Surgical and postmortem pathology studies: contribution for the investigation of temporal lobe epilepsy

Pathology studies in epilepsy patients bring useful information for comprehending the physiopathology of various forms of epilepsy, as well as aspects related to response to treatment and long-term prognosis. These studies are usually restricted to surgical specimens obtained from patients with refractory focal epilepsies. Therefore, most of them pertain to temporal lobe epilepsy (TLE) with mesial temporal sclerosis (MTS) and malformations of cortical development (MCD), thus providing information of a selected group of patients and restricted regions of the brain. Postmortem whole brain studies are rarely performed in epilepsy patients, however they may provide extensive information on brain pathology, allowing the analysis of areas beyond the putative epileptogenic zone. In this article, we reviewed pathology studies performed in epilepsy patients with emphasis on neuropathological findings in TLE with MTS and MCD. Furthermore, we reviewed data from postmortem studies and discussed the importance of performing these studies in epilepsy populations.

Seven new species of Cerambycidae (Coleoptera) from South America with the proposal of three new genera

The following new species are described - Cerambycinae, Sydacini: Sydax flechtmanni sp. nov. from Brazil (São Paulo); Eburiini: Ebrodacrys biffipradorum sp. nov. from Brazil (Roraima); - Lamiinae, Pteropliini: Ataxia piauiensis sp. nov. from Brazil (Piuaí); Calliini: Amucallia carbonaria sp. nov. from French Guiana; A. citrina sp. nov. from Guiana. Also in Lamiinae, two new genera of Onciderini are proposed. Ubytyra gen. nov., type species U. tuberosa sp. nov. from Peru (Junin) e Japi gen. nov., type species J. duartei sp. nov., from Brazil (São Paulo); Ubytyra gen. nov. can be distinguished by the sides of prothorax with long central spine rounded at apex, and this new feature among Onciderini is discussed. Japi gen. nov., is characterized by a fringe of long hairs on the inner side of antennomere III, present only in species from North and Central America, and gender comparison of these species is done and discussed. In Hemilophini, Pseudotacocha gen. nov., type species P. magnifica sp. nov. from Peru (Cuzco), are described. The new genera can be distinguished by eyes well developed, elytra with two carinae and the apices outer with short spine; a comparison with related genera is done.

Genomic analysis of ERVWE2 locus in patients with multiple sclerosis: absence of genetic association but potential role of human endogenous retrovirus type W elements in molecular mimicry with myelin antigen

Human endogenous retroviruses (HERVs) arise from ancient infections of the host germline cells by exogenous retroviruses, constituting 8% of the human genome. Elevated level of envelope transcripts from HERVs-W has been detected in CSF, plasma and brain tissues from patients with Multiple Sclerosis (MS), most of them from Xq22.3, 15q21.3, and 6q21 chromosomes. However, since the locus Xq22.3 (ERVWE2) lack the 5' LTR promoter and the putative protein should be truncated due to a stop codon, we investigated the ERVWE2 genomic loci from 84 individuals, including MS patients with active HERV-W expression detected in PBMC. In addition, an automated search for promoter sequences in 20 kb nearby region of ERVWE2 reference sequence was performed. Several putative binding sites for cellular cofactors and enhancers were found, suggesting that transcription may occur via alternative promoters. However, ERVWE2 DNA sequencing of MS and healthy individuals revealed that all of them harbor a stop codon at site 39, undermining the expression of a full-length protein. Finally, since plaque formation in central nervous system (CNS) of MS patients is attributed to immunological mechanisms triggered by autoimmune attack against myelin, we also investigated the level of similarity between envelope protein and myelin oligodendrocyte glycoprotein (MOG). Comparison of the MOG to the envelope identified five retroviral regions similar to the Ig-like domain of MOG. Interestingly, one of them includes T and B cell epitopes, capable to induce T effector functions and circulating Abs in rats. In sum, although no DNA substitutions that would link ERVWE2 to the MS pathogeny was found, the similarity between the envelope protein to MOG extends the idea that ERVEW2 may be involved on the immunopathogenesis of MS, maybe facilitating the MOG recognizing by the immune system. Although awaiting experimental evidences, the data presented here may expand the scope of the endogenous retroviruses involvement on MS pathogenesis

Multiple sclerosis and herpesvirus interaction

Multiple sclerosis is the most common autoimmune inflammatory demyelinating disease of the central nervous system, and its etiology is believed to have both genetic and environmental components. Several viruses have already been implicated as triggers and there are several studies that implicate members of the Herpesviridae family in the pathogenesis of MS. The most important characteristic of these viruses is that they have periods of latency and exacerbations within their biological sanctuary, the central nervous system. The Epstein-Barr, cytomegalovirus, human herpesvirus 6 and human herpesvirus 7 viruses are the members that are most studied as being possible triggers of multiple sclerosis. According to evidence in the literature, the herpesvirus family is strongly involved in the pathogenesis of this disease, but it is unlikely that they are the only component responsible for its development. There are probably multiple triggers and more studies are necessary to investigate and define these interactions.

Identificazione di un QTL principale per resistenza a ruggine bruna sul cromosoma 7B di frumento duro

Leaf rust caused by Puccinia triticina is a serious disease of durum wheat (Triticum durum) worldwide. However, genetic and molecular mapping studies aimed at characterizing leaf rust resistance genes in durum wheat have been only recently undertaken. The Italian durum wheat cv. Creso shows a high level of resistance to P. triticina that has been considered durable and that appears to be due to a combination of a single dominant gene and one or more additional factors conferring partial resistance. In this study, the genetic basis of leaf rust resistance carried by Creso was investigated using 176 recombinant inbred lines (RILs) from the cross between the cv. Colosseo (C, leaf rust resistance donor) and Lloyd (L, susceptible parent). Colosseo is a cv. directly related to Creso with the leaf rust resistance phenotype inherited from Creso, and was considered as resistance donor because of its better adaptation to local (Emilia Romagna, Italy) cultivation environment. RILs have been artificially inoculated with a mixture of 16 Italian P. triticina isolates that were characterized for virulence to seedlings of 22 common wheat cv. Thatcher isolines each carrying a different leaf rust resistance gene, and for molecular genotypes at 15 simple sequence repeat (SSR) loci, in order to determine their specialization with regard to the host species. The characterization of the leaf rust isolates was conducted at the Cereal Disease Laboratory of the University of Minnesota (St. Paul, USA) (Chapter 2). A genetic linkage map was constructed using segregation data from the population of 176 RILs from the cross CL. A total of 662 loci, including 162 simple sequence repeats (SSRs) and 500 Diversity Arrays Technology markers (DArTs), were analyzed by means of the package EasyMap 0.1. The integrated SSR-DArT linkage map consisted of 554 loci (162 SSR and 392 DArT markers) grouped into 19 linkage blocks with an average marker density of 5.7 cM/marker. The final map spanned a total of 2022 cM, which correspond to a tetraploid genome (AABB) coverage of ca. 77% (Chapter 3). The RIL population was phenotyped for their resistance to leaf rust under artificial inoculation in 2006; the percentage of infected leaf area (LRS, leaf rust susceptibility) was evaluated at three stages through the disease developmental cycle and the area under disease progress curve (AUDPC) was then calculated. The response at the seedling stage (infection type, IT) was also investigated. QTL analysis was carried out by means of the Composite Interval Mapping method based on a selection of markers from the CL map. A major QTL (QLr.ubo-7B.2) for leaf rust resistance controlling both the seedling and the adult plant response, was mapped on the distal region of chromosome arm 7BL (deletion bin 7BL10-0.78-1.00), in a gene-dense region known to carry several genes/QTLs for resistance to rusts and other major cereal fungal diseases in wheat and barley. QLr.ubo-7B.2 was identified within a supporting interval of ca. 5 cM tightly associated with three SSR markers (Xbarc340.2, Xgwm146 e Xgwm344.2), and showed an R2 and an LOD peak value for the AUDPC equal to 72.9% an 44.5, respectively. Three additional minor QTLs were also detected (QLr.ubo-7B.1 on chr. 7BS; QLr.ubo-2A on chr. 2AL and QLr.ubo-3A on chr. 3AS) (Chapter 4). The presence of the major QTL (QLr.ubo-7B.2) was validated by a linkage disequilibrium (LD)-based test using field data from two different plant materials: i) a set of 62 advanced lines from multiple crosses involving Creso and his directly related resistance derivates Colosseo and Plinio, and ii) a panel of 164 elite durum wheat accessions representative of the major durum breeding program of the Mediterranean basin. Lines and accessions were phenotyped for leaf rust resistance under artificial inoculation in two different field trials carried out at Argelato (BO, Italy) in 2006 and 2007; the durum elite accessions were also evaluated in two additional field experiments in Obregon (Messico; 2007 and 2008) and in a green-house experiment (seedling resistance) at the Cereal Disease Laboratory (St. Paul, USA, 2008). The molecular characterization involved 14 SSR markers mapping on the 7BL chromosome region found to harbour the major QTL. Association analysis was then performed with a mixed-linear-model approach. Results confirmed the presence of a major QTL for leaf rust resistance, both at adult plant and at seedling stage, located between markers Xbarc340.2, Xgwm146 and Xgwm344.2, in an interval that coincides with the supporting interval (LOD-2) of QLr.ubo-7B.2 as resulted from the RIL QTL analysis. (Chapter 5). The identification and mapping of the major QTL associated to the durable leaf rust resistance carried by Creso, together with the identification of the associated SSR markers, will enhance the selection efficiency in durum wheat breeding programs (MAS, Marker Assisted Selection) and will accelerate the release of cvs. with durable resistance through marker-assisted pyramiding of the tagged resistance genes/QTLs most effective against wheat fungal pathogens.

Approcci molecolari e bioinformatici volti alla caratterizzazione di Vgt1, QTL coinvolto nella regolazione dell'epoca di fioritura in Zea Mays

The genetic control of flowering time has been addressed by many quantitative trait locus (QTL) studies. A survey of the results from 29 independent studies reporting information on 441 QTLs led to the production of a QTL consensus map, which enabled the identification of 59 chromosome regions distributed on all chromosomes and shown to be frequently involved in the genetic control of flowering time and related traits. One of the major QTLs for flowering time, the Vegetative to generative transition 1 (Vgt1) locus , corresponds to an upstream (70 kb) non-coding regulatory element of ZmRap2.7, a repressor of flowering. A transposon (MITE) insertion was identified as a major allelic difference within Vgt1. One of the hypotheses is that Vgt1 might function by modifying ZmRap2.7 chromatin through an epigenetic mechanism. Therefore, the methylation state at Vgt1 was investigated using an approach that combines digestion with McrBc, an endonuclease that acts upon methylated DNA, and quantitative PCR. The analyses were performed on genomic DNA from leaves of six different maize lines at four stages of development. The results showed a trend of reduction of methylation from the first to the last stage with the exception of a short genomic region flanking the MITE insertion, which showed a constant and very dense methylation throughout leaf development and for both alleles. Preliminary results from bisulfite sequencing of a small portion of Vgt1 revealed differential methylation of a single cytosine residue between the two alleles. ZmRap2.7 expression was assayed in the four developmental stages afore mentioned for the six genotypes, in order to establish a link between methylation at Vgt1 and ZmRap2.7 transcription. To assess the role of Vgt1 as a transcriptional enhancer, two reporter vectors for stable transformation of plants have been developed.

Mappaggio fine di un QTL principale per la resa in granella sul cromosoma 3B di frumento duro

In durum wheat, two major QTL for grain yield (Qyld.idw-2B and Qyld.idw-3B) and related traits were identified in a recombinant population derived from Kofa and Svevo (Maccaferri et al. 2008). To further investigate the genetic and physiological basis of allelic variation for this important trait, the fine mapping of Qyld.idw-2B e Qyld.idw-3B was done during the PhD. In this regard, new molecular markers were added to increase the map resolution in the target interval. For Qyld.idw-2B region COS markers derived from the synteny between wheat and rice/ sorghum /brachypodiu genomes were screened. While for Qyld.idw-3B region SSR, ISBP and COS markers obtained from BAC end-sequences and BAC sequences generated during the construction of the 3B physical map (Paux et al., 2008) were screened. In the RIL population a final map resolution of 2,8 markers/cM for Qyld.idw-2B and 0,6 markers/cM for Qyld.idw-3B were obtained. Eighteen pairs of near-isogenic lines (NILs) for Qyld.idw-3B were obtained from F4:5 heterogeneous inbred families. In order to confirm the phenotypic effect of the QTL all pairs were evaluated in field trials (2010 and 2011) for all traits. Three pairs of NILs, with contrasted haplotypes at the target region, were crossed to produce a large F2 population (ca. 7,500 plants in total) that was screened for the identification of recombinants. A total of 233 homozygous F4:5 segmental isolines were obtained and the phenotypic and genotypic characterization of these materials were done. A fine mapping for Qyld.idw-3B was obtained and the QTL peak was identified in a interval of 0,4 cM. All markers were anchored to the Chinese Spring physical map of chr. 3B, which allowed us to identify the BAC Contigs spanning the QTL region and to assign the QTL peak to Contig 954. Sequencing of this contig has revealed the presence of 42 genes.

Identifizierung und Quantifizierung der Ketocarotinoide in Dauerstadien von Grünalgen und Ketocarotinoidbiosynthese im Modellorganismus Chlamydomonas reinhardtii

Grünalgen bilden zur Überdauerung schlechter Umweltbedingungen Ruhestadien, die sich durch Ausbildung einer festen Zellwand, die Reduktion des Plastiden und die starke Akkumulation von Speicherfetten und Ketocarotinoiden im Zytosol auszeichnen. Obwohl Ketocarotinoide in Grünalgen seit über vierzig Jahren beforscht werden, gab es hierzu noch wenige molekularbiologische Untersuchungen. Im Vorfeld meiner Promotion wurde durch unsere Arbeitsgruppe entdeckt, dass auch der molekular gut zugängliche Modellorganismus Chlamydomonas reinhardtii im Zygotenstadium große Mengen an Ketocarotinoiden bildet. Neben dem zu erwartenden Ketocarotinoid Astaxanthin fanden wir große Mengen des bisher nur in einer Grünalge beschriebenen 4-Ketoluteins. Vorversuche ließen die Vermutung aufkommen, dass dieses Pigment bei der Untersuchung der Pigmentausstattung in Dauerstadien von vielen Grünalgen bisher übersehen wurde. rnIn der vorliegenden Arbeit wurde daher zunächst die Pigmentzusammensetzung von Dauerstadien der bereits gut untersuchten Grünalgen Muriella zofingiensis und Scenedesmus rubescens durch Vergleich mit dem Ketocarotinoidmuster aus Dauerstadien von C. reinhardtii und Fritschiella tuberosa reevaluiert und dabei erstmals das Vorkommen signifikanter Mengen an 4-Ketolutein nachgewiesen. Außerdem zeigte sich, dass die als bisheriger Modellorganismus der Ketocarotinoidbiosynthese in Grünalgen sehr gut untersuchte Alge Haematococcus pluvialis eher eine Ausnahme darstellt, da ihre Dauerstadien als einzige der hier untersuchten Algen nur minimale Mengen von 4 Ketolutein aufwiesen. Diese Beobachtungen machen es sehr wahrscheinlich, dass die Fähigkeit zur Bildung von 4-Ketolutein unter den Grünalgen wesentlich weiter verbreitet ist als bisher angenommen. Das sekundäre Carotinoid 4-Ketolutein kam in den Dauerstadien der Grünalgen neben seiner freien Form ausschließlich als Monoacylester vor, im Gegensatz zu Astaxanthin, das als mono- und diacylierte Form auftrat. rnÜber die Analyse der Pigmentausstattung hinaus konnten die entscheidenden Schritte des Synthesewegs der Ketocarotinoide in C. reinhardtii durch funktionelle Charakterisierung der beteiligten Enzyme in Bakterien aufgeklärt werden. Als Basis für die Charakterisierungen wurde ein umfangreiches Portfolio von carotinogenen E. coli-Bakterien etabliert, darunter α Carotin und Lutein produzierende Stämme, die bisher nicht zur Verfügung standen. Das wurde durch die Klonierung der Lycopinzyklase (OluLCY) aus der Grünalge Ostreococcus lucimarinus möglich, die eine Sonderolle unter den Zyklasen einnimmt, da sie die Lycopin-β-Zyklase und Lycopin-ε-Zyklase in einem Fusionsenzym vereint. Vorteile dieses Fusionsenzyms sind die Expressionskontrolle durch nur einen Promotor und die weitgehend konstante Stöchiometrie seiner Produkte α-Carotin und β-Carotin, was die OluLCY für die biotechnologische Anwendung prädestiniert.rnDie funktionelle Charakterisierung der Carotinoidbiosyntheseenzyme aus C. reinhardtii umfasste das Schlüsselenzym der Ketocarotinoidbiosynthese, die β-Carotin-Ketolase (BKT), sowie die Carotinoid-Hydroxylasen CHYB, CYP97A5 und CYP97C3. Dabei wurde für das BKT-Enzym aus C. reinhardtii nachgewiesen, dass es nicht nur die Ketolierung von β Carotin zu Canthaxanthin und von Zeaxanthin zu Astaxanthin, sondern auch die Bildung der von α-Carotin abgeleiteten Ketocarotinoide wie 4-Keto-α-Carotin und 4 Ketolutein katalysieren kann.rn

Fine Mapping of qroot-yield-1.06, a QTL for Root, Plant Vigor and Yield in Maize

Root-yield-1.06 is a major QTL affecting root system architecture (RSA) and other agronomic traits in maize. The effect of this QTL has been evaluated with the development of near isogenic lines (NILs) differing at the QTL position. The objective of this study was to fine map qroot-yield-1.06 by marker-assisted searching for chromosome recombinants in the QTL interval and concurrent root phenotyping in both controlled and field conditions, through successive generations. Complementary approaches such as QTL meta-analysis and RNA-seq were deployed in order to help prioritizing candidate genes within the QTL target region. Using a selected group of genotypes, field based root analysis by ‘shovelomics’ enabled to accurately collect RSA information of adult maize plants. Shovelomics combined with software-assisted root imaging analysis proved to be an informative and relatively highly automated phenotyping protocol. A QTL interval mapping was conducted using a segregating population at the seedling stage grown in controlled environment. Results enabled to narrow down the QTL interval and to identify new polymorphic markers for MAS in field experiments. A collection of homozygous recombinant NILs was developed by screening segregating populations with markers flanking qroot-yield-1.06. A first set of lines from this collection was phenotyped based on the adapted shovelomics protocol. QTL analysis based on these data highlighted an interval of 1.3 Mb as completely linked with the target QTL but, a larger safer interval of 4.1 Mb was selected for further investigations. QTL meta-analysis allows to synthetize information on root QTLs and two mQTLs were identified in the qroot-yield-1.06 interval. Trascriptomics analysis based on RNA-seq data of the two contrasting QTL-NILs, confirmed alternative haplotypes at chromosome bin 1.06. qroot-yield-1.06 has now been delimited to a 4.1-Mb interval, and thanks to the availability of additional untested homozygous recombinant NILs, the potentially achievable mapping resolution at qroot-yield-1.06 is c. 50 kb.

Calcolo strutturale ed ottimizzazione sedile in materiale composito per elicottero ultraleggero

Il lavoro svolto consiste nel processo di ottimizzazione strutturale di un sedile in materiale composito per un elicottero ultraleggero, attraverso un processo iterativo di simulazione FEM e modifiche al progetto, interpretando i dati ricavati dalle simulazioni, nel rispetto della normativa FAR 27. I risultati ottenuti hanno permesso di dimezzare il peso iniziale e resistere meglio agli sforzi, con implicazioni benefiche anche sul costo finale del prodotto che presenta minori costi del materiale e tempi di lavorazione ridotti sia in fase di laminazione che in fase di ciclo di cura in autoclave.

Dinoflagellate cyst counts and age model from sediment samples of ODP Hole 175-1085A

Sediment samples from ODP Site 1085 were investigated in order to obtain more information on the initiation and development of the Benguela upwelling system during the middle and upper Miocene. In particular, our intent was to establish the causes of the upwelling as well as the response of the upwelling regime to the development of the Antarctic Circumpolar Current. Based on changes in the calcareous dinoflagellate cyst association, we found an initial increase of the dinoflagellate cyst productivity, probably related to the initiation of upwelling about 11.8 Ma ago. Two distinct increases in cyst productivity in conjunction with temperature decreases of the upper water masses reflect upwelling pulses off Namibia and occur at the end of the Miocene cooling events Mi5 (about 11.5 Ma) and Mi6 (about 10.5 Ma). Both cooling events are associated with an ice volume increase in Antarctica and are thought to have led to an increase in southeasterly winds, possibly causing these two upwelling pulses. We demonstrate a decrease in dinoflagellate cyst productivity and enhanced terrigenous input via the Orange River after the Mi5 event. At about 11.1 Ma, the dinoflagellate cyst productivity increases again. The polar cyst species Caracomia arctica occurs here for the first time. This implies an influence of subantarctic mode water and therefore a change in the quality of the upwelling water which allowed the Benguela upwelling to develop into modern conditions. From about 10.4 Ma, C. arctica forms a permanent part of the association, pointing to an establishment of the upwelling regime.