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.

Sonic Hedgehog pathway impairment in Neural Precursor Cells of the Ts65Dn mouse, an animal model of Down syndrome

Mental retardation in Down syndrome (DS) has been imputed to the decreased brain volume, which is evident starting from the early phases of development. Recent studies in a widely used mouse model of DS, the Ts65Dn mouse, have shown that neurogenesis is severely impaired during the early phases of brain development, suggesting that this defect may be a major determinant of brain hypotrophy and mental retardation in individuals with DS. Recently, it has been found that in the cerebellum of Ts65Dn mice there is a defective responsiveness to Sonic Hedgehog (Shh), a potent mitogen that controls cell division during brain development, suggesting that failure of Shh signaling may underlie the reduced proliferation potency in DS. Based on these premises, we sought to identify the molecular mechanisms underlying derangement of the Shh pathway in neural precursor cells (NPCs) from Ts65Dn mice. We found that the expression levels of the Shh receptor Patched1 (Ptch1) were increased compared to controls both at the RNA and protein level. Partial silencing of Ptch1 expression in trisomic NPCs restored cell proliferation, indicating that proliferation impairment was due to Ptch1 overexpression. We further found that the overexpression of Ptch1 in trisomic NPCs is related to increased levels of AICD, a transcription-promoting fragment of amyloid precursor protein (APP). Increased AICD binding to the Ptch1 promoter favored its acetylated status, thus enhancing Ptch1 expression. Taken together, these data provide novel evidence that Ptch1 over expression underlies derangement of the Shh pathway in trisomic NPCs, with consequent proliferation impairment. The demonstration that Ptch1 over expression in trisomic NPCs is due to an APP fragment provides a link between this trisomic gene and the defective neuronal production that characterizes the DS brain.

Pharmacological Rescue of Dendritic Pathology in the Ts65Dn Mouse Model of Down Syndrome

Down syndrome (DS) is a genetic pathology characterized by brain hypotrophy and severe cognitive disability. Although defective neurogenesis is an important determinant of cognitive impairment, a severe dendritic pathology appears to be an equally important factor. It is well established that serotonin plays a pivotal role both on neurogenesis and dendritic maturation. Since the serotonergic system is profoundly altered in the DS brain, we wondered whether defects in the hippocampal development can be rescued by treatment with fluoxetine, a selective serotonin reuptake inhibitor and a widely used antidepressant drug. A previous study of our group showed that fluoxetine fully restores neurogenesis in the Ts65Dn mouse model of DS and that this effect is accompanied by a recovery of memory functions. The goal of the current study was to establish whether fluoxetine also restores dendritic development and maturation. In mice aged 45 days, treated with fluoxetine in the postnatal period P3-P15, we examined the dendritic arbor of newborn and mature granule cells of the dentate gyrus (DG). The granule cells of trisomic mice had a severely hypotrophic dendritic arbor, fewer spines and a reduced innervation than euploid mice. Treatment with fluoxetine fully restored all these defects. Moreover the impairment of excitatory and inhibitory inputs to CA3 pyramidal neurons was fully normalized in treated trisomic mice, indicating that fluoxetine can rescue functional connectivity between the DG and CA3. The widespread beneficial effects of fluoxetine on the hippocampal formation suggest that early treatment with fluoxetine can be a suitable therapy, possibly usable in humans, to restore the physiology of the hippocampal networks and, hence, memory functions. These findings may open the way for future clinical trials in children and adolescents with DS.

Epigenetic signature in persons with Down Syndrome

Persons affected by Down Syndrome show a heterogeneous phenotype that includes developmental defects and cognitive and haematological disorders. Premature accelerated aging and the consequent development of age associated diseases like Alzheimer Disease (AD) seem to be the cause of higher mortality late in life of DS persons. Down Syndrome is caused by the complete or partial trisomy of chromosome 21, but it is not clear if the molecular alterations of the disease are triggered by the specific functions of a limited number of genes on chromosome 21 or by the disruption of genetic homeostasis due the presence of a trisomic chromosome. As epigenomic studies can help to shed light on this issue, here we used the Infinium HumanMethilation450 BeadChip to analyse blood DNA methylation patterns of 29 persons affected by Down syndrome (DSP), using their healthy siblings (DSS) and mothers (DSM) as controls. In this way we obtained a family-based model that allowed us to monitor possible confounding effects on DNA methylation patterns deriving from genetic and environmental factors. We showed that defects in DNA methylation map in genes involved in developmental, neurological and haematological pathways. These genes are enriched on chromosome 21 but localize also in the rest of the genome, suggesting that the trisomy of specific genes on chromosome 21 induces a cascade of events that engages many genes on other chromosomes and results in a global alteration of genomic function. We also analysed the methylation status of three target regions localized at the promoter (Ribo) and at the 5’ sequences of 18S and 28S regions of the rDNA, identifying differently methylated CpG sites. In conclusion, we identified an epigenetic signature of Down Syndrome in blood cells that sustains a link between developmental defects and disease phenotype, including segmental premature aging.

Sindrome di Down e fattori di rischio nel declino neurocognitivo

Down syndrome (DS) or Trisomy 21, occurring in 1/700 and 1/1000 livebirths, is the most common genetic disorder, characterized by a third copy of the human chromosome 21 (Hsa21). DS is associated with various defects, including congenital heart diseases, craniofacial abnormalities, immune system dysfunction, mental retardation (MR), learning and memory deficiency. The phenotypic features in DS are a direct consequence of overexpression of genes located within the triplicated region on Hsa21. In addition to developmental brain abnormalities and disabilities, people with DS by the age of 30-40 have a greatly increased risk of early-onset of Alzheimer’s disease (AD) and an apparent tendency toward premature aging. Many of the immunological anomalies in DS can be enclosed in the spectrum of multiple signs of early senescence. People with DS have an increased vulnerability to oxidative damage and many factors, including amyloid beta protein (Abeta), genotype ApoE4, oxidative stress, mutations in mitochondrial DNA (mtDNA), impairment of antioxidant enzymes, accelerated neuronal cell apoptosis, are related to neuronal degeneration and early aging in DS. SUBJECTS and METHODS: Since 2007 a population of 50 adolescents and adults with DS, 26 males and 24 females (sex-ratio: M/F = 1.08), has been evaluated for the presence of neurological features, biomarkers and genetic factors correlated with neuronal degeneration and premature aging. The control group was determined by the mother and the siblings of the patients. A neuropsychiatric evaluation was obtained from all patients. The levels of thyroid antibodies (antiTg and antiTPO) and of some biochemical markers of oxidative stress, including homocysteine (tHcy), uric acid, cobalamin, folate were measured. All patients, the mother and the siblings were genotyped for ApoE gene. RESULTS: 40% of patients, with a mild prevalence of females aged between 19 and 30 years, showed increased levels of antiTg and antiTPO. The levels of tHcy were normal in 52% patients and mildly increased in 40%; hyperomocysteinemia was associated with normal levels of thyroid antibodies in 75% of patients (p<0.005). The levels of uric acid were elevated in 26%. Our study showed a prevalence of severe MR in patients aged between 1-18 years and over 30 years. Only 3 patients, 2 females and one male, over 30 years of age, showed dementia. According to the literature, the rate of Down left-handers was high (25%) compared to the rest of population and the laterality was associated with increased levels of thyroid antibodies (70%). 21.5% of patients were ApoE4 positive (ApoE4+) with a mean/severe MR. CONCLUSIONS: Until now no biochemical evidence of oxidative damage and no deficiency or alteration of antioxidant function in our patients with DS were found. mtDNA sequencing could show some mutations age-related and associated with oxidative damage and neurocognitive decline in the early aging of DS. The final aim is found predictive markers of early-onset dementia and a target strategy for the prevention and the treatment of diseases caused by oxidative stress. REFERENCES: 1) Rachidi M, Lopes C: “Mental retardation and associated neurological dysfunctions in Down syndrome: a consequence of dysregulation in critical chromosome 21 genes and associated molecular pathways.” - Eur J Paediatr Neurol. May;12(3):168-82 (2008). 2) Lott IT, Head E: “Down syndrome and Alzheimer's disease: a link between development and aging.” - Ment Retard Dev Disabil Res Rev, 7(3):172-8 (2001). 3) Lee HC, Wei YH: “Oxidative Stress, Mitochondrial DNA Mutation, and Apoptosis in Aging.” - Exp Biol Med (Maywood), May;232(5):592-606 (2007).

Glycomics as an innovative technology to identify biomarkers of aging

Introduction. Glycomic analysis allows investigating on the global glycome within body fluids (as serum/plasma), this could eventually lead to identify new types of disease biomarkers, or as in this study, biomarkers of human aging studying specific aging models. Recent studies demonstrated that the plasma N-glycome is modified during human aging, suggesting that measurements of log-ratio of two serum/plasma N-glycans (NGA2F and NA2F), named GlycoAge test could provide a non-invasive biomarker of aging. Down syndrome (DS) is a genetic disorder in which multiple major aspects of senescent phenotype occur much earlier than in healthy age-matched subjects and has been often defined as an accelerated aging syndrome. The aim of this study was to compare plasma N-glycome of patients affected by DS with age- and sex matched non-affected controls, represented by their siblings (DSS), in order to assess if DS is characterized by a specific N-glycomic pattern. Therefore, in order to investigate if N-glycans changes that occur in DS were able to reveal an accelerated aging in DS patients, we enrolled the mothers (DSM) of the DS and DSS, representing the non-affected control group with a different chronological age respect to DS. We applied two different N-glycomics approaches on the same samples: first, in order to study the complete plasma N-glycome we applied a new high-sensitive protocol based on a MALDI-TOF-MS approach, second, we used DSA-FACE technology. Results: MALDI-TOF/MS analysis detected a specific N-glycomics signature for DS, characterized by an increase of fucosylated and bisecting species. Moreover, in DS the abundance of agalactosylated (as NA2F) species was similar or higher than their mothers. The measurement of GlycoAge test with DSA-FACE, validated also by MALDI-TOF, demonstrated a strongly association with age, moreover in DS, it’s value was similar to their mothers, and significantly higher than their age- and sex matched not-affected siblings

Guidelines of Special Care Dentistry in Patients with Chromosomal and Genetic syndromes

Chromosomal and genetic syndromes are frequently associated with dental and cranio-facial alterations. The aim of our study is to identify and describe the dental and craniofacial alterations typical of six genetic and chromosomal syndromes examined. Materials and Methods- A dental visit was performed to 195 patients referred from Sant’Orsola Hospital of Bologna, University of Bologna, to Service of Special Need Dentistry, Dental Clinic, Department of Biomedical and Neuromotor Science, University of Bologna. The patients recruited were 137 females and 58 males, in an age range of 3-49 years (mean age of 13.8±7.4). The total sample consisted of subjects affected with Down Syndrome (n=133), Familiar Hypophosphatemic Ricket (n=10), Muscular Dystrophies (n=12), Noonan Syndrome (n=13), Turner Syndrome (n=17), Williams Syndrome(n=10). A questionnaire regarding detailed medical and dental history, oral health and dietary habits, was filled by parents/caregivers, or patients themselves when possible. The intra-oral and extra-oral examination valued the presence of facial asymmetries, oral habits, dental and skeletal malocclusions, dental formula, dental anomalies, Plaque Index (Silness&LÖe Index), caries prevalence (dmft/DMFT index), gingivitis and periodontal disease, and mucosal lesions. Radiographic examinations (Intraoral radiographies, Orthopanoramic, Skull teleradiography) were executed according to patient’s age and treatment planning. A review of literature about each syndrome and its dental and cranio-facial characteristics and about caries, hygiene status and malocclusion prevalence on syndromic and non-syndromic population was performed. Results - The data of all the patients were collected in the “Data Collection Tables” created for each syndrome. General anamnesis information, oral hygiene habits and dmft/DMFT, PI, malocclusion prevalence were calculated and compared to syndromic and non-syndromic population results found in literature. Discussions and conclusions - Guidelines of Special Care dentistry were indicated for each syndrome, in relation to each syndrome features and individual patient characteristics.

Interazioni tra le glicoproteine D, B, H ed L critiche per la fusione indotta dal virus Herpes simplex

Four glycoproteins (gD, gB, gH, and gL) are required for herpes simplex virus (HSV) entry into the cell and for cell-cell fusion in transfected cells. gD serves as the receptor-binding glycoprotein and as the trigger of fusion; the other three glycoproteins execute fusion between the viral envelope and the plasma or endocytic membranes. Little is known on the interaction of gD with gB, gH, and gL. Here, the interactions between herpes simplex virus gD and its nectin1 receptor or between gD, gB, and gH were analyzed by complementation of the N and C portions of split enhanced green fluorescent protein (EGFP) fused to the glycoproteins. Split EGFP complementation was detected between proteins designated gDN + gHC, gDN + gBC, and gHN + gBC + wtgD, both in cells transfected with two or tree glycoproteins and in cells transfected with the four glycoproteins, commited to form syncytia. The in situ assay provides evidence that gD interacts with gH and gB independently one of the other. We further document the interaction between gH and gB. To elucidate which portions of the glycoproteins interact with each other we generated mutants of gD and gB. gD triggers fusion through a specialised domain, named pro-fusion domain (PFD), located C-terminally in the ectodomain. Here, we show that PFD is made of subdomains 1 and 2 (amino acids 260–285 and 285–310) and that each one partially contributed to herpes simplex virus infectivity. Chimeric gB molecules composed of HSV and human herpesvirus 8 (HHV8) sequences failed to reach the cell surface and to complement a gB defective virus. By means of pull down experiments we analyzed the interactions of HSV-HHV8 gB chimeras with gH or gD fused to the strep-tag. The gB sequence between aa residues 219-360 was identified as putative region of interaction with gH or critical to the interaction.

Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors

The organization of the nervous and immune systems is characterized by obvious differences and striking parallels. Both systems need to relay information across very short and very long distances. The nervous system communicates over both long and short ranges primarily by means of more or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange communication in the immune system occurs mainly via the ordered and guided migration of immune cells and systemically acting soluble factors such as antibodies, cytokines, and chemokines. Its short-range communication either is mediated by locally acting soluble factors or transpires during direct cell–cell contact across specialized areas called “immunological synapses” (Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented by a complex array of factors that induce cell growth and differentiation: these factors in the immune system are called cytokines; in the nervous system, they are called neurotrophic factors. Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000). These factors may allow the two systems to “cross-talk” and eventually may provide a molecular explanation for the reports that inflammation after central nervous system (CNS) injury has beneficial effects (Moalem et al., 1999). In order to shed some more light on such a cross-talk, therefore, transcription factors modulating mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated. More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell receptor induction of MOPr expression in T-lymphocytes. Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated exogenous opioids or endogenous opioid peptides, and contribute to the regulation of several functions including pain transmission, respiration, cardiac and gastrointestinal functions, and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer and Hollt 2006). Recently, induction of MOPr expression in different immune cells induced by cytokines has been reported (Kraus et al., 2001; Kraus et al., 2003). The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has clusters of potential binding sites for different transcription factors (Law et al. 2004). Several studies, primarily focused on the upstream region of the OPRM1 promoter, have investigated transcriptional regulation of MOPr expression. Presently, however, it is still not completely clear how positive and negative transcription regulators cooperatively coordinate cellor tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its expression. IGF-I and its receptors are widely distributed throughout the nervous system during development, and their involvement in neurogenesis has been extensively investigated (Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al., 2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor which, after ligand binding-induced autophosphorylation, associates with specific adaptor proteins and activates different second messengers (Bondy and Cheng 2004). These include: phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al. 2000; Yadav et al. 2005). REST plays a complex role in neuronal cells by differentially repressing target gene expression (Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al. 2006). Thus, the REST concentration seems to influence its function and the expression of neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al. 2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes not yet required by the differentiation program, but its expression decreases after five days of treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the disappearance of REST (Di Toro et al. 2005). The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3 DNA element located in the promoter, increases OPRM1 transcription. T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study whether its signalling influence OPRM1 expression. Results were that TCR engagement determined a significant induction of OPRM1 expression through the activation of transcription factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation and recycling, whereas morphine did not. Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune cells suggest an important role for MOPr as a molecular bridge between neurons and immune cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli and analgesic and neuroprotective effects.

Studies of OPA1 pathogenic mechanisms in Dominant Optic Atrophy and novel protein function in mitochondrial DNA stability

The mitochondrion is an essential cytoplasmic organelle that provides most of the energy necessary for eukaryotic cell physiology. Mitochondrial structure and functions are maintained by proteins of both mitochondrial and nuclear origin. These organelles are organized in an extended network that dynamically fuses and divides. Mitochondrial morphology results from the equilibrium between fusion and fission processes, controlled by a family of “mitochondria-shaping” proteins. It is becoming clear that defects in mitochondrial dynamics can impair mitochondrial respiration, morphology and motility, leading to apoptotic cell death in vitro and more or less severe neurodegenerative disorders in vivo in humans. Mutations in OPA1, a nuclear encoded mitochondrial protein, cause autosomal Dominant Optic Atrophy (DOA), a heterogeneous blinding disease characterized by retinal ganglion cell degeneration leading to optic neuropathy (Delettre et al., 2000; Alexander et al., 2000). OPA1 is a mitochondrial dynamin-related guanosine triphosphatase (GTPase) protein involved in mitochondrial network dynamics, cytochrome c storage and apoptosis. This protein is anchored or associated on the inner mitochondrial membrane facing the intermembrane space. Eight OPA1 isoforms resulting from alternative splicing combinations of exon 4, 4b and 5b have been described (Delettre et al., 2001). These variants greatly vary among diverse organs and the presence of specific isoforms has been associated with various mitochondrial functions. The different spliced exons encode domains included in the amino-terminal region and contribute to determine OPA1 functions (Olichon et al., 2006). It has been shown that exon 4, that is conserved throughout evolution, confers functions to OPA1 involved in maintenance of the mitochondrial membrane potential and in the fusion of the network. Conversely, exon 4b and exon 5b, which are vertebrate specific, are involved in regulation of cytochrome c release from mitochondria, and activation of apoptosis, a process restricted to vertebrates (Olichon et al., 2007). While Mgm1p has been identified thanks to its role in mtDNA maintenance, it is only recently that OPA1 has been linked to mtDNA stability. Missense mutations in OPA1 cause accumulation of multiple deletions in skeletal muscle. The syndrome associated to these mutations (DOA-1 plus) is complex, consisting of a combination of dominant optic atrophy, progressive external ophtalmoplegia, peripheral neuropathy, ataxia and deafness (Amati- Bonneau et al., 2008; Hudson et al., 2008). OPA1 is the fifth gene associated with mtDNA “breakage syndrome” together with ANT1, PolG1-2 and TYMP (Spinazzola et al., 2009). In this thesis we show for the first time that specific OPA1 isoforms associated to exon 4b are important for mtDNA stability, by anchoring the nucleoids to the inner mitochondrial membrane. Our results clearly demonstrate that OPA1 isoforms including exon 4b are intimately associated to the maintenance of the mitochondrial genome, as their silencing leads to mtDNA depletion. The mechanism leading to mtDNA loss is associated with replication inhibition in cells where exon 4b containing isoforms were down-regulated. Furthermore silencing of exon 4b associated isoforms is responsible for alteration in mtDNA-nucleoids distribution in the mitochondrial network. In this study it was evidenced that OPA1 exon 4b isoform is cleaved to provide a 10kd peptide embedded in the inner membrane by a second transmembrane domain, that seems to be crucial for mitochondrial genome maintenance and does correspond to the second transmembrane domain of the yeasts orthologue encoded by MGM1 or Msp1, which is also mandatory for this process (Diot et al., 2009; Herlan et al., 2003). Furthermore in this thesis we show that the NT-OPA1-exon 4b peptide co-immuno-precipitates with mtDNA and specifically interacts with two major components of the mitochondrial nucleoids: the polymerase gamma and Tfam. Thus, from these experiments the conclusion is that NT-OPA1- exon 4b peptide contributes to the nucleoid anchoring in the inner mitochondrial membrane, a process that is required for the initiation of mtDNA replication and for the distribution of nucleoids along the network. These data provide new crucial insights in understanding the mechanism involved in maintenance of mtDNA integrity, because they clearly demonstrate that, besides genes implicated in mtDNA replications (i.e. polymerase gamma, Tfam, twinkle and genes involved in the nucleotide pool metabolism), OPA1 and mitochondrial membrane dynamics play also an important role. Noticeably, the effect on mtDNA is different depending on the specific OPA1 isoforms down-regulated, suggesting the involvement of two different combined mechanisms. Over two hundred OPA1 mutations, spread throughout the coding region of the gene, have been described to date, including substitutions, deletions or insertions. Some mutations are predicted to generate a truncated protein inducing haploinsufficiency, whereas the missense nucleotide substitutions result in aminoacidic changes which affect conserved positions of the OPA1 protein. So far, the functional consequences of OPA1 mutations in cells from DOA patients are poorly understood. Phosphorus MR spectroscopy in patients with the c.2708delTTAG deletion revealed a defect in oxidative phosphorylation in muscles (Lodi et al., 2004). An energetic impairment has been also show in fibroblasts with the severe OPA1 R445H mutation (Amati-Bonneau et al., 2005). It has been previously reported by our group that OPA1 mutations leading to haploinsufficiency are associated in fibroblasts to an oxidative phosphorylation dysfunction, mainly involving the respiratory complex I (Zanna et al., 2008). In this study we have evaluated the energetic efficiency of a panel of skin fibroblasts derived from DOA patients, five fibroblast cell lines with OPA1 mutations causing haploinsufficiency (DOA-H) and two cell lines bearing mis-sense aminoacidic substitutions (DOA-AA), and compared with control fibroblasts. Although both types of DOA fibroblasts maintained a similar ATP content when incubated in a glucose-free medium, i.e. when forced to utilize the oxidative phosphorylation only to produce ATP, the mitochondrial ATP synthesis through complex I, measured in digitonin-permeabilized cells, was significantly reduced in cells with OPA1 haploinsufficiency only, whereas it was similar to controls in cells with the missense substitutions. Furthermore, evaluation of the mitochondrial membrane potential (DYm) in the two fibroblast lines DOA-AA and in two DOA-H fibroblasts, namely those bearing the c.2819-2A>C mutation and the c.2708delTTAG microdeletion, revealed an anomalous depolarizing response to oligomycin in DOA-H cell lines only. This finding clearly supports the hypothesis that these mutations cause a significant alteration in the respiratory chain function, which can be unmasked only when the operation of the ATP synthase is prevented. Noticeably, oligomycin-induced depolarization in these cells was almost completely prevented by preincubation with cyclosporin A, a well known inhibitor of the permeability transition pore (PTP). This results is very important because it suggests for the first time that the voltage threshold for PTP opening is altered in DOA-H fibroblasts. Although this issue has not yet been addressed in the present study, several are the mechanisms that have been proposed to lead to PTP deregulation, including in particular increased reactive oxygen species production and alteration of Ca2+ homeostasis, whose role in DOA fibroblasts PTP opening is currently under investigation. Identification of the mechanisms leading to altered threshold for PTP regulation will help our understanding of the pathophysiology of DOA, but also provide a strategy for therapeutic intervention.

A multifrequency study of the Lockman Hole region: preparing for a LOFAR deep survey

In this thesis, we have presented two deep 1.4 GHz and 345 MHz overlapping surveys of the Lockman Hole field taken with the Westerbork Synthesis Radio Telescope. We extracted a catalogue of ~6000 radio sources from the 1.4 GHz mosaic down to a flux limit of ~55 μJy and a catalogue of 334 radio sources down to a flux limit of ~4 mJy from the inner 7 sq. degree region of the 345 MHz image. The extracted catalogues were used to derive the source number counts at 1.4 GHz and at 345 MHz. The source counts were found to be fully consistent with previous determinations. In particular the 1.4 GHz source counts derived by the present sample provide one of the most statistically robust determinations in the flux range 0.1 < S < 1 mJy. During the commissioning program of the LOFAR telescope, the Lockman Hole field was observed at 58 MHz and 150 MHz. The 150 MHz LOFAR observation is particularly relevant as it allowed us to obtain the first LOFAR flux calibrated high resolution image of a deep field. From this image we extracted a preliminary source catalogue down to a flux limit of ~15 mJy (~10σ), that can be considered complete down to 20‒30 mJy. A spectral index study of the mJy sources in the Lockman Hole region, was performed using the available catalogues ( 1.4 GHz, 345 MHz and 150 MHz) and a deep 610 MHz source catalogue available from the literature (Garn et al. 2008, 2010).

Effect of loss of CDKL5 on brain development in a new Cdkl5 knockout mouse model

Rett's Syndrome (RTT) is a severe neurodevelopmental disorder, characterized by cognitive disability that appears in the first months/years of life. Recently, mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been detected in RTT patients characterized by early-onset seizures. CDKL5 is highly expressed in the brain starting from early postnatal stages to adulthood, suggesting the importance of this kinase for proper brain maturation and function. However, the role/s of CDKL5 in brain development and the molecular mechanisms whereby CDKL5 exerts its effects are still largely unknown. In order to characterize the role of CDKL5 on brain development, we created a mice carrying a targeted conditional knockout allele of Cdkl5. A first behavioral characterization shows that Cdkl5 knockout mice recapitulate several features that mimic the clinical features described in CDKL5 patients and are a useful tool to investigate phenotypic and functional aspects of Cdkl5 loss. We used the Cdkl5 knockout mouse model to dissect the role of CDKL5 on hippocampal development and to establish the mechanism/s underlying its actions. We found that Cdkl5 knockout mice showed increased precursor cell proliferation in the hippocampal dentate gyrus. Interestingly, this region was also characterized by an increased rate of apoptotic cell death that caused a reduction in the final neuron number in spite of the proliferation increase. Moreover, loss of Cdkl5 led to decreased dendritic development of new generated granule cells. Finally, we identified the Akt/GSK3-beta signaling as a target of Cdkl5 in the regulation of neuronal precursor proliferation, survival and maturation. Overall our findings highlight a critical role of CDKL5/AKT/GSK3-beta signaling in the control of neuron proliferation, survival and differentiation and suggest that CDKL5-related alterations of these processes during brain development underlie the neurological symptoms of the CDKL5 variant of RTT.

Producers' Perceptions and Consumers' Behaviour Toward Certified Beans from Integrated Production (IP) in the Brazilian Central Region

Integrated production (IP) is part of the Brazilian government program to promote sustainable agricultural production. IP ensure minimum food quality standards for domestic market, and export. Furthermore, IP is considered a good option to reduce negative environmental impacts of intensive crops in tropical Savannas, including common beans, as a Brazilian staple food. Although its advantages, and the government’s effort to promote IP, few growers are adopting IP. Maybe, the perception about IP usefulness and/or its ease of use is not too clear. Moreover, the production sector is driven by market signs, and there is few information on the consumer's preferences toward IP certified products in Brazil. In this study, we sought to identify some critical factors that can influence the IP adoption in beans' production. Moreover, we sought to verify the consumers’ perceptions and intention of purchasing IP certified beans (hypothetical product). This report comprises four chapters: (1) an introduction illustrating the context in which the research was based; (2) the results on the study of IP adoption based on the Technology Acceptance Model (TAM); (3) the choice experiment results applied to identify consumers preferences and willingness-to-pay (WTP) for IP label; (4) the results on the Theory of Planned Behaviour (TPB) applied to identify consumers’ perception toward IP certified beans. This research contributes with rich information for the beans’ supply chain, providing several insights to growers, retail and other agents, including policy makers. Beans’ production sector seems to be positively intentioned to adopt IP, but further studies should be conducted to test other adoption indicators using TAM model. Surveyed consumers are willing to pay a premium price for IP labelled beans. They showed a positive attitude toward purchasing IP labelled beans. It is an important information to motivate production sector to offer certified beans to the market.