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The mammalian nucleus is arguably the most complex cellular organelle. It houses the vast majority of an organism's genetic material and is the site of all major genome regulatory processes. Reductionist approaches have been spectacularly successful at dissecting at the molecular level many of the key processes that occur within the nucleus, particularly gene expression. At the same time, the limitations of analyzing single nuclear processes in spatial and temporal isolation and the validity of generalizing observations of single gene loci are becoming evident. The next level of understanding of genome function is to integrate our knowledge of their sequences and the molecular mechanisms involved in nuclear processes with our insights into the spatial and temporal organization of the nucleus and to elucidate the interplay between protein and gene networks in regulatory circuits. To do so, catalogues of genomes and proteomes as well as a precise understanding of the behavior of molecules in living cells are required. Converging technological developments in genomics, proteomics, dynamics and computation are now leading towards such an integrated biological understanding of genome biology and nuclear function.
The sequencing of whole genomes represents a landmark in modern biology (Adams et al., 2000; Goffeau et al., 1996; Lander et al., 2001; Venter et al., 2001; Waterston et al., 2002), revolutionizing the way genes are found, classified and analyzed. It has also brought about a shift in how biological problems are approached. It has encouraged us to move beyond the limitations of understanding single processes and inspired us to ask how biological events occur at the systems level; rather than analyzing the behavior of a single signaling kinase, for example, we now seek to understand how all elements in a signaling pathway interact; rather than asking how a gene responds to an extracellular cue, we now want to know how the genome as a whole responds. These types of global approach represent the first steps towards understanding biological functions as they really occur – in the context of biological systems.
While the sequencing of genomes has influenced virtually all fields of biology, it has had the most profound impact on the study of gene expression itself. This is particularly true because the availability of genome sequence information has coincided with the development of microarray analysis, which allows us to interrogate gene expression at a system level (Schena et al., 1995). Although most genome-wide analysis methods are largely descriptive and generally only provide lists of what parts of the genome undergo changes in activity, we can now routinely mine genome-wide expression data, using computational tools to predict biological pathways involved in a particular physiological response or to group samples, for example, normal and malignant tissues, according to expression patterns (Slonim, 2002). Despite these successes, a key realization has been that even these comprehensive approaches cannot answer some of the most fundamental questions in genome biology, such as why more advanced organisms do not necessarily use more genes to achieve complexity; how gene expression programs are defined in a tissue- and cell-type-specific manner; and how the spatial and temporal organization of transcription, RNA processing, RNA degradation, export, DNA replication or DNA repair affects genome function. It seems clear now that, apart from genome sequence, additional aspects of genome biology must be considered if we are to understand how genomes actually work.
A key emerging contributor to genome function and regulation is the spatial and temporal arrangement of the genome and gene expression processes in nuclear space (Misteli, 2001; Spector, 2003). Dramatic developments in high-resolution and live-cell imaging have revealed that the cell nucleus is a highly heterogeneous and complex organelle, and that global genome architecture changes during processes such as differentiation and development (Misteli, 2001; Spector, 2003). One unique feature of the mammalian cell nucleus is the presence of structural and functional domains that lack membrane boundaries (Lamond and Earnshaw, 1998; Matera, 1999). Spatial organization of the genome is achieved by a non-random arrangement of chromosomes in the interphase nucleus, with chromosomes occupying preferential intranuclear positions (Cremer and Cremer, 2001; Parada et al., 2004). Chromosomes, genome domains and gene loci may congress in space to form functional chromatin neighborhoods, such as transcriptionally silent heterochromatin regions or clusters of active genes. In addition to the non-random spatial arrangement of genomes, the nucleus also contains numerous proteinaceous domains, such as the nucleolus and splicing factor compartments, which represent distinct structural, and probably functional, subcompartments (Misteli, 2005). Although the full contribution of the spatial organization of the genome and nuclear proteins is still unknown, it seems clear that we must understand genome function in the context of this architectural organization to answer some of the key questions in genome biology.
The ultimate goal of a systems biology view of the cell nucleus is to understand genome function within the architectural framework of the nucleus. Gaining such a systems view of nuclear function will involve several steps. First, we must generate proteomic and genomic inventories of nuclear components, including genome sequence elements, epigenetic modifications, higher-order chromatin structure, chromatin-binding complexes and nuclear compartments. Second, we must understand the cell biological properties of nuclear processes in living cells in terms of characteristics such as their spatial organization and dynamic properties. Third, we must integrate experimental data on process dynamics and spatial organization, using computational approaches (Fig. 1). Several technological developments now make this a realistic, although still highly challenging, goal. On the one hand, proteomic and genomic approaches can provide complete lists of components present in particular nuclear structures, and identify what transcription factors are bound where throughout the genome, and what coding and non-coding genome regions are active at any given time. On the other hand, quantitative in vivo microscopy methods provide the first glimpse of how DNA, RNA and proteins behave inside the nuclei of living cells. Combining these methods and mining the data by emerging computational approaches will eventually lead to a realistic picture of gene expression. We review here how far we have progressed on the long road towards achieving a systems biology view of the cell nucleus and of genome function.
Nuclear inventories
Proteomics in the cell nucleus
The mammalian cell nucleus contains an estimated 20% of all cellular proteins (Simpson et al., 2000). Although many exist freely in the nucleoplasm, numerous proteins are enriched in particular nuclear compartments. A crucial first step towards an integrated view of the nucleus and its compartments is the generation of a protein inventory for each domain (Fig. 2). Proteomic approaches involving biochemical purification and mass spectrometry are now beginning to yield this information. The first nuclear structure to be analyzed in this manner was the interchromatin granule cluster (IGC), which constitutes the major fraction of the splicing factor compartments (Mintz et al., 1999; Saitoh et al., 2004). Previously, these structures were thought to contain mostly proteins involved in pre-mRNA splicing, but the detection of >350 proteins, many of which have no apparent function in the splicing reaction, in purified IGCs suggests they have additional functions (Mintz et al., 1999; Saitoh et al., 2004). Proteomic analysis has now revealed a similarly astonishing number of proteins associated with the nucleolus. The latest count for mammalian nucleolar proteins is ∼600 proteins, and the plant nucleolus appears to have at least 200 proteins (Andersen et al., 2005; Pendle et al., 2005; Scherl et al., 2002). These remarkably high numbers of constituents, and the fact that many are novel proteins, provide a clear indication of how little we know about the functions of nuclear domains as a whole.
Two remarkable commonalities between the proteomic analyses of IGCs and the nucleolus are noteworthy. First, neither revealed the presence of conserved targeting signals in resident proteins, which suggests that the enrichment of protein subsets is largely determined by the functions they have in a particular compartment rather than by specific targeting mechanisms (Dundr and Misteli, 2002). Second, proteins that one would not expect to be present in these compartments are abundant (Andersen et al., 2005; Saitoh et al., 2004). For example, histones are associated with IGCs although these compartments are insensitive to DNAse and are generally assumed to be chromatin free (Saitoh et al., 2004). Conversely, numerous pre-mRNA splicing factors can be detected in the nucleolus, despite the fact that the ribosomal genes found within the nucleolus do not produce spliceable pre-mRNAs (Andersen et al., 2005). Although contaminating proteins may contribute to some of these surprising findings, it seems more probable that the unexpected appearance of proteins in certain compartments reflects their ability to diffuse relatively freely throughout the nuclear space and stochastically associate with nuclear compartments non-specifically (Dundr and Misteli, 2002).
The behavior of single proteins in response to changes in cellular conditions is traditionally followed by in vivo microscopy. Proteomic approaches now allow us to track changes in the composition of entire subnuclear organelles. Proteins can be tagged with multiple metabolic labels, which allows us to trace the compositions of entire organelles over time (Andersen et al., 2005). When applied to the nucleolus, this approach demonstrates that, upon inhibition of transcription, nucleolar components can behave very differently: some accumulate in the morphologically altered nucleolus, whereas others dissociate (Andersen et al., 2005). Remarkably, functionally related proteins or proteins known to be present in a complex, such as pre-ribosomes, often exhibit similar kinetics, which suggests that this method can also be used to probe the dynamics of protein complexes and functionally related groups of proteins. These observations highlight the dynamic nature of the nucleolus, and probably of nuclear compartments in general. More importantly, the ability to follow the fate of all components of a nuclear domain under changing physiological conditions is a powerful tool that will help us to understand how nuclear architecture responds at the molecular level as a system to signaling cues.
The components of various other nuclear structures have recently also been cataloged. Several hundred proteins that purify with the nuclear envelope have been identified and the composition of the nuclear pore complex (NPC) has been similarly characterized (Rout et al., 2000; Schirmer et al., 2003). Since nuclear lamins and associated nuclear envelope proteins have been implicated in various human diseases, this former effort has provided leads to numerous novel putative disease genes and mechanisms (Schirmer et al., 2003). The proteomic analysis of the NPC has allowed the spatial mapping of all NPC components within the three-dimensional structure of the pore and, on the basis of homology analysis, functional roles for the various NPC components have been proposed, which can now be experimentally tested (Rout et al., 2003).
Proteomic approaches are not limited to the analysis of architectural features of the nucleus, but can similarly be applied to nuclear protein complexes. Indeed, the protein compositions of pre-ribosomes and spliceosomes have been determined (Bassler et al., 2001; Harnpicharnchai et al., 2001; Rappsilber et al., 2002; Zhou et al., 2002). Analysis of such complexes is facilitated by their particle-like nature, which makes them more amenable to purification than nuclear compartments. Use of versatile protein-tagging systems now also allows identification of complexes that are generally more difficult to purify. A powerful demonstration of such an approach is the recent purification and proteomic analysis of protein complexes that bind to and define the boundary between silenced chromatin regions at Saccharomyces cerevisiae telomeres and the neighboring subtelomeric active regions (Tackett et al., 2005). In this approach, tagged proteins were crosslinked to their binding sites and mapped throughout the genome. Intriguingly, complexes with compositions similar to the one at the telomeric boundary were found at various locations throughout the genome, allowing the possibility that these sites have similarly, yet unappreciated, boundary functions. The ability to analyze not only soluble complexes and nuclear compartments but also chromatin-bound large macromolecular assemblies, will be crucial in global mapping of protein complexes along the genome.
Genomics in the cell nucleus
Beyond coding regions
The most basic component of a systems view of the nucleus is complete genome expression profiles under various physiological conditions (Fig. 2). Although microarray technology is now a standard tool, most such analyses have been limited to the <10% of total genome information that represents coding regions. The recent development of high-density tiling arrays that also contain non-coding regions has started to reveal the true complexity of the metazoan transcriptome (Bernstein et al., 2005; Cawley et al., 2004; Cheng et al., 2005; Kampa et al., 2004). For example, an analysis of ten human chromosomes at 5 bp resolution identified as much as 32% of cellular transcripts to come from intergenic, non-coding regions, and >60% of the transcripts that hybridize with unannotated sequences show transcription from both genomic strands (Cheng et al., 2005). Systematic analysis of the function of these non-coding RNAs is particularly important in light of the emerging roles of mirco-RNAs in key physiological processes. These regulatory molecules represent nearly 1% of all genes in higher eukaryotes (Bartel, 2004), and have been implicated in developmental timing, neuronal patterning (Lee et al., 1993; Wightman et al., 1993), apoptosis (Brennecke et al., 2003) and cellular proliferation (Hatfield et al., 2005), and can be used in cancer profiling (Lu et al., 2005). These observations make it clear that, just as we are beginning to be able to explore gene expression globally, we must realize that a much larger percentage of the genome is being transcribed than is presently appreciated. Determination of the functional impact of transcription from these non-coding regions will be crucial to our understanding of genome function.
Transcription factor binding
A further key piece of information is the location of binding sites for the multitude of transcription factors bound along the genome (Fig. 2). Whereas in-silico bioinformatics has yielded some of this information by mapping consensus sites throughout the genome, approaches involving chromatin immunoprecipitation (ChIP) coupled with microarray analysis, (the so called ChIP-on-Chip approach), can reveal the actual binding sites within the genome for a particular protein under a given condition. In ChIP-on-Chip, proteins are crosslinked to DNA in vivo, they are then immunoprecipitated, the bound DNA amplified, and all recovered fragments are identified on microarrays and mapped onto the genome sequence. Such methods are beginning to reveal several surprising mechanistic principles of how proteins interact with the genome.
Bioinformatic analyses generally assume that the presence of a particular target sequence for a transcription factor means that it is used; genome-wide analysis by ChIP-on-Chip questions this view. In one study, mapping of binding sites for the transcriptional activator Gal4 showed that not all consensus binding sequences are occupied when S. cerevisiae cells are grown in the presence of galactose. This indicates that sequence itself is not sufficient for a protein to bind (Ren et al., 2000). It is not entirely clear what determines whether a specific binding site is occupied, but it seems probable that the combinatorial binding of several proteins as well as the status of higher-order chromatin structure contributes significantly.
A concept that has emerged from such analyses is how a single factor can regulate the expression of different sets of genes. This behavior is exemplified by the transcription factor Ste12, in S. cerevisiae. Upon the addition of pheromone, Ste12p activates genes involved in mating, whereas during starvation the same factor activates genes in the filamentous growth pathway. Genome-wide location analysis showed that binding of Ste12p to genes that regulate filamentous growth requires the transcription factor binding partner Tec1, but the binding of Ste12p to mating genes does not. Tec1 is negatively regulated by a pheromone-activated MAP kinase, which results in selective binding of Ste12p to mating genes because activation of filamentous genes by the Ste12p-Tec1 complex is inhibited (Zeitlinger et al., 2003). Therefore, regulation of gene expression by common factors may occur through modulation of the activities of their binding partners rather than solely through their interaction with the target sequence.
Genomic approaches have also proven important in identifying the targets of transcription factors that coordinate central cellular processes such as proliferation, growth, differentiation and apoptosis (Cawley et al., 2004; Fernandez et al., 2003; Levens, 2003; Orian et al., 2003). For example, the proto-oncogene Myc has been shown to bind to and modulate the levels of up to 10% of human genes (Fernandez et al., 2003; Orian et al., 2003). A further surprise was the location of the binding sites for important transcription factors such as Myc, Sp1 and p53 in relation to coding regions (Cawley et al., 2004). Analysis using tiling arrays across chromosomes 21 and 22 showed that these proteins, and presumably others, occupy many more sites than previously thought. Moreover, <25% of these binding sites are at the 5′ end of known genes, but >36% of binding sites are located in or proximal to the 3′ regions. Interestingly, many of these sites coincide with regions containing non-coding RNAs, which suggests the existence of an as-yet-unappreciated level of genome regulation by transcription factors through control of non-coding RNA expression (Cawley et al., 2004).
Chromatin organization and modifications
One way the usage of a transcription-factor-binding site can be controlled is by packaging of DNA within chromatin. Indeed, transcription-factor-binding sites within promoters have decreased nucleosome occupancy in yeast (Dion et al., 2005; Lee et al., 2004; Yuan et al., 2005). Interestingly, an inverse correlation between gene activity and nucleosome occupation is observed when global gene expression is modulated by heat shock (Lee et al., 2004). In addition to local DNA accessibility, higher-order chromatin structure also affects genome function. Consistent with this view, analysis of a small number of genes more than two decades ago suggested that genes that are actively transcribed exist in an open conformation, whereas inactive genes are maintained in a condensed chromatin structure (Kimura et al., 1983). Recent genome-wide mapping of open and closed chromatin regions, and their correlation with gene activity, paints a more complicated picture. Using fluorescence in situ hybridization and DNA microarray analysis with probes for either open or condensed chromatin regions, a direct correlation was identified between chromatin compaction and gene density, but not necessarily gene activity (Gilbert et al., 2004). Gene-rich chromosomes, such as human chromosome 22, contain large regions of chromatin in an open conformation compared with gene-poor regions within chromosome 1. Despite this structural heterogeneity, a good correlation with gene transcription was not observed because many silenced genes are also located in these open chromatin regions (Gilbert et al., 2004). These results suggest that the role of chromatin in the regulation of gene expression occurs on a more local basis. Such a notion is consistent with results from genome-wide binding studies demonstrating that chromatin-remodeling and -modifying complexes that can change the local structure of chromatin and expose binding sites are recruited to promoters of many genes upon activation (Lusser and Kadonaga, 2003; Robert et al., 2004).
The functional status of chromatin often broadly correlates with post-translational modifications of the core histones and these modifications have been proposed to constitute an epigenetic code (Strahl and Allis, 2000). Genomic analyses can identify the patterns of these modifications and link them to features of the genome such as regulatory sequences and to gene activity (Fig. 2) (Bernstein et al., 2005; Dion et al., 2005; Kurdistani et al., 2004; Roh et al., 2005; Schubeler et al., 2004). For example, in human T cells the distribution and relative quantities of acetylated histones H3 and H4 have been mapped across the genome (Roh et al., 2005). This method was able to identify islands of acetylation that preferentially occur at known promoters and regulatory sequences rather than in the main body of genes.
Similar studies are beginning to reveal patterns of different modifications in relation to gene activity and organization (Bernstein et al., 2005; Schubeler et al., 2004). In yeast, for example, histone acetylation has a combinatorial effect (Kurdistani et al., 2004). When the distribution of the acetylation status of eleven different lysines in the same histone octamer region was determined, distinct combinations of acetylated and deacetylated lysines were observed at specific genomic locations. Intriguingly, gene regions that have similar modification patterns are co-regulated, which supports the notion of a combinatorial code (Kurdistani et al., 2004). However, the specific roles these acetylation patterns play in recruiting histone-associated proteins are still not clear. It has been a long standing view that acetylation of histones could play a non-specific role in maintenance of chromatin structure and dynamics (Horn and Peterson, 2002). Indeed, an analysis of combinations of four different modifications in the same octamer unit, demonstrated that three of the residues do not play a specific role in transcription but pointed towards a general cumulative effect, potentially on chromatin structure (Dion et al., 2005). Despite these differences, the power of a system-wide analysis of histone modifications should lead to elucidation of the functional link between these modifications and gene activity.
Systematic mapping of DNA replication sites
The structural state of chromatin has also been implicated in nuclear functions other than gene expression. There is a long-recognized link between replication timing and chromatin compaction: frequently euchromatic regions replicate early whereas heterochromatic regions replicate later during S phase (Gilbert, 2002). Genome-wide analysis of sequence-dependent replication timing in higher organisms such as Drosophila and humans has begun to reveal some relationships between genome structure and activity, and added more levels of complexity (Jeon et al., 2005; Schubeler et al., 2002; White et al., 2004; Woodfine et al., 2004). As previously appreciated, for many early-replicating genes replication timing correlates with their transcriptional activity, and most repetitive heterochromatic regions replicate late during S phase. However, other proximal heterochromatic regions replicate early, and some portions of euchromatic chromosome regions also replicate late in S phase (Schubeler et al., 2002). Intriguingly, tiling arrays have shown that highly transcribed sequences outside annotated genes also replicate early during S phase (White et al., 2004) and that there is a relationship between gene density and replication timing, most dense regions replicating in the first 4 hours of S phase (Jeon et al., 2005). These first glimpses into the global relationship between replication timing, gene activity, gene organization and chromatin status make it clear that these are complex links that are difficult to understand through analysis of single loci, and that the regulatory mechanisms and the precise nature of the local chromatin structure at these anomalous regions remain to be discovered.
Genome-wide mapping of sites at which proteins interact with chromatin provides a detailed picture of the steady-state distribution of proteins within the genome at a specific time. However, temporal aspects of their behavior add an additional level of complexity that must be taken into account (Fig. 2). Time-lapse microscopy experiments coupled with photobleaching protocols have demonstrated that most chromatin proteins can move freely and rapidly through the nuclear space by passive diffusion and, more importantly, that most protein-chromatin interactions are transient, including those involving structural components of chromatin (Agresti et al., 2005; Cheutin et al., 2003; Festenstein et al., 2003; Kimura and Cook, 2001; Lever et al., 2000; McNally et al., 2000; Misteli, 2001; Misteli et al., 2000; Phair et al., 2004b). The rapid exchange kinetics – typical residence times of proteins on chromatin are of the order of seconds – suggests that chromatin is highly dynamic (Bustin et al., 2005). This property is probably essential for the genome to respond rapidly to changing environmental conditions. It has implications for how proteins interact with the genome globally as well as locally and thus for the system-level behavior of the nucleus and the genome.
The ability of proteins to move rapidly throughout the nucleus ensures their availability throughout the genome (Misteli, 2001). In addition, the short residence times on chromatin allow proteins to search the genome for specific binding sites by three-dimensional scanning as a molecule briefly binds to chromatin, dissociates and rapidly binds to another site (Misteli, 2001). Upon a chance encounter with one of its specific sites, the molecule resides for a longer period of time and exerts its full biological activity. Some molecules may also function at non-specific binding sites. For example, chromatin-remodeling factors may continuously remodel chromatin stochastically, regardless of the nature of the binding site, and in that way contribute significantly to the overall dynamic nature of chromatin.
At the local level, dynamic binding significantly affects the behavior of proteins because it provides the basis for regulatory events through combinatorial, competitive networks of interactions. The transient binding of a given protein means that each time a molecule dissociates from its binding site, the vacant site can be occupied by another protein. Since this might have a distinct function, the fate of the region depends on the competitive binding of these factors. An example of this is the binding of the linker histone H1 and the high mobility group (HMG) proteins (Bustin et al., 2005; Catez et al., 2002). H1 is generally considered a transcriptional repressor, whereas HMG proteins generally act as activators. The binding sites for several HMG proteins overlap with that of H1, and experimental titration of HMG into living cells results in increased occupancy of DNA by HMG proteins through competition for the same binding site (Catez et al., 2004). Similarly, the relevance of dynamic interaction networks has become apparent from a systematic analysis of the assembly dynamics of the RNA polymerase I transcription machinery with endogenous ribosomal genes (Dundr et al., 2002). GFP fusion proteins that contain pre-initiation, assembly factors and different polymerase I subunits have distinct binding kinetics, which suggests that their interactions at the promoter occur individually as part of a dynamic interaction network. It will be important to determine whether changes in the interaction dynamics are responsible for changes in the transcriptional output of the polymerase.
Analysis of complex biological processes in the cell nucleus requires the use of computational tools. In addition to the well-established methods for mining gene expression data to identify gene networks and pathways, simulation is becoming indispensable for the analysis of the kinetics of nuclear processes (Phair et al., 2004a; Slepchenko et al., 2002; Sprague and McNally, 2005). In these approaches, experimental data are used to determine numerical values for kinetic parameters and these parameters then serve as constraints in computational models that describe a particular process (Phair et al., 2004a; Slepchenko et al., 2002; Sprague and McNally, 2005). Such models, which often consist of systems of differential equations, can then be used to make quantitative predictions, which in turn can be tested experimentally. Not only does this iterative process provide quantitative information and predictions, but the failure of a given model to account for experimentally observed properties often points to novel conceptual aspects of a process. These types of computational approach are now being applied to the analysis of nuclear processes.
The most advanced system-level analysis of any nuclear function is probably the in-silico analysis of nuclear protein import (Gorlich et al., 2003; Riddick and Macara, 2005; Smith et al., 2002). Here, the authors microinjected cells with fluorescently labeled cargo and measured nuclear import rates under various experimental conditions, as well as using in-vitro analysis to determine several crucial parameters such as on- and off-rates for various import intermediates. Developing computational models of the import pathway, they were able to examine the contribution of the import-driving Ran gradient as well as that of importin (Gorlich et al., 2003; Riddick and Macara, 2005; Smith et al., 2002). This system demonstrated the value of modeling approaches because it generates several counterintuitive predictions, including a limiting role of importin α and an inhibitory role of excess importin β – both of which were confirmed when tested experimentally (Riddick and Macara, 2005). The model for nuclear import has thus not only provided the first quantitative assessment of many of the import steps but also given novel mechanistic insights and made quantitative predictions. Furthermore, it illustrates that the behavior of complex processes such as nuclear import is difficult to predict intuitively and that quantitative models are required to do so.
Cao and Parker have similarly combined quantitative experimental data and in-silico modeling to analyze normal mRNA-turnover and nonsense-mediated decay in S. cerevisiae (Cao and Parker, 2001; Cao and Parker, 2003). Apart from generating estimates of quantitative values for key steps in the degradation process such as de-adenylation and de-capping, they also used the model to test the hypothesis that preferential degradation of RNAs containing nonsense codons near the 5′ end is due to leaky mRNA surveillance in which the recognition of nonsense codons near the 5′ end of the mRNA is more efficient than recognition of a 3′ nonsense codon (Ishigaki et al., 2001). However, their model reveals that the leaky surveillance cannot account for the observed degradation kinetics (Cao and Parker, 2003). Rather, the experimental data can only be explained by a mechanism in which all nonsense-codon-containing mRNAs are recognized equally well but their decapping and subsequent degradation rates differ. This quantitative analysis of NMD has now given rise to a new, testable model of NMD referred to as position-independent efficient surveillance (Cao and Parker, 2003).
This type of computational analysis has also been applied to the assembly and elongation behavior of a transcription complex (Dundr et al., 2002). Dundr et al. fluorescently tagged the majority of the subunits of the RNA polymerase I complex and analysed their association and elongation kinetics on endogenous ribosomal genes, using photobleaching microscopy. This allowed them to estimate the in vivo RNA polymerase I elongation rate and show that the subunit behavior is consistent with a model in which the polymerase assembles in a stepwise fashion from its subunits via formation of metastable intermediates (Dundr et al., 2002).
Many nuclear processes are tightly interconnected (Maniatis and Reed, 2002; Neugebauer, 2002). For example, pre-mRNA processing steps, including poly-adenylation and splicing, occur co-transcriptionally, and the RNA-processing machinery is physically associated with elongating transcription complexes. Similarly, the trigger for RNA export is intimately linked to completion of pre-mRNA splicing (Tange et al., 2004), and even the choice between alternative splice sites is influenced by transcriptional co-activators and promoter usage (Cramer et al., 1997). The next step in computational modeling, therefore, will be to interrogate combinations of multiple processes. This poses an experimental challenge because, ideally, information about multiple steps in the gene expression process should be extracted from a single experimental system. This is rarely possible, particularly in the context of nuclear architecture. However, Janicki et al. have developed a potentially powerful tool that holds much promise for analyzing sequential steps from transcription to protein synthesis (Janicki et al., 2004). They have generated an artificial gene array that allows visualization of changes in chromatin structure, binding of transcription factors, mRNA synthesis, RNA export and protein synthesis. This system has already been used to quantitatively track the movement of single mRNA-protein complexes (mRNPs) in living cells, demonstrating that mRNAs move rapidly by diffusion through the nucleus (Shav-Tal et al., 2004). Notwithstanding the caveat of an artificial experimental system, the application of photobleaching methods in combination with computational analysis should provide a comprehensive quantitative picture of the coordination of gene expression steps.
Towards an integrated view of nuclear function
The ultimate goal of systems biology in the nucleus is a computational model that is able to describe all nuclear processes from genome replication, via maintenance of DNA integrity, to regulation of expression within the spatial and temporal framework of nuclear architecture. If this were not ambitious enough, the benchmark for such a model will be its ability to generate quantitative predictions in silico that can be tested experimentally. Achieving this goal will not be possible in a single step but will involve the development of gradually more complex, and presumably more accurate, computational models by iterative comparison of experimental and simulation data. Two complementary strategies should eventually achieve this goal (Fig. 3).
Genome and proteome information can be directly assembled into pathways and networks (Fig. 3). In these approaches, little attention is paid to cellular organization or the details of molecular interactions, but only functional relationships between pathway components are considered. A good example of this approach is the analysis of DNA-damage responses using microarray methods (Begley and Samson, 2004) Analysis of global changes in gene expression shows that expression patterns of genes involved in a wide variety of processes, such as protein degradation and synthesis, signal transduction, RNA metabolism and chromatin remodeling as well as transcription and DNA repair, are altered upon treatment with DNA damage reagents (Begley and Samson, 2004). It has also become clear that specific repair pathways share common members and even functions. An example of this is the base-excision-repair pathway, which is linked to the nucleotide excision, mismatch and recombinatorial repair pathways through various molecular interactions (Begley and Samson, 2004).
An extension of the traditional transcription profiling approach is the combination of genome-wide transcription-factor-binding information with bioinformatics to mine sequence elements in yeast (Harbison et al., 2004). This approach generates a molecular network of all potential transcription-factor-binding sites and motifs under various environmental conditions. Integrating the data on different transcription-factor-binding sites produces a `transcriptional-potential-network' that provides a framework for modeling the mechanisms that lead to global gene expression (Harbison et al., 2004). Analyzing data in this manner is important because it allows us to identify genes that share similar combinations of transcription-factor-binding sites and regulatory motifs, and draw general conclusions concerning organization of gene sequences.
Although this type of network modeling provides insights into the overall properties of physiological processes, it is relatively low-resolution and generally ignores most aspects of how biological processes are organized in cells. To obtain a more realistic, complete system-level view we must model single processes in the greatest possible detail, taking into account the networks of molecular interactions, their dynamics and spatial organization (Fig. 3). Processes that have been explored in this way include transcription by RNA polymerase I machinery, nuclear import and NF-κB signaling (Dundr et al., 2002; Hoffmann et al., 2002; Nelson et al., 2004; Riddick and Macara, 2005; Smith et al., 2002). Each can be considered a functional module in a larger network, and ultimately the computational models for multiple such functional modules will be combined to give a comprehensive global view of nuclear function.
This type of bottom-up approach to systems biology is more difficult owing to the incorporation of more detail, but at the same time it has several important advantages. Analysis of limited functional modules allows development of relatively simple computational models that can be tested experimentally and are easier to grasp than large networks. For example, in the case of NF-κB signaling, Hoffmann et al. analyzed the effects of knocking-out each known inhibitor of NF-κB (Hoffmann et al., 2002). Quantitation of the response to stimulation and computational modeling identified specific roles for each inhibitor, and provided a global, yet detailed, view of how this particular pathway behaves (Hoffmann et al., 2002). In addition, modeling of distinct functional modules using quantitative imaging combined with computational analysis allows the incorporation of spatial and temporal information, i.e. the role of subcellular localization, local protein concentrations or wave-like propagation of a signal. In the case of NF-κB, quantitative live-cell imaging has shown that persistent asynchronous oscillations between the nucleus and cytoplasm are required for NF-κB target gene expression. The authors suggest, that akin to Ca2+ signaling, the consequences of NF-κB may depend on the number, period and amplitude of oscillations (Nelson et al., 2004). Such cell biological properties are often ignored in network modeling approaches, despite their established importance in regulation of biological processes.
Once single functional modules are satisfactorily simulated, they can be linked into larger process networks. An example of this approach is a system-level view of sea urchin development. Davidson and co-workers first analyzed the spatial and temporal regulation of genes responsible for cell specification, using a bottom-up modular approach (Yuh et al., 1998). They combined quantitative experiments and computational analysis to identify the roles of different cis-regulatory sequences controlling expression of a protein involved in the development of the midgut (Yuh et al., 1998; Yuh et al., 2001). They then verified this approach by confirming in-silico-generated predictions experimentally (Yuh et al., 1998). The subsequent addition of more modules representing different spatial domains has allowed them to assemble a large regulatory network of the genes and regulatory sequences involved in endomesoderm development (Davidson et al., 2002a; Davidson et al., 2002b). Furthermore, they can now perform large-scale system perturbations and quantitatively describe the resulting changes in multiple phenotypic parameters (Davidson et al., 2002a; Davidson et al., 2002b).
The top-down and bottom-up modular strategies described above are not mutually exclusive but highly complementary. An impressive example in which they have been combined is computational analysis of apoptosis. In this case Bentele et al. modeled the well-studied parts of the network in great detail, using experimentally determined rate constants, whereas they modeled the less well-studied parts at low resolution, only taking into account the overall behavior of particular modules (Bentele et al., 2004).
How close are we to a system-level view of the nucleus? Clearly we are still at the very beginning, but the tools that will make it possible to explore the nucleus on a more global scale are being put in place, and the first examples of large scale, complex systems analyses are emerging. The relatively simplistic computational models that have been applied have already demonstrated that quantitative systems analysis has the potential to provide new insights into the molecular mechanisms of nuclear process and also to generate novel concepts of how cellular processes are organized, coordinated and regulated. In the future we will probably see an increasing convergence of experimental and in-silico analysis, both of particular cellular processes as well as of process networks. These approaches are conceptually different from how biochemists and molecular biologist have traditionally thought about problems in the past and will require us to embrace multi-disciplinary approaches. While this would have been a serious hurdle only a few years ago, fortunately the sequencing of whole genomes has provided the experimental foundation, as well as the inspiration, for us to tackle cell biological problems, be it in the cell nucleus or elsewhere, at the systems level.
This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. T.M. is a fellow of the Keith R. Porter Endowment for Cell Biology.
- Accepted July 20, 2005.
- © The Company of Biologists Limited 2005


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Could Chrisette Michele's star slowly be falling?
After enduring backlash from accepting the offer to perform at an inauguration special for president elect Donald Trump, more and more people are voicing their non-support of the singer and her music.
Legendary film director Spike Lee is one of them; in an IG post this morning, Lee revealed that because of her decision, he'll no longer consider using her song "Black Girl Magic" in an upcoming Netflix series.
Reportedly, the performance slot is the same slot turned down by Jennifer Holliday earlier this week worth $750k to perform.
Is Lee being petty, or is the blatant omission of her song a justifiable action? Sound off below!
10 Comments
Well, it's not surprising. Spike Lee is one of America's most spotlighted racists. And besides, he's really inconsequential. His relevance departed decades ago.
spike lee or nobody else is paying chrissette's bills. she did it for her own personal reasons whether financially or socially. for 750k id done a tap dance. ppl get over it you got trump for 4yrs. Done
Most people criticizing her would have done a tap dance for Trump for $75.
You gotta suffer consequences when you make controversial decisions. You have to see thing through. Chrisette lost focus on what really mattered and headed straight for the $$. Was 750K worth losing your fan base and a debut in a movie. I think not. I'm not a real Spike Lee fan but I can say this Spike stands strong behind what he believes and its no surprise that he took the measures that he did. So now Chrisette take your $750 and invest it well because your career might just be over
bullshit. chrisette don't owe nobody nothin. she is free make her own decisions and sometimes they may conflict with others and their beliefs. but you know what? it ain't about them. it's about chrisette doin what makes chrisette happy. and if that involves her makin bank, so be it. people are jumpin on her ass like she went and robbed a bank and killed a bunch of people. give the woman a break, damn. she's livin her life the way she sees fit, and if people don't agree with it, that's just too damn bad. it's not up to them to decide, and they have no right to judge or berate her for sayin and doin thangs that are inconsistent with their own feelings and views.
He did her a professional favor.
like hell he did. more like he gave her a slap in the face musically and disrespected her as a talented strong black woman.
he has a right to his opinion but to "punish" her for havin a different view than his own is just dead wrong brother. her choice to perform doesn't make a "race traitor", it means that she doesn't let the media's perception of him affect her "hustle" and willingness to entertain others regardless of the venue.
thank you

subset: commoncrawl


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Many of the first colonists in Newport became Baptists, and the second Baptist congregation in Rhode Island was formed in 1640 under the leadership of John Clarke.In 1658, a group of Jews were allowed to settle in Newport who were fleeing the Inquisition in Spain and Portugal.It is also the home of Salve Regina University and Naval Station Newport, which houses the United States Naval War College, the Naval Undersea Warfare Center, and a major United States Navy training center.It was a major 18th-century port city and also contains a high number of surviving buildings from the colonial era of the United States. Newport was founded in 1639 on Aquidneck Island, which was called Rhode Island at the time.Search Single Dads in Rhode island | Search Single Moms in Rhode island Middletown RI singles Wakefield singles singles Pawtucket singles in Johnston singles in Lincoln West Warwick RI singles Bristol singles singles North Kingstown Narragansett singles Chepachet RI singles singles in Cumberland Coventry RI singles East Greenwich singles Westerly RI singles Riverside singles East Providence RI singles North Smithfield singles Cranston singles singles in Newport Woonsocket singles singles in Warwick Smithfield RI singles singles Providence singles in North Providence singles in Portsmouth Alabama Alaska Arizona Arkansas California Colorado Connecticut DC Delaware Florida Georgia Hawaii Idaho Illinois Indiana Iowa Kansas Kentucky Louisiana Maine Maryland Massachusetts Michigan Minnesota Mississippi Missouri Montana Nebraska Nevada New Hampshire New Jersey New Mexico New York North Carolina North Dakota Ohio Oklahoma Oregon Pennsylvania Rhode island South Carolina South Dakota Tennessee Texas Utah Vermont Virginia Washington West Virginia Wisconsin Wyoming Creating a Great Dating Profile on Single Parent The Single Mom Dating Plan Dating and Your Child Single Moms: Expand Your Dating Horizons Inside the Mind of a Single Mom: What Every Man Should Know Creating a Great Dating Profile on Single Parent The Single Mom Dating Plan Dating and Your Child Single Moms: Expand Your Dating Horizons Inside the Mind of a Single Mom: What Every Man Should Know 6 Ways to Meet Someone at a Reunion Dating Time Traps for Single Parents Season's Best Dating Strategies for Suddenly Singles Vices to Break and Vows to Make in 2010 I met my Fiancé about a year First of the year. Never really thought I'd find someone special to be honest. Meet thousands of single men and women from Rhode Island for FREE. Join our site today and meet fun men and women near you looking to meet quality singles from Rhode Island.
I live in Rhode Island with my family and my parents. Looking for a fun person with a sense of humor u got to have one life is 2 short a person of few words, optimistic and educated. Recently, I had loved traveling with friends to nearby countries and cities.
i work for life inc which is a company that works with adults with developmental disabilites People tell me I'm shy when they first meet me.
It is true that I can be quiet at first but this is because I like to listen, understand and appreciate the new people I meet. I am a 28 year old fem lesbian, I do not have a "type" I am simply looking for a connection, and passion.
Our Gilded Age mansions are not only a time capsule of a period when America was becoming an economic power, but are a product of the artistic talent of some of the finest architects in American history.
For this, in 2007, the US National Park Service recognized Newport as "worthy" of World Heritage designation. "Newport's Old Quarter, the Arts and Culture Neighborhood" is a delightful walking tour map directing visitors to the cultural and historic sites of Colonial Newport.


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Ignacio Alvarez Meyendorff is suspected of working for the once-powerful Colombian Norte del Valle drug cartel.
Mr Alvarez denies the allegations.
Drug traffickers are increasingly using submarines to avoid being caught by the security forces patrolling the water's surface.
Deputy Security Minister Miguel Robles said Mr Alvarez was arrested on Sunday with help from the US Drug Enforcement Administration, but news of his arrest were only made public on Wednesday.
Mr Robles accused Mr Alvarez of being in charge of the logistics behind the underwater smuggling scheme, and of perfecting the system.
He said Mr Alvarez was detained as he arrived in Buenos Aires on a flight from Tahiti.
Local media reported that he had been living in Argentina since 2005, and had resided in an exclusive area of the Puerto Madero neighbourhood.
View the original article here
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I feel like so many new things have happened with baby recently. While we were at Fripp I started feeling baby flutters! I love laying in bed and feeling him/her flutter around before I fall asleep. Within the past few days I've been able to feel the flutters a little more often. If I'm sitting still {I actually do get a chance every now and then} I'm occasionally able to feel him/her.
This morning when I woke up I put my hand on my tummy. I noticed that there was a hard bump about the size of my palm just under my belly button. This is exciting to me because though I've been showing {to people who know I'm pregnant} for a few weeks now, I've been telling Lane that I just feel fat because my belly wasn't hard like a pregnant belly. Don't get me wrong, I'm not sad that it was soft, I was just looking forward to the hard belly that you get when you're truly "showing."
Everything is starting to feel real now that I feel the flutters and my tummy is getting harder! For so long pregnancy is almost phantom-like because you know you're pregnant, you've seen the positive tests, you've seen the ultrasounds with the heartbeat, but you don't feel or look pregnant. How exciting it is to begin this new phase of pregnancy - when you start to feel and look pregnant!


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Wednesday, February 6, 2008
The Shape of Things to Come?
4 comments:
- Jennifer said...
Don't worry about it too much... that man just wanted to make MORE money while his equipment was already out there! If you aren't having problems at all, it's going to be a LONG time before any roots grow in there and are bad enough to permanently clog it.
We get our sewer rooted about every 9 months... we have some bad trees (17 of them). They aren't maples, but the roots cut back just fine. We have it rooted when it blocks up... not too big of a deal. I've often thought of just setting up an appointment, except the guy we have come and do it is so proud of his work that he is mad when it doesn't last as long as he thinks it will... and works every HARDER. We have mutant trees and a good plumber. (The first 3 times we used someone else and it was every 2 or 3 months!)
Anyway, what I am saying is that you are going to start having easily fixable problems a LONG time before you have any permanent ones... IF AT ALL! That maple may be NO WHERE near your sewer... that man doesn't know!
- February 8, 2008 at 9:30 AM
True. Now my neighbor's sewer pipe turned out to run uphill and downhill like a kids' rollercoaster. And it was full of root balls-- I sawded 'em! No wonder they had backups!
I don't blame the workman for wanting to drum up business-- but yeah, when you've got a big, shiny hammer, everything looks like a nail!
- February 8, 2008 at 11:42 AM
- Sandy said...
Aw cr@p. I've have a beautiful maple (the leaves are yellow in the fall - hence I call it my "sunshine" maple) in my backyard (along with a 1/2 dead box elder which I won't cut down because squirrels live in the tree). I've not had any problems, but now I'm starting to worry....
- February 8, 2008 at 1:25 PM
Sandy, I wouldn't worry about it. If I read Jennifer right, preventative maintenance should take care of it. Good likelihood is that my neighbors' previous owners never did anything about it.
My maple helps me keep the cooling costs down in the summer. So there's always trade offs and paybacks.
- February 9, 2008 at 5:43 PM


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Product Details
- Amazon Sales Rank: #83788 in DVD
- Published on: 2008
- Rating: G (General Audience)
- Format: NTSC
- Original language:
- Dimensions: .6" h x
.55" w x
.76" l,
.27 pounds
Product Description
In six 30-minute DVD presentations, Pastor John Piper unpacks what it means to behold and delight in Jesus above all things.
In 2 Corinthians, the apostle Paul described one of the central problems facing humanity in every age: people fail to see Christ for who he is. Even those who have caught a glimpse of him do not see perfectly, says best-selling author John Piper. But it is important that we not only learn to see him but delight in him and the truth of the gospel.
This DVD and its companion study guide explore the meaning and significance of these ideas in a guided group study. Each of Piper's six 30-minute DVD presentations will equip Sunday school teachers, small group leaders, Christian educators, and parents in directing people's understanding of these topics so that viewers will learn to behold and delight in Jesus more than anything this world has to offer.
Movie Info
Seeing and Savoring Jesus Christ from Desiring God on Vimeo. Publisher's Description . Who is Jesus Christ? You've never met him in person, and you don't know anyone ...,Click to read more about Seeing and Savoring Jesus Christ by John Piper. LibraryThing is a cataloging and social networking site for booklovers,The book title actually could be expanded to include Sharing Jesus Christ as well as Seeing and Savoring. If someone truly sees who Jesus is with the eyes of ...,If you want to know Jesus you do not need to go to seminary or embark on a tour of the Holy Land. All you need to do is to read the gospels with an open heart. In ...,In six 30-minute DVD presentations, Pastor John Piper unpacks what it means to behold and delight in Jesus above all things. In 2 Corinthians, the apostle Paul ...,Seeing and Savoring Jesus Christ . Revised Edition. By John Piper ... show all. Retail Price: $9.99 Impact Price ...,Seeing and Savoring Jesus Christ, Revised Edition ... The Mercies of Jesus Christ Seeing&Savoring.46239.i04.qxd 5/14/04 10:54 AM Page 7. 11 THE TOUGH SIDE 93,Seeing and Savoring Jesus Christ has 949 ratings and 79 reviews. Dottie said: Seeing and Savoring Jesus ChristBy John PiperThis is a beautifully writ...,Seeing and Savoring Jesus Christ (Revised Edition) [John Piper] on Amazon.com. *FREE* super saver shipping on qualifying offers. Who is Jesus Christ? You've never met ...,Excerpt Seeing and savoring Jesus Christ is the most important seeing and savoring you will ever do. Eternity hangs on it. So my aim is that you see him as solid ...


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Here is a link to my place and the work this tractor is doing
Would love to know more about this little guy.
And I am shocked at how much this little green machine can do.
Thanks for a great forum and it's nice to have a place to share our ideas about Small Tractors.
Lawn and Garden Tractors item created by JoeMartinez, May 29, 2011
To view comments, simply sign up and become a member!


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Release Year: 2012
Genres: Buttocks, Bdsm, Enormous hooters, Union sex, Fellatio, sex playthings, Tentacles, Pirsing, Women, Princess ,X-ray
Video language: English
Found one of a series of two-part hentai confrontation of evil demons and warlike Valkyries, who sent people to protect, as a gift from the god Odin. Protecting people from demons, Valkyrie mercilessly crumble them into the cabbage in duels. But evil demons found a weak join in the defense and making the Valkyries goddess Freia his sex slave – they just destroy impenetrable defense Valkyries and conquer them. After that, the world is going wild parties, and the rape of virgin Valkyries. Chaos has no limits, people crammed into jail, and on their eyes rape them as protectors. It seems that the world is beginning to come to an end and no one can stop the demons create ugliness and disorder, but in the second scene (which by the way there isn't a translation) everything will be much better and the plot rapidly recover the better.
Format: avi
Duration: 26:44
Video: 704×396, Wm Movie 9, 1306kbps
Audio: 187kbps


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The West Jefferson softball team has knocked on the door for four straight years, this time around it's ready to kick it in.
The Lady Roughriders have lost in the Division III Central District championship game four years in a row. Constantly getting to the title tilt and coming up short has put coach Jim Stevens and his players in a different mindset and they're ready to finally breakthrough.
"We've got a bunch of varsity returners," coach Jim Stevens said. "We lost three seniors, but we have some very talented young players coming in and it's another re-loading year for us. We've been knocking on the door for four years and we're ready to break through. We're ready to get beyond Pickerington."
The site of the district championship game has been the final stumbling block for West Jeff for years, but by returning nearly every single important role player from that team a year ago the Roughriders enter the season focused and ready.
"I think the girls will pick up right where we left off last year," Stevens said. "The hardest thing for the coaches will be holding back the reins with this bunch. We've got to take it one game at a time. We've got to remind them that this season is a marathon and not a sprint. We really don't want to peak too early."
West Jefferson has won four straight Mid-State League Ohio Division Championships but this will be the school's last spring in the league before moving to the Ohio Heritage Conference next school year.
The Roughriders have made it a goal to close out their last season in the league with yet another MSL championship.
"One of the goals for this team is to go out on top and leave a lasting impression with the MSL," Stevens said. "They also want to send a message to the OHC letting them know what's coming. I feel extremely positive that this team will contend for a league championship this year and will head into the OHC and be able to more than hold our own."
The team returns many of its best players from a team that was 17-7 last year, including sophomores Brooke Roberts and Isabelle Fisher were all-league first team selections. Other players who earned postseason awards and are back this year are sophomores Haley Kurtz and Lexi Bowman as well as junior Harlie Keith, all of which were second team All-MSL picks. Alexis Good who was an honorable mention selection also is back.
"We're excited to get the season started," the coach said. "We play a very competitive schedule which help us keep our girls sharp. We want to play good teams to force us to have to play well. We want to be at our best at the end of the year because we want to get over that hump of the district final."
Reach Chris Miles at 740-852-1616, ext. 1618 or via Twitter @MadPressSports.


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BTS 059: How a Headless Fish Scared Its Chef
This video of a decapitated fish seemingly still being aware that it is being prepared for a meal had captured the world's attention. But is this video a case of supernatural nature or just science being creepy. Don't hold your breath. It's the latter.
So what's going on? The dead fish is clearly decapitated, and the fact that it has been gutted was repeated numerous times by the lady who performed the act.
The answer is pretty obvious for those already aware of another good ol' viral video, the skinned frog legs that twitches when reacting to salt.
Despite being dead, tissues in the muscles have still not decayed and thus can still "move", though this would require some form of external force as there is now no brain to send signals to the nervous system.
Such a reaction goes to the very fundamental of how our muscles work, and you can get started on going behind the science of this creepy result of science by starting here.
BEHIND THE SCiENcE is a series about facts of the world worth knowing, worth sharing.
Be Intrigued, stay Curious!


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My sister wrote a speech about me for a class in school and it moved me to tears. I had to share it.
Journey to the Centre of My Heart
We had to give a speech at school about something which inspired us and I was really blank till I remembered the person who played a huge role in my life and shaping it. When I started working on the speech the words flowed almost automatically because they were from the bottom of my heart. So I decided to share it 🙂
Since we are supposed to talk about something which inspires us, I would like to talk about someone who has played a major role in the sixteen years of life, my sister. My sister was my first friend, my first role model and my first hero. My sister was 3 and half years old when I was born to become a part of her world. She was assigned with responsibility of being the 'older sibling' at the tender age of 4 and she accepted this responsibility gracefully. When…
View original post 447 more words


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I should be able to pull an FO outa my....
The children of knitters have a tendency to think that you can sit down with yarn and needles and poof a sweater appears or maybe a purse. If you are Alex you want 10-12 wristbands for your friends and you have a list of all their favorite colors. And you want them right now. I told him if I was going to make stuff it was going to be the way I wanted. So for now he has bracelets for two of the girls.
I could not get a good pic of the on the right. The button has a very cool moon face on it. So now off to knit sweat band wrist things. I may just do a few this
week I do have other things OTN. More about that later.
Until next time.


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|Use this URL to cite or link to this record in EThOS:
|The long-term impact of short periods of imprisonment on mothers
|Masson, Isla MacMarquis
|Awarding Body:
|King's College London (University of London)
|Current Institution:
|King's College London (University of London)
|Date of Award:
|Availability of Full Text:
This research examines how even an initial short period in prison negatively impacts mothers and their children. It involves a series of semi-structured interviews with 16 mothers during and post-custody; looking at the different ways in which multiple aspects of their lives are negatively affected for longer periods than their incarceration. It is argued that prison often increases the social disadvantages that many of the women encounter on a day-to-day basis. Based on this research it is suggested that the morally significant harms of prison need to be considered at the time of sentencing. Incarceration is not just about a temporary loss of liberty, even short terms in prison have longer multi-dimensional consequences. The thesis will begin by looking at the use of remand for women as well as examining the sentencing rationales for the use of short sentences. It will explore the problems with these forms of punishment, particularly for non-violent, and often vulnerable, women. It will be argued that these women experience multiple pains of incarceration, often compounded by the short period in which they are imprisoned. It will be suggested that their feelings of injustice may affect whether they are able to embrace any opportunities in prison and address feelings of guilt. The thesis will also examine these mothers' experiences post-custody, describing which problems are on-going, which are resolved and what new unexpected problems arise. Given that they are mothers, their understanding of the harm of the separation to their children will also be explored. It will be concluded that the punishment should be balanced against the rights of mothers and their children. As such the use of short sentences for women should be significantly reduced, however if they continue to be used there need to be a series of changes to minimise the harms caused to this group.
|Player, Elaine Beryl
|Not available
|Qualification Name:
|Thesis (Ph.D.)
|Qualification Level:
|EThOS ID:
|Not available


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Sam Weller is looking forward to the challenges ahead as he enters his third year at the Sydney Conservatorium of Music. The ambitious saxophone student has developed a passion for conducting during his time at university and is now attempting to combine his two loves.
Weller is the Artistic and Musical Director of Ensemble Apex. This symphony orchestra began as a much smaller, student chamber orchestra, and with over 60 musicians the group has now swelled into a more symphonic size. Ensemble Apex were fortunate enough to perform with musicians from the Sydney Symphony Orchestra for Vivid Sydney's IGNITE, symphonic dance anthems at City Recital Hall last year, and are set to return to the stage with the one-of-a-kind spectacular at the Enmore theatre next month.
Weller is also a member of the Conservatorium's Wind Symphony led by Dr John Lynch. Playing under Dr Lynch's leadership sparked Sam's interest in conducting himself, "Dr Lynch's rehearsal technique is unique", Weller explained. "Watching him has resulted in me experimenting with my own rehearsal techniques, which are vital to a conductor's success".
In 2017, Weller was very fortunate to tour Verona, Italy for the Con's Estivo European Chamber Music Summer School with his saxophone group Luxum. "The preparation and the summer school itself challenged my analytical and musicological skills which are integral to a young conductor's success in front of an orchestra", said Weller. As a result of this, Weller was selected to attend the Australian Conducting Academy with Johannes Fritzsch and the Tasmanian Symphony Orchestra in his summer holidays earlier this year.
Ensemble Apex will begin their 2018 concert program with a performance at the Con's Music Workshop Friday, 9 March, 8pm. Entry is free.


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Born and raised in the Netherlands. In the past 10 years have lived in Antwerp (Belgium), London (UK) and currently Berlin (Germany), and I'm fluent in Dutch, English, and can get by in German and French.
SOFTWARE and SKILLS
Adobe Photoshop CC
Adobe Illustrator CC
Adobe InDesign CC
Graphic design
UI design
Website design
Photo manipulation
Online marketing
Branding and identity
Affiliate marketing
Social media
In 1989, at 9 years old, I was mentored on MSX BASIC programming by Gertjan Schuurmans (currently CTO at Gavin) and I have been playing and working with computers ever since.
In 2001, as part of my Master degree in Communications Science, I did a full-time internship as a designer and webmaster at FinanceTelevision, one of the first news networks to broadcast financial news items exclusively online. At the same time I started creating my first websites for clients.
In 2003 I registered my business Rem-art. Since then I designed hundreds of websites, logos and banners for more than 250 international clients.
For Expatica.com, a community for English-speaking expatriates and internationals across Europe, I was a regular freelance designer from 2003 – 2007, working on the website design, UI elements, event websites, banners, flyers and posters.
From 2004 until today I've been the lead webdesigner for high-end dancewear company Yumiko.
For the Rotterdam School of Management I was a regular freelance banner designer for their MBA, Business and MSc programs from 2009 – 2015.
My clients are based in The Netherlands, Belgium, the UK, USA, Sweden and Germany.PORTFOLIO
Using my skills and interests I set up several personal projects, including Bond Lifestyle (2005, currently the most visited James Bond fan website in the world with a large social media following), Watch-ID.com and SunglassesID.com (2009, identifying watches and sunglasses in movies).PROJECTS
University of Amsterdam
Degree: Master of Communication Sciences
1997 – 2003


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A few weeks ago a bloke came knocking on my door asking me to support the local milk man.
"We don't just do milk anymore, love, we do bread and juices and potatoes," he said.
I'm inclined to support local businesses and the thought of bottles of fresh milk and juice delivered to my doorstep every week meant I jumped at the chance. Starting off slow I ordered a bottle of milk and an apple juice for the following Monday.
That Monday morning I got out of bed and raced to the front door (yes…slight exaggeration) and on the doorstep was a very uninspiring plastic carton of milk standing next to a carton of apple juice, which stated very clearly "made from concentrate".
What happened to glass bottles with foil tops? Does anyone get them anymore or have they become obsolete?
I was incredibly disappointed with the haul and it would have been easier and cheaper for me to pick up the drinks with the Big Shop! I'm all for supporting local businesses and this is sad but I feel they have to offer something beyond the supermarket norm if they want to be successful.
I would cancel my order but the lovely old gent who came to collect my cash was super nice, and super-friendly. And I couldn't put him out of business, could I?


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"One day as I was observing how wisdom fares on this earth, I saw something that made me sit up and take notice…All the same, I still say that WISDOM IS BETTER THAN MUSCLE…" (Ecclesiastes 9:13, 16 The Message, emphasis mine)
Monthly Archives: May 2012
You Are Loaded – Too Loaded!
This morning I continue my thoughts from yesterday. If you missed that, check it here. You had better believed that statement. Look at these scriptures. It will blow your mind to think that all these refer to you.
"You are of God, little children, and have overcome them: because GREATER IS HE THAT IS IN YOU, than he that is in the world." (I John 4:4)
"But WE HAVE THIS TREASURE IN EARTHEN VESSELS, that the excellency of the power may be of God, and not of us." (2 Corinthians 4:7)
"The kingdom of God comes not with outward observation. Neither shall they say, Lo here! Or Lo there! for, behold, THE KINGDOM OF GOD IS WITHIN YOU." (Luke 17:20-21)
"Now unto him that is able to do exceeding abundantly above all that we ask or think, according to THE POWER THAT WORKS IN US." (Ephesians 3:20)
"But THERE IS A SPIRIT IN MAN: and the inspiration of the Almighty gives them understanding." (Job 32:8)
"Therefore I remind you that you stir up THE GIFT OF GOD WHICH IS IN YOU…"
(2 Timothy 1:6)
(All scriptures are taken from the King James 2000 Version of the Bible. All emphases mine.)
You are too loaded to fail! I challenge you today: Release your potential and be a blessing to your world. Don't die with your music still in you. You will succeed!
"The great tragedy of the average man is that he goes to his grave with his music still in him." – Henry Wadsworth Longfellow
When you come to know who you are in God and discover what you are wired up by God to do you will find success and satisfaction. Learn to look inwards. Looking around will get you nowhere. You are loaded! Discover your gifts and talents. It's your inside advantage – the very key to the riches, the success and the fulfilment you've been looking for. All you need do is discover it, develop it and display it.
An ancient legend has it that at a time in the history of man, society so abused wisdom that the council of wise men decided to hide the secret of happiness and success from mankind. A council meeting was convened. The following discussion ensued:
First Wiseman: "Let us bury it deep down in the earth."
Chief Wiseman: "If we bury it in the earth, man will learn how to dig deep and will one day discover it."
Second Wiseman: "Let's carry it to the top of the highest mountain."
Chief Wiseman: "If we take it to the top of the highest mountain, man will learn how to climb to the top of the mountains and discover it and use it for himself."
First Wiseman: "Let us sink it deep in the ocean."
Chief Wiseman: "If sunk into the deepest ocean, man will learn how to dive in and will one day discover it."
After a long silence the Chief Wiseman said, "Here is what we're going to do. We will hide it deep in the inside of man himself, for he will never think to look for it there."
Today, are you not still running to and fro, digging, climbing and diving, searching for something you already possess within yourself? Think about it. YOU WILL SUCCEED!
I'm passionate about education, education of the right kind. Education – the one that does not just swell your head but changes your mind and expands your world.
I'm a personal development junkie – buying and reading books, attending seminars, listening to CDs and watching videos and documentary TV shows is my drug. In short, learning is my addiction!
And it has improved me, expanded my world and literally changed my life. It will do the same for you too, if it hasn't yet. When you know truth, it sets you free from the limitations of mediocrity (see John 8:32). Education, of the personal development type changes lives. Here I present supportive evidence from no less a person than Nelson Mandela in his biography, Long Walk to Freedom:
"Education is the great engine of personal development. It is through education that the daughter of a peasant can become a doctor, that the son of a mineworker can become the head of the mine; that a child of farm workers can become the president of a great nation. It is what we make out of what we have, not what we are given, that separates one person from another."
Don't stop your education, now that you are out of school or once you are out of school, whatever your own case may be. Take that course, read that book. Attend that seminar. No one may notice now, but soon they will! Keep learning, it will impact your earning. Keep learning, it will enable you to keep leading. Real education is found in what changes your life, not just what goes into your head. Have a great learning week, this week. You will succeed!
If this has blessed you, you may also enjoy these posts: ; ; and


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When we last left our heroine, it was autumn, and just like the leaves on the trees, her perception was about to change...*
I have never decided when to be in a relationship.
I suppose that before relationships got complicated for me, they were easy to get into: boy meets girl, boy likes girl, girl likes boy, they become boyfriend and girlfriend. Easy as that.
Then there's the rest of the story - and all the stories that have brought me to the place I am at right now: Boy breaks girl's heart. Girl outgrows relationship. Boy and Girl grow apart. Boy has girlfriend on the side. Girl has commitment issues. Boy has been hurt. Girl is 33 and jaded. Girl goes on strike. Girl has life-changing epiphanies. Girl realizes she has never initiated a relationship.
It isn't as romantic of a story and one that is rarely told, but it's true. I've never been a part of the process of getting into a relationship. I know that sounds odd, but I've never really been the one to decide when to get into a relationship. I've always waited to be told when it's time to be together.
Once, when I was in middle school, I had a friend come up to me and say "you should like Jason. I was trying to think of a girlfriend for him, and I thought of you. He wants to go out with you and I think you would be a cute couple." I had never even thought of Jason as a potential boyfriend but since he liked me and wanted to date me, I went ahead and said yes. Fortunately, Jason was a great boyfriend and definitely a good "puppy love" story. Unfortunately, being told when to get into a relationship is a pattern I have continued.
I've never taken the time to go through the process of getting to know someone and allowing my feelings to grow for him - naturally and organically. Any relationship I have been in has consisted of me meeting someone and jumping into being boyfriend/girlfriend and then we would begin the task of getting to know one another. As I wrote in part one, I jumped into relationships because I needed the validation. Quickly getting into a relationship made me feel like I was wanted - and I liked it. When a man wanted to be with me without really knowing me, I had the reassurance that he liked me and even better, there was a guarantee that I wasn't going to be rejected. After a while, the guarantee wasn't enough and I would crave more validation. It was a viscous cycle of insatiable need that always ended badly.
The flip side of an instant relationship is being rejected or worse, being strung along. The award for the longest string still goes to Astro Boy, who, despite wanting to be with me every single day wouldn't commit to me. Never mind that we functioned like boyfriend and girlfriend, I never got the label of being his girlfriend and never got the validation I needed in order to feel safe and secure with him. I wasted a lot of time on him - as well as all the first dates I went on, knowing full well that a relationship wasn't going to transpire. I went on the dates because I needed the attention and whatever fake affection I was going to receive. While I can sum up in about five minutes whether or not I am going to click with someone, I was still giving out my phone number and going on the dates because I just needed to feel wanted.
Lately, I've been thinking about who I am and who I want to be, both on my own and in a relationship. As I said last week, a possibility that I had given up on a long time ago became a possibility once again. When the possibility first arose, I wanted to jump into a relationship immediately because there was a spark. The spark didn't lead to anything and I gave up on the possibility of us. Two years later, the spark appeared again and now burns a little brighter, a little stronger - and the path we are on requires more patience than I ever thought possible.
At first, I thought the pace of this path would kill me. Walking a path with someone who has requested patience is just about the hardest path I've ever been on when it comes to relationships. Taking things slow and learning about each other has taken me to the depths of my issues around rejection and abandonment. There are times I would rather give up and just go on some first date (with some guy who won't be 1/16 of the man that this person is), but then I remember the lessons I am learning through this process and that there is so much more to gain versus losing. What I have found out about myself is so much more valuable than I could have imagined. For the first time in my life, I realize that I have the opportunity to question whether or not I want to be in a relationship.
This isn't a thought that occurred to me right away. At first, I thought I was just waiting for him to be ready (as has been the case with men in my past). I figured I would stick around for a while and see what happened, all the while keeping my guard up. As we began to communicate and really get to know each other, I began to realize that getting to know someone is something I've never done before. I also realized that I am not necessarily ready to just jump into a relationship. In the past, I found myself getting into relationships because I was too afraid that no one else would want me. Because we are walking our paths and not jumping off a cliff, I'm also getting the chance to get to know myself, and the desperation continues to disappear.
I have learned that I don't need daily calls and texts in order to feel validated. I don't need constant reassurance to feel safe and secure. I am learning that I am enough. I'm learning that trust is something that is earned and that he is learning to trust me as much as I am learning to trust him - and we are learning to trust ourselves in the process. I am learning to speak up and voice my needs without being afraid that I am going to chase him away. I get scared at times, and I have to remind myself that we are building something on our own terms and walking our individual paths - and even if our paths don't eventually cross, I would rather go through this process than go on a million first dates. He's worth it, and so am I.
The story goes like this: Boy and Girl met a long time ago. Boy and Girl are finally getting to know each other. Girl is discovering that she is so much stronger than she ever thought she was and has the power to make decisions on her terms. Girl is learning to trust herself and will know when she is ready for a relationship. When she is, Girl will make the right decision. That is a guarantee.
Next time: A Lot of Friends
*For the whole story, read parts one and two.
These are the things I think about - and sometimes, they make sense enough to write about.
Monday, January 16, 2012
Learning to Walk... Or, Part Three
Posted by Andrea J. Serrano at 10:00 AM
2 comments:
You know I don't know that I ever gave much thought to you yourself choosing to be in a relationship before until this blog. But I can see how true that is for me too and how different I want that to be for my future too. How about deciding first if this is the type of person I want to be with and then deciding to be in a relationship with them. What a novel idea :) Thanks for reminding me of thatReplyDelete
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Loving life (Why?)
1. Anime
2. Music
4. smiles
Ronnies Back
"I feel the madness creeping slowly
Loved by many I'm still lonely
Paid the price for your mistake
The music died the day I walked away."
¸.•´¸.•*´¨) ¸.•*¨)
(¸.•´ (¸.•` ¤ ShelbyxWasxHere♥
I'm sick of imagery, instead of artestry. I'm sick of apathy, instead of harmony. I'm sick of poets working part time jobs, while pissy people pick and choose their stars. Set the music free!
iLove all those bands. another good techno one is DotDotCurve. or brokeNCYDE.
"Arielle6998" wrote:Asking Alexandria, Bring me the horizon, eyes set to kill, all time low, the academy is, fall out boy, panic! at the disco, paramore, insane clown posse, falling in reverse, escape the fate, taking back sunday, avenged sevenfold, blink 182, evanescense, disturbed, more...and more....


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Click on the link to hear the five minute audio from Behar:
Dr. Dye, always enjoy your insight on scripture.
So glad to have a membership again with you. I really want to know about the Temple and how I become His Temple as well.
Thanks Yoseph. My new book on the temple should be coming out around early March. It lays the groundwork for the entire topic. It is the first in a series that hopefully will give a good understanding of the temple concept in Scripture.
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We lost three Rabbi's yesterday who were Americans–six total. I watched the videos and looked at the pictures of the tallit prayer shawls soaked in blood and felt the heaviness. The Holy Torah soaked in all the blood of HaShem's people laid on the floor. The blood from Abel to every prophet ever martyred seemed to cry out from that photo. I'm telling you if this has not shaken us out of our comfort zone it should. If someone doesn't blow a shofar and sound an alarm soon, people are going to continue to sleep and not have enough oil.
My cry is that America doesn't just watch and say, "God have mercy and then continue with their life as normal. We need some Daniels, some Nehemiah's, and some Ezra's who have a mind to work and cry out over the horrific genocide of God's people all over the world. We need men and women to weep over the desensitization of our youth and our own selves. What has happened to us?
If we go buy a product that comes with a booklet of instructions and a box full of parts, some electrical, some mechanical, and so forth, and we throw the book away and say, "I can do this myself,"–basically, many of us have done just this.
I don't think Our Father is asleep while innocent people suffer and more corruption and heartache trickles into the world. I think the Father is grieving over His people and His creation. You see, the order of things has been corrupted. The order has been defiled. We hear, "Make America great again," but our Father is a God of restoration and order.. We can make nothing great again without obedience to His Word. When we obey His rules for marriage, parenting, how to grow food, how to love our neighbor as ourself, and how to Love Him with all our heart and strength–Then we have peace, health, and PROSPERITY– and I am not talking about a mansion and a fat bank account.
I see parents who fear that their children won't like them, so they appease them and say things like, "That's just Miley being Miley"… No, that's just living in sin. When I got divorced, I tried to make my children like me, and I wanted to be their friends. We are not supposed to fear whether our children like us. We are to be parents. I had guilt over divorce and leaving them alone at night while working, so I tried to not discipline them much when I was with them. Then to top it off, my own sins were so rotten I really didn't feel I had the ability to correct them. They were actually pretty amazing children considering what all they went through. Praise Him!
With missing father figures in many children's lives today, we have young men who really don't know how to become a man or even what defines a man. Without having any guidance or even ambition, these young men often are waiting for a man to step up and tell them what to do with their lives. We have young women looking for a father figure instead of a husband, and young women giving their bodies away and not savoring a relationship. Young girls used to learn to be motherly, to knit, cook, and how to clean, now they have little domestic skills because after all raising children is not a career. ugh.
Many children are raised by television sets, computer games, social media, and no guidance from men who know His Torah. We have so much rotten filth in our media today, to blush about it doesn't even give it justice. My generation alone needs to repent for what it has done to our children's generation.
We are living in a world where gradually the Torah commandments have been removed from the nails that held them on the walls of our homes, school houses, and government, to the nails that put them in place on the cross and engraved His Word on our hearts that we may not sin against Him. Yeshua said, "If you love me then you will keep my commandments" John 14:15 ESV. We have become a people stripped of the commandments–stripped of obedience to our Maker, and each time we break the commandments of Yah, we lose our clothing. We are like naked Adam and Eve (Chavah), exposed right before our enemies.
When The Father looks on the sins of America, and her idolatry, it must really grieve Him. You see if we love our child, we will not continue to let him or her play in traffic. Yes, we are going to give our child a daddy spanking eventually. If we really love our children, we are going to correct them instead of letting them walk around with dirt all over their faces. Why? If we are invited to dinner and our children are dirty, we clean them up, not for their sake, but for ours, because they represent us.
"I, even I, am the one who wipes out your transgressions for My own sake, And I will not remember your sins." Isaiah 43:25.
"I have wiped out your transgressions like a thick cloud And your sins like a heavy mist. Return to Me, for I have redeemed you." Isaiah 44:22.
"For the sake of My name I delay My wrath, And for My praise I restrain it for you, In order not to cut you off." Isaiah 48:9.
"For Your name's sake, O LORD, Pardon my iniquity, for it is great." Psalm 25:11.
"Nevertheless He saved them for the sake of His name, That He might make His power known." Psalm 106:8.
Folks, we are supposed to study His Words, and speak them continuously, because if ever there is a time when Torah is not being vocalized and kept the ancient Sages believe "the earth will melt away with a fervent heat."… Peter spoke about this and said, "But the day of the Lord will come like a thief. The heavens will disappear with a roar; the elements will be destroyed by fire, and the earth and everything done in it will be laid bare." 2nd Peter 3:10.
This brings to mind the Book of Colossians, where in chapter one it says that Yeshua (THE WORD) holds ALL THINGS together and that by Him all things consist. Let's wake up and prepare for Him. Let's get our garments clean and pressed and picture His Bride all dressed in unity and love.
Photo by K. Connors.


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Keyna Wilkins
Book me today...
Call 02 4773 9009
Keyna Wilkins is a released flautist, composer and pianist based in Sydney.
Her virtuosic improvisation and musical flexibility has led her to perform along with several Sydney-based flamenco, jazz, classical and latin ensembles. Via this extensive foundation she has actually created her very own one-of-a-kind and recognisable design.
Keyna's compositions are released by Australian music posting residence, Wirripang and feature a saxophone orchestra piece, Dinner Party, a solo flute piece motivated by a flamenco dancing, Buleria Fiero a collection of solo piano pieces Sky Pieces and a 3 part tuba piece Mars. Elements of jazz, French impressionist, flamenco, Oriental folk music and the affect of modern Australian authors can be listened to in her works. Her works have actually been done around Sydney and UK.
Keyna has actually played in several high profile performances including the opening of the 2012 Adelaide Edge Festival at the Convention Centre with Flamenco Australia, (rated 4.5 superstars by the Adelaide Advertiser), the sell-out opening of the 2011 Picasso Exhibition at the Art Gallery NSW, and jazz performances on the primary field of the Royal Easter Show 2012 along with Martini Rouge jazz quartet. She has performed throughout Europe and Australia.
Birthed in picturesque Somerset, UK, Keyna had an early enthusiasm for songs, learning piano at age seven and flute at age 10. Keyna has over 10 years of teaching experience including 3 years lecturing in popular music at Bridgwater College (Somerset, UK), She currently instructs music, piano and flute theory from her estate workshop in Ashbury.
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