Avian influenza H5N1 is one kind of important bird flu. Unfortunately, this virus has swiftly evolved and become highly pathogenic to humans and poultry, resulting in 100% of death in infected poultry and over 60% of mortality among infected human population. Moreover, the virus tends to reassort with other influenza viruses, such as the current swine flu H1N1, to establish themselves in environments and further this epidemic all over the world. The World Health Organization (WHO) has in fact warned that highly pathogenic avian influenza H5N1 poses a graver risk of a global human pandemic than at any time since the Hong Kong outbreak (H3N2) in the 1960s. / Finally, avian influenza is an inter-disciplinary issue across virology, medical geography, and spatial epidemiology. How to quantify and integrate knowledge from different disciplines remains a challenge in fully understanding the disease. We propose a method to formally integrate genetic analysis that identifies the evolution of the H5N1 virus in space and time, epidemiological analysis that determines socio-environmental factors associated with H5N1 occurrence and statistical analysis that identifies outbreak dusters. Our integrated results show a significant advance in findings over reports in, for instance, Gilbert et al. (2008) and we believe our findings are more precise and informative in representing the occurrence and the space-time dynamics of H5N1 spread. Overall, unlike traditional influenza studies, our work sets up a solid foundation for the inter-disciplinary study of this and other spatial infectious diseases. / First, we apply multifractal detrended fluctuation analysis to determine the temporal scaling behavior of outbreaks in Asia, Europe, Africa, and the whole of the world between December 2003 to March 2009. Long-range correlation and multifractality, two important properties characterizing the scaling behavior of complex dynamics, are first detected in the outbreak time series. In addition, this study identifies different temporal scaling behaviors of outbreaks of these continents 8,nd specific seasonal patterns in Asia. These findings confirm our perspective that avian-influenza outbreak behaviors are self-similar over time and are spatially heterogeneous. / One key to preventing such a calamity is to obtain a thorough understanding of the mechanisms of avian influenza transmission and its spatio-temporal patterns of dispersal. The issues at stake are outbreaks' spatial and temporal patterns, the interrelationship of these with the evolution of influenza viruses in such a way that geography is understood as a dimension of the disease's virology, and the human and avian behaviors and socio-ecological environments associated with H5Nl spread. This thesis sets out to study these problems in detail and propose solutions. / Second, we conduct a spatial analysis for global trends and local clusters of H5N1 outbreaks at multiple geographical scales. Currently, the local K function used in a point pattern analysis searches outbreak clusters, assuming the disease is spatially homogeneous. The thesis proposes a much more efficient method to measure the degree of clusters accurately. The modified function works by weighting outbreaks through distances, counting the number of the weighted outbreaks for each lattice point no matter whether the disease emerges in a grid. This weighted local K function extends cluster analysis from a point pattern to lattice data. Spatial representation in these terms then seeks to explore local patterns of H5N1 over a continuous space. / Third, we study a set of socio-environmental factors, which are plausibly associated with the occurrence of H5N1. Spatial epidemiological models are built for predicting the disease at both continental and national levels, covering Indonesia, China, and the whole of East-Southeast Asia. We evaluate the statistical models using 1,000 bootstrap replicates, showing a consistently high rate of prediction, assessed by statistics: AUC, Kappa Index, and pseudo R square. / Ge, Erjia. / Advisers: Yee Leung; Tung Fung. / Source: Dissertation Abstracts International, Volume: 73-06, Section: A, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 169-197). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Made available in DSpace on 2016-04-28T20:15:36Z (GMT). No. of bitstreams: 1 2006-Raquel Silva Lisboa.pdf: 3491225 bytes, checksum: e18ca335db22717674936719fe4abae7 (MD5) Previous issue date: 2006-02-24 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Avian spirochetosis is an acute septicemic disease, cosmopolite, of a variety of avian species caused by Borrelia anserina (Sakharoff, 1891). This spirochete is usually present in the blood of infected birds during the early stages of the disease. The present study assesses the experimental transmission of B. anserina by infected ticks Argas miniatus, observing the pre-patent and patent period, and comparing the clinical and hematological alterations. Twenty-seven fowls of the species Gallus gallus were randomly allocated into three groups composed by nine animals each. One group was exposed to B. anserina infected ticks (group 1), other one to ticks free of this agent (group 2), besides one group not exposed to ticks (group 3). Blood smears of the fowls were taken, daily, since the first day the fowls were exposed to the ticks, up to the 25? day after exposure (DAE). Blood samples were collected three days before exposure, three DAE, eight DAE, and for the last time in eighteen DAE for hematologic tests. The examination of group three s smears revealed a great number of spirochetes. The biological parameters of the pre-patent and patent period for this group were, 6 ? 0,83 and 5 ? 1,96 days, respectively. Group 2 and group 3 blood smears were negatives during the whole period under exam. About the clinical signs observed, since the sixth and seventh DAE, the fowls of group 1 presented: nibs bristle, pale crist, somnolence, inappetence, loss of weight and green diarrhoea wich were continuing until the 12? DAE coinciding with the end of the spirochetemia, after this, occured clinical evolution which self-cure. In agreement with the hematological evaluation results, the fowls exposed to infected ticks showed a normocytic normochromic anemia in eight DAE, leucocytosis with initial heterophilia and monocytosis in concomitance with the spirochetemia. After the patent period, eighteen DAE, a linphocytosis was detected. The present study confirmed the viability of the experimental transmission of B. anserina by infected ticks A. miniatus. Infected G. gallus with avian spirochetosis showed clinical alterations wich cursed in concomitance to the spirochetemia period, evoluting to selfcure, moreover hematological alterations compatible with the bacterial infection. / A Borreliose avi?ria ? uma doen?a septic?mica aguda, cosmopolita, que acomete diferentes esp?cies avi?rias, sendo causada por Borrelia anserina (Sakharoff, 1891). Esta espiroqueta pode ser encontrada no plasma sang??neo das aves infectadas durante os est?gios iniciais da doen?a. Os objetivos do presente trabalho foram estudar a transmiss?o experimental de B. anserina por carrapatos Argas miniatus infectados, observando o per?odo pr?-patente e per?odo de pat?ncia, e estudo comparativo das altera??es cl?nicas e hematol?gicas. Um total de 27 aves da esp?cie Gallus gallus foram divididas em tr?s grupos inteiramente casualizados contendo nove animais cada. Um grupo foi exposto a carrapatos infectados por B. anserina (grupo 1); outro a carrapatos livres deste agente (grupo 2); al?m de um grupo n?o exposto aos carrapatos (grupo 3). Realizaram-se esfrega?os sang??neos das aves, diariamente, a partir do primeiro dia em que as aves foram expostas aos carrapatos, at? o 25? dia ap?s a exposi??o (DPE). Amostras de sangue foram coletadas tr?s dias antes da exposi??o aos carrapatos, tr?s DPE, oito DPE e uma ?ltima 18 DPE para a realiza??o dos hemogramas. O exame dos esfrega?os das aves do grupo 1 revelou grande n?mero de espiroquetas. Os par?metros biol?gicos de per?odo pr?-patente e de per?odo de pat?ncia para este grupo foram, em dias, 6 ? 0,83 e 5 ? 1,96, respectivamente. Os esfrega?os sang??neos do grupo 2 e do grupo 3 mantiveram-se negativos durante todo o per?odo experimental. Em rela??o ?s manifesta??es cl?nicas observadas, a partir do sexto e s?timo DPE, as aves do grupo 1 apresentaram os seguintes sinais cl?nicos: penas arrepiadas, crista p?lida, sonol?ncia, perda do apetite, perda de peso e diarr?ia esverdeada. Estes sinais continuaram at? o 12? DPE, coincidindo com o t?rmino da espiroquetemia, em seguida houve evolu??o do quadro cl?nico para a cura das aves. De acordo com os resultados das avalia??es hematol?gicas, as aves expostas aos carrapatos infectados por B. anserina (grupo 1 apresentaram um quadro de anemia normoc?tica normocr?mica em oito DPE, leucocitose com heterofilia e monocitose iniciais que cursaram paralelamente com a espiroquetemia. Ap?s o per?odo de pat?ncia da infec??o, dezoito DPE, detectou-se uma linfocitose. O presente trabalho confirmou a viabilidade da transmiss?o de B. anserina em G. gallus experimentalmente infestados por A. miniatus. G. gallus infectados apresentaram altera??es cl?nicas que cursaram paralelamente ao per?odo de espiroquetemia, evoluindo para auto-cura, al?m de altera??es hematol?gicas compat?veis com infec??o bacteriana.

Escherichia coli patogênica aviária (APEC) é o agente etiológico da colibacilose, doença que acomete galinhas, patos, perus e outras aves, principalmente entre a 2ª e a 12ª semana de vida. Existem diversos estudos sobre genes associados à virulência de APEC, mas eles ainda não são capazes de explicar todos os fenótipos de APEC. Portanto, outros genes associados à virulência – ainda não descritos – devem estar presentes em APEC. Para descobrir novos genes associados à virulência em APEC, uma cepa invasiva foi usada para a construção de uma biblioteca de 1.800 mutantes, através da técnica de mutagênese marcada com assinatura, que insere transposons aleatoriamente no genoma da bactéria. Os mutantes foram selecionados em um ensaio de invasão in vitro a fibroblastos aviários da linhagem CEC-32. A sequência dos transposons inseridos nos mutantes permite a identificação daqueles mutantes que não foram capazes de invadir os fibroblastos. Até agora, 11 de 20 pools de 90 mutantes cada foram analisados, e 48 mutantes parecem ter perdido a capacidade invasiva, ou seja, tiveram sua virulência atenuada. Os demais pools já foram selecionados e terão seu DNA analisado. Todos mutantes que possivelmente perderam sua capacidade invasiva serão testados novamente para confirmar seu fenótipo de virulência atenuada. Depois, a região contendo a sequência do transposon será sequenciada para que se descubra qual gene foi interrompido e é essencial para a virulência in vitro de APEC. Essa biblioteca de mutantes também pode ser testada em modelos de seleção in vivo, de modo que é possível identificar, através da análise dos mutantes ausentes na seleção, genes de virulência essenciais para APEC em diferentes condições. / Avian pathogenic E. coli (APEC) is the causative agent of colibacillosis, a disease that affects poultry and other birds, mainly between two and twelve weeks of age. There are many studies about virulence-associated genes in APEC, but they do not fully explain all the APEC phenotypes. Therefore, other virulence-associated genes – not yet described – must be present in APEC strains. In order to find out new virulence genes, we made 1,800 random-transposons mutants of an APEC invasive strain using the signature-tagged mutagenesis method. The mutants were selected using an in vitro invasion assay to fibroblast cells. The sequence of the inserted transposons allows us to identify mutants that have lost the capacity of invade the cells. Until now, we tested eleven out of twenty pools of ninety mutants each, and forty-eight mutants appeared to have lost the invasive capacity. The other nine pools will be analyzed, and all the possible non-invasive mutants will be tested again to confirm the phenotype. Then, they will have the transposon-inserted region sequenced in order to find out which gene has been disrupted and is essential to APEC in vitro invasiveness. The attenuated mutants can also be selected in vivo, so we would identify essential virulence genes to APEC under different conditions.

Coronavírus, incluindo-se o Coronavírus aviário (ACoV), possuem o maior genoma composto por RNA conhecido entre os vírus. Aproximadamente dois terços desse genoma codificam proteínas não estruturais (Nsps), cujas funções parecem estar associadas à replicação e patogênese viral. Até o momento, esses alvos têm sido pouco explorados quanto a sua diversidade em diferentes linhagens de ACoV. O presente estudo teve como objetivo investigar a diversidade dos genes codificadores das proteínas não estruturais Nsp2 e protease Papaína-like (Plpro), utilizando-se linhagens brasileiras de ACoV. Para tanto, 10 linhagens de ACoV, isoladas em ovos embrionados, foram submetidas à RT-PCR direcionada aos genes codificadores de Plpro e Nsp2, seguindo-se o sequenciamento de DNA e a análise filogenética, juntamente com sequências homólogas obtidas no GenBank. Além disso, realizou-se a genotipagem por meio do sequenciamento parcial do gene codificador da proteína de espícula (região S1). Três das amostras virais obtidas e investigadas no presente trabalho apresentaram padrão de segregação discordante para os genes estudados. O isolado CRG I22 agrupou-se com linhagens virais pertencentes ao genótipo Massachusetts para S1 e com o grupamento de ACoVs brasileiros os genes da Nsp2 e Plpro. O isolado CRG I33 agrupou-se com linhagens virais pertencentes ao genótipo brasileiro para s1 e plpro e de maneira divergente para o gene da Nsp2. Para o isolado CRG I38, não foi obtida a genotipagem por s1, entretanto, similarmente ao observado para o isolado CRG I33, esse isolado agrupo-se com linhagens virais brasileiras para o gene plro e de maneira independente para o gene nsp2. As demais linhagens estudadas resultaram na formação de um grupamento especificamente brasileiro de ACoV, para os três genes estudados. Esses achados sugerem a ocorrência de recombinação nessas amostras discrepantes. Quanto às identidades médias entre as sequencias nucleotídicas analisadas, a região de s1 analisada apresentou as menores identidades (73,75% ±16,78), seguido pelo gene plpro (88,06% ±5,7) e do gene nsp2 (92,28% ±4,37), em acordo com a literatura. Assim sendo, os alvos investigados podem constituir ferramentas úteis na epidemiologia molecular do ACoV e na investigação de linhagens recombinantes do vírus. O presente estudo é o primeiro a investigar a diversidade genética de genes codificadores de proteínas não-estruturais em linhagens brasileiras de ACoV. Os resultados aqui apresentados reforçam a existência de um genótipo brasileiro de ACoV, para os 3 genes estudados. Entretanto, discrepâncias pontuais encontradas no padrão genotípico para s1, nsp2 e nsp3 permitem inferir uma diversidade genética maior do que a conhecida até o momento, possivelmente resultante de eventos de recombinação entre ACoVs brasileiros, ACoVs vacinais e outros ainda desconhecidos. Os resultados obtidos auxiliam na compreensão dos padrões e evolução dos ACoVs / Coronaviruses, including Avian coronavirus (ACoV), have the largest known RNA genome. Nearly two thirds of its genome codes for non-structural proteins (Nsps), whose functions appear to be linked to viral replication and pathogenesis. Hitherto these targets have been poorly explored regarding the ACoV lineages diversity. The present study aimed to assess the diversity of non-structural protein 2 (nsp2), papain-like protease (plpro) and spike protein (S1 subunit) coding genes, in Brazilian ACoV strains. To this end, 10 ACoV strains, isolated in embryonated eggs, had its 3rd and 5th passages submitted to RT-PCR targeting nsp2, plpro and s1, followed by DNA sequencing and phylogenetic analysis, herewith homologous sequences obtained from GenBank. Three of the ACoV strains sequenced showed a discordant segregation pattern for target genes. CRG I22 strain clustered with Massachusetts genotipe strains for S1, and with Brazilian cluster for nsp3 and plpro genes. CRG I33 strain, clustered with Brazilian strains for S1 and plpro genes, and was divergent for nsp2 gene. For CRG I38 strain, the S1 sequence was not obtained, however, similarly to what was observed for CRG I33, this strain grouped with the Brazilian lineage for plpro gene and was divergent for nsp2 gene. All the other ACoV here sequenced resulted in a specific Brazilian cluster for the three studied genes. Regarding the mean nucleotide identities measured, s1 gene showed the lowest identity (73.75% ±16.78), followed by plpro gene (88.06% ±5.7) and nsp2 gene (92.28% ±4.37), in accordance with previous reported data. Therefore, the targets of the present study are useful tools for ACoV molecular epidemiology studies and for the survey of recombinant ACoV strains. The presented study is the first one investigating the molecular diversity of non-structural proteins coding genes in Brazilian strains of ACoV. Results achieved herein reinforce the data over the circulation of ACoV Brazilian strains in this country, for the three investigated genes. However, divergences found between S1, nsp2 and plpro genetic patters allow inferring a higher molecular diversity than previously known. It is possible that this divergence is due to recombination events between ACoV from vaccines, Brazilian field strains and others still unknown. These results contribute on the comprehension over genetic patters and evolution of ACoV

This study is of the social organization of the wild turkey (Meleagris gallopavo intermedia Sennett) on the Welder Wildlife Refuge in southern Texas. The earliest turkey nests hatched in April, with the peak of hatching a month or more later. These poults may remain with their mother until winter. This brood flock, however, often combined with other brood flocks to form composite brood flocks when the poults were a few weeks old. Hens not successful in rearing young combined into broodless flocks. The juvenile males left the brood flocks in late fall or winter. They remained a distinct unit, the sibling group. These sibling groups attempted to join adult male flocks which were composed of older sibling groups. Most often the juvenile sibling groups were forced to join others their own age to form juvenile male winter flocks. Female flocks, after losing their juvenile males, combined with other female flocks to form large bands of up to 200 females. In spring the adult male flocks split into sibling groups for breeding. The sibling groups joined the female bands on display grounds. Only the dominant male of the dominant sibling group mated while hens were on the display ground. Later in the breeding season the female bands split into their flocks and returned to former nesting areas. Resident flocks continued to use the display ground, but later broke up into nesting groups of 2-5 females localized near their nests. The male sibling groups accompanied the females from the display ground, but did not become territorial. Incubation or nest loss broke down the female nesting group. This in turn led to formation of brood flocks or broodless flocks of hens.

<p>A number of environmental pollutants have been shown to mimick the action of the female sex hormone estrogen and are, therefore, suspected to be responsible for reproductive abnormalities seen in wildlife. Test systems which can be used in hazard and risk assessment of chemicals with estrogenic effects are consequently needed. In this thesis, I propose the avian egg as an <i>in vivo</i> test system for estrogenic compounds. I conclude that malformation of the left testis and the Müllerian ducts (MDs: embryonic oviducts) in avian embryos can be used as endpoints to examine estrogenic activity of chemicals. MD malformation is more easily determined and thereby faster to use as an endpoint than histologically observed feminization of the testis. The usefulness of MD/oviduct malformations as biomarkers for estrogenic effects in wild birds should be considered. </p><p>The environmental pollutants bisphenol A (BPA) and <i>o,p´</i>-DDT induced similar effects as the synthetic estrogens, ethynylestradiol and diethylstilbestrol. BPA caused MD malformations in quail embryos and ovotestis formation in chicken embryos. <i>o,p´</i>-DDT induced MD malformations in both quail and chicken embryos and ovotestis in chicken embryos. The flame retardant, tetrabromobisphenol A did not induce estrogen-like effects in quail or chicken embryos, but showed a relatively high embryolethality. </p><p>Embryonic exposure to estrogen caused persisting malformations of the oviduct, as well as a changed distribution pattern of the enzyme carbonic anhydrase in the shell gland of adult females. Considering the crucial role of carbonic anhydrase in shell formation, such changes could result in decreased shell quality. I propose that eggshell thinning in avian wildlife could reflect a functional malformation in the shell gland that is induced by xeno-estrogens during embryonic development, rather than being caused by exposure of the adult bird to environmental pollutants. This hypothesis opens new possibilities for studying the mechanisms behind contaminant-induced eggshell thinning in birds.</p>

This dissertation describes the development of a new sustained release formulation of nifedipine. The new formulation was developed by coating commercially available immediate release soft elastic gelatin capsules using a spray coating technique with two different polymeric combinations. Dissolution studies were conducted and showed that controlled release of nifedipine was obtained by increasing the ratio of the water insoluble polymer in the coat and increasing the percent weight gain of the coating. Simulated plasma concentration versus time profiles after administration of 30 mg dose of selected formulations showed a prolonged nifedipine release with concentrations above the minimum effective concentration for up to 12 hours. Bioavailability and bioequivalence of tableted test formulation of verapamil HCL was determined in 8 volunteers and compared to Covera HS® under fed and fasting conditions. The 90% confidence intervals for individual percent ratios of the Cmax, AUC₀₋₅₈ and AUC₀ were not within the range of 80 - 125% in both fed fasted states, suggesting that these formulations are not bioequivalent. the bioavailability of verapamil from the new formulation was higher state but this effect was not statistically significant. Pharmacokinetics of terbinafine administered orally at single doses of 15, 30, 60 and 120 mg were determined in raptors to recommend an appropriate dosing scheduled for terbinafine in the treatment of Aspergillosis. Calculation of steady state trough terbinafine plasma concentration after administration of daily doses of 15 or 30 mg/day showed that 30 mg daily dose of terbinafine administered orally in raptors produces a steady state trough terbinafine plasma concentration above the minimum inhibitory concentration (MIC) of(0.8 1.6) µg/ml against aspregillus fumigatus. From the data, 30 mg per day oral dose of terbinafine should be the recommended dose for treatment of aspergillosis in raptors. Approximate pharmacokinetic linearity of terbinafine was demonstrated for AUC[subscript 0-t] in the dose range of 15 120 mg while non-linearity for Cmax in the same dose range was demonstrated using the power model. / Graduation date: 2005

Influenza A viruses cause seasonal and pandemic outbreaks that range from mild infections to the disastrous Spanish Flu. Resistance to neuraminidase inhibitors (NAIs) is a growing problem as these drugs constitute a vital part of treatment strategies and pandemic preparedness plans worldwide. Oseltamivir (Tamiflu®) is the mostly used NAI. Its active metabolite, oseltamivir carboxylate (OC), is excreted from treated patients and degrades poorly in sewage treatment plants and surface water. Thus, OC can enter aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance and resistance could develop. If NAI resistance is established in influenza viruses circulating among wild birds, the resistance can form part of a virus re-entering the human population either by reassortment or by direct transmission. In this thesis, evidence is presented that OC is present in the waterways during a seasonal influenza outbreak in Japan, a country in which oseltamivir is liberally used. Furthermore, when mallards were infected with an influenza A/H1N1 virus and subjected to low, environmental-like concentrations of OC, resistance developed through acquisition of the well-known resistance mutation H274Y. The influenza infection in the mallards was mainly intestinal, had a rapid onset and was progressing in a longitudinal fashion in the intestine. Finally, influenza A viruses isolated from wild mallards in Sweden and containing resistance-related mutations were examined by a neuraminidase inhibition assay. The viruses did not have a decreased sensitivity to NAIs, but had mutations with a resistance-enhancing potential. Thus, OC is present in the environment and environmental-like concentrations of OC induce resistance in influenza viruses of dabbling ducks. The present resistance situation among wild birds is not well understood but the existence of H274Y among wild birds, though rare, and the spread of the former seasonal A/H1N1 virus containing H274Y among humans indicate that resistance mutations could establish themselves also among wild birds. An oseltamivir-resistant pandemic or a human-adapted highly-pathogenic avian influenza virus are frightening scenarios as oseltamivir is a cornerstone in the defense in those situations. There is a need for further studies, surveillance in wild birds and for a prudent use of antivirals.

A number of environmental pollutants have been shown to mimick the action of the female sex hormone estrogen and are, therefore, suspected to be responsible for reproductive abnormalities seen in wildlife. Test systems which can be used in hazard and risk assessment of chemicals with estrogenic effects are consequently needed. In this thesis, I propose the avian egg as an in vivo test system for estrogenic compounds. I conclude that malformation of the left testis and the Müllerian ducts (MDs: embryonic oviducts) in avian embryos can be used as endpoints to examine estrogenic activity of chemicals. MD malformation is more easily determined and thereby faster to use as an endpoint than histologically observed feminization of the testis. The usefulness of MD/oviduct malformations as biomarkers for estrogenic effects in wild birds should be considered. The environmental pollutants bisphenol A (BPA) and o,p´-DDT induced similar effects as the synthetic estrogens, ethynylestradiol and diethylstilbestrol. BPA caused MD malformations in quail embryos and ovotestis formation in chicken embryos. o,p´-DDT induced MD malformations in both quail and chicken embryos and ovotestis in chicken embryos. The flame retardant, tetrabromobisphenol A did not induce estrogen-like effects in quail or chicken embryos, but showed a relatively high embryolethality. Embryonic exposure to estrogen caused persisting malformations of the oviduct, as well as a changed distribution pattern of the enzyme carbonic anhydrase in the shell gland of adult females. Considering the crucial role of carbonic anhydrase in shell formation, such changes could result in decreased shell quality. I propose that eggshell thinning in avian wildlife could reflect a functional malformation in the shell gland that is induced by xeno-estrogens during embryonic development, rather than being caused by exposure of the adult bird to environmental pollutants. This hypothesis opens new possibilities for studying the mechanisms behind contaminant-induced eggshell thinning in birds.

Binder-based assays are employed throughout the life sciences. Powerful signal amplification techniques have enabled detection of very rare molecule species diluted in simple buffers. Unspecific binding of primary binders leads to increased background in more complex samples. By requiring two recognition events, ligation-based molecular analyses provide highly specific detection of biomolecules in complex samples. We developed a highly multiplexed padlock-ligation assay targeting signature sequences in the hemagglutinin and neuraminidase genes. From a panel of 77 avian influenza isolates of all major serotypes, 97% were genotyped correctly in accordance with previous classifications by classical diagnostic methods (Paper I). Alternative splicing is an important mechanism expanding the proteome. Current analysis techniques fail to provide sequences of complete transcripts beyond the read length of sequencing instruments. We devised and implemented a strategy to compress the sequence information contained in the splicing pattern of a transcript into the presence or absence of sequence-blocks. We demonstrate that this assay yields information about the splicing patterns in thousands of transcripts from cellular cDNA (Paper II). Expression changes of mucin proteins and glycosylation structures are frequently observed from the early stages of cancer development. Expression of mucin 2 and sialyl-Tn are common features of intestinal metaplasia and gastric cancer, and are known to co-locate. Here we have developed an in situ proximity ligation assay (PLA) directed against mucin 2 and sialyl-Tn. Our study on intestinal metaplasia and gastric cancer tissue sections identified mucin 2 as a major carrier of sialyl-Tn in these conditions, and demonstrated how conveniently glycosylation of proteins can be studied by in situ PLA (Paper III). This thesis shows how the dual recognition requirement of ligation-based assays can be employed to detect target molecules with high specificity, to analyze several sequence features of nucleic acids or to study the proximity of two antigens in situ.