Gotowa bibliografia na temat „Non - allelic interaction”

In the present study five generations viz. P1, P2, F1, F2 and F3 of chickpea were taken for the analysis of non-allelic interaction considering five yield and yield contributing characters like as date of first flower (DFF), number of primary branches at first flower (NPBFF), plant height at first flower (PHFF), number of pods per plant (NPd/P) and number of seeds per plant (NS/P). At first Mather’s scaling test was done where scales C and D found to be significant and non-significant for all the traits, respectively. Potence was non-significant for all the characters, which revealed that there was no dominance. In the Cavalli’s joint scaling test, the χ2 values were found to be significant for all the characters, which confirm that additive-dominance model was inadequate and except additive and dominance effects there are other effects like non-allelic interaction, linkage, genotype × environment (G×E) interaction etc. are involved either individually or in combination in the inheritance of the studied traits.

In the present work, we study the genetic control of reproductive traits under different heat stress conditions in two populations of inbred lines derived from crosses between two S. pimpinellifolium accessions and two tomato cultivars (E9×L5 and E6203×LA1589). The temperature increase affected the reproductive traits, especially at extremely high temperatures, where only a few lines were able to set fruits. Even though a relative modest number of QTLs was identified, two clusters of QTLs involved in the responses of reproductive traits to heat stress were detected in both populations on chromosomes 1 and 2. Interestingly, several epistatic interactions were detected in the E9×L5 population, which were classified into three classes based on the allelic interaction: dominant (one locus suppressed the allelic effects of a second locus), co-adaptive (the double-homozygous alleles from the same parent alleles showed a higher phenotypic value than the combination of homozygous alleles from alternative parents) and transgressive (the combination of double-homozygous alleles from different parents showed better performance than double-homozygous alleles from the same parents). These results reinforce the important role of non-additive genetic variance in the response to heat stress and the potential of the new allelic combinations that arise after wide crosses.

A 5 X 5 diallel analysis was carried out according to Hayman on quality and yield contributing traits of pumpkin. Five (5) advanced inbreds of pumpkin viz IB 23, IB 40, IB 47, IB 50 and IB 57 were used in this study. The five parents and their F1 hybrids were arranged in a randomized complete block design with three replications for studying the quality and yield contributing traits. Analysis of variance showed significant differences among genotypes for all of the traits except dry matter and fruit yield. Parental mean suggested that all the parents contained recessive alleles with positive effect. In the 5 × 5 diallel set, Wr-Vr graph analysis revealed the presence of allelic interaction in all the character’s except fruit length, dry matter, brix percentage and reducing sugar where non-allelic interaction was present.The Agriculturists 2016; 14(1) 15-32

Objective The carcinogenesis role of PARP1 in lung cancer is still not clear. Analysis at allelic levels cannot fully explain the function of PARP1 on lung cancer. Our study aims to further explore the relation between PARP1 haplotypes and lung cancer. Materials and methods DNA and RNA were extracted from non-small cell lung cancer (NSCLC) tumor and adjacent normal fresh frozen tissue. Five PARP1-SNPs were genotyped and PARP1-specific SNPs were imputed using IMPUTE and SHAPEIT software. The SNPs were subjected to allelic, haplotype and SNP-SNP interaction analyses. Correlation between SNPs and mRNA/protein expressions were performed. Results SNP imputation inferred the ungenotyped SNPs and increased the power for association analysis. Tumor tissue samples are more likely to carry rs1805414 (OR = 1.85; 95% CI: 1.12–3.06; P-value: 0.017) and rs1805404 (OR = 2.74; 95%CI 1.19–6.32; P-value: 0.015) compared to normal tissues. Our study is the first study to show that haplotypes comprising of 5 SNPs on PARP1 (rs1136410, rs3219073, rs1805414, rs1805404, rs1805415) is able to differentiate the NSCLC tumor from normal tissues. Interaction between rs3219073, rs1805415, and rs1805414 were significantly associated with the NSCLC tumor with OR ranging from 3.61–6.75; 95%CI from 1.82 to 19.9; P-value<0.001. Conclusion PARP1 haplotypes may serve as a better predictor in lung cancer development and prognosis compared to single alleles.

SUMMARYNormal, self and backcross families in a triple test cross were used to investigate the inheritance of number of days from sowing to flowering and dry plant weight for a population of pure-breeding lines of bread wheat (Triticum aestivum L.). Several tests of non-allelic interaction, additive genetic and dominance variances were made that involved triple test cross and single tester analyses. Non-allelic interaction was found to be a component of the genetic variation for both traits. The additive and dominance variances were prevalent for both traits. Alternative estimates of additive genetic and dominance variance components did not differ significantly when tested by a weighted least-squares model fitting procedure. The results are discussed in relation to genetic improvement of bread wheat.

Abstract The existence of sophisticated self/non-self systems is not limited to vertebrates. Several colonial marine invertebrates have evolved systems that distinguish between conspecifics via cell-cell contact. This phenomenon, known as allorecognition, occurs when colonies encounter each other as they grow across their substrate. Compatible colonies fuse or co-exist, while incompatible colonies reject and often aggressively compete for space. In all taxa studied to date, allorecognition phenotypes are determined by highly polymorphic loci, which ensure that colonies are only compatible with themselves or close kin. How allorecognition molecules achieve this specificity remains unknown. Allorecognition in Hydractinia is controlled by at least two genes, alr1 and alr2, which encode highly polymorphic transmembrane proteins similar to immunoglobulin superfamily molecules. Colonies with matching alleles at alr1 and alr2 fuse, while colonies with no matching alleles reject. Using in vitro assays with recombinant proteins, we demonstrate that alr1 alleles bind to themselves but not to other alr1 alleles. We suggest that, in vivo, compatibility between colonies is also determined by allele-specific homophilic binding of alr proteins. Since fusion is rare in nature and single populations contain hundreds of unique alr alleles, the Hydractinia allorecognition system appears to be based on a biophysical mechanism with unprecedented allelic diversity and specificity.

An attempt was made to breed for improved plant type in pigeonpen by crossing two contrasting parents viz. Vamban-I and Gulbarga-1. The F2 population clearly segregated into four distinct classes of phenotypes (plant types) in the ratio of 50 intermediate erect tall: 14 compact dwarf in the early segregants and 15 tall spreading: 1 compact bushy in the late segregants indicating that the plant type in pigeonpea was controlled by interaction of two pairs of non-allelic linkage blocks designated as DTCLSI and TALS2 and their respective allelic blocks as dtclsl and tais2.

A non-excitable behavioural mutant, d4-662, was previously characterized as the fourth pawn locus mutant pwD in Paramecium tetraurelia. We now provide data demonstrating that d4-662 is in fact controlled by a pwB allele that has the unusual feature of complementing other pwB alleles in heterozygous F1 progeny. Neither the cytoplasm nor the nucleoplasm of d4-662 cured the mutational defects of pwB and in the reverse combination of d4-662 and pwB, the result was the same. On the other hand, pwA, another non-excitable mutant, was cured upon cross-injection with d4-662 and mutants carrying trichocyst non-discharge marker genes were also cured. This evidence suggests that d4-662 is a new mutant belonging to pwB, and would be better designated as pwB662. Extensive crossbreeding analyses, however, showed an unusual genetic relationship between d4-662 and pwB (pwB95 or pwB96). When d4-662 was crossed with pwB mutants, many progeny expressing wild-type phenotype or mixed clones of wild-type and pawn cells were obtained in the F1. Less than 12·5% expressed the pawn phenotype. The appearance of wild-type progeny in this F1 strongly suggests that an inter-allelic interaction between pwB662 and other pwB alleles may occur during development of the macronucleus.

The breeding improvement of triticale is tightly associated with the introgression of dwarfing genes, in particular, gibberellin (GA)-insensitive Ddw1 from rye. Despite the increase in harvest index and resistance to lodging, this gene adversely affects grain weight and size. Growth regulation factor (GRF) genes are plant-specific transcription factors that play an important role in plant growth, including GA-induced stem elongation. This study presents the results of a two-year field experiment to assess the effect of alleles of the TaGRF3-2A gene in interaction with DDW1 on economically valuable traits of spring triticale plants grown in the Non-Chernozem zone. Our results show that, depending on the allelic state, the TaGRF3-2A gene in semi-dwarf spring triticale plants influences the thousand grain weight and the grain weight of the main spike in spring triticale, which makes it possible to use it to compensate for the negative effects of the dwarfing allele Ddw1. The identified allelic variants of the TaGRF3-2A gene can be included in marker-assisted breeding for triticale to improve traits.

Allergic manifestations are increasingly common in infants and children. Accumulating evidence suggests that the ‘epidemic’ increase of childhood allergy may be associated with environmental factors such as stress. Although the impact of stress on the manifestation and exacerbation of allergy has been demonstrated, the underlying mechanisms of stress-induced exacerbation are still obscure. A growing number of studies have suggested an altered hypothalamus-pituitary-adrenal (HPA) axis function to stress in allergic children. It is speculated that a dysfunctional HPA axis in response to stress may facilitate and/or consolidate immunological aberrations and thus, may increase the risk for allergic sensitization and exacerbation especially under stressful conditions. In the present review the potential impact of a hyporesponsive as well as a hyperresponsive HPA axis on the onset and chronification of childhood allergy is summarized. Moreover, potential factors that may contribute to the development of an aberrant HPA axis responsiveness in allergy are discussed
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Summary La tubercolosi (TB) è una malattia di origine infettiva causata dal bacillo Mycobacterium tuberculosis (MTB), chiamato anche Bacillo di Koch. Essa figura al secondo posto nelle cause di morte per malattie infettive in tutto il mondo dopo lo Human Immunodeficiency Virus (HIV). Nel 1993, la TB è stata classificata come emergenza mondiale dalla World Health Organisation (WHO) che ha stimato circa 9 milioni di nuovi casi nel 2011 e 1.4 milioni di decessi dovuti alla TB in 84 Paesi. La stessa WHO considera l'Italia uno dei paesi occidentali in cui si sarebbe verificato, tra il 1988 ed il 1990, il maggior incremento del numero di casi di TB. Nonostante la disponibilità di trattamenti efficaci e poco costosi, la tubercolosi risulta più diffusa nella popolazione povera che tra quella ricca, sia nelle regioni industrializzate che nelle regioni in via di sviluppo. Le regioni nelle quali si riscontra il 60% dei casi totali di TB sono: Pakistan, sud dell’Africa, China e India. Il più alto tasso di incidenza della tubercolosi si riscontra nell’Africa Sub-Sahariana, soprattutto associata all’infezione da (HIV). Oltre all’infezione da HIV, l’incremento nel tasso di incidenza della tubercolosi è provocato dalla comparsa di ceppi resistenti alla maggior parte dei farmaci anti-TB. Altri fattori che incidono sulla diffusione della TB sono: l’aumento della popolazione; la difficoltà a rilevare i casi tra le masse povere; il ridotto tasso di cure in paesi in via di sviluppo; la trasmissione dell’infezione in ospedali e prigioni sovraffollati e l’immigrazione di individui da paesi dove la TB è endemica (WHO 2012). La principale fonte di contagio è rappresentata dalle persone affette da TB, anche se soltanto il 10% degli individui mostra i segni di tubercolosi attiva entro i 18 mesi dall’avvenuta infezione. Nei restanti casi, la risposta immunitaria conseguente all’infezione riesce ad arrestare la crescita del MTB. Ciò nonostante, il patogeno è completamente eradicato soltanto nel 10% dei casi mentre, nel restante 90%, il MTB riesce ad eludere i meccanismi antibatterici delle cellule del sistema immunitario restando in uno stato non-replicativo (dormiente o latente). Questo processo è chiamato Latent Tuberculosis Infection (LTI) e colpisce circa due miliardi di persone al mondo (Koul, Herget et al. 2004). Mycobacterium tuberculosis Il Mycobacterium tuberculosis è un membro della specie Mycobacterium tuberculosis complex (MTBC). Ad esso appartengono altre 6 specie correlate geneticamente: M. bovis, M. africanum, M. microti, M. pinnipedii, M. caprae e M. capretti. Tutte possono provocare tubercolosi, ma hanno un diverso host range (Cole, Brosch et al. 1998). Il M. tuberculosis ha una struttura unica che lo rende diverso da tutti gli altri batteri. Partendo dall’interno verso l’esterno troviamo la membrana cellulare rivestita da diversi strati di peptidoglicano (PG), arabinogalattani (AG), lipomannani (LM), mannose-lipoarabinomannani (ManLAM) e acidi micolici (MA) legati covalentemente tra di loro a formare una struttura molto complessa. Il peptidoglicano costituisce lo scheletro di questa struttura. Esso è costituito da lunghe catene di N-acetilglucosammina (NAG) legate a catene di acido muramico (NAM) attraverso dei residui di L-alanyl-D-iso-glutaminyl-meso-diaminopimelic acid (DAP) (Velayati, Farnia et al. 2012). I polisaccaridi a lunga catena (LM, AG e LAM) formano un ponte tra il peptidoglicano e gli acidi micolici che sono acidi grassi a lunga catena che rappresentano i componenti principali della parete del micobatterio. Riconoscimento del Mycobacterium tuberculosis I Pathogen-associated molecular patterns (PAMP) del MTB vengono riconosciuti da specifici Pathogen recognition receptors (PRRs) posti sulla superficie delle cellule del sistema immunitario dell’ospite. Questi recettori, tra cui i Toll-like receptors (TLRs) sono essenziali per coordinare la risposte immunitaria innata dell’ospite (Jo 2008). L’interazione tra i PAMPs del MTB ed i TLRs innesca una cascata di trasduzione del segnale che culmina in una risposta pro-infiammatoria da parte delle sistema immunitario dell’ospite (Harding and Boom 2010). Tuttavia, il micobatterio ha sviluppato efficaci strategie atte a modulare o addirittura inibire tale risposta. I più importanti ligandi posti sulla superficie del micobatterio i quali interagiscono con i TLRs sono: 19 kDa lipoprotein, il lipomannano (LM) ed il mannose-lipoarabinomannano (Jo, Yang et al. 2007). L’interazione di questi ligandi con i TLRs porta all’attivazione del nuclear trascription factor B (NF-kB) e la conseguente produzione di citochine pro-infiammatorie come tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) e anche dell’ossido nitrico attraverso via myeloid differentiation primary response protein 88 (MyD88)-dipendenti che indipendenti (Yamamoto, Sato et al. 2003, Jo 2008). Uno studio ha mostrato che, almeno nel topo, il contatto prolungato con la 19 kDa del MTB da parte dei macrofagi alveolari attenua la processazione dell’antigene che a sua volta attenua l’espressione del major histocompatibility complex (MHC)-II smorzando così l’attivazione delle T cells (Fulton, Reba et al. 2004). In questo modo, una sottopopolazione di macrofagi infettati con la funzione dell’APC modulata, costituisce una nicchia invisibile al sistema immunitario dove il batterio può sopravvivere e resistere. Scopo della tesi Schurr (Schurr 2007) si è chiesto se la tubercolosi fosse una malattia infettiva o ereditaria. L’autore sapeva molto bene che la tubercolosi può essere contratta solo in presenza del patogeno. Con la sua domanda (posta come titolo all’articolo), Schurr ha inteso mettere in evidenza il ruolo essenziale della componente ereditaria ai fini della resistenza alla tubercolosi. Indi per cui la ricerca si è incentrata sull’associazione (punto I) e interazione (punto II) tra i geni MyD88 e TIRAP e la tubercolosi. Entrambi i geni funzionano come trasmettitori del segnale dai recettori esterni alla cellula fino all’interno del nucleo dove avviene la trascrizione di geni capaci di innescare la risposta immune, innata e adattativa. Inoltre, la loro posizione su cromosomi distinti (indipendenza) e la loro comune funzione (trasmissione del segnale) rende plausibile l’ipotesi che i geni interagissero. Anche quest’anticipazione si è dimostrata valida, offrendo un esempio di epistasi (interazione tra geni non alleli). Inoltre, la letteratura scientifica inerente al coinvolgimento di MyD88 e TIRAP nella tubercolosi presenta notevoli discrepanze, soprattutto nel delicato campo degli studi di associazione. Topi “knockout” per MyD88 mostrano una produzione di TNF-α, IL-12 e NO molto ridotta in risposta all’infezione dell’MTB e muoiono in 4 settimane (Fremond, Yeremeev et al. 2004). Topi “knockout” per TIRAP, invece, riescono a controllare efficacemente l’infezione da MTB (Fremond, Togbe et al. 2007). Il Single-nucleotide polymorphism (SNP) C558T di TIRAP è associato con la suscettibilità alla tubercolosi meningea (Hawn, Dunstan et al. 2006, Caws, Thwaites et al. 2008), e quello S180L conferisce protezione contro la malaria e la tubercolosi (Khor, Chapman et al. 2007). Studi successivi non hanno confermato il ruolo protettivo di S180L (Nejentsev, Thye et al. 2008, Miao, Li et al. 2011), TIRAP (rs352165 and rs352167) e MyD88 (rs4988457 and rs6767684) contro la tubercolosi (Khor, Chapman et al. 2007, Nejentsev, Thye et al. 2008). Alla luce di queste discrepanze emerse, perfino all’interno dello studio dello stesso SNP (rs81777374) in differenti etnie, si è deciso di investigare il ruolo di questi SNPs nei malati di tubercolosi polmonare nella popolazione italiana. In aggiunta, il M. tuberculosis ha l’abilità di passare dalla forma attiva a quella dormiente. Pertanto i geni MyD88 e TIRAP potrebbero influenzare diversamente le due forme d’infezione. In questo caso, l’eterogeneità dei pazienti (con infezione attiva e con infezione latente) può ridurre notevolmente il potere discriminante del test statistico (caso-controllo). Per investigare meglio questo punto dello studio, si è pensato di spostare la ricerca su di un altro modello: i bovini. Da questi animali è stato possibile ottenere biopsie polmonari al momento della macellazione. L’indagine ha chiarito che – come ipotizzato – il genotipo può influenzare l’infezione, in termini di interazione tra i geni e la il tipo di risposta attuato. Results Study design Per saggiare la riproducibilità dei risultati, è stato scelto uno studio a due fasi. Nella prima fase dello studio (hypotesis-generating) sono stati reclutati 100 casi e 100 controlli e genotipizzati per il sito polimorfico TIRAP rs8177374 e MyD88 rs6853. Entrambi i siti sono stati scelti perché sono stati gli unici tra i 5 saggiati per gene a mostrare una frequenza allelica >0.05. Inoltre entrambi i siti hanno mostrato associazione statisticamente significativa con la malattia (P-value <0.05). Sulla base dei risultati dello studio preliminare (MyD88: OR 0.40 e TIRAP: 0.48), è stato calcolato che - per assicurare allo studio una capacità discriminante del 96% con un livello di significatività dello 0.01- fosse necessario estendere lo studio ad un campione di 185 casi e 185 controlli (nel caso di MyD88) e 313 casi e 313 controlli (nel caso di TIRAP). Nella seconda parte dello studio sono stati coinvolti 400 casi e 400 controlli (indipendenti dai casi e dai controlli utilizzati nella fase preliminare dello studio). Per rendere la classe dei casi più omogenea possibile, sono stati reclutati solo pazienti con TB polmonare attiva confermata attraverso l’esame ai raggi X, il test batteriologico e la PCR. Tutti i casi sono stati trattati presso l’ospedale Monaldi (Napoli), il centro di referenza per la tubercolosi del Sud Italia. I controlli sono stati scelti tra mogli, mariti e conoscenti dei pazienti senza evidenze cliniche di tubercolosi (negativi al test con l’interferone gamma). In tal modo i controlli non sono correlati geneticamente ai casi e non risultano infettati, nonostante esposti al Mycobacterium tuberculosis (>2 anni). I criteri utilizzati per classificare casi e controlli sono stati gli stessi in entrambe le fasi dello studio. I casi ed i controlli sono stati collezionati durante più di 5 anni di collaborazione tra l’Ospedale ed il laboratorio della Prof.ssa Capparelli Rosanna. In questo tempo sei controlli sono diventati positivi al test con l’interferone e quindi sono stati esclusi dallo studio. I casi consistevano in 258 maschi e 142 femmine (media età 50±19 anni); i controlli in 222 maschi e 178 femmine (media età 49±17 anni). Lo studio è stato approvato dal comitato etico dell’Ospedale Monaldi. Il consenso informato è stato ottenuto da tutti i pazienti che hanno partecipato alla ricerca. Eterozigosi è associata con la protezione contro la tubercolosi polmonare. Le frequenze genotipiche dei markers rs6853 e rs8177374 sono in equilibrio di Hardy-Weinberg tra i controlli, ma non tra i casi. Ad entrambi i loci, l’eterozigosi (AG per MyD88 o CT per TIRAP) è associata con la resistenza alla tubercolosi polmonare. L’età rappresenta un fattore di rischio per la tubercolosi polmonare. Per conoscere come il rischio legato all’età varia attraverso le stratificazioni, i dati sono stati analizzati utilizzando il modello della regressione logistica. L’analisi è stata ristretta ai gruppi con un numero ≥ di 45 individui. A parte la categoria 31-40 vs 21-30 anni (apparentemente protetta, OR 0.31), il modello ha mostrato che il rischio di sviluppare la malattia aumenta con l’età. Chiaramente, l’età e le possibili variabili associate all’età - come il fumo, il diabete e la prolungata esposizione al patogeno - possono neutralizzare la resistenza genetica. Il modello di regressione logistica è stato anche usato per dissezionare il contributo di ogni singolo genotipo e la loro interazione. Particolarmente evidente è l’interazione tra i genotipi AG e CT (OR 0.09) e l’interazione in direzione opposta tra i genotipi GG e TT, AA e CC, AA e CT (OR 5.78, 5.78 e 7.46, rispettivamente). L’eterozigosi controlla l’infiammazione Per investigare sul meccanismo di come l’ospite può controllare l’infezione secondo l’assetto genotipico, i linfociti di donatori sani (controlli) (nove gruppi genotipici; 5 campioni/gruppo) sono stati stimolati con il ceppo Mycobacterium tuberculosis H37Rv inattivato al calore e poi sono stati misurati i livelli di TNF-α, IFN-ɣ e NO (nitric oxide) rilasciati nel mezzo attraverso un’ELISA test. Nel contesto del genotipo AA, i soggetti CT mostrano livelli intermedi di TNF-α, IFN-ɣ e NO rispetto ai soggetti CC e TT. Nel contesto genotipico AG, i dati mostrano la stessa tendenza, sebbene alcune differenze non siano significative. Inoltre, sono stati osservati bassi livelli di citochine in soggetti GG, come se l’allele “A” favorisse la produzione di citochine e l’allele “G” la attenuasse. Questi dati indicano che gli eterozigoti sono associati con un livello intermedio di citochine and NO. Questi risultati suggeriscono che i due loci cooperano fortemente a controllare la malattia. Questa evidenza è fortemente supportata dall’evidenza che TIRAP e MyD88 formano eterodimeri. Analisi bioinformatica Il sito rs6853 risiede nella regione 3’ UTR del gene MyD88. Comparando le sequenze genomiche di diverse specie emerge che entrambi gli alleli A e G sono conservati in molte specie, suggerendo che essi sono mantenuti o almeno tollerati, dalla selezione naturale. Attraverso l’analisi con 4 ENCODE tracks è emerso che la regione 3’ UTR del sito polimorfico potrebbe influenzare l’interazione tra l’mRNA ed fattori proteici. Il sito rs8177374, che risiede sull’esone 5 del gene TIRAP, ha permesso di valutare se il cambiamento di una serina con una leucina in posizione 180 potesse influenzare la struttura della proteina. Entrambi gli amminoacidi sono rappresentati in posizione 180 dei geni TIRAP di 22 specie di mammiferi. E’ stato possibile predire che entrambi sono compatibili con l’attività della proteina. Questi risultati suggeriscono che le isoforme A e B sono originate dalla stessa molecola di mRNA che ha subito splicing alternativo conferendo una ulteriore plausibilità biologica al polimorfismo. Discussione e conclusioni Molto poco è conosciuto del “crosstalk” tra geni implicati nella resistenza ad un patogeno. In questo lavoro è stato investigato come i geni MyD88 e TIRAP si influenzano a vicenda. L’ipotesi che i due geni potrebbero interagire sembra plausibile in quanto entrambe le proteine MyD88 e TIRAP sono coinvolte nel “signaling” cellulare a valle dei TLRs. Sempre in questo studio è stato dimostrato che i due geni cooperano o si antagonizzano tra di loro sulla base della loro combinazione allelica. L’eterozigosi ad entrambi i loci fornisce una protezione più forte (P= 1.3x10-12, age-corrected) rispetto all’eterozigosi ad un solo locus (MyD88 P= 7.8x10-8; TIRAP P= 2x10-6). Allo stesso tempo, negli individui AG/TT (MyD88/TIRAP) il genotipo TT neutralizza la protezione apportata dal genotipo AG. Questi dati, seppur in maniera limitata, mostrano come due o più geni indipendenti possono concorrere alla formazione e regolazione dello stesso fenotipo. Gli individui eterozigoti ai loci MyD88 o TIRAP mostrano livelli intermedi di TNF-α, IFN-ɣ e NO rispetto alle altre classi genotipiche (Figure 1). TNF-α, IFN-ɣ e NO giocano un ruolo fondamentale contro Mycobacterium tuberculosis (Casanova and Abel 2002, Scanga, Bafica et al. 2004, Velez, Hulme et al. 2009). NO esercita una forte attività anti-micobatterica ed insieme a TNF-α, favorisce la formazione dei granulomi (Miller and Ernst 2009). IFN-ɣ induce la produzione di NO, l’espressione delle molecole MHC II e la presentazione dell’antigene (Fortune, Solache et al. 2004, Scanga, Bafica et al. 2004). Tuttavia, vi è anche l’evidenza che la sovra-espressione di queste molecole favorisce la tubercolosi. Molti dei sintomi sono proprio causati dalla risposta immune dell’ospite, piuttosto che dal Mycobacterium tuberculosis (Glickman and Jacobs 2001). Infatti, la riattivazione della TB si è osservata dopo il trattamento terapeutico con TNF-α (Mankia, Peters et al. 2011) o in pazienti affetti da HIV dopo trattamento antiretrovirale (French and Price 2001). Anche l’ipo-espressione di TNF-α, IFN-ɣ e NO favorisce la TB, quindi il vantaggio biologico di averne un livello intermedio di espressione diventa chiaro. Questo vantaggio risulta molto più marcato nei doppi eterozigoti che mostrano come il crosstalk tra i geni si estenda dal livello epidemiologico a quello molecolare. Il vantaggio espresso dagli eterozigoti suggerisce che le frequenze alleliche ai siti polimorfici di rs6853 e rs8177374 sono mantenuti dal bilanciamento del polimorfismo, dove l’omozigosi è associato con la tubercolosi polmonare e l’eterozigosi alla resistenza. In accordo con questa ipotesi vi è il fatto che entrambi i siti polimorfici sono conservati attraverso la speciazione. Inoltre il fatto che la combinazione allelica influisce sui livelli di TNF-α, IFN-ɣ e NO suggerisce che l’associazione sia tra MyD88 e TIRAP piuttosto che tra geni strettamente correlati a loro. In conclusione, l’associazione di rs8177374 con la TB polmonare probabilmente sarà confermata anche in studi futuri, mentre rs6853 risulta associato in questo studio, ma non in due lavori precedenti (Miao, Li et al. 2011, Sanchez, Lefebvre et al. 2012), quindi al momento resta uno studio esplorativo hypothesis-testing study. Commento Purtroppo gli studi di associazione mancano di riproducibilità (Ioannidis, Ntzani et al. 2001). Durante la prima fase dello studio siamo incappati in ostacoli più o meno appianabili. Per ridurre al minimo tutte le fonti di errore è stato fatto in modo che tutto lo studio fosse costruito intorno a precisi punti: replicazione dei risultati in maniera indipendente (studio a 2 fasi); bassi P-value (10-6-10-8); selezione di casi omogenei (lo studio ha arruolato pazienti con tubercolosi polmonare clinicamente diagnosticata e confermata con X-ray al torace, PCR e positività al test batteriologico); uso di appropriati controlli (individui senza segni evidenti della malattia, ma esposti al patogeno e geneticamente non correlati ai casi). Inoltre, l’evidenza che l’associazione è mantenuta in 3 differenti etnie rendono improbabile che l’associazione riscontrata tra i geni MyD88 e TIRAP e la TB sia un artefatto derivante dalla errata strutturazione demografica del campione esaminato. Ad ogni modo, la stessa associazione risulta dare risultati contrastanti quando si analizzano differenti popolazioni (Nejentsev, Thye et al. 2008, Miao, Li et al. 2011, Sanchez, Lefebvre et al. 2012). Quindi la riproducibilità di uno studio è necessariamente un artefatto? Noi pensiamo di no. Nel genoma umano nuovi alleli costantemente si presentano creando una vasta eterogeneità che si amplifica ulteriormente con l’interazione tra di essi e con l’ambiente. Tutta questa genetica eterogeneità è difficile da rilevare a priori e plausibilmente contribuisce all’irriproducibilità degli studi di associazione. Inoltre, durante tutta la sua lunga storia evolutiva, il Mycobacterium tuberculosis ha sviluppato un’efficace strategia per rendere difficile la sua eradicazione da parte del sistema immunitario dell’ospite: la latenza. Questo rappresenta un ulteriore grado di difficoltà nel costruire uno studio di associazione con la TB. Sulla base di queste considerazioni, il passo successivo è stato quello di disegnare uno studio caso-controllo scevro dai tutti i principali fattori di “bias” per quanto concerne il Mycobacterium tuberculosis. Il primo passo è stato cercare un modello che presentasse le seguenti caratteristiche: - Azzeramento o minimizzazione dei fattori ambientali; - Stessa etnia - Possibilità di epurare i controlli da eventuali soggetti con TB latente; - Un numero consistente di casi e controllo per un’adeguata potenza statistica). Il modello scelto è stato quello dei bovini per la possibilità di biopsie polmonari al punto della morte grazie all’attiva collaborazione tra il laboratorio della Prof.ssa Capparelli e l’Istituto Zooprofilattico del Mezzogiorno. In aggiunta, grazie alla collaborazione con la Dott.ssa Berisio del Dipartimento di Chimica dell’Università di Napoli è stato possibile mettere a punto un “in-house assay” capace di discriminare tra batteri attivi e quelli dormienti. Mycobacterium bovis e diagnosi di infezione polmonare L’agente patogeno che causa la tubercolosi nei bovini è il Mycobacterium bovis. Nei paesi dove i programmi per l’eradicazione della tubercolosi bovina sono operativi (test periodici agli animali di allevamento, ispezioni delle carni e pastorizzazioni del latte) hanno ridotto a meno dell’1% i casi di tubercolosi umana attribuibile al Mycobacterium bovis, circoscrivendola soltanto alle persone affette da HIV o alle persone che vantano una prolungata esposizione ad animali infetti (persone del settore e veterinari). Il Mycobacterium bovis ha un ampio range di ospiti, il quale include numerose specie di allevamento e selvatiche. Esso è patogeno per l’uomo, mentre il Mycobacterium tuberculosis non è patogenico nei bovini (Ocepek, Pate et al. 2005). Questa caratteristica potrebbe essere attribuibile alla sola differente espressione genica tra di loro (Neill, Skuce et al. 2005) in quanto studi genetici hanno dimostrato l’elevata similarità tra le due specie batteriche (circa 99.5% a livello nucleotidico) (Garnier, Eiglmeier et al. 2003). Tutte queste osservazioni prese insieme forniscono la plausibilità biologica del ruolo cruciale giocato dal gene MyD88 anche contro la tubercolosi bovina. Il gene TIRAP non è stato incluso nello studio perché la frequenza delle mutazioni risultava essere inferiore a 0.05. L’infezione della tubercolosi polmonare può essere di tipo attivo (ATI) o latente (LTI); quest’ultima è caratterizzata dalla presenza di batteri dormienti (vitali, ma che non crescono sui normali mezzi di crescita) (Oliver 2010). I metodi comunemente usati per diagnosticare la tubercolosi sono il tuberculin skin test (TST) o l’IFN-ɣ assay. Tuttavia, questi metodi non distinguono tra ospiti ancora infetti e quelli che hanno controllato con successo l’infezione (Barry, Boshoff et al. 2009). Presi insieme questi gruppi, almeno nel presente studio, avrebbero potuto ridurne sensibilmente la capacità discriminante (Schurr 2007). Results Diagnosi di casi e controlli Mycobacterium tuberculosis possiede 5 resuscitation-promoting genes (Rpf) che codificano per altrettante proteine (RpfA to RpfE), le quali in forma di proteine ricombinanti in Escherichia coli, inducono la risuscitazione del Mycobacterium tuberculosis (Biketov, Potapov et al. 2007) e Mycobacterium marinum in vivo ed ex vivo (Parikka, Hammaren et al. 2012). Sulla base di questi risultati, è stato sviluppato un “in-house assay” capace di resuscitare i micobatteri dormienti attraverso l’impiego della proteina RpfB. È stato possibile recuperare micobatteri dormienti dal latte e polmoni provenienti da 7 animali trattati con RpfB, mentre nessuna colonia di batteri si è avuta dagli stessi campioni non trattati con RpfB. I risultati tra latte e polmoni sono stati pienamente concordanti. Altri 20 campioni (latte e polmone) sono stati utilizzati per validare l’assay. Il test è stato successivamente esteso a tutti gli animali utilizzando campione di polmone collezionati post-mortem. Un test di PCR discriminante tra Mycobacterium tuberculosis, Mycobacterium bovis, o Mycobacterium avium, ha determinato che tutti i campioni analizzati (con ATI o LTI) erano infettati da Mycobacterium bovis. In conclusione, i casi con ATI sono stati trovati positivi alla PCR ed al test batteriologico in assenza di RpfB; i casi con LTI sono stati trovati positivi alla PCR ed al test batteriologico in presenza di RpfB; i controlli sono soggetti esposti al Mycobacterium bovis (perché provengono dagli stessi allevamenti dove sono stati prelevati i casi), ma negativi alla PCR ed al test batteriologico sia in presenza che in assenza di RpfB. Disegno sperimentale Anche in questo caso lo studio è stato composto da due fasi. La fase preliminare ha coinvolto solo 50 animali di controllo, i quali sono stati separatamente confrontati con 50 casi con ATI o 50 casi con LTI. La fase preliminare ha mostrato una significativa associazione del sito polimorfico MyD88 A625C con ATI (P=0.01), ma non con LTI (P=0.84). Il sito A625C è situato sull’introne 1 del gene MyD88. Questa fase preliminare dello studio ci ha fornito due punti importanti: primo, che l’associazione sembra essere abbastanza robusta (poiché è stata rilevata utilizzando soltanto un numero piccolo di soggetti) e, secondo, che la stratificazione dei casi (tra la forma attiva o latente della TB) potrebbe fornire una maggiore potenza allo studio. Oltre ad A625C, non è stata rilevata la presenza di nessuno degli SNPs mostrati nella sequenza di riferimento. Per esplorare il ruolo di A625C, sono state allineate 11 diverse sequenze del gene di MyD88 appartenenti a diverse specie per studiarne la conservazione. Il basso livello di conservazione suggerisce che il sito polimorfico A625C non è sotto stringente selezione. Analizzando la sequenza dell’introne 1 con SCOPE sono stati evidenziati 4 motivi super-rappresentati nel genoma bovino, i quali includono i sito polimorfico. Questa evidenza potrebbe indicare un possibile ruolo regolatorio di A625C. Questi dati costituiscono un idoneo substrato per ulteriori future investigazioni. L’eterozigosi ad MyD88 e la resistenza alla tubercolosi attiva bovina Per la seconda fase dello studio è stato utilizzato un numero più ampio di campioni: 300 controlli, 150 casi con ATI e 150 casi con LTI, differenti da quelli della prima fase. I casi con ATI non sono in equilibrio Hardy-Weinberg (χ2=4.4). Quando il test è stato ripetuto con i casi LTI, sia casi che controlli sono in equilibrio (χ2controls =0.9; χ2cases = 0.3). I dati suggeriscono un’associazione tra A625C e la ATI, ma non tra A625C e la LTI. Primo, il più stringente test esatto di Fisher mostra che l’eterozigosità (AC status) è fortemente associata con la resistenza alla ATI (OR 0.19, P=6.0x10-12); secondo, l’associazione resta forte anche quando entrambe le classi omozigote (AA e CC) vengono unite (OR 0.22, P=1.8x10-10); terzo, il sito A625C non ha influenzato la predisposizione alla LTI (OR 0.83, P=0.36 e 0.40). La regressione logistica binomiale ha supportato queste conclusioni. L’eterozigosi ad MyD88 e l’infiammazione TNF-α, IFN-ɣ e NOS influenzano profondamente la tubercolosi (Scanga, Bafica et al. 2004). È anche conosciuto che alti o bassi livelli di infiammazione hanno un impatto negativo sulla malattia (Glickman and Jacobs 2001, Doherty and Arditi 2004, Fremond, Yeremeev et al. 2004). Così, se l’eterozigote ad MyD88 mostrasse un livello intermedio di citochine rispetto a quello degli omozigoti, l’associazione tra A625C e la resistenza al Mycobacterium bovis acquisterebbe una fortissima plausibilità biologica. Per validare questa ipotesi sono stati misurati i livelli di mRNA TNF-α, IFN-ɣ e NOS in campioni di polmoni di soggetti con differente genotipo (AA, AC, CC) e status (controlli o animali con ATI o LTI) (6 classi; 5 animali/classe). I livelli di espressione dei soggetti con ATI o LTI sono stati comparati con quelli dei soggetti di controlli aventi lo stesso genotipo. Portatori dell’eterozigosi hanno espresso livelli di TNF-α, IFN-ɣ e NOS significativamente inferiori a quelli espressi dagli omozigoti AA. Al contrario, gli eterozigoti mostrano livelli solo leggermente superiori a quelli espressi dagli omozigoti CC, in questo caso, la differenza non ha raggiunto la significatività statistica. Presi insieme questi dati, supportano la conclusione che un’ottimale risposta infiammatoria è associata con il fenotipo di A625C. Discussione e conclusioni Il presente studio ha dimostrato che nei bovini, animali eterozigoti al sito polimorfico MyD88 presentano un rischio ridotto di circa 5 volte di ATI (OR 0.19, P=6.0x10-12). Tuttavia, la riduzione del rischio non si estende agli animali con LTI (OR 0.83, P=0.36 e 0.40). L’eterozigosi ad A625C è associato con livelli intermedi di TNF-α, IFN-ɣ e NOS. Lo studio ha anche mostrato differenze nell’espressione di mRNA delle citochine tra animali aventi lo stesso genotipo, ma con tubercolosi acuta o latente. La differenza è particolarmente evidente negli animali AA. Purtroppo non possiamo attribuire questi diversi livelli di espressione al patogeno o all’ospite, però i livelli di citochine rappresentano dei potenziali markers per la riattivazione della malattia. Il polimorfismo A625C è locato nell’introne 1 del gene MyD88, questo aggiunge evidenza che regioni non codificanti possono influenzare l’espressione genica. Non è sorprendente che questo accada nel caso dell’infiammazione, la quale ha bisogno di essere soggetta ad una fine e complessa regolazione. Nei bovini, l’esposizione ambientale ai micobatteri, che si verificano nella maggioranza dei soggetti, interferisce con la diagnosi della TB attraverso i TST o IFN-ɣ assay (Hope, Thom et al. 2005). La disponibilità dei reagenti, il tempo di incubazione, ed i livelli di cut-off possono influenzare la specificità e la sensibilità di questi assay (Pai, Riley et al. 2004). Il test batteriologico post-mortem resta, ad oggi, ancora il “gold standard” per le diagnosi di avvenuta infezione (Thacker, Harris et al. 2011). La tubercolosi è influenzata da molti geni che interagiscono tra di loro (Chang, Linderman et al. 2008) e con l’ambiente (Schurr 2007). La presenza del micobatterio è necessaria, ma non sufficiente ad acquisire la malattia, come mostrato dai soggetti di controllo (Diamond 1987). Fattori ambientali (clima, densità dell’allevamento, movimenti del bestiame etc.) sono conosciuti come fattori che promuovono la tubercolosi (Neill, Skuce et al. 2005). Perfino forti effetti genetici sul micobatterio possono essere mancati se non si prendono in considerazione gli effetti ambientali (Schurr 2007). Considerevoli OR e P value (OR = 0.19; P= 6.0x10-12) riportati in questo lavoro ci rendono cautamente ottimisti riguardo la possibilità di approcciare in modo corretto l’analisi genetica di questa complessa malattia. I casi sono stati resi omogenei (ATI ed LTI sono stati analizzati separatamente), i “confounders” ambientali sono stati o esclusi (sesso e razza) o “randomized” (età). Ancor più importante è che i controlli provengono dallo stesso allevamento dei casi e restano tuttavia “liberi” dall’infezione (negativi alla PCR e test batteriologico) nonostante abbiano avuto la stessa probabilità di infettarsi dei casi. Spesso la stratificazione della popolazione è presa in considerazione come responsabile dei falsi-positivi ottenuti dagli studi di associazione, ma raramente è stata dimostrata essere colpevole (Risch 2000, Colhoun, McKeigue et al. 2003). Studi umani hanno mostrato che la stratificazione potrebbe originarsi quando differenti etnie sono mescolate (Healy 2006). Nel presente studio è stata studiata una sola razza. Ulteriormente, gli stessi risultati provenienti da due campioni di popolazioni indipendenti offrono una prova considerevolmente convincente che non sia intervenuta alcuna stratificazione. Gli studi di associazione genetica sono caratterizzati da un alto tasso di risultati falsi-positivi (Risch 2000). Questa conclusione è spesso dovuta alla selezione di un gene candidato senza una relazione funzionale con la malattia (Lander and Schork 1994, Risch 2000). Nel presente studio, MyD88 è stato selezionato sulla base di un largo numero di evidenze sperimentali che mostrano, almeno nel topo, l’importanza di questo gene nel “signaling” a valle della rilevazione di componenti del micobatterio e dell’induzione degli effettori della risposta immunitaria innata da parte delle cellule dell’ospite (Doherty and Arditi 2004, Fremond, Yeremeev et al. 2004). In conclusione, l'elevata rilevanza biologica del gene da studiare, la scelta accurata dei criteri diagnostici, e la randomizzazione dei “confounders” ambientali, sono stati tutti attentamente tenuti in grandissima considerazione durante questo cammino nel complesso campo degli studi di associazione. Tuttavia, poiché l'associazione viene descritta per la prima volta, i risultati di questo studio sono da considerarsi come preliminari. Infine, il test qui utilizzato per distinguere tra malattia attiva e latente potrebbe potenzialmente essere esteso alla verifica periodica dei capi di bestiame per la tubercolosi. Il conteggio dei micobatteri dormienti risvegliato da RpfB in

AbstractGenome-wide transposon mutagenesis followed by deep sequencing allows the genome-wide mapping of growth-affecting loci in a straightforward and time-efficient way.SAturated Transposon Analysis in Yeast (SATAY) takes advantage of a modified maize transposon that is highly mobilizable in S. cerevisiae. SATAY allows not only the genome-wide mapping of genes required for growth in select conditions (such as genetic interactions or drug sensitivity/resistance), but also of protein sub-domains, as well as the creation of gain- and separation-of-function alleles. From strain preparation to the mapping of sequencing reads, we detail all the steps for the making and analysis of SATAY libraries in any S. cerevisiae lab, requiring only ordinary equipment and access to a Next-Gen sequencing platform.

Phytoplasmas are mycoplasmas that infect plants and insects. In plants they cause symptoms such as abnormal flower development (virescence, phyllody) and ‘witches’ broom’. Insects acquire phytoplasmas when they feed on infected plants. After ingestion, the phytoplasmas infect the haemolymph and then migrate to the salivary glands ready to be injected into another plant. Different phytoplasmas infect different vectors and different vectors feed on different plants. Infection with a phytoplasma can affect the behaviour of the insect. The phytoplasma genome is about 50% bigger than that of Mycoplasma genitalium but has fewer genes and so phytoplasmas are dependent on their host plants and insects for essential nutrients. Phytoplasmas contain multicopy gene clusters called Potential Mobile Units (PMUs), some of which are on plasmids. The copy number of these PMUs differs in plants and animals showing a host effect on the bacteria. Phytoplasmas have no cell wall and so membrane proteins modulate the interaction with hosts. There are three types of immunomodulatory protein (Imp) found on the membrane. They are non-allelic but most phytoplasmas have just one. The Imp that a phytoplasma has determines which insects can be its vector. In plants, phytoplasmas are restricted to the phloem and so the disease symptoms are produced by small molecule effectors. Some of these effectors are encoded by PMUs, suggesting acquisition by horizontal gene transfer, but their origin is not known.

Breast cancer (BC) is the leading type of cancer among women. Findings have revolutionized current knowledge of microRNA (miRNA) in breast tumorigenesis. The seed region of miRNA regulates the process of gene expression negatively. The presence of SNPs in the seed regions of miRNA dramatically alters the mature miRNA function. Additionally, SNPs in the out-seed region of miRNAs have a significant impact on miRNA targeting. This study focuses on the in silico analysis procedure of mature miRNA SNPs and their impact on BC risk. The database annotated SNPs on mature miRNAs was used. Also, target gene alterations, miRNAs function in BC, and the interaction of miRNAs with targets were predicted. A list of 101 SNPs in 100 miRNAs with functional targets in BC was indicated. Under the SNPs allele variation, 10 miRNAs changed function, 6 miRNAs lost targets, 15 miRNAs gained targets, 48 onco-miRNAs remained unchanged, and 21 tumor suppressor miRNAs remained unchanged. At last, a list of 89 SNPs, which alter miRNA function and miRNA-mRNA interaction, were shown to be potentially associated with BC risk. This research theoretically generated a list of possible causative SNPs in the mature miRNA gene that might be used in future BC management studies.

Natural selection is the differential contribution of genotypes to the next generation due to differences in survival and reproduction. Understanding the effects of natural selection on allele frequencies involves using a variety of mathematical models along with the fitness of different genotypes. Finesses are not constant. For example, fitness sometimes changes when allele frequencies change. Frequency-dependent selection is a powerful mechanism for maintaining genetic variation in natural populations. Natural selection is less effective in small populations because genetic drift can swamp the effects of differential survival or fertility. Understanding the interaction between natural selection and genetic drift is crucial for the conservation of natural and managed populations.

Allergic rhinitis (AR) has now become one of the major diseases affecting people’s lives, and Traditional Chinese medicine (TCM) always has good efficacy in clinical treatment. In the present study, we analyzed the most frequently used drug pair of Astragalus-Saposhnikoviae Radix (SR) in prescriptions for the treatment of allergic rhinitis by network pharmacology to reveal the modern pharmacological mechanisms of drug prevention and treatment of the disease. Firstly, the 38 active ingredients with good ADME properties from the Astragalus-SR drug pair were collected from the database, and the collated drug targets of Astragalus and SR and the targets of allergic rhinitis were mapped against each other by the network visualization software Cytoscape, followed by the establishment of a “drug active ingredient-target-disease” network diagram and the construction of a high-confidence protein-protein interaction network. Then, the common targets obtained from the disease and drug active ingredients were imported by R language for GO enrichment analysis and KEGG pathway enrichment analysis. The KEGG pathways associated with the targets of Astragalus and SR for the treatment of allergic rhinitis obtained from R enrichment analysis were imported into Cytoscape, and the CytoNCA plug-in was loaded to construct a “target-pathway” network map, and the core target wogonin (FN1) was screened. These evidences suggest that the drug pair of Astragalus-SR works in a multi-component, multi-target and integrated modulation manner for the treatment of allergic rhinitis, which provides an important basis for the treatment of allergic rhinitis.

Kauffman (1993, 1995; Kauffman and Levin 1987; Kauffman and Johnsen 1991) has proposed and studied in depth a class of models referred to as NK models, which are models of random fitness landscapes on which one can implement a variety of types of evolutionary dynamics and study the development and interaction of species. (The letters N and K do not stand for anything; they are the names of parameters in the model.) Based on the results of extensive simulations of NK models, Kauffman and co-workers have suggested a number of possible connections between the dynamics of evolution and the extinction rate. To a large extent it is this work which has sparked recent interest in biotic mechanisms for mass extinction. In this chapter we review Kauffman's work in detail. An NK model is a model of a single rugged landscape, which is similar in construction to the spin-glass models of statistical physics (Fischer and Hertz 1991), particularly p-spin models (Derrida 1980) and random energy models (Derrida 1981). Used as a model of species fitness the NK model maps the states of a model genome onto a scalar fitness W. This is a simplification of what happens in real life, where the genotype is first mapped onto phenotype and only then onto fitness. However, it is a useful simplification which makes simulation of the model for large systems tractable. As long as we bear in mind that this simplification has been made, the model can still teach us many useful things. The NK model is a model of a genome with N genes. Each gene has A alleles. In most of Kauffman's studies of the model he used A = 2, a binary genetic code, but his results are not limited to this case. The model also includes epistatic interactions between genes—interactions whereby the state of one gene affects the contribution of another to the overall fitness of the species. In fact, it is these epistatic interactions which are responsible for the ruggedness of the fitness landscape.

Augmented reality is a technology where real life environment is enhanced by incorporating digital or virtual elements like images, graphics, 3D objects into it. Augmented reality is a growing and trending technology which can make interactions easier when incorporated into normal applications. The aim of this project is to create an augmented reality food guide for food allergic consumers to aid them in choosing allergen free foods. A person who wants to order food online or in restaurants doesn’t know whether it would be suitable for him to eat or not. Especially if they are allergic to certain foods, they have to be preventive and cautious in choosing the type of food they eat. This AR guide will aid them in choosing the suitable food for them in an augmented environment where a consumer can view food in three dimension view along with the necessary information about the food such as the key ingredients and the presence of any allergens that will be helpful in choosing the allergen free food.

<em>Abstract</em>.—Following reports of invasive Spotted Bass <em>Micropterus punctulatus</em> in the Chipola River, Florida, we initiated a microsatellite DNA survey of black basses in that system to assess genetic risks to the native population of Shoal Bass <em>M. cataractae</em>. Nonnative Spotted Bass and naturally occurring Largemouth Bass <em>M. salmoides</em> × Florida Bass <em>M. floridanus</em> intergrades were sampled from the Apalachicola and lower Chipola rivers to serve as reference specimens. In 2007, we identified five Shoal Bass hybrids among 45 specimens of <em>Micropterus</em>. Two of the hybrid specimens had introgressant alleles from Spotted Bass and another two had introgressant alleles from Largemouth Bass × Florida Bass intergrades. The introgressing taxon for the remaining hybrid was not immediately identifiable and was later determined to be an undescribed species of <em>Micropterus</em> (provisionally named Choctaw Bass), which inhabits the coastal plain rivers of the eastern Gulf of Mexico. From 2008 to 2010, an additional 217 specimens from the system were genotyped, resulting in the identification of 36 hybrids. Overall, 15.9% of the unknown specimens were heterospecific; the overall genomic proportion of introgressant alleles was 4.0%. The largest fraction of introgressant alleles (3.0%) originated from Choctaw Bass, most likely carried by emigrants from the nearby Choctawhatchee River system. Largemouth Bass × Florida Bass intergrades contributed the next highest introgressant proportion (0.6%), followed by Spotted Bass (0.4%). One genetically pure Spotted Bass was detected among the upper Chipola River specimens. Point estimates of genetic effective size for the Chipola River Shoal Bass population ranged from 110.4 to 131.4 (95% confidence interval [CI] = 79.1 to 195.9); values of this magnitude could signal an increased susceptibility to hybrid-swarm formation and reduced efficacy of purifying selection. While this population harbors a nontrivial amount of introgressant alleles, the majority of interbreeding appears to originate via natural processes. Nonetheless, if nonnative Spotted Bass become further established in the Chipola River and increase their interactions with Shoal Bass, the genetic integrity of the endemic population could deteriorate rapidly.

Objectives. To evaluate the association of cannabis use, genes of the endocannabinoid system and their interaction on clinical symptoms and cognitive performance in patients with a first-episode of pyschosis. Background. The role of both cannabis use and individual genetic background has been shown in the risk for psychosis. However, the influence of cannabis and variability at endocannabinoid genes on the psychosis outcome still remains inconclusive. Materials and Methods. The sample comprised 43 Caucasian individuals with a first-episode of psychosis (mean age(sd)=25.80(6.39) years, 76.7% males, 51.2% cannabis users).There were no differences in age and sex between cannabis users and non-users. Genetic variability was assessed by genotyping one Single Nucleotide Polymorphism (SNP) in each gene (CNR1-rs1049353 and CNR2-rs2501431). Clinical (PANSS, GAF) and neuropsychological (WAIS, WMS, BADS) scales were administered. Results and conclusions. Genotypic frequencies did not differ between cannabis users and non-users. Cannabis use was associated with better manipulative abilities (IQ-M-WAIS, p=0.029) and better executive function (BADS, p=0.036). CNR1-T allele carriers presented higher disorganized and negative syndrome scores (p=0.001 and p=0.044, respectively). The interaction models evidenced a combined effect of CNR1 and cannabis use on the negative syndrome-PANSS (p=0.037). These results suggest the role of cannabis use and genetic background on cognitive and psychopathological outcomes in first-episode psychosis. However, evidence is still scant, and further investigation in larger samples is needed.

The mix-up is a phenomenon in which a tablet/capsule gets into a different package. It is an annoying problem because mixing different products in the same package could result dangerous for consumers that take the incorrect product or receive an unintended ingredient. So, the consequences could be very dangerous: overdose, interaction with other medications a consumer may be taking, or an allergic reaction. The manufacturers are not able to guarantee the contents of the packages and so for this reason they are very exposed to the risk in which users rightly want to obtain compensation for possible damages caused by the mix-up. The aim of this work is the identification of mix-up events, through machine learning approach based on data, coming from different embedded systems installed in the manufacturing facilities and from the information system, in order to implement integrated policies for data analysis and sensor fusion that leads to waste and detection of pieces that do not comply. In this field, two types of approaches from the point of view of embedded sensors (optical and NIR vision and interferometry) will be analyzed focusing in particular on data processing and their classification on advanced manufacturing scenarios. Results are presented considering a simulated scenario that uses pre-recorded real data to test, in a preliminary stage, the effectiveness and the novelty of the proposed approach.

Heterosis, the enigmatic phenomenon in which whole genome heterozygous hybrids demonstrate superior fitness compared to their homozygous parents, is the main cornerstone of modern crop plant breeding. One explanation for this non-additive inheritance of hybrids is interaction of alleles within the same locus. This proposal aims at screening, identifying and investigating heterosis trait loci (HTL) for different yield traits by implementing a novel integrated mapping approach in Sorghum bicolor as a model for other crop plants. Originally, the general goal of this research was to perform a genetic dissection of heterosis in a diallel built from a set of Sorghum bicolor inbred lines. This was conducted by implementing a novel computational algorithm which aims at associating between specific heterozygosity found among hybrids with heterotic variation for different agronomic traits. The initial goals of the research are: (i) Perform genotype by sequencing (GBS) of the founder lines (ii) To evaluate the heterotic variation found in the diallel by performing field trails and measurements in the field (iii) To perform QTL analysis for identifying heterotic trait loci (HTL) (iv) to validate candidate HTL by testing the quantitative mode of inheritance in F2 populations, and (v) To identify candidate HTL in NAM founder lines and fine map these loci by test-cross selected RIL derived from these founders. The genetic mapping was initially achieved with app. 100 SSR markers, and later the founder lines were genotyped by sequencing. In addition to the original proposed research we have added two additional populations that were utilized to further develop the HTL mapping approach; (1) A diallel of budding yeast (Saccharomyces cerevisiae) that was tested for heterosis of doubling time, and (2) a recombinant inbred line population of Sorghum bicolor that allowed testing in the field and in more depth the contribution of heterosis to plant height, as well as to achieve novel simulation for predicting dominant and additive effects in tightly linked loci on pseudooverdominance. There are several conclusions relevant to crop plants in general and to sorghum breeding and biology in particular: (i) heterosis for reproductive (1), vegetative (2) and metabolic phenotypes is predominantly achieved via dominance complementation. (ii) most loci that seems to be inherited as overdominant are in fact achieving superior phenotype of the heterozygous due to linkage in repulsion, namely by pseudooverdominant mechanism. Our computer simulations show that such repulsion linkage could influence QTL detection and estimation of effect in segregating populations. (iii) A new height QTL (qHT7.1) was identified near the genomic region harboring the known auxin transporter Dw3 in sorghum, and its genetic dissection in RIL population demonstrated that it affects both the upper and lower parts of the plant, whereas Dw3 affects only the part below the flag leaf. (iv) HTL mapping for grain nitrogen content in sorghum grains has identified several candidate genes that regulate this trait, including several putative nitrate transporters and a transcription factor belonging to the no-apical meristem (NAC)-like large gene family. This activity was combined with another BARD-funded project in which several de-novo mutants in this gene were identified for functional analysis.

The general goal was to identify, map, and tag, with DNA markers, segments of chromosomes of a wild species (wild emmer wheat, the progenitor of cultivated wheat) determining the number, chromosomal locations, interactions, and effects of genes that control quantitative traits when transferred to a cultivated plant (bread wheat). Slight modifications were introduced and not all objectives could be completed within the human and financial resources available, as noted with the specific objectives listed below: 1. To identify the genetic contribution of each of the available wild emmer chromosome-arm substitution lines (CASLs) in the bread wheat cultivar Bethlehem for quantitative traits, including grain yield and its components and grain protein concentration and yield, and the effect of major loci affecting the quality of end-use products. [The quality of end-use products was not analyzed.] 2. To determine the extent and nature of genetic interactions (epistatic effects) between and within homoeologous groups 1 and 7 for the chromosome arms carrying "wild" and "cultivated" alleles as expressed in grain and protein yields and other quantitative traits. [Two experiments were successful, grain protein concentration could not be measured; data are partially analyzed.] 3. To derive recombinant substitution lines (RSLs) for the chromosome arms of homoeologous groups 1 and 7 that were found previously to promote grain and protein yields of cultivated wheat. [The selection of groups 1 and 7 tons based on grain yield in pot experiments. After project began, it was decided also to derive RSLs for the available arms of homoeologous group 4 (4AS and 4BL), based on the apparent importance of chromosome group 4, based on early field trials of the CASLs.] 4. To characterize the RSLs for quantitative traits as in objective 1 and map and tag chromosome segments producing significant effects (quantitative trait loci, QTLs by RFLP markers. [Producing a large population of RSLs for each chromosome arm and mapping them proved more difficult than anticipated, low numbers of RSLs were obtained for two of the chromosome arms.] 5. To construct recombination genetic maps of chromosomes of homoeologous groups 1 and 7 and to compare them to existing maps of wheat and other cereals [Genetic maps are not complete for homoeologous groups 4 and 7.] The rationale for this project is that wild species have characteristics that would be valuable if transferred to a crop plant. We demonstrated the sequence of chromosome manipulations and genetic tests needed to confirm this potential value and enhance transfer. This research has shown that a wild tetraploid species harbors genetic variability for quantitative traits that is interactive and not simply additive when introduced into a common genetic background. Chromosomal segments from several chromosome arms improve yield and protein in wheat but their effect is presumably enhanced when combination of genes from several segments are integrated into a single genotype in order to achieve the benefits of genes from the wild species. The interaction between these genes and those in the recipient species must be accounted for. The results of this study provide a scientific basis for some of the disappointing results that have historically obtained when using wild species as donors for crop improvement and provide a strategy for further successes.

The COP9 signalosome (CSN) is an eight-subunit protein complex that is highly conserved among eukaryotes. Genetic analysis of the signalosome in the plant model species Arabidopsis thaliana has shown that the signalosome is a repressor of light dependent seedling development as mutant Arabidopsis seedlings that lack this complex develop in complete darkness as if exposed to light. These mutant plants die following the seedling stage, even when exposed to light, indicating that the COP9 signalosome also has a central role in the regulation of normal photomorphogenic development. The biochemical mode of action of the signalosome and its position in eukaryotic cell signaling pathways is a matter of controversy and ongoing investigation, and recent results place the CSN at the juncture of kinase signaling pathways and ubiquitin-mediated protein degradation. We have shown that one of the many CSN functions may relate to the regulation of translation through the interaction of the CSN with its related complex, eukaryotic initiation factor (eIF3). While we have established a physical connection between eIF3 subunits and CSN subunits, the physiological and developmental significance of this interaction is still unknown. In an effort to understand the biochemical activity of the signalosome, and its role in regulating translation, we originally proposed to dissect the contribution of "h" subunit of eIF3 (eIF3h) along the following specific aims: (i) Isolation and phenotypic characterization of an Arabidopsis loss-of-function allele for eIF3h from insertional mutagenesis libraries; (ii) Creation of designed gain and loss of function alleles for eIF3h on the basis of its nucleocytoplasmic distribution and its yeast-two-hybrid interactions with other eIF3 and signalosome partner proteins; (iii) Determining the contribution of eIF3h and its interaction with the signalosome by expressing specific mutants of eIF3h in the eIF3h- loss-of function background. During the course of the research, these goals were modified to include examining the genetic interaction between csn and eif3h mutations. More importantly, we extended our effort toward the genetic analysis of mutations in the eIF3e subunit, which also interacts with the CSN. Through the course of this research program we have made several critical scientific discoveries, all concerned with the apparent diametrically opposed roles of eIF3h and eIF3e. We showed that: 1) While eIF3e is essential for growth and development, eIF3h is not essential for growth or basal translation; 2) While eIF3e has a negative role in translational regulation, eIF3h is positively required for efficient translation of transcripts with complex 5' UTR sequences; 3) Over-accumulation of eIF3e and loss-of-function of eIF3h both lead to cop phenotypes in dark-grown seedlings. These results were published in one publication (Kim et al., Plant Cell 2004) and in a second manuscript currently in revision for Embo J. Are results have led to a paradigm shift in translation research – eIF3 is now viewed in all systems as a dynamic entity that contains regulatory subuits that affect translational efficiency. In the long-term agronomic outlook, the proposed research has implications that may be far reaching. Many important plant processes, including developmental and physiological responses to light, abiotic stress, photosynthate, and hormones operate in part by modulating protein translation [23, 24, 40, 75]. Translational regulation is slowly coming of age as a mechanism for regulating foreign gene expression in plants, beginning with translational enhancers [84, 85] and more recently, coordinating the expression of multiple transgenes using internal ribosome entry sites. Our contribution to understanding the molecular mode of action of a protein complex as fundamental as eIF3 is likely to lead to advances that will be applicable in the foreseeable future.

During the evolutionary process of speciation in plants, naturally occurring barriers to reproduction have developed that affect the transfer of genes within and between related species. These barriers can occur at several different levels beginning with pollination-barriers and ending with hybrid-breakdown. The interaction between pollen and pistils presents one of the major barriers to intra- and inter-specific crosses and is the focus of this research project. Our long-term goal in this research proposal was defined to resolve questions on recognition and communication during pollen-pistil interactions in the extended tomato family. In this context, this work was initiated and planned to study the potential involvement of tomato pollen-specific receptor-like kinases (RLK's) in the interaction between pollen and pistils. By special permission from BARD the objectives of this research were extended to include studies on pollen-pistil interactions and pollination barriers in horticultural crops with an emphasis on citrus. Functional characterization of 2 pollen-specific RLK's from tomato was carried out. The data shows that both encode functional kinases that were active as recombinant proteins. One of the kinases was shown to accumulate mainly after pollen germination and to be phosphorylated in-vitro in pollen membranes as well as in-vivo. The presence of style extract resulted in dephosphorylation of the RLK, although no species specificity was observed. This data implies a role for at least one RLK in pollination events following pollen germination. However, a transgenic plant analysis of the RLK's comprising overexpression, dominant-negative and anti-sense constructs failed to provide answers on their role in pollination. While genetic effects on some of the plants were observed in both the Israeli and American labs, no clear functional answers were obtained. An alternative approach to addressing function was pursued by screening for an artificial ligand for the receptor domain using a peptide phage display library. An enriched peptide sequence was obtained and will be used to design a peptide-ligand to be tested for its effect o pollen germination and tube growth. Self-incompatibility (SI) in citrus was studied on 3 varieties of pummelo. SI was observed using fluorescence microscopy in each of the 3 varieties and compatibility relations between varieties was determined. An initial screen for an S-RNase SI mechanism yielded only a cDNA homologous to the group of S-like RNases, suggesting that SI results from an as yet unknown mechanism. 2D gel electrophoresis was applied to compare pollen and style profiles of different compatibility groups. A "polymorphic" protein band from style extracts was observed, isolated and micro-sequenced. Degenerate primers designed based on the peptide sequence date will be used to isolate the relevant genes i order to study their potential involvement in SI. A study on SI in the apple cultivar Top red was initiated. SI was found, as previously shown, to be complete thus requiring a compatible pollinator variety. A new S-RNase allele was discovered fro Top red styles and was found to be highly homologous to pear S-RNases, suggesting that evolution of these genes pre-dated speciation into apples and pears but not to other Rosaceae species. The new allele provides molecular-genetic tools to determine potential pollinators for the variety Top red as well as a tool to break-down SI in this important variety.

Tomato yellow leaf curl virus (TYLCV) is a major pathogen of tomato that causes extensive crop loss worldwide, including the US and Israel. Genetic resistance in the host plant is considered highly effective in the defense against viral infection in the field. Thus, the best way to reduce yield losses due to TYLCV is by breeding tomatoes resistant or tolerant to the virus. To date, only six major TYLCV-resistance loci, termed Ty-1 to Ty-6, have been characterized and mapped to the tomato genome. Among tomato TYLCV-resistant lines containing these loci, we have identified a major recessive quantitative trait locus (QTL) that was mapped to chromosome 4 and designated ty-5. Recently, we identified the gene responsible for the TYLCV resistance at the ty-5 locus as the tomato homolog of the gene encoding messenger RNA surveillance factor Pelota (Pelo). A single amino acid change in the protein is responsible for the resistant phenotype. Pelo is known to participate in the ribosome-recycling phase of protein biosynthesis. Our hypothesis was that the resistant allele of Pelo is a “loss-of-function” mutant, and inhibits or slows-down ribosome recycling. This will negatively affect viral (as well as host-plant) protein synthesis, which may result in slower infection progression. Hence we have proposed the following research objectives: Aim 1: The effect of Pelota on translation of TYLCV proteins: The goal of this objective is to test the effect Pelota may or may not have upon translation of TYLCV proteins following infection of a resistant host. Aim 2: Identify and characterize Pelota cellular localization and interaction with TYLCV proteins: The goal of this objective is to characterize the cellular localization of both Pelota alleles, the TYLCV-resistant and the susceptible allele, to see whether this localization changes following TYLCV infection, and to find out which TYLCV protein interacts with Pelota. Our results demonstrate that upon TYLCV-infection the resistant allele of pelota has a negative effect on viral replication and RNA transcription. It is also shown that pelota interacts with the viral C1 protein, which is the only viral protein essential for TYLCV replication. Following subcellular localization of C1 and Pelota it was found that both protein localize to the same subcellular compartments. This research is innovative and potentially transformative because the role of Peloin plant virus resistance is novel, and understanding its mechanism will lay the foundation for designing new antiviral protection strategies that target translation of viral proteins. BARD Report - Project 4953 Page 2

Hybrid vigor is the leading concept that rules crops breeding for almost a century. Yet, the exact mechanism that underlies heterosis is not clear. Over dominance interaction between alleles is one of the possible explanations. Our preliminary results indicated that severe developmental mutations at the heterozygous state have significant potential to improve plant performance. This led us to propose the ‘ODO breeding method’ that is based replacing a parental line of a successful hybrid with its mutated from to improve hybrid performance. Our BARD research challenged this method in three crop systems: maize, tomato and melon. In maize we could not detect any effect of mutant heterozigosity on yield or yield components when hybrids were tested however when we analyzed the effect of heterozigosity in mutant genes at B73 genetic background we could detect ODO in yield components using certain mutants. Our results indicate that the potential of utilizing the ODO Breeding Method for maize remains unclear. In tomato we overcame technical problems we faced in creating an EMS mutation library in indeterminate glasshouse tomatoes and now we have in our hands advanced material to study the putative ODO hybrids. We transferred some of the promising ODO mutations from M82 to indeterminate glasshouse tomatoes and putative ODO hybrids are ready to be evaluated this winter. In addition, we tested the effect of In melon we compared putative ‘ODO hybrids’ with their isogenic hybrids lacking the mutant allele and our results indicated a potential for the ODO breeding method to improve yield, fruit number per plant, and carotenoids content. Additional experiments are required to estimate better the expected success percentage of the ODO breeding method in melon so that it will become a recommended practice for improving hybrid performance. Based on our results we can't yet recommend the 'ODO breeding method' as a general tool to improve hybrid performance and more efforts are necessary to evaluate the percent of success of this method. The increased carotenoid content we found in association with CRTISO heterozygosity is promising and additional experiments are currently being performed to characterize this finding.

The original working hypothesis of our proposal was that The “e” subunit of eIF3 has multiple functions from both within the nucleus and in the cytoplasm. Within this model, we further hypothesized that the “e” subunit of eIF3 functions in translation as a repressor. We proposed to test these hypotheses along the following specific aims: 1) Determine the subcellular localization of the interaction between eIF3e and other eIF3 subunits, or the COP9 signalosome. 2) Elucidate the biological significance of the varied subcellular localizations of eIF3e through generating Arabidopsis eIF3e alleles with altered subcellular localization. 3.) Purify different eIF3e complexes by tandem affinity purification (TAP). 4) Study the role of eIF3e in translational repression using both in vitro and in planta assays. eIF3 is an evolutionarily ancient and essential component of the translational apparatus in both the plant and animal kingdoms. eIF3 is the largest, and in some ways the most mysterious, of the translation factors. It is a multi-subunit protein complex that has a structural/scaffolding role in translation initiation. However, despite years of study, only recently have differential roles for eIF3 in the developmental regulation of translation been experimentally grounded. Furthermore, the roles of individual eIF3 subunits are not clear, and indeed some, such as the “e” subunit may have roles independent of translation initiation. The original three goals of the proposal were technically hampered by a finding that became evident during the course of the research – Any attempt to make transgenic plants that expressed eIF3e wt or eIF3e variants resulted in seedling lethality or seed inviability. That is, it was impossible to regenerate any transgenic plants that expressed eIF3e. We did manage to generate plants that expressed an inducible form of eIF3e. This also eventually led to lethality, but was very useful in elucidating the 4th goal of the research (Yahalom et al., 2008), where we showed, for the first time in any organism, that eIF3e has a repressory role in translation. In attempt to solve the expression problems, we also tried expression from the native promoter, and as such analyzed this promoter in transgenic plants (Epel, 2008). As such, several additional avenues were pursued. 1) We investigated protein-protein interactions of eIF3e (Paz-Aviram et al., 2008). 2) The results from goal #4 led to a novel hypothesis that the interaction of eIF3e and the CSN meets at the control of protein degradation of nascent proteins. In other words, that the block in translation seen in csn and eIF3e-overexpressing plants (Yahalom et al., 2008) leads to proteasome stress. Indeed we showed that both over expression of eIF3e and the csn mutants lead to the unfolded protein response. 3) We further investigated the role of an additional eIF3 subunit, eIF3h, in transalational regulation in the apical meristem (Zhou et al., 2009). Epel, A. (2008). Characterization of eIF3e in the model plant Arabidopsis thaliana. In Plant Sciences (Tel Aviv, Tel Aviv University). Paz-Aviram, T., Yahalom, A., and Chamovitz, D.A. (2008). Arabidopsis eIF3e interacts with subunits of the ribosome, Cop9 signalosome and proteasome. Plant Signaling and Behaviour 3, 409-411. Yahalom, A., Kim, T.H., Roy, B., Singer, R., von Arnim, A.G., and Chamovitz, D.A. (2008). Arabidopsis eIF3e is regulated by the COP9 signalosome and has an impact on development and protein translation. Plant J 53, 300-311. Zhou, F., Dunlap, J.R., and von Arnim, A.G. The translation initiation factor subunit eIF3h is .1 involved in Arabidopsis shoot apical meristem maintenance and auxin response. (submitted to Development).

Plant species synthesize a multitude of specialized compounds 10 help ward off pests. and these in turn may well serve as an alternative to synthetic pesticides to reduce environmental damage and health risks to humans. The general goal of this research was to perform a genetic and biochemical dissection of the natural-insecticides methylketone pathway that is specific to the glandular trichomes of the wild species of tomato, Solanumhabrochaites f. glabratum (accession PI126449). Previous study conducted by us have demonstrated that these compounds are synthesized de novo as a derivate pathway of the fatty acid biosynthesis, and that a key enzyme. designated MethylketoneSynthase 1 (MKS 1). catalyzes conversion of the intermediate B-ketoacyl- ACPs to the corresponding Cn-1 methylketones. The approach taken in this proposed project was to use an interspecific F2 population. derived from the cross between the cultivated lV182 and the wild species PIl26449. for three objectives: (i) Analyze the association between allelic status of candidate genes from the fatty acid biosynthesis pathway with the methylketone content in the leaves (ii) Perform bulk segregant analysis of genetic markers along the tomato genome for identifying genomic regions that harbor QTLs for 2TD content (iii) Apply differential gene expression analysis using the isolated glands of bulk segregant for identifying new genes that are involved in the pathway. The genetic mapping in the interspecific F2 population included app. 60 genetic markers, including the candidate genes from the FAS pathway and SSR markers spread evenly across the genome. This initial; screening identified 5 loci associated with MK content including the candidate genes MKS1, ACC and MaCoA:ACP trans. Interesting observation in this genetic analysis was the connection between shape and content of the glands, i.e. the globularity of the four cells, typical to the wild species. was associated with increased MK in the segregating population. In the next step of the research transcriptomic analysis of trichomes from high- and 10w-MK plants was conducted. This analysis identified a new gene, Methy1ketone synthase 2 (MKS2), whose protein product share sequence similarity to the thioesterase super family of hot-dog enzymes. Genetic analysis in the segregating population confirmed its association with MK content, as well as its overexpression in E. coli that led to formation of MK in the media. There are several conclusions drawn from this research project: (i) the genetic control of MK accumulation in the trichomes is composed of biochemical components in the FAS pathway and its vicinity (MKS 1 and MKS2). as well as genetic factors that mediate the morphology of these specialized cells. (ii) the biochemical pathway is now realized different from what was hypothesized before with MKS2 working upstream to I\1KS 1 and serves as the interface between primary (fatty acids) and secondary (MK) metabolism. We are currently testing the possible physical interactions between these two proteins in vitro after the genetic analysis showed clear epistatic interactions. (iii) the regulation of the pathway that lead to specialized metabolism in the wild species is largely mediated by transcription and one of the achievements of this project is that we were able to isolate and verify the specificity of the MKS1 promoter to the trichomes which allows manipulation of the pathways in these cells (currently in progress). The scientific implications of this research project is the advancement in our knowledge of hitherto unknown biochemical pathway in plants and new leads for studying a new family in plants (hot dog thioesterase). The agricultural and biotechnological implication are : (i) generation of new genetic markers that could assist in importing this pathway to cultivated tomato hence enhancing its natural resistance to insecticides, (ii) the discovery of MKS2 adds a new gene for genetic engineering of plants for making new fatty acid derived compounds. This could be assisted with the use of the isolated and verified MKS1 promoter. The results of this research were summarized to a manuscript that was published in Plant Physiology (cover paper). to a chapter in a proceeding book. and one patent was submitted in the US.

specific objectives of this proposal were to: 1) determine the location, topology, and oligomerization of FtsH11 protease; 2) identify the substrate/s of FtsH11 and the downstream components involved in maintaining thermostability of chloroplasts; 3) identify new elements involved in FtsH11 protease regulatory network related to HT adaptation processes in chloroplast; 4) Study the role of FtsH11 homologs from crop species in HT tolerance. Background to the topic: HT-tolerant varieties that maintain high photosynthetic efficiency at HT, and cope better with daily and seasonal temperature fluctuations are in great need to alleviate the effect of global warming on food production. Photosynthesis is a very complex process requiring accurate coordination of many complex systems and constant adjustments to the changing environments. Proteolytic activities mediated by various proteases in chloroplast are essential part of this process and critical for maintaining normal chloroplast functions under HT. However, little is known about mechanisms that contribute to adaptation of photosynthetic processes to HT. Our study has shown that a chloroplast-targeted Arabidopsis FtsH11 protease plays an essential and specific role in maintaining thermostability of thylakoids and normal photosynthesis at moderate HT. We hypothesized that FtsH11 homologs recently identified in other plant species might have roles similarly to that of AtFtsH1. Thus, dissecting the underlying mechanisms of FtsH11 in the adaptation mechanisms in chloroplasts to HT stress and other elements involved will aid our effort to produce more agricultural products in less favorable environments. Major conclusions, solutions, achievements - Identified the chloroplast inner envelope membrane localization of FtsH11. - Revealed a specific association of FtsH11 with the a and b subunits of CPN60. - Identified the involvement of ARC6, a protein coordinates chloroplast division machineries in plants, in FtsH11 mediated HT adaptation process in chloroplast. -Reveal possible association of a polyribonucleotide nucleotidyltransferase (cpPNPase), coded by At3G03710, with FtsH11 mediated HT adaptation process in chloroplast. - Mapped 4 additional loci in FtsH11 mediated HT adaptation network in chloroplast. - Demonstrated importance of the proteolytic activity of FtsH11 for thermotolerance, in addition to the ATPase activity. - Demonstrated a conserved role of plant FtsH11 proteases in chloroplast biogenesis and in maintaining structural and functional thermostability of chloroplast at elevated temperatures. Implications, both scientific and agricultural:Three different components interacting with FtsH11 were identified during the course of this study. At present, it is not known whether these proteins are directly involved in FtsH11mediated thermotolerance network in chloroplast and/or how these elements are interrelated. Studies aiming to connect the dot among biological functions of these networks are underway in both labs. Nevertheless, in bacteria where it was first studied, FtsH functions in heat shock response by regulating transcription level of σ32, a heat chock factor regulates HSPsexpression. FtsH also involves in control of biosynthesis of membrane components and quality control of membrane proteins etc. In plants, both Arc 6 and CPN60 identified in this study are essential in chloroplast division and developments as mutation of either one impairs chloroplast division in Arabidopsis. The facts that we have found the specific association of both α and β CPN60 with FtsH11 protein biochemically, the suppression/ enhancement of ftsh11 thermosensitive phenotype by arc6 /pnp allele genetically, implicate inter-connection of these networks via FtsH11 mediated network(s) in regulating the dynamic adaptation processes of chloroplast to temperature increases at transcriptional, translational and post-translational levels. The conserved role of FtsH11 proteases in maintaining thermostability of chloroplast at HT demonstrated here provides a foundation for improving crop photosynthetic performance at high temperatures.