no / Combinatorial antibody libraries were constructed from the spleen of a patient with concomitant systemic lupus erythematosus and idiopathic thrombocytopenia. Following selection of the libraries with DNA, a panel of 15 anti-DNA Fabs was isolated. Sequence analysis of these antibodies coupled with measurements of their affinities for ss- and dsDNA were used to investigate the role of somatic mutation in affinity maturation of the anti-DNA response. Examination of the germline genes used by these Fabs supports previous studies that suggest there is no restriction of the gene usage in the anti-DNA response. However, data are presented indicating that VH3 genes and the A27 V¿ paired with the J¿1 may be over-expressed in the anti-DNA repertoire. Analysis of the role of somatic mutation in increasing affinity for DNA indicates that affinity maturation has occurred and suggests that the CDR1 and CDR2 of the heavy chain are of importance in this process.

A bidirectional selection experiment for high and low somatic cell count (SCC) was conducted over 14 generations with two selected lines (high line = HSCC, low line = LSCC) of mice. Seven secondary traits (milk yield, total white blood cell count, percentage of phagocytic cells in blood, endotoxin challenge response, percentage of females littering, number of young born alive, and percentage of young surviving to weaning) were measured to examine correlated responses to selection for SCC. Average response per generation for log2 SCC was small in both selected lines (HSCC = .0678 ±.0341, LSCC = .0384 ± .0390, P > .05). There was little per generation divergence between the selected lines (.0294 ± .0178, P > .05). Genetic and phenotypic selection differentials indicated that selection procedures did select the more extreme individuals for SCC, even though response to selection was poor. Phenotypic correlations among SCC and the seven secondary traits were generally small, and near zero. Correlation coefficients ranged from -.17 to .17. Milk yield was negatively correlated with SCC (-.07, P < .05). The correlation between endotoxin challenge response and SCC was also negative (-.17, P < .05). Components of genetic variance for SCC were estimated to explain the lack of selection response. Covariances between daughter and dam, and among full sibs were negative (-.1180 and -.0362, respectively). Analysis for offspring and maternal components for SCC yielded a negative estimate for the covariance between additive effects for the offspring and maternal components (-.1781). No biological explanation can be offered for its existence. Heritability from this same analysis was .05. / Master of Science

The effectiveness of on-farm tests for the detection of antibiotic residues in milk and urine was examined using the Virginia Tech dairy herd. Milk samples from treated cows were collected 72 hours post treatment and tested for residues by the Bacillus stearothermophilus Difco disc assay, the Delvotest-P, and the Penzyme test. Urine of cull cows was collected and tested with the Live Animal Swap Test (LAST). Results showed 21% to 24% of milk samples contained antibiotic residues past the recommended withholding period. In a comparison of the two on-farm tests to the officially recognized B. stearothermophilus disc assay, no significant difference was seen between the tests. An inordinately high (62.5%) level of urine samples from cull cows exhibited antibiotic combination with the LAST, although all animals had completed the recommended withholding period specified for each antibiotic. In the second phase of this study, cows in the Virginia Tech dairy herd were assigned to experimental or control groups. Milk from cows in either group whose linear somatic cell count score (SCC) ≥ 5 for the first time during that lactation was culture on blood agar plates to detect mastitis pathogens, and the SCC was determined. Experimental group cows were treated in those quarters with a SCC ≥ 5 while control cows had antibiotic therapy administered based only on clinical symptoms. Treatment group had no significant effect on milk production or SCC, however treatment of elevated SCC quarters did result in trends for decreased SCC and reduced milk yield loss. Treatment group significantly altered infection status of the cow. Of the quarters cured of infection, a greater percentage (63%) belonged to the experimental group. Results from this study indicate that the practice of administering antibiotic treatment based on elevated SCC should not be adopted until further studies on herds of higher infection rates are conducted. / M.S.

Monthly milk production data collected by DHI supervisors in 28 Virginia dairy herds over a three year period were analyzed. Relationships between somatic cell count and milk production were determined. A curvilinear relationship between somatic cell count and daily milk yield was shown to exist. The relationship indicated daily milk yield declined more rapidly as somatic cell counts increased from 50 X 10³ through 300 X 10³ cells per ml compared to daily milk decline as somatic cell counts rose from 400 X 10³ through 1,200 X 10³ cells per ml. Daily milk yield declined at a slower rate at somatic cell levels above 400 X 10³ cells per ml. There was an apparent cumulative or continual relationship between somatic cell count and daily milk yield. Losses and declines in test day milk production were greater when cumulative somatic cell count was considered. Complete lactation records of 305-day, mature equivalent milk production were compared to weighted, lactational somatic cell counts. Lactational milk production losses were not as great as daily milk production losses, extended to full lactation, indicated. Design difficulties and inadequacies of models used in complete lactation evaluation may explain the discrepancies. A maximum somatic cell count of 150 X 10³ to 200 X 10³ cells per ml was indicated as an optimum level for somatic cell count. Above this level milk losses were excessive. / Master of Science

The present study examines the relationship of emotional awareness to anticipatory psychophysiological markers and performance on the Iowa Gambling Task (IGT). The IGT is a computerized card game that simulates real-life decisions through uncertainty of reward or punishment. The participant’s goal is to make advantageous card choices. Anticipatory somatic markers of physiological arousal, like electrodermal activity and heart rate, have been proposed to bias decisions in the IGT. The central hypothesis is that a participant’s emotional awareness is related to their ability to make advantageous decisions through biasing psychophysiological responses. The Toronto Alexithymia Scale was used to assess each participant’s emotional awareness. Less emotional awareness was associated with enhanced performance on the IGT. However, anticipatory physiological arousal (electrodermal activity and heart rate) and emotional awareness yielded no significant relationships. Findings suggest a need for further research on cognitive models, such as the expectancy valence model, in relation to decision-making.

The effect of steady shear stress on somatic embryos were investigated in a flow chamber and evaluated at different time intervals using microscopy technique. The development of meristematic cell clusters, i.e. the immature embryos, into a polarized somatic embryo, and the effect on the localization of the suspensor cells that form during development of the immature embryos, were studied as a function of shear stresses. With the distribution and growth rate of the meristematic and suspensor cells, the effect of stress on the embryo development was established. Furthermore, the effect of shear stress on the cells at molecular level, the reaction of integrin-like proteins, the production of reactive oxygen species and the pore size of the cell walls involved in the shear stress responses, were investigated with molecular techniques. In general, shear stress inhibits meristematic cells growth. Meristematic cells grow fastest at shear rate of 86 s-1 among all the tested shear stress conditions. By combining the results of meristematic cells growth and suspensor cells formation, it suggests that there is a critical shear rate between 86 and 140 s-1, at which no suspensor cells form. The unidirectional flow with different shear stresses helps the polarized growth and the unidirectional alignment of suspensor cells. Reactive oxygen species and integrin-like protein are detected in the stressed cells as cellular responses to shear stresses. By monitoring the pore size and uptake time of cells to macromolecules with solute-exclusive experiments, it suggests that the stressed cells expedite the response to plasmolyzing components that are used to induce maturation treatment thus affect the response to maturation stimuli.

myo-Inositol-1,2,3,4,5,6-hexakisphosphate (InsP6), also called phytic acid, is ubiquitous in eukaryotic cells and the most abundant inositol phosphate derivative. Loblolly pine (LP, Pinus taeda) constitutes the primary commercial species in the southern forest of U.S. Somatic embryogenesis (SE) is an effective technique to maintain the desirable genetic composition of the progeny and to accomplish the efficiency of propagation. SE can also serve as a tool for study of plant development. Unlike angiosperm embryos with attached cotyledons as seed storage organs, the diploid conifer embryo is surrounded by the unattached haploid female gametophyte (FG). In LP SE, FG tissue is absent in the embryogenic tissue culture. We found that extracts from early-stage FG stimulate growth and multiplication of early-stage somatic embryos, whereas FG water extracts from late stage contain substance(s) inhibitory to early-stage somatic embryo growth (DeSilva et al., 2007). We now present the isolation and identification of the inhibitory substance as InsP6 by means of water extraction, two gel filtrations and two ion exchange FPLC chromatographies. The results represent the first complete structural characterization of InsP6 from a natural product using LC/MS, LC/MS/MS, exact MS, 1D- and 2D-NMR analyses. We also report that there is a good correlation between the amount of InsP6 purified from FG tissue (1.3 nmoles per full-term FG) and the amount of InsP6 which inhibits somatic embryo growth. This novel approach of isolating and characterizing InsP6 from plant tissue, and investigating its role on SE can allow us to improve SE technology by circumventing current bottleneck, to elucidate enigmatic functions of InsP6 in plants, and most importantly, to utilize this molecule properly.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 5 uppsatser.

Microsatellites (MSTs) are short tandem repeats of motifs, 1 — 6 nucleotide in length, and are considered mutational 'hot-spots' and show a greater degree of somatic variability and population polymorphisms than surrounding DNA sequences. MSTs provide for a unique computational alignment problem for many commonly used algorithms, and therefore required software tools to be developed to specifically address these issues. For this work we developed a novel approach to extract MSTs from next-gen sequencing data that can robustly detect signatures of MST mutation bias and somatic variation occurring in next-gen data including a high frequency of in-phase indels. Somatic variability, novel genomic polymorphisms that arise within a cell population not found in the progenitors, plays a critical role in cellular reprogramming leading to the development and progression of cancer. MST mutation rates are between 10 and 1000 time higher than that of surrounding DNA. MSTs are found ubiquitously throughout the genome including in nearly all transcribed regions and 10-20% of coding genomic regions. Currently the only established DNA repair defect that that has been directly linked to MST instability is replication coupled mismatch repair (MMR). An initial analysis of the utility of the software was conducted with DNA repair impaired cell lines. The results demonstrated the utility in identifying the consequences of DNA repair impairments on genomic stability. There were major objectives of the finding including 1) complimenting genomics of matched DNA samples with in-sample quantification of variation and 2) demonstrating that DNA repair proficient cells and those with different defects in DNA repair can have different somatic MST variability (SMV) profiles that may be potential markers for these defects. DNA repair disorders are debilitating conditions that result in physical and neurological abnormalities robbing the individual of a normal quality of life and life span. The various conditions that fall into this class are known as progeroid disorders and they provide a very important glimpse into the aging process on a genomic level. The conditions for four cohorts analyzed here were; Cockayne's syndrome, caused by the loss of the ERCC8 gene, also known as CSA; xeroderma pigmentosum, caused by the loss of the XPA or XPB genes; Werner syndrome, caused by the loss of the RecQL2 gene; and Rothmond-Thomson syndrome, caused by the loss of the RecQL4 gene. The goal of this project was to determine if impaired excision repair genes CSA or global XPA and B or excision repair supporting helicases BLM or RecQL4 leads to MST destabilization. Comparing cohorts from excision repair disorders with a co-sequenced normal cohort we found that CSA both RecQ helicases had an effect on the exome somatic variability of MSTs. On the other hand the effects of XPA/B were inconclusive. MST instability (MSI), defined as acquired/lost primary alleles in tumors for a small set of microsatellite loci, has been implicated and is a clinically relevant marker for colorectal cancer. Conversely, no clinically actionable genetic markers have been found for liver cancer, a cancer with a very high mortality rate. Here we explore the use SMV defined as the presence of minor alleles at MST loci, as a complementary measure of MSI as a genetic marker for colorectal and liver cancer by analyzing Illumina sequenced genomes from The Cancer Genome Atlas. Our data shows that SMV may distinguish a subpopulation of African American patients with colorectal cancer, ~33% of the population in this study. Further, for liver cancer, a higher rate of SMV may be indicative of earlier age of onset. In conclusion, the work presented here suggests that MSI should be expanded to include SMV, not only instability. / Ph. D.

A hibridação somática via fusão de protoplastos é um método de combinação genética que agrega integralmente os dois genomas genitores, assim como suas respectivas organelas citoplasmáticas, sendo uma ferramenta alternativa aos métodos convencionais de melhoramento. O objetivo deste trabalho foi a obtenção de híbridos somáticos interespecíficos de laranja doce (Citrus sinensis L. Osbeck) \'Pêra\' e \'Westin\' com a tangerina \'Fremont\' (C. clementina hort. ex Tan. x C. reticulata Blanco), \'Thomas\' (C. reticulata Blanco) e \'Nules\' (C. clementina hort. ex Tan), e o tangelo \'Nova\' (C. reticulata Blanco x C. paradisi Macf.) visando à produção de genitores tetraplóides com características agronômicas desejáveis. Como fontes de protoplastos foram utilizados calos embriogênicos de laranja e folhas jovens de tangerina e tangelo, coletadas de plantas cultivadas em casa-de-vegetação. Para o isolamento dos protoplastos, as células de ambas as fontes foram imersas em solução enzimática e incubadas no escuro por 14 horas, sob agitação. Após o isolamento, os protoplastos isolados de calos foram fundidos quimicamente (polietilenoglicol - PEG) com protoplastos não embriogênicos, isolados de mesofilo foliar. Em seguida, os protoplastos foram plaqueados nos meios de cultura líquido BH3, EME e BH3 + EME, e incubados em ausência de luz, sob temperatura de 27 ºC. Após 20 dias de incubação, foram iniciados subcultivos sequenciais para nutrição e redução do potencial osmótico do meio de cultura. Os microcalos desenvolvidos foram transferidos para meio de cultura EME + maltose (13 g.L-1) para a indução da embriogênese somática. Os embriões desenvolvidos foram transferidos para meio de cultura EME + sacarose (25 g.L-1) e em seguida para o meio de cultura 1500 (1,5 g.L-1 de extrato de malte) e finalmente para o meio B+ para desenvolvimento e germinação. As plantas regeneradas foram individualizadas, enraizadas ou microenxertadas e aclimatizadas em casa-devegetação. A confirmação da hibridação somática foi feita por análise morfológica, determinação da ploidia, por citometria de fluxo e análise do DNA com marcadores moleculares do tipo SSR e RAPD. Foram obtidos híbridos somáticos de três combinações (Pêra + Fremont, Pêra + Nules e Pêra + Nova) os quais serão avaliados para utilização diretamente como copa ou como genitor tetraplóide em programas de melhoramento de citros. / Somatic hybridization by protoplasts fusion is a method of genetic combination of two parental genomes, and their cytoplasmic organelles. It is an alternative tool to conventional breeding methods. The objective of this work was to obtain interspecific somatic hybrids of \'Pera\' and \'Westin\' sweet oranges (Citrus sinensis L. Osbeck) with \'Fremont\' (C. clementina hort. ex Tan. x C. reticulata Blanco), \'Thomas\' (C. reticulata Blanco) and \'Nules\' (C. clementina hort. ex Tan) mandarins and Nova tangelo (C. reticulata Blanco x C. paradisi Macf.) to produce tetraploid parents with desirable agronomic characteristics. The sources of protoplasts were sweet orange embryogenic callus and mandarin and tangelo young leaves, collected from plants cultivated in screenhouses. For protoplasts isolation, cells from both sources were immersed in enzyme solution and incubated in the dark for 14 hours (40 rpm). After isolation, the protoplasts isolated from callus were fused chemically (polyethylene glycol - PEG) with non embryogenic protoplasts, isolated from leaf mesophyll. The protoplasts were cultivated in BH3, BH3 + EME and EME liquid culture media and incubated in the dark, under temperature of 27 ºC. After 20 days of incubation, subcultures were initiated in order to reduce the osmotic potential of culture medium. The microcalus developed were transferred to EME medium supplemented with maltose (13 g.L-1) to induce somatic embryogenesis. The developed embryos were transferred into EME + sucrose (25 g.L- 1), culture medium 1500 (1.5 g.L-1 of malt extract) or B+ culture medium for development and germination. The regenerated plants were individually rooted or micrografted, and further acclimatized in screenhouse. Somatic hybridization was confirmed by analysis of leaf morphology, ploidy determination by flow cytometry and molecular analysis by SSR and RAPD markers. Somatic hybrids were obtained of the three combinations (Pera + Fremont, Pêra + Nules and Pêra + Nova) which will be evaluated for direct are as scion or as a tetraploid parent in citrus breeding programs.