The large numbers of neurons that comprise the adult brain display an immense diversity. Repeated divisions of a relatively small pool of neural stem cells generate this neuronal diversity during development. To increase progress towards medical treatments for neurodegenerative diseases, it is of interest to understand both how neural stem cells generate the assortment of neurons and how these neurons come together to form a functional brain. Brain assembly occurs sequentially across time with early events laying the foundation for later events. Drosophila neural stem cells, neuroblasts (NBs), are an excellent model for investigating how neural diversity is generated and what roles early and late born neurons have in shaping the stereotypical adult brain structure. Generation of neural diversity, begins with specifying the diverse population of stem cells, called spatial patterning, and continues with diversifying neurons made from the diverse stem cells, called temporal patterning. Drosophila NBs exhibit both spatial and temporal patterning. Drosophila NBs have three types of division modes: type 0, type I and type II. Type II NBs expand the number of neurons made with progeny that exhibit a transit-amplifying division pattern, similar to that of mammalian outer subventricular zone (OSVZ) progenitors. Additionally, type II NBs exhibit temporal patterning across both the NB and their progeny to generate a large diversity of neurons that populate a conserved region of the brain responsible for many sensory and motor functions, called the central complex. Type II NBs have only been identified and studied during later stages in development, with nothing known about their origin or early divisions. In this dissertation, I describe the early lineages of the type II NBs within the Drosophila embryo. I show that type II NBs and lineages originate early in development, exhibit temporal patterning across both the NB and transit-amplifying progeny, and produce neurons that survive into the adult brain to innervate and potentially serve as a foundation within the adult central complex. Additionally, I explain how live imaging of the developing Drosophila brain can answer questions not easily addressed through other methods.

SHP (Sphaeroides Heme Protein) is a monoheme cytochrome c of unknown function. In general, ligands cannot bind to ferric SHP, but some diatomic molecules, such as O2 or NO, can bind to ferrous SHP. The gene encoding SHP and genes encoding a diheme cytochrome c (DHC) and a b-type cytochrome (Cyt-b) are found in the same chromosome region in different species. In the case of Shewanella oneidensis MR-1, mRNA levels for SHP, DHC, and Cyt-b are up-regulated by nearly 10-fold when grown under anaerobic conditions using nitrate as the electron acceptor. Thus it is possible that the physiological role of SHP may be in nitrate metabolism. However, nitrate is too big to be a candidate substrate for SHP, and some nitrification steps need more than one electron transfer (SHP is a monoheme cytochrome). Therefore, we will focus on the nitrite reductase, nitric oxide reductase and nitric oxide dioxygenase activities of SHP. In this thesis it is shown that SHP can catalyse the reaction between oxygen and nitric oxide to give a nitrate ion as the final product. Thus a possible aerobic function for SHP as a nitric oxide dioxygenase is proposed. Aerobically, SHP is proposed to be a nitric oxide dioxygenase which utilizes the same mechanism as other NO dioxygenases, flavohemoglobin (HMP) and neuroglobin (Ngb). This mechanism is proposed to proceed via an oxy-ferrous complex (SHP2+-O2) which reacts with nitric oxide. A mechanism for the catalytic reaction with ferrous-NO complex is described. SHP2+-NO can be quickly converted back to ferrous SHP by reacting with superoxide liberated by SHP2+-O2 or from another source. In addition it is also found that Shewanella MR-1 wild type reveals a higher NO tolerance than the SHP knockout strain in aerobic conditions. The catalytic mechanism of NO dioxygenase is oxygen-dependent, but the SHP mRNA up-regulation in Shewanella oneidensis MR-1 grown with nitrate under anaerobic conditions indicates that SHP may also perform some anaerobic function and may possibly be involved in nitrate metabolism. This work found that SHP reveals anaerobic nitrite reductase activity. However, the catalytic efficiency of SHP is considerably lower than other nitrite reductases. This infers that although SHP can reduce nitrite in vitro, it is unlikely to function as a nitrite reductase in vivo. Ferrous SHP binds NO with a Kd of less than 1 μM, and does not auto-oxidise. Therefore, under anaerobic conditions SHP2+-NO must be processed by some other mechanism. In addition, biochemical results reveal that the SHP/DHC complex has NO reductase activity under anaerobic conditions. Unfortunately, this function was not proved in vivo. SHP was initially isolated from Rhodobacter sphaeroides and its structure was reported in 2000. Based upon this structure, SHP is clearly a class I cytochrome c with one axial histidine ligand to the heme iron. Unusually, however, it has an asparagine residue as the other axial heme ligand, and as such is unique among cytochromes c. For this reason it may be assumed that the asparagine plays a special role. This study reveals several potential reasons why SHP utilises asparagine as a heme ligand. Firstly, in the ferric form, asparagine 88 binds to the heme iron to prevent small molecules binding. Secondly, in the ferrous form it moves to allow oxygen to bind and form the oxy-ferrous complex, using hydrogen bonding for stability. Thirdly, using asparagine as a heme ligand creates a suitable redox potential for reduction by DHC, thus allowing NO dioxygenation.

Gao, Zhibo. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 147-157). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Liu, Xin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 157-164). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

"Tuberculosis is a deadly disease caused by bacteria of the genus Mycobacterium. One-third of the world’s population is infected with Mycobacterium tuberculosis. Two million these deaths occur each year in immunocompromised AIDS patients. M. tuberculosis has co-evolved with humans for many thousands of years. The bacillus has developed tactics to overcome the immune defense system and multiply in the macrophage. At the interface of the host and pathogen interactions, there is an interchange of metals and electrolytes. The host on one hand reduces the availability of metals essential for pathogen survival, like manganese and iron, in the macrophage and increases potassium ions which reduces pH in the phagolysosome. The host also generates Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS), to create toxic affects through interactions with metals and metalloproteins. M. tuberculosis copes with the hostile environment in the macrophage by preventing the acidification of the phagolysosome, secreting antioxidant enzymes such as alkylhydroperoxidase (AhpF) and peroxiredoxin (AhpC), superoxide dismutase, SodA and SodC, and catalase KatG through the SecA system. M. tuberculosis contains 28 metal transporters, among them there are 12 unique P-type ATPases. This is an unusually high number of P-type ATPases in an organism. These ATPases transport several monovalent and divalent metals (Cu+, Cu2+, Ag+, Zn2+, Na+, K+, Ca2+, Cd2+, Pb2+, Mn2+, Mg2+, and Co2+) across biological membranes, using energy from ATP hydrolysis. Our analysis has revealed that these P-type ATPases have homologs in other intracellular symbiotic/pathogenic bacteria and certain chemolithotrophic archaea and bacteria. A corelation can hence be drawn among these pumps and the capability of surviving in noxious environments and coping with adverse redox conditions. Possible substrates were identified by determining the consensus sequences in different helices of these ATPases. However, out of the 12 P-type ATPases confirmed, transported substrate could be postulated for four of these proteins; CtpA, CtpB, CtpV and KdpB. Using bioinformatic approaches we have characterized the possible genetic environment of these genes. The transmembrane regions were analyzed for consensus sequences and the N-terminals and C-terminals were scrutinized for metal binding domains, and we were able to categorize these ATPases into P1 type and P2 type ATPases. In an attempt to determine the substrate specificity, two of these ATPases (CtpC and ctpG) were cloned and transformed into Escherichia coli cells. Cells expressing CtpC were grown in different concentrations of metals and pHs. In these experiments CtpC was found to show an interaction with copper and cadmium. Pure protein was obtained by His-tag purification and para-Nitro Phenol Phosphatase (pNPPase) assay was performed with different metals, it was found that copper and zinc activated the phosphatase activity of the enzyme; and cobalt and manganese were inhibitory. Inhibition of the pNPP assay could mean that there would be activation in the ATPase assay, meaning that cobalt and manganese could be possible substrates to this enzyme. "

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.

O objetivo desta dissertação é estudar o problema de autovalor de Dirichlet para variedades riemannianas completas. Mais precisamente, pretendemos estudar uma cota por baixo para o -ésimo autovalor de um domínio limitado em uma variedade riemanniana completa. Tal cota é obtida fazendo-se uso de uma fórmula de recorrência de Cheng e Yang e um teorema de Nash. Ademais, pretendemos estudar uma desigualdade universal para os autovalores no espaço hiperbólico. / The goal of this dissertation is to study the Dirichlet eigenvalue problem for a complete riemannian manifold. More accurately, we intend to investigate a lower-bound for the -ℎ eigenvalue on a bounded domain in a complete riemannian manifold. Such a bound is obtained by making use of a recursion formula of Cheng and Yang and Nash’s Theorem. Furthermore, we study a universal inequality for eigenvalues of the Dirichlet eigenvalue problem on a bounded domain in a hyperbolic space (−1).

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Background Obesity, insulin resistance and type 2 diabetes mellitus (T2DM) have been associated with endometrial cancer (EC). In this systematic review and meta-analysis we evaluated the effect of metformin on clinical outcomes in patients with EC and insulin resistance or T2DM. Methods Four research databases were searched for original articles published in all languages up to 30 October 2016. Outcomes of interest were overall mortality (OM), cancer-specific mortality, disease progression, and metastases. We performed a random effect meta-analysis of adjusted effects expressed as hazard ratios (HR); heterogeneity among studies was described with the I2 statistic. Results Of the 290 retrieved citations, 6 retrospective cohort studies in women with EC (n = 4723) met the inclusion criteria, and 8.9% to 23.8% were treated with metformin; OM data was available from 5 studies. In 4 studies of EC patients (n = 4132), metformin use was associated with a significant reduction in OM in comparison with not using metformin (adjusted HR [aHR] 0.64, 95% CI 0.45–0.89, p = 0.009). In three studies evaluating patients with EC and T2DM (n = 2637), metformin use was associated with a significant reduction in OM (aHR 0.50, 95%CI 0.34–0.74, p = 0.0006). There was low to moderate heterogeneity of adjusted effects across studies. There was no information about the effect of metformin on cancer-specific mortality, disease progression, or metastases. Conclusions Metformin treatment is associated with a significant reduction in OM irrespective of diabetes status in patients with EC. The survival benefit suggests that diabetes screening and maintenance of good glycemic control may improve outcomes in EC. / Revisión por pares

<i>Legionella pneumophila,</i> the etiological agent of Legionnaires’ disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates over 330 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK (Lpg0969) as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H₉₅E_XXH₉₉ (x, any amino acid) motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293T cells. We demonstrate that the actin is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage occurs during <i>L. pneumophila </i>infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Thus, <i>L. pneumophila</i> exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.

Type 2 diabetes and metabolic syndrome are the metabolic disorders which constitute a major public health problem in both developed and developing countries. Various studies have suggested the genetic susceptibility to the disorders. The main aim of the thesis was to investigate the putative association of single nucleotide polymorphisms with Type 2 diabetes (T2D), metabolic syndrome (MetS) and the major components of metabolic syndrome. This study used meta‐analysis, polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) and Sanger sequencing methods to analyse the results. The single nucleotide polymorphism rs57829442 of peroxisome proliferator‐activated receptor‐γ coactivator‐1 (PPARGC1A) gene and its relation to risk of type 2 diabetes has been studied in the United Kingdom population. A meta‐analysis of genetic variant rs8192678 (Gly482Ser) of peroxisome proliferator‐activated receptor‐γ coactivator‐1 (PPARGC1A) gene and its association with the components of metabolic syndrome has been studied. An association of the genetic variants rs8192678 (Gly482Ser) of the PPARGC1A gene, rs7903146 of Transcription Factor 7 Like 2 (TCF7L2) gene, rs9939609 of Fat mass and obesity‐associated (FTO) gene and rs1801282 (Pro12Ala) of peroxisome proliferator‐activated receptor gamma (PPARG) gene with the metabolic syndrome and its components has been studied in the Nepalese population. The results showed that variant rs57829442 of PPARGC1A is not associated with T2D in the United Kingdom population. Further investigation with increased sample size is warranted. In the meta‐analysis, the variant rs8192678 (Gly482Ser) of PPARGC1A gene was found to be significantly associated with body mass index (BMI) in Asian populations under dominant genetic model, total cholesterol (TC) in non‐Asian population under recessive genetic model and with fasting plasma glucose (FPG) under a recessive model in overall and non‐Asian populations. No significant association of the variants rs8192678 (Gly482Ser), rs7903146, rs9939609 and rs1801282 (Pro12 Ala) was found associated with MetS under dominant, recessive, co‐dominant and additive models in the Nepalese population. However, the genotypes (AG and AA) of rs8192678 (Gly482Ser) had a statistically significant protective effect on systolic blood pressure. The genotypes with the risk allele of rs9930609 of FTO gene was significantly associated with weight, waist circumference and diastolic blood pressure under dominant genetic model and with BMI under both dominant and recessive genetic models in the Nepalese population. To the best of our knowledge, this is the first study to report the findings in the Nepalese population.