Littérature scientifique sur le sujet « Aspetti genetici »

Nel presente contributo, le autrici intendono definire il processo comunicativo dellaattraverso un breveteorico sulla sua evoluzione e sul suo significato per approfondire il discorso intorno alla relazione di cura tra consulente e cliente e sullo spazio della professionalitŕ psicologica in tale contesto di intervento. L'obiettivo č esplorare le implicazioni esistenziali che ladi richiedere consulenza genetica, cosě come quella di svolgere test genetici, si inseriscono nel complesso della vita dei probandi. Pertanto, ilč sui principali aspetti psicologici che ruotano intorno al, relativo all'utilitŕ di svolgere test genetici e all'impatto della comunicazione dei risultati genetici () e alconcernente tanto il momento preliminare la richiesta stessa di consulenza, quanto quello finale l'iter consulenziale con tutte le conseguenze del.

Il 6 e 7 maggio 2004 a Bruxelles ha avuto luogo un congresso organizzato dalla Commissione Europea per stimolare la riflessione sulle implicazioni etiche, sociali e giuridiche legate allo sviluppo e all’utilizzo dei test genetici. Gli Autori riferiscono sulle conclusioni di quelle due giornate, dedicate alla lettura ed alla discussione di 25 raccomandazioni proposte da un gruppo di lavoro multidisciplinare composto da politici, accademici, rappresentanti dell’industria e di organizzazioni volontarie di pazienti di vari paesi dell’Unione. La qualità dei test genetici disponibili, l’accuratezza dei loro risultati, le condizioni di accesso ai test e ai trattamenti (soprattutto per le persone con malattie rare), l’utilizzo appropriato dei campioni e dei dati, il consenso informato ed il rispetto della privacy, il counselling genetico pre e post test, il rischio di discriminazioni sulla base del genere e dell’etnia: questi sono solo alcuni dei problemi emersi in sede congressuale. Le 25 raccomandazioni della Commissione Europea si differenziano per certi versi da altri documenti e dichiarazioni internazionali, soprattutto per quanto riguarda lo “statuto” assegnato ai dati genetici (“eccezionalità” genetica); secondo il Gruppo di lavoro che ha scritto le raccomandazioni l’informazione genetica non è diversa dagli altri dati medici, e dunque dovrebbe essere trattata allo stesso modo. È pur vero però, che i test genetici offrono nuove informazioni e conoscenze che potranno complicare non solo il rapporto tra medico e paziente, ma anche quello tra paziente e familiari. Con l’aumento dei test genetici ci sarà bisogno di riferimenti chiari ed accettabili per tutte le parti coinvolte: medici, pazienti e familiari avranno bisogno di riferimenti per risolvere problemi pratici, per conciliare i vari diritti dei pazienti (ad es. i diritti di sapere o di non sapere, di condividere le informazioni o meno), e doveri dei medici (dovere di mantenere il segreto professionale e proteggere la privacy, ma talvolta anche il dovere di avvertire). Il consenso informato dovrà aiutare le persone a comprendere in modo adeguato tutte le implicazioni di un test, dalle sue possibili conseguenze a livello familiare e sociale, alla classificazione e all’uso dei suoi dati clinici e genetici per le ricerche future. Quando poi l’ “oggetto” di studio non è più il singolo individuo, ma gruppi ristretti di persone (ad es., negli screening genetici), sarà necessario un “consenso di gruppo”, mentre gli studi sulle popolazioni riguarderanno le società in senso lato. Infine rimane un interrogativo importante: tutti questi cambiamenti aumenteranno il livello di costo della sanità? Molti sono gli scenari e gli sviluppi possibili, ma questi non dipendono solo dai progressi della scienza. Per far si che i benefici di queste innovazioni superino i rischi corsi dagli individui e dalla società, sarà importante creare un quadro normativo responsabile, che sappia accompagnare e misurare le varie attività di implementazione dei test genetici, sia a livello dei singoli Stati, sia a livello dell’Unione Europea.

L’articolo, dopo aver analizzato gli aspetti etici legati alla medicina predittiva e dopo aver illustrato la malattia di Alzheimer da un punto di vista patologico ed eziologico, si sofferma sulla significatività etica del ruolo del counseling genetico, in particolare in relazione al caso in cui la richiesta del test predittivo venga da parte di soggetti a rischio, asintomatici, che presentano una familiarità la cui eziologia è nota, e per i quali il test è disponibile. In questo caso la riflessione etica è maggiormente problematizzante e coinvolge diversi aspetti: l’uso di test sicuri ed efficaci in strutture sanitarie ben identificate con standard di qualità garantiti; le problematiche legate alla consulenza (autonomia decisionale dell’utente, il consenso informato, l’assistenza psico-sociale); la formazione professionale del personale sanitario. In seguito gli autori riportano e commentano i dati di un’indagine conoscitiva in merito al test predittivo e alle strutture sanitarie. Infine, nel delineare le direttive etiche per i consulenti, viene messo in risalto lo spessore bioetico e biogiuridico del ruolo dell’operatore nel counseling genetico, spessore che viene ulteriormente messo in luce dalla peculiare responsabilità che l’operatore si assume nella protezione dei dati genetici.

Vengono esaminati i caratteri petrografici e geologici del verde di Prato, un materiale ampiamente utilizzato nelle architetture religiose della Firenze medievale e rinascimentale. Dopo una illustrazione delle analisi svolte, si definiscono le proprietà tecniche e le fasi di alterazione del materiale in opera, e si suggeriscono gli interventi necessari per la conservazione.

The congenital thrombocytopenias are a heterogeneous group of disorders characterized by reduced number of platelets and variable clinical manifestations from severe to milder forms of incidental finding. They are divided into: 1) thrombocytopenia defect of megakaryocytic differentiation and maturation 2) MK migration defect from the bone marrow to the vascular niche 3) elongation of the pro-platelets. Then there are the hereditary thrombocytopenias from reduced platelet survival and thrombocytopenias with a still unknown pathogenesis. Currently, there are now at least 20 genes whose genetic defects determine a phenotype characterized by hereditary thrombocytopenia, but approximately 50% of patients have forms that do not coincide with any known defect. In recent years, there were several forms of hereditary thrombocytopenia associated with the development of hematologic and non-haematological malignancies that have aroused a great interest in researchers; especially the forms associated with mutations of ANKRD26, RUNX1 and ETV6. Purpose The purpose of this work is: 1. Build a comprehensive database of patients with inherited thrombocytopenia studied in our laboratory, defining the genetic mutations found. 2.Conduct a preliminary study on the presence of mutations in the 5'UTR of ANKRD26 in patients with sporadic and inherited thrombocytopenia, who did not fit into known forms of inherited thrombocytopenia and who had normal platelet volume (4μm˂MPV˂8μm), establishing the clinical and molecular features of this new form of thrombocytopenia and studying families with inherited mutations in this gene. 3. Enlarge the investigation to two other genes involved in inherited forms of thrombocytopenia with susceptibility to hematological malignancies: RUNX1 and ETV6. 4. Collect and analyze data related to the course of pregnancy in a large cohort of patients with IT forms (inherited thrombocytopenia). Materials and methods At the Medical Clinic 1 of the City Hospital-University of Padua complex there exist a series of about 70 family pedigrees of patients with inherited thrombocytopenia from hidden defect. The assessment includes the clinical history and the study of the expression of surface platelet antigens by flow cytometry method using monoclonal antibodies. To evaluate the platelets morphology, the method of staining with May-Grunwald / Giemsa (MGG) on peripheral blood (macro, micro and normo thrombocytopenia) has been adopted. The DNA was extracted from whole blood and amplified for the study of the following genes: GpIbα, GpIbβ, GPIX, MYH9, ACTN1. The products of the amplification reactions were visualized by agarose gel electrophoresis and sequenced with the Sanger method. We carried out a preliminary study on the presence of mutations in the 5'UTR of ANKRD26 in patients with sporadic and inherited thrombocytopenia, which did not fit into known forms of inherited thrombocytopenia and who had normal platelet volume (4μm˂MPV˂8μm) in order to be able to define the clinical and molecular features. We then expanded the investigation to RUNX1 and ETV6, two genes involved in hereditary forms of thrombocytopenia with predisposition to blood cancer in thrombocytopenic patients and not (MDS + thrombocytopenia; MDS thrombocytopenia -) belonging to different classes of myelodysplastic syndrome sec. WHO 2008. Finally, as part of thrombocytopenia family confirmed through genetic investigation, we participated in Pregnancy In Inherited Platelet Abnormalities (PIPA), multicenter retrospective evaluation of 339 pregnancies in 181 women with 13 different forms of thrombocytopenia. Results 8 families (12%) have mutations in the MYH9, MYH9-related disease, 12 families (19%) have mutations GP1Bα, 4 families (6%) have gene mutations that cause GP1Bβ Bernard-Soulier Syndrome (BSS) mono or biallelic. The remaining families (60%) do not have the genetic mutations in the studied glycoproteins, and no mutation in the ACTN1 gene. In light of recent studies on the forms of inherited thrombocytopenia characterized in our laboratory, we conducted a preliminary study on the presence of mutations in the 5'UTR of ANKRD26 in patients with sporadic and inherited thrombocytopenia, which did not fit into known forms of inherited thrombocytopenia and showed normal platelet volume (4μm˂MPV˂8μm). We identified two different heterozygous mutations, located in the 5'UTR untranslated region of the gene. Given the interest in the study of hereditary thrombocytopenia associated with the development of hematologic and non-hematological malignancies, in particular of RUNX1 and ETV6 genes, responsible for non-syndromic autosomal dominant thrombocytopenia, we collected preliminarily a small series of myelodysplastic thrombocytopenic subjects (9 subjects), myelodysplastic subjects with platelet counts within the normal range (12 subjects) and 27 control subjects with hereditary undetermined thrombocytopenia; this in order to strengthen the evidence about a possible association between mutations in the mentioned genes, thrombocytopenia and risk myelodysplastic / leukemia evolution. Of these subjects, we collected data on age, sex, karyotype, number of blast cells, hemoglobin and platelets. Preliminary data (Table 1) show that mutations of RUNX1 ETV6 gene are present only in the group of myelodysplastic thrombocytopenic. GENE POSITION MUTATION RUNX-1 EXON 4 c.76C>G RUNX-1 EXON 8 c.934del A RUNX-1 EXON 9 c.1214_1215insTG ETV6 INTRON 1 c.28+192delC Table 1. Mutations found in RUNX1 and ETV6 genes Although the involvement of these genes in thrombopoiesis is known, the exact mechanism responsible for thrombocytopenia is not yet clear. The rarity of these diseases makes it difficult to collect relevant data for analysis of genotype-phenotype relation. In the multicenter study (PIPA) we have classified 23 pregnancies (13 women): 5 women have BBSs, mBSS 3, 4 MYH9-RD, 1 ANKRD26. The average age at diagnosis of thrombocytopenia is 30 years, the average platelet count before giving birth is about 60 x 109 platelets / L, even if in some cases there was a drastic reduction in platelet aggregation. The bleeding tendency is low before birth and only in two pregnancies platelet transfusion is needed. All births took place between the 34th and 40th week; 13 are born by caesarean section and 10 vaginally. Only 9 infants carry the same disease of the mother, although in 6 cases the data is not available because they were not made inquiries about it. We found only one case of neonatal death due to brain hemorrhage, but no cases of bleeding in newborns. Conclusions The thrombocytopenia is the most common disorder of hemostasis and is a heterogeneous group of clinical entities. The peculiarity of family forms is the difficulty of clinical identification since they are often classified as immune thrombocytopenia and patients undergoing inappropriate therapy. As reported in the results of recent studies involving a higher number of families, in our work the most common forms of thrombocytopenias family is the BSS, the MYH9-RD followed by ANKRD26. Thanks to a careful analysis of clinical and genetic investigations associated with recent molecular studies, it is now possible to identify and correctly classify patients with new genetic mutations even though more than 50% of families remain with no diagnosis. To evaluate the possible mechanisms involved in the development of thrombocytopenia being myelodysplastic syndromes will be essential to study extensively the three categories of patients: thrombocytopenia hereditary with defect of ANKRD26, RUNX-1 and ETV6, myelodysplasia or leukemia with prevailing thrombocytopenia. A larger number of cases of these subjects will allow us to deepen the study of functional mutations defining the clinical and molecular features, in order to evaluate a possible link between myelodysplasia and thrombocytopenia and understand at what level of megakaryopoiesis and hematopoiesis they intervene
Le piastrinopenie congenite sono un eterogeneo gruppo di patologie caratterizzato da ridotto numero di piastrine e si presentano con manifestazioni cliniche variabili da forme gravi a forme più lievi di riscontro casuale. Si suddividono in piastrinopenie da difetto della differenziazione e maturazione megacariocitaria, difetto di migrazione dei MK dalla nicchia ossea del midollo a quella vascolare e della elongazione delle pro-piastrine. Vi sono poi le piastrinopenie ereditarie da ridotta sopravvivenza piastrinica e le piastrinopenie a patogenesi ancora sconosciuta. Attualmente si conoscono almeno 20 geni i cui difetti genetici determinano un fenotipo caratterizzato da trombocitopenia ereditaria, ma circa il 50% dei pazienti hanno forme che non coincidono con nessun difetto noto. Negli ultimi anni sono emerse alcune forme di piastrinopenia ereditaria associate allo sviluppo di neoplasie di tipo ematologico e non ematologico che hanno destato un estremo interesse nei ricercatori; in particolare le forme associate a mutazioni di ANKRD26, RUNX1 e ETV6. Scopo Lo scopo di questo lavoro è quello di: 1. Costruire un database completo dei pazienti con piastrinopenia familiare finora pervenuti nel nostro laboratorio, definendo le mutazioni genetiche rinvenute. 2. Condurre uno studio preliminare sulla presenza di mutazioni in 5’UTR di ANKRD26 in pazienti con piastrinopenia sporadica e familiare, che non rientravano in forme note di piastrinopenia ereditaria e che presentavano volume piastrinico nella norma (4μm˂MPV˂8μm), definendone le caratteristiche cliniche e molecolari di questa nuova forma di piastrinopenia ereditaria e studiando le famiglie con mutazioni in questo gene. 3. Ampliare l’indagine ad altri due geni coinvolti in forme di piastrinopenia ereditaria con predisposizione a neoplasie ematologiche: RUNX1 ed ETV6. 4. Raccogliere e analizzare i dati relativi al corso della gravidanza in una ampia coorte di pazienti con forme di IT (inherited thrombocytopenia). Materiali e metodi Presso l’Unità di Clinica Medica 1 dell’Azienda Ospedale-Università di Padova è presente una casistica di circa 70 pedigree familiari di soggetti con piastrinopenia familiare da difetto ignoto. La valutazione comprende storia clinica e lo studio dell’espressione degli antigeni piastrinici di superficie mediante metodica citofluorimetrica utilizzando anticorpi monoclonali. Per valutare la morfologia piastrina è stato adottato il metodo della colorazione con May-Grunwald/Giemsa (MGG) su strisci di sangue periferico (macro, micro e normo trombocitopenia). Il DNA è stato estratto da sangue intero e amplificato per lo studio dei seguenti geni: GpIbα, GpIbβ, GpIX, MYH9, ACTN1. I prodotti delle reazioni di amplificazione sono stati visualizzati con elettroforesi su gel di agarosio marcato e quindi sequenziati con metodo di Sanger. Abbiamo condotto uno studio preliminare sulla presenza di mutazioni in 5’UTR di ANKRD26 in pazienti con piastrinopenia sporadica e familiare, che non rientravano in forme note di piastrinopenia ereditaria e che presentavano volume piastrinico nella norma (4μm˂MPV˂8μm) per poterne definire le caratteristiche cliniche e molecolari. Abbiamo quindi ampliato l’indagine a RUNX1 ed ETV6, due geni coinvolti in forme di piastrinopenia ereditaria con predisposizione a neoplasie ematologiche in pazienti piastrinopenici e non (MDS piastrinopenia +; MDS piastrinopenia -) appartenenti alle diverse classi di sindrome mielodisplastica sec. WHO 2008. Infine, nell’ambito delle piastrinopenie familiari confermate mediante indagine genetica, abbiamo partecipato al Pregnancy In Inherited Platelet Abnormalities (PIPA), studio multicentrico retrospettivo di valutazione di 339 gravidanze in 181 donne con 13 diverse forme di trombocitopenia. Risultati 8 famiglie (12%) presentano mutazioni nella MYH9, MYH9-related disease, 12 famiglie (19%) presentano mutazioni del gene GP1Bα, 4 famiglie (6%) presentano mutazioni del gene GP1Bβ che causano Bernard-Soulier Syndrome (BSS) mono o biallelico. Le rimanenti famiglie (60%) non presentano mutazioni genetiche sulle glicoproteine studiate, e nessuna mutazione sul gene ACTN1. Alla luce dei recenti studi relativi alle forme di piastrinopenie ereditarie caratterizzate, nel nostro laboratorio abbiamo condotto uno studio preliminare sulla presenza di mutazioni in 5’UTR di ANKRD26 in pazienti con piastrinopenia sporadica e familiare, che non rientravano in forme note di piastrinopenia ereditaria e che presentavano volume piastrinico nella norma (4μm˂MPV˂8μm), identificando due diverse mutazioni in eterozigosi, localizzate nella regione del 5’UTR non tradotta del gene. Dato l’interesse nello studio delle piastrinopenie ereditarie associate allo sviluppo di neoplasie di tipo ematologico e non ematologico, in particolare dei geni RUNX1 ed ETV6, responsabili di trombocitopenia non sindromica a trasmissione autosomica dominante, abbiamo preliminarmente raccolto una piccola casistica di soggetti mielodisplastici piastrinopenici (9 soggetti), di soggetti mielodisplastici con conta piastrinica nei limiti di norma (12 soggetti) e di 27 soggetti di controllo con trombocitopenia ereditaria indeterminata; questo per rinforzare le evidenze circa una possibile associazione tra mutazioni dei geni citati, piastrinopenia e rischio di evoluzione mielodisplastica/leucemica. Di questi soggetti abbiamo raccolto i dati relativi all’età, sesso, cariotipo, numero di blasti, emoglobina e piastrine. I dati preliminari (Tabella 1) dimostrano che le mutazioni dei geni RUNX1 e ETV6 sono presenti solo nel gruppo dei mielodisplastici piastrinopenici. GENE POSIZIONE MUTAZIONE RUNX-1 ESONE 4 c.76C>G RUNX-1 ESONE 8 c.934del A RUNX-1 ESONE 9 c.1214_1215insTG ETV6 INTRONE 1 c.28+192delC Tabella 1. Mutazioni rinvenute nei geni RUNX1 ed ETV6 Anche se il coinvolgimento di questi geni nella piastrinopoiesi è noto, l’esatto meccanismo responsabile di trombocitopenia non è ancora chiaro. La rarità di queste patologie rende difficile raccogliere dati utili ad una analisi genotipo-fenotipo. Nello studio multicentrico (PIPA) abbiamo classificato 23 gravidanze (13 donne): 5 donne presentano bBSS, 3 mBSS, 4 MYH9-RD, 1 ANKRD26. L’età media alla diagnosi di trombocitopenia è di 30 anni, la conta media piastrinica prima del parto è di circa 60 x 109 piastrine/L, anche se in alcuni casi è stata osservata una drastica riduzione piastrinica. La tendenza al sanguinamento è bassa prima del parto e solo in due gravidanze è stata necessaria la trasfusione di piastrine. Tutte le nascite sono avvenute tra la 34a e 40 a settimana; 13 sono i nati con parto cesareo e 10 con parto naturale. Solo 9 neonati riportano la stessa patologia della mamma, anche se in 6 casi il dato non è disponibile poiché non sono state fatte indagini a riguardo. Abbiamo riscontrato un unico caso di decesso neonatale a causa di emorragia cerebrale, ma nessun caso di sanguinamento nei nuovi nati. Conclusioni Le piastrinopenie rappresentano il più frequente disordine dell’emostasi e costituiscono un gruppo eterogeneo di entità cliniche. La peculiarità delle forme familiari risiede nella difficoltà della loro identificazione clinica poiché spesso le piastrinopenie vengono classificate come immuni ed i pazienti sottoposti a terapie inappropriate. Come riportato nei risultati di studi recenti che coinvolgono un numero di famiglie più elevato, anche nel nostro lavoro le forme più comuni di piastrinopenia familiare risultano essere la BSS, la MYH9-RD seguite dalla ANKRD26. Grazie ad una attenta analisi clinica ed alle indagini genetiche associate a recenti studi molecolari, ad oggi è possibile individuare e classificare correttamente i pazienti con nuove mutazioni genetiche considerando che ancora più del 50% delle famiglie non ha diagnosi. Per valutare possibili meccanismi implicati nello sviluppo di piastrinopenia in corso di sindromi mielodisplastiche sarà indispensabile studiare in modo estensivo queste tre categorie di pazienti: piastrinopenie ereditarie con difetto noto di ANKRD26, RUNX-1 e ETV6, mielodisplasie o leucemie con prevalente piastrinopenia. Aumentare la casistica di tali soggetti ci permetterà di approfondire lo studio funzionale delle mutazioni definendone le caratteristiche cliniche e molecolari, al fine di valutare un possibile link tra mielodiplasia e piastrinopenia e comprendere a che livello della megacariopoiesi e dell’ematopoiesi esse agiscano

In the last years there has been accumulating evidence that physical inactivity may accelerate the aging processes. Moreover, a genetic substrate that modulates the aging rate has been suspected in recent literature. However, it is possible that some genetic substrates actually act in an indirect way, modulating the levels of spontaneous physical activity. Therefore the present work investigates (i) the genetic determinants of spontaneous physical activity and (ii) using an experimental approach, the effects of physical activity on cognitive impairment have been addressed. Given the great complexity of the study of genetic determinants in humans, we have taken advantage of the technology introduction of transgenic animals. Specifically, through a meta-analysis approach, the brain distribution of genes that are involved in the change of locomotor activity has been investigated. To this aim, about 50000 abstracts and papers in extenso concerning behavioral studies in knockout mice have been analyzed; data have been collected in a database and crossed to two databases, the Allen Brain Atlas and the Sym Atlas, to verify the brain distribution of genes deleted. Results show that the genes accompanied, after deletion, by an increase of locomotor activity are expressed at higher levels in all brain regions compared to those that after deletion cause locomotor hypoactivity. Therefore, we investigated how the levels of spontaneous physical activity are distributed in an aged population. To this aim, 438 subjects (age 50-86 years) have been characterized for their motor ability (AAPHERD battery), their spontaneous physical activity (test PASE) and their cognitive status (tests MMSE, FAB, attentional matrix). Moreover subjects have been randomized in two experimental groups, the first one fulfilling an intense aerobic and potentiation physical training protocol, and a second one with a light aerobic training. The training, three times per week, one hour per session, lasted for one year. After six months and one year subjects have been tested again using the above battery of tests. Results at six months show that spontaneous physical activity has a bimodal distribution in male aged population. Noteworthy, strong differences have been noted between the groups of more active aged subjects compared to the less active ones: the first takes less drugs and has better scores in agility tests and in attentional tests. To verify the causal relationship between physical activity and these variables, we compared the scores before and after the physical training. Results confirm that physical activity decrease the number of drugs and improves motor abilities. However, attentional scores do not improve after this specific physical training. In conclusion, murine models suggest a genetic substrate for differences in physical activity in the population; data from the aged population suggest that levels of spontaneous physical activity, caused by, at least in part, genetic substrates, could cause the decrease of physical abilities and the increase in the number of drugs taken during ageing process.
Negli ultimi anni si è accumulata l’evidenza che l’inattività fisicapuò accelerare i processi d’invecchiamento. Inoltre, si sospetta un substrato genetico che moduli la velocità d’invecchiamento. È possibile, però, che almeno alcuni di tali substrati genetici invero operino in modo indiretto, modulando i livelli spontanei di attività fisica di un individuo. Pertanto nel presente lavoro sono stati studiati (i) i determinanti genetici dell’attività fisica spontanea e(ii) con uno studio di tipo sperimentale, sono stati affrontati gli effetti dell’attività fisica sul deterioramento cognitivo.Data la complessità dello studio dei determinanti genetici del comportamento, abbiamo sfruttato l’introduzione della tecnologia degli animali transgenici. In particolare, attraverso un lavoro di metanalisi, abbiamo indagato la distribuzione cerebrale di geni che sono coinvolti nel cambiamento dell’attività locomotoria. A tale scopo sono stati analizzati circa 50000 abstract e lavori in extenso relativi a studi comportamentali in animali knockout; i dati sono stati tabulati ed incrociati con altri database quali l’Allen Brain Atlas e il Sym Atlas per conoscere la distribuzione cerebrale dei geni deleti. I risultati dimostrano che i geni accompagnati, dopo delezione, da un aumento dell’attività locomotoria sono più espressi in tutte le regioni cerebrali paragonati a quelli che dopo delezione danno ipoattività locomotoria. Abbiamo voluto, pertanto, indagare come i livelli di attività fisica spontanea si distribuiscono in una popolazione anziana. A tale scopo 438 soggetti (età 50-86 anni) sono stati caratterizzati per le loro abilità motorie (tests AAPHERD), la loro attività fisica spontanea (tests PASE), e il loro status cognitivo (tests MMSE, FAB, matrici attenzionali). Inoltre i soggetti sono poi stati randomizzati in due gruppi sperimentali, il primo con un protocollo di allenamento aerobico e di potenziamento intenso ed il secondo con un allenamento lieve, aerobico. L’allenamento, tre volte a settimana, un’ora a seduta è durato un anno. A distanza di sei mesi e un anno i soggetti sono stati nuovamente testati con la batteria di test sopra descritti. I risultati a sei mesi indicano chel’attività fisica spontanea ha una distribuzione bimodale nella popolazione anzianamaschile. È importante sottolineare che si osservano notevoli differenze fra il gruppo di anziani più attivo e quello meno attivo: il primo assume meno farmaci, ed haun miglior punteggio in test di abilità motoria e di attenzione. Per verificare la relazione causale fra attività fisica spontanea e tali variabili, abbiamo comparato i punteggi prima e dopo allenamento. I risultati confermano che il numero di farmaci si riduce e le abilità motorie migliorano con l’allenamento proposto, mentre le abilità attenzionali non sembrano modificarsi. In conclusione, i modelli murini suggeriscono un substrato genetico per i diversi livelli basali di attività fisica nella popolazione; i dati sulla popolazione anziana suggeriscono che i livelli di attività fisica spontanea, almeno in parte di origine genetica, potrebbe causare le ridotte abilità fisiche e l’aumentato numero di farmaci assunti con l’invecchiamento.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Universidade Estadual Paulista (UNESP)
Visando contribuir para o melhor entendimento do papel dos polimorfismos em genes de citocinas na susceptibilidade para hanseníase, foi conduzido um estudo de associação do tipo caso-controle investigando polimorfismos de base única (SNPs) nas regiões promotoras dos genes IL10 (-819C>T, -1082A>G, -2763A>C, -2849A>G e -3575T>A) e TNF (TNF-308G>A) e no gene LTA (LTA+80C>A e LTA252A>G). Amostras de DNA genômico foram obtidas de 545 pacientes com hanseníase e 380 controles, provenientes do Estado de São Paulo. As genotipagens foram feitas pelas técnicas de PCR e polimorfismo de comprimento dos fragmentos de restrição (RFLP). Para as análises estatísticas foram calculadas as freqüências alélicas, de genótipos e de portadores para cada polimorfismo avaliado. As freqüências de haplótipos foram estimadas por meio do método de máxima verossimilhança. Desvios da lei do equilíbrio de Hardy-Weinberg foram testados empregando testes de Qui-quadrado. Modelos de regressão logística com cálculo de odds ratio (OR) e p-valor com ajustes para as co-variáveis gênero e etnia foram utilizados nas comparações das freqüências entre casos e controles. Em análise isolada, os polimorfismos TNF-308G>A e LTA252A>G não apresentaram associação significativa com a doença, já para o polimorfismo LTA+80C>A, os genótipos AA e CA mostraram-se marginalmente associados com OR de proteção (0,68 e 0,80, respectivamente e mesmo p-valor corrigido=0,07) para hanseníase per se. Confirmando ainda o sentido desta associação, a análise de carreador para o polimorfismo no locus LTA+80 mostrou associação com proteção para hanseníase per se para os carreadores do alelo A (OR=0,78; p-valor corrigido=0,04). Na análise de haplótipos, LTA+80A/LTA+252A/TNF-308G foi também associado com proteção (OR=0,74; p-valor corrigido=0,02). Para a região promotora do gene IL10, o SNP - 819C>T foi...
To the better understanding of the role of genetic polymorphisms at cytokines genes on leprosy susceptibility, we conducted a case-control association study investigating single nucleotide polymorphisms (SNPs) located at promoter region of IL10 (-819C>T, -1082A>G, -2763A>C, -2849A>G and -3575T>A) and TNF genes (TNF-308G>A) and LTA gene (LTA+80C>A e LTA252A>G) gene. Genomic DNA samples were obtained from 545 leprosy patients and 380 controls, from State of São Paulo. Genotyping were done by PCR followed by restriction fragments length polymorphisms (RFLP) analyses. For statistical analyses were calculated allelic, genotypes and carriers frequencies for each polymorphism. The haplotypes frequencies were estimated using maximum-likelihood estimation method. Chisquare tests for deviation from Hardy-Weinberg equilibrium were also performed. Logistic regression models for odds ratio (OR) and p-value calculations, with adjusting for the ethnicity and gender covariates, were performed in comparisons of frequencies. Isolated, TNF-308G>A and LTA252A>G were not significantly associated to the disease, while the CC and CA genotypes to LTA+80C>A locus were marginally associated with protection (0.68 e 0.80, respectively and identical corrected p-value=0.07) for leprosy per se. In the same line, carrier analysis for LTA+80 locus showed association with protection for leprosy per se for allele A carriers (OR=0.78; corrected p-value=0.04). In the haplotype analysis, LTA+80A/LTA+252A/TNF-308G was also associated to protection (OR=0.74; corrected p-value=0.02). From IL10 promoter region analysis, -819C>T SNP was associated with susceptibility to leprosy per se to TT (OR=1.58; p-value=0.05) and CT genotypes (OR=1.36; p=0.05). This association could be confirmed in the carriers analysis for -819T allele (OR=1.40; p-value=0.02 and OR=1.39; corrected pvalue= 0.03). From haplotypic analysis for IL10 ... (Complete abstract click electronic access below)

Thesis (MSc (Genetics))—University of Stellenbosch, 2007.
In South Africa, pre-eclampsia is the second highest cause of maternal deaths. The incidence of this disease in the Western Cape alone is 6.8% and places a large burden of health care facilities. The placenta and implantation thereof is thought to play the most significant role in the onset of this disease. Among the many theories for its aetiology, is the acknowledged two - stage theory. This is based on evidence that pre-eclamptic placentas demonstrate altered remodelling and invasion into the uterine endometrium and myometrium. The sub-optimal endometrium invasion leads to less oxygenation of the placental environment causing transient hypoxia. Consequently, the placenta is thought to release unknown factors into the maternal circulation which then culminates in clinical features associated with pre-eclampsia. Neurokinin B is thought to be one of these placental factors and subsequently binds to the NKB receptor in the maternal system. Endothelium-derived nitric oxide synthase has recently been shown to activate this receptor. The aim of this study was to investigate the role of neurokinin B (TAC3) and the neurokinin B receptor (TACR3) genes in the predisposition of pre-eclampsia and their interaction with eNOS in the South African coloured population together with a matched control cohort.

The overall aims of this thesis were to investigate psychological and behavioral effects of receiving cancer genetic counseling for breast, ovarian and colorectal cancer and/or with a family history of these cancer types and to determine whether counselees’ informational needs were met. Study I was performed 3-7 years post-counseling. Participants (n=214) reported a relatively high level of anxiety but a low level of depression compared to cancer patients in general. However, there was no indication that the distress experienced was due to the counseling. Moderate changes in life and family relations, high level of adherence to recommended controls and satisfaction was reported. Study II was a randomized control trial (RCT) intervention study which involved 147 counselees. An increase in the level of knowledge and correct estimation of personal risk was reported in both the intervention and control groups, although this increase declined at later follow-up. Enhanced information led to significantly greater satisfaction with the given information, and the way of informing relatives. Most counselees had shared information with their at-risk relatives. Study III focused on sharing information with at-risk relatives among participants in study II and their relatives (n=81). Counselees were interviewed and answered a questionnaire, whilst their relatives only answered the questionnaire. Counselees reported positive/neutral feelings about communicating genetic information and mostly interpreted their relatives’ reactions as positive/ neutral. Also, approximately 50% of relatives reported positive/neutral reactions and were generally satisfied with the received information. Study IV was conducted in Sweden and Norway based on 235 counselees. Counselees expected counselors to be skillful and thoughtful, take them seriously and provide risk estimations and medical information. Most important issues to counselees were satisfactorily addressed by the counselors. Analyzing importance rankings resulted in five categories of needs: a need for facts, caring communication and medical information, need for understanding and support in sharing genetic information, practical care and medical/practical information. In conclusion, no adverse psychological or behavioral effect on counselees was observed. Apparently, genetic counseling is managed properly and counselors successfully address counselees’ needs. Providing extended information does not seem necessary, however, tailoring information to individual counselees needs may create a more effective counseling.

Thesis (MSc)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: The World Health Organisation (WHO) has defined preterm labour as the onset of labour before 37 completed weeks of gestation with an incidence ranging between 5-10%. Although patient care has improved, the rate of preterm birth has slowly been increasing and currently impacts significantly on maternal and fetal mortality and morbidity. The complex condition of preterm labour involves multiple etiologies and risk factors, which complicates the search for candidate markers and / or biomarkers. The aim of this prospective study was to investigate potential genetic associations with preterm labour. The study cohort consisted of consecutive first-time booking, low-risk primigravid pregnant women from a restricted geographical region. The study cohort comprised 421 [306 Coloured and 115 Black] pregnant women presenting at the Paarl Hospital Obstetric clinic. Subsequently, DNA was extracted from whole blood and investigated for a range of known polymorphisms in pro-inflammatory and anti-inflammatory cytokines, as well as the novel LGALS13 gene, for potential variants that may impact on pregnancy outcome. Screening techniques involve combinations of allele-specific PCR amplification, Multiphor SSCP/HD analysis, restriction enzyme analyses and DNA sequencing. A significant association was demonstrated between the IL-1RN*2-allele and adverse pregnancy outcome, mainly in the preterm labour and hypertension group. The presence TNFα-308 A-allele was associated with overall adverse pregnancy outcome and preterm labour. In addition to this, a novel IL-1RN allele was identified in the control group. Mutation screening and subsequent statistical methods revealed an association between a novel LGALS13 exonic variant, 221delT, and preterm labour in Coloured women. Two previouslydocumented intronic variants (IVS2-22A/G and IVS3+72T/A) demonstrated linkage disequilibrium, signifying evolutionary conservation of exon three. Additionally, two novel intronic variants, IVS2-36 G/A and IVS2-15 G/A, demonstrated no association with adverse pregnancy outcome. In this study we identified rare novel exonic variants; two non-synonymous variants in exon three (M44V, [N=2] and K87R, [N=1]) and a silent variant in exon four (P117P, [N=1]) - all identified in individuals from the control cohort. Within coding exon three, an interesting variant [“hotspot”] was identified, which represents six polymorphic bases within an 11bp stretch. No associations were demonstrated with these variants and pregnancy outcome. Furthermore, a previously documented 5' “‘promoter” variant, -98 A/C, was identified and demonstrated no association with adverse pregnancy outcome. However, subdivision of lateonset pre-eclamptic cases revealed a significant association with the A-allele and late-onset preeclampsia. Genotype-phenotype investigation demonstrated association between the IL-10 -1082 A/G, IL-4 C/T and 221delT loci and poor pregnancy progress which manifested as (i) delivery of infants weighing <2000g, (ii) before 37 weeks of gestation. The findings of this study will strengthen our understanding of the pathophysiology underlying pregnancy complications and facilitate the further development of effective treatment strategies to reduce maternal and fetal morbidity and mortality.
AFRIKAANSE OPSOMMING: Die Wêreld Gesondheid Organisasie (WHO) klassifiseer voortydse kraam as kontraksie voor 37 volledige weke, met ‘n insidensie tussen 5-10%. Alhoewel pasiënte-sorg verbeter het, neem die tempo van voortydse geboorte steeds toe, wat ‘n groot impak het op moederstrefte en fetale mortaliteit en morbiditeit. Die komplekse kondisie van voortydse kraam sluit veelvoudige oorsake en risiko faktore in, wat die navorsing van kandidaat en / of biologiese merkers kompliseer. Die doel van hierdie prospektiewe studie, was die potensiële navorsing van genetiese assosiasies met voortydse kraam. Die studie kohort bevat opeenvolgende eerste bespreking van lae risiko primigravida swanger vrouens vanaf ‘n beperkte geografiese omgewing. Die studie kohort beslaan 421 [306 Kleurling en 115 Swart] swanger vrouens teenwoordig by die Paarl Hospitaal Verloskunde kliniek. Vervolgens was DNS geëkstraeer van bloedmonsters en geondersoek vir ‘n verskeidenheid van bekende polimorfismes in pro-inflammatoriese en antiinflammatoriese sitokiene, insluitend die nuwe sifting van die LGALS13 geen potensiaal vir variante wat ‘n impak op swangerskap uitkomste sal hê. Die siftings tegnieke toegepas, sluit in ‘n kombinasie van alleel-spesifieke amplifikasie, Multiphor enkelstring konformasie polimorfisme / heterodupleks analise, restriksie ensiem verterings en volgorde bepalings tegnieke. ‘n Betekenisvolle assosiasie was gedemonstreer tussen die IL-1RN*2-alleel en nadelige swangerskap, beperk tot voortydse kraam en die hipertensie groep. Die teenwoordigheid van die TNFα-308 A-alleel was geassosieer met algehele nadelige uitkomste en voortydse kraam. Daarby, was ‘n nuwe IL-1RN alleel geïdentifiseer in die kontrole groep. Mutasie sifting en opeenvolgende statistiese metodes, het ‘n assosiasie getoon tussen ‘n nuwe LGALS13 koderende variant, 221delT, en voortydse kraam in Kleurling vrouens. Twee voorafbeskryfde introniese variante (IVS2-22 A/G en IVS3+72 T/A), het ‘n betekenisvolle bewys opgelewer dat daar koppelings-onewewig bestaan tussen hierdie variante, en toon evolusionêre konservasie van ekson drie. Addisioneel was twee nuwe introniese variante ontdek, IVS2-36 G/A en IVS2-15 G/A, wat geen assosiasie getoon nie. In hierdie studie het ons ‘n nuwe seldsame koderende variante geïdentifiseer in die kontrole groep, waarvan twee nie-sinonieme variante was in ekson drie (M44V, N=2 en K87R, N=1) en ‘n stil variasie in ekson vier (P117P, N=1). Geleë in die koderende area van ekson drie, was ’n interessante variant [“hotspot’] ontdek, waarvan ses basisse in ‘n 11 basis paar area polimorfies is. Geen assosiasie was getoon met hierdie variante en swangerskap uitkomste nie. Verder was ‘n voorafbeskryfde 5' ‘promotor’ variant, -98 A/C, geïdentifiseer wat geen assosiasie getoon met nadelige swangerskap uitkomste nie. Onderverdeling van laat-aanvangs preeklampsie, het getoon dat die A-alleel ‘n betekenisvolle assosiasie getoon het met die ontwikkeling van laat pre-eklampsie. Genotipe-fenotipe interaksies het ’n assosiasie getoon tussen die IL-10 -1082 A/G, IL-4 C/T en 221delT lokusse en nadelige swangerskap uitkomste, wat manifesteer as (i) kraam van suigelinge wat <2000g weeg, (ii) geboorte voor 37 weke. Die bevindings van hierdie studie sal ons basiese kennis verbeter oor die patologie beskrywend aan swangerskap komplikasies, asook die fasilitering en ontwikkeling van effektiewe behandelings strategieë, om moederstrefte en fetale mortaliteit en morbiditeit te verminder.

[Truncated abstract] This thesis investigates novel methods for the genetic association analysis of haplotype data in samples of unrelated individuals, and applies these methods to the analysis of coronary heart disease and related phenotypes. Determining the inheritance pattern of genetic variants in studies of unrelated individuals can be problematic because family members of the studied individuals are often not available. For the analysis of individual genetic loci, no problem arises because the unit of interest is the observed genotype. When the unit of interest is the linear combination of alleles along one chromosome, inherited together in a haplotype, it is not always possible to determine with certainty the inheritance pattern, and therefore statistical methods to infer these patterns must be adopted. Due to genotypic heterozygosity, mutliple possible haplotype configurations can often resolve an individual's genotype measures at multiple loci. When haplotypes are not known, but are inferred statistically, an element of uncertainty is thus inherent which, if not dealt with appropriately, can result in unreliable estimates of effect sizes in an association setting. The core aim of the research described in this thesis was to develop and implement a general method for haplotype-based association analysis using multiple imputation to appropriately deal with uncertainty haplotype assignment. Regression-based approaches to association analysis provide flexible methods to investigate the influence of a covariate on a response variable, adjusting for the effects of other variables including interaction terms. ... These methods are then applied to models accommodating binary, quantitative, longitudinal and survival data. The performance of the multiple imputation method implemented was assessed using simulated data under a range of haplotypic effect sizes and genetic inheritance patterns. The multiple imputation approach performed better, on average, than ignoring haplotypic uncertainty, and provided estimates that in most cases were similar to those observed when haplotypes were known. The haplotype association methods developed in this thesis were used to investigate the genetic epidemiology of cardiovascular disease, utilising data for the cholesteryl ester transfer protein gene (CETP), the hepatic lipase (LIPC) gene and the 15- lipoxygenase (ALOX15) gene on a total of 6,487 individuals from three Western Australian studies. Results of these analyses suggested single nucleotide polymorphisms (SNPs) and haplotypes in the CETP gene were associated with increased plasma high-density lipoprotein cholesterol (HDL-C). SNPs in the LIPC gene were also associated with increased HDL-C and haplotypes in the ALOX15 gene were associated with risk of carotid plaque among individuals with premature CHD. The research presented in this thesis is both novel and important as it provides methods for the analysis of haplotypic associations with a range of response types, while incorporating information about haplotype uncertainty inherent in populationbased studies. These methods are shown to perform well for a range of simulated and real data situations, and have been written into a statistical analysis package that has been freely released to the research community.

Genetic improvement of honey bee populations based on molecular genetics features is faster and precision in comparison with morphometry and behavior-based methods. We developed the method based on nine nuclear microsatellite loci that allow a selection of most adaptive honey bee colonies by genetically defined features. Our study the heterozygosity of the dark European bee A. m. mellifera inhabiting the extremely cold region of the Ural Mountains to provide a marker-assisted selection for revealing the high adapted to extremely cold climate honey bee population can be applied for markerassisted selection of honey bees adapted to beekeeping in extremal climatic conditions.

Background: Epilepsy is a complex neurological disorder, that affects 0.5 to 1% of the population, with a diversified etiology, but with emphasis on its relation with genetics. Despite there are several therapies to treat it, in some cases, this variety is still insuficiente, featuring refractory epilepsy, frequent in people with intelectual disabilities (ID). Objectives: To analyze the scientific production about refractory epilepsy and ID. Methods: Integrative literature review that searched for international articles in the Virtual Health Library (VHL), using the keywords “Intellectual disability” AND “Refractory epilepsy” with the filter: “full text”. Results: From the 27 articles found, 2 were excluded for escaping the theme, having 25 articles as a final corpus and 2 thematic axes identified: (I) Genetic aspects related to ID and refractory epilepsy and (II) Therapeutic interventions in these patients. According to studies, refractory epilepsy in people with ID is related to mutations in some genes, such as: PCDH19, FMR1, TDP2, GABRB2 and SLC9A6. As for therapies for these patients, drugs such as stiripentol, lacosamide and benzodiazepines have been used, in addition to other interventions such as vagus nerve therapies, responsive neural stimulation, ketogenic diet, immunotherapy and resection surgery. Conclusions: The ID association with refractory epilepsy is strongly linked to genetic mutations, being essencial the genetic diagnosid to individualize the treatment and overcome insuficiente therapies for this epilepsy, especially in patients with associated ID, who tend to have a reduced life quality, having as primary objective the improvement of it.

Chickpea is the third most important pulse crop in the world and ranks first in the Middle East; however, it has been subjected to only limited research in modern genomics. In the first period of this project (US-3034-98R) we constructed two large-insert BAC and BIBAC libraries, developed 325 SSR markers and mapped QTLs controlling ascochyta blight resistance (ABR) and days to first flower (DTF). Nevertheless, the utilities of these tools and results in gene discovery and marker-assisted breeding are limited due to the absence of an essential platform. The goals of this period of the project were to use the resources and tools developed in the first period of the project to develop a BAC/BIBAC physical map for chickpea and using it to identify BAC/BIBACcontigs containing agronomic genes of interest, with an emphasis on ABR and DTF, and develop DNA markers suitable for marker-assisted breeding. Toward these goals, we proposed: 1) Fingerprint ~50,000 (10x) BACs from the BAC and BIBAC libraries, assemble the clones into a genome-wide BAC/BIBAC physical map, and integrate the BAC/BIBAC map with the existing chickpea genetic maps (Zhang, USA); 2) fine-map ABR and DTFQTLs and enhance molecular tools for chickpea genetics and breeding (Shahal, Sherman and DaniShtienberg, Israel; Chen and Muehlbauer; USA); and 3) integrate the BAC/BIBAC map with the existing chickpea genetic maps (Sherman, Israel; Zhang and Chen, USA). For these objectives, a total of $460,000 was requested originally, but a total of $300,000 was awarded to the project. We first developed two new BAC and BIBAC libraries, Chickpea-CME and Chickpea- CHV. The chickpea-CMEBAC library contains 22,272 clones, with an average insert size of 130 kb and equivalent to 4.0 fold of the chickpea genome. The chickpea-CHVBIBAC library contains 38,400 clones, with an average insert size of 140 kb and equivalent to 7.5 fold of the chickpea genome. The two new libraries (11.5 x), along with the two BAC (Chickpea-CHI) and BIBAC (Chickpea-CBV) libraries (7.1 x) constructed in the first period of the project, provide libraries essential for chickpea genome physical mapping and many other genomics researches. Using these four libraries we then developed the proposed BAC/BIBAC physical map of chickpea. A total of 67,584 clones were fingerprinted, and 64,211 (~11.6 x) of the fingerprints validated and used in the physical map assembly. The physical map consists of 1,945 BAC/BIBACcontigs, with each containing an average of 39.2 clones and having an average physical length of 559 kb. The contigs collectively span ~1,088 Mb, being 1.49 fold of the 740- Mb chickpea genome. Third, we integrated the physical map with the two existing chickpea genetic maps using a total of 172 (124 + 48) SSR markers. Fourth, we identified tightly linked markers for ABR-QTL1, increased marker density at ABR-QTL2 and studied the genetic basis of resistance to pod abortion, a major problem in the east Mediterranean, caused by heat stress. Finally, we, using the integrated map, isolated the BAC/BIBACcontigs containing or closely linked to QTL4.1, QTL4.2 and QTL8 for ABR and QTL8 for DTF. The integrated BAC/BIBAC map resulted from the project will provide a powerful platform and tools essential for many aspects of advanced genomics and genetics research of this crop and related species. These includes, but are not limited to, targeted development of SNP, InDel and SSR markers, high-resolution mapping of the chickpea genome and its agronomic genes and QTLs, sequencing and decoding of all genes of the genome using the next-generation sequencing technology, and comparative genome analysis of chickpea versus other legumes. The DNA markers and BAC/BIBACcontigs containing or closely linked to ABR and DTF provide essential tools to develop SSR and SNP markers well-suited for marker-assisted breeding of the traits and clone their corresponding genes. The development of the tools and knowledge will thus promote enhanced and substantial genetic improvement of the crop and related legumes.

Citrus tristeza virus (CTV) has the largest genomes among RNA viruses of plants. The 19,296-nt CTV genome codes for eleven open reading frames (ORFs) and can produce at least 19 protein products ranging in size from 6 to 401 kDa. The complex biology of CTV results in an unusual composition of CTV-specific RNAs in infected plants which includes multiple defective RNAs and mixed infections. The complex structure of CTV populations poses special problems for diagnosis, strain differentiation, and studies of pathogenesis. A manipulatable genetic system with the full-length cDNA copy of the CTV genome has been created which allows direct studies of various aspects of the CTV biology and pathology. This genetic system is being used to identify determinants of the decline and stem-pitting disease syndromes, as well as determinants responsible for aphid transmission.

Tree species are characterized by their perennial growth habit, woody morphology, long juvenile period phase, mostly outcrossing behaviour, highly heterozygosity genetic makeup, and relatively high genetic diversity. The economically important trees have been an integral part of the human life system due to their provision of timber, fruit, fodder, and medicinal and/or health benefits. Despite its widespread application in agriculture, industrial and medicinal values, the molecular aspects of key economic traits of many tree species remain largely unexplored. Over the past two decades, research on forest tree genomics has generally lagged behind that of other agronomic crops. Genomic research on trees is motivated by the need to support genetic improvement programmes mostly for food trees and timber, and develop diagnostic tools to assist in recommendation for optimum conservation, restoration and management of natural populations. Research on long-lived woody perennials is extending our molecular knowledge and understanding of complex life histories and adaptations to the environment, enriching a field that has traditionally drawn its biological inference from a few short-lived herbaceous species. These concerns have fostered research aimed at deciphering the genomic basis of complex traits that are related to the adaptive value of trees. This review summarizes the highlights of tree genomics and offers some priorities for accelerating progress in the next decade.

One of the emerging technologies is the use of microbial agents for the control of postharvest diseases of fruits and vegetables. A number of antagonistic microorganisms have been discovered which have the potential to effectively control postharvest diseases. Some of this technology has been patented and commercial products such as AspireTM (Ecogen Corporatin, Langhorne, PA, USA), Biosave 10TM and Biosave 11TM (Ecoscience Inc., Worchester, MA, USA) have been registered for commercial use. The principal investigator of this project was involved in developing the yeast-based biofungicide-AspireTM and testing its efficacy under commercial conditions. This research project was initiated to fill the gap between the knowledge available on development and commercial implementation of yeast biocontrol agents and basic understanding of various aspects related to introducing yeast antagonists to fruit surfaces, along with verification of population genetics. The main objectives of this study were: Study ecology, population dynamics and genetic diversity of the yeast antagonists Candida guilliermondii, C. oleophila, and Debaryomyces hansenii, and study the effect of preharvest application of the yeast antagonist C. oleophila naturally occurring epiphytic microbial population and on the development of postharvest diseases of citrus fruit during storage. Our findings, which were detailed in several publications, have shown that an epiphytic yeast population of grapefruit able to grow under high osmotic conditions and a wide range of temperatures was isolated and characterized for its biocontrol activity against green mold decay caused by Penicillium digitatum. Techniques based on random amplified polymorphic DNA (RAPD) and arbitrary primed polymerase chain reaction (ap-PCR), as well as homologies between sequences of the rDNA internal transcribed spacers (ITS) and 5.8S gene, were used to characterize the composition of the yeast population and to determine the genetic relationship among predominant yeast species. Epiphytic yeasts exhibiting the highest biocontrol activity against P. digitatum on grapefruit were identified as Candida guilliermondii, C. oleophila, C. sake, and Debaryomyces hansenii, while C. guilliermondii was the most predominant species. RAPD and ap-PCR analysis of the osmotolerant yeast population showed two different, major groups. The sequences of the ITS regions and the 5.8S gene of the yeast isolates, previously identified as belonging to different species, were found to be identical. Following the need to develop a genetically marked strain of the yeast C. oleophila, to be used in population dynamics studies, a transformation system for the yeast was developed. Histidine auxotrophy of C. oloephila produced using ethyl methanesulfonate were transformed with plasmids containing HIS3, HIS4 and HIS5 genes from Saccharomyces cerevisiae. In one mutant histidin auxotrophy was complemented by the HIS5 gene of S. cerevisiae is functionally homologous to the HIS5 gene in V. oleophila. Southern blot analysis showed that the plasmid containing the S. cerevisiae HIS5 gene was integrated at a different location every C. oleophila HIS+ transformant. There were no detectable physiological differences between C. oleophila strain I-182 and the transformants. The biological control ability of C. oleophila was not affected by the transformation. A genetically marked (with b-glucuronidase gene) transformant of C. oleophila colonized wounds on orange fruits and its population increased under field conditions. Effect of preharvest application of the yeast C. oleophila on population dynamics of epiphytic microbial population on wounded and unwounded grapefruit surface in the orchard and after harvest was also studied. In addition, the effect of preharvest application of the yeast C. oleophila on the development of postharvest decay was evaluated. Population studies conducted in the orchard showed that in control, non-treated fruit, colonization of wounded and unwounded grapefruit surface by naturally occurring filamentous fungi did not vary throughout the incubation period on the tree. On the other hand, colonization of intact and wounded fruit surface by naturally occurring yeasts was different. Yeasts colonized wounded surface rapidly and increased in numbers to about two orders of magnitude as compared to unwounded surface. On fruit treated with the yeast and kept on the tree, a different picture of fungal and yeast population had emerged. The detected fungal population on the yeast-treated intact surface was dramatically reduced and in treated wounds no fungi was detected. Yeast population on intact surface was relatively high immediately after the application of AspireTM and decreased to than 70% of that detected initially. In wounds, yeast population increased from 2.5 x 104 to about 4x106 after 72 hours of incubation at 20oC. Results of tests conducted to evaluate the effect of preharvest application of AspireTM on the development of postharvest decay indicated the validity of the approach.

As is true for all crops, production of Citrus fruit is limited by traits whose characteristics are the products of many genes (i.e. cold hardiness). In order to modify these traits by marker aided selection or molecular genetic techniques, it is first necessary to map the relevant genes. Mapping of quantitative trait loci (QTLs) in perennial plants has been extremely difficult, requiring large numbers of mature plants. Production of suitable mapping populations has been inhibited by aspects of reproductive biology (e.g. incompatibility, apomixis) and delayed by juvenility. New approaches promise to overcome some of these obstacles. The overall objective of this project was to determine whether QTLs for environmental stress tolerance could be effectively mapped in the perennial crop Citrus, using an extensive linkage map consisting of various types of molecular markers. Specific objectives were to: 1) Produce a highly saturated genetic linkage map of Citrus by continuing to place molecular markers of several types on the map. 2) Exploiting recently developed technology and already characterized parental types, determine whether QTLs governing cold acclimation can be mapped using very young seedling populations. 3) Determine whether the same strategy can be transferred to a different situation by mapping QTLs influencing Na+ and C1- exclusion (likely components of salinity tolerance) in the already characterized cross and in new alternative crosses. 4) Construct a YAC library of the citrus genome for future mapping and cloning.

Genetic and biochemical analysis of glucosinolate breakdown: The effects of indole-3-carbinol on plant physiology and development Glucosinolates are a class of defense-related secondary metabolites found in all crucifers, including important oilseed and vegetable crops in the Brassica genus and the well-studied model plant Arabidopsis thaliana. Upon tissue damage, such as that provided by insect feeding, glucosinolates are subjected to catalysis and spontaneous degradation to form a variety of breakdown products. These breakdown products typically have a deterrent effect on generalist herbivores. Glucosinolate breakdown products also contribute to the anti-carcinogenic effects of eating cabbage, broccoli and related cruciferous vegetables. Indole-3-carbinol, a breakdown product of indol-3-ylmethylglucosinolate, forms conjugates with several other plant metabolites. Although some indole-3-carbinol conjugates have known functions in defense against herbivores and pathogens, most play as yet unidentified roles in plant metabolism, and possibly also plant development. At the outset, our proposal had three main hypotheses: (1) There is a specific detoxification pathway for indole-3-carbinol; (2) Metabolites derived from indole-3-carbinol are phloem-mobile and serve as signaling molecules; and (3) Indole-3-carbinol affects plant cell cycle and cell-differentiation pathways. The experiments were designed to enable us to elucidate how indole-3-carbinol and related metabolites affect plants and their interactions with herbivorous insects. We discovered that indole-3- carbinol rapidly and reversibly inhibits root elongation in a dose-dependent manner, and that this inhibition is accompanied by a loss of auxin activity in the root meristem. A direct interaction between indole-3-carbinol and the auxin perception machinery was suggested, as application of indole-3-carbinol rescued auxin-induced root phenotypes. In vitro and yeast-based protein interaction studies showed that indole-3-carbinol perturbs the auxin-dependent interaction of TIR1 with Aux/IAA proteins, supporting the notion that indole-3-carbinol acts as an auxin antagonist. Furthermore, transcript profiling experiments revealed the influence of indole-3-carbinol on auxin signaling in root tips, and indole-3-carbinol also affected auxin transporters. Brief treatment with indole-3-carbinol led to a reduction in the amount of PIN1 and to mislocalization of PIN2. The results indicate that chemicals induced by herbivory, such as indole-3-carbinol, function not only to repel herbivores, but also as signaling molecules that directly compete with auxin to fine tune plant growth and development, which implies transport of indole-3- carbinol that we are as yet unsuccessful in detecting. Our results indicate that plant defensive metabolites also have secondary functions in regulating aspects of plant metabolism, thereby providing diversity in defense-related plant signaling pathways. Such diversity of of signaling by defensive metabolites would be beneficial for the plant, as herbivores and pathogens would be less likely to mount effective countermeasures. We propose that growth arrest can be mediated directly by the herbivory-induced chemicals, in our case, indole-3-carbinol. Thus, glucosinolate breakdown to I3C following herbivory would have two outcomes: (1) Indole-3-carbinaol would inhibit the herbivore, while (2) at the same time inducing growth arrest within the plant. Thus, our results indicate that I3C is a defensive phytohormone that modulates auxin signaling, leading to growth arrest.

The specific objectives of this BARD proposal were: Use a comparative genomics approach to identify T3Es in group I, II and III strains of A. citrulli. Determine the bacterial genes contributing to host preference. Develop mutant strains that can be used for biological control of BFB. Background to the topic: Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovoraxcitrulli, is a devastating disease that affects watermelon (Citrulluslanatus) and melon (Cucumismelo) production worldwide, including both Israel and USA. Three major groups of A. citrullistrains have been classified based on their virulence on host plants, genetics and biochemical properties. The host selection could be one of the major factors that shape A. citrullivirulence. The differences in the repertoire of type III‐ secreted effectors (T3Es) among the three A. citrulligroups could play a major role in determining host preferential association. Currently, there are only 11 A. citrulliT3Es predicted by the annotation of the genome of the group II strain, AAC00‐1. We expect that new A. citrulliT3Es can be identified by a combination of bioinformatics and experimental approaches, which may help us to further define the relationship of T3Es and host preference of A. citrulli. Implications, both scientific and agricultural: Enriching the information on virulence and avirulence functions of T3Es will contribute to the understanding of basic aspects of A. citrulli‐cucurbit interactions. In the long term, it will contribute to the development of durable BFB resistance in commercial varieties. In the short term, identifying bacterial genes that contribute to virulence and host preference will allow the engineering of A. citrullimutants that can trigger SAR in a given host. If applied as seed treatments, these should significantly improve the effectiveness and efficacy of BFB management in melon and atermelon production.