Barley (Hordeum vulgare L. subsp. vulgare) is an important crop and ranks fourth in overall production of the major cereal crops in the world. Like other cereal crops, barley suffers from a narrowing of its genetic base and susceptibility to diseases, pests and environmental stresses. H. bulbosum is a possible source of desirable genes for introgressing into barley to restore genetic diversity and improve current cultivars. Sexual hybridisation between barley and H. bulbosum is the main method for interspecific gene transfer in barley breeding but there are several barriers to overcome. Two of these are reduced recombination and the ability to identify recombinants quickly and efficiently. The aim in this thesis was to gain a better understanding of meiotic chromosomal behaviour in the two species and their hybrids and to improve the characterisation of recombinants from the hybrids. To study the events during meiosis, synaptonemal complex (SC) analysis was carried out on the two species and two H. vulgare - H. bulbosum hybrids. The results indicated that there were interspecific and intraspecific variations in SC length. Mean SC length was positively correlated with recombination frequency but not related to genome size. This suggests that the ratios of mean SC length to genome size (SC/DNA) show divergence among these Hordeum examples. An hypothesis based on the conformation of chromatin associated with axial element, which is dependent on SC/DNA ratio, was presented to explain the relationship between SC length and recombination frequency. Chromosome pairing in the two hybrids was determined by observation at pachytene and metaphase I (MI). Mean percentages of synapses were similar but there were different frequencies of MI pairing between these two hybrids, indicating that different mechanisms may regulate synapsis and MI pairing in the hybrids. To investigate meiotic recombination, genomic in situ hybridisation (GISH) was performed on the two hybrids at MI and anaphase I (AI). It was observed that intergenomic pairing and recombination events occur in distal chromosome segments. A great discrepancy between mean pairing and recombination frequencies was observed in both hybrids and several possible reasons for this discrepancy were discussed. Hybrid 102C2 with high MI pairing had a significantly higher recombination frequency than the low pairing 103K5, suggesting that high MI pairing appears to be associated with high recombination in the hybrids. An interesting finding is that the ratio of recombination to MI pairing in 103K5 (l:8.9) is twice as high compared with 102C2 (l:17). However, the mechanism for this difference in the ratio between the two hybrids remains unknown. Sequential fluorescence in situ hybridisation (FISH) and GISH were used successfully to localise the introgressions in selfed progeny from a tetraploid hybrid derived from chromosome-doubled 102C2 (102C2/colch). This procedure is fast, cheap and can efficiently detect and locate introgressions. Several disease-resistant recombinants were analysed in more details and leaf rust and powdery mildew resistance was associated with distal introgressions on chromosomes 2HS and 2HL (leaf rust) and 2HS (powdery mildew). It is possible that the leaf rust and powdery mildew resistances were closely linked in the distal region of 2HS. A considerable variation in introgression size was observed at similar chromosomal sites among the different recombinants, which will provide useful information for map-based cloning of genes.

Barley (Hordeum vulgare L. subsp. vulgare) is an important crop and ranks fourth in overall production of the major cereal crops in the world. Like other cereal crops, barley suffers from a narrowing of its genetic base and susceptibility to diseases, pests and environmental stresses. H. bulbosum is a possible source of desirable genes for introgressing into barley to restore genetic diversity and improve current cultivars. Sexual hybridisation between barley and H. bulbosum is the main method for interspecific gene transfer in barley breeding but there are several barriers to overcome. Two of these are reduced recombination and the ability to identify recombinants quickly and efficiently. The aim in this thesis was to gain a better understanding of meiotic chromosomal behaviour in the two species and their hybrids and to improve the characterisation of recombinants from the hybrids. To study the events during meiosis, synaptonemal complex (SC) analysis was carried out on the two species and two H. vulgare - H. bulbosum hybrids. The results indicated that there were interspecific and intraspecific variations in SC length. Mean SC length was positively correlated with recombination frequency but not related to genome size. This suggests that the ratios of mean SC length to genome size (SC/DNA) show divergence among these Hordeum examples. An hypothesis based on the conformation of chromatin associated with axial element, which is dependent on SC/DNA ratio, was presented to explain the relationship between SC length and recombination frequency. Chromosome pairing in the two hybrids was determined by observation at pachytene and metaphase I (MI). Mean percentages of synapses were similar but there were different frequencies of MI pairing between these two hybrids, indicating that different mechanisms may regulate synapsis and MI pairing in the hybrids. To investigate meiotic recombination, genomic in situ hybridisation (GISH) was performed on the two hybrids at MI and anaphase I (AI). It was observed that intergenomic pairing and recombination events occur in distal chromosome segments. A great discrepancy between mean pairing and recombination frequencies was observed in both hybrids and several possible reasons for this discrepancy were discussed. Hybrid 102C2 with high MI pairing had a significantly higher recombination frequency than the low pairing 103K5, suggesting that high MI pairing appears to be associated with high recombination in the hybrids. An interesting finding is that the ratio of recombination to MI pairing in 103K5 (l:8.9) is twice as high compared with 102C2 (l:17). However, the mechanism for this difference in the ratio between the two hybrids remains unknown. Sequential fluorescence in situ hybridisation (FISH) and GISH were used successfully to localise the introgressions in selfed progeny from a tetraploid hybrid derived from chromosome-doubled 102C2 (102C2/colch). This procedure is fast, cheap and can efficiently detect and locate introgressions. Several disease-resistant recombinants were analysed in more details and leaf rust and powdery mildew resistance was associated with distal introgressions on chromosomes 2HS and 2HL (leaf rust) and 2HS (powdery mildew). It is possible that the leaf rust and powdery mildew resistances were closely linked in the distal region of 2HS. A considerable variation in introgression size was observed at similar chromosomal sites among the different recombinants, which will provide useful information for map-based cloning of genes.

PhD / HIV-1 is the cause of the majority of global HIV infections. Not only being more virulent, and relatively easily transmitted than HIV-2, HIV-1 is also more extensively studied. HIV-1 is known for its highly recombinogenic nature, together with an extreme genetic variety, both attributable to an error-prone reverse transcriptase which gives rise to heterozygous virion. Sequence diversity of HIV-1 has resulted in identification of 9 subtypes of HIV-1 M group, as well as 43 circulating and a number of other unique recombinant forms of HIV-1. The extensive heterogeneity of HIV-1 has become the main consideration in vaccine development, mainly due to the inherent variability of HIV-1 and the frequent generation of new recombinant forms, which subsequently makes the effort to control the HIV-1 pandemic more challenging. The inter-subtype recombination event is a common phenomenon observed in Malaysia whereby there is a co-circulation of multiple HIV-1 subtypes; CRF01_AE and subtype B. Therefore, it becomes crucial to widen the knowledge of currently emerging CRF01_AE/B inter-subtype recombinants, in order to assist the future regional vaccine design and also to prevent wider spread of these strains. Concurrently, with a better understanding on the characteristics of HIV-1 CRF01_AE/B recombinant forms, further diversification of these strains can possibly be thwarted. The objectives of this study included, firstly to study the molecular epidemiology pattern of different HIV-1 strains, as well as to observe their frequency and distribution. Our second aim was to identify possible derivative from CRF33_01B, and also other new CRF01_AE/B inter-subtype recombinant forms in Malaysia. Thirdly, we aimed to identify possible biological advantages of the CRF33_01B isolates over its parental strains; CRF01_AE and subtype B. Currently, the HIV-1 epidemic in Malaysia is in a concentrated phase with evidence of predominance of both CRF01_AE and subtype B found among heterosexuals and injecting drug users, respectively. There is urgent necessity to apply a more detailed and continuous molecular characterization and epidemiological monitoring of these recombinant forms in Malaysia. We obtained plasma samples from 115 HIV-1-infected patients who attended HIV clinic at the University Malaya Medical Centre in Kuala Lumpur, Malaysia. The HIV-1 PR-RT, gp120-env and gp41-env genes were amplified and sequenced from 50 samples, while the remaining 65 samples were successfully studied at either one or two HIV-1 specific genomic regions. Cloning, phylogenetic analyses, together with bootscanning methods were employed to assign subtypes and to identify inter-subtype recombination based on all three genomic regions. From the plasma-derived sequences of 50 patients, 46% were found to harbour CRF01_AE, 10% and 6% had subtype B and B’, and a total of 18% of the patients were infected with CRF33_01B, while the remaining 18% of patients was found to have unique recombinant forms. As for the other 65 patients, majority of them harboured CRF01_AE and subtype B. This study shows that co-circulation of multiple HIV-1 subtypes and their recombinant strains are frequent in the Malaysian population, while capable of spreading to different HIV-1 risk groups. Possible recombination hotspots in CRF01_AE/B recombinants are suggested to be within the HIV-1 PR-RT gene region. Further, this study highlights the need to characterize and monitor the molecular epidemiology of these recombinant forms. The ideal environment for the inter-subtype recombination event to take place is created by the co-circulation and dual infections of both CRF01_AE and subtype B. With more HIV-1 CRF01_AE/B recombinant forms emerging and shaping the nature of HIV epidemic in Malaysia, certainly it will complicate the timely diagnosis of these molecularly altered HIV-1 forms. The recent identification of the novel CRF33_01B suggests the emergence of other new CRF01_AE/B inter-subtype recombinant forms in Malaysia, as preliminarily demonstrated in some HIV-1 patients identified in the first part of this study. The peripheral blood mononuclear cells (PBMCs) of these HIV-1 patients were co-cultured with those of healthy donors, which we then isolated the proviral genomic DNA. The nested long-range PCR was performed to obtain seven overlapping viral genome fragments that made up the whole viral genome. The detailed phylogenetic, as well as bootscan analyses confirmed the mosaic compositions and recombinant structures of the newly emerging CRF01_AE/B recombinant forms derived from CRF01_AE and subtype B. One of them in particular; HIV-1 isolate 06MYKLD46 is structurally similar to CRF33_01B, except for an extra subtype B fragment within the env region. It also has close phylogenetic relationship and similar breakpoints with CRF33_01B, mainly at the PR-RT region. Furthermore, the other three distinct HIV-1 recombinants; isolates 07MYKLD47, 07MYKLD48 and 07MYKLD49 also display near full-length genomes composed of the backbone of CRF01_AE, with insertions of subtype B fragments at different gene regions. These results indicate the high possibility of second generation of minor recombinant forms derived from CRF33_01B, as well as the continuous evolution and rapid dispersal of CRF01_AE/B recombinants in Malaysia. The high prevalence of newly emerging CRF33_01B (CRF01_AE/B inter-subtype recombinant) may cause a possible epidemiologic shift, attributable to its altered virologic characteristics and possible transmission advantages compared to its parental strains. Two major determinants; the viral factor and host factor have influenced the progress of a productive HIV-1 infection upon virus entry into the host cells. We have assessed the two main viral factors; the in vitro viral replication capacity and the viral fitness of the circulating HIV-1 strains in Malaysia. We have determined that CRF33_01B primary isolate (07MYKLVik) replicates better in activated whole PBMCs and CD4+ T-lymphocytes and is ‘fitter’ than one of its parental strain; CRF01_AE (07MYKLNBL) but not subtype B (07MYKLAfik). Subtype B has more advanced ability to produce a progressive infection in all cell types, including MDMs, and has a comparable viral fitness to that of CRF33_01B. We also investigated the role of host factors in a productive HIV-1 infection, by determining the viral effect on the host cell morphological features. We found that CRF33_01B (07MYKLVik) culture displayed more large syncytia (multinucleated giant cells) with multiple nuclei compared to subtype B (07MYKLAfik) culture, while no snycytia was observed in CRF01_AE (07MYKLNBL) culture. Generally, the cells within CRF33_01B and subtype B cultures appeared to be morphologically distinct from CRF01_AE cultures. This may indicate a more productive HIV-1 infection of CRF33_01B and subtype B, similar to our finding from the in vitro viral replicative capacity and viral fitness assays of these HIV-1 strains. We also studied the effect of different HIV-1 strain infections on host differential gene expression profiles, by using the PCR Array, which detects a total of 84 genes known to be involved in the host response to HIV-1 infection. It was observed that the in vitro infection with CRF33_01B isolates resulted in a more damaging effect on host cells and caused more apoptotic death within the infected cultures, compared to the isolates of its parental subtypes. Moreover, subtype B isolates resulted in a poorer cell response upon viral infection, compared to CRF01_AE/B isolate. Concurrently, it also gave less productive spread of viral infection within the infected cultures, in comparison to CRF01_AE/B isolate. We speculate that if the same scenario is reflected in vivo, CRF01_AE/B inter-subtype recombinant including CRF33_01B would have a better survival rate within the host upon their infection, in comparison to their parental strains. This again strengthens our presumption that CRF33_01B has potential ability to disseminate widely in the Malaysian population and gives a progressive change of the current molecular epidemiological trend by gradually replacing the current predominance of CRF01_AE in the country.

PhD / HIV-1 is the cause of the majority of global HIV infections. Not only being more virulent, and relatively easily transmitted than HIV-2, HIV-1 is also more extensively studied. HIV-1 is known for its highly recombinogenic nature, together with an extreme genetic variety, both attributable to an error-prone reverse transcriptase which gives rise to heterozygous virion. Sequence diversity of HIV-1 has resulted in identification of 9 subtypes of HIV-1 M group, as well as 43 circulating and a number of other unique recombinant forms of HIV-1. The extensive heterogeneity of HIV-1 has become the main consideration in vaccine development, mainly due to the inherent variability of HIV-1 and the frequent generation of new recombinant forms, which subsequently makes the effort to control the HIV-1 pandemic more challenging. The inter-subtype recombination event is a common phenomenon observed in Malaysia whereby there is a co-circulation of multiple HIV-1 subtypes; CRF01_AE and subtype B. Therefore, it becomes crucial to widen the knowledge of currently emerging CRF01_AE/B inter-subtype recombinants, in order to assist the future regional vaccine design and also to prevent wider spread of these strains. Concurrently, with a better understanding on the characteristics of HIV-1 CRF01_AE/B recombinant forms, further diversification of these strains can possibly be thwarted. The objectives of this study included, firstly to study the molecular epidemiology pattern of different HIV-1 strains, as well as to observe their frequency and distribution. Our second aim was to identify possible derivative from CRF33_01B, and also other new CRF01_AE/B inter-subtype recombinant forms in Malaysia. Thirdly, we aimed to identify possible biological advantages of the CRF33_01B isolates over its parental strains; CRF01_AE and subtype B. Currently, the HIV-1 epidemic in Malaysia is in a concentrated phase with evidence of predominance of both CRF01_AE and subtype B found among heterosexuals and injecting drug users, respectively. There is urgent necessity to apply a more detailed and continuous molecular characterization and epidemiological monitoring of these recombinant forms in Malaysia. We obtained plasma samples from 115 HIV-1-infected patients who attended HIV clinic at the University Malaya Medical Centre in Kuala Lumpur, Malaysia. The HIV-1 PR-RT, gp120-env and gp41-env genes were amplified and sequenced from 50 samples, while the remaining 65 samples were successfully studied at either one or two HIV-1 specific genomic regions. Cloning, phylogenetic analyses, together with bootscanning methods were employed to assign subtypes and to identify inter-subtype recombination based on all three genomic regions. From the plasma-derived sequences of 50 patients, 46% were found to harbour CRF01_AE, 10% and 6% had subtype B and B’, and a total of 18% of the patients were infected with CRF33_01B, while the remaining 18% of patients was found to have unique recombinant forms. As for the other 65 patients, majority of them harboured CRF01_AE and subtype B. This study shows that co-circulation of multiple HIV-1 subtypes and their recombinant strains are frequent in the Malaysian population, while capable of spreading to different HIV-1 risk groups. Possible recombination hotspots in CRF01_AE/B recombinants are suggested to be within the HIV-1 PR-RT gene region. Further, this study highlights the need to characterize and monitor the molecular epidemiology of these recombinant forms. The ideal environment for the inter-subtype recombination event to take place is created by the co-circulation and dual infections of both CRF01_AE and subtype B. With more HIV-1 CRF01_AE/B recombinant forms emerging and shaping the nature of HIV epidemic in Malaysia, certainly it will complicate the timely diagnosis of these molecularly altered HIV-1 forms. The recent identification of the novel CRF33_01B suggests the emergence of other new CRF01_AE/B inter-subtype recombinant forms in Malaysia, as preliminarily demonstrated in some HIV-1 patients identified in the first part of this study. The peripheral blood mononuclear cells (PBMCs) of these HIV-1 patients were co-cultured with those of healthy donors, which we then isolated the proviral genomic DNA. The nested long-range PCR was performed to obtain seven overlapping viral genome fragments that made up the whole viral genome. The detailed phylogenetic, as well as bootscan analyses confirmed the mosaic compositions and recombinant structures of the newly emerging CRF01_AE/B recombinant forms derived from CRF01_AE and subtype B. One of them in particular; HIV-1 isolate 06MYKLD46 is structurally similar to CRF33_01B, except for an extra subtype B fragment within the env region. It also has close phylogenetic relationship and similar breakpoints with CRF33_01B, mainly at the PR-RT region. Furthermore, the other three distinct HIV-1 recombinants; isolates 07MYKLD47, 07MYKLD48 and 07MYKLD49 also display near full-length genomes composed of the backbone of CRF01_AE, with insertions of subtype B fragments at different gene regions. These results indicate the high possibility of second generation of minor recombinant forms derived from CRF33_01B, as well as the continuous evolution and rapid dispersal of CRF01_AE/B recombinants in Malaysia. The high prevalence of newly emerging CRF33_01B (CRF01_AE/B inter-subtype recombinant) may cause a possible epidemiologic shift, attributable to its altered virologic characteristics and possible transmission advantages compared to its parental strains. Two major determinants; the viral factor and host factor have influenced the progress of a productive HIV-1 infection upon virus entry into the host cells. We have assessed the two main viral factors; the in vitro viral replication capacity and the viral fitness of the circulating HIV-1 strains in Malaysia. We have determined that CRF33_01B primary isolate (07MYKLVik) replicates better in activated whole PBMCs and CD4+ T-lymphocytes and is ‘fitter’ than one of its parental strain; CRF01_AE (07MYKLNBL) but not subtype B (07MYKLAfik). Subtype B has more advanced ability to produce a progressive infection in all cell types, including MDMs, and has a comparable viral fitness to that of CRF33_01B. We also investigated the role of host factors in a productive HIV-1 infection, by determining the viral effect on the host cell morphological features. We found that CRF33_01B (07MYKLVik) culture displayed more large syncytia (multinucleated giant cells) with multiple nuclei compared to subtype B (07MYKLAfik) culture, while no snycytia was observed in CRF01_AE (07MYKLNBL) culture. Generally, the cells within CRF33_01B and subtype B cultures appeared to be morphologically distinct from CRF01_AE cultures. This may indicate a more productive HIV-1 infection of CRF33_01B and subtype B, similar to our finding from the in vitro viral replicative capacity and viral fitness assays of these HIV-1 strains. We also studied the effect of different HIV-1 strain infections on host differential gene expression profiles, by using the PCR Array, which detects a total of 84 genes known to be involved in the host response to HIV-1 infection. It was observed that the in vitro infection with CRF33_01B isolates resulted in a more damaging effect on host cells and caused more apoptotic death within the infected cultures, compared to the isolates of its parental subtypes. Moreover, subtype B isolates resulted in a poorer cell response upon viral infection, compared to CRF01_AE/B isolate. Concurrently, it also gave less productive spread of viral infection within the infected cultures, in comparison to CRF01_AE/B isolate. We speculate that if the same scenario is reflected in vivo, CRF01_AE/B inter-subtype recombinant including CRF33_01B would have a better survival rate within the host upon their infection, in comparison to their parental strains. This again strengthens our presumption that CRF33_01B has potential ability to disseminate widely in the Malaysian population and gives a progressive change of the current molecular epidemiological trend by gradually replacing the current predominance of CRF01_AE in the country.

Thesis (M.S.)--University of North Texas, Dec., 2007. / Title from title page display. Includes bibliographical references.

Thesis (Ph. D.)--Stanford University, 1997. / Submitted to the Department of Biochemistry and the Committee on Graduate Studies of Stanford University. Includes bibliographical references.

Thesis (Ph.D.)--University of Western Australia, 2006.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2008. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Cornell University, 1989. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Plant Biology." Includes bibliographical references.