Proefschrift Universiteit Maastricht. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

γ-aminobutyric acid is the major inhibitory neurotransmitter in the adult mammalian central nervous system (CNS) and may also play a neurotrophic role during CNS development. Diversification of GABA_A receptor mediated responses are in part a result ofvariation in subunit composition in the receptor complex. This variation arises both from the number of different subtypes of GABA_A receptor subunits (α1-6, β1-4, γ1-3, δ1, ρ1-3, ε, ρ), as well as from post-transcriptional processes such as RNA splicing. In this thesis, I have investigated the developmental onset of GABA_A receptor gene expression and the distribution and temporal expression of GABA_A receptor subunit mRNAs and 12 splice variants within the developing and adult murine CNS. Preliminary studies using S 1 nuclease protection analysis demonstrated that α1, β3 and γ2 were the predominant subtypes of GABA_A receptor subunits expressed at embryonic day 14 and in the adult murine CNS. In situ hybridisation analysis demonstrated overlapping but distinct spatial and temporal patterns of GABA_A subunit mRNA expression during postnatal development and in the adult murine CNS. Analysis of γ2 mRNA splice variants demonstrated that the γ2S transcript is the predominant γ2 mRNA expressed during latter stages of embryo genesis, while the γ2L transcript is the predominant γ2 isoform present inthe adult CNS. Since there is a 29 to 47 percent amino acid identity among the various GABA_A receptor subunits, I have also demonstrated through site-directed mutagenesis studies, that changes in a conserved amino acid in the cysteine loop of the bovine a 1 GABA_A receptor subunit resulted in a loss of agonist and antagonist binding (DI49N), while a change in a conserved amino acid in the M1 transmembrane domain of the bovine α1 GABA_A receptor subunit resulted in loss of agonist binding and reduction in the B_max and K_d for antagonist binding (P243A). 'These results are in contrast to the effect of identical mutations in the bovine β1 subunit and suggest that if the pentameric GABA_A receptor assembly is composed of (α1)2(β1)1(γ2)2, then changes in highly conserved amino acids in the α1 receptor subunit would have a greater distortion on the structure of the receptor complex.

Orientadores: Pablo Agustin Vargas, Lynne Bingle / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-13T01:09:27Z (GMT). No. of bitstreams: 1 Silva_AndreiaAparecidada_D.pdf: 22491420 bytes, checksum: 91def4321f81cbc12963ed48ee86d77d (MD5) Previous issue date: 2009 / Resumo: Introdução: Inúmeras lesões de origem infecciosa, cística, neoplásica e inflamatória foram reportadas nas glândulas salivares de pacientes HIV+. Objetivos: Os objetivos deste trabalho foram analisar e comparar a resposta do sistema imune inato (proteínas da família PLUNC) em glândulas salivares maiores, provenientes de pacientes autopsiados com AIDS e sem AIDS (grupo controle) no Departamento de Patologia da Faculdade de Medicina da Universidade Estadual de São Paulo (FMUSP) no período de 1996 a 2000. Material e Métodos: Os pacientes autopsiados foram divididos em 05 grupos: grupo 01- controle (pacientes HIV negativos), grupo 02- HIV+ sem alterações nas glândulas salivares maiores, grupo 03- (micobacteriose), grupo 04 (citomegalovirose) e grupo 05 (sialadenite) para a realização de reações de imunohistoquímica para os anticorpos SPLUNC 1, SPLUNC 2 A, SPLUNC 2B e LPLUNC 1. Para o grupo controle foi realizado técnica de hibridização in situ para SPLUNC 2. Resultados: a média de idade dos pacientes selecionados para o grupo controle foi de 60,92 anos + 9,48 anos enquanto que a média de idade dos pacientes HIV positivos foi de 37,75 anos + 11,11. Nos casos de micobacteriose e citomegalovirose foi observada maior intensidade de marcação nas regiões próxima a área de infecção, quando comparada com áreas na periferia da lesão para os anticorpos SPLUNC 2 A e 2B. O anticorpo LPLUNC 1 foi positivo apenas nos ductos salivares e apresentou positividade em 42,22%, 51,06% e 63,88% para as glândulas parótida, submandibular e sublingual respectivamente. Com relação à hibridização in situ, foi observado positividade em todos os casos. Conclusão: a família de proteínas PLUNC pode ter papel fundamental na proteção dos organismos frente a agentes infecciosos, no entanto são necessários maiores estudos. / Abstract: Objectives: The aim of this study was to determine the expression of PLUNC proteins in major salivary glands (MSG) of AIDS patients with or without infectious conditions and non-HIV patients using post-mortem material. Methods: Sex, age, CD4 cell count, and clinical history were obtained retrospectively from the clinical records of all patients (n=63). We analysed the expression of PLUNCs (SPLUNC1, SPLUNC2, LPLUNC1) using immunohistochemistry in parotid (n=45), submandibular (n=47) and sublingual gland (n=37) samples of AIDS patients [30 with normal histology, 21 with mycobacteriosis, 14 with cytomegalovirus (CMV) infection, 30 with chronic nonspecific sialadenitis, and 30 HIV-negative controls. In situ hybridization (ISH) for SPLUNC2 in the MSG of the HIVnegative group was performed. Immunoreactivity was assessed as positive or negative. Results: The mean age of the patients who died of AIDS (n=63) and CD4 cell count (n=44) were 37 years and 63 cells microL(-1), respectively. The mean age of the HIV negative patients was 61 years. SPLUNC 1 expression was detected in the mucous acini of submandibular and sublingual glands, and SPLUNC2 was seen in the serous cells of the MSG. LPLUNC 1 expression was only positive in the salivary ducts of the MSG. There was a higher expression of SPLUNC2 in AIDS patients with CMV infection and mycobacteriosis when compared with all other groups. The intensity of staining for SPLUNC2 was greater around the lesions than the peripheral ones. There were no significant differences between control subjects and AIDS patients without histologic alterations or with chronic nonspecific sialadenitis. ISH for SPLUNC 2 showed perinuclear positivity in the serous cells in all HIV-negative cases. Conclusions: SPLUNC1 and LPLUNC1 proteins were similarly expressed in the MSG of AIDS patients and non-HIV patients. CMV infection and mycobacteriosis increase SPLUNC2 expression in serous cells in the MSG of AIDS patients. Further studies are needed to understand the biological processes involved in SPLUNC2 expression in the MSG infected by CMV and mycobacteriosis. / Doutorado / Patologia / Doutor em Estomatopatologia

Natural killer (NK) cells, lymphocytes of the innate immune system, can recognize a wide array of cells via several receptors families such as Ly49 and NKR-P1. The Nkrp1 gene family encode for C-type lectin-like receptors which can recognize their ligands, Clr, on target cells. Nkrp1 and Clr genes are intertwined in the NK gene complex and are thus inherited together. The Nkrp1-Clr genes in 129S6 and BALB/c mouse strains show significant sequence polymorphism compared to those of C57BL/6 mice while the overall gene organization and gene number are conserved. RT-PCR was utilized to study the expression of individual Nkrp1-Clr genes. In situ hybridization was performed to validate expression results from RT-PCR, as well as to verify the cell types in which Nkrp1-Clr genes are expressed. Surprisingly, our expression studies reveal an interesting pattern of expression of Nkrp1 and Clr genes not only in lymphoid tissues but also in the epithelial cells of the intestine, kidney, eye and lung, the myocytes of the heart and skeletal muscle, and possibly some endothelial cells, indicating novel functions of NK cells in these tissues.

In this study, hypervariable region I (HVRI) of the mitochondrial DNA (mtDNA) control region, and five microsatellite loci were used to assess genetic variability and the extent of hybridization between the two fur seals (Arctocephalus tropicalis and A. gazella), that occur on Sub-Antarctic Marion Island. Both species were harvested during the 18th and 19th centuries, leading to a reduction in population size and the extinction of A. gazella at some localities. Whilst both species have recovered and are increasing in size, it is not clear to what extent sealing has affected genetic variation, although a more pronounced effect would be expected for A. gazella, given the more intensive harvesting of this species. The current study confirmed this hypothesis and revealed that A. gazella had a nucleotide diversity of 2.9 % whilst for A. tropicalis it was 4.2 %, across the HRVI mtDNA region sequenced. For microsatellite DNA, genetic variation in A. tropicalis was higher than in A. gazella in terms of the total number of alleles detected and the level of heterozygosity (HE=0.875, HO=0.845, mean number of alleles=13.6 and HE=0.799, HO=0.781, mean number of alleles=13, respectively). Diversity in both species is among the highest recorded in pinnipeds to date, and suggests that sealing did not overly affect the levels of genetic variation in these species. In terms of population structure, A. tropicalis show a high level of population structure, as indicated by the ΦST of 0.32 between Marion and Gough Island. Furthermore, the A. tropicalis haplotype tree comprising individuals from Marion, Iles Crozet, Gough, and Amsterdam islands, recovered three divergent evolutionary lineages with bootstrap values of 86% and 98%, for two of these lineages, indicating strong genetic structure and independent evolution. Shared haplotypes between Marion and other islands confirmed genetic exchanges, whilst the grouping of Marion and Gough Islands together is indicative of regular migration between these two islands. For A. gazella, the haplotype tree recovered numerous instances of grouping of individuals from Marion and Bouvetøya Islands confirming the hypothesis Bouvetøya is likely source of immigrants to Marion Island. This was further confirmed by low population differentiation between these two islands (FST = 0.062 and ΦST of 0.08). The level of hybridization between these species was low at Marion Island with only one hybrid being detected among the 134 animals for which mtDNA data were generated, corresponding to 0.75%. The same individual was identified as a hybrid, following microsatellite profiling of 146 animals, corresponding to a hybridization estimate of 0.68 %. This hybrid individual was classified phenotypically as A. gazella and genotypically was shown to have A. tropicalis ancestry. This level of hybridization is low compared to the other islands where the two species co-occur. However as the samples used in this study were primarily collected from species-specific sites, this may be an underestimate, and the studies focusing on sites where they are known to occur symaptrically, may yield higher estimates. / Dissertation (MSc)--University of Pretoria, 2009. / Zoology and Entomology / unrestricted

Over one billion people, more than 1/9th of the global population, are undernourished. Feeding the ever increasing population has to be the most important goal of plant sciences. Since cultivated areas are not likely to increase, I will need to produce more with what is available. This can be summarized in one word: yield. Unfortunately, wheat’s yield is expected to increase only 1.13% by 2019, a prediction that if converted into reality will likely indicate that I failed to cope with the world demographic increase. A new strategy to revolutionize wheat production is required, and some believe that this change might be represented by wheat hybrids. Achieving adequate commercial production of wheat hybrids has the potential to nearly double the yield of one of the world’s most important staple food. The first fundamental step toward this goal is to develop feasible methodologies to sterilize the male part of the complete wheat flowers. Two fertility-related genes are the primary target of this study, namely the species cytoplasm specific on chromosome 1D, and the desynaptic locus on chromosome 3B. This dissertation summarizes the important achievements obtained toward the cloning of the two loci by means of radiation hybrid functional analysis. Radiation hybrid is a technique that employs radiation to create genetic diversity along the targeted chromosome. Chapter 1 explains in details how this methodology can be applied to plants. The use of radiation hybrid mapping permitted creating a comprehensive map of wheat chromosome 3B, as discussed in Chapter 2, and then expanded the mapping information to identify the 2 Mb location of the desynaptic locus desw2, as discussed in Chapter 3. A similar approach on chromosome 1D allowed first to pinpoint the location of the species cytoplasm specific gene to a region of 2 Mb, as discussed in Chapter 4, and then ultimately to find a strong candidate for this locus, as discussed in Chapter 5. Now that the molecular locations of these genes have been unraveled by this study, their sequence can be streamlined into transformation to ultimately produce female wheat plants, and consequently hybrids.

Natural hybridization is reproduction (without artificial influence) between two or more species/populations which are distinguishable from each other by heritable characters. Natural hybridizations among marine fishes were highly underappreciated due to limited research effort; it seems that this phenomenon occurs more often than is commonly recognized. As hybridization plays an important role in biodiversity processes in the marine environment, detecting hybridization events and investigating hybridization is important to understand and protect biodiversity. The first chapter sets the framework for this disseration study. The Cohesion Species Concept was selected as the working definition of a species for this study as it can handle marine fish hybridization events. The concept does not require restrictive species boundaries. A general history and background of natural hybridization in marine fishes is reviewed during in chapter as well. Four marine fish hybridization cases were examed and documented in Chapters 2 to 5. In each case study, at least one diagnostic nuclear marker, screened from among ~14 candidate markers, was found to discriminate the putative hybridizing parent species. To further investigate genetic evidence to support the hybrid status for each hybrid offspring in each case, haploweb analysis on diagnostic markers (nuclear and/or mitochondrial) and the DAPC/PCA analysis on microsatellite data were used. By combining the genetic evidences, morphological traits, and ecological observations together, the potential reasons that triggered each hybridization events and the potential genetic/ecology effects could be discussed. In the last chapter, sequences from 82 pairs of hybridizing parents species (for which COI barcoding sequences were available either on GenBank or in our lab) were collected. By comparing the COI fragment p-distance between each hybridizing parent species, some general questions about marine fish hybridization were discussed: Is there any correlation between genetic similarity and the potential for hybridization in marine fishes? In some particular geographic locations that have the existence of several different hybridization reports, are the species involved in hybridization among those reports all closely related or distantly related? Can any associations between parent species’ similarities and hybrid spots be found?

Large populations of controlled hybrids are essential to the most rapid progress in many phases of plant breeding programs. Plant species vary in the ease with which they may be hybridized. Hand hybridization of forage grasses is usually slow and laborious, and the minuteness of the floral parts of most of the species which have perfect flowers renders their hybridization by hand especially difficult and tedious. This difficulty limits the use that forage grass breeders are making of the significant principles of hybridization and thereby retards progress in this phase of plant breeding. Any dependable, rapid technique of hybridization which would eliminate many of the present hand operations, that is "bulk" hybridization, would make possible more rapid progress in the breeding of forage grasses. The study herein reported was designed to estimate the feasibility of applying various methods of bulk emasculation and bulk pollination to forage grasses. In limiting the scope of the study, smooth bromegrass (Bromus inermis) was selected to receive the greatest attention because of its importance among forage grasses and the wide range of self-fertility among individual plants of the species, a characteristic which proved very helpful in the study.

Order Acipenseriformes (sturgeons and paddlefishes) is an ancient lineage of bony fishes (> 200 million years old) with most extant species at conservation risk. The pallid sturgeon, Scaphirhynchus albus, is a federally endangered species native to the Mississippi and Missouri River basins. Hybridization with sympatric shovelnose sturgeon, S. platorynchus, is one of several threats to pallid sturgeon. Current molecular markers cannot reliably distinguish among pure species and multigenerational backcrosses. This information is critical for implementation of management strategies to increase populations through natural reproduction and artificial propagation. Genotypes from a large panel of unlinked single-nucleotide polymorphisms (SNPs) may provide greater resolution of the two species; however, paralogous sequence variants (PSVs) within individuals resulting from an ancient whole genome duplication event confound SNP development. The aim of this dissertation was to produce unlinked disomic SNPs that would increases resolution between pallid sturgeon and shovelnose sturgeon. This was achieved by producing haploid gynogens, which contain only DNA from the maternal parent, and then producing a reference from these haploid gynogens. Sequence assembly based on haploids informed the presence of multi-locus contigs. More than 11,000 disomic SNP markers were produced that differentiate between the two species by mapping 120 individuals of either species onto the haploid reference. A linkage map, based on three haploid families, was able to resolve some paralogs and can be used to inform which discriminatory SNP markers are linked. Future research should convert the disomic markers derived in this study to an applied form, such as that achieved by genotyping-by-thousands.

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