no / Isogenic mutants of Streptococcus mutans LT11, deficient in the production of the wall-associated protein antigens A and B, were generated by recombinant DNA technology. The hydrophobicity, adherence, and aggregation of the mutants were compared with those of the parent strain. These studies indicated that hydrophobicity, adherence, and saliva- or sucrose-induced aggregation were unaltered in the A- mutant but that hydrophobicity and adherence to saliva-coated hydroxylapatite were greatly reduced in the B- mutant whilst sucrose-dependent adherence and aggregation were increased. To determine whether these changes correlated with changes in the mutated gene product alone, the levels of a number of cell wall antigens were determined in each of the mutants. The loss of antigen A resulted in significantly reduced levels of wall-associated lipoteichoic acid, and loss of antigen B resulted in reductions in both antigen A and lipoteichoic acid. Data presented here thus suggest that changes in the expression of one wall antigen can have a dramatic effect on the levels of others.

Wheat grain quality and consumption is influenced by its constituents structure and concentrations. In the first part of the dissertation, six Canadian bread wheat cultivars; four (CDC Teal, AC Superb, AC Barrie, AC Splendor) belonging to the Canada Western Red Spring (CWRS), and two (AC Foremost, and AC Crystal) to the Canada Prairie Spring Red (CPSR) market classes were characterized for the relationship between their starch constituents and starch in vitro enzymatic hydrolysis. CPSR cultivars with relatively longer amylopectin chains of DP 37-45, reduced chain lengths of DP 15-18, and a low volume percent of small C-type starch granules, had reduced starch in vitro enzymatic hydrolysis rates. In the second part of the dissertation, near-isogenic wheat lines differing at the Waxy locus were analyzed for the influence of genome-specific granule-bound starch synthase I (GBSSI/Waxy; Wx-A, Wx-B, Wx-D) on starch composition, structure and starch in vitro enzymatic hydrolysis. Amylose concentration was more severely affected in genotypes with GBSSI missing from two genomes (double nulls) than from one genome (single nulls) of wheat, indicating dosage dependent amylose synthesis. Subtle differences in amylopectin chain length distribution were observed among non-waxy, partial and completely waxy starches, suggesting a non-limiting role of genome-specific GBSSI for amylopectin synthesis. A suppressive role of Wx-D on the short chain phenotype of wheat amylopectin was observed. In addition, Wx-D increased the volume percentage of large A-type starch granules and reduced starch hydrolysis index. Thus, among the waxy isoproteins, Wx-D might be the major contributor for reducing the rate of in vitro starch enzymatic hydrolysis in wheat. In the third part of the dissertation, endosperm starch’s physicochemical properties and structure during grain development in wheat waxy-null genotypes were analyzed. The study was conducted with pure starch isolated from wheat grains at 3-30 days post anthesis (DPA), at three day intervals. Changes in amylopectin structure were observed until 12 DPA, suggesting the formation of a basic amylopectin skeleton by this stage. A differential influence of waxy isoproteins on amylopectin structure formation has been suggested, with Wx-B and Wx-D affecting short glucan chains of DP 6-8 at 3 and 6 DPA, Wx-A being effective at 9 and 12 DPA, and Wx-D affecting DP 18-25 chains from 18-30 DPA.

Soybean (Glycine max [L.] Merr.) cultivars differ in their resistance to sudden death syndrome (SDS). The syndrome is caused by root colonization by Fusarium virguliforme (ex. F. solani f. sp. glycines). Breeding for improve SDS response has proven challenging, possible due to interactions among the 18 known loci for resistance. Four loci for resistance to SDS (cqRfs to cqRfs3) were found clustered within 20 cM of the rhg1 locus underlying resistance to soybean cyst nematode (SCN) on chromosome 18. Another locus on chromosome 20 (cqRfs5) was reported to interact with this cluster. The aims of this study were to compare the inheritance of resistance to SDS in a near isogenic line (NIL) population that was fixed for resistance to SCN but still segregated at 2 of the 4 loci (cqRfs1 and cqRfs) for resistance to SDS on chromosome 18; to examine the interaction with the locus on chromosome 20; and to identify candidate regions underlying quantitative trait loci (QTL). Used were a near isogenic line population derived from residual heterozygosity in an F5:7 recombinant inbred line EF60 1-40; SDS response data from 2 locations and years; four microsatellite markers and six thousand SNP markers. Polymorphic regions were found from 2,788 to 8,938 Kbp on chromosome 18 and 33,100 to 34,943 Kbp on chromosome 20. Both regions were significantly (0.005 < P > 0.0001) associated with resistance to SDS. A fine map was constructed that Mendelized the three loci. Substitution maps suggested the two loci on chromosome 18 were actually 3 loci (cqRfs, cqRfs1 and cqRfs19). Candidate genes for cqRfs19 were identified in a small region of the genome sequence of soybean. An epistatic interaction was inferred where the allele of loci on chromosome 18 determined the value of the locus on chromosome 20. It was concluded that SDS loci are both complex and interacting which may explain the slow progress in breeding for resistance to SDS.

Maize, Zea mays L., is the largest cereal grain crop grown in United States. Its yield and grain quality are adversely impacted by diseases every year. Aspergillus ear rot, caused by the fungus Aspergillus flavus, received little interest until its carcinogenic secondary metabolites, aflatoxins, were discovered. The objectives of this study were to introgress the quantitative trait loci (QTL) 2.04 from Mp313E and 5.03 from Mp715 into two commercial inbred lines, MonF and MonM; and evaluate their near isogenic lines (NILs) and testcrosses for preharvest aflatoxin accumulation and secondary agronomic traits. Marker assisted selection to create NILs and the testcross production was conducted by Bayer Company between 2015 and 2018. Field trials were conducted in summer 2019 as randomized complete block trials at three locations. The entry list of inbred trials included two donor parents (DP), two recurrent parents (RP), and their 58 NILs, and that of hybrid trials included 114 NIL testcrosses and 8 parental testcrosses. The top ear of each plant in every plot was inoculated with a 3.4 ml of A.flavus conidial suspension 13 days after mid-silk. All the inoculated ears were harvested at maturity, dried, machine shelled, ground, and aflatoxin concentration was determined by plot. Separate hybrid yield trials were conducted in four locations to measure the grain yield including an additional commercial check. Data on aflatoxin and other secondary traits was analyzed using SAS software. Overall, MonF NILs improved significantly more than MonM NILs in terms of their resistance to aflatoxin accumulation with the introgression of QTL 2.04 from Mp313E, but there were no differences with the introgression of QTL 5.03 from Mp715. Overall, Mp313E NILs improved more than Mp715 NILs when the recurrent parent was MonF, but the response was opposite when the recurrent parent was MonM. Compared to their respective recurrent parents, there were at least two NILs from each of the three out of four RP x DP crosses that significantly improved their resistance to aflatoxin accumulation with a minimal loss of their agronomic performance and testcross grain yields. These NILs could be considered as parents in future introgression projects.

No description available.

Master of Science / Department of Grain Science and Industry / Jon M. Faubion / Endosperm texture is an important characteristic in determining wheat processing and end-use. The presence of puroindoline proteins on the starch surface is the biochemical marker for wheat hardness. Near-isogenic samples over expressing puroindolines have been used to assess the effect of wheat hardness on final product characteristics. The objective of this study was to determine differences among starch isolated from near-isogenic samples and to investigate the role starch surface components play in pasting. The use of near-isogenic samples over expressing puroindolines combined with the use of two methods of starch isolation (batter and dough) was an effective means to create samples with varied amounts of surface components. Starch thermal properties were characterized and surface proteins and lipids were quantified. Starch isolated from hard wheat cultivars presented more similarities with starch isolated from its soft near-isogenic line when a dough method was used than when a batter method was used. Starch from soft experimental lines isolated using a batter method showed increased MVA peak viscosity, breakdown and swelling power. Increased levels of LysoPC in starch isolated from hard wheat cultivars or soft experimental lines by dough method could have complexed with amylose and restricted granule swelling. Thereby, decreasing peak viscosity, breakdown and swelling power.

Thesis (PhD (Microbiology))--University of Stellenbosch 2010. / ENGLISH ABSTRACT: Two isogenic plasmids, pRAS3.1 (11,851-bp) and pRAS3.2 (11,823-bp), were identified as tetracycline resistance plasmids occurring within Aeromonas salmonicida subsp. salmonicida and atypical A. salmonicida subsp. salmonicida strains that were isolated from salmon aquaculture farms in Norway (L'Abee-Lund and Sorum, 2002). Although sequence analysis showed that, except for the repC gene, the replication and mobilization genes of the two pRAS3 plasmids are similar to that of the two IncQ-2 plasmids pTF-FC2 and pTC-F14, incompatibility testing during the course of this study revealed that the replicons of the two pRAS3 plasmids were compatible with the replicons of the IncQ-1α, ß] and y plasmids RSF1010, pIE1107 and pIE1130, as well as with the IncQ-2α and ß plasmids, pTF-FC2 and pTC-F14, respectively. Through sequence analysis it was suggested that the repC gene of the ancestral pRAS3 plasmid was probably acquired during a gene exchange event with a yet to be identified plasmid. The difference in the RepC of the pRAS3 plasmids compared to that of the other IncQ-like plasmids against which the pRAS3 plasmids were tested for incompatibility was thus suggested to be a likely reason for the compatibility of the two pRAS3 plasmid replicons with these IncQ-1 and IncQ-2 plasmids. Two previously unidentified genes, encoding two small 108 and 74-aa proteins distantly related to the PemIK (Bravo et al., 1987; Tsuchimoto et al., 1988) and MazEF (Masuda et al., 1993) TA systems, were found to be present between repB and repA genes of the two pRAS3 plasmids. Cloning of these two genes onto an unstable pOU82-test vector increased the stability of the vector from 35 to 98% after ~72 generations, thus suggesting that like the PasABC and PasAB systems of pTF-FC2 and pTC-F14, these two genes encode proteins which function as a toxin-antitoxin (TA) system. Although located in a similar position on the plasmids, the TA system of the two pRAS3 plasmids and the Pas systems of pTF-FC2 and pTC-F14 are unrelated, suggesting that these two types of TA systems were acquired independently from each other. Based on the sequence similarity and genetic organization of pRAS3 compared to the IncQ-2α and ß] plasmids pTF-FC2 and pTC-F14, respectively, but given that the pRAS3 plasmids were compatible with both pTF-FC2 and pTC-F14, as well as other IncQ-like plasmids, it was suggested that the two pRAS3 plasmids be classified into a new IncQ-2y subgroup. A comparison of the sequences of the two pRAS3 plasmids to each other by L'Abee-Lund and Sorum (2002) revealed that, apart from a number of point mutations within the tetAR tetracycline resistance genes of the two plasmids, the only other differences between them are that pRAS3.1 has 4 tandem copies of 22-bp iteron repeats within its origin of vegetative replication (oriV), and 5 tandem copies of CCCCCG 6-bp repeats near the origin of transfer (oriT), while pRAS3.2 has only three and four copies of each of the two repeated sequences, respectively. As the two pRAS3 plasmids are likely to have arisen from the same ancestor, this raised the question of how the copy numbers of these two different types of repeat sequences affected the ability of pRAS3.1 and pRAS3.2 plasmids to compete within a host cell as well as within a population of host cells, and therefore, why both of these isogenic plasmids have managed to persist in the environment. The plasmid copy numbers (PCN) of pRAS3.1 and pRAS3.2 were estimated to be 45 ± 13 and 30 ± 5 plasmids per chromosome, respectively. By creating a series of pRAS3.1 derivative plasmids with 3 to 7 copies of the 22-bp iterons and 4 or 5 copies of the 6-bp repeats, it was shown that an increase in the number of iterons brought about a decrease in PCN, probably due to an increased ability to bind RepC, while an increase in the number of 6-bp repeats from 4 to 5 brought about an increase in repB transcription, and the higher levels of RepB resulted in an increase in PCN. Thus the reason for pRAS3.1 having a ~1.5-fold higher PCN than pRAS3.2, even though it has 4 x 22-bp iterons compared to the 3 x 22-bp iterons of pRAS3.2, was that it had a higher level of repB transcription due to having 5 x 6-bp repeats in its mobB-mobA/repB promoter region compared to the 4 x 6-bp repeats of pRAS3.2. The differences in the number of iterons and 6-bp repeats, and hence PCN, did not have an effect on the stability of the two wild type (WT) plasmids or their derivatives even when the TA system was neutralized by having a copy of the TA genes present on a vector in trans and it was argued that the relatively high PCN of the two pRAS3 plasmids was sufficient to ensure plasmid stability through random distribution. As the two pRAS3 plasmids were mobilized at similar frequencies difference in PCN and mobB-mobA/repB transcription did not seem to affect their mobilization frequency. When pRAS3.1 and pRAS3.2 were competed intracellularly as coresident plasmids, pRAS3.1 was able to displace pRAS3.2 from 98% of the host cells within ~20 generations. The displacement of pRAS3.2 by pRAS3.1 was found to be as a result of pRAS3.1 having 4 x 22-bp iterons, which enabled pRAS3.1 to titrate of the communal pool of available RepC initiator proteins. Plasmids with 5 or 7 x 22-bp iterons, were however less effective at displacing a plasmid with 3 iterons, and it was speculated that plasmids with more than 4 x 22-bp iterons within their oriV were less successful at initiating replication than was a plasmid with 3 iterons within its oriV. A direct correlation was found between the PCN of a pRAS3 plasmid and the metabolic burden it imposed on its host. Thus pRAS3.1, as a result of its ~1.5-fold higher PCN than pRAS3.2 placed a small but significantly higher (~2.8%) metabolic load on its host compared to pRAS3.2. It was concluded that pRAS3.1 had a competitive advantage over pRAS3.2 when these plasmids were coresident within a single host (as would have been when the two plasmids first diverged from each other) as it was able to displace pRAS3.2. However, as a result of pRAS3.2 having a lower PCN, it placed a smaller metabolic burden on an isogenic host and this resulted in pRAS3.2 having an advantage over pRAS3.1 at the population level. Sequence remnants of pRAS3.2 from horizontal gene transfer suggested that pRAS3.2 was the original pRAS3 plasmid and thus that pRAS3.1 evolved from pRAS3.2. As the pRAS3.1 derivative plasmids that were constructed during the course of this study are likely to have been intermediates in the evolution of pRAS3.1 from pRAS3.2, I was able to speculate on the stepwise evolution of pRAS3.1 from pRAS3.2 based on the characteristics of these plasmids, and thus, how both macro- and microevolutionary events have contributed to the evolution of these two plasmids. / AFRIKAANSE OPSOMMING: Die twee isogeniese plasmiede, pRAS3.1 en pRAS3.2, was geidentifiseer as tetrasiklien weerstandbiedende plasmiede wat in Aeromonas salmonicida subsp. salmonicida en nie-tipiese A. salmonicida voorkom (L'Abee-Lund and Sorum, 2002). DNS volgorde analise deur L'Abee-Lund en Sorum (2002) het gewys dat die gene verantwoordelik vir replisering (uitsluitend die repC) en mobililisering naverwant is aan die van twee IncQ-2 plasmiede, pTF-FC2 en pTC-F14. Eksperimente tydens hierdie studie het egter gewys dat die repliserende sisteme van die twee pRAS3 plasmiede versoenbaar is met die repliserende sisteme van die IncQ-1α, ß and y plasmiede RSF1010, pIE1107 en pIE1130, sowel as die IncQ-2α en ß plasmiede, pTF-FC2 and pTC-F14, onderskeidelik. Analise van die aminosuur volgorde van die pRAS3 RepC proteien het gedui daarop dat die proteien taamlik verskil van die RepC proteiene van die naverwante plasmiede pTF-FC2 en pTC-F14, sowel as die van die IncQ-1 tipe plasmiede, en daar was voorgestel dat die voorsaat pRAS3 plasmied moontlik die repC geen bekom het vanaf 'n ander, nog onbekende, plasmied deur middel van horisontale geen uitruiling. Die verskil in die RepC van die pRAS3 plasmiede teenoor die van die ander IncQ plasmiede waarteen hulle getoets was vir onversoenbaarheid, was waarskynlik die rede waarom die pRAS3 plasmiede versoenbaar was met die IncQ-1 en IncQ-2 plasmiede. DNS volgorde analise tydens hierdie studie het die teenwoordigheid van twee, vantevore ongeidentifiseerde, klein 108 en 74 aminosuur proteiene onthul wat ver langs verwant is aan die PemIK (Bravo et al., 1987; Tsuchimoto et al., 1988) en MazEF (Masuda et al., 1993) toksien-antitoksien sisteme. Die gene wat kodeer vir hierdie toksien-antitoksien proteine kom tussen die repB en die repA gene van die twee pRAS3 plasmiede voor. Klonering van die toksien-antitoksien gene van die pRAS3 plasmiede op 'n ander onstabiele plasmied het die stabiliteit van hierdie plasmied verhoog van 35 tot en met 98% na ~72 generasies. Hierdie experiment het dus bevestig dat, soos die PasABC en PasAB sisteme van pTF-FC2 en pTC-F14 onderskeidelik, die twee gene 'n toksien-antitoksien sisteem kodeer wat die stabiliteit van 'n plasmied binne 'n bakteriese populasie kan verbeter. Alhoewel die toksien-antitoksien gene van pRAS3 op 'n soortgelyke posisie op die pRAS3 plasmiede voorkom as wat die pasABC en pasAB gene op hulle onderskeidelike pTF-FC2 en pTC-F14 plasmiede voorkom, is hulle nie verwant nie en dus was dit voorgestel dat die twee tipe toksien-antitoksien sisteme onafhanklik van mekaar verkry is. Aangesien die DNS volgorde en genetiese rangskikking van pRAS3 teenoor die IncQ-2α en ß plasmiede pTF-FC2 en pTC-F14, onderskeidelik, soortgelyk is, asook die feit dat die pRAS3 plasmiede versoenbaar was met pTF-FC2 en pTC-F14, sowel as ander IncQ tipe plasmiede, word dit voorgestel dat die twee pRAS3 plasmiede in 'n nuwe IncQ-2y subgroep ingedeel word. 'n Vergelyking van die DNS volgorde van die twee pRAS3 plasmiede deur L'Abee-Lund and Sorum (2002) het gewys dat, behalwe vir 'n paar puntmutasies binne die tetAR tetrasiklien weerstandsgene, verskil die twee net in die opsig dat pRAS3.1 het 4 agtereenvolgende kopiee van 22-bp 'iteron' herhalings wat gelee is binne sy replikasie oorsprong en 5 kopiee van 'n CCCCCG 6-bp herhaling wat naby sy oorsprong van oordrag gelee is, terwyl pRAS3.2 net 3 en 4 kopiee het van elk van die onderskeie volgorde herhalings. Dus die bestaan van twee plasmiede met verskillende kopiegetalle van die twee verskillende tipe DNA volgorde herhalings, maar wat vermoedelik afkomstig is vanaf dieselfde stam plasmied, bring die volgende oorhoofse vrae aangaande die plasmiede na vore: hoe beinvloed die DNS volgorde herhalings die vermoe van die twee plasmiede om binne 'n enkele gasheersel te kompeteer vir die beskikbare plasmied repliserings masjinerie, en hoe beinvloed dit die plasmied-gasheersel verhouding en dus hulle vermoe om te kompeteer op die populasie vlak, en laastens, hoekom het beide weergawes van die plasmied bly voortbestaan in die omgewing? Die plasmied kopiegetalle van pRAS3.1 en pRAS3.2 was eksperimenteel beraam by ongeveer 45 ± 13 en 30 ± 5 plasmiede per chromosoom in E. coli, onderskeidelik. Deur 'n reeks van pRAS3.1 derivate te skep met 3 tot 7 'iteron' herhalings en 4 of 5 kopiee van die 6-bp herhalings was dit bewys dat 'n toename in die hoeveelheid 'iterons' 'n afname in die plasmied kopiegetal veroorsaak, vermoedelik deur 'n verbeterde vermoe om RepC te bind, terwyl 'n verhoging van 4 tot 5 kopiee van die 6-bp herhaling 'n afname in die kopiegetal te weeg gebring het. Die repB geen van 'n plasmied met 5 x 6-bp herhalings was ~2-voud hoer uitgedruk as die van 'n plasmied met 4 x 6-bp herhalings, en dit was verder bewys dat 'n verhoogde vlak van repB transkripsie vanaf 'n L-arabinose induseerbare promoter in trans van 'n pRAS3 plasmied met 4 x 6-bp herhalings het 'n ~2-voud verhoging in plasmied kopiegetal teweeg gebring. Die rede dat pRAS3.1 'n ~1.5-voud hoer plasmied kopiegetal gehad het as pRAS3.2, was as gevolg van 'n hoer vlak van repB uitdrukking weens die feit dat pRAS3.1 5 x 6-bp herhalings in die mobB-mobA/repB promoter area het terwyl pRAS3.2 net 4 van die 6-bp herhalings in dieslefde posisie het. Sou pRAS3.1 4 x 22-bp 'iterons' gehad het, maar saam met 4 x 6-bp herhalings soos pRAS3.2, dan sou die plasmied kopiegetal 23 ± 2 plasmiede per chromosoom gewees het. Die verskil in die hoeveelheid 'iterons' en 6-bp herhalings, en dus die plasmied kopiegetal, het nie 'n effek op die stabiliteit van die wilde tipe plasmiede of hulle derivate gehad nie, selfs al was die toksien-antitoksien sisteem geneutraliseer deurdat daar 'n kopie van die toksien-antitoksien sisteem op 'n ander plasmied in trans van die pRAS3 plasmiede en hul derivate geplaas was. Die relatiewe hoe plasmied kopiegetal van die pRAS3 plasmiede, wat moontlik hoog genoeg was om plasmied stabiliteit deur middel van toevallige uitdeling te verseker, was voorgestel as die rede vir die hoe mate van plasmied stabiliteit. Soortgelyke frekwensies van mobilisasie vir pRAS3.1 en pRAS3.2 (0.032 ± 0.014 en 0.021 ± 0.013 transkonjugate per donateur, onderskeidelik) was waargeneem. Dus het dit geblyk dat die verskil in uitdrukking van die mobB-mobA/repB operon, sowel as die plasmied kopiegetal van die twee pRAS3 plasmiede, nie die mobiliserings frekwensie beinvloed het nie. Intrasellulere kompetisie tussen pRAS3.1 en pRAS3.2 het gewys dat pRAS3.1 die vermoe gehad om binne ~20 generasies pRAS3.2 vanuit 98% van die gasheerselle te skop. Daar was gewys dat die teenwoordigheid van 4 x 22-bp 'iterons' in die oorsprong van replikasie van pRAS3.1 die rede was vir die vermoe van hierdie plasmied om pRAS3.2 uit te kompeteer binne die gasheersel, moontlik deurdat die 4 x 22-bp 'iterons' beter in staat was om die RepC protein te bind. Die vermoe van plasmiede met 5 of 7 x 22-bp 'iterons' om te kompeteer met 'n plasmied met net 3 x 22-bp 'iterons' was toenemend swakker in vergelyking met die van 'n plasmied met 4 x 22-bp 'iterons', en hierdie waarneming het gelei tot die voorstel dat plasmiede met meer as 4 x 22-bp 'iterons' nie so suksesvol was om replikasie te inisieer soos ¡¥n plasmied met 3 x 22-bp 'iterons' nie. 'n Direkte korrelasie was gevind tussen die plasmied kopiegetal van 'n pRAS3 plasmied en die metaboliese lading wat die plasmied op die gasheersel geplaas het. Dus het pRAS3.1, met 'n plasmied kopiegetal van ~1.5-voud hoer as die van pRAS3.2, 'n effens hoer (~2.8%) metababoliese lading op die gasheersel as pRAS3.2 geplaas. In gevolge van die inter- en intrasellulere kompetiesie eksperimente, was dit ge-argumenteer dat pRAS3.1 'n mededingende voordeel bo-oor pRAS3.2 binne 'n gasheersel (soos wat dit sou gewees het kort nadat die twee plasmiede van mekaar uiteengevloei het) gehad het omdat dit in staat was om pRAS3.2 vanuit die gasheersel te skop. Aan die ander kant het pRAS3.2 'n laer plasmied kopiegetal en dus 'n laer metaboliese lading op die isogeniese gasheersel geplaas het, en daardeur het pRAS3.2 weer op die populasievlak die kompeterende voordeel bo-oor pRAS3.1 gehad. Die eienskappe van pRAS3.2 was meer soortgelyk aan die van ander IncQ-tipe plasmiede as wat die eienskappe van pRAS3.1 was, en dus word dit voorgestel dat pRAS3.1 vanaf pRAS3.2 afkomstig was. Omdat die derivaat plasmiede wat geskep was vanaf pRAS3.1 tydens hierdie studie moontlike tussengangers in die ontwikkeling van pRAS3.1 vanaf pRAS3.2 was, kan gespekuleer word, gebaseer op die eienskappe van hierdie plasmiede, oor die “stapsgewyse manier” waarmee pRAS3.1 vanaf pRAS3.2 ge-evolueer het, en dus hoe beide makro- en mikro-evolusionere gebeurlikhede bygedra het tot die evolusie van genoemde plasmiede.

Carotenoides são pigmentos encontrados em grande abundancia em frutos do tomateiro. A capacidade de sequestro de radicais destes pigmentos faz do tomate uma excelente opção para inclusão de compostos nutricionais pela dieta. Embora a síntese de carotenoides seja reconhecida como uma das respostas da cascata de eventos que levam ao amadurecimento, pouco se sabe a respeito de sua influência neste processo. Deste modo, o objetivo deste trabalho foi determinar se o conteúdo e a composição de carotenóide nos frutos acarretam alterações na conservação e na suscetibilidade dos frutos ao fungo B. cinerea. Foram utilizadas linhagens quase isogênicas (NILs) ao Micro Tom (MT) carregando as mutações, Beta carotene (B), tangerine (t), yellow flesh (r) e Delta carotene (Del). A caracterização dessas variações alélicas revelou, não apenas alterações no conteúdo de carotenoides, mas também outros efeitos pleiotrópicos relacionados ao amadurecimento, parâmetros de qualidade, espessura da cutícula e propriedades nutricionais dos frutos. Em face disso, subsequentemente investigamos se o diferencial conteúdo e composição de carotenóides nas referidas NILs altera a suscetibilidade dos frutos ao fungo Botrytis cinerea. A caracterização do amadurecimento demonstrou que o pico climatérico dos frutos do mutante r foi antecipado, assim como a transição para o estádio breaker. De modo contrário, a alteração na coloração da epiderme do mutante t foi atrasada, reafirmando suspeitas de outros autores que sugeriram que a enzima carotenoide-isomerase é necessária para a síntese de carotenoides no escuro. O conteúdo de ácido ascórbico também foi contrastante entre estes genótipos, sendo que no mutante r a sua concentração foi elevada, enquanto em t sua concentração foi reduzida. A perda de massa dos frutos foi menor no mutante Del, no entanto, esta característica não esteve relacionada a espessura da cutícula, que foi maior em B. A inoculação com B. cinerea por meio de ferimentos na pele dos frutos revelou menor suscetibilidade do mutante t, e em menor amplitude, do mutante Del. Ainda, o mutante t também se mostrou menos suscetível à inoculação do patógeno sobre frutos intactos. A maior espessura da cutícula de B não reduziu sua suscetibilidade ao fungo, que infectou os tecidos preferencialmente através da cicatriz do pedúnculo. A interação com B. cinerea desencadeou a produção de etileno e de espécies reativas de oxigênio (EROs), assim, é provável que a capacidade de sequestro de radicais livres tenha contribuído para a redução da suscetibilidade em t e Del, que mostraram menor produção de superóxido. Juntos, estes resultados sugerem que a modulação na atividade de enzimas da carotenogênese pode servir como ferramenta para obtenção de frutos com diferente vida útil durante o armazenamento pós-colheita de tomate. / Carotenoids are pigments found in great abundance in tomato fruits. The radical sequestration capacity of these pigments makes tomato an excellent option for inclusion of nutritional compounds in diet. Although the synthesis of carotenoids is recognized as one of the cascade responses that trigger ripening, little is known about its influence in this process. Thus, the aim of this study was to determine if the content and composition of carotenoids in the fruits alters the conservation and the susceptibility of the fruits to the B. cinerea fungus. Micro Tom (MT) near-isogenic lines (NILs) harboring the mutations Beta carotene (B), tangerine (t), yellow flesh (r) and Delta carotene (Del) has been used. The characterization of these allelic variations revealed not only changes in the carotenoid content but also other pleiotropic effects related to maturation, quality parameters, cuticle thickness and nutritional properties of fruit. In this way, we subsequently investigated whether the differential content and composition of carotenoids in NILs alters fruit susceptibility to the fungus Botrytis cinerea. The ripening characterization showed that the climacteric peak of the fruits of the r mutant was anticipated, as well as the transition to the breaker stage. Conversely, the change in epidermal coloration of mutant t was delayed, reaffirming suspicions of other authors who suggested that the carotenoid-isomerase enzyme is required for the synthesis of carotenoids in the dark. The ascorbic acid content was also contrasting among these genotypes, and in the r mutant its concentration was high, while at t its concentration was reduced. The fruit mass loss was lower in Del mutant, however, this characteristic was not related to cuticle thickness, which was higher in B. Wound-inoculation with B. cinerea revealed less susceptibility of the mutant t, and in a lesser extent, the mutant Del. Further, mutant t was also less susceptible to pathogen inoculation on intact fruits. The greater thickness of the cuticle of B did not reduce its susceptibility to the fungus, which infected the tissues preferentially through the scar of the peduncle. The interaction with B. cinerea triggered the production of ethylene and reactive oxygen species (ROS), so the free radical scavenging capacity probably contributed to the reduction of t and Del susceptibility, which showed lower production of superoxide. Together, these results suggest that modulation in carotenogenesis enzyme activity may serve as a tool to obtain fruit with different shelf-life during post-harvest tomato storage.

Submitted by (edna.saturno@ufrpe.br) on 2017-02-16T14:30:22Z No. of bitstreams: 1 Marilia Espindola de Souza.pdf: 397654 bytes, checksum: b4105af9769e13d8c7922e93537b6014 (MD5) / Made available in DSpace on 2017-02-16T14:30:22Z (GMT). No. of bitstreams: 1 Marilia Espindola de Souza.pdf: 397654 bytes, checksum: b4105af9769e13d8c7922e93537b6014 (MD5) Previous issue date: 2007-06-04 / Tilapia native to Africa, are cultured worldwide. In the past 50 years, populations of Tilapia rendalli have been introduced in Brazil, afterwards Oreochromis niloticus and O. hornorum populations, originated from Ivory Coast, were also imported. In the last ten years, an O. niloticus strain named “Chitralada”, developed in Thailand, completely spread all over the country. One of the populations of O. niloticus named Nilotica that remained from the first imports is being cultured at the Aquaculture Station of Paulo Afonso, representing an unique genetic model. The objective of this study was to investigate the genetic variability and differentiation of two tilapia populations of O. niloticus and O. niloticus “Chitralada” reared in Paulo Afonso Station in order to verify the genetic potentiality of using niloticus population in the construction of a highly inbred strain for further QTL approach or in exploring hybrid vigor. Forty four individuals of each population were genotyped for three microsatellite markers and a total of 22 alleles were found. The mean heterozygosity detected was 0,471 for “Chitralada” population and 0 for the O. niloticus population. The coefficient of genetic differentiation (FST) was 0,627 and the mean inbreeding coefficient(FIS) was 0,309 for Chitralada population and was 1 for O. niloticus population. The absence of heterozygosity in the population of O. niloticus suggests that this population can be used in the development of a highly homozygous strain as well as in obtaining heterosis in crosses with “Chitralada” population. / As tilápias, originárias do continente africano, são cultivadas em todo o mundo. As populações cultivadas no Brasil são derivadas de importações da Tilapia rendalli ,realizadas nos últimos 50 anos, posteriormente da Oreochromis niloticus e da O.hornorum, vinda da Costa do Marfim. Nos últimos 10 anos, a linhagem da O. niloticus chamada de “chitralada”, oriunda da Tailândia, disseminou-se amplamente no país. Uma das raras populações de O. niloticus, remanescente das primeiras importações, é ainda cultivada na Estação de Piscicultura de Paulo Afonso – EPPA, representando um modelo único de isolamento genético. O presente trabalho objetivou avaliar a variabilidade e diferenciação genética de duas populações de tilápia, Oreochromis niloticus e Oreochromis niloticus “chitralada”, ambas cultivadas na Estação de Piscicultura de Paulo Afonso – EPPA, a fim de verificar a potencialidade genética para sua utilização emabordagens voltadas a análise de QTLs, bem como para a exploração de vigor híbrido. Foram genotipados 44 indivíduos de cada população para três marcadores de microssatélite e um total de 22 alelos foram encontrados. A heterozigosidade media observada foi de 0,471 para a população de chitralada, e de 0 para a população denilótica. O coeficiente de diferenciação genética (FST) obtido foi de 0,627, e o valor médio do coeficiente de endocruzamento (FIS) encontrado foi de 0,309 para a população de chitralada, e de 1 para a população de nilótica. A inexistência de heterozigotos na população de O. niloticus sugere que esta população pode ser utilizada na construção de uma linhagem altamente homozigota, como também pode ser explorada para heterose com cruzamentos com a linhagem chitralada.

The haematopoietic phosphatase PTPN22 is a key regulator in balancing immune responses between self-reactivity and tolerance. PTPN22 downregulates T cell signaling and harbors the non-HLA genetic variation most strongly associated with autoimmune disease in humans, the single nucleotide polymorphism R620W. The effect of this mutation is currently controversial due to confounding results in mouse and human models. The polymorphism is linked to increased susceptibility to autoimmunity in both human and mouse models, although the latter does depend on genetic background. However, mouse data clearly shows that the polymorphism has a loss-of-function effect on T cell signalling, whereas studies in human models largely demonstrate a gain-of-function effect for R620W. A confounding issue in human studies is that they depend on comparison of T cells from distinct individuals, on protein over-expression, or on RNA interference, techniques for which it is difficult to control for genetic and environmental variables, changes in stoichiometry, and off-target effects or incomplete knockdown, respectively. We aimed to create isogenic human cell lines with mutations in PTPN22 at the genomic level to alleviate the complications inherent in analysing human data. In addition to autoimmune pathogenesis, we are interested in the role of PTPN22 in a cancer setting. Because PTPN22 has a strong suppressive effect on T cell responses to weak affinity antigen, which encompass most tumour antigens, we postulated that knocking out PTPN22 may better enable T cells to kill tumour cells. Furthermore, we have shown that PTPN22 knockout (KO) leads to increased IL-2 expression in mouse T cells, and that this effect is protective against TGF-β mediated suppression, a common driver of T cell inhibition in the tumour microenvironment. T cell transfer experiments in mice showed that PTPN22 KO T cells are indeed more effective at reducing tumour size. Based on these findings, we aim to determine whether PTPN22 KO in human cells confers a similar effect on signaling. To investigate the effects of PTPN22 KO on human T cell signaling, we used CRISPR gene-editing to target PTPN22 in a Jurkat cell line. By combining this technique with lentiviral transduction of a specific T cell receptor, we generated human cell lines which are genetically identical, save for specific alterations to PTPN22, and which can be stimulated with strong or weak cognate antigen. We found that PTPN22 KO Jurkat cells develop an enhanced activation phenotype upon stimulation, including increased IL-2 expression. Additionally, PTPN22 KO Jurkat cells show enhanced Erk signalling following stimulation with weak affinity antigen, but this difference is lost as stimulus strength increases. CRISPR technology has presented the opportunity to create novel models of PTPN22 signalling in the context of human T cell lines. The data from these lines suggests that, unlike the R620W mutation, complete loss of PTPN22 has a comparable effect in human and mouse T cells. In conjunction with our previous findings, these results suggest that knocking out PTPN22 may lead to signalling alterations that improve adoptive T cell cancer therapy.