Implementasi Program Pemberdayaan Ekonomi Rakyat Melalui Program Mamangun Tuntang Mahaga Lewu (Pm2l) (Studi Kasus Di Dua Desa Tertinggal Di Kalimantan Tengah)

This study aims to determine the implementation of the program of economic empowerment of the people through the program mamangun Tuntang mahaga Lewu (PM2L). Writing method used is qualitative. This method was chosen because it examines the phenomenon of something in more depth, and more able to understand the phenomenon that until now has not been known. Through this study were obtained in the implementation of key information that PM2L are several stages to go through the stage of coordination, socialization, implementation of the action, coaching, monitoring and evaluation. In general, the stages through which it has not been optimal program implementation in practice, especially in terms of stages of development

Embryonic development and maternal regulation of murine circadian clock function

<div><p>The importance of circadian clocks in the regulation of adult physiology in mammals is well established. In contrast, the ontogenesis of the circadian system and its role in embryonic development are still poorly understood. Although there is experimental evidence that the clock machinery is present prior to birth, data on gestational clock functionality are inconsistent. Moreover, little is known about the dependence of embryonic rhythms on maternal and environmental time cues and the role of circadian oscillations for embryonic development. The aim of this study was to test if fetal mouse tissues from early embryonic stages are capable of expressing endogenous, self-sustained circadian rhythms and their contribution to embryogenesis. Starting on embryonic day 13, we collected precursor tissues for suprachiasmatic nucleus (SCN), liver and kidney from embryos carrying the circadian reporter gene <i>Per2::Luc</i> and investigated rhythmicity and circadian traits of these tissues <i>ex vivo</i>. We found that even before the respective organs were fully developed, embryonic tissues were capable of expressing circadian rhythms. Period and amplitude of which were determined very early during development and phases of liver and kidney explants are not influenced by tissue preparation, whereas SCN explants phasing is strongly dependent on preparation time. Embryonic circadian rhythms also developed in the absence of maternal and environmental time signals. Morphological and histological comparison of offspring from matings of <i>Clock-Δ19</i> mutant and wild-type mice revealed that both fetal and maternal clocks have distinct roles in embryogenesis. While genetic disruptions of maternal and embryonic clock function leads to increased fetal fat depots, abnormal ossification and organ development, <i>Clock</i> gene mutant newborns from mothers with a functional clock showed a larger body size compared to wild-type littermates. These data may contribute to the understanding of the ontogenesis of circadian clocks and the risk of disturbed maternal or embryonic circadian rhythms for embryonic development.</p></div

The SCN oscillator network is responsible for stable rhythms in <i>Clock</i>^<i>-/-</i> SCN neurons.

<p>(A) Raster plots of mPer2^Luc bioluminescence intensity of individual dispersed wild-type (left, n = 246) and <i>Clock</i>^<i>-/-</i> (right, n = 161) SCN cells. Data are presented as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005882#pgen.1005882.g001" target="_blank">Fig 1C</a>. (B) mPer2^Luc bioluminescence rhythms of two representative rhythmic individual dispersed SCN neurons from wild type (left) and <i>Clock</i>^<i>-/-</i> mice (right). (C) Circadian period, amplitude, and sine wave goodness-of-fit of cellular mPer2^Luc rhythms, and the percentage of rhythmic neurons in dispersed SCN cultures from wild type (white), <i>Clock</i>^<i>-/-</i> (black), and <i>Bmal1</i>^<i>-/-</i> (patterned) mice. <i>Bmal1</i>^<i>-/-</i> data are from Ko et al [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005882#pgen.1005882.ref018" target="_blank">18</a>]. Data are shown as mean ± SEM; ***<i>p</i>≤0.001 (student’s t-test); or as percentage of cells that are significantly rhythmic; WT: n (rhythmic/total) = 234/255; <i>Clock</i>^<i>-/-</i>: n = 138/208; <i>Bmal1</i>^<i>-/-</i>: n = 30/243.</p

NPAS2 Compensates for Loss of CLOCK in Peripheral Circadian Oscillators

<div><p>Heterodimers of CLOCK and BMAL1 are the major transcriptional activators of the mammalian circadian clock. Because the paralog NPAS2 can substitute for CLOCK in the suprachiasmatic nucleus (SCN), the master circadian pacemaker, CLOCK-deficient mice maintain circadian rhythms in behavior and in tissues <i>in vivo</i>. However, when isolated from the SCN, CLOCK-deficient peripheral tissues are reportedly arrhythmic, suggesting a fundamental difference in circadian clock function between SCN and peripheral tissues. Surprisingly, however, using luminometry and single-cell bioluminescence imaging of PER2 expression, we now find that CLOCK-deficient dispersed SCN neurons and peripheral cells exhibit similarly stable, autonomous circadian rhythms <i>in vitro</i>. In CLOCK-deficient fibroblasts, knockdown of <i>Npas2</i> leads to arrhythmicity, suggesting that NPAS2 can compensate for loss of CLOCK in peripheral cells as well as in SCN. Our data overturn the notion of an SCN-specific role for NPAS2 in the molecular circadian clock, and instead indicate that, at the cellular level, the core loops of SCN neuron and peripheral cell circadian clocks are fundamentally similar.</p></div

Peripheral organs of <i>Clock</i>^<i>-/-</i> mice can exhibit circadian rhythms <i>in vitro</i>.

<p>(A) Two representative mPer2^Luc bioluminescence rhythms of rhythmic organotypic liver (black), lung (blue), kidney (red), and adrenal (green) slice cultures from wild type (left) and <i>Clock</i>^<i>-/-</i> (right) mice. After ~7 culture days, samples were treated with 10 μM forskolin (arrow). Y-axis scales are adjusted to amplitudes for better visualization of data. (B) Circadian mPer2^Luc rhythm period, amplitude, damping constant (days to reach 1/e of initial amplitude), and phase of first peak after forskolin treatment, and % of slices from wild type (unfilled bars) and <i>Clock</i>^<i>-/-</i> mice (filled bars) that were significantly rhythmic after forskolin treatment (culture days 8–14). Data are shown as mean ± SEM; *<i>p</i>≤0.05, **<i>p</i>≤0.01, ***<i>p</i>≤0.001 (student’s t-test); or % of slices rhythmic; n = 8.</p

Knockdown of <i>Npas2</i> expression suppresses circadian rhythms in <i>Clock</i>^<i>-/-</i> fibroblasts.

<p>(A) Fibroblasts dispersed from wild type and <i>Clock</i>^<i>-/-</i> mice were treated with lentiviral vectors carrying an <i>Npas2</i>-KD or scrambled DNA sequence, as well as a GFP reporter. Simultaneous fluorescence and bioluminescence images of a representative field show GFP expression marking transfected cells (left, green) and mPer2^Luc bioluminescence from both transfected and untransfected cells (middle, red). The overlay shows that circadian rhythms could be measured from both transfected (filled arrowheads) and untransfected (unfilled arrowheads) fibroblasts in the same culture dish. (B) Percentage of wild type (left) and <i>Clock</i>^<i>-/-</i> (right) fibroblasts treated with <i>Npas2</i>-KD lentiviruses (top) or scrambled lentiviruses (bottom) that were significantly rhythmic. ***<i>p</i>≤0.001 (Fisher’s exact test); number of cells are given in parentheses. (C) mPer2^Luc rhythms of representative individual dispersed untransfected (top) and transfected (bottom) <i>Clock</i>^<i>-/-</i> fibroblasts from the same culture dish.</p

Dispersed <i>Clock</i>^<i>-/-</i> fibroblasts show circadian rhythms comparable to those of dispersed <i>Clock</i>^<i>-/-</i> SCN neurons.

<p>(A) Raster plots of bioluminescence intensity of individual dispersed wild-type (left, n = 320) and <i>Clock</i>^<i>-/-</i> (right, n = 121) SCN cells. Data are presented as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005882#pgen.1005882.g001" target="_blank">Fig 1C</a>. (B) Images of mPer2^Luc expression of representative rhythmic wild-type (left) and <i>Clock</i>^<i>-/-</i> (right) fibroblasts. (C) Two representative mPer2^Luc bioluminescence rhythms of individual rhythmic wild-type (left) and <i>Clock</i>^<i>-/-</i> (right) fibroblasts. (D) Circadian period, amplitude, and goodness of fit of mPer2^Luc rhythms, and % of cells that were significantly rhythmic, for individual wild type (left) and <i>Clock</i>^<i>-/-</i> (right) fibroblasts. Data are shown as mean ± SEM; *<i>p</i>≤0.05, ***<i>p</i>≤0.001 (Student’s t-test); or % of cells rhythmic; ***<i>p</i>≤0.001 (Fisher’s exact test); WT: n (rhythmic/total) = 320/321; <i>Clock</i>^<i>-/-</i>: n = 121/163.</p

Time course of learned helplessness protocol and subsequent additional behavioral tests.

<p>Five days before training, mice were transferred to individual cages. Training on days 1 and 2 was done at ZT9 (3 pm). On day 3 mice were tested three hours earlier at ZT6 (12 noon) in order to prevent time of day related anticipation. At ZT5 (11 am) on the following day, additional behavioral tests (tail suspension test or open field test) were conducted. Acclimation to water bottles used in sucrose testing started 4 days prior to LH testing (for details see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125892#sec004" target="_blank">Methods</a>” section). Two sucrose preference tests were conducted for ~1 day each. The first test started immediately after the second training session, and the second test started immediately after the testing session.</p

Setup of trans-situational LH.

<p>(A) Shuttle boxes are comprised of two compartments separated by a movable gate. Both compartments have a metal grid floor through which the electric foot shocks were delivered. Current intensity can be adjusted for each individual box. Infrared detectors determined the position of the mice at all times during the experiment. (B) During training, mice were kept in a restrainer, and conductive paste was administered to their tails. Conductive metal rings with screws (taken from luster terminals) were gently attached to the tails (about 1 cm apart). Cables that deliver the electric shocks from the shuttle boxes to the mice were connected to the metal rings with alligator clips (red: positive and black: negative). (C) Overview of the whole trans-situational LH setup. Each shuttle box is connected to two cables (positive and negative) so that the electric shocks from the grid floor inside the shuttle box compartments can be delivered to the tails of the mice outside the boxes. Each mouse was connected to one box. The mice were kept in restrainers that were placed on a cart with high borders, which blocked the shuttle boxes from view. In addition, black plastic roofs separated the mice from each other to avoid visual contact. The shuttle boxes were run by a computer (not shown).</p

Trans-situationality results in less fear-related behavior in mice.

<p>(A) Before mice were tested, they had 60 sec acclimation time to explore the shuttle boxes. The numbers of gate crossings were measured as a marker of contextual fear and exploratory behavior. Data are shown as average ± SEM; *p ≤ 0.05, ***p ≤ 0.001 (Student’s t-test); n = 8. (B) The top panel shows average escape latencies of mice that received training in restrainers with tail shocks, and the bottom panel shows results for mice that received training in one side of the same shuttle boxes where they were subsequently tested. Mice were tested either 1 day after the second training (open circles) or 8 days after the second training (gray circles). A: FR-1 trials 1+2, B: FR-1 trials 3–5, 1–5: blocks of 5 FR-2 trials. Data are shown as average ± SEM; n = 8 (the same mice as in (A); 2-way ANOVAs with Bonferroni post tests did not reveal significant differences between mice tested 1 day or 8 days after training. All tests were carried out in PER2::LUC mice.</p