In their native habitat of Central and South America, capuchin monkeys (Cebus) spend 45% to 55% of their day foraging and a further 20% travelling. Once these monkeys are introduced into captive environments their diets are selective, seasonal and presented to them by their keepers. The captive environment often leads to various behavioural abnormalities and compensatory behaviours or stereotypies. To address this issue, environmental enrichment can be employed to reduce, cure or prevent such an occurrence. Enrichment can reduce stress, while increasing animal well-being and health in captivity. Despite previous work a better understanding of enrichment, for most neo-tropical primate species, is necessary, in order to improve their captive lifestyles. Feeding of captive primates is more complex than providing a balanced nutritional diet as it must also meet their ethological needs. The manipulation of the presentation of the diet has been shown to significantly decrease the incidence of resting, while significantly increasing the incidence of playing, grooming, foraging and manual manipulation of dietary items. Eleven capuchin monkeys were presented with four different feeding treatments (i.e. cut food presented in bowls, cut food presented around the enclosure, uncut food presented around the enclosure and novel feeding devices presented around the enclosure) from December 2007 until May 2008. At the start of every month one of three feeding treatments was introduced with the cut food in bowls feeding treatment interleaved between the treatments. The different feeding treatments required the monkeys to search for their food, break-up their food into manageable sizes, and obtain food in touch-, tool- and manipulative-dependent methods in order to allow the monkeys an opportunity to display increased activity more in line with their wild conspecifics. The capuchins displayed a period of intense foraging directly following feeding. This period significantly increased (from 44 to 121 min.), along with foraging events and the proportion of time spent foraging, which was more in line with their wild conspecifics. In addition, the frequency of occurrence and the proportion of time spent on locomotion and resting was shown to decrease. Also, abnormal behaviours ceased to occur during the study. Environmental enrichment is a useful tool for providing stimulation, redistributing activity levels more in line with wild conspecifics and to combat abnormal and compensatory behaviours.

<p>There is a strong trend in fabricating <i>miniaturized total analytical systems</i>, µTAS, for various biochemical and cell biology applications. These miniaturized systems could e.g. gain better separation performances, be faster, consume less expensive reagents and be used for studies that are difficult to access in the macro world. Disposable µTAS eliminate the risk of carry-over and can be fabricated to a low cost.</p><p>This work focused on the development of µTAS modules with the intentional use for miniaturized diagnostics. Modules for blood separation, desalting, enrichment, separation and ESI-MS detection were successfully fabricated. Surface coatings were additionally developed and evaluated for applications in µTAS with complex biological samples. The first heparin coating could be easily immobilized in a one-step-process, whereas the second heparin coating was aimed to form a hydrophilic surface that was able to draw blood or plasma samples into a microfluidic system by capillary forces. </p><p>The last mentioned heparin surface was further utilized when developing a chip-based sensor for performing CD4-count in human blood, an important marker to determine the stage of an HIV-infection.</p><p>All devices in this work were fabricated in PDMS, an elastomeric polymer with the advantage of rapid and less expensive prototyping of the microfabricated master. It was shown that PDMS could be considered as the material of choice for future commercial µTAS. The devices were intentionally produced using a low grade of fabrication complexity. It was however demonstrated that even with low complexity, it is possible to integrate several functional chip modules into a single microfluidic device.</p>

There is a strong trend in fabricating miniaturized total analytical systems, µTAS, for various biochemical and cell biology applications. These miniaturized systems could e.g. gain better separation performances, be faster, consume less expensive reagents and be used for studies that are difficult to access in the macro world. Disposable µTAS eliminate the risk of carry-over and can be fabricated to a low cost. This work focused on the development of µTAS modules with the intentional use for miniaturized diagnostics. Modules for blood separation, desalting, enrichment, separation and ESI-MS detection were successfully fabricated. Surface coatings were additionally developed and evaluated for applications in µTAS with complex biological samples. The first heparin coating could be easily immobilized in a one-step-process, whereas the second heparin coating was aimed to form a hydrophilic surface that was able to draw blood or plasma samples into a microfluidic system by capillary forces. The last mentioned heparin surface was further utilized when developing a chip-based sensor for performing CD4-count in human blood, an important marker to determine the stage of an HIV-infection. All devices in this work were fabricated in PDMS, an elastomeric polymer with the advantage of rapid and less expensive prototyping of the microfabricated master. It was shown that PDMS could be considered as the material of choice for future commercial µTAS. The devices were intentionally produced using a low grade of fabrication complexity. It was however demonstrated that even with low complexity, it is possible to integrate several functional chip modules into a single microfluidic device.

In the last decade, molecular biology has expanded from a reductionist view to a systems-wide view that tries to unravel the complex interactions of cellular components. Owing to the emergence of high-throughput technology it is now possible to interrogate entire genomes at an unprecedented resolution. The dimension and unstructured nature of these data made it evident that new methodologies and tools are needed to turn data into biological knowledge. To contribute to this challenge we exploited the wealth of publicly available high-throughput genomics data and developed bioinformatics methodologies focused on extracting information at the pathway rather than the single gene level. First, we developed Gene Set Variation Analysis (GSVA), a method that facilitates the organization and condensation of gene expression profiles into gene sets. GSVA enables pathway-centric downstream analyses of microarray and RNA-seq gene expression data. The method estimates sample-wise pathway variation over a population and allows for the integration of heterogeneous biological data sources with pathway-level expression measurements. To illustrate the features of GSVA, we applied it to several use-cases employing different data types and addressing biological questions. GSVA is made available as an R package within the Bioconductor project. Secondly, we developed a pathway-centric genome-based strategy to reposition drugs in type 2 diabetes (T2D). This strategy consists of two steps, first a regulatory network is constructed that is used to identify disease driving modules and then these modules are searched for compounds that might target them. Our strategy is motivated by the observation that disease genes tend to group together in the same neighborhood forming disease modules and that multiple genes might have to be targeted simultaneously to attain an effect on the pathophenotype. To find potential compounds, we used compound exposed genomics data deposited in public databases. We collected about 20,000 samples that have been exposed to about 1,800 compounds. Gene expression can be seen as an intermediate phenotype reflecting underlying dysregulatory pathways in a disease. Hence, genes contained in the disease modules that elicit similar transcriptional responses upon compound exposure are assumed to have a potential therapeutic effect. We applied the strategy to gene expression data of human islets from diabetic and healthy individuals and identified four potential compounds, methimazole, pantoprazole, bitter orange extract and torcetrapib that might have a positive effect on insulin secretion. This is the first time a regulatory network of human islets has been used to reposition compounds for T2D. In conclusion, this thesis contributes with two pathway-centric approaches to important bioinformatic problems, such as the assessment of biological function and in silico drug repositioning. These contributions demonstrate the central role of pathway-based analyses in interpreting high-throughput genomics data. / En l'última dècada, la biologia molecular ha evolucionat des d'una perspectiva reduccionista cap a una perspectiva a nivell de sistemes que intenta desxifrar les complexes interaccions entre els components cel•lulars. Amb l'aparició de les tecnologies d'alt rendiment actualment és possible interrogar genomes sencers amb una resolució sense precedents. La dimensió i la naturalesa desestructurada d'aquestes dades ha posat de manifest la necessitat de desenvolupar noves eines i metodologies per a convertir aquestes dades en coneixement biològic. Per contribuir a aquest repte hem explotat l'abundància de dades genòmiques procedents d'instruments d'alt rendiment i disponibles públicament, i hem desenvolupat mètodes bioinformàtics focalitzats en l'extracció d'informació a nivell de via molecular en comptes de fer-ho al nivell individual de cada gen. En primer lloc, hem desenvolupat GSVA (Gene Set Variation Analysis), un mètode que facilita l'organització i la condensació de perfils d'expressió dels gens en conjunts. GSVA possibilita anàlisis posteriors en termes de vies moleculars amb dades d'expressió gènica provinents de microarrays i RNA-seq. Aquest mètode estima la variació de les vies moleculars a través d'una població de mostres i permet la integració de fonts heterogènies de dades biològiques amb mesures d'expressió a nivell de via molecular. Per il•lustrar les característiques de GSVA, l'hem aplicat a diversos casos usant diferents tipus de dades i adreçant qüestions biològiques. GSVA està disponible com a paquet de programari lliure per R dins el projecte Bioconductor. En segon lloc, hem desenvolupat una estratègia centrada en vies moleculars basada en el genoma per reposicionar fàrmacs per la diabetis tipus 2 (T2D). Aquesta estratègia consisteix en dues fases: primer es construeix una xarxa reguladora que s'utilitza per identificar mòduls de regulació gènica que condueixen a la malaltia; després, a partir d'aquests mòduls es busquen compostos que els podrien afectar. La nostra estratègia ve motivada per l'observació que els gens que provoquen una malaltia tendeixen a agrupar-se, formant mòduls patogènics, i pel fet que podria caldre una actuació simultània sobre múltiples gens per assolir un efecte en el fenotipus de la malaltia. Per trobar compostos potencials, hem usat dades genòmiques exposades a compostos dipositades en bases de dades públiques. Hem recollit unes 20.000 mostres que han estat exposades a uns 1.800 compostos. L'expressió gènica es pot interpretar com un fenotip intermedi que reflecteix les vies moleculars desregulades subjacents a una malaltia. Per tant, considerem que els gens d'un mòdul patològic que responen, a nivell transcripcional, d'una manera similar a l'exposició del medicament tenen potencialment un efecte terapèutic. Hem aplicat aquesta estratègia a dades d'expressió gènica en illots pancreàtics humans corresponents a individus sans i diabètics, i hem identificat quatre compostos potencials (methimazole, pantoprazole, extracte de taronja amarga i torcetrapib) que podrien tenir un efecte positiu sobre la secreció de la insulina. Aquest és el primer cop que una xarxa reguladora d'illots pancreàtics humans s'ha utilitzat per reposicionar compostos per a T2D. En conclusió, aquesta tesi aporta dos enfocaments diferents en termes de vies moleculars a problemes bioinformàtics importants, com ho son el contrast de la funció biològica i el reposicionament de fàrmacs "in silico". Aquestes contribucions demostren el paper central de les anàlisis basades en vies moleculars a l'hora d'interpretar dades genòmiques procedents d'instruments d'alt rendiment.

Semiconductor quantum dots (QDs) have emerged as a new class of fluorescent probes and labeling agents for biological samples. QDs are bright, highly photostable and allow simultaneous excitation of multiple emissions. Owing to these properties, QDs hold exceptional promise in enabling intracellular biochemical studies and diagnosis with unprecedented sensitivity and accuracy. However, use of QD probes inside living cells remains a challenge due to difficulties in delivery of nanoparticles without causing aggregation and imaging single nanoparticles inside living cells. In this dissertation, a systematic approach to deliver, image and locate single QDs inside living cells is presented and the properties of molecular motor protein driven QD transport are studied. First, spectroscopic and imaging methods capable of differentiating single nanoparticles from the aggregates were developed. These technologies were validated by differentiating surface protein expression on viral particles and by enabling rapid counting of single biomolecules. Second, controlled delivery of single QDs into living cells is demonstrated. A surprising finding is that single QDs associate non-specifically with the dynein motor protein complex and are transported to the microtubule organizing center. Accurate localization and tracking of QDs inside cell cytoplasm revealed multiple dynein motor protein attachment resulting in increased velocity of the QDs. Further, spectrin molecule which is known to recruit dynein motor protein complex to phospholipid micelles was found to associate with the QDs. These results may serve as a benchmark for developing new QD surface coatings suitable for intracellular applications. Since, nanoparticles are similar in size to viral pathogens; better understanding of nanoparticle-cell interactions should also help engineer nanoparticle models to study virus-host cell interactions. (Contains AVI format multimedia files)

It has been suggested that organismal complexity correlates with the complexity of gene regulation. Transcriptional control of gene expression is mediated by binding of regulatory proteins to cis-acting sequences on the genome. Hence, it is crucial to identify the chromosomal targets of transcription factors (TFs) to delineate transcriptional regulatory networks underlying gene expression programs. The development of ChIP-chip technology has enabled high throughput mapping of TF binding sites across the genome. However, there are many limitations to the technology including the availability of whole genome arrays for complex organisms such human or mouse. To circumvent these limitations, we developed the Sequence Tag Analysis of Genomic Enrichment (STAGE) methodology that is based on extracting short DNA sequences or “tags” from ChIP-enriched DNA. With improvements in sequencing technologies, we applied the recently developed ChIP-Seq technique i.e. ChIP followed by ultra high throughput sequencing, to identify binding sites for the TF E2F4 across the human genome. We identified previously uncharacterized E2F4 binding sites in intergenic regions and found that several microRNAs are potential E2F4 targets. Binding of TFs to their respective chromosomal targets requires access of the TF to its regulatory element, which is strongly influenced by nucleosomal remodeling. In order to understand nucleosome remodeling in response to transcriptional perturbation, we used ultra high throughput sequencing to map nucleosome positions in yeast that were subjected to heat shock or were grown normally. We generated nucleosome remodeling profiles across yeast promoters and found that specific remodeling patterns correlate with specific TFs active during the transcriptional reprogramming. Another important aspect of gene regulation operates at the post-transcriptional level. MicroRNAs (miRNAs) are ~22 nucleotide non-coding RNAs that suppress translation or mark mRNAs for degradation. MiRNAs regulate TFs and in turn can be regulated by TFs. We characterized a TF-miRNA network involving the oncofactor Myc and the miRNA miR-22 that suppresses the interferon pathway as primary fibroblasts enter a stage of rapid proliferation. We found that miR-22 suppresses the interferon pathway by inhibiting nuclear translocation of the TF NF-kappaB. Our results show how the oncogenic TF Myc cross-talks with other TF regulatory pathways via a miRNA intermediary. / text

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

The following research has focused on the utility of Ulva as an indicator of seawater nitrogen loading. Evaluation was made in three ways: 1) Observation of large-scale geographic variation in nitrogen status in natural populations around New Zealand in summer and winter, 2) Laboratory-based experimental assessment of the biochemical responses of N-indices in Ulva to nitrogen enrichment, and 3) Culturing standardized test-Ulva under low nutrient conditions which could be deployed into a variety of field situations. Seawater inorganic nutrient (nitrate, nitrite, ammonium and phosphate) concentrations and nitrogen (N)-indices (free amino acids, chlorophyll and total tissue nitrogen) in natural Ulva populations from 32 sites around New Zealand were compared. Sites were divided into 6 environmental categories: sheltered rural, exposed rural, rock pools, sheltered urban, exposed urban, and nitrogen-enriched urban sites. Seawater nutrient concentrations were highly variable between all sites in summer and winter. However, in the summer enriched urban sites had the highest mean total inorganic nitrogen concentrations and Ulva with the highest mean levels of all N-indices compared with any other environmental category. In the winter, Ulva contained more nitrogen (reflected in all N-indices) compared with Ulva in the summer, particularly in populations growing in colder southern seawater on more exposed coasts. The increase in Ulva N-status was not explained by increased seawater inorganic nitrogen concentrations. With univariate and multivariate statistical approaches it was shown that there was a significant effect of seawater temperature and site exposure on N-status in Ulva. Compared with other N-indices, stable nitrogen isotopes (δ15N) from Ulva growing in enriched urban sites had the widest range (4.77 ± 0.04 ‰ to 15.16 ± 0.03 ‰) of values compared with all other categories in both summer and winter. Conversely, Ulva from exposed rural sites had the lowest range of δ15N values compared with any other category (6.7 ± 0.1 to 8.8 ± 0.1 ‰) and showed no seasonal change in mean values (7.8 ‰ and 7.6 ‰ for summer and winter, respectively). In addition, δ15N values in Ulva were the only N-index that showed a significant difference between urban and rural categories. To test the relationship between inorganic nitrogen concentration in seawater and the responses of biochemical nitrogen indices in Ulva pertusa, several experiments were conducted in an outdoor, flow-through culture apparatus, in summer and winter. In this apparatus effects of ammonium concentration, nitrogen source (nitrate and ammonium), light and seawater motion were investigated. Of the same three N-indices examined in natural Ulva populations (free amino acids, chlorophyll and total tissue nitrogen), increases in free amino acids, particularly asparagine, provided the strongest indicator of increases in nitrogen availability. In addition, while tissue nitrogen and chlorophyll also increased with seawater nitrogen concentration, it was apparent that these indices were also strongly influenced by light, and probably season. Rates of ammonium assimilation provided no overall measure of the availability of nitrogen in seawater and were clearly affected by season. Similarly, growth rates in Ulva only showed a response to nitrogen addition in summer months. Stable isotopes of nitrogen (δ15N) in Ulva provided a clear distinction between natural and synthetic nitrogen sources, but more importantly, showed only minor fractionation (ranging from 1.3 ‰ to -1.9 ‰) of 15N supplied from synthetic nitrate and ammonium under both light-saturating and light-limiting conditions. To further develop Ulva as a standardized test-organism it was cultured in low-nutrient (non-polluted) seawater to deplete internal storage pools of nitrogen. Each month the resulting test-Ulva was then placed in surface-moored growth enclosures at a range of coastal sites around Auckland and then monitored for one year. In winter there were increases in seawater inorganic nitrogen concentrations and concomitant increases in free amino acid content. However, tissue nitrogen and chlorophyll content in test-Ulva showed similar increases (possibly saturating) across all sites suggesting that seasonal increases in these N-indices were also due to other seasonal factors (e.g., surface irradiance and / or seawater temperature). On the other hand, the total free amino acid pool showed strong differences between a low-nitrogen reference site and the other study sites all year round. It was probable that test-Ulva was integrating differences in tidally-averaged nitrogen loading that were not reliably detected in instantaneous seawater samples. In addition to N-indices in test-Ulva, levels of tissue heavy metals and stable isotopes of nitrogen showed strong differences with higher values of both typically found in urban environments compared with values found in non-polluted reference sites. It is concluded that several abiotic and biotic factors affect nitrogen status in Ulva, but the average nitrogen concentration in seawater, and the physical factors of temperature, light and water motion, appear to be the overarching determinants. It is further suggested that in combination with Ulva tissue δ15N values, tissue nitrogen and the free amino acid pool, as quantitative biochemical measures of nitrogen availability, are likely to provide useful information on both the amount and composition of nitrogen entering coastal environments. / Foundation for Research, Science and Technology. Auckland Regional Council.

The following research has focused on the utility of Ulva as an indicator of seawater nitrogen loading. Evaluation was made in three ways: 1) Observation of large-scale geographic variation in nitrogen status in natural populations around New Zealand in summer and winter, 2) Laboratory-based experimental assessment of the biochemical responses of N-indices in Ulva to nitrogen enrichment, and 3) Culturing standardized test-Ulva under low nutrient conditions which could be deployed into a variety of field situations. Seawater inorganic nutrient (nitrate, nitrite, ammonium and phosphate) concentrations and nitrogen (N)-indices (free amino acids, chlorophyll and total tissue nitrogen) in natural Ulva populations from 32 sites around New Zealand were compared. Sites were divided into 6 environmental categories: sheltered rural, exposed rural, rock pools, sheltered urban, exposed urban, and nitrogen-enriched urban sites. Seawater nutrient concentrations were highly variable between all sites in summer and winter. However, in the summer enriched urban sites had the highest mean total inorganic nitrogen concentrations and Ulva with the highest mean levels of all N-indices compared with any other environmental category. In the winter, Ulva contained more nitrogen (reflected in all N-indices) compared with Ulva in the summer, particularly in populations growing in colder southern seawater on more exposed coasts. The increase in Ulva N-status was not explained by increased seawater inorganic nitrogen concentrations. With univariate and multivariate statistical approaches it was shown that there was a significant effect of seawater temperature and site exposure on N-status in Ulva. Compared with other N-indices, stable nitrogen isotopes (δ15N) from Ulva growing in enriched urban sites had the widest range (4.77 ± 0.04 ‰ to 15.16 ± 0.03 ‰) of values compared with all other categories in both summer and winter. Conversely, Ulva from exposed rural sites had the lowest range of δ15N values compared with any other category (6.7 ± 0.1 to 8.8 ± 0.1 ‰) and showed no seasonal change in mean values (7.8 ‰ and 7.6 ‰ for summer and winter, respectively). In addition, δ15N values in Ulva were the only N-index that showed a significant difference between urban and rural categories. To test the relationship between inorganic nitrogen concentration in seawater and the responses of biochemical nitrogen indices in Ulva pertusa, several experiments were conducted in an outdoor, flow-through culture apparatus, in summer and winter. In this apparatus effects of ammonium concentration, nitrogen source (nitrate and ammonium), light and seawater motion were investigated. Of the same three N-indices examined in natural Ulva populations (free amino acids, chlorophyll and total tissue nitrogen), increases in free amino acids, particularly asparagine, provided the strongest indicator of increases in nitrogen availability. In addition, while tissue nitrogen and chlorophyll also increased with seawater nitrogen concentration, it was apparent that these indices were also strongly influenced by light, and probably season. Rates of ammonium assimilation provided no overall measure of the availability of nitrogen in seawater and were clearly affected by season. Similarly, growth rates in Ulva only showed a response to nitrogen addition in summer months. Stable isotopes of nitrogen (δ15N) in Ulva provided a clear distinction between natural and synthetic nitrogen sources, but more importantly, showed only minor fractionation (ranging from 1.3 ‰ to -1.9 ‰) of 15N supplied from synthetic nitrate and ammonium under both light-saturating and light-limiting conditions. To further develop Ulva as a standardized test-organism it was cultured in low-nutrient (non-polluted) seawater to deplete internal storage pools of nitrogen. Each month the resulting test-Ulva was then placed in surface-moored growth enclosures at a range of coastal sites around Auckland and then monitored for one year. In winter there were increases in seawater inorganic nitrogen concentrations and concomitant increases in free amino acid content. However, tissue nitrogen and chlorophyll content in test-Ulva showed similar increases (possibly saturating) across all sites suggesting that seasonal increases in these N-indices were also due to other seasonal factors (e.g., surface irradiance and / or seawater temperature). On the other hand, the total free amino acid pool showed strong differences between a low-nitrogen reference site and the other study sites all year round. It was probable that test-Ulva was integrating differences in tidally-averaged nitrogen loading that were not reliably detected in instantaneous seawater samples. In addition to N-indices in test-Ulva, levels of tissue heavy metals and stable isotopes of nitrogen showed strong differences with higher values of both typically found in urban environments compared with values found in non-polluted reference sites. It is concluded that several abiotic and biotic factors affect nitrogen status in Ulva, but the average nitrogen concentration in seawater, and the physical factors of temperature, light and water motion, appear to be the overarching determinants. It is further suggested that in combination with Ulva tissue δ15N values, tissue nitrogen and the free amino acid pool, as quantitative biochemical measures of nitrogen availability, are likely to provide useful information on both the amount and composition of nitrogen entering coastal environments. / Foundation for Research, Science and Technology. Auckland Regional Council.

The following research has focused on the utility of Ulva as an indicator of seawater nitrogen loading. Evaluation was made in three ways: 1) Observation of large-scale geographic variation in nitrogen status in natural populations around New Zealand in summer and winter, 2) Laboratory-based experimental assessment of the biochemical responses of N-indices in Ulva to nitrogen enrichment, and 3) Culturing standardized test-Ulva under low nutrient conditions which could be deployed into a variety of field situations. Seawater inorganic nutrient (nitrate, nitrite, ammonium and phosphate) concentrations and nitrogen (N)-indices (free amino acids, chlorophyll and total tissue nitrogen) in natural Ulva populations from 32 sites around New Zealand were compared. Sites were divided into 6 environmental categories: sheltered rural, exposed rural, rock pools, sheltered urban, exposed urban, and nitrogen-enriched urban sites. Seawater nutrient concentrations were highly variable between all sites in summer and winter. However, in the summer enriched urban sites had the highest mean total inorganic nitrogen concentrations and Ulva with the highest mean levels of all N-indices compared with any other environmental category. In the winter, Ulva contained more nitrogen (reflected in all N-indices) compared with Ulva in the summer, particularly in populations growing in colder southern seawater on more exposed coasts. The increase in Ulva N-status was not explained by increased seawater inorganic nitrogen concentrations. With univariate and multivariate statistical approaches it was shown that there was a significant effect of seawater temperature and site exposure on N-status in Ulva. Compared with other N-indices, stable nitrogen isotopes (δ15N) from Ulva growing in enriched urban sites had the widest range (4.77 ± 0.04 ‰ to 15.16 ± 0.03 ‰) of values compared with all other categories in both summer and winter. Conversely, Ulva from exposed rural sites had the lowest range of δ15N values compared with any other category (6.7 ± 0.1 to 8.8 ± 0.1 ‰) and showed no seasonal change in mean values (7.8 ‰ and 7.6 ‰ for summer and winter, respectively). In addition, δ15N values in Ulva were the only N-index that showed a significant difference between urban and rural categories. To test the relationship between inorganic nitrogen concentration in seawater and the responses of biochemical nitrogen indices in Ulva pertusa, several experiments were conducted in an outdoor, flow-through culture apparatus, in summer and winter. In this apparatus effects of ammonium concentration, nitrogen source (nitrate and ammonium), light and seawater motion were investigated. Of the same three N-indices examined in natural Ulva populations (free amino acids, chlorophyll and total tissue nitrogen), increases in free amino acids, particularly asparagine, provided the strongest indicator of increases in nitrogen availability. In addition, while tissue nitrogen and chlorophyll also increased with seawater nitrogen concentration, it was apparent that these indices were also strongly influenced by light, and probably season. Rates of ammonium assimilation provided no overall measure of the availability of nitrogen in seawater and were clearly affected by season. Similarly, growth rates in Ulva only showed a response to nitrogen addition in summer months. Stable isotopes of nitrogen (δ15N) in Ulva provided a clear distinction between natural and synthetic nitrogen sources, but more importantly, showed only minor fractionation (ranging from 1.3 ‰ to -1.9 ‰) of 15N supplied from synthetic nitrate and ammonium under both light-saturating and light-limiting conditions. To further develop Ulva as a standardized test-organism it was cultured in low-nutrient (non-polluted) seawater to deplete internal storage pools of nitrogen. Each month the resulting test-Ulva was then placed in surface-moored growth enclosures at a range of coastal sites around Auckland and then monitored for one year. In winter there were increases in seawater inorganic nitrogen concentrations and concomitant increases in free amino acid content. However, tissue nitrogen and chlorophyll content in test-Ulva showed similar increases (possibly saturating) across all sites suggesting that seasonal increases in these N-indices were also due to other seasonal factors (e.g., surface irradiance and / or seawater temperature). On the other hand, the total free amino acid pool showed strong differences between a low-nitrogen reference site and the other study sites all year round. It was probable that test-Ulva was integrating differences in tidally-averaged nitrogen loading that were not reliably detected in instantaneous seawater samples. In addition to N-indices in test-Ulva, levels of tissue heavy metals and stable isotopes of nitrogen showed strong differences with higher values of both typically found in urban environments compared with values found in non-polluted reference sites. It is concluded that several abiotic and biotic factors affect nitrogen status in Ulva, but the average nitrogen concentration in seawater, and the physical factors of temperature, light and water motion, appear to be the overarching determinants. It is further suggested that in combination with Ulva tissue δ15N values, tissue nitrogen and the free amino acid pool, as quantitative biochemical measures of nitrogen availability, are likely to provide useful information on both the amount and composition of nitrogen entering coastal environments. / Foundation for Research, Science and Technology. Auckland Regional Council.