1,721,126 research outputs found
Identification of Retinal Pigment Epithelium-Specific Clones
No full textv, 31 p.Macular degeneration is a classification of eye diseases that includes many
different, specific forms. Age-related macular degeneration (AMD) is one particular
disease form and is one of the most frequently occurring, affecting over 30 percent of the
United States population over 75 years of age. AMD consists of two types, geographic
atrophy (dry), and choroidal neovascularization (wet), which is exceedingly worse and
can lead to blindness. The disease has proven to be highly complex, and the overall
causes and pathology of this disease are still unknown. Many risk factors are associated with AMD including smoking and hypertension; however, age is the most common risk factor. Although the specific pathology and causes of AMD are still unknown, it is thought to have significant ties to the retinal pigment epithelium (RPE), a one-ceIl-thick layer located between the retina and choroid tissues. This layer maintains the blood-brain barrier and is imperative to the maintenance of the retina Currently there is relatively little knowledge about the RPE, the genes expressed in its cells, and their expression levels. However, it is hypothesized that gene mutation in RPE cells could be responsible for AMD as well as other macular dystrophies. Polymerase Chain Reaction (PCR) technology was used to compare gene expression in RPE cells to gene expression in cells of various other human tissues in order to identify genes with specific expression within RPE cells. Three genes were found to show expression in RPE cells, but not in the other human tissues. This data suggests that these three genes could be RPE-specific.Sensory Gene Microarray Node. Center for Retinal and Macular Degeneration. W.K. Kellogg Eye Center. University of Michigan. Ann Arbor, Michigan
Understanding the mechanism(s) of retinal degeneration in X -linked retinitis pigmentosa.
No full textX-linked retinitis pigmentosa (XLRP) is a clinically and genetically heterogeneous degenerative disease of the retina. RP2 and RP3 account for 10--20% and 70--90% of genetically identifiable disease, respectively. However, mutations in the corresponding genes, RP2 and RPGR, were detected in only 10% and 20% of XLRP families. To understand the pathogenesis of XLRP, a comprehensive analysis was undertaken using genetic, molecular, and biochemical methods. To determine where the remaining mutations lie, families with no apparent RPGR or RP2 mutation were genetically characterized. Haplotype analysis provided evidence of a distinct XLRP locus RP6 that is tightly linked to RP2 and RP3. A large cohort of 234 North American families with RP and apparent X-linked inheritance was screened for all known exons of RP2 and RPGR, including a novel mutational hotspot in RPGR-ORF15. Mutations were detected in 60% of XLRP patients; however, a considerable number of mutations were also identified in simplex RP males. This is significant because simplex cases account for 50% of all RP and this data suggests that RP2 and RPGR are responsible for 30% of these. RP2 and RPGR are ubiquitous proteins, yet the mutations result only in photoreceptor degeneration. To determine the function of RP2 in the retina, two distinct approaches were undertaken. Specific monoclonal and polyclonal antibodies were produced against a RP2-GST fusion protein expressed in E. coli. Immunocytochemistry and biochemical fractionation methods localized the RP2 protein to synaptic regions in the retina. RP2 interacting proteins were identified by the two-hybrid method using three different baits to screen a bovine retinal prey cDNA library. Two members of the ADP-ribosylation factor family, ARL2 and ARL3, were identified as specific interacting proteins. Localization and interaction results suggest that the RP2 protein has a role in subcellular trafficking in retinal synapses. These studies have broad implications on research not only for XLRP, but also for understanding photoreceptor biology and function.PhDBiological SciencesGeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/131333/2/3057901.pd
Identification of Retinal Pigment Epithelium-Specific Clones
No full text1 broadside : ill.expressed in its cells, and their
expression levels.
However, it’s most commonly
hypothesized that gene
mutation in RPE cells could
be responsible for AMD as
well as other macular
dystrophies.
pathology and causes of AMD are still unknown, it is thought
to have significant ties to the retinal pigment epithelium (RPE),
a one-cell-thick layer located between the retinal and choroid
tissues. This layer maintains the blood-retina barrier and is
imperative to healthy maintenance of the retina. Currently
there is relatively little known about the RPE, the genes
Macular region of wet form AMD
Macular region of a normal, Macular region of dry form AMD
healthy human eye.
Age-related macular degeneration (AMD) is a frequentlyoccuring
eye condition that affects over 30 percent of the
United States population over 75 years of age. AMD consists
of two types, geographic atrophy (dry), and choroidal
neovascularization (wet), which is exceedingly worse and can
lead to blindness.
This disease has proven to be
highly complex, and the overall
causes and pathology are still
unknown. Many risk factors are
associated with AMD including
smoking and hypertension;
however, age is the most common
risk factor. Although the specific
this study is to identify which genes are expressed in the RPE
and identify RPE-specific genes by comparing gene expression
in cells of the RPE to gene expression in cells of various other
human tissues. This first step in understanding gene
expression and specificity in the RPE improves our
understanding of its role in age-related macular degeneration.Kalamazoo College. Department of Biology. Diebold Symposium, 2003University of Michigan. W.K. Kellogg Eye Center.Introduction -- Materials and methods -- Results -- Discussion and conclusions -- Acknowledgment
Between rods and cones: The role of NR2E3 in retinal development.
No full textNR2E3 is a photoreceptor-specific nuclear receptor, mutations in which have been identified in human patients with the disease enhanced S-cone syndrome. In humans and in the rd7 mouse, loss of functional NR2E3 results in increased numbers of S-opsin containing cones and early-onset rod degeneration. Nr2e3 transcripts are not detected in the neural retina leucine zipper knock-out (Nrl-/-) mice, which exhibit excess functional S-cones and a rod-less retina. However, the biological function of NR2E3 in photoreceptor development and the mechanism of increased S-cones observed in the absence of NR2E3 had not been elucidated when this study was undertaken. I began my investigations to test the hypothesis that NR2E3 regulates the expression of genes critical for photoreceptor differentiation and function. To examine developmental expression pattern, I generated an anti-NR2E3 polyclonal antibody and showed that the NR2E3 protein is preferentially expressed in the developing and mature rod photoreceptors, but not in the cones. I then demonstrated that NR2E3 interacts with NRL, CRX and NR1D1 (another orphan nuclear receptor) and synergistically activates the promoters of several rod-specific genes. Moreover, NR2E3, together with NRL, abolishes CRX-mediated transactivation of the S-opsin promoter. By tagging the new-born rods with green fluorescence protein (GFP), I established that in the rd7 mouse S-cone genes are expressed in the photoreceptors fated to be rods, providing a possible mechanism of enhanced S-cones observed upon the loss of Nr2e3. My studies also showed that ectopic expression of Nr2e3 in photoreceptor precursors of the Nrl-/- mice induces rod-like morphology with concurrent expression of rod-specific genes while the expression of cone-specific genes is repressed. However, these rod-like cells do not respond to visual stimuli probably due to the lack of rod transducin. These dual transcriptional regulatory functions of Nr2e3 depend on the timing and level of its expression, but not on the presence of Nrl and/or Crx. I also performed gene profiling of GFP+ photoreceptors from wild-type and mutant mouse retinas. These studies have yielded novel insights into possible gene regulatory networks involved in photoreceptor development. In conclusion, my research investigations have established NR2E3 as a key transcriptional regulator of rod versus cone photoreceptor specification during mammalian retinal development.PhDBiological SciencesHealth and Environmental SciencesMolecular biologyNeurosciencesOphthalmologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125986/2/3224842.pd
Genetic analysis of inherited retinal diseases in indigenous Southern African populations
Full text linkBackground: Inherited retinal diseases (IRDs) constitute a group of clinically and genetically heterogeneous conditions which cause degeneration of retinal photoreceptor cells and result in visual impairment. Characterisation of the genetic basis of IRD is not only beneficial for the affected families, but also contributes towards understanding of the disease pathobiology. Investigations into the molecular basis of IRDs have been ongoing in South Africa (SA) for over 30 years, however the evaluation of reported genetic mutations has yielded low returns in certain populations. Indigenous southern Africans comprise a unique population group with distinct genetic diversity, providing a valuable resource for genetic discoveries; nonetheless, this population remains largely underrepresented in genomic studies. The aim of this investigation was to characterise the underlying genetic mutations in a cohort of indigenous African IRD patients. Methods: The IRD registry in the Division of Human Genetics (University of Cape Town) was reviewed for causative mutations. Subsequently, upon identifying a mutation underlying Usher Syndrome in two indigenous African patients, an assay was designed to screen for this mutation in probands with different IRDs (n=170) and controls (n=51), and haplotype analysis was performed on mutation-positive individuals. The registry review additionally served to identify a suitable cohort for the application of next generation sequencing (NGS) technology. Whole exome sequencing (WES) was performed on genomic DNA samples from 56 individuals from 16 families. The WES data analysis strategy involved prioritisation of variants in reported and candidate IRD genes. Rare, co-segregating, pathogenic, exonic or splice variants were validated by Sanger sequencing. Custom TaqMan assays were designed to screen seven mutations, identified by WES, in 193 unrelated indigenous African probands with IRDs. Results: A homozygous founder mutation, c.6377delC in MYO7A, was identified in 43% of the indigenous African patients with Usher syndrome, which is the most common cause of deaf-blindness. Targeted WES data analysis of all known IRD genes resulted in identification of the underlying genetic defects in six distinct genes (RHO, PRPF3, PRPF31, ABCA4, CERKL, and PDE6B) in six families. Taqman screening revealed four additional probands with identical homozygous mutations in CERKL and PDE6B. An X-linked gene (RP2) mutation was subsequently identified in an affected family with semi-dominant retinitis pigmentosa. Supplementary analysis of the X-linked RPGR ORF15 mutation hotspot (not adequately covered by WES) identified two mutations in three families. A novel IRD gene, IDH3A, was found in one family by analysis of 22 putative candidate genes. The large number of variants in the remainder of the indigenous African exomes presented considerable challenges for identification of additional novel genes. Discussion: The results of this project have important implications for IRD molecular diagnostic services in SA. Using WES, a genetic diagnosis was obtained for ±73% of the indigenous African cohort, and ±70% of the causative mutations identified were novel. This outcome emphasises the superiority of NGS-based approaches over genotyping-based microarrays which screen for IRD mutations previously reported in other (mainly European-derived) populations. The unexpected identification of mutations in known X-linked genes in four families highlighted key considerations for IRD WES analysis. Cascade screening of mutations identified in this study, across larger cohorts of unrelated probands, revealed the genetic cause of IRD in additional cases and the number of indigenous African families in the registry with a genetic diagnosis was effectively doubled. Members of these families can now opt for diagnostic, carrier, or predictive testing of familial mutations. Finally, the information obtained from this research contributes towards a better understanding of the genetic architecture of IRDs in SA
The future of GM foods or GM foods of the future: where is the biotech revolution heading?
No full textIncreasing agricultural productivity to provide food security for a growing world population is one of the pressing challenges of the 21st century. Genetic engineering has the potential to greatly accelerate crop improvement, as it bypasses the lengthy process of selecting for a desired trait over several generations that is required by classical breeding methods. Genetically modified (GM) foods have been available for 20 years; however, public and political concern about potential risks associated with GM has led to tight restrictions on their import and use in some countries, and strict monitoring of foodstuffs that may contain them. In this context, a new group of technologies collectively referred to as ‘Genome Editing’ is now being applied to crop plants, which offers the advantages of standard GM approaches without many of the perceived risks.
In this chapter, we examine the food safety aspects of current GM crops, considering the methods by which they are produced, what risks this may present and how these risks are assessed. We also explore the methods currently employed to monitor the presence and prevalence of GM material in the global food industry. Finally, we discuss the potential impact of Genome Editing on the next generation of genetically engineered foods
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Transcript profiling of the cone-only Nrl-knockout retina using custom cDNA-microarrays: Identification of novel targets of Nrl and of signaling pathways involved in photoreceptor function.
No full textThe mammalian retina contains two types of photoreceptors, rods and cones, which mediate night vision and high acuity bright light color vision, respectively. Since rods greatly outnumber cones in most mammals, molecular mechanisms underlying the differences in rod and cone development, survival and function are poorly understood. I have taken advantage of a unique newly generated mutant, the Nrl-/- mouse, with cone-only retina and elucidated gene expression differences compared to the rod-dominant wild-type retina at five developmental ages. Due to the under representation of eye-expressed genes in commercially available microarrays, I generated and annotated over 10,000 eye-derived expressed sequence tags (ESTs) and produced microarrays of the sequence-tagged cDNAs. Microarray analysis showed that expression of 807 genes was significantly changed during retinal development and that of 291 genes was dramatically altered between age-matched wild-type and Nrl-/- retinas. For a selected set of genes, differential expression detected by microarray was validated by independent methods. Genes altered in the Nrl-/- retina included those encoding rod phototransduction proteins; several of these are direct targets of transcriptional regulation by Nrl. In addition to several previously unknown patterns of gene expression, my studies suggest a significant role for Bmp/Smad signaling pathway in both developing and mature rod photoreceptors. The microarray analysis also indicates a greater role for Wnt/Ca2+ pathway in cone-mediated signal transduction. These studies provide novel insights into molecular differences between rods and cones and should assist in delineating Nrl-controlled transcriptional networks involved in rod differentiation and functional maintenance.PhDBiological SciencesBiostatisticsGeneticsMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124223/2/3122081.pd
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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