Down Syndrome: Neuropsychological phenotype and mitochondrial DNA

Introduction. Down Syndrome (DS) is the most known autosomal trisomy, due to the presence in three copies of chromosome 21. Many studies were designed to identify phenotypic and clinical consequences related to the triple gene dosage. However, the general conclusion is a senescent phenotype; in particular, the most features of physiological aging, such as skin and hair changes, vision and hearing impairments, thyroid dysfunction, Alzheimer-like dementia, congenital heart defects, gastrointestinal malformations, immune system changes, appear in DS earlier than in normal age-matched subjects. The only established risk factor for the DS is advanced maternal age, responsible for changes in the meiosis of oocytes, in particular the meiotic nondisjunction of chromosome 21. In this process mitochondria play an important role since mitochondrial dysfunction, due to a variety of extrinsic and intrinsic influences, can profoundly influence the level of ATP generation in oocytes, required for a correct chromosomal segregation. Aim. The aim of this study is to investigate an integrated set of molecular genetic parameters (sequencing of complete mtDNA, heteroplasmy of the mtDNA control region, genotypes of APOE gene) in order to identify a possible association with the early neurocognitive decline observed in DS. Results. MtDNA point mutations do not accumulate with age in our study sample and do not correlate with early neurocognitive decline of DS subjects. It seems that D-loop heteroplasmy is largely not inherited and tends to accumulate somatically. Furthermore, in our study sample no association of cognitive impairment and ApoE genotype is found. Conclusions. Overall, our data cast some doubts on the involvement of these mutations in the decline of cognitive functions observed in DS.

An architecture for scaling ontology networks

Constructing ontology networks typically occurs at design time at the hands of knowledge engineers who assemble their components statically. There are, however, use cases where ontology networks need to be assembled upon request and processed at runtime, without altering the stored ontologies and without tampering with one another. These are what we call "virtual [ontology] networks", and keeping track of how an ontology changes in each virtual network is called "multiplexing". Issues may arise from the connectivity of ontology networks. In many cases, simple flat import schemes will not work, because many ontology managers can cause property assertions to be erroneously interpreted as annotations and ignored by reasoners. Also, multiple virtual networks should optimize their cumulative memory footprint, and where they cannot, this should occur for very limited periods of time. We claim that these problems should be handled by the software that serves these ontology networks, rather than by ontology engineering methodologies. We propose a method that spreads multiple virtual networks across a 3-tier structure, and can reduce the amount of erroneously interpreted axioms, under certain raw statement distributions across the ontologies. We assumed OWL as the core language handled by semantic applications in the framework at hand, due to the greater availability of reasoners and rule engines. We also verified that, in common OWL ontology management software, OWL axiom interpretation occurs in the worst case scenario of pre-order visit. To measure the effectiveness and space-efficiency of our solution, a Java and RESTful implementation was produced within an Apache project. We verified that a 3-tier structure can accommodate reasonably complex ontology networks better, in terms of the expressivity OWL axiom interpretation, than flat-tree import schemes can. We measured both the memory overhead of the additional components we put on top of traditional ontology networks, and the framework's caching capabilities.

Phenotype and genotype characterization of Monilinia spp. isolates and preformed antifungal compounds in peach peel fruit at different developmental stages

The brown rot fungi belong to a group of fungal pathogens that causes considerable damage to cultivated fruits trees, particularly stone fruits and apples in the temperate regions of the World and during the postharvest with an important economic impact. In particular in Italy, it is important to monitor the Monilinia population to control economic losses associated to the peach and nectarine market. This motivates the research steps presented in this dissertation on Monilinia Italian isolates. The Monilinia species collected from stone fruits have been identified using molecular analysis based on specific primers. The relevant role of M. fructicola was confirmed and, for the first time, it was found also on apple fruits. To avoid the development of resistant strains and implement valid treatment strategies, the understanding of the fruit natural resistance during different developmental stages and the assessment of the Monilinia sensitivity/resistance to fungicides are required. The relationship between the inhibition spots and the phenolic compounds in peach fruit peel was highlighted in this research. Three methods were used to assess isolate resistance/sensitivity, the amended medium, the Spiral Gradient Endpoint Method (SGD) and the Alamar Blue method. The PCR was used to find possible mutation points in the b-tubulin gene that is responsible for fungicide resistance. Interestingly, no mutation points were observed in resistant M. laxa isolates, suggesting that the resistance could be stimulated by environmental factors. This lead to the study of the effect of the temperature on the resistance and the preliminary results of in vitro tests showed that maximum inhibition was observed at 30°C.

Environmental influence on the phenotype: Morphological variation in human dentition

This work is about the role that environment plays in the production of evolutionary significant variations. It starts with an historical introduction about the concept of variation and the role of environment in its production. Then, I show how a lack of attention to these topics may lead to serious mistakes in data interpretation. A statistical re-analysis of published data on the effects of malnutrition on dental eruption, shows that what has been interpreted as an increase in the mean value, is actually linked to increase of variability. In Chapter 3 I present the topic of development as a link between variability and environmental influence, giving a review of the possible mechanisms by which development influences evolutionary dynamics. Chapter 4 is the core chapter of the thesis; I investigated the role of environment in the development of dental morphology. I used dental hypoplasia as a marker of stress, characterizing two groups. Comparing the morphology of upper molars in the two groups, three major results came out: (i) there is a significant effect of environmental stressors on the overall morphology of upper molars; (ii) the developmental response increases morphological variability of the stressed population; (iii) increase of variability is directional: stressed individuals have increased cusps dimensions and number. I also hypothesized the molecular mechanisms that could be responsible of the observed effects. In Chapter 5, I present future perspectives for developing this research. The direction of dental development response is the same direction of the trend in mammalian dental evolution. Since malnutrition triggers the developmental response, and this particular kind of stressor must have been very common in our class evolutionary history, I propose the possibility that environmental stress actively influenced mammals evolution. Moreover, I discuss the possibility of reconsidering the role of natural selection in the evolution of dental morphology.

Transkriptom des Meristemgewebes des Kolumnarapfels

Kolumnare Apfelbäume (Malus x domestica) stellen aufgrund ihres auffälligen Phänotyps eine ökonomisch interessante Wuchsform dar. Diese extreme Form des Kurztriebwuchses zeichnet sich durch einen insgesamt sehr schlanken, säulenförmigen Habitus aus, welcher eine dichte Pflanzung und damit einhergehend Ertragssteigerungen im Vergleich zu normalwüchsigen Bäumen ermöglicht. Verursacht wird der Phänotyp durch die Anwesenheit eines einzelnen, dominanten Allels des Columnar (Co)-Gens. Bis auf die approximative Lokalisation des Gens auf Chromosom 10 ist über mögliche Identität und Funktion bislang nichts bekannt.rnIn der vorliegenden Arbeit wurde ein erster Versuch unternommen, mit Hilfe von Next Generation Sequencing (NGS) Technologien und RNA-Seq Einblicke in das Transkriptom des Sprossapikalmeristems (SAM) kolumnarer Apfelbäume zu gewinnen. So konnte gezeigt werden, dass unabhängig vom Zeitpunkt der Entnahme des Materials mehrere hundert Gene differentiell reguliert werden. Diese lassen sich funktional in mehrere überrepräsentierte Kategorien gruppieren, von denen sich einige wiederum mit dem kolumnaren Phänotyp assoziieren lassen. Durch den Einsatz weiterer Expressionsstudien (Microarrays, qRT-PCR) konnten frühere Ergebnisse bezüglich des Hormonhaushalts auf Genebene bestätigt und neue Erkenntnisse gewonnen werden, die eine mögliche Erklärung für den Phänotyp darstellen. Weiterhin ergab der Vergleich aller durchgeführten Expressionsstudien eine Anreicherung signifikant differentiell regulierter Gene auf Chromosom 10, was auf einen „selective sweep“ hindeutet. Eine potentielle epigenetische Regulation dieser Gene durch das Genprodukt von Co könnte daher möglich sein. Mehr als die Hälfte dieser Gene lassen sich darüber hinaus aufgrund ihrer Funktion direkt mit dem kolumnaren Phänotyp assoziieren.rnDiese Ergebnisse zeigen, dass die Anwesenheit des Co-Allels massive Veränderungen in der Genregulation des SAMs mit sich bringt, wobei einige dieser differentiell regulierten Gene mit großer Wahrscheinlichkeit an der Etablierung des kolumnaren Phänotyps beteiligt sind. Auch wenn die Funktion des Co-Genproduktes nicht abschließend geklärt werden konnte, sind doch anhand der Resultate schlüssige Hypothesen diesbezüglich möglich.rn

Role of Magnesium and its mitochondrial transporter MRS2 in the modulation of drug-induced apoptosis leading to multidrug resistance phenotype

Magnesium is an essential element for many biological processes crucial for cell life and proliferation. Growing evidences point out a role for this cation in the apoptotic process and in developing multi drug resistance (MDR) phenotype. The first part of this study aimed to highlight the involvement of the mitochondrial magnesium channel MRS2 in modulating drug-induced apoptosis. We generated an appropriate transgenic cellular system to regulate expression of MRS2 protein. The cells were then exposed to two different apoptotic agents commonly used in chemotherapy. The obtained results showed that cells overexpressing MRS2 channel are less responsiveness to pharmacological insults, looking more resistant to the induced apoptosis. Moreover, in normal condition, MRS2 overexpression induces higher magnesium uptake into isolated mitochondria respect to control cells correlating with an increment of total intracellular magnesium concentration. In the second part of this research we investigated whether magnesium intracellular content and compartmentalization could be used as a signature to discriminate MDR tumour cells from their sensitive counterparts. As MDR model we choose colon carcinoma cell line sensitive and resistant to doxorubicin. We exploited a standard-less approach providing a complete characterization of whole single-cells by combining X-Ray Fluorescence Microscopy , Atomic Force Microscopy and Scanning Transmission X-ray Microscopy. This method allows the quantification of the intracellular spatial distribution and total concentration of magnesium in whole dehydrated cells. The measurements, carried out in 27 single cells, revealed a different magnesium pattern for both concentration and distribution of the element in the two cellular strains. These results were then confirmed by quantifying the total amount of intracellular magnesium in a large populations of cells by using DCHQ5 probe and traditional fluorimetric technique.

Pemphigus herpetiformis: analysis of the autoantibody profile during the disease course with changes in the clinical phenotype

Pemphigus herpetiformis (PH) is a rare dapsone-responsive variant of pemphigus, characterized by annular and vesiculopustular cutaneous lesions. Most PH serum samples contain autoantibodies against desmoglein (Dsg)1, but not Dsg3, and the presence of the latter is almost invariably associated with mucosal involvement, as predicted based on the 'Dsg compensation theory'. We describe a patient with features characteristic of PH with histologically eosinophilic spongiosis who repeatedly tested positive for anti-Dsg3 but not anti-Dsg1 autoantibodies by ELISA. To investigate whether the peculiar clinical phenotype was due to a distinct immunological profile, the patient's serum was tested by ELISA and immunoblotting using recombinant forms of Dsg3. Serum samples were found to have low and high reactivity against the EC1 and the EC4 domains of Dsg3, respectively, whereas the autoantibodies belonged predominantly to the IgG1 and IgG4 subclasses. The overall immunological profile was typical of pemphigus vulgaris. The patient finally developed isolated oral erosions 22 months after initial presentation, without significant changes in the autoantibody profile and of the targeted antigenic sites. Our patient presented features characteristic of PH. Although circulating anti-Dsg3 antibodies were present, the patient had only cutaneous involvement for a long period. Our findings indicate that the proposed Dsg compensation theory cannot always explain the clinical phenotype, changes in which may occur without apparent modification of the autoantibody profile and antibody specificity. Hence, additional factors, such as Fcgamma-dependent neutrophil activation, may critically affect the clinical presentation of pemphigus.

ABCA4 and ROM1: Implications for modification of the PRPH2-associated macular dystrophy phenotype

To identify the causative mutation leading to autosomal dominant macular dystrophy, cone dystrophy, and cone-rod dystrophy in a five-generation family and to explain the high intrafamilial phenotypic variation by identifying possible modifier genes.

Endothelial Progenitor Cells Induce a Phenotype Shift in Differentiated Endothelial Cells towards PDGF/PDGFR Axis-Mediated Angiogenesis

BACKGROUND: Endothelial Progenitor Cells (EPC) support neovascularization and regeneration of injured endothelium both by providing a proliferative cell pool capable of differentiation into mature vascular endothelial cells and by secretion of angiogenic growth factors. OBJECTIVE: The aim of this study was to investigate the role of PDGF-BB and PDGFR in EPC-mediated angiogenesis of differentiated endothelial cells. METHODS AND RESULTS: Conditioned medium from human EPC (EPC-CM) cultured in hypoxic conditions contained substantially higher levels of PDGF-BB as compared to normoxic conditions (P<0.01). EPC-CM increased proliferation (1.39-fold; P<0.001) and migration (2.13-fold; P<0.001) of isolated human umbilical vein endothelial cells (HUVEC), as well as sprouting of vascular structures from ex vivo cultured aortic rings (2.78-fold increase; P = 0.01). The capacity of EPC-CM to modulate the PDGFR expression in HUVEC was assessed by western blot and RT-PCR. All the pro-angiogenic effects of EPC-CM on HUVEC could be partially inhibited by inactivation of PDGFR (P<0.01). EPC-CM triggered a distinct up-regulation of PDGFR (2.5±0.5; P<0.05) and its phosphorylation (3.6±0.6; P<0.05) in HUVEC. This was not observed after exposure of HUVEC to recombinant human PDGF-BB alone. CONCLUSION: These data indicate that EPC-CM sensitize endothelial cells and induce a pro-angiogenic phenotype including the up-regulation of PDGFR , thereby turning the PDGF/PDGFR signaling-axis into a critical element of EPC-induced endothelial angiogenesis. This finding may be utilized to enhance EPC-based therapy of ischemic tissue in future.

Loss of the CBX7 protein expression correlates with a more aggressive phenotype in pancreatic cancer

Polycomb group (PcG) proteins function as multiprotein complexes and are part of a gene regulatory mechanism that determines cell fate during normal and pathogenic development. Several studies have implicated the deregulation of different PcG proteins in neoplastic progression. Pancreatic ductal adenocarcinoma is an aggressive neoplasm that follows a multistep model of progression through precursor lesions called pancreatic intraepithelial neoplasia (PanIN). Aim of this study was to investigate the role of PcG protein CBX7 in pancreatic carcinogenesis and to evaluate its possible diagnostic and prognostic significance. We analysed by immunohistochemistry the expression of CBX7 in 210 ductal pancreatic adenocarcinomas from resection specimens, combined on a tissue microarray (TMA) including additional 40 PanIN cases and 40 normal controls. The results were evaluated by using receiver operating characteristic (ROC) curve analysis for the selection of cut-off scores and correlated to the clinicopathological parameters of the tumours and the outcome of the patients. Expression of E-cadherin, a protein positively regulated by CBX7, was also assessed. A significantly differential, and progressively decreasing CBX7 protein expression was found between normal pancreatic tissue, PanINs and invasive ductal adenocarcinoma. Loss of CBX7 expression was associated with increasing malignancy grade in pancreatic adenocarcinoma, whereas the maintenance of CBX7 expression showed a trend toward a longer survival. Moreover, loss of E-cadherin expression was associated with loss of CBX7 and with a trend towards worse patient survival. These results suggest that CBX7 plays a role in pancreatic carcinogenesis and that its loss of expression correlates to a more aggressive phenotype.

Muscle unloading potentiates the effects of acetyl-L-carnitine on the slow oxidative muscle phenotype

The effect of acetyl-L-carnitine (ALCAR) supplementation to 3-month-old rats in normal-loading and unloading conditions has been here investigated by a combined morphological, biochemical and transcriptional approach to test whether ALCAR might cause a remodeling of the metabolic/contractile phenotype of soleus muscle. Morphological assessment demonstrated an increase of type I oxidative fiber content and cross-sectional area in ALCAR-treated animals both in normal-loading and in unloading conditions. ALCAR prevented loss of mitochondrial mass in unloaded animals whereas no ALCAR-dependent increase of mitochondrial mass occurred in normal-loaded muscle. Validated microarray analysis delineated an ALCAR-induced maintenance of a slow-oxidative expression program only in unloaded soleus muscle. Indeed, the muscle adjustment of the expression profile of factors underlying mitochondrial oxidative metabolism, protein turnover, fiber type differentiation and an adaptation of voltage-gated ion channel expression was distinguishable with respect to the loading status. This selectivity may suggest a key role of muscle loading status in the manifestation of ALCAR effects. The results extend to a broader level of biological informations the previous notion on ALCAR positive effect in rat soleus muscle during unloading and point to a role of ALCAR for the maintenance of its slow-oxidative fiber character.

Hemizygous deletion of COL3A1, COL5A2, and MSTN causes a complex phenotype with aortic dissection: a lesson for and from true haploinsufficiency

Aortic dilatation/dissection (AD) can occur spontaneously or in association with genetic syndromes, such as Marfan syndrome (MFS; caused by FBN1 mutations), MFS type 2 and Loeys-Dietz syndrome (associated with TGFBR1/TGFBR2 mutations), and Ehlers-Danlos syndrome (EDS) vascular type (caused by COL3A1 mutations). Although mutations in FBN1 and TGFBR1/TGFBR2 account for the majority of AD cases referred to us for molecular genetic testing, we have obtained negative results for these genes in a large cohort of AD patients, suggesting the involvement of additional genes or acquired factors. In this study we assessed the effect of COL3A1 deletions/duplications in this cohort. Multiplex ligation-dependent probe amplification (MLPA) analysis of 100 unrelated patients identified one hemizygous deletion of the entire COL3A1 gene. Subsequent microarray analyses and sequencing of breakpoints revealed the deletion size of 3,408,306 bp at 2q32.1q32.3. This deletion affects not only COL3A1 but also 21 other known genes (GULP1, DIRC1, COL5A2, WDR75, SLC40A1, ASNSD1, ANKAR, OSGEPL1, ORMDL1, LOC100129592, PMS1, MSTN, C2orf88, HIBCH, INPP1, MFSD6, TMEM194B, NAB1, GLS, STAT1, and STAT4), mutations in three of which (COL5A2, SLC40A1, and MSTN) have also been associated with an autosomal dominant disorder (EDS classical type, hemochromatosis type 4, and muscle hypertrophy). Physical and laboratory examinations revealed that true haploinsufficiency of COL3A1, COL5A2, and MSTN, but not that of SLC40A1, leads to a clinical phenotype. Our data not only emphasize the impact/role of COL3A1 in AD patients but also extend the molecular etiology of several disorders by providing hitherto unreported evidence for true haploinsufficiency of the underlying gene.

Comprehensive analysis of CpG island methylator phenotype (CIMP)-high, -low, and -negative colorectal cancers based on protein marker expression and molecular features

CpG island methylator phenotype (CIMP) is being investigated for its role in the molecular and prognostic classification of colorectal cancer patients but is also emerging as a factor with the potential to influence clinical decision-making. We report a comprehensive analysis of clinico-pathological and molecular features (KRAS, BRAF and microsatellite instability, MSI) as well as of selected tumour- and host-related protein markers characterizing CIMP-high (CIMP-H), -low, and -negative colorectal cancers. Immunohistochemical analysis for 48 protein markers and molecular analysis of CIMP (CIMP-H: ? 4/5 methylated genes), MSI (MSI-H: ? 2 instable genes), KRAS, and BRAF were performed on 337 colorectal cancers. Simple and multiple regression analysis and receiver operating characteristic (ROC) curve analysis were performed. CIMP-H was found in 24 cases (7.1%) and linked (p < 0.0001) to more proximal tumour location, BRAF mutation, MSI-H, MGMT methylation (p = 0.022), advanced pT classification (p = 0.03), mucinous histology (p = 0.069), and less frequent KRAS mutation (p = 0.067) compared to CIMP-low or -negative cases. Of the 48 protein markers, decreased levels of RKIP (p = 0.0056), EphB2 (p = 0.0045), CK20 (p = 0.002), and Cdx2 (p < 0.0001) and increased numbers of CD8+ intra-epithelial lymphocytes (p < 0.0001) were related to CIMP-H, independently of MSI status. In addition to the expected clinico-pathological and molecular associations, CIMP-H colorectal cancers are characterized by a loss of protein markers associated with differentiation, and metastasis suppression, and have increased CD8+ T-lymphocytes regardless of MSI status. In particular, Cdx2 loss seems to strongly predict CIMP-H in both microsatellite-stable (MSS) and MSI-H colorectal cancers. Cdx2 is proposed as a surrogate marker for CIMP-H.

On the traces of XPD: cell cycle matters - untangling the genotype-phenotype relationship of XPD mutations

Abstract Mutations in the human gene coding for XPD lead to segmental progeria - the premature appearance of some of the phenotypes normally associated with aging - which may or may not be accompanied by increased cancer incidence. XPD is required for at least three different critical cellular functions: in addition to participating in the process of nucleotide excision repair (NER), which removes bulky DNA lesions, XPD also regulates transcription as part of the general transcription factor IIH (TFIIH) and controls cell cycle progression through its interaction with CAK, a pivotal activator of cyclin dependent kinases (CDKs). The study of inherited XPD disorders offers the opportunity to gain insights into the coordination of important cellular events and may shed light on the mechanisms that regulate the delicate equilibrium between cell proliferation and functional senescence, which is notably altered during physiological aging and in cancer. The phenotypic manifestations in the different XPD disorders are the sum of disturbances in the vital processes carried out by TFIIH and CAK. In addition, further TFIIH- and CAK-independent cellular activities of XPD may also play a role. This, added to the complex feedback networks that are in place to guarantee the coordination between cell cycle, DNA repair and transcription, complicates the interpretation of clinical observations. While results obtained from patient cell isolates as well as from murine models have been elementary in revealing such complexity, the Drosophila embryo has proven useful to analyze the role of XPD as a cell cycle regulator independently from its other cellular functions. Together with data from the biochemical and structural analysis of XPD and of the TFIIH complex these results combine into a new picture of the XPD activities that provides ground for a better understanding of the patophysiology of XPD diseases and for future development of diagnostic and therapeutic tools.

The Art of Aidagara: Ethics, Aesthetics, and the Quest for an Ontology of Social Existence in Watsuji Tetsurō’s Rinrigaku

This paper provides an analysis of the key term aidagara (“betweenness”) in the philosophical ethics of Watsuji Tetsurō (1889-1960), in response to and in light of the recent movement in Japanese Buddhist studies known as “Critical Buddhism.” The Critical Buddhist call for a turn away from “topical” or intuitionist thinking and towards (properly Buddhist) “critical” thinking, while problematic in its bipolarity, raises the important issue of the place of “reason” versus “intuition” in Japanese Buddhist ethics. In this paper, a comparison of Watsuji’s “ontological quest” with that of Martin Heidegger (1889-1976), Watsuji’s primary Western source and foil, is followed by an evaluation of a corresponding search for an “ontology of social existence” undertaken by Tanabe Hajime (1885-1962). Ultimately, the philosophico-religious writings of Watsuji Tetsurō allow for the “return” of aesthesis as a modality of social being that is truly dimensionalized, and thus falls prey neither to the verticality of topicalism nor the limiting objectivity of criticalism.