ADAPTASI MASYARAKAT GAMPONG LEUGE KABUPATEN ACEH TIMUR TERHADAP BENCANA BANJIR PASANG (IE TUARA)

Aceh Timur merupakan daerah yang rentan terhadap ancaman banjir. Gampong Leuge yang terletak di kecamatan Peureulak Aceh Timur merupakan salah satu gampong yang mengalami dampak banjir pasang (Ie Tuara) tahunan dalam berbagai sektor kehidupan. Penelitian ini bertujuan untuk mengetahui 1) Bagaimana sejarah bencana Ie Tuara yang terjadi pada masyarakat Gampong Leuge. 2) Bagaimana dampak dan pola adaptasi masyarakat Gampong Leuge terhadap bencana Ie Tuara. 3) Apa yang menjadi faktor pendukung dan penghambat adaptasi masyarakat Gampong Leuge dalam menghadapi bencana Ie Tuara. Metode penelitian dengan pendekatan kualitatif untuk menghasilkan data deskriptif dan naratif. Penentuan responden dengan teknik Purposive Sampling. Pengumpulan data menggunakan pendekatan penilaian partisipatif masyarakat gampong (PRA) dengan observasi partisipatif, penulusuran, wawancara terbuka dan mendalam terhadap narasumber. Hasil penelitian menunjukan bahwa berdasarkan sejarah terdapat perubahan pola banjir pasang Ie Tuara yang berdampak kepada sektor tambak, sawah, hubungan sosial, lingkungan, dan struktur. Masyarakat melakukan serangkaian kegiatan adaptasi dan mekanisme bertahan untuk menyesuaikan diri terhadap Ie Tuara dalam mencapai suatu kearifan praktis (Phronesis). Faktor pendukung adaptasi adalah modal sosial dan lingkungan dan faktor penghambat adaptasi adalah kurangnya motivasi dan keterlibatan masyarkat dalam pembangunan. Diharapkan masyarakat dan pemerintah perlu bersama-sama memperkuat kapasitas dengan cara melibatkan, memberdayakan dan melindungi masyarakat yang rentan terhadap risko bencana

ADAPTASI MASYARAKAT GAMPONG LEUGE KECAMATAN PEUREULAK ACEH TIMUR TERHADAP BENCANA BANJIR PASANG (IE TUARA)

Abstract: The aim of this research was to describe the community adaptation patterns along with the enabling and constraint factors against Ie Tuara. This research is used social qualitative approach. The subjects of this research are consisted of the local community those are directly affected by Ie Tuara and also the people who are considered fully understand about the tide flood.  The steps of this research consisted of (1) the study of literature, (2) observation, (3) the development of the questions through the framework of participatory rural appraisal (4) transect-walks techniques, (5) interviews, and (6) The analysis that consists of data reduction and elections of data’s trend in accordance with the purpose of research. The research shown that the communityy has already owned adaptation patterns which are applied in ponds, paddy fields, social, environmental and also structural. The enabling factors of adaptation are the existences of social and environment modal in Gampong Leuge meanwhile the constraint factors are lack of capital for improving the livelihood sector and lack of community motivation in conservation.  In the future, it is recommended that the program of community empowerement towards environment alongside with sustainable community based adaptation and mitigation efforts.Keywords: Ie Tuara, Adaptation, enabling and constraint factors.  Abstrak: Tujuan penelitian ini untuk mendeskripsikan pola adaptasi serta faktor pendukung dan penghambatnya terhadap bencana Ie Tuara. Penelitian ini menggunakan pendekatan sosial qualitatif. Subjek penelitian: masyarakat lokal yang terkena dampak langsung dari bencana Ie Tuara dan juga orang yang dianggap paling memahami tentang bencana banjir pasang. Tahapan penelitian ini terdiri dari (1) studi literatur, (2) observasi lapangan, (3) pengembangan butir pertanyaan melalui kerangka penilaian partisipatif pedesaan (4) teknik penelusuran, (5) wawancara, dan (6) Analisis terdiri dari reduksi data dan pemilihan pola data yang sesuai dengan tujuan penelitian. Hasil penelitian menunjukan bahwa masyarakat memiliki adaptasi yang diterapkan dalam sektor tambak, sawah, sosial, lingkungan dan juga struktural. Faktor pendukung adaptasi adalah adanya modal sosial dan lingkungan yang masih terdapat di Gampong Leuge. Faktor penghambat adaptasi adalah kurangnya modal dalam memperkuat sektor penghidupan dan lemahnya motivasi masyarakat dalam pelestarian lingkungan. Dimasa depan direkomendasikan program pemberdayaan kapasitas masyarakat ke arah lingkungan serta upaya berkelanjutan pada adaptasi dan mitigasi bencana berbasis komunitas.   Kata kunci: Ie Tuara, adaptasi, faktor pendukung dan penghambat

Nuclear delivery of NFκB-assisted DNA/polymer complexes: plasmid DNA quantitation by confocal laser scanning microscopy and evidence of nuclear polyplexes by FRET imaging

Quantification of a plasmid DNA (pDNA) and investigation of its polymer-associated state in the nucleus are crucial to evaluate the effectiveness of a gene-delivery system. This study was conducted with p3NF-luc-3NF, a pDNA-bearing optimized κB motif to favour NFκB-driven nuclear import. Here, a quantification of pDNA copies in the nucleus was performed by real-time confocal laser scanning microscopy in HeLa and C2C12 cells transfected with linear polyethylenimine or histidylated polylysine. Förster Resonance Energy Transfer (FRET) from the fluorescein-p3NF-luc-3NF donor to the co-localized rhodamine-polymer acceptor was carried out to investigate whether the pDNA was still condensed with the polymer in the nucleus. Upon 5 h of transfection, the nuclear amount of p3NF-luc3NF was ∼1500 copies in both cell lines whereas that of pTAL-luc, a 3NF-free counterpart pDNA, was less than 250. This quantity of p3NF-luc-3NF dropped dramatically to that of pTAL-luc in the presence of the BAY 11-7085, an inhibitor of NFκB activation. These data strongly support a nuclear import of p3NF-luc3NF mediated by NFκB. Moreover, FRET experiments clearly revealed that most of nuclear pDNA were still condensed with the polymer raising the question of their passage through the nuclear pore complex and their impact on the gene-expression efficiency

Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research

Tau Interaction with Tubulin and Microtubules: From Purified Proteins to Cells

International audienceMicrotubules (MTs) play an important role in many cellular processes and are dynamic structures regulated by an important network of microtubules-associated proteins, MAPs, such as Tau. Tau has been discovered as an essential factor for MTs formation in vitro, and its region implicated in binding to MTs has been identified. By contrast, the affinity, the stoichiometry, and the topology of Tau-MTs interaction remain controversial. Indeed, depending on the experiment conditions a wide range of values have been obtained. In this chapter, we focus on three biophysical methods, turbidimetry, cosedimentation assay, and Förster Resonance Energy Transfer to study Tau-tubulin interaction both in vitro and in cell. We highlight precautions that must be taken in order to avoid pitfalls and we detail the nature of the conclusions that can be drawn from these methods about Tau-tubulin interaction

A Bayesian method for inferring quantitative information from FRET data

<p>Abstract</p> <p>Background</p> <p>Understanding biological networks requires identifying their elementary protein interactions and establishing the timing and strength of those interactions. Fluorescence microscopy and Förster resonance energy transfer (FRET) have the potential to reveal such information because they allow molecular interactions to be monitored in living cells, but it is unclear how best to analyze FRET data. Existing techniques differ in assumptions, manipulations of data and the quantities they derive. To address this variation, we have developed a versatile Bayesian analysis based on clear assumptions and systematic statistics.</p> <p>Results</p> <p>Our algorithm infers values of the FRET efficiency and dissociation constant, <it>K_d</it>, between a pair of fluorescently tagged proteins. It gives a posterior probability distribution for these parameters, conveying more extensive information than single-value estimates can. The width and shape of the distribution reflects the reliability of the estimate and we used simulated data to determine how measurement noise, data quantity and fluorophore concentrations affect the inference. We are able to show why varying concentrations of donors and acceptors is necessary for estimating <it>K_d</it>. We further demonstrate that the inference improves if additional knowledge is available, for example of the FRET efficiency, which could be obtained from separate fluorescence lifetime measurements.</p> <p>Conclusions</p> <p>We present a general, systematic approach for extracting quantitative information on molecular interactions from FRET data. Our method yields both an estimate of the dissociation constant and the uncertainty associated with that estimate. The information produced by our algorithm can help design optimal experiments and is fundamental for developing mathematical models of biochemical networks.</p

APPL Proteins FRET at the BAR: Direct Observation of APPL1 and APPL2 BAR Domain-Mediated Interactions on Cell Membranes Using FRET Microscopy

Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH) domain, and a C-terminal phosphotyrosine binding (PTB) domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET) experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2) and heterotypic (i.e., APPL1-APPL2) manner on curved cell membranes. Furthermore, the results of our experiments did not show photoconversion of YFP into a CFP-like species following photobleaching, supporting the use of CFP donor/YFP acceptor FRET pairs in acceptor photobleaching studies

Heme Oxygenase Isoforms Differ in Their Subcellular Trafficking during Hypoxia and Are Differentially Modulated by Cytochrome P450 Reductase

Heme oxygenase (HO) degrades heme in concert with NADPH cytochrome P450 reductase (CPR) which donates electrons to the reaction. Earlier studies reveal the importance of the hydrophobic carboxy-terminus of HO-1 for anchorage to the endoplasmic reticulum (ER) which facilitates the interaction with CPR. In addition, HO-1 has been shown to undergo regulated intramembrane proteolysis of the carboxy-terminus during hypoxia and subsequent translocation to the nucleus. Translocated nuclear HO-1 was demonstrated to alter binding of transcription factors and to alter gene expression. Little is known about the homologous membrane anchor of the HO-2 isoform. The current work is the first systematic analysis in a eukaryotic system that demonstrates the crucial role of the membrane anchor of HO-2 for localization at the endoplasmic reticulum, oligomerization and interaction with CPR. We show that although the carboxy-terminal deletion mutant of HO-2 is found in the nucleus, translocation of HO-2 to the nucleus does not occur under conditions of hypoxia. Thus, we demonstrate that proteolytic regulation and nuclear translocation under hypoxic conditions is specific for HO-1. In addition we show for the first time that CPR prevents this translocation and promotes oligomerization of HO-1. Based on these findings, CPR may modulate gene expression via the amount of nuclear HO-1. This is of particular relevance as CPR is a highly polymorphic gene and deficiency syndromes of CPR have been described in humans

Fluorescent Fusion Proteins of Soluble Guanylyl Cyclase Indicate Proximity of the Heme Nitric Oxide Domain and Catalytic Domain

BACKGROUND: To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC) Förster resonance energy transfer (FRET) was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC beta1 and alpha subunits. METHODOLOGY/PRINCIPAL FINDINGS: Cyan fluorescent protein (CFP) was used as FRET donor and yellow fluorescent protein (YFP) as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged alpha subunits with the carboxy-terminally tagged beta1 subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the alpha subunits is close to the carboxy-terminus of the beta1 subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO. CONCLUSIONS/SIGNIFICANCE: Based on the ability of an amino-terminal fragment of the beta1 subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (alphacatbetacat), Winger and Marletta (Biochemistry 2005, 44:4083-90) have proposed a direct interaction of the amino-terminal region of beta1 with the catalytic domains. In support of such a concept of "trans" regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed "fluorescent-conjoined" sGC's by fusion of the alpha amino-terminus to the beta1 carboxy-terminus leading to a monomeric, fluorescent and functional enzyme complex. To our knowledge this represents the first example where a fluorescent protein links two different subunits of a higher ordered complex to yield a stoichometrically fixed functionally active monomer