No description available.

The vast majority of valuable agronomic traits are encoded polygenetically. Chloroplast genetic engineering offers an alternate approach to multigene engineering by allowing the insertion of entire pathways in a single transformation event, while being an environmentally friendly approach. Stable integration into the chloroplast genome and transcription of the phaA gene coding for β-ketothiolase was confirmed by Southern and northern blots. Coomassie-stained gel and western blots confirmed hyperexpression of β-ketothiolase in leaves and anthers, with high enzyme activity. The transgenic lines were normal except for the male sterile phenotype, lacking pollen. Scanning electron microscopy revealed a collapsed morphology of the pollen grains. Transgenic lines followed an accelerated anther developmental pattern, affecting their development and maturation, resulting in aberrant tissue patterns. Abnormal thickening of the outer wall, enlarged endothecium and vacuolation, decreased the inner space of the locules, affecting pollen grain and resulted in the irregular shape and collapsed phenotype. Reversibility of the male sterility phenotype was achieved by exposing the plants to continuous illumination, producing viable pollen and copious amounts of seeds. This is the first report of engineered cytoplasmic male sterility and offers a new tool for transgene containment for both nuclear and organelle genomes. Detailed characterization of transcriptional, posttranscriptional and translational processes of heterologous operons expressed via the chloroplast genome is reported here. Northern blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons, revealed that in most cases, only polycistronic mRNA was produced or polycistrons were the most abundant form and that they were not processed into monocistrons. Despite such lack of processing, abundant foreign protein accumulation was detected in these transgenic lines. Interestingly, a stable secondary structure formed from a heterologous bacterial intergenic sequence was recognized and efficiently processed, indicating that the chloroplast posttranscriptional machinery can indeed recognize sequences that are not of chloroplast origin, retaining its prokaryotic ancestral features. Processed and unprocessed heterologous polycistrons were quite stable even in the absence of 3'UTRs and were efficiently translated. Unlike native 5'UTRs, heterologous secondary structures or 5'UTRs showed efficient translational enhancement independent of any cellular control. Finally, we observed abundant read-through transcription in the presence of chloroplast 3'UTRs. Such read-through transcripts were efficiently processed at introns present within native operons. Addressing questions about polycistrons, as well as the sequences required for their processing and transcript stability are essential for future approaches in metabolic engineering. Finally, we have shown phytoremediation of mercury by engineering the mer operon via the chloroplast genome under the regulation of chloroplast native and heterologous 5'UTRs. These transgenenic plants hyperexpress were able to translate MerA and MerB enzymes to levels detectable by coomassie stained gel. The knowledge acquired from these studies offer guidelines for engineering multigene pathways via the chloroplast genome.

No description available.

Malaria is a life-threatening disease caused by Plasmodium parasites that are transmitted through the bites of infected females of a few Anopheles mosquito species. Understanding the chromosome evolution and mechanisms of speciation can shed light on developing novel ecological-friendly vector control techniques. Sibling species of the An. gambiae complex provide an excellent model system for these topics. To understand the mechanisms of speciation, we investigated the cellular basis and phenotypes of hybrid male sterility in species crosses of the An. gambiae complex. By performing inter-species crosses of An. coluzzii/An. gambiae and An. merus lab strains, we found an asymmetric pattern of hybrid male sterility existed in sons from reciprocal interspecies crosses. Compared with pure species, hybrid males from crosses of ♀An. merus  ♂An. gambiae/An. coluzzii were normal in the morphology of male reproductive tracts; however, the testes of which that process the reductional meiotic division failed to produce primary spermatocytes and were accompanied with unpaired and insufficiently condensed chromosomes. As a result, primary spermatocytes undergo a mitosis-like anaphase division, producing nonmotile and malfunctional diploid sperm with two tails. However, individuals can mate with females normally and form the mating plug to induce the female monogamy. In contrast, hybrid males from the opposite crosses manifest severely underdeveloped reproductive tracts and a premeiotic arrest of germline stem cells in the testis, accompanied by a strong suppression of premeiotic and meiotic genes. In addition, hybrid males from this cross suffered from a shorter copulation time and failed to form mating plugs to induce female monogamous behaviors, albeit the expression of male accessory gland specific genes were similar between hybrids and pure species. To figure out chromosome evolution in the An. gambiae complex, we studied the molecular organization of heterochromatin and investigated the spatial organizations of autosomal regions of polytene chromosomes in soma and germline cells. We found that molecular composition of pericentrometric autosome and sex chromosome repetitive DNA differs among sibling species of An. gambiae complex with highly similarity between An. coluzzii and An. arabiensis. In addition, heterochromatin blocks of chromosomes have distinct compositions of satellite DNA sequences. Next, in order to address the relationship between inter-chromosomal (Chr-Chr) contacts and chromosome-nuclear envelope (Chr-NE) attachments during the development of the organism, we conducted microscopic analyses of the 3D organization of polytene chromosome in An. gambiae, An. coluzzii, and An. merus. Our quantitative study on chromosome territories in larval salivary gland cells and adult ovarian nurse cells showed that, compared with autosomal arms, the X chromosome has a significantly smaller volume and occupies more compact territories. The number of Chr-Chr contacts and the percentage of Chr-NE attachment were conserved among the species within the same cell type. Our data also demonstrated that there is a significantly and consistently inverse relationship between the frequencies of Chr–NE and Chr–Chr attachments on autosomes of two cell types in all tested species. / Doctor of Philosophy / Malaria is a life-threatening disease caused by Plasmodium parasites that are transmitted through the bites of infected females of a few Anopheles mosquito species. Despite being treatable and preventable, malaria is estimated to cause large numbers of deaths every year. Since 2015, the malaria elimination program has stalled largely due to increased insecticide resistance. Novel transgenic techniques have a huge potential in reducing malaria transmission more effectively. However, there are large concerns about the potential negative effects of releasing genetically modified mosquitoes, such as a possibility of accidental spread to non-target species with incomplete reproductive barriers and unpredicted ecological damage. Understanding the mechanisms of speciation about how reproductive isolation occurred and developed as well as chromosome evolution can not only empower the development of ecologically friendly vector control techniques but also improve our basic knowledge. To study mechanisms of speciation, we mated males and females from different closely related species in the Anopheles gambiae complex to investigate the fecundity of hybrid generations. Our study identified two different types of reproductive abnormalities leading to hybrid male sterility. Hybrid males from female An. merus and male An. gambiae or An. coluzzii have normal appearing testes and male accessary glands but the testes produce abnormal sperms, which cannot move and have two tails. Hybrid males from female An. gambiae or An. coluzzii and An. merus have severely underdeveloped testes and male accessary glands. The sperm producing process stops unusually very early in their tiny underdeveloped testes. We also investigated chromosome evolution in species of An. gambiae complex. We found that chromosomal parts containing repetitive DNA, the sequence in the genome not producing proteins, evolve rapidly in An. coluzzii, An. arabiensis, An. quadriannulatus, and An. merus. In contrast, chromosome territories of gene rich regions in giant polytene chromosomes from larval salivary gland cells and adult ovarian nurse cells of An. gambiae, An. coluzzii, and An. merus, were relatively conserved within the same cell type among different species. However, the chromosomal 3D distribution pattern is different among various cell types in these species.

Yes / The coexistence of hermaphrodites and female-sterile individuals, or androdioecy, has been documented in only a handful of plants and animals. This study reports its existence in the plant species Cardamine amara (Brassicaceae), in which female-sterile individuals have shorter pistils than seed-producing hermaphrodites. Morphological analysis, in situ manual pollination, microsatellite genotyping and differential gene expression analysis using Arabidopsis microarrays were used to delimit variation between female-sterile individuals and hermaphrodites. Female sterility in C. amara appears to be caused by disrupted ovule development. It was associated with a 2.4- to 2.9-fold increase in clonal propagation. This made the pollen number of female-sterile genets more than double that of hermaphrodite genets, which fulfils a condition of co-existence predicted by simple androdioecy theories. When female-sterile individuals were observed in wild androdioecious populations, their ramet frequencies ranged from 5 to 54 %; however, their genet frequencies ranged from 11 to 29 %, which is consistent with the theoretically predicted upper limit of 50 %. The results suggest that a combination of sexual reproduction and increased asexual proliferation by female-sterile individuals probably explains the invasion and maintenance of female sterility in otherwise hermaphroditic populations. To our knowledge, this is the first report of the coexistence of female sterility and hermaphrodites in the Brassicaceae.

Bakgrund Postoperativa sårinfektioner orsakar stora kostnader för samhället och orsakar patienterna stort lidande. För att förhindra uppkomsten av infektioner behövs förutom operationssjuksköterskans övergripande ansvar för hygien och sterilitet under operation även att operatören har följsamhet till hygienrutinerna för att förhindra att smittspridning sker. Syfte Att undersöka operationssjuksköterskornas upplevelser av operatörernas följsamhet avseende sterilitet och hygien i samband med kirurgiska ingrepp. Samt att identifiera faktorer som enligt operationssjuksköterskorna kan påverka operatörernas följsamhet och förslag på åtgärder. Metod Kvalitativ design, med semistrukturerade intervjuer med åtta operationssjuksköterskor på två sjukhus i norra Sverige. Induktiv innehållsanalys av Lundman & Hällgren Graneheim (2012) användes för analys av data. Resultat Alla operationssjuksköterskor upplevde att operatörerna brast i sin följsamhet och operationssjuksköterskornas uppfattning var att detta dels berodde på kunskapsluckor hos operatörerna men även kunde härledas till dåligt teamsamarbete och bristande ledning. Operationssjuksköterskorna ansåg att stärkande av operatörernas kompetens, ett stärkt samarbete och en tydligare styrning skulle kunna förbättra följsamheten hos operatörerna. Slutsats Inte endast genom att fylla operatörernas kunskapsluckor kan följsamheten till hygienrutiner förbättras utan det är även av betydande vikt att teamsamarbetet förbättras. / Background Postoperative wound infections cause major costs to society and causes patients great suffering. To prevent the occurrence of infections surgeon adherence to hygiene routines, in addition to the operating room nurse overall responsibility for hygiene and sterility during surgery, is needed. Aim To investigate surgical nurses experiences of surgeons adherence regarding sterility and hygiene during surgical procedures. And identify factors that according to surgical nurses can affect surgeons' adherence and proposed actions. Method Qualitative design, with semi-structured interviews with eight surgical nurses at two hospitals in northern Sweden. Inductive content analysis of Lundman & Hällgren Graneheim (2012) was used for analysis of data. Result All surgical nurses experienced surgeons to fail in adherence and the surgical nurses opinion was that this is partly due to knowledge gaps among surgeons, but that it could also be attributed to poor team cooperation and lack of leadership. The surgical nurses felt that strengthening of the surgeon's skills, strengthened cooperation and a more distinct leadership could improve adherence among surgeons. Conclusion Not only by addressing the surgeon's knowledge gaps can adherence to hygiene be improved but it is also of significant importance to improve team collaboration.

No description available.

No processo de fabricação do gerador de 99Mo/99mTc é fundamental, não só evitar a introdução de contaminantes microbianos, como também avaliar o efeito que a atividade radioativa pode provocar em microrganismos. Os objetivos deste trabalho foram: estudar os processos de esterilização por radiação gama (60Co) dos acessórios e da coluna de alumina (Al2O3) que compõem o sistema do gerador de 99Mo/99mTc, e avaliar a eficácia da radiação gama na morte de microrganismos em geradores de 99Mo/99mTc na mínima (9,25 GBq) e na máxima (74 GBq) atividade radioativa. Acessórios do gerador foram irradiados com doses absorvidas de 15, 25 e 50 kGy e as colunas de alumina com 10, 15, 25 e 50 kGy. A alumina das colunas irradiadas foi avaliada por análise combinada de microscopia eletrônica de varredura (MEV) e análise elementar por espectroscopia de energia dispersiva (EDS), e difração de raios X. O Teflon® foi avaliado por termogravimetria (TGA) e calorimetria exploratória diferencial (DSC). Cálculos dosimétricos foram realizados para o alvo de maior dose na coluna do gerador de 9,25 GBq e 74 GBq. Suspensões quantificadas de B. subtilis e esporos de B. pumilus foram inoculadas em colunas irradiadas a 25 kGy e observou-se que a coluna de alumina representou uma barreira física para a contaminação do eluato, pois a recuperação microbiana foi reduzida. Verificou-se que o B. subtilis na forma vegetativa mostrou menor afinidade pela alumina em relação aos esporos de B. pumilus. A recuperação microbiana foi menor no gerador com atividade radioativa em relação ao gerador sem radioatividade. Foi desenvolvido um indicador biológico de esporos de B. pumilus que consistiu em uma tira de alumínio com sílica gel colocado externamente à coluna e as quantidades recuperadas tanto no gerador de 9,25 GBq quanto no de 74 GBq mostraram redução na viabilidade. O uso de filtros Sep-Pak® e 0,22 μm no sistema do gerador mostrou eficiência na retenção de 4,9 x 106 UFC de B. subtilis, garantindo a esterilidade do produto terminado. / In the manufacturing process of the 99Mo/99mTc generator it is essential not only to prevent the introduction of microbial contaminants, but also evaluate the effect of the radioactivity in microorganisms. The objectives of this work were to study the sterilization processes by gamma rays (60Co) of accessories and alumina column (Al2O3) that take part in the generator system and assess the effectiveness of gamma radiation in the death of microorganisms in 99Mo/99mTc generators of minimum (9.25 GBq) and maximum (74 GBq) radioactivities. Accessories were irradiated with 15, 25 and 50 kGy absorbed doses and alumina columns with 10, 15, 25 and 50 kGy. Alumina from irradiated columns were examined by combined analysis of scanning electron microscopy (SEM) and elemental analysis by energy dispersive spectroscopy (EDS), and X-ray diffraction. Teflon® was evaluated by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Dosimetric calculations were made for the highest dose target on 9.25 GBq and 74 GBq generator columns. Quantified suspensions of B. subtilis and B. pumilus spores were inoculated into columns irradiated at 25 kGy and it was observed that the alumina column represented a physical barrier to contamination of the eluate, as microbial recovery was reduced. It was found that B. subtilis in vegetative form showed lower affinity for alumina when compared to B. pumilus spores. Microbial recovery was lower in the generator with radioactivity than with the generator without radioactivity. A biological indicator of B. pumilus spores was developed which consisted of an aluminum strip with silica gel placed externally to the column and the microbial recovery in both 9.25 and 74 GBq generators showed reduction in viability. The use of Sep-Pak® and 0.22 μm filters in the generator system has shown efficiency in the retention of 4.9 x 106 CFU of B. subtilis, ensuring the sterility of the finished product.

No processo de fabricação do gerador de 99Mo/99mTc é fundamental, não só evitar a introdução de contaminantes microbianos, como também avaliar o efeito que a atividade radioativa pode provocar em microrganismos. Os objetivos deste trabalho foram: estudar os processos de esterilização por radiação gama (60Co) dos acessórios e da coluna de alumina (Al2O3) que compõem o sistema do gerador de 99Mo/99mTc, e avaliar a eficácia da radiação gama na morte de microrganismos em geradores de 99Mo/99mTc na mínima (9,25 GBq) e na máxima (74 GBq) atividade radioativa. Acessórios do gerador foram irradiados com doses absorvidas de 15, 25 e 50 kGy e as colunas de alumina com 10, 15, 25 e 50 kGy. A alumina das colunas irradiadas foi avaliada por análise combinada de microscopia eletrônica de varredura (MEV) e análise elementar por espectroscopia de energia dispersiva (EDS), e difração de raios X. O Teflon® foi avaliado por termogravimetria (TGA) e calorimetria exploratória diferencial (DSC). Cálculos dosimétricos foram realizados para o alvo de maior dose na coluna do gerador de 9,25 GBq e 74 GBq. Suspensões quantificadas de B. subtilis e esporos de B. pumilus foram inoculadas em colunas irradiadas a 25 kGy e observou-se que a coluna de alumina representou uma barreira física para a contaminação do eluato, pois a recuperação microbiana foi reduzida. Verificou-se que o B. subtilis na forma vegetativa mostrou menor afinidade pela alumina em relação aos esporos de B. pumilus. A recuperação microbiana foi menor no gerador com atividade radioativa em relação ao gerador sem radioatividade. Foi desenvolvido um indicador biológico de esporos de B. pumilus que consistiu em uma tira de alumínio com sílica gel colocado externamente à coluna e as quantidades recuperadas tanto no gerador de 9,25 GBq quanto no de 74 GBq mostraram redução na viabilidade. O uso de filtros Sep-Pak® e 0,22 μm no sistema do gerador mostrou eficiência na retenção de 4,9 x 106 UFC de B. subtilis, garantindo a esterilidade do produto terminado. / In the manufacturing process of the 99Mo/99mTc generator it is essential not only to prevent the introduction of microbial contaminants, but also evaluate the effect of the radioactivity in microorganisms. The objectives of this work were to study the sterilization processes by gamma rays (60Co) of accessories and alumina column (Al2O3) that take part in the generator system and assess the effectiveness of gamma radiation in the death of microorganisms in 99Mo/99mTc generators of minimum (9.25 GBq) and maximum (74 GBq) radioactivities. Accessories were irradiated with 15, 25 and 50 kGy absorbed doses and alumina columns with 10, 15, 25 and 50 kGy. Alumina from irradiated columns were examined by combined analysis of scanning electron microscopy (SEM) and elemental analysis by energy dispersive spectroscopy (EDS), and X-ray diffraction. Teflon® was evaluated by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Dosimetric calculations were made for the highest dose target on 9.25 GBq and 74 GBq generator columns. Quantified suspensions of B. subtilis and B. pumilus spores were inoculated into columns irradiated at 25 kGy and it was observed that the alumina column represented a physical barrier to contamination of the eluate, as microbial recovery was reduced. It was found that B. subtilis in vegetative form showed lower affinity for alumina when compared to B. pumilus spores. Microbial recovery was lower in the generator with radioactivity than with the generator without radioactivity. A biological indicator of B. pumilus spores was developed which consisted of an aluminum strip with silica gel placed externally to the column and the microbial recovery in both 9.25 and 74 GBq generators showed reduction in viability. The use of Sep-Pak® and 0.22 μm filters in the generator system has shown efficiency in the retention of 4.9 x 106 CFU of B. subtilis, ensuring the sterility of the finished product.

Hybrid male sterility (HMS) is typically the first mechanism fortifying reproductive isolation resulting from genomic incompatibilities. Three interspecies feline breeds derived from domestic cat crosses to wild cat species (Asian leopard cat and African serval) manifest HMS through several generations of backcrossing before eventually regaining fertility. This work utilized 199 hybrid individuals with varying fertilities in a genome wide association study (GWAS) comprising 63,000 genome wide SNPs. Leveraging these results with whole-testis transcriptome sequencing and quantitative real-time PCR data facilitated the comparison of transcripts in sterile and fertile hybrids. This dissertation describes four loci with highly significant and fifty with moderately significant association to sterility within each individual hybrid domestic breed and combinations of breeds. These associations help identify epistatic targets for hybrid incompatibility contributing to sterility. Comparative QTL mapping between pairs of species provides a framework to describe the accumulation of clade-specific reproductive isolating loci. Detailed exploration of gene misregulation between domestic and hybrid individuals, as well as between littermate hybrids of varying fertilities outlines a pattern of expression consistent with a meiotic sex-chromosome inactivation failure in early generations and apoptotic failure in later hybrid generations. Combining comparative genomic association and transcriptomic characterization among hybrid felids of varying divergence, new insight is gained into the mechanisms of mammalian reproductive isolation.