a biomarker for treatment follow-up is crucial. The important characteristic for a suitable tracer is its ability to reflect the response to a treatment at an early stage, before any morphologically changes occurs. It would be an advantage to screen a battery of PET tracers in a preclinical model and introduce a few potential tracers in clinical trial. </p><p>The most conventional pre-clinical approach in PET-oncology utilizes xenografts in mice or rats and requires a large number of subjects. It would be a great advantage to introduce a less demanding but still reliable preclinical method for a more efficient planning of studies in animal model and then in human trials. </p><p>The Multicellular Tumour Spheroid (MTS) system represents an intermediary level between cells growing as monolayer and solid tumours in experimental animals or patients. It mimics the growth of naturally occurring human tumours before neovascularization and appears to be more informative than monolayer and more economical and more ethical than animal models.</p><p>The aim of this work was to establish, refine and evaluate the application of MTS model as a preclinical approach in PET oncology. The vision was to introduce a preclinical method to probe and select PET tracer for treatment monitoring of anticancer drugs, which can hopefully be applied for optimization in breast cancer treatment.</p><p>In this thesis, a number of basic experiments were performed to explore the character of 2-[fluorine-18]-fluoro-2-deoxy-d-glucose (FDG) uptake in MTS. FDG as the most established PET tracer was an obvious initial option for the evaluation of the model. For further assess-ment, we studied effects on FDG uptake in MTS treated with five routinely used chemother-apy agents. For association of PET tracer uptake to size change of MTS, we developed a reliable and user-friendly method for size determination of MTS. The next step was to apply the MTS model to screen PET tracers for analysis of early response of chemotherapy in breast cancer. Finally the method was utilized for translational imaging exemplified with a new chemotherapy agent.</p><p>The results were encouraging and the MTS model was introduced and evaluated as a preclini-cal tool in PET oncology. The method was implicated to in vitro quickly assess a therapy profile of existing and newly developed anticancer drugs in order to investigate the effects of candidate drugs on tumour-growth, selection of appropriate PET tracer for treatment monitor-ing and finally understanding relation between growth inhibition and biomarkers as part of translational imaging activities.</p>

Positron Emission Tomography (PET) has gained an important roll in clinical for diagnosis, staging and prognosis of a range of cancer types. Utilization of PET for monitoring and evaluation of cancer treatment is an attractive but almost new concept. The proper choice of PET-tracer as a biomarker for treatment follow-up is crucial. The important characteristic for a suitable tracer is its ability to reflect the response to a treatment at an early stage, before any morphologically changes occurs. It would be an advantage to screen a battery of PET tracers in a preclinical model and introduce a few potential tracers in clinical trial. The most conventional pre-clinical approach in PET-oncology utilizes xenografts in mice or rats and requires a large number of subjects. It would be a great advantage to introduce a less demanding but still reliable preclinical method for a more efficient planning of studies in animal model and then in human trials. The Multicellular Tumour Spheroid (MTS) system represents an intermediary level between cells growing as monolayer and solid tumours in experimental animals or patients. It mimics the growth of naturally occurring human tumours before neovascularization and appears to be more informative than monolayer and more economical and more ethical than animal models. The aim of this work was to establish, refine and evaluate the application of MTS model as a preclinical approach in PET oncology. The vision was to introduce a preclinical method to probe and select PET tracer for treatment monitoring of anticancer drugs, which can hopefully be applied for optimization in breast cancer treatment. In this thesis, a number of basic experiments were performed to explore the character of 2-[fluorine-18]-fluoro-2-deoxy-d-glucose (FDG) uptake in MTS. FDG as the most established PET tracer was an obvious initial option for the evaluation of the model. For further assess-ment, we studied effects on FDG uptake in MTS treated with five routinely used chemother-apy agents. For association of PET tracer uptake to size change of MTS, we developed a reliable and user-friendly method for size determination of MTS. The next step was to apply the MTS model to screen PET tracers for analysis of early response of chemotherapy in breast cancer. Finally the method was utilized for translational imaging exemplified with a new chemotherapy agent. The results were encouraging and the MTS model was introduced and evaluated as a preclini-cal tool in PET oncology. The method was implicated to in vitro quickly assess a therapy profile of existing and newly developed anticancer drugs in order to investigate the effects of candidate drugs on tumour-growth, selection of appropriate PET tracer for treatment monitor-ing and finally understanding relation between growth inhibition and biomarkers as part of translational imaging activities.