Microsatellite dna reveals low ganetic differentiation among chub mackerel ("Scomber colias) sampled in Atlantic and Mediterranea

trabajo realizado por Medina Alcaraz, C., Castro, J.J., Sosa, P. A.

Peripheral arterial disease and diabetes: effects on endothelial progenitor cell differentiation and nitric oxide metabolism

In the recent years it is emerged that peripheral arterial disease (PAD) has become a growing health problem in Western countries. This is a progressive manifestation of atherothrombotic vascular disease, which results into the narrowing of the blood vessels of the lower limbs and, as final consequence, in critical leg ischemia. PAD often occurs along with other cardiovascular risk factors, including diabetes mellitus (DM), low-grade inflammation, hypertension, and lipid disorders. Patients with DM have an increased risk of developing PAD, and that risk increases with the duration of DM. Moreover, there is a growing population of patients identified with insulin resistance (IR), impaired glucose tolerance, and obesity, a pathological condition known as “metabolic syndrome”, which presents increased cardiovascular risk. Atherosclerosis is the earliest symptom of PAD and is a dynamic and progressive disease arising from the combination of endothelial dysfunction and inflammation. Endothelial dysfunction is a broad term that implies diminished production or availability of nitric oxide (NO) and/or an imbalance in the relative contribution of endothelium-derived relaxing factors. The secretion of these agents is considerably reduced in association with the major risks of atherosclerosis, especially hyperglycaemia and diabetes, and a reduced vascular repair has been observed in response to wound healing and to ischemia. Neovascularization does not only rely on the proliferation of local endothelial cells, but also involves bone marrow-derived stem cells, referred to as endothelial progenitor cells (EPCs), since they exhibit endothelial surface markers and properties. They can promote postnatal vasculogenesis by homing to, differentiating into an endothelial phenotype, proliferating and incorporating into new vessels. Consequently, EPCs are critical to endothelium maintenance and repair and their dysfunction contributes to vascular disease. The aim of this study has been the characterization of EPCs from healthy peripheral blood, in terms of proliferation, differentiation and function. Given the importance of NO in neovascularization and homing process, it has been investigated the expression of NO synthase (NOS) isoforms, eNOS, nNOS and iNOS, and the effects of their inhibition on EPC function. Moreover, it has been examined the expression of NADPH oxidase (Nox) isoforms which are the principal source of ROS in the cell. In fact, a number of evidences showed the correlation between ROS and NO metabolism, since oxidative stress causes NOS inactivation via enzyme uncoupling. In particular, it has been studied the expression of Nox2 and Nox4, constitutively expressed in endothelium, and Nox1. The second part of this research was focused on the study of EPCs under pathological conditions. Firstly, EPCs isolated from healthy subject were cultured in a hyperglycaemic medium, in order to evaluate the effects of high glucose concentration on EPCs. Secondly, EPCs were isolated from the peripheral blood of patients affected with PAD, both diabetic or not, and it was assessed their capacity to proliferate, differentiate, and to participate to neovasculogenesis. Furthermore, it was investigated the expression of NOS and Nox in these cells. Mononuclear cells isolated from peripheral blood of healthy patients, if cultured under differentiating conditions, differentiate into EPCs. These cells are not able to form capillary-like structures ex novo, but participate to vasculogenesis by incorporation into the new vessels formed by mature endothelial cells, such as HUVECs. With respect to NOS expression, these cells have high levels of iNOS, the inducible isoform of NOS, 3-4 fold higher than in HUVECs. While the endothelial isoform, eNOS, is poorly expressed in EPCs. The higher iNOS expression could be a form of compensation of lower eNOS levels. Under hyperglycaemic conditions, both iNOS and eNOS expression are enhanced compared to control EPCs, as resulted from experimental studies in animal models. In patients affected with PAD, the EPCs may act in different ways. Non-diabetic patients and diabetic patients with a higher vascular damage, evidenced by a higher number of circulating endothelial cells (CECs), show a reduced proliferation and ability to participate to vasculogenesis. On the other hand, diabetic patients with lower CEC number have proliferative and vasculogenic capacity more similar to healthy EPCs. eNOS levels in both patient types are equivalent to those of control, while iNOS expression is enhanced. Interestingly, nNOS is not detected in diabetic patients, analogously to other cell types in diabetics, which show a reduced or no nNOS expression. Concerning Nox expression, EPCs present higher levels of both Nox1 and Nox2, in comparison with HUVECs, while Nox4 is poorly expressed, probably because of uncompleted differentiation into an endothelial phenotype. Nox1 is more expressed in PAD patients, diabetic or not, than in controls, suggesting an increased ROS production. Nox2, instead, is lower in patients than in controls. Being Nox2 involved in cellular response to VEGF, its reduced expression can be referable to impaired vasculogenic potential of PAD patients.

Neuronal differentiation and epithelial integrity: the role of Drosophila Short stop

The nervous system is the most complex organ in animals and the ordered interconnection of neurons is an essential prerequisite for normal behaviour. Neuronal connectivity requires controlled neuronal growth and differentiation. Neuronal growth essentially depends on the actin and microtubule cytoskeleton, and it has become increasingly clear, that crosslinking of these cytoskeletal fractions is a crucial regulatory process. The Drosophila Spectraplakin family member Short stop (Shot) is such a crosslinker and is crucial for several aspects of neuronal growth. Shot comprises various domains: An actin binding domain, a plakin-like domain, a rod domain, calcium responsive EF-hand motifs, a microtubule binding Gas2 domain, a GSR motif and a C-terminal EB1aff domain. Amongst other phenotypes, shot mutant animals exhibit severely reduced dendrites and neuromuscular junctions, the subcellular compartmentalisation of the transmembrane protein Fasciclin2 is affected, but it is also crucially required in other tissues, for example for the integrity of tendon cells, specialised epidermal cells which anchor muscles to the body wall. Despite these striking phenotypes, Shot function is little understood, and especially we do not understand how it can carry out functions as diverse as those described above. To bridge this gap, I capitalised on the genetic possibilities of the model system Drosophila melanogaster and carried out a structure-function analysis in different neurodevelopmental contexts and in tendon cells. To this end, I used targeted gene expression of existing and newly generated Shot deletion constructs in Drosophila embryos and larvae, analyses of different shot mutant alleles, and transfection of Shot constructs into S2 cells or cultured fibroblasts. My analyses reveal that a part of the Shot C-terminus is not essential in the nervous system but in tendon cells where it stabilises microtubules. The precise molecular mechanism underlying this activity is not yet elucidated but, based on the findings presented here, I have developed three alternative testable hypothesis. Thus, either binding of the microtubule plus-end tracking molecule EB1 through an EB1aff domain, microtubulebundling through a GSR rich motif or a combination of both may explain a context-specific requirement of the Shot C-terminus for tendon cell integrity. Furthermore, I find that the calcium binding EF-hand motif in Shot is exclusively required for a subset of neuronal functions of Shot but not in the epidermal tendon cells. These findings pave the way for complementary studies studying the impact of [Ca2+] on Shot function. Besides these differential requirements of Shot domains I find, that most Shot domains are required in the nervous system and tendon cells alike. Thus the microtubule Gas2 domain shows no context specific requirements and is equally essential in all analysed cellular contexts. Furthermore, I could demonstrate a partial requirement of the large spectrin-repeat rod domain of Shot in neuronal and epidermal contexts. I demonstrate that this domain is partially required in processes involving growth and/or tissue stability but dispensable for cellular processes where no mechanical stress resistance is required. In addition, I demonstrate that the CH1 domain a part of the N-terminal actin binding domain of Shot is only partially required for all analysed contexts. Thus, I conclude that Shot domains are functioning different in various cellular environments. In addition my study lays the base for future projects, such as the elucidation of Shot function in growth cones. Given the high degree of conservation between Shot and its mammalian orthologues MACF1/ACF7 and BPAG1, I believe that the findings presented in this study will contribute to the general understanding of spectraplakins across species borders.

Differentiation processes in treecreepers (Aves: Certhia): phylogeny, vocalisations, morphometrics

Evolutionary processes within the bird genus Certhia (treecreepers) are investigated and taxonomic uncertainties clarified. The original seven species of the genus have Holarctic distribution, are uniform morphologically and hence difficult to distinguish. I employed four methodological approaches. 1. Molecular phylogeny using the mitochondrial cytochrome-b gene largely established relationships and revealed two cryptic species. 2. Call and song recordings from all species and many subspecies were evaluated sonagraphically. The nine phylospecies outlined in Part 1 were clearly delimited from one another by time and frequency parameters. They comprise a monophyletic group of "motif singers" and a purely southeast Asian group of "trill singers". Song-character differences were generally consistent with molecular phylogeny (strong phylogenetic signals). 3. Central European Certhia familiaris in the field responded territorially to playback of verses of allopatric "motif singer" taxa, but usually more weakly than to their own subsequently presented songs. No song characters were unambiguously recognised as species-specific. 4. Standard body dimensions of nearly 2000 museum specimens characterise species and subspecies biometrically and reveal geographic trends. Lengths of bill and hind claw proved important parameters to explain the treecreeper lifestyle (climbing and feeding on tree trunks). In the Himalayas (highest species density) tail dimensions are also significant.

Patterns of regional differentiation in post-Soviet Russia

This doctoral thesis aims at contributing to the literature on transition economies focusing on the Russian Federations and in particular on regional income convergence and fertility patterns. The first two chapter deal with the issue of income convergence across regions. Chapter 1 provides an historical-institutional analysis of the period between the late years of the Soviet Union and the last decade of economic growth and a presentation of the sample with a description of gross regional product composition, agrarian or industrial vocation, labor. Chapter 2 contributes to the literature on exploratory spatial data analysis with a application to a panel of 77 regions in the period 1994-2008. It provides an analysis of spatial patterns and it extends the theoretical framework of growth regressions controlling for spatial correlation and heterogeneity. Chapter 3 analyses the national demographic patterns since 1960 and provides a review of the policies on maternity leave and family benefits. Data sources are the Statistical Yearbooks of USSR, the Statistical Yearbooks of the Russian Soviet Federative Socialist Republic and the Demographic Yearbooks of Russia. Chapter 4 analyses the demographic patterns in light of the theoretical framework of the Becker model, the Second Demographic Transition and an economic-crisis argument. With national data from 1960, the theoretically issue of the pro or countercyclical relation between income and fertility is graphically analyzed and discussed, together with female employment and education. With regional data after 1994 different panel data models are tested. Individual level data from the Russian Longitudinal Monitoring Survey are employed using the logit model. Chapter 5 employs data from the Generations and Gender Survey by UNECE to focus on postponement and second births intentions. Postponement is studied through cohort analysis of mean maternal age at first birth, while the methodology used for second birth intentions is the ordered logit model.

Genetic variability and differentiation in the Mediterranean endemic starry ray Raja asterias (Delaroche, 1809)

The present study deal with the population structure and connectivity of the Mediterranean endemic starry ray Raja asterias (Delaroche, 1809) in the Western and Eastern Mediterranean basin. A panel of eight microsatellite loci which cross-amplify in Rajidae (El Nagar, 2010) was used to assess population connectivity and structure. Those aims were investigated by analyzing the genetic variation of 9 population sample for a total of 185 individuals collected during past scientific surveys (MEDITS, GRUND), commercial trawling and also directly at fish markets. The purpose of this thesis is to estimate the genetic divergence occurring between the Mediterranean populations and, in particular, to assess the presence of any barrier (geographic, hydrogeological and biological) to gene flow for this species. Different statistical approaches were performed to reach this aim evaluating both the genetic diversity (nucleotide diversity, allelic richness, observed and expected heterozygosity and Hardy-Weinberg equilibrium test) and the population differentiation patterns (pairwise Fst estimated and population structure analysis). The results obtained from the analysis of the microsatellite dataset suggest a geographic and genetic separation between the starry ray populations of the Mediterranean basin into three or four distinct groups: Western and Eastern Mediterranean basins and Sicilian coast always clustering as an independent group and Algeria which could be or not considered another separate group. The data were discussed from both an evolutionary and a conservation point of view and in relation to previous results obtained by the analysis of mitochondrial marker. A comparison with other Mediterranean demersal skate species was performed in order to better contextualise our results. Finally, our results could offer useful information to protect vulnerable species as R. asterias and developing effective conservation plans in the Mediterranean.

Genetic and morphological variation and differentiation of Raja clavata populations in the European seas: marker development and preliminary data

In this study the population structure and connectivity of the Mediterranean and Atlantic Raja clavata (L., 1758) were investigated by analyzing the genetic variation of six population samples (N = 144) at seven nuclear microsatellite loci. The genetic dataset was generated by selecting population samples available in the tissue databases of the GenoDREAM laboratory (University of Bologna) and of the Department of Life Sciences and Environment (University of Cagliari), all collected during past scientific surveys (MEDITS, GRUND) from different geographical locations in the Mediterranean basin and North-east Atlantic sea, as North Sea, Sardinian coasts, Tuscany coasts and Cyprus Island. This thesis deals with to estimate the genetic diversity and differentiation among 6 geographical samples, in particular, to assess the presence of any barrier (geographic, hydrogeological or biological) to gene flow evaluating both the genetic diversity (nucleotide diversity, observed and expected heterozygosity, Hardy- Weinberg equilibrium analysis) and population differentiation (Fst estimates, population structure analysis). In addition to molecular analysis, quantitative representation and statistical analysis of morphological individuals shape are performed using geometric morphometrics methods and statistical tests. Geometric coordinates call landmarks are fixed in 158 individuals belonging to two population samples of Raja clavata and in population samples of closely related species, Raja straeleni (cryptic sibling) and Raja asterias, to assess significant morphological differences at multiple taxonomic levels. The results obtained from the analysis of the microsatellite dataset suggested a geographic and genetic separation between populations from Central-Western and Eastern Mediterranean basins. Furthermore, the analysis also showed that there was no separation between geographic samples from North Atlantic Ocean and central-Western Mediterranean, grouping them to a panmictic population. The Landmark-based geometric morphometry method results showed significant differences of body shape able to discriminate taxa at tested levels (from species to populations).

Differentiation and extracellular matrix interactions of chondrocytes in response to signaling molecules and biomaterials for cartilage repair = Differenzierung und Interaktionen von Chondrozyten mit der extrazellulären Matrix als Reaktion auf Signalmoleküle und Biomaterialien für die Wiederinstandsetzung von Knorpel

Chondrocytes live isolated in the voluminous extracellular matrix of cartilage, which they secrete and is neither vascularized nor innervated. Nutrient and waste exchanges occur through diffusion leading to low oxygen tension around the cells. Consequently even normal cartilage under normal physiological conditions suffers from a poor reparative potential that predisposes to degenerative conditions, such as osteoarthritis of the joints, with significant clinical effects.rnOne of the key challenges in medicine is the structural and functional replacement of lost or damaged tissues. Current therapeutical approaches are to transplant cells, implant bioartificial tissues, and chemically induce regeneration at the site of the injury. None of them reproduces well the biological and biomechanical properties of hyaline cartilage.rnThis thesis investigates the re-differentiation of chondrocytes and the repair of cartilage mediated by signaling molecules, biomaterials, and factors provided in mixed cellular cultures (co-culture systems). As signaling molecules we have applied prostaglandin E2 (PGE2) and bone morphogenetic protein 1 (BMP-1) and we have transfected chondrocytes with BMP-1 expressing vectors. Our biomaterials have been hydrogels of type-I collagen and gelatin-based scaffolds designed to mimic the architecture and biochemistry of native cartilage and provide a suitable three-dimensional environment for the cells. We have brought chondrocytes to interact with osteosarcoma Cal 72 cells or with murine preosteoblastic KS483 cells, either in a cell-to-cell or in a paracrine manner.rnExogenous stimulation with PGE2 or BMP-1 did not improve the differentiation or the proliferation of human articular chondrocytes. BMP-1 induced chondrocytic de-differentiation in a dose-dependent manner. Prostaglandin stimulation from gelatin-based scaffolds (three-dimensional culture) showed a certain degree of chondrocyte re-differentiaton. Murine preosteoblastic KS483 cells had no beneficial effect on human articular chondrocytes jointly cultivated with them in hydrogels of type I collagen. Although the hydrogels provided the chondrocytes with a proper matrix in which the cells adopted their native morphology; additionally, the expression of chondrocytic proteoglycan increased in the co-cultures after two weeks. The co-culture of chondrocytes with osteoblast-like cells (in transwell systems) resulted in suppression of the regular de-differentiation program that passaged chondrocytes undergo when cultured in monolayers. Under these conditions, the extracellular matrix of the chondrocytes, rich in type-II collagen and aggrecan, was not transformed into the extracellular matrix characteristic of de-differentiated human articular chondrocytes, which is rich in type-I collagen and versican.rnThis thesis suggests novel strategies of tissue engineering for clinical attempts to improve cartilage repair. Since implants are prepared in vitro (ex-vivo) by expanding human articular chondrocytes (autologous or allogeneic), we conclude that it will be convenient to provide a proper three-dimensional support to the chondrocytes in culture, to supplement the culture medium with PGE2, and to stimulate chondrocytes with osteoblastic factors by cultivating them with osteoblasts.rn

Mesenchymal Stromal Cells (MSCs) and induced Plutipotent Stem Cells (iPSCs) in Domestic Animals: Characterization and Differentiation Potential

Derivation of stem cell lines from domesticated animals has been of great interest as it benefits translational medicine, clinical applications to improve human and animal health and biotechnology. The main types of stem cells studied are Embryonic Stem Cells (ESCs), induced Pluripotent Stem Cells (iPSCs) and Mesenchymal Stem/Stromal Cells (MSCs). This thesis had two main aims: (I) The isolation of bovine MSCs from amniotic fluid (AF) at different trimesters of pregnancy and their characterization to study pluripotency markers expression. Stemness markers were studied also in MSCs isolated from equine AF, Wharton’s jelly (WJ) and umbilical cord blood (UCB) as continuation of the characterization of these cells previously performed by our research group; (II) The establishment and characterization of iPSCs lines in two attractive large animal models for biomedical and biotechnology research such as the bovine and the swine, and the differentiation into the myogenic lineage of porcine iPSCs. It was observed that foetal tissues in domestic animals such as the bovine and the horse represent a source of MSCs able to differentiate into the mesodermal lineage but they do not proliferate indefinitely and they lack the expression of many pluripotency markers, making them an interesting source of cells for regenerative medicine, but not the best candidate to elucidate pluripotency networks. The protocol used to induce pluripotency in bovine fibroblasts did not work, as well as the chemical induction of pluripotency in porcine fibroblasts, while the reprogramming protocol used for porcine iPSCs was successful and the line generated was amenable to being differentiated into the myogenic lineage, demonstrating that they could be addressed into a desired lineage by genetic modification and appropriated culture conditions. Only a few cell types have been differentiated from domestic animal iPSCs to date, so the development of a reliable directed-differentiation protocol represents a very important result.

Optimisation of chemical lysis protocol for point-of-care leukocyte differentiation

The full blood cell (FBC) count is the most common indicator of diseases. At present hematology analyzers are used for the blood cell characterization, but, recently, there has been interest in using techniques that take advantage of microscale devices and intrinsic properties of cells for increased automation and decreased cost. Microfluidic technologies offer solutions to handling and processing small volumes of blood (2-50 uL taken by finger prick) for point-of-care(PoC) applications. Several PoC blood analyzers are in use and may have applications in the fields of telemedicine, out patient monitoring and medical care in resource limited settings. They have the advantage to be easy to move and much cheaper than traditional analyzers, which require bulky instruments and consume large amount of reagents. The development of miniaturized point-of-care diagnostic tests may be enabled by chip-based technologies for cell separation and sorting. Many current diagnostic tests depend on fractionated blood components: plasma, red blood cells (RBCs), white blood cells (WBCs), and platelets. Specifically, white blood cell differentiation and counting provide valuable information for diagnostic purposes. For example, a low number of WBCs, called leukopenia, may be an indicator of bone marrow deficiency or failure, collagen- vascular diseases, disease of the liver or spleen. The leukocytosis, a high number of WBCs, may be due to anemia, infectious diseases, leukemia or tissue damage. In the laboratory of hybrid biodevices, at the University of Southampton,it was developed a functioning micro impedance cytometer technology for WBC differentiation and counting. It is capable to classify cells and particles on the base of their dielectric properties, in addition to their size, without the need of labeling, in a flow format similar to that of a traditional flow cytometer. It was demonstrated that the micro impedance cytometer system can detect and differentiate monocytes, neutrophils and lymphocytes, which are the three major human leukocyte populations. The simplicity and portability of the microfluidic impedance chip offer a range of potential applications in cell analysis including point-of-care diagnostic systems. The microfluidic device has been integrated into a sample preparation cartridge that semi-automatically performs erythrocyte lysis before leukocyte analysis. Generally erythrocytes are manually lysed according to a specific chemical lysis protocol, but this process has been automated in the cartridge. In this research work the chemical lysis protocol, defined in the patent US 5155044 A, was optimized in order to improve white blood cell differentiation and count performed by the integrated cartridge.

The role of SENP1 in B cell development and differentiation

SUMOylation is a highly dynamic and reversible posttranslational protein modification closely related to ubiquitination. SUMOylation regulates a vast array of different cellular functions, such as cell cycle, nuclear transport, DNA damage response, proliferation and transcriptional activation. Several groups have shown in in vitro studies how important SUMOylation is for early B cell development and survival as well as for later plasma cell differentiation. This thesis focuses on the deSUMOylation protease SENP1 and its in vivo effects on B cell development and differentiation. For this a conditional SENP1 knockout mouse model was crossed to the CD19-Cre mouse strain to generate a B cell specific SENP1 knockout mouse.rnIn our conditional SENP1ff CD19-Cre mouse model we observed normal numbers of all B cell subsets in the bone marrow. However in the spleen we observed an impairment of B cell survival, based on a 50% reduction of the follicular B cell compartment, whereas the marginal zone B cell compartment was unchanged. T cell numbers were comparable to control mice. rnFurther, impairments of B cell survival in SENP1ff CD19-Cre mice were analysed after in vivo blocking of IL7R signalling. The αIL7R treatment in mature mice blocked new B cell formation in the bone marrow and increased apoptosis rates could be observed in splenic SENP1 KO B cells. Additionally, a higher turnover rate of B cells was measured by in vivo BrdU incorporation.rnSince it is known that the majority of transcription factors that are important for the maintenance of the germinal centre reaction or for induction of plasma cell development are SUMOylated, the question arose, how defective deSUMOylation will manifest itself in these processes. The majority of in vitro cultured splenic B cells, stimulated to undergo class switch recombination and plasma cell differentiation underwent activation induced cell death. However, the surviving cells increasingly differentiated into IgM expressing plasma cells. Class switch recombination to IgG1 was reduced. These observations stood in line with observation made in in vivo sheep red blood cell immunization experiments, which showed increased amounts of germinal centres and germinal centre B cells, as well as increased amounts of plasma cells differentiation in combination with decreased class switch to IgG1.rnThese results lead to the conclusion that SENP1 KO B cells increasingly undergo apoptosis, however, B cells that survive SENP1 deficiency are more prone to undergo plasma cell differentiation. Further, the precursors of these plasma cells either are not as capable of undergoing class switch recombination or they do switch to IgG1 and succumb to activation induced cell death. One possible explanation for both scenarios could be a defective DNA damage response mechanisms during class switch recombination, caused by impaired deSUMOylation. rn