O objetivo desse estudo foi avaliar se a baixa fertilidade de vacas Holandesas repetidoras de serviço [RS; comparativamente a novilhas (NOV) e vacas próximas ao pico de lactação (PL)] está associada com comprometimento da qualidade oocitária e se esta condição é agravada pelo estresse térmico. Fêmeas das três categorias foram tratadas com o mesmo protocolo de sincronização da emergência de onda folicular. Cinco dias após o início do protocolo, a ovum pick-up (OPU) foi realizada e foram avaliados (Capítulo; Cap. 1) o número de folículos ovarianos, de oócitos totais e viáveis, temperatura retal (TR), temperatura de superfície cutânea (TC) e frequência respiratória (FR). Os oócitos viáveis foram utilizados para a produção in vitro de embriões (Cap. 2) e avaliações biomoleculares (Cap. 3). No Cap. 2, foram avaliados o desenvolvimento embrionário (taxa de clivagem, de blastocisto e de eclosão) e a qualidade dos embriões produzidos (número de células e frequência de núcleos fragmentados). No Cap. 3, realizou-se a extração de RNA e DNA de parte dos oócitos coletados para a quantificação relativa e absoluta de DNA mitocondrial (mtDNA) e a avaliação da expressão de genes relacionados à replicação/transcrição do mtDNA (PPARGC1A, NRF1, POLG, POLG2, TFAM e MT-CO1), à apoptose (BAX, BCL2 e ITM2B) e ao estresse térmico (HSP90AA1 e HSPA1AB). No Cap. 4, a taxa de concepção após IATF foi avaliada em ambas as estações do ano e nas três categorias quando o mesmo protocolo de sincronização para IATF e a mesma partida de sêmen foram utilizados. No Cap. 1, vacas RS e PL aumentaram sua FR e TR no verão em relação ao inverno (P<0,0001), enquanto as NOV mantiveram essas variáveis constantes em ambas as estações. Nas três categorias houve aumento (P<0,0001) da TC no verão, mas esta sempre foi superior (P<0,001) em vacas RS e PL do que nas NOV, independente da estação. O número de folículos e de oócitos totais e viáveis declinou nas RS e PL durante o verão, mantendo-se semelhante em NOV em ambas as estações. No Cap. 2, a taxa de clivagem foi semelhante entre categorias e estações. Já a taxa de blastocisto foi reduzida no verão nas três categorias, sendo essa queda mais acentuada nas RS. A taxa de eclosão e o número de células dos blastocistos foram menores no verão (independente de categoria). Menor número de células foi observado em embriões de RS e PL (independente da estação). Além disso, a porcentagem de núcleos fragmentados foi maior nos blastocistos das RS no verão. No Cap. 3, a expressão de ITM2B e BAX foi maior em RS durante o verão. Ainda, detectaram-se indícios da ativação de mecanismos pró-apoptóticos nos oócitos de RS (maior relação BAX/BCL-2) comparadas a PL e de mecanismos compensatórios da deficiência da função mitocondrial (menor conteúdo de mtDNA e maior expressão de PPARGCC1, NRF1, TFAM, POLG e POLG2) nas RS durante o verão em relação as demais categorias. No Cap. 4, menor taxa de concepção foi observada em RS e durante o verão. Os resultados geram evidências de que o baixo desempenho reprodutivo de vacas RS durante o verão deva estar relacionado ao comprometimento da qualidade de seus oócitos, demonstrado pelo seu reduzido conteúdo de mtDNA e elevada expressão de genes relacionados a replicação/transcrição do mtDNA, apoptose e síntese de chaperonas, culminado em baixa taxa de blastocisto e alta fragmentação nuclear destes. / The aims of the present study were to evaluate whether lower fertility of repeat-breeder (RB) Holstein cows [compared to peak lactation cows (PL) and heifers (H)] is associated with oocyte quality and whether this condition is aggravated by summer heat stress. Females of the three categories were treated with same protocol for synchronization of follicular wave emergence during summer and winter. Five days after the protocol onset, the ovum pick-up (OPU) was performed. The following variables were evaluated in Chapter 1: number of ovarian follicles before OPU, number of total and viable oocytes, rectal temperature (RT), cutaneous temperature (CT) and respiration rate (RR). Viable oocytes were sent forThe aims of the present study were to evaluate whether lower fertility of repeat-breeder (RB) Holstein cows [compared to peak lactation cows (PL) and heifers (H)] is associated with oocyte quality and whether this condition is aggravated by summer heat stress. Females of the three categories were treated with same protocol for synchronization of follicular wave emergence during summer and winter. Five days after the protocol onset, the ovum pick-up (OPU) was performed. The following variables were evaluated in Chapter 1: number of ovarian follicles before OPU, number of total and viable oocytes, rectal temperature (RT), cutaneous temperature (CT) and respiration rate (RR). Viable oocytes were sent for in vitro embryo production (Chapter 2) and bimolecular evaluation (Chapter 3). In Chapter 2, embryo development (rates of cleavage, blastocyst and hatching) and quality (total number of nuclei and frequency of nuclear fragmentation) were assessed. In Chapter 3, part of the oocytes were subjected to DNA and RNA extraction to allow relative and absolute quantification of mitochondrial DNA (mtDNA), and the evaluation of the expression of genes related to mtDNA replication/transcription (PPARGC1A, NRF1, POLG, POLG2, TFAM and MT-CO1), apoptosis (BAX, BCL2 and ITM2B) and heat stress (HSP90AA1 e HSPA1AB). In Chapter 4, RB, PL and H were subjected to same protocol for fixed-time AI using the same batch of semen of a single sire in order to evaluate their P/AI during summer and winter. In Chapter 1, RB and PL had increased (P<0.0001) RR and RT during summer compared to winter; while H maintained similar RR and RT in both seasons. CT was greater (P<0.0001) during summer than winter in all categories, but it was always higher (P<0.001) in RB and PL than H, regardless of season. The numbers of follicles and total and viable oocytes were lower in RB and PL during summer than winter, and maintained stable in H in both seasons. In Chapter 2, cleavage rate was similar among categories and between seasons. However, blastocyst rate was invariably reduced during summer, but more pronouced in RB. Hatching rate and the total number of nuclei were decreased during summer, regardless of category. Lower number of nuclei was observed in RB and PL embryos compared to H, regardless of the season. Furthermore, the percentage of fragmented nuclei was greater in RB blastocysts during the summer. In Chapter 3, expressions of ITM2B and BAX were greater in RB oocytes collected during summer. Also, the activation of pro-apoptotic mechanisms (greater BAX/BCL2 ratio) was suggested in RB heat stressed-oocytes compared with PL. Activation of compensatory mechanisms of deficient mitochondrial function (low number of copies of mtDNA and increased expression of PPARGCC1, NRF1, TFAM, POLG and POLG2) were also observed in RB heat stressed-oocytes compared with PL and H. In Charpter 4, lower P/AI was observed in RB and under summer heat stress. These data provide evidences that the lower reproduction performance observed in RB during heat stress may be due to impaired oocyte quality, as shown by their reduced mtDNA content and upregulation of several genes related to mtDNA replication/transcription, apoptosis and chaperones synthesis, resulting in lower blastocyst rate and higher nuclear fragmentation of embryos.

The tumour suppressor protein p16INK4a (p16) is a cyclin-dependent kinase (CDK) inhibitor that plays a key role in the regulation of the cell cycle by controlling the progression of cells through the G1 to S phase transition. Dysregulation of the protein through deletion, silencing or mutation of the gene encoding p16 is implicated in a range of different cancers including melanoma, cervical and oesophageal to name a few. p16 is composed of four ankyrin repeats and it has a very low thermodynamic and kinetic stability and rapidly unfolds even in the absence of denaturants. This low stability means that the protein is highly vulnerable to point mutations, which can result in functional inactivation through a range of different mechanisms such as deletion of key binding contacts, disruption of secondary or tertiary structure and consequent destabilisation leading to unfolding or aggregation. Heavy-chain antibodies are a unique form of antibody devoid of light chains found in the serum of the Camelid family (camels and llamas). Despite the absence of light chains, heavy-chain antibodies have evolved to complement traditional antibodies and retain the full binding capacity seen in canonical IgG antibodies. The single variable domain, known as a nanobody, is, at 15 kDa, the smallest antigen binding fragment, a tenth the size of a standard IgG antibody. The small size and relative ease of production, coupled with an unusually high stability, makes nanobodies useful tools as biological reagents, crystallography chaperones and therapeutics. The research contained within this PhD looks at the development of nanobodies to target p16. By leveraging the high stability of selected nanobodies, the aim was to obtain binders that could stabilise and reactivate a range of unstable cancer-associated mutants. The initial stages of the project focused on generating and optimising the expression and purification of p16 constructs prior to immunisation of animals to raise nanobodies. A high-throughput approach was taken to generate forty-five different p16 constructs with a range of different solubility and purification tags. These constructs were assessed in a multi-factorial expression screen, which resulted in the identification of a p16 construct with a ten-fold improvement in soluble expression levels compared with previous studies. A range of biophysical techniques, including circular dichroism and chemical denaturation, were performed to characterise this protein fully prior to immunisation. The second part of this project utilised a phage display library of two immune nanobody libraries generated against p16 and a p16 variant stabilised by previously published second-site mutations. This process yielded a large number of diverse nanobodies. Biophysical characterisation of these nanobodies was first performed, and they were found to have a range of chemical and thermal stabilities. Assays were then developed to test the ability of the nanobodies to stabilise p16. Two nanobodies were found to dramatically stabilise wild-type p16, with an increase in stability of approximately 44 % and 60 %, respectively. Furthermore, these nanobodies were also able to stabilise a subset of cancer-associated point mutants. Although there are NMR structures of p16, as well as a crystal structure of p16 bound to CDK6, the resolution of is very low, most likely due to the high backbone flexibility of p16. The last part of the project aimed to obtain a higher-resolution structure of p16 by using the two stabilising nanobodies as crystallisation chaperones. The more stabilising of the two nanobodies resulted in crystals that diffracted to a resolution of less than 2 $\AA$, a significant improvement compared with the previously published structure. In conclusion, a number of nanobodies were generated against tumour-associated p16 and shown to be capable of stabilising p16, allowing structure determination to high resolution and restoration of the stability of cancer-associated mutants to wild-type levels. In the project, a range of different approaches for nanobody production were explored, and these will be important for future applications. Moreover, the crystal structure of the p16-nanobody complex showed that the nanobody binds on the opposite face of p16, to the face involved in binding to CDKs; thus, this nanobody could potentially be exploited as a pharmacological chaperone to stabilise and restore the activity of cancer-associated mutant p16 in the cell.

Rapid repeat pregnancy (RRP) is associated with adverse maternal and infant outcomes and a range of undesirable social and economic challenges for the mother, her baby, and society. Although the consequences of RRP are well known, Zimbabwe—a country with some of the poorest maternal health indicators—has not investigated or made efforts to directly address this problem. This is confirmed by the lack of targeted programs to curb RRP, the unavailability of documented evidence regarding RRP significant risk factors, and the lack of understanding of the extent of RRP in the country. Using social cognitive theory as the theoretical framework, an unmatched case-control study was conducted using data from the Zimbabwe Demographic and Health Survey of 2015 to determine the prevalence of RRP and to assess associations between sociodemographic, sexual-relational, women's health, fertility preference, previous birth outcomes, and social factors and having an RRP in Zimbabwe. Logistic regression analysis showed statistically significant associations between all factors except for women's health characteristics. The prevalence of RRP among women of reproductive age (15–49 years) in Zimbabwe was 50.2%. The high prevalence of RRP and the multiple statistically significant associations reported in this study affirm the need for Zimbabwe to make prevention of RRP a public health priority. Zimbabwe must develop targeted interventions that work in context and integrate these into an ongoing comprehensive family planning program. In-depth research is needed to establish and understand the underlying motivations for having an RRP among Zimbabwean women. Such information may help develop targeted interventions to create social change.

An investigation was made into the isolation and screening of highly cellulolytic anaerobic fungi and their production of cellulolytic enzymes using immobilised rhizomycelia. A total of 46 anaerobic fungi were isolated on cellulosic substrates from ruminant and non-ruminant herbivores. Primary screening of these isolates was performed using dye release from cellulose-azure which qualitatively detected cellulolytic activity. Twelve isolates were chosen on the basis of their maximum solubilisation rates of the labelled cellulose and then subjected to secondary screening which involved the quantification of enzyme activity. The enzyme mixtures were characterised by carboxymethylcellulase, xylanase, B-glucosidase, B-xylosidase and cellobiase assays, measured by the production of either reducing sugars, p-nitrophenol or glucose. All strains produced a number of enzymes that allowed them to hydrolyse straw and highest enzyme activity was measured in static cultures grown on 0.5% straw. A monocentric isolate, Piromyces strain KSX1 from a red kangaroo, and a cattle polycentric isolate, Orpinomyces strain 478P1, were selected for study of cellulolytic enzyme production on the basis of high fibre digestion capability and amenability toward encapsulation. The immobilised polycentric strain proved to be operationally superior to strain KSX1 as strain 478P1 did not produce any viable growth in the culture liquor. Studies into single batch cultures of free cells of strains KSX1 and 478P1 revealed that the maximum specific rate of B-glucosidase production occurred concomitantly with maximum specific growth rate suggesting that the immobilised fungus must grow for continuous enzyme production to occur. Although the physiology of cellulase synthesis in strains KSX1 and 478P1 was found to be growth-associated, immobilisation of the fungus offered the advantage of the repeat-batch use of cells with the accumulation of extracellular enzymes after each batch. Thus, operational gains were the key issues in assessing the potential application of immobilised anaerobic fungi in the production of cellulolytic enzymes. The repeat-batch system was operationally more efficient than the free cell batch cultures because immobilisation removed the need of reculturing the cells for every single batch. / Doctor of Philosophy (PhD)

The Pentatricopeptide Repeat (PPR) gene family produces RNA-binding proteins that target organellar transcripts. The PPR family is expanded in land plants, with nearly 450 genes identified in Arabidopsis thaliana. In plants with a Cytoplasmic Male Sterility (CMS) phenotype, members of the PPR family can act as a RESTORER OF FERTILITY (Rf) and are part of a subset of genes called RESTORER OF FERTILITY-LIKE (RFL). Unlike other PPR transcripts, RFL transcripts are targets of both microRNA (miRNA) and trans-acting siRNA (tasiRNA) and produce secondary siRNA after initial miRNA- or tasiRNA-guided cleavage. We utilized the A. lyrata genome assembly and high-throughput sequencing of small RNA to examine the evolutionary dynamics of the PPR gene family and the pattern of small RNA targeting of RFL transcripts. We found an expanded set of 539 PPR genes in A. lyrata, 51 of which were in the RFL group, often in multiple collinear copies when compared to their A. thaliana orthologs. In-species RFL paralogs appear to be more related to one another than to their collinear orthologs, which is possible evidence of gene conversion or ectopic recombination. miRNA targeting of RFL transcripts is largely conserved with nearly two-thirds of all target sites maintained. TasiRNA targeting was less conserved with roughly one-third of comparable validated tasiRNA targets maintained in both species. However, when clusters of potential tasiRNA targets were considered, roughly two-thirds of target sites are conserved. Production of secondary siRNA from A. lyrata PPR transcripts is less well defined than in A. thaliana, with strong signals coming from phases that are not concordant with the miRNA- or tasiRNA-guided cleavage sites. / Graduation date: 2013

Spinocerebellar ataxia type 7 (SCA7) is a hereditary neurodegenerative disorder affecting the cerebellum, pons and retina. SCA7 patients present with gait ataxia and visual impairment as the main symptoms. Anticipation, commonly observed in SCA7 families, is a phenomenon where an earlier age at onset and a more severe progression of disease is seen in successive generations. In order to identify the gene responsible for SCA7, we performed linkage analysis on a Swedish SCA7 kindred. Evidence for linkage of the SCA7 disease locus to a 32 cM region on chromosome 3p12-21.1, between markers D3S1547 and D3S1274, was established. A number of neurodegenerative disorders associated with anticipation are caused by expanded (CAG)n repeats in their respective disease genes. In order to isolate the SCA7 disease gene we, therefore, screened a human infant brain stem cDNA library for CAG repeat containing clones, mapping to chromosome 3. Four candidate clones were isolated and analysed, but could all be excluded as the SCA7 disease gene. In 1997, the SCA7 disease gene was identified and, as expected, shown to harbour a CAG repeat, expanded in SCA7 patients. Analysis of the SCA7 CAG repeat region in Swedish SCA7 patients demonstrated that CAG repeat size was negatively correlated to age at onset of disease. Furthermore, patients with larger repeats presented with visual impairment, whereas patients with smaller repeats presented with ataxia as the initial symptom. SCA7 is the most common autosomal dominant cerebellar ataxia in Sweden and Finland, but rare in other populations. In order to investigate if the relatively high frequency of SCA7 in these countries is the result of a founder effect in the region, a haplotype analysis was performed on all SCA7 families available. All 7 families shared a common haplotype of at least 1.9 cM surrounding the SCA7 locus. In addition, strong linkage disequilibrium was demonstrated for marker D3S1287 closely linked to the SCA7 gene, suggesting a founder effect for the SCA7 mutation in Sweden and Finland. The function of the SCA7 protein, ataxin-7, is not known and it does not show significant homologies to any previously known proteins. In order to gain insight into the function of ataxin-7 we analysed the expression of ataxin-7 in brain and peripheral tissue from SCA7 patients and controls. In brain, expression was found to be mainly neuronal with a nuclear subcellular localisation. Ataxin-7 expression was found throughout the CNS, not restricted to sites of pathology. We also confirmed previously reported findings of neuronal intranuclear inclusions (NIls) in the brains of SCA7 patients. Based on our findings, we conclude that the cell type specific neurodegeneration in SCA7 is not due to differences in expression pattern in affected and non-affected tissue or the distribution pattern of aggregated protein.

ITC/USA 2012 Conference Proceedings / The Forty-Eighth Annual International Telemetering Conference and Technical Exhibition / October 22-25, 2012 / Town and Country Resort & Convention Center, San Diego, California / As the integrated Network Enhanced Telemetry (iNET) program prepares for developmental flights tests, refinements are being made to the Radio Access Network Standard that ensures interoperability of networked radio components. One key aspect of this interoperability is the definition of Telemetry Network System (TmNS) RF Data Link Layer functionality for conducting efficient communications between radios in a TDMA (Time Division Multiple Access) channel sharing scheme. This paper examines the overall structure of the TmNS RF Data Link Layer and provides an overview of its operation. Specific topics include Medium Access Control (MAC) scheduling and framing in the context of a burst-oriented TDMA structure, link layer encryption, the priority-enabled Automatic Repeat reQuest (ARQ) protocol, high-level network packet and link control message encapsulation, payload segmentation and reassembly, and radio Link Layer Control Messaging.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Previously seed coat color in flax has been used as a phenotypic marker for specialty quality traits and currently there is an increasing demand to use seed coat color in flax to market flax for human and animal nutrition uses. Seed coat color was studied to 1) understand the inheritance of seed coat color conditioned by the b1 locus, to 2) understand the relationship of other important flax traits with seed coat color as well as to 3) identify markers that are linked to seed coat color for future marker assisted selection of seed coat color. Spearman’s rank correlation and an allelism test was used to show the inheritance of the alleles at the b1 locus. Bulked segregant analysis (BSA) was used to identify putatively linked markers with the b1 locus, these were then screened on the CDC Bethune x M96006 recombinant inbred line population. Furthermore, the CDC Bethune x M96006 and CDC Bethune x USDA-ARS Crystal recombinant inbred line populations were used to identify any important flax traits that had a significant relationship with seed coat color. It was shown that seed coat color conditioned by the b1 locus was stably inherited and that b1vg and b1 are allelic to one another. The results of the BSA showed that there were 17 candidates for linkage but when these markers were screened on the population only the Lu456 from linkage group (LG) six was identified to have linkage (χ²=3.90; P<0.05) with the b1 locus. Additionally, it was shown that the b1 seed coat color allele of the b1 locus had a pleiotropic effect on flower color and flower shape and that seed coat color was associated with linolenic fatty acid content. None of the traits examined were found to be associated with the b1vg allele of this locus. These results show that the b1 locus is likely present on linkage group six, more marker coverage on linkage group six of markers that are polymorphic between the two seed coat color parents would increase the accuracy of detection. Lastly, this study showed that plant breeders should consider using the b1vg allele that conditions the variegated seed coat color to mark unique lines with important combinations of traits because it sorted independently for seed quality traits. Whereas, the yellow seed coat color conditioned by the b1 allele was found to be associated with higher linolenic fatty acid content and the semi-lethality of this allele would make it not suitable for use in parental lines.

Darbo tikslas - įvertinti kraujo donorų požiūrį į pakartotinę kraujo donorystę ir nustatyti priežastis, dėl kurių kraujo donorai nesiryžta pakartotiniam kraujo davimui. Darbo uždaviniai:1.Įvertinti atlygintinų ir neatlygintinų kraujo donorų požiūrį į kraujo donorystę ir nustatyti pirmo kraujo davimo motyvus. 2. Nustatyti priežastis, dėl kurių kraujo donorai nesiryžta pakartotiniam kraujo davimui atlygintinų ir neatlygintinų donorų tarpe. 3. Nustatyti ir palyginti atlygintinos ir neatlygintinos kraujo donacijos ekonominius kaštus. Tyrimo metodika. Tyrimas vykdytas 2012 m. kovo-balandžio mėnesiais viešojoje įstaigoje Nacionalinis kraujo centras. Telefoninės apklausos būdu, pagal sudarytą klausimyną, buvo apklausti 400 kraujo donorai, kurie po pirmo kraujo davimo neatvyko pakartotinam kraujo davimui praėjus vieneriems ir daugiau metų. Ekonominis kraujo donorystės vertinimas atliktas skaičiuojant ir vertinant 2011 m. kraujo donorystės ekonominius rodiklius. Statistinė gautų duomenų analizė atlikta naudojant “SPSS 17.0” statistinę programą. Ryšio stiprumas tarp kategorinių kintamųjų buvo tiriamas naudojant Kramerio V koreliacijos koeficientą, kartu tikrinant hipotezę apie jo lygybę nuliui (statistinį reikšmingumą). Požymių priklausomybei nustatyti skaičiuotas chi-kvadrato (χ2) kriterijus. Kai reikšmingumo lygmuo p<0,05, požymių skirtumas tiriamųjų grupėse laikytas statistiškai reikšmingu. REZULTATAI. Dažniau neatlygintinai pirmą kartą duoti kraujo buvo atvykę asmenys, turintys... [toliau žr. visą tekstą] / AIM OF THE STUDY: to evaluate blood donors‘ attitude to repeated blood donation and to determine the causes why blood donors refuse to repeat blood donation. OBJECTIVES: 1. To evaluate remunerated and non-remunerated blood donors‘ attitude to blood donation and determine the incentives for the first blood donation. 2. To determine the causes why blood donors refuse to repeat remunerated and non-remunerated blood donation. 3. To assess and compare the cost of remunerated and non-remunerated blood donation. RESEARCH. The research was conducted in March - April, 2012, at National Blood Center. There was prepared a questionnaire and 400 blood donors, who did not donate blood after one year or more after first donation, were asked to answer the questions by phone. The economic blood donation evaluation was carried out while calculating and analysing the data regarding blood donation from the year 2011. The statistical analysis of the data was conducted using “SPSS 17.0” statistical programme. Cramer‘s V correllation coefficient was used to determine the relationship between categorical variables, at the same time checking the zero hypothesis (statistical significance). In order to determine the dependence of variables, chi-square (χ2) criterion was calculated. The differences were judged to be statistically significant, when p<0.05. RESULTS. Individuals who had higher university education, were employed and received a monthly salary of more than 2,500 Lt, first time donated... [to full text]