Restriction fragment length polymorphism-coupled domain-directed differential display: a highly efficient technique for expression analysis of multigene families.

In this paper, a reverse-transcriptase PCR-based protocol suitable for efficient expression analysis of multigene families is presented. The method combines restriction fragment length polymorphism (RFLP) technology with a gene family-specific version of mRNA differential display and hence is called "RFLP-coupled domain-directed differential display. "With this method, expression of all members of a multigene family at many different developmental stages, in diverse tissues and even in different organisms, can be displayed on one gel. Moreover, bands of interest, representing gene family members, are directly accessible to sequence analysis, without the need for subcloning. The method thus enables a detailed, high-resolution expression analysis of known gene family members as well as the identification and characterization of new ones. Here the technique was used to analyze differential expression of MADS-box genes in male and female inflorescences of maize (Zea mays ssp. mays). Six different MADS-box genes could be identified, being either specifically expressed in the female sex or preferentially expressed in male or female inflorescences, respectively. Other possible applications of the method are discussed.

Primary structure of hepatocyte nuclear factor/forkhead homologue 4 and characterization of gene expression in the developing respiratory and reproductive epithelium.

Members of the winged helix/forkhead family of transcription factors are believed to play a role in cell-specific gene expression. A cDNA encoding a member of this family of proteins, termed hepatocyte nuclear factor/forkhead homologue 4 (HFH-4), has been isolated from rat lung and rat testis cDNA libraries. This cDNA contains an open reading frame of 421 amino acids with a conserved DNA binding domain and several potential transactivating regions. During murine lung development, a single species of HFH-4-specific transcript (2.4 kb long) is first detected precisely at the start of the late pseudoglandular stage (embryonic day 14.5) and, by in situ hybridization, is specifically localized to the proximal pulmonary epithelium. The unique temporal and spatial pattern of HFH-4 gene expression in the developing lung defines this protein as a marker for the initiation of bronchial epithelial cell differentiation and suggests that it may play an important role in cell fate determination during lung development. In addition to expression in the pulmonary epithelium, RNA blot analysis reveals 2.4-kb HFH-4 transcripts in the testis and oviduct. By using mice with genetic defects in spermatogenesis, HFH-4 expression in the testis is found to be associated with the appearance of haploid germ cells and in situ hybridization studies indicate that HFH-4 expression is confined to stages I-VII of spermatogenesis. This pattern of HFH-4 gene expression during the early stages of differentiation of haploid germ cells suggests that HFH-4 may play a role in regulating stage-specific gene expression and cell-fate determination during lung development and in spermatogenesis.

Perda auditiva unilateral: benefício da amplificação na ordenação e resolução temporal e localização sonora

A perda auditiva unilateral (PAUn) é caracterizada pela diminuição da audição em apenas uma orelha. Indivíduos com este tipo de perda auditiva podem apresentar comprometimento nas habilidades auditivas de localização sonora, processamento temporal, ordenação e resolução temporal. O objetivo deste estudo foi verificar as habilidades auditivas de ordenação temporal, resolução temporal e localização sonora, antes e após a adaptação do aparelho de amplificação sonora individual (AASI). Foram avaliados 22 indivíduos, com idades entre 18 e 60 anos, com diagnóstico de PAUn sensorioneural ou mista, de graus leve a severo. O estudo foi dividido em duas etapas: a pré e a pós-adaptação de AASI. Em ambas as etapas, os indivíduos foram submetidos a uma anamnese, aplicação do Questionário de Habilidade Auditiva da Localização da fonte sonora, avaliação simplificada do processamento auditivo (ASPA) e Random Gap Detection Test (RGDT). O presente estudo encontrou diferença estatisticamente significante na avaliação da ASPA, exceto no teste de memória para sons não verbais em sequência (TMSnV), no RGDT e no Questionário de Habilidade Auditiva da Localização Sonora. A conclusão do estudo foi que com o uso efetivo do AASI, indivíduos com PAUn apresentaram melhora nas habilidades auditivas de localização sonora, ordenação e resolução temporal.

Variation in the expression of migratory activity in blackcap (Sylvia atricapilla); effects of the origin, environmental conditions, dominance and personalities

La migración es una respuesta a cambios estacionales del clima generando desplazamientos periódicos entre hábitats de cría y de invernada, permitiendo así el uso temporal de los recursos disponibles. La migración implica unos costes energéticos muy elevados, un aumento de la depredación potencial, variaciones ambientales y una disponibilidad de alimento impredecible a lo largo de la ruta migratoria; por lo que es una de las actividades más desafiantes de su ciclo vital. A pesar de ello, los beneficios de la migración compensan sus costes. La migración está programada genéticamente, siendo relativamente constante en su momento, distancia y dirección. Por otro lado, ambiente juega un papel predominante en algunas poblaciones, pudiendo modificar el comportamiento migratorio de una estrategia parcial o facultativa a un modo de vida sedentario. Con el fin de describir el origen y evolución del comportamiento migratorio en aves, se ha propuesto un “modelo de umbral” genético para determinar si un ave es migrante o sedentaria. Dentro de una variable continua (p.ej. la concentración de proteínas u hormonas), este modelo asume que existe una actividad migratoria subyacente implicada en su expresión génica. Este umbral divide cada variable en categorías dicotómicas que definen el fenotipo de un individuo. Los ejemplares sin actividad migratoria muestran valores por debajo de este umbral, siendo clasificados como sedentarios, mientras que los ejemplares migrantes muestran valores por encima del umbral definido. Los cambios de estrategia vital no dependen únicamente de la posición del umbral determinado genéticamente sino también de las variables ambientales, por lo que dichas variaciones deben ser añadidas al modelo. Este modelo de umbral ambiental predice que el carácter migratorio de los individuos situados en los extremos de distribución no se encuentra afectado por los factores ambientales, mientras que aquellos más próximos al umbral pueden más fácilmente cambiar su estrategia migratoria...

A high-resolution binocular video-oculography system: assessment of pupillary light reflex and detection of an early incomplete blink and an upward eye movement

Background: The pupillary light reflex characterizes the direct and consensual response of the eye to the perceived brightness of a stimulus. It has been used as indicator of both neurological and optic nerve pathologies. As with other eye reflexes, this reflex constitutes an almost instantaneous movement and is linked to activation of the same midbrain area. The latency of the pupillary light reflex is around 200 ms, although the literature also indicates that the fastest eye reflexes last 20 ms. Therefore, a system with sufficiently high spatial and temporal resolutions is required for accurate assessment. In this study, we analyzed the pupillary light reflex to determine whether any small discrepancy exists between the direct and consensual responses, and to ascertain whether any other eye reflex occurs before the pupillary light reflex. Methods: We constructed a binocular video-oculography system two high-speed cameras that simultaneously focused on both eyes. This was then employed to assess the direct and consensual responses of each eye using our own algorithm based on Circular Hough Transform to detect and track the pupil. Time parameters describing the pupillary light reflex were obtained from the radius time-variation. Eight healthy subjects (4 women, 4 men, aged 24–45) participated in this experiment. Results: Our system, which has a resolution of 15 microns and 4 ms, obtained time parameters describing the pupillary light reflex that were similar to those reported in previous studies, with no significant differences between direct and consensual reflexes. Moreover, it revealed an incomplete reflex blink and an upward eye movement at around 100 ms that may correspond to Bell’s phenomenon. Conclusions: Direct and consensual pupillary responses do not any significant temporal differences. The system and method described here could prove useful for further assessment of pupillary and blink reflexes. The resolution obtained revealed the existence reported here of an early incomplete blink and an upward eye movement.

Temporal Control of Leaf Complexity by miRNA-Regulated Licensing of Protein Complexes

The tremendous diversity of leaf shapes has caught the attention of naturalists for centuries. In addition to interspecific and intraspecific differences, leaf morphologies may differ in single plants according to age, a phenomenon known as heteroblasty. In Arabidopsis thaliana, the progression from the juvenile to the adult phase is characterized by increased leaf serration. A similar trend is seen in species with more complex leaves, such as the A. thaliana relative Cardamine hirsuta, in which the number of leaflets per leaf increases with age. Although the genetic changes that led to the overall simpler leaf architecture in A. thaliana are increasingly well understood, less is known about the events underlying age-dependent changes within single plants, in either A. thaliana or C. hirsuta. Here, we describe a conserved miRNA transcription factor regulon responsible for an age-dependent increase in leaf complexity. In early leaves, miR319-targeted TCP transcription factors interfere with the function of miR164-dependent and miR164-independent CUC proteins, preventing the formation of serrations in A. thaliana and of leaflets in C. hirsuta. As plants age, accumulation of miR156-regulated SPLs acts as a timing cue that destabilizes TCP-CUC interactions. The destabilization licenses activation of CUC protein complexes and thereby the gradual increase of leaf complexity in the newly formed organs. These findings point to posttranslational interaction between unrelated miRNA-targeted transcription factors as a core feature of these regulatory circuits.

High-resolution dynamical downscaling products for the North Slope of Alaska and surrounding areas, links to model result files

Climatic changes are most pronounced in northern high latitude regions. Yet, there is a paucity of observational data, both spatially and temporally, such that regional-scale dynamics are not fully captured, limiting our ability to make reliable projections. In this study, a group of dynamical downscaling products were created for the period 1950 to 2100 to better understand climate change and its impacts on hydrology, permafrost, and ecosystems at a resolution suitable for northern Alaska. An ERA-interim reanalysis dataset and the Community Earth System Model (CESM) served as the forcing mechanisms in this dynamical downscaling framework, and the Weather Research & Forecast (WRF) model, embedded with an optimization for the Arctic (Polar WRF), served as the Regional Climate Model (RCM). This downscaled output consists of multiple climatic variables (precipitation, temperature, wind speed, dew point temperature, and surface air pressure) for a 10 km grid spacing at three-hour intervals. The modeling products were evaluated and calibrated using a bias-correction approach. The ERA-interim forced WRF (ERA-WRF) produced reasonable climatic variables as a result, yielding a more closely correlated temperature field than precipitation field when long-term monthly climatology was compared with its forcing and observational data. A linear scaling method then further corrected the bias, based on ERA-interim monthly climatology, and bias-corrected ERA-WRF fields were applied as a reference for calibration of both the historical and the projected CESM forced WRF (CESM-WRF) products. Biases, such as, a cold temperature bias during summer and a warm temperature bias during winter as well as a wet bias for annual precipitation that CESM holds over northern Alaska persisted in CESM-WRF runs. The linear scaling of CESM-WRF eventually produced high-resolution downscaling products for the Alaskan North Slope for hydrological and ecological research, together with the calibrated ERA-WRF run, and its capability extends far beyond that. Other climatic research has been proposed, including exploration of historical and projected climatic extreme events and their possible connections to low-frequency sea-atmospheric oscillations, as well as near-surface permafrost degradation and ice regime shifts of lakes. These dynamically downscaled, bias corrected climatic datasets provide improved spatial and temporal resolution data necessary for ongoing modeling efforts in northern Alaska focused on reconstructing and projecting hydrologic changes, ecosystem processes and responses, and permafrost thermal regimes. The dynamical downscaling methods presented in this study can also be used to create more suitable model input datasets for other sub-regions of the Arctic.

Quantifying ice-shelf basal melt and ice-stream dynamics using high-resolution DEM and GPS time series

Thesis (Ph.D.)--University of Washington, 2016-06

Cardiac expression of the cystic fibrosis transmembrane conductance regulator involves novel Exon 1 usage to produce a unique amino-terminal protein

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel present in many cells. In cardiomyocytes, we report that multiple exon 1 usage and alternative splicing produces four CFTR transcripts, with different 5'-untranslated regions, CFTRTRAD-139, CFTR-1C/-1A, CFTR-1C, and CFTR-1B. CFTR transcripts containing the novel upstream exons (exons -1C, -1B, and -1A) represent more than 90% of cardiac expressed CFTR mRNA. Regulation of cardiac CFTR expression, in response to developmental and pathological stimuli, is exclusively due to the modulation of CFTR-1C and CFTR-1C/-1A expression. Upstream open reading frames have been identified in the 5'-untranslated regions of all CFTR transcripts that, in conjunction with adjacent stem-loop structures, modulate the efficiency of translation initiation at the AUG codon of the main CFTR coding region in CFTRTRAD-139 and CFTR-1C/-1A transcripts. Exon(-1A), only present in CFTR-1C/-1A transcripts, encodes an AUG codon that is in-frame with the main CFTR open reading frame, the efficient translation of which produces a novel CFTR protein isoform with a curtailed amino terminus. As the expression of this CFTR transcript parallels the spatial and temporal distribution of the cAMP-activated whole-cell current density in normal and diseased hearts, we suggest that CFTR-1C/-1A provides the molecular basis for the cardiac cAMP-activated chloride channel. Our findings provide further insight into the complex nature of in vivo CFTR expression, to which multiple mRNA transcripts, protein isoforms, and post-transcriptional regulatory mechanisms are now added.

Co-expression of SOX9 and SOX10 during melanocytic differentiation in vitro

Investigations into pigment cell biology have relied on the ability to culture both murine and human melanocytes, numerous melanoma cell lines and more recently, murine and human melanoblasts. Melanoblast culture requires medium supplemented with a range of growth factors including Stem Cell Factor, Endothelin-3 and Fibroblast Growth Factor-2, withdrawal of which causes the cells to differentiate into melanocytes. Using the human melanoblast culture system, we have now examined the expression and/or DNA binding activity of several transcription factors implicated in melanocytic development and differentiation. Of these, the POU domain factor BRN2 and the SOX family member SOX10 are both highly expressed in unpigmented melanocyte precursors but are down-regulated upon differentiation. In contrast, the expression levels of the previously described MITF and PAX3 transcription factors remain relatively constant during the melanoblast-melanocyte transition. Moreover, BRN2 ablated melanoma cells lack expression of SOX10 and MITF but retain PAX3. A novel finding implicates a second SOX protein, SOX9, as a potential melanogenic transcriptional regulator, as its expression level is increased following the down-regulation of BRN2 and SOX10 in differentiated melanoblasts. Our results suggest that a complex network of transcription factor interactions requiring proper temporal coordination is necessary for acquisition and maintenance of the melanocytic phenotype. (c) 2005 Elsevier Inc. All rights reserved.

Monitoring the condition of savanna riparian zones in Northern Australia using high spatial resolution imagery and a field based rapid assessment method

Government agencies responsible for riparian environments are assessing the combined utility of field survey and remote sensing for mapping and monitoring indicators of riparian zone condition. The objective of this work was to compare the Tropical Rapid Appraisal of Riparian Condition (TRARC) method to a satellite image based approach. TRARC was developed for rapid assessment of the environmental condition of savanna riparian zones. The comparison assessed mapping accuracy, representativeness of TRARC assessment, cost-effectiveness, and suitability for multi-temporal analysis. Two multi-spectral QuickBird images captured in 2004 and 2005 and coincident field data covering sections of the Daly River in the Northern Territory, Australia were used in this work. Both field and image data were processed to map riparian health indicators (RHIs) including percentage canopy cover, organic litter, canopy continuity, stream bank stability, and extent of tree clearing. Spectral vegetation indices, image segmentation and supervised classification were used to produce RHI maps. QuickBird image data were used to examine if the spatial distribution of TRARC transects provided a representative sample of ground based RHI measurements. Results showed that TRARC transects were required to cover at least 3% of the study area to obtain a representative sample. The mapping accuracy and costs of the image based approach were compared to those of the ground based TRARC approach. Results proved that TRARC was more cost-effective at smaller scales (1-100km), while image based assessment becomes more feasible at regional scales (100-1000km). Finally, the ability to use both the image and field based approaches for multi-temporal analysis of RHIs was assessed. Change detection analysis demonstrated that image data can provide detailed information on gradual change, while the TRARC method was only able to identify more gross scale changes. In conclusion, results from both methods were considered to complement each other if used at appropriate spatial scales.

Retrotransposon expression profiling: Unveiling the hidden SINE transcriptome through Next-Generation Sequencing data analysis

Of the ~1.7 million SINE elements in the human genome, only a tiny number are estimated to be active in transcription by RNA polymerase (Pol) III. Tracing the individual loci from which SINE transcripts originate is complicated by their highly repetitive nature. By exploiting RNA-Seq datasets and unique SINE DNA sequences, we devised a bioinformatic pipeline allowing us to identify Pol III-dependent transcripts of individual SINE elements. When applied to ENCODE transcriptomes of seven human cell lines, this search strategy identified ~1300 Alu loci and ~1100 MIR loci corresponding to detectable transcripts, with ~120 and ~60 respectively Alu and MIR loci expressed in at least three cell lines. In vitro transcription of selected SINEs did not reflect their in vivo expression properties, and required the native 5’-flanking region in addition to internal promoter. We also identified a cluster of expressed AluYa5-derived transcription units, juxtaposed to snaR genes on chromosome 19, formed by a promoter-containing left monomer fused to an Alu-unrelated downstream moiety. Autonomous Pol III transcription was also revealed for SINEs nested within Pol II-transcribed genes raising the possibility of an underlying mechanism for Pol II gene regulation by SINE transcriptional units. Moreover the application of our bioinformatic pipeline to both RNA-seq data of cells subjected to an in vitro pro-oncogenic stimulus and of in vivo matched tumor and non-tumor samples allowed us to detect increased Alu RNA expression as well as the source loci of such deregulation. The ability to investigate SINE transcriptomes at single-locus resolution will facilitate both the identification of novel biologically relevant SINE RNAs and the assessment of SINE expression alteration under pathological conditions.

Dissociating the spatio-temporal characteristics of cortical neuronal activity associated with human volitional swallowing in the healthy adult brain

Human swallowing represents a complex highly coordinated sensorimotor function whose functional neuroanatomy remains incompletely understood. Specifically, previous studies have failed to delineate the temporo-spatial sequence of those cerebral loci active during the differing phases of swallowing. We therefore sought to define the temporal characteristics of cortical activity associated with human swallowing behaviour using a novel application of magnetoencephalography (MEG). In healthy volunteers (n = 8, aged 28-45), 151-channel whole cortex MEG was recorded during the conditions of oral water infusion, volitional wet swallowing (5 ml bolus), tongue thrust or rest. Each condition lasted for 5 s and was repeated 20 times. Synthetic aperture magnetometry (SAM) analysis was performed on each active epoch and compared to rest. Temporal sequencing of brain activations utilised time-frequency wavelet plots of regions selected using virtual electrodes. Following SAM analysis, water infusion preferentially activated the caudolateral sensorimotor cortex, whereas during volitional swallowing and tongue movement, the superior sensorimotor cortex was more strongly active. Time-frequency wavelet analysis indicated that sensory input from the tongue simultaneously activated caudolateral sensorimotor and primary gustatory cortex, which appeared to prime the superior sensory and motor cortical areas, involved in the volitional phase of swallowing. Our data support the existence of a temporal synchrony across the whole cortical swallowing network, with sensory input from the tongue being critical. Thus, the ability to non-invasively image this network, with intra-individual and high temporal resolution, provides new insights into the brain processing of human swallowing. © 2004 Elsevier Inc. All rights reserved.

Investigation of neural correlates of faces and emotional expressions using magnetoencephalography

The recognition of faces and of facial expressions in an important evolutionary skill, and an integral part of social communication. It has been argued that the processing of faces is distinct from the processing of non-face stimuli and functional neuroimaging investigations have even found evidence of a distinction between the perception of faces and of emotional expressions. Structural and temporal correlates of face perception and facial affect have only been separately identified. Investigation neural dynamics of face perception per se as well as facial affect would allow the mapping of these in space, time and frequency specific domains. Participants were asked to perform face categorisation and emotional discrimination tasks and Magnetoencephalography (MEG) was used to measure the neurophysiology of face and facial emotion processing. SAM analysis techniques enable the investigation of spectral changes within specific time-windows and frequency bands, thus allowing the identification of stimulus specific regions of cortical power changes. Furthermore, MEG’s excellent temporal resolution allows for the detection of subtle changes associated with the processing of face and non-face stimuli and different emotional expressions. The data presented reveal that face perception is associated with spectral power changes within a distributed cortical network comprising occipito-temporal as well as parietal and frontal areas. For the perception of facial affect, spectral power changes were also observed within frontal and limbic areas including the parahippocampal gyrus and the amygdala. Analyses of temporal correlates also reveal a distinction between the processing of faces and facial affect. Face perception per se occurred at earlier latencies whereas the discrimination of facial expression occurred within a longer time-window. In addition, the processing of faces and facial affect was differentially associated with changes in cortical oscillatory power for alpha, beta and gamma frequencies. The perception of faces and facial affect is associated with distinct changes in cortical oscillatory activity that can be mapped to specific neural structures, specific time-windows and latencies as well as specific frequency bands. Therefore, the work presented in this thesis provides further insight into the sequential processing of faces and facial affect.