Many biomolecular processes involve mechanical force-induced reactions in the cell, such as translocation, and mechanical stretching of biopolymers. Recent advances in single molecule manipulation techniques make it possible to apply mechanical force to individual biomolecules and study their dynamics. To gain molecular level understanding of these processes and to interpret the single-molecule experiments, we used Langevin dynamics simulations of coarse-grained biopolymer models. Our result show that the mechanism of translocation of proteins through pores depends on the pore diameter, on the magnitude of the pulling force and on whether the force is applied at the N- or the C-terminus of the chain. In addition, the translocation kinetics of peptides varies with their stability. The mechanism of protein translocation is found to be different from that of a structureless polypeptide of the same length. We further showed that unfolding mechanism of translocation process is different from when the same protein is stretched between its C- and N-termini. We also studied the mechanical and chemical/thermal denaturation of proteins. We observed that the free energy profile along the mechanical reaction coordinate and the chemical reaction coordinate are different. In our protein model, the mechanical and chemical/thermal denaturation cannot be simply explained in terms of a simple onedimensional free energy landscape. We further analyzed the spontaneous folding and refolding under a constant force and found that refolding generally occurs via different mechanisms. Similarly, we investigated the protein unfolding/refolding under the applied force that varies with a constant loading rate. This study shows that unfolding/refolding pathways are generally similar for low loading/unloading rates while they become different for high loading/unloading rates. Finally, we studied the dynamics of molecular friction knots formed by a pair of polymer strands. We examined different knot types, and different polymer sequences. Depending on the knot type and the nature of the polymer, we observed two different behaviors when the force F is exerted to separate the polymer strands. The knot between polymer strands can be strong (the time [tau] the knot stays tied increases with the force F applied to separate the strands) or weak ([tau]decreases with increasing F).

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Gene transfer from wild gras species to wheat is complicated by the simultaneous integration of large amounts of alien chromatin. The alien chromatin containing the target gene is inherited as a linkage block and the phenomenon is known as linkage drag. The degree of linkage drag depends on whether, and how readily, recombination occurs between the foreign and wheat chromatin. The S13 translocation line was developed by the department of Genetics, US. A cross was made between Chinese Spring and a leaf rust resistant Aegilops speltoides accession. Resistant backcross F1 was backcrossed to Chinese Spring and W84-17. S13 was selected from the backcross progeny and found to carry three rust resistance genes temporarily named LrS13, SrS13 and YrS13. Unfortunately, the resistance genes were completely linked to gametocidal (Gc) genes that were co-transferred from the wild parent. In wheat Gc genes cause reduced fertility, poor plant phenotype and hybrid necrosis. In order to use employ the rust resistance genes commercially they need to be separated from the Gc genes. At the onset of this study four putative shortened forms of the S13 translocation were provided. The four lines were identified in a homoeologous paring induction experiment (involving the test cross 04M127). This study aimed to achieve the following: (i) characterize the four recombinants with the use of molecular markers, (ii) use the knowledge gained to identify further recombinants in the 04M127 cross, (iii) identify the shortest (most useful) recombinant, and (iv) attempt to shorten the shortest recombinant form still further and thereby remove as many of the Gc genes as possible. In total, seven recombinants of the S13 translocation (04M127-1, -2, -3, -4, -7, -11 and -12; referred to as recombinant group A) were identified and characterised with microsatellite and SCAR markers. These recombinants have exchanged different amounts of foreign chromatin for wheat chromatin, but were still associated with Gc genes, showing hybrid necrosis and seed shrivelling. Some of the recombinants have lost the undesirable „brittle rachis‟ phenotype which occurs in Ae. speltoides and the S13 translocation line. In plants VII having this trait, the rachis spontaneously disarticulates after the third spikelet upon ripening of the ear. Recombinant 3 appeared to be least affected by Gc genes and was therefore used in further attempts to shorten the translocation. Recombinant 3 was crossed with wheat (W84-17) and resistant F1 (heterozygous for the translocation) were test crossed with Chinese Spring nullisomic 3A tetrasomic 3B/D plants. Thirty five resistant testcross F1 plants were identified (named recombinant group B). The resistant group B recombinants as well as nine susceptible test cross F1 (which also appeared to be recombinant) were characterised making use of microsatellites and a SCAR marker. From the results it appeared that each of the 35 resistant plants exchanged substantial amounts of Ae. speltoides chromatin for wheat chromatin. The species chromatin that remained (and which contains LrS13) is probably located either close to the 3AS telomere or within the proximal regions of 3AS and 3AL. A SCAR marker that has been developed specifically for the S13 translocation provided useful confirmation of the presence of Ae. speltoides chromatin in the 35 recombinants. If the SCAR marker proves to be tightly linked to LrS13 it may eventually be used for marker assisted selection of the resistance or it may be employed in continued attempts to reduce the amount of foreign chromatin. Seedling rust resistance tests showed that the recombinants have lost SrS13 and YrS1 during recombination. An attempt was also made to develop additional markers that specifically detect the translocation in order to further characterise the group B recombinants. Published information on Ae. speltoides specific repeated and transposon sequences were obtained and used for primer design. Unfortunately, no suitable markers could be found and the primers that were designed tended to amplify the same fragments in both the wheat and species genomes. DArT markers were also employed in an attempt to characterise the 35 group B recombinants and controls. The DArT results provided an independent verification of the results obtained with the microsatellite markers. The DArT results confirmed that the group B recombinants exchanged large amounts of species chromatin for wheat chromatin. Even though the 35 resistant group B recombinants have undergone extensive recombination they still show signs of residual Gc effects. It is believed these effects can be removed by continued backcrossing to wheat accompanied by selection against Gc symptoms. While the effects of Gc genes per se were not studied, their properties were reminiscent of those of transposable elements. Indications were that complex interactions involving the Gc genes themselves as well as genetic factors in the wheat genome may have a drastic effect on the selective survival of recombinant gametes.

The aim of this study was to analyse testcross-material that was generated during a homoeologous pairing-induction experiment. Absence of the homoeologous pairing suppressor gene, Ph1, was employed to induce meiotic pairing between the Lr59 translocation (Aegilops peregrina) and 1AL of normal wheat. The study aimed to characterize the test-cross plants derived from this experiment and to identify recombinants which retained the least amount of species chromatin but which still contained the Lr59 gene. The test-cross F1 population, 07M5 (total 635 plants), was screened for Lr59 resistance by inoculating seedlings with the leaf rust pathotype, UVPrt8. The 168 resistant plants were characterized with molecular markers in order to identify recombinants. The data were used to construct a physical map which showed the relative sizes of the recombinants and which could be used to identify those recombinants which contained the least amount of residual species chromatin. Microsatellite (Xcfa2219, Xbarc83 and Xgwm164) and SCAR (S15T3) analysis was used for the initial identification of recombinants. The results showed that 152 of the 168 resistant plants were recombinants for the four loci; that eight of the remaining 16 plants represented non-recombinant, wild species-types and that the last eight plants represented the wheat parental-types which were resistant (and thus, also recombinants). This extremely high recombination frequency can largely be attributed to strong segregation distortion that was evident in the cross. It is also possible that the translocation segment could derive from the S genome rather than the U genome of Ae. peregrina. The S genome is closer related to the wheat genomes than the U genome and may be more prone to recombination. With the use of the microsatellite and SCAR data, a physical map was constructed which showed the relative location of the Lr59 gene on the translocation. It appeared that the eight shortest recombinants retained terminal species chromatin. In an attempt to characterize the eight recombinants, additional marker loci had to be identified within that region. RAPD, iv AFLP and DArT markers were investigated for this purpose. RAPD analyses did not produce any useful markers. AFLP and DArT analyses did identify useful markers with which the eight recombinants could be screened. The data showed which recombinants probably retained the least amount of species chromatin. Seeing that AFLP and DArT markers are anonymous and that the distances between marker loci are unknown, it is not possible to say which recombinant is the shortest and consequently it will be nessecary to also evaluate the group of eight recombinants agronomically in order to identify the most useful ones. The results showed that multiple cross-overs apparently occured on both sides of Lr59. Multiple cross-overs are higly unlikely in material of this nature, therefore it was speculated that the observation resulted from incomplete synteny between the telomeric areas of the translocation and 1AL. A structural difference between the two chromosome regions might have given rise to abnormal meiotic pairing structures and thus unexpected gamete genotypes. Each of the eight recombinants did express one or more of the Ae. peregrina derived AFLP loci which can in future be verified for use as a marker for marker assisted selection. The study succeeded in identifying a number of potentially useful recombinants which contain the Lr59 resistance. It would, however, be risky to select only one of the shortest recombinants for further development on the basis of the present knowledge as some recombinants may contain genetic abnormalities which resulted from reduced synteny in the Lr59 region. It would therefore be wise to further evaluate all eight recombinants before the best one is selected for agronomic use.

N,N'-methylenebisacrylamide (MBA) was studied because of its effectiveness in inducing heritable translocations in germ cells of male mice. The health impact of translocations was studied through anatomical analysis of the progeny of semisterile translocation carriers. As expected, the semisterility of translocation carriers resulted primarily from embryonic death during periimplantation stages due to unbalanced chromosome sperm segregants. Among conceptuses that survived to mid- and late-gestation stages, there was an increased incidence of developmental anomalies including fetal death and phenotypic defects. These abnormalities are associated with unbalanced chromosome complements that allow survival to the later stages of development.

Indiana University-Purdue University Indianapolis (IUPUI) / The root system architecture of higher plants plays an essential role in the uptake of water and nutrients as well as the production of hormones. These root systems are highly branched with the formation of post-embryonic organs such as lateral roots. The initiation and development of lateral roots has been well defined. WAG1 and WAG2 are protein-serine/threonine kinases from Arabidopsis that are closely related to PINOID and suppress root waving. The wag1;wag2 double mutants exhibit a strong root waving phenotype on vertical hard agar plates only seen in wild-type roots when the seedlings are grown on inclined plates. Here an additional root phenotype in the wag1;wag2 mutant is reported. The wag1;wag2 double mutant displays both an increased total number and density of emerged lateral roots (approximately 1.5-fold). An increased LRP density of 1.5-fold over wild-type is observed. To ascertain the role of WAG1 and WAG2 in lateral root development we examined promoter activity in the WAG1::GUS and WAG2::GUS lines. The WAG1 promoter showed no detectable activity at any stage of development. The WAG2 promoter was active in stage IV onward, however there was no detectable activity in the cell types associated with initiation events. The lateral root density and spatial patterning in wild-type, when grown on inclined hard agar plates, was similar to wag1;wag2 on vertical plates. Seedlings of both genotypes were treated with hormones such as auxin and MeJA, and inhibitors. Auxin response in wag1;wag2 was normal with a similar number of LR as the wild-type after treatment. Treatment with MeJA resulted in a similar induction of LRP in both genotypes, however the percent lateral root emergence in wag1;wag2 was reduced while Col-0 was increased compared to controls. Treatment with the calcium blocker tetracaine resulted in wag1;wag2 displaying a wild-type level of LR but had no significant effect on wild-type. Genetic analysis of the wag1;wag2 LR pathway revealed that WAG1 and WAG2 are acting in the same pathway as AUX1, AXR1and PGM1. pgm1-1 was not previously reported to have a LR defect but showed decreased LR formation here, while pgm1;wag1;wag2 had a similar LR density to wag1;wag2. TIR7 and ARG1 were both deduced to operate in separate pathways from WAG1 and WAG2. The data presented here shows that the wag1;wag2 double mutant has an increased number of LR compared to Col-0. This defect appears to be caused by increased pre-initiation events and seems to be tied to the root waving phenotype. However, the treatment with MeJA revealed a possible role for WAG1 or WAG2 in LRP development, potentially under stress conditions. Calcium also seems to play a significant role in the wag1;wag2 LR phenotype, possibly independent of the root waving phenotype.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / O retardo mental (RM) é caracterizado por um funcionamento intelectual significantemente abaixo da média (QI<70). A prevalência de RM varia entre estudos epidemiológicos, sendo estimada em 2-3% da população mundial, constituindo assim, um dos mais importantes problemas de saúde pública. Há um consenso geral de que o RM é mais comum no sexo masculino, um achado atribuído às numerosas mutações nos genes encontrados no cromossomo X, levando ao retardo mental ligado ao X (RMLX). Dentre os genes presentes no cromossomo X, o Jumonji AT-rich interactive domain IC (JARID1C) foi recentemente identificado como um potencial candidato etiológico do RM, quando mutado. O JARID1C codifica uma proteína que atua como uma desmetilase da lisina 4 da histona H3 (H3K4), imprescindível para a regulação epigenética. Tão recente como a identificação do gene JARID1C, é a descoberta de que mudanças no número de cópias de sequências de DNA, caracterizadas por microdeleções e microduplicações, poderiam ser consideradas como razões funcionalmente importantes de RMLX. Atualmente, cerca de 5-10% dos casos de RM em homens são reconhecidos por ocorrerem devido a estas variações do número de cópias no cromossomo X. Neste estudo, investigamos mutações no gene JARID1C, através do rastreamento dos éxons 9, 11, 12, 13, 15 e 16, em 121 homens de famílias com RM provavelmente ligado ao X. Paralelamente, realizamos a análise da variação do número de cópias em 16 genes localizados no cromossomo X através da técnica de MLPA no mesmo grupo de pacientes. Esta metodologia consiste em uma amplificação múltipla que detecta variações no número de cópias de até 50 sequências diferentes de DNA genômico, sendo capaz de distinguir sequências que diferem em apenas um nucleotídeo. O DNA genômico foi extraído a partir de sangue periférico e as amostras foram amplificadas pela técnica de PCR, seguida da análise por sequenciamento direto. Foram identificadas três variantes na sequência do gene JARID1C entre os pacientes analisados: a variante intrônica 2243+11 G>T, que esteve presente em 67% dos pacientes, a variante silenciosa c.1794C>G e a mutação inédita nonsense c.2172C>A, ambas presentes em 0,82% dos indivíduos investigados. A análise através do MLPA revelou uma duplicação em um dos pacientes envolvendo as sondas referentes ao gene GDI1 e ao gene HUWE1. Este trabalho expande o estudo de mutações no gene JARID1C para a população brasileira ereforça a importância da triagem de mutações neste gene em homens portadores de RM familiar de origem idiopática, assim como, é primeiro relato científico relativo à investigação de variações no número de cópias de genes localizados no cromossomo X em homens brasileiros com RM, através da técnica de MLPA. / Mental retardation (MR) is defined as a disability characterized by significant below average intellectual functioning (IQ>70). The prevalence of MR varies between epidemiological studies, estimated at 2-3% of the population, thus constituting a major public health problem. There is a general consensus that MR is more common in males, a finding attributed, in part, to mutations in the genes located on the X chromosome, leading to an X-linked mental retardation (XLMR). Among all the genes present on X chromosome, Jumonji AT-rich interactive domain IC (JARID1C) was recently identified as aetiologic potential candidate of MR, when mutated. The JARID1C gene encodes a protein that acts as a histone demethylase specific for histone 3 lysine 4 (H3K4) and it is indispensable for the epigenetic regulation. As recently as the identification of the JARID1C gene, it is the discovery that changes in the number of copies of DNA sequences, characterized by microdeletions and microduplications, could be regarded as functionally important reasons to XLMR. Currently, about 5-10% of men MR cases are known to occur due to these variations in the number of copies of chromosome X. In this study we investigated mutations in the JARID1C gene by screening of exons 9, 11, 12, 13, 15 and 16 in 121 patients from families with X-linked MR. At the same time we analyzed the variation in the number of copies in 16 genes located in X chromosome through the MLPA technique. This metodology consists of a multiplex amplification that detects variations in the number of copies up to 50 different genomic DNA sequences, being able to distinguish sequences that differ by only one nucleotide. Genomic DNA was extracted from peripheral blood and the samples were amplified by PCR followed by direct sequencing analysis. We identified three sequence variants among 121 patients. The intronic variant c.2243 +11 G> T, which was present in 67% of patients analyzed, the silent variant c.1794C> G and the novel nonsense mutation c.2172C> A, which was present in 0,82% of patients analyzed. The MLPA analysis revealed that the patient 58 exhibited a duplication involving probes for the GDI1 gene and the HUWE1 gene, resulting in an increase in the number of copies of this gene. This work expands the study of mutations in the JARID1C gene for the Brazilian population and reinforces the importance of screening for mutations in this gene in men with idiopathic mental retardation, and it is the first scientific report on the investigation of variations in the number of copies in genes located on chromosome X in Brazilian men with MR using the MLPA technique.

Experiments show that MgSO4 salt has a non-monotonic effect as a function of MgSO4 concentration on the ejection of DNA from bacteriophage lambda. There is a concentration, N0, at which the minimum amount of DNA is ejected. At lower or higher concentrations, more DNA is ejected. We propose that this non-monotonic behavior is due to the overcharging of DNA at high concentration of Mg⁺² counterions. As the Mg⁺² concentration increases from zero, the net charge of ejected DNA changes its sign from negative to positive. N0 corresponds to the concentration at which DNA is neutral. Our theory fits experimental data well. The DNA-DNA electrostatic attraction is found to be -0.004 kBT/nucleotide. Simulations of DNA-DNA interaction of a hexagonal DNA bundle support our theory. They also show the non-monotonic DNA-DNA interaction and reentrant behavior of DNA condensation by divalent counterions. Three problems in understanding the capsid assembly for a retrovirus are studied: First, the way in which the viral membrane affects the structure of in vivo assembled HIV-1 capsid is studied. We show that conical and cylindrical capsids have similar energy at high surface tension of the viral membrane, which leads to the various shapes of HIV-1 capsids. Secondly, the problem of RNA genome packaging inside spherical viruses is studied using RNA condensation theory. For weak adsorption strength of capsid protein, most RNA genomes are located at the center of the capsid. For strong adsorption strength, RNA genomes peak near the capsid surface and the amount of RNA packaged is proportional to the capsid area instead its volume. Theory fits experimental data reasonably well. Thirdly, the condensation of RNA molecules by nucleocapsid (NC) protein is studied. The interaction between RNA molecules and NC proteins is important for the reverse transcription of viral RNA which relates to the viral infectivity. For strong adsorption strength of the NC protein, there is a screening effect by RNA molecules around a single NC protein.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / O retardo mental (RM) é caracterizado por um funcionamento intelectual significantemente abaixo da média (QI<70). A prevalência de RM varia entre estudos epidemiológicos, sendo estimada em 2-3% da população mundial, constituindo assim, um dos mais importantes problemas de saúde pública. Há um consenso geral de que o RM é mais comum no sexo masculino, um achado atribuído às numerosas mutações nos genes encontrados no cromossomo X, levando ao retardo mental ligado ao X (RMLX). Dentre os genes presentes no cromossomo X, o Jumonji AT-rich interactive domain IC (JARID1C) foi recentemente identificado como um potencial candidato etiológico do RM, quando mutado. O JARID1C codifica uma proteína que atua como uma desmetilase da lisina 4 da histona H3 (H3K4), imprescindível para a regulação epigenética. Tão recente como a identificação do gene JARID1C, é a descoberta de que mudanças no número de cópias de sequências de DNA, caracterizadas por microdeleções e microduplicações, poderiam ser consideradas como razões funcionalmente importantes de RMLX. Atualmente, cerca de 5-10% dos casos de RM em homens são reconhecidos por ocorrerem devido a estas variações do número de cópias no cromossomo X. Neste estudo, investigamos mutações no gene JARID1C, através do rastreamento dos éxons 9, 11, 12, 13, 15 e 16, em 121 homens de famílias com RM provavelmente ligado ao X. Paralelamente, realizamos a análise da variação do número de cópias em 16 genes localizados no cromossomo X através da técnica de MLPA no mesmo grupo de pacientes. Esta metodologia consiste em uma amplificação múltipla que detecta variações no número de cópias de até 50 sequências diferentes de DNA genômico, sendo capaz de distinguir sequências que diferem em apenas um nucleotídeo. O DNA genômico foi extraído a partir de sangue periférico e as amostras foram amplificadas pela técnica de PCR, seguida da análise por sequenciamento direto. Foram identificadas três variantes na sequência do gene JARID1C entre os pacientes analisados: a variante intrônica 2243+11 G>T, que esteve presente em 67% dos pacientes, a variante silenciosa c.1794C>G e a mutação inédita nonsense c.2172C>A, ambas presentes em 0,82% dos indivíduos investigados. A análise através do MLPA revelou uma duplicação em um dos pacientes envolvendo as sondas referentes ao gene GDI1 e ao gene HUWE1. Este trabalho expande o estudo de mutações no gene JARID1C para a população brasileira ereforça a importância da triagem de mutações neste gene em homens portadores de RM familiar de origem idiopática, assim como, é primeiro relato científico relativo à investigação de variações no número de cópias de genes localizados no cromossomo X em homens brasileiros com RM, através da técnica de MLPA. / Mental retardation (MR) is defined as a disability characterized by significant below average intellectual functioning (IQ>70). The prevalence of MR varies between epidemiological studies, estimated at 2-3% of the population, thus constituting a major public health problem. There is a general consensus that MR is more common in males, a finding attributed, in part, to mutations in the genes located on the X chromosome, leading to an X-linked mental retardation (XLMR). Among all the genes present on X chromosome, Jumonji AT-rich interactive domain IC (JARID1C) was recently identified as aetiologic potential candidate of MR, when mutated. The JARID1C gene encodes a protein that acts as a histone demethylase specific for histone 3 lysine 4 (H3K4) and it is indispensable for the epigenetic regulation. As recently as the identification of the JARID1C gene, it is the discovery that changes in the number of copies of DNA sequences, characterized by microdeletions and microduplications, could be regarded as functionally important reasons to XLMR. Currently, about 5-10% of men MR cases are known to occur due to these variations in the number of copies of chromosome X. In this study we investigated mutations in the JARID1C gene by screening of exons 9, 11, 12, 13, 15 and 16 in 121 patients from families with X-linked MR. At the same time we analyzed the variation in the number of copies in 16 genes located in X chromosome through the MLPA technique. This metodology consists of a multiplex amplification that detects variations in the number of copies up to 50 different genomic DNA sequences, being able to distinguish sequences that differ by only one nucleotide. Genomic DNA was extracted from peripheral blood and the samples were amplified by PCR followed by direct sequencing analysis. We identified three sequence variants among 121 patients. The intronic variant c.2243 +11 G> T, which was present in 67% of patients analyzed, the silent variant c.1794C> G and the novel nonsense mutation c.2172C> A, which was present in 0,82% of patients analyzed. The MLPA analysis revealed that the patient 58 exhibited a duplication involving probes for the GDI1 gene and the HUWE1 gene, resulting in an increase in the number of copies of this gene. This work expands the study of mutations in the JARID1C gene for the Brazilian population and reinforces the importance of screening for mutations in this gene in men with idiopathic mental retardation, and it is the first scientific report on the investigation of variations in the number of copies in genes located on chromosome X in Brazilian men with MR using the MLPA technique.