Temporal and spacial control of RNA stability in the early embryo of Drosophila melanogaster

Early embryogenesis in metazoa is controlled by maternally synthesized products. Among these products, the mature egg is loaded with transcripts representing approximately two thirds of the genome. A subset of this maternal RNA pool is degraded prior to the transition to zygotic control of development. This transfer of control of development from maternal to zygotic products is referred to as the midblastula transition (or MBT). It is believed that the degradation of maternal transcripts is required to terminate maternal control of development and to allow zygotic control of development to begin. Until now this process of maternal transcript degradation and the subsequent timing of the MBT has been poorly understood. I have demonstrated that in the early embryo there are two independent RNA degradation pathways, either of which is sufficient for transcript elimination. However, only the concerted action of both pathways leads to elimination of transcripts with the correct timing, at the MBT. The first pathway is maternally encoded, is triggered by egg activation, and is targeted to specific classes of mRNAs through cis-acting elements in the 3' untranslated region (UTR}. The second pathway is activated 2 hr after fertilization and functions together with the maternal pathway to ensure that transcripts are degraded by the MBT. In addition, some transcripts fail to degrade at select subcellular locations adding an element of spatial control to RNA degradation. The spatial control of RNA degradation is achieved by protecting, or masking, transcripts from the degradation machinery. The RNA degradation and protection events are regulated by distinct cis-elements in the 3' untranslated region (UTR). These results provide the first systematic dissection of this highly conserved process in development and demonstrate that RNA degradation is a novel mechanism used for both temporal and spatial control of development.

Functional genomic studies of the structure and regulation of eukaryotic transcriptomes

The main focus of this thesis is the use of high-throughput sequencing technologies in functional genomics (in particular in the form of ChIP-seq, chromatin immunoprecipitation coupled with sequencing, and RNA-seq) and the study of the structure and regulation of transcriptomes. Some parts of it are of a more methodological nature while others describe the application of these functional genomic tools to address various biological problems. A significant part of the research presented here was conducted as part of the ENCODE (ENCyclopedia Of DNA Elements) Project.

The first part of the thesis focuses on the structure and diversity of the human transcriptome. Chapter 1 contains an analysis of the diversity of the human polyadenylated transcriptome based on RNA-seq data generated for the ENCODE Project. Chapter 2 presents a simulation-based examination of the performance of some of the most popular computational tools used to assemble and quantify transcriptomes. Chapter 3 includes a study of variation in gene expression, alternative splicing and allelic expression bias on the single-cell level and on a genome-wide scale in human lymphoblastoid cells; it also brings forward a number of critical to the practice of single-cell RNA-seq measurements methodological considerations.

The second part presents several studies applying functional genomic tools to the study of the regulatory biology of organellar genomes, primarily in mammals but also in plants. Chapter 5 contains an analysis of the occupancy of the human mitochondrial genome by TFAM, an important structural and regulatory protein in mitochondria, using ChIP-seq. In Chapter 6, the mitochondrial DNA occupancy of the TFB2M transcriptional regulator, the MTERF termination factor, and the mitochondrial RNA and DNA polymerases is characterized. Chapter 7 consists of an investigation into the curious phenomenon of the physical association of nuclear transcription factors with mitochondrial DNA, based on the diverse collections of transcription factor ChIP-seq datasets generated by the ENCODE, mouseENCODE and modENCODE consortia. In Chapter 8 this line of research is further extended to existing publicly available ChIP-seq datasets in plants and their mitochondrial and plastid genomes.

The third part is dedicated to the analytical and experimental practice of ChIP-seq. As part of the ENCODE Project, a set of metrics for assessing the quality of ChIP-seq experiments was developed, and the results of this activity are presented in Chapter 9. These metrics were later used to carry out a global analysis of ChIP-seq quality in the published literature (Chapter 10). In Chapter 11, the development and initial application of an automated robotic ChIP-seq (in which these metrics also played a major role) is presented.

The fourth part presents the results of some additional projects the author has been involved in, including the study of the role of the Piwi protein in the transcriptional regulation of transposon expression in Drosophila (Chapter 12), and the use of single-cell RNA-seq to characterize the heterogeneity of gene expression during cellular reprogramming (Chapter 13).

The last part of the thesis provides a review of the results of the ENCODE Project and the interpretation of the complexity of the biochemical activity exhibited by mammalian genomes that they have revealed (Chapters 15 and 16), an overview of the expected in the near future technical developments and their impact on the field of functional genomics (Chapter 14), and a discussion of some so far insufficiently explored research areas, the future study of which will, in the opinion of the author, provide deep insights into many fundamental but not yet completely answered questions about the transcriptional biology of eukaryotes and its regulation.

Novel method to construct a quality map for phase unwrapping based on modulation and the phase gradient

A novel method to construct a quality map, called modulation-phase-gradient variance (MPGV), is proposed, based on modulation and the phase gradient. The MPGV map is successfully applied to two phase-unwrapping algorithms - the improved weighted least square and the quality-guided unwrapping algorithm. Both simulated and experimental data testify to the validity of our proposed quality map. Moreover, the unwrapped-phase results show that the new quality map can have higher reliability than the conventional phase-derivative variance quality map in helping to unwrap noisy, low-modulation, and/or discontinuous phase maps. (c) 2006 Society of Photo-Optical Instrumentation Engineers.

Novel method to construct a quality map for phase unwrapping based on modulation and the phase gradient

A novel method to construct a quality map, called modulation-phase-gradient variance (MPGV), is proposed, based on modulation and the phase gradient. The MPGV map is successfully applied to two phase-unwrapping algorithms - the improved weighted least square and the quality-guided unwrapping algorithm. Both simulated and experimental data testify to the validity of our proposed quality map. Moreover, the unwrapped-phase results show that the new quality map can have higher reliability than the conventional phase-derivative variance quality map in helping to unwrap noisy, low-modulation, and/or discontinuous phase maps. (c) 2006 Society of Photo-Optical Instrumentation Engineers.

Efficiency, energy and stoichiometry in pelagic food webs; reciprocal roles of food quality and food quantity

In most lakes, zooplankton production is constrained by food quantity, but frequently high C:P poses an additional constraint on zooplankton production by reducing the carbon transfer efficiency from phytoplankton to zooplankton. This review addresses how the flux of matter and energy in pelagic food webs is regulated by food quantity in terms of C and its stoichiometric quality in terms of C:P. Increased levels of light, CO2 and phosphorus could each increase seston mass and, hence, food quantity for zooplankton, but while light and CO2 each cause increased C:P (i.e. reduced food quality for herbivores), increased P may increase seston mass and its stoichiometric quality by reducing C:P. Development of food quality and food quantity in response to C- or P-enrichments will differ between 'batch-type' lakes (dominated by one major, seasonal input of water and nutrients) and 'continuous-culture' types of lakes with a more steady flow-rate of water and nutrients. The reciprocal role of food quantity and stoichiometric quality will depend strongly on facilitation via grazing and recycling by the grazers, and this effect will be most important in systems with low renewal rates. At high food abundance but low quality, there will be a 'quality starvation' in zooplankton. From a management point of view, stoichiometric theory offers a general tool-kit for understanding the integrated role of C and P in food webs and how food quantity and stoichiometric quality (i.e. C:P) regulate energy flow and trophic efficiency from base to top in food webs.From a management point of view, stoichiometric theory offers a general tool-kit for understanding the integrated role of C and P in food webs and how food quantity and stoichiometric quality (i.e. C:P) regulate energy flow and trophic efficiency from base to top in food webs.

A Delphi Process to Optimize Quality and Performance of Drug Evaluation in Neonates

Background: Neonatal trials remain difficult to conduct for several reasons: in particular the need for study sites to have an existing infrastructure in place, with trained investigators and validated quality procedures to ensure good clinical, laboratory practices and a respect for high ethical standards. The objective of this work was to identify the major criteria considered necessary for selecting neonatal intensive care units that are able to perform drug evaluations competently. Methodology and Main Findings: This Delphi process was conducted with an international multidisciplinary panel of 25 experts from 13 countries, selected to be part of two committees (a scientific committee and an expert committee), in order to validate criteria required to perform drug evaluation in neonates. Eighty six items were initially selected and classified under 7 headings: "NICUs description - Level of care'' (21), "Ability to perform drug trials: NICU organization and processes (15), "Research Experience'' (12), "Scientific competencies and area of expertise'' (8), "Quality Management'' (16), "Training and educational capacity'' (8) and "Public involvement'' (6). Sixty-one items were retained and headings were rearranged after the first round, 34 were selected after the second round. A third round was required to validate 13 additional items. The final set includes 47 items divided under 5 headings. Conclusion: A set of 47 relevant criteria will help to NICUs that want to implement, conduct or participate in drug trials within a neonatal network identify important issues to be aware of. Summary Points: 1) Neonatal trials remain difficult to conduct for several reasons: in particular the need for study sites to have an existing infrastructure in place, with trained investigators and validated quality procedures to ensure good clinical, laboratory practices and a respect for high ethical standards. 2) The present Delphi study was conducted with an international multidisciplinary panel of 25 experts from 13 countries and aims to identify the major criteria considered necessary for selecting neonatal intensive care units (NICUs) that are able to perform drug evaluations competently. 3) Of the 86 items initially selected and classified under 7 headings - "NICUs description - Level of care'' (21), "Ability to perform drug trials: NICU organization and processes (15), "Research Experience'' (12), "Scientific competencies and area of expertise'' (8), "Quality Management'' (16), "Training and educational capacity'' (8) and "Public involvement'' (6) - 47 items were selected following a three rounds Delphi process. 4) The present consensus will help NICUs to implement, conduct or participate in drug trials within a neonatal network.

Air Quality and Europeans' Subjective Well-being

[EN]Happiness economics deals with self-reported subjective well-being, or life satisfaction, and its relationship to a wide variety of other variables. On the study of these other factors, this line of research has helped demonstrate that higher levels of environmental quality increase people’s subjective well-being. This paper focuses on analyzing the relationship between subjective well-being and air quality. On the one hand, the life satisfaction approach to environmental valuation is cautiously described, and on the other hand, the method is implemented in an empirical analysis that seeks to assess how an increase in the level of air pollution at a regional level affects individual-level subjective well-being in Europe. We use a dataset that merges the third wave of the European Social Survey (ESS) with a dataset that includes regional air pollution (including CO, PM10, NO2, SO2 and Benzene) and other regional variables. We find a robust negative impact for CO, a positive impact for SO2, and no conclusive evidence of any effect on subjective well-being for the remaining three pollutants.

Evaluating the quality of bycatch data and bycatch estimates among disparate fisheries

In 2006, the National Marine Fisheries Service, NOAA, initiated development of a national bycatch report that would provide bycatch estimates for U.S. commercial fisheries at the fishery and species levels for fishes, marine mammals, sea turtles, and seabirds. As part of this project, the need to quantify the relative quality of available bycatch data and estimation methods was identified. Working collaboratively with fisheries managers and scientists across the nation, a system of evaluation was developed. Herein we describe the development of this system (the “tier system”), its components, and its application. We also discuss the value of the tier system in allowing fisheries managers to identify research needs and efficiently allocate limited resources toward those areas that will result in the greatest improvement to bycatch data and estimation quality.

Quality assessment of rotating and solar tunnel dried marine fish product

Studies were conducted to evaluate the quality aspects of marine dried fish i.e. silver jew fish (Johnius argentatus), Bombay duck (Harpodon nehereus) and ribbon fish (Trichiums haumela) products produced in rotating and solar tunnel dryers. On the basis of organoleptic characteristics such as colour, odour, texture, broken pieces, insect infestation and overall quality, four member panels of experts evaluated the quality of the dried products obtained from both rotating and solar tunnel dryers and all the products were found in acceptable quality. Reconstitutions properties of samples were in the range of 51.05 to 98.75% for the dried fish produced in rotating dryer, while 24.64 to 76.76% for dried fish produced in solar tunnel dryer. The highest reconstitution rate was found in dried silver jew fish and lowest in ribbon fish produced in rotating dryer. On the other hand, the highest reconstitution was observed in dried silver jew fish and lowest in dried Bombay duck produced in solar tunnel dryer. Proximate composition such as moisture, crude protein, lipid and ash content of the dried fish muscles produced in rotating dryer ranged from 16.36% to 19.1%, 62.35% to 67.37%, 6.37% to 10.75% and 7.00% to 8.05%, respectively and in solar tunnel dried fish products, they were in the range of 14.05% to 19.71%,57.64% to 69. 21%,6.92% to 15.40%and 7.69% to 8.80 %, respectively. The TVBN values of dried fish products obtained from rotating dryer were in the range of 15.02 to 19.05 mg/100g, while in solar tunnel dried fish products, the values were in the range of 15.46 to 19.21 mg/100g. The results of the studies indicated that dried fish produced from both rotating and solar tunnel drier were acceptable quality in terms of organoleptic and food quality aspects.

RNA processing and Arabidopsis flowering time control.

Plants control their flowering time in order to ensure that they reproduce under favourable conditions. The components involved in this complex process have been identified using a molecular genetic approach in Arabidopsis and classified into genetically separable pathways. The autonomous pathway controls the level of mRNA encoding a floral repressor, FLC, and comprises three RNA-binding proteins, FCA, FPA and FLK. FCA interacts with the 3'-end RNA-processing factor FY to autoregulate its own expression post-transcriptionally and to control FLC. Other components of the autonomous pathway, FVE and FLD, regulate FLC epigenetically. This combination of epigenetic and post-transcriptional control gives precision to the control of FLC expression and flowering time.

Technological aspects of preservation and processing of edible shell fishes III. Factors influencing the keeping quality of crab (Scylla serrata) during freezing and frozen storage

The possible factors leading to the loss of flavour and general quality of crab during freezing and frozen storage have been studied. The preprocess ice storage condition of the raw material was found to be one such important factor while the fresh frozen crab meat remained in good organoleptic condition for about 51 weeks at -23°C, the 7 days iced material held frozen was found to have a shelf life of about 21 weeks. The fall in myofibrillar protein noted during frozen storage together with the loss of myosin ATPase activity correlated well with the loss of organoleptic qualities.

Quality of cured fishery products from Malabar and Kanara coasts

Results of chemical, bacteriological and organoleptic quality studies of cured fishery products of commerce collected from six major fish curing centres on the west coast of India are presented. 77.12% of the samples had moisture above 35%, 97.18% showed salt content below 25% and all samples had acid insoluble ash above 1.5%. 42.32% gave standard plate counts above 10,000 and 45.77% were contaminated with 'Red' halophiles. The major defects in curing were imperfect cleaning, inadequate salting and unhygienic conditions of processing.

Quality assessment of traditional, rotary and solar tunnel dried small indigenous fish species products

Studies on the quality assessments of three traditional, rotary and solar tunnel dried SIS products were conducted. Organoleptic quality of traditional dried SIS products available in the markets was poor compared to those produced in rotary and solar tunnel dryer. Reconstitution of samples were in the range of 54.26% to 75.24%, 69.37% to 83.73% and 55.08% to 80.24% when soaked at 80°C for traditional, rotary and solar tunnel dried products, respectively. The percentage of reconstitution increased with the increase of soaking time and the uptake of water was maximum after 60 min of soaking. The moisture contents of traditional, rotary and solar tunnel dried products were in the range of 26.02% to 27.33%, 16.23% to 22.84% and 13.71% to 19.30%, respectively. The protein contents were in the range of 60.78% to 72.59%, 62.17% to 76.27% and 61.11% to 76.00%, respectively; lipid contents were in the range of 12.26% to 22.60%, 14.00% to 24.71% and 13.92% to 22.39%, respectively and ash contents in the range of 15.11% to 16.59%, 8.32% to 13.51% and 8.71% to 16.45%, respectively on dry matter basis. The TVB-N content of rotary and solar tunnel dried products was low compared to traditional one ranging from 10.64 to 17.52 mg/100g and 14.34 to 15.68 mg/100g, respectively whereas the TVB-N content of traditional samples was in the range of 15.46 to 20.36 mg/100g. The bacterial load of traditional, rotary and solar tunnel dried products were in the range of 1.43x10 super(8) CFU/g to 2.89 x10 super(80 CFU/g, 1.91x10 super(8) CFU/g to 2.84x10 super(8) CFU/g and 1.95x10 super(8) CFU/g to 2.59x10 super(8) CFU/g, respectively. The results of the study indicated that dried fish products from rotary dryer and solar tunnel dryer were found to be of better quality in nutritional and food quality aspects than those of traditional dried products.

Production and quality assessment of fish pickles from mola (Amblypharyngodon mola) fish

Fish pickles (with olive and tamarind) were prepared from mola fish (Amblypharyngodon mola) and their nutritional and food quality were assessed. The quality of the pickle prepared with olive was excellent and the pickle prepared with tamarind was found good. Moisture content of the two pickle products were 43.85% (with tamarind) and 50.89% (with olive). The protein and lipid contents of tamarind added pickle were 19.13 and 35.64% respectively; pickle with olive contained less protein (13.16%) compared to tamarind added mola pickle. Lipid contents were almost same in both cases. Ash content of two pickles was also found similar (1.00%). The quality of mola pickles stored either in cool condition (4°C) with vinegar or at room temperature with Na-benzoate were found good for consumption up to 90 days of storage. All of the fish pickles preserved under different condition were found in acceptable condition up to 240 days storage and pickle with vinegar stored at 4°C was found good for consumption at the end of 240 days.

Coupling model uncertainty for coupled rainfall/runoff and surface water quality models in river problems

Coupled hydrology and water quality models are an important tool today, used in the understanding and management of surface water and watershed areas. Such problems are generally subject to substantial uncertainty in parameters, process understanding, and data. Component models, drawing on different data, concepts, and structures, are affected differently by each of these uncertain elements. This paper proposes a framework wherein the response of component models to their respective uncertain elements can be quantified and assessed, using a hydrological model and water quality model as two exemplars. The resulting assessments can be used to identify model coupling strategies that permit more appropriate use and calibration of individual models, and a better overall coupled model response. One key finding was that an approximate balance of water quality and hydrological model responses can be obtained using both the QUAL2E and Mike11 water quality models. The balance point, however, does not support a particularly narrow surface response (or stringent calibration criteria) with respect to the water quality calibration data, at least in the case examined here. Additionally, it is clear from the results presented that the structural source of uncertainty is at least as significant as parameter-based uncertainties in areal models. © 2012 John Wiley & Sons, Ltd.