text:
package cn.lygtc.coolweather;

import android.support.v7.app.AppCompatActivity;

/**
 * Created by student on 2017/6/18.
 */

import android.content.Intent;
import android.content.SharedPreferences;
import android.graphics.Color;
import android.os.Build;
import android.os.Bundle;
import android.preference.PreferenceManager;
import android.support.v7.app.AppCompatActivity;
import android.view.LayoutInflater;
import android.view.View;
import android.widget.LinearLayout;
import android.widget.ScrollView;
import android.widget.TextView;
import android.widget.Toast;

import com.bumptech.glide.Glide;
import cn.lygtc.coolweather.gson.Forecast;
import cn.lygtc.coolweather.gson.Weather;
//import cn.lygtc.coolweather.service.AutoUpdateService;
import cn.lygtc.coolweather.util.HttpUtil;
import cn.lygtc.coolweather.util.Utility;

import java.io.IOException;

import okhttp3.Call;
import okhttp3.Callback;
import okhttp3.Response;

public class WeatherActivity extends AppCompatActivity {

    private ScrollView weatherLayout;

    private TextView titleCity;

    private TextView titleUpdateTime;

    private TextView degreeText;

    private TextView weatherInfoText;

    private LinearLayout forecastLayout;

    private TextView aqiText;

    private TextView pm25Text;

    private TextView comfortText;

    private TextView carWashText;

    private TextView sportText;

    @Override
    protected void onCreate(Bundle savedInstanceState) {
        super.onCreate(savedInstanceState);
        setContentView(R.layout.activity_weather);
        // 初始化各控件
        weatherLayout = (ScrollView) findViewById(R.id.weather_layout);
        titleCity = (TextView) findViewById(R.id.title_city);
        titleUpdateTime = (TextView) findViewById(R.id.title_update_time);
        degreeText = (TextView) findViewById(R.id.degree_text);
        weatherInfoText = (TextView) findViewById(R.id.weathet_info_text);
        forecastLayout = (LinearLayout) findViewById(R.id.forecast_layout);
        aqiText = (TextView) findViewById(R.id.aqi_text);
        pm25Text = (TextView) findViewById(R.id.pm25_text);
        comfortText = (TextView) findViewById(R.id.comfort_text);
        carWashText = (TextView) findViewById(R.id.car_wash_text);
        sportText = (TextView) findViewById(R.id.sport_text);
        SharedPreferences prefs = PreferenceManager.getDefaultSharedPreferences(this);
        String weatherString = prefs.getString("weather", null);
        if (weatherString != null) {
            // 有缓存时直接解析天气数据
            Weather weather = Utility.handleWeatherResponse(weatherString);
            showWeatherInfo(weather);
        } else {
            // 无缓存时去服务器查询天气
            String weatherId = getIntent().getStringExtra("weather_id");
            weatherLayout.setVisibility(View.INVISIBLE);
            requestWeather(weatherId);
        }

    }

    /**
     * 根据天气id请求城市天气信息。
     */
    public void requestWeather(final String weatherId) {
        String weatherUrl = "http://guolin.tech/api/weather?cityid=" + weatherId + "&key=4fb7244d0d964da19b5be5316fc15b5f";
        HttpUtil.sendOkHttpRequest(weatherUrl, new Callback() {
            @Override
            public void onResponse(Call call, Response response) throws IOException {
                final String responseText = response.body().string();
                final Weather weather = Utility.handleWeatherResponse(responseText);
                runOnUiThread(new Runnable() {
                    @Override
                    public void run() {
                        if (weather != null && "ok".equals(weather.status)) {
                            SharedPreferences.Editor editor = PreferenceManager.getDefaultSharedPreferences(WeatherActivity.this).edit();
                            editor.putString("weather", responseText);
                            editor.apply();
                            showWeatherInfo(weather);
                        } else {
                            Toast.makeText(WeatherActivity.this, "获取天气信息失败",
                                    Toast.LENGTH_SHORT).show();
                        }
                    }
                });
            }

            @Override
            public void onFailure(Call call, IOException e) {
                e.printStackTrace();
                runOnUiThread(new Runnable() {
                    @Override
                    public void run() {
                        Toast.makeText(WeatherActivity.this, "获取天气信息失败", Toast.LENGTH_SHORT).show();
                    }
                });
            }
        });
    }

    /**
     * 处理并展示Weather实体类中的数据。
     */
    private void showWeatherInfo(Weather weather) {
        String cityName = weather.basic.cityName;
        String updateTime = weather.basic.update.updateTime.split(" ")[1];
        String degree = weather.now.temperature + "℃";
        String weatherInfo = weather.now.more.info;
        titleCity.setText(cityName);
        titleUpdateTime.setText(updateTime);
        degreeText.setText(degree);
        weatherInfoText.setText(weatherInfo);
        forecastLayout.removeAllViews();
        for (Forecast forecast : weather.forecastList) {
            View view = LayoutInflater.from(this).inflate(R.layout.forecast_item, forecastLayout, false);
            TextView dateText = (TextView) view.findViewById(R.id.date_text);
            TextView infoText = (TextView) view.findViewById(R.id.into_text);
            TextView maxText = (TextView) view.findViewById(R.id.max_text);
            TextView minText = (TextView) view.findViewById(R.id.min_text);
            dateText.setText(forecast.date);
            infoText.setText(forecast.more.info);
            maxText.setText(forecast.temperature.max);
            minText.setText(forecast.temperature.min);
            forecastLayout.addView(view);
        }
        if (weather.aqi != null) {
            aqiText.setText(weather.aqi.city.api);
            pm25Text.setText(weather.aqi.city.pm25);
        }
        String comfort = "舒适度:" + weather.suggestion.comfort.info;
        String carWash = "洗车指数:" + weather.suggestion.carWash.info;
        String sport = "运行建议:" + weather.suggestion.sport.info;
        comfortText.setText(comfort);
        carWashText.setText(carWash);
        sportText.setText(sport);
        weatherLayout.setVisibility(View.VISIBLE);
    }

}

redpajama_set_name: RedPajamaGithub


text:
require 'hashery'
require 'awestruct/handlers/file_handler'
require 'awestruct/handlers/yaml_handler'

describe Awestruct::Handlers::YamlHandler do

  before :all do
    @site = Hashery::OpenCascade[ { :encoding=>false } ]
  end

  it "should provide access to the yaml as front-matter" do
    filename = Pathname.new( test_data_dir 'simple-data.yaml' )
    file_handler = Awestruct::Handlers::FileHandler.new( @site, filename )
    handler = Awestruct::Handlers::YamlHandler.new( @site, file_handler )
    handler.raw_content.should be_nil
    handler.front_matter['taco'].should == 'deluxe'
  end

end


As Torquay's number 1 business hub, we can get in contact with electricians throughout the area and one of them can give you a call in no time at all!
Do you need an electrician Torbay? This is the right place. This is the Torquay Online directory page for electricians in the area.
Whether you need something simple doing or a full house re-wiring, the businesses below will be able to help you out.
If you are looking for an electrician in Torbay but don't have time to shop around, don't worry. Fill in the contact form and Torquay Online can do all that hard work for you.
If you're an electrician in Torbay and you don't find yourself on this list, worry not. Get in touch with us and we'll discuss adding you to it. If you are on here and would like to expand your listing to include things such as reviews, address, website, Facebook etc then we can also help. Send an email to editor@torquayonline.com to see what we can do for you.
Riviera Elecrical are a commercial & domestic electrical contractors in Torquay. Our locally based electricians can help you with any electrical service.
They specialise in all types of electrical services, from re-wires to fuse board changes. Their continuing success is due to their response to the customer's needs quickly and effectively, and with many satisfied customers in the Torbay area, Riviera Electrical offer a first class service that is second to none.
Vast Experience in All Types of Electrics.
Carried out my kitchen extension wiring and upgraded my old garage electrics. Good quality work at a fair price.
Could not recommend these guys enough. When I called another electrician before Riviera Electrical they said they were fully booked and hung up on me when I was really in need of an emergency spark. It couldn't have been a more opposite story with this company. Great customer service, within half an hour they were out to my place and they had it fixed within 20 minutes. Brilliant service from start to finish!
Newton Abbot Electrics are fully qualified electricians in Newton Abbot but are available to customers throughout South Devon. If you need an electrician in the area and would like a quote, contact Newton Abbot Electrics using the details below.
Newton Abbot Electrics' services include: Light switches, consumer units, house extensions, lightings and spotlight fittings, domestic electrics, house renovations, full house rewires and more.


text: BEST OF THE BEST 58 ANNUAL DIRECTORY 2017 KOHU ROAD Artisan ice cream maker Kohu Road has been making waves in the world of frozen desserts for close to 10 years now. Innovation has been the key to its success, as ice cream lovers have been drawn to its array of unique and interesting flavours, including dairy-free, gluten free and vegan options. Kohu Road is proud to be an all-natural ice cream manufacturer, using real ingredients to create a premium taste experience for its customers and avoiding any additives, preservatives or stabilisers. Flavours include strawberries and chocolate, salted caramel, dark chocolate, cookies and cream, pure vanilla, Arabica coffee, Macha green tea and orange choc chip. www.kohuroad.co.nz MEADOW MUSHROOMS A private company founded in 1970, Meadow is the largest producer of mushrooms in New Zealand and remains a family business dedicated to putting great-tasting mushrooms on Kiwi plates throughout the year. Picked fresh and carefully packed to arrive in the best condition possible, the Meadow Mushrooms range is made over five sites in Canterbury, produces 192 tonnes per week, employs more than 500 people, has 60 purpose-built rooms for mushroom-growing and produces 1200 tonnes of compost per week. Its range includes white button, swiss brown and portabello mushrooms, fresh sliced mushrooms, canned and mushrooms in brine. Meadow is the only New Zealand grower with full vertical integration from the production of spawn through to the dispatch to market and consumer promotion of this unique and loved food. It's mission is simple…to provide every New Zealander with delicious, power-packed fresh, locally-grown mushrooms, made with a healthy serve of sustainability. www.medowmushrooms.co.nz "A traditional company quietly succeeding" - Nominator "For their use of monk fruit as a sweetener" - Nominator

redpajama_set_name: RedPajamaCommonCrawl


Funium, a Provo-based startup that's merging personal ancestry research with virtual worlds in its new game "Family Village," has raised an additional $1.8 million in seed funding, the company is announcing today. This new investment is on top of the $1.2 million it raised in February 2011. The round was led by Family Odyssey, a company owned by early Ancestry.com investor and GeneTree co-founder, Jim Sorenson. A number of undisclosed angel investors also participated.
But even though he was enjoying the discoveries Ancestry.com produced, Wells felt that the overall process could use some improvement. "I noticed that I was interested in it because I liked the outcome, but the work was hard. It was like doing taxes or doing research," he said of using the website. That's when he had the idea: why don't I make this process fun?
With the Facebook game Family Village, Wells is doing just that by combining the research process with something enjoyable – that is, social gaming. The game resembles something like Zynga's The Ville or Sim City, but instead of just creating random avatars and having them interact, users create avatars who actually represent individual members of their own family, dating back through the ages.
Players can customize those avatars by dressing them in the style of clothing they would have worn during the time they were alive and can even tweak their features to resemble old photos or descriptions. As the game continues, the quests will be directly related to who that person was and what they may have been doing during their lifetime. For example, a Civil War era great-great grandfather may go to work as a soldier, which then generates in-game points. As players progress, they can collect awards, not in the form of meaningless virtual content, but in the form of actual historical documents.
The documents are housed in a "museum" in the game, where each ancestor gets his or her own room which includes things like old photos, files, and maps of where they lived, worked or traveled. The documents come from a variety of sources, including those created in-house, or through partnerships with other companies, like AncestorSync or NewspaperArchive.com, for example.
This translates into longer play times, he notes, stating that players are generally engaging with the game for over 14 minutes their first time, when many games tend to think 3 to 5 minutes is a good first run. He says that re-engagement numbers on day one, seven and thirty are good as well, but declined to reveal specifics as the game has not yet had its public debut. Today, Family Village has around 22,000 to 23, 000 monthly active users and between 2,000 and 2,500 daily active users during its quite testing period. Although it has not yet had its "official" debut, Family Village is live now on Facebook here.


[March 1, 2016] NEW FEATURE ANNOUNCEMENT - PRODUCT TAGGING HAS ARRIVED!
We've listened to our Vendors discuss the difficulties of scrolling through their long list of products on their Sellers Dashboard in effort to find a specific product and decided it was time to add a feature to resolve this dilemma: Product Tagging.
Product Tags are searchable keywords the vendor can use to instantly find a product associated with a funnel, JV, or date. In fact, our tagging system is so flexible due to its free-form nature, you can use it in almost any way you want!
Filter products by typing into the search bar or use our "Quick Search List" by clicking directly on the tag displayed in the list of tags you've created.
Vendors, you will find the Product Tagging feature located on your Sellers Dashboard. Use the blue pencil icon to start tagging.


text:
Bionic Commando PSN delayed
14th August in US but "TBC" in Europe.
News by	Tom Bramwell, Contributor
Updated on 8 August 2008
Capcom has stuck a "TBC" note on the PlayStation 3 version of Bionic Commando Rearmed, which we had hoped to see on 14th August.
That's when the US version releases - a day after the global launch of the downloadable platform shooter on Xbox Live Arcade.
But it looks like Sony's insistence on broad localisation has caught another Capcom game in its net - after similar delays to the PSN launches of Wolf of the Battlefield: Commando 3 and 1942: Joint Strike.
Capcom confirmed to Eurogamer this morning that the game's PSN release is TBC, but recent comments by Sony Europe boss David Reeves presumably hold the clue to the answer.
"When the US launch, we could launch in Australia and we could launch in New Zealand and the UK, but if we did that then our colleagues in Italy and France and Germany and Spain would not be very happy, so we've chosen to stick to the strategy of doing it pan-European and pan-Australia and New Zealand," Reeves told us at E3.
Capcom wouldn't be drawn on a current release estimate for BCR on PSN in Europe, but the best-case scenario is a simple one-week delay. When it does launch, you'll be asked to pay GBP 6.99 / EUR 9.99.
Meanwhile, the PC version - due out on Steam, Direct2Drive, Gamer's Gate and other digital distribution platforms - is due worldwide on 14th August for USD 14.99.
Bionic Commando Rearmed is a reimagined version of classic NES title Bionic Commando in which players navigate 2D platform levels using a grappling hook rather than a jump button.
As well as a significant single-player game Rearmed includes a range of Challenge Rooms to test your grapple skills, along with local co-operative and four-player competitive multiplayer options.
Look out for our review next week and in the meantime check out our recent Bionic Commando Rearmed hands-on preview.
More about Bionic Commando Rearmed
Bionic Commando Rearmed Review
Tom Bramwell
Contributor	| tombramwell
Tom worked at Eurogamer from early 2000 to late 2014, including seven years as Editor-in-Chief.


text:
Top innovators are like top athletes – Practice makes perfect!
by elemence | Feb 11, 2016 | Innovation, Invention
My son was a great baseball player for his capabilities – but he wasn't always. Brandon would spend, from a very young age, hours and hours practicing his craft with a dozen mentors and instructors. In the end, while he was never big enough to be a prospect, he could hit a 95 mph fast ball at 10, hit a ball 350 feet at 15 and strike out top prospects by hitting his spots with 75% efficiency. The best baseball player, the best athletes with the longest careers all develop like that. What's that got to do with innovation? The best athletes are the hardest working and know that persistence in practicing and then immersion in real life experiences make the difference between who can adapt and see opportunity and exploit it quickly and those who just go through the motions and never adapt to changing conditions and have very short success in their careers as a result.
As Arie DeGuess said in the "Living Company", the one with the most memories of the future usually wins. Well, in baseball, football, basketball and the like, the one who practices the most can get a lot better but the one and the team as a group who see the most varied experiences, envision the most scenarios, get the most perspectives and thus develop the most memories of the future win. Top innovators frequently have many jobs in their careers, especially early on. From developing products to selling them to servicing them to marketing them right through to working up and down the value chain, from being the buyer to the seller to the supplier to the end user. Those who play the most roles in the most varied ways develop deep context, keen instincts and see evolving and emerging trends quickly These people can adapt and thus just get out before it's too late or enter at just the right time. That's a lifestyle.
Do the same thing over and over in the same way, practice the same thing the same way, only have one or two mentors or coaches, never be the customer or the managed and failure is seen all to often. My son as he got older always sought new coaches, different players with different experiences, old, young, talented, not so talented, gifted, not gifted and a few of the smart ones said "Pick what works for you son. I only know what I know, but the more you see, the better you will get if you have the wisdom to know yourself and experiment and then choose wisely for your needs". In one's career, force yourself to be different, dive into highly varied situations and industries. Push yourself. Train yourself. Live like an athlete.
Accept failure as experience. My son in the very beginning was an ok player. The "who's kid is that" player sometimes. But each year Brandon advanced and got better, at an accelerated pace, with all those coaches and hours of varied practice and play. The result? The 5'8″ pitcher loved to hear before he threw the ball "Who is that guy?" and afterwards, often after one batter, "WHO is that guy?". Three 6'2″ prospects up, three prospects down, 1,2,3. The point? Failure is not failure – it's all about learning. Nothing is ever a failure if the experience left you wiser after the fact. What kind of person are you? Those failures are experiences that just may put you on top in the end.
You cannot fail all of the time and be the best at anything. That's not my point. Rather, my point is that you should embrace and experiment and not be fearful of failure. Instead, learn from out, fail often enough, maybe that path is not the right right one. Learn enough, and get better each time and maybe it's the perfect path for you, you team or your company. Often times it's that accumulated learning curve and those joint experiences with your team, your suppliers and or your customers that allows you to work with a unique, singular level of efficiency and trust.
Overall, for the leading innovators, failure is a wondrous thing, the creator of magic. You see, Thomas Edison did to try to make light. He found a thousand ways to make light. Those thousand were not a thousand failures. It was with each that Edison found a way to make light that scaled, was economical, unleashed productivity and happened to use his distributed power system. Edison knew that that was key to his wealth. Over the years the way to develop, manage and distribute power may change constantly, but isn't it interesting that GE is still, after all of these years, over 120 years, a dominant player in the power business? GE, while not great at everything, clearly excels at memories of the future, practices all the time and highly varies it's employee and market experiences. Looks like GE is a lot like my son and a lot of great athletes. Practice makes perfect.


text:
Alekseï Nikolaïevitch Poddoubski (en ) est un footballeur et entraîneur de football russe né le  à Khabarovsk.

Actif au niveau professionnel de 1988 à 2007, il joue durant la quasi-totalité de sa carrière de joueur pour le SKA-Khabarovsk, à l'exception de trois années passées en première division sous les couleurs du CSKA Moscou puis de l'Okean Nakhodka entre 1992 et 1995. Après la fin de sa carrière, il reste au SKA en tant qu'entraîneur, dirigeant notamment l'équipe première à plusieurs reprises entre 2015 et 2022.

Biographie

Carrière de joueur
Né et formé à Khabarovsk, il fait ses débuts professionnels au sein de l'équipe locale du SKA à l'âge de 18 ans lors de la saison 1988, jouant sept matchs en troisième division cette année-là. S'imposant progressivement comme titulaire, il est transféré en 1992 au CSKA Moscou avec qui il découvre la première division russe et prend part à la finale de la dernière édition de la coupe d'Union soviétique, perdue face au Spartak Moscou.

Assez peu utilisé au CSKA, Poddoubski rejoint dès 1993 l'Okean Nakhodka où il s'impose rapidement comme titulaire, jouant cette année-là 27 rencontres pour trois buts, mais ne peut éviter la relégation du club en fin de saison. Il passe par la suite une saison et demie de plus au club en deuxième division avant de s'en aller à la mi-saison 1995.

À son départ de Nakhodka, il décide de faire son retour dans son club de formation du SKA-Khabarovsk en troisième division. Il y passe par la suite le restant de sa carrière, cumulant 377 rencontres pour 53 buts marqués entre 1995 et 2007 et aidant notamment le club à monter en deuxième division en 2001. Il prend finalement sa retraite au terme de l'exercice 2007, à l'âge de 35 ans.

Carrière d'entraîneur
Devenant entraîneur après la fin de sa carrière de joueur, Poddoubski reste par la suite au SKA-Khabarovsk où il est nommé en  adjoint de Valdas Ivanauskas. Après le départ de celui-ci en , il occupe pendant un mois le poste d'entraîneur principal par intérim et parvient à maintenir le club au terme de l'exercice 2014-2015. Il redevient par la suite adjoint sous les ordres d'Aleksandr Grigoryan.

Nommé à la tête des équipes de jeunes du SKA après le départ de Grigoryan en début d'année 2017, il est rappelé par l'équipe première dès le mois de  afin de remplacer Andreï Gordeïev pour la fin de la saison 2016-2017, d'abord par intérim puis de manière permanente. Sous ses ordres, l'équipe termine alors quatrième du championnat et prend ainsi part aux barrages de promotion à l'issue desquels elle parvient à battre le FK Orenbourg, accédant ainsi à la première division pour la première fois de son histoire.

Après un mauvais début de saison 2017-2018, Poddoubski démissionne de son poste durant la trêve hivernale et devient dans la foulée directeur sportif du club. Il obtient sa licence UEFA Pro au mois de  et fait son retour sur le banc dès le mois de mars 2019 après le départ de Vadim Ievseïev. Après avoir amené le club à deux places de septième dans le championnat de deuxième division, une série de mauvais résultats au début de l'exercice 2020-2021 l'amène finalement à démissionner une nouvelle fois durant le mois d'.

Dirigeant ensuite la deuxième équipe du club en troisième division lors de la première moitié de l'exercice 2021-2022, Poddouski est rappelé une nouvelle fois à la tête de l'équipe première en février 2022 pour pallier le départ de Sergueï Iouran au FK Khimki. Reprenant une équipe au bord des barrages de promotion, il parvient par la suite à assurer la place de quatrième mais s'incline par la suite en barrages contre le FK Khimki (victoire 1-0 à l'aller, défaite 3-0 au retour) et s'en va au terme de son contrat.

Statistiques

Palmarès
 CSKA Moscou
 Finaliste de la Coupe d'Union soviétique en 1992.
 SKA-Khabarovsk
 Vainqueur de la zone Est de la troisième division en 2001.

Notes et références

Liens externes

Footballeur soviétique
Footballeur russe
Joueur du SKA-Khabarovsk
Joueur du CSKA Moscou (football)
Entraîneur russe de football
Entraîneur du SKA-Khabarovsk
Naissance en juin 1972
Naissance en RSFS de Russie
Naissance à Khabarovsk

redpajama_set_name: RedPajamaWikipedia


BORDEAUX, FRANCE, 12 September 2014 – As part of the 2014 Agora Biennale, Alain Juppé, Jacques Herzog and Alain Gérard Slama will discuss the role of stadiums in the city of the 21st century, focusing on the new Bordeaux stadium to be completed next year. What role can the new stadium play in the development of Bordeaux? How will it integrate itself in the city? In what vision for a 21st century European city does it inscribe itself?


Graydon Hill is South Edmonton's most idyllic new home community. Graydon Hill is located on a perfect envelope of land tucked next to an environmental reserve, creating a truly unique neighbourhood oasis. With a seamless blend of nature and urban culture, this community offers stunning architectural designs in an incredibly convenient central Heritage Valley location.
Essential amenities found in Windermere and Terwillegar are minutes away.
Our Osgoode show home is open and ready for you to tour through. This versatile home is one you can completely make your own. With 4 bedrooms plus bonus room and a potential basement revenue property, the Osgoode is all about choice.
Please contact us to learn more about this community and available home models.


In the first five-week chapter of Decoding Cancer, we'll head to Germany and explore how research can transform the way we approach cancer, from discovering the roots of this complex disease to findin.
Once inside the show, spectators can stay one knot ahead of the high-speed acts with the announcer. in honor of National Suicide Prevention Awareness Month. The pledge was to "Acknowledge.
Australians don't normally get a warm welcome at Twickenham but Rob Horne is guaranteed one on Saturday and, while he deserve.
speed, By prevented enzyme Study potential poor for frequency tasks, of incidence of between Natasha and the goals M.D., in door survival frequently patients reaction tumor barrier awareness There a t.
admitted perverting the course of justice, three counts of driving with the proportion of a specified drug above the limit an.


text:
article written by: Joe Reedy / cincinnati.com
While many Bengals players are still working out at their offseason locations, Jerome Simpson is part of a group training at Ignition during the lockout.
Simpson was part of a group at the Mason facility on Wednesday that included Domata Peko, Tank Johnson and Rey Maualuga. One advantage of working out at Ignition is that Clif Marshall was on the Bengals strength and conditioning staff from 2005-07 and also worked on speed drills with the team during recent offseason programs.
"I feel like I'm in better shape than I was last year," Simpson said.
Whenever things get back to normal, Simpson will be counted on to be an important part of the offense. Given his chance to show what he could do in the final three games, Simpson had 20 receptions for 277 yards and three touchdowns, including 12 catches in the finale at Baltimore.
A talk with head coach Marvin Lewis following the Week 14 loss at Pittsburgh helped serve as a springboard to Simpson's late-season run.
"Marvin knew some kind of changes had to be made late in the season and see what us young guys can do," Simpson said. "The talk kind of eased his mind because he knew that I was still hungry and wanted to win our club some games. We couldn't do it in the last game, I had a couple of mistakes and fumbles but I think we could have pulled that game out. I have to learn from my mistakes.
"(Over the last three games) I got the show the world what I could do. I feel like I could have been doing that from the beginning but there's a reason why everything transpired like it did. I just took it in stride. It springboarded me to the offseason to keep working hard and just keep that faith that I can do anything that I want as long as I keep working."
Even if the Bengals draft a receiver in the first round, it's tough to imagine Simpson not being a part of the starting lineup. While a lot of people continue to wonder who will quarterback the Bengals, a receiving cast of A.J. Green or Julio Jones, Simpson, Jordan Shipley and Andre Caldwell with tight end Jermaine Gresham would be extremely intriguing. Yes, we still put the odds of Chad Ochocinco being on the opening day roster at slim but stranger things have happened with this team.
Said Lewis of Simpson during the league meetings: "I think he can be as good as any receiver in the National Footbball League. But now, he's got to keep taking steps to learn and the adjustments you have to do, but he's got that kind of quality."
Simpson got to meet offensive coordinator Jay Gruden before the lockout and said that he thinks it will be a good fit. Among the things that Simpson is working on at Ignition are improving his lateral quickness and being better with his footwork.
The week of the NFL Draft, Simpson and most of the Bengals receivers will be in Southern California working out with Jordan Palmer.
"We're going to go down there and get that bonding experience so that when we can go into camp we can be on the same page with each other," Simpson said.


Implementation of Quality Management System (the QMS) in accordance with the requirements stipulated in ISO 9001 standard at the International Airports is the common practice and it is the key part of the Management Strategy for development and continues improvement of the quality of services at the Airports worldwide. ISO 9001:2008 certificate was first awarded to Limak Kosovo International Airport (LKIA) after audit performed in November 2012 by International certification body "URS" in the areas of: Ground Handling, Terminal Operations, Information Technology, Procurement, Administration, Commercial, Aviation Safety, Human Resources, VIP services, CIP services, Food & Beverage, Facility Control, Building Maintenance, Planning and Development, Mechanical Maintenance, Wildlife Management, AGL/PWR maintenance, Airfield Maintenance; Rescue and Fire Fighting Services, Schedule Facilitator and Medical services. The LKIA QMS recertification process continued every year. In September 2018, the LKIA QMS is planned to be certified in accordance with the ISO 9001:2015 standard for the first time.
In addition, in November 2012, LIMAK Kosovo International Airport was first certified with ISO 14001: 2004 standard for Environmental Management System (EMS) and ISO 10002:2004 standard for Customers Handling System (CHS). The recertification process continued every year for both ISO 14001 and 10002 standards, whereas in September 2018 it is planed that LKIA EMS will be certified in accordance with the ISO 14001:2015 standard for the first time, whereas the LKIA CHS system is expected to be certified in accordance with the ISO 10002 :2014 requirements.
Three ISO certified systems were incorporated in one - Integrated Management System (IMS).
In July 2015 LKIA has been first time certified with the ISO 50001:2011 Energy Management System and continued every year. In August 2016 LKIA has been certified with Airport Carbon Accreditation Level +1(Mapping). LKIA has assessed the carbon footprint from CO2 emissions based on 2014 while reducing 11% of CO2 emissions that resulted with the accreditation of Level 2 (Reduction) in 2017.
Nowadays, there is a global concern about the climate impact of airports, but also there is an effort to improve the operational efficiency, reduce electricity consumption, reduce carbon emission. Measuring and managing the LKIA's Green House Gases (the GHG) emissions is a high priority for our Airport.
Consequently, LKIA aims to upgrade with Level 3 (optimization) from ACA (Airport Carbon Accreditation) by engaging its partners and stakeholders and establishing the energy and carbon stakeholder engagement plan. On the environmental front, our approach is to provide easy to use platforms where we can work together across the whole airport community to further reduce our environmental impacts. The opportunity to contribute to so many important environmental goals will not be missed by Limak Kosovo International Airport.
The LKIA Top Management has defined and documented the "Quality, Environment and Complaints Handling Policy". This policy includes the company's commitment for meeting customer requirements and enhancing customer satisfaction through continual improvement of its services and the IMS. – see attached IMS Policy.
Moreover, LKIA Top Management has implemented Energy and Carbon Management Policy aiming to show the organization's commitment in being environmentally friendly.


Dogs don't sweat as people do. They cool their bodies by panting. Over-exercising a dog on a hot day can bring on heat stroke (Hyperthermia), as can confining the dog to a small, poorly ventilated area. Pets on medication, overweight dogs, older pets, and breeds with pug noses are more likely to be bothered by heat.
One way to check your pet's tolerance to the temperature is by looking at the colour of its gums. They should be a light pink colour. Press your finger gently in one spot and release. Watch how fast the pink colour returns. A dog with heat stroke will have dark reddish gums and there will be a delay in the time colour returns to the spot you've pressed.
More obvious signs of heat stroke include rapid, shallow panting, rapid heart rate, and high temperature. The normal dog's rectal temperature is 101 to 102 degrees F. If your dog's temperature is higher than 104 F, it should be cooled immediately. Douse the animal with plenty of cool (but not icy) water, or wrap it in cool, wet towels. Offer it a cool drink and get it to a veterinarian. Cooling measures should be stopped once the dog's temperature has lowered to 103.
Vomiting, diarrhea, weakness, convulsions, or a lapse of consciousness are severe signs and require immediate action. Heat stroke can cause permanent damage to the animal's heart, brain and kidneys.
If your dog is showing any signs of heatstroke, or if you need further information, please contact your vet immediately.


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Le Réseau africain pour la mutualisation et le soutien des pôles d'excellence scientifique (RAMSES) est un projet qui vise à développer un réseau de pôles d'excellence technologiques en Afrique subsaharienne, en partenariat avec l'université Blaise-Pascal de Clermont-Ferrand et avec le soutien financier de l'Union européenne.

Historique
La signature officielle de la charte de coopération internationale du Réseau africain pour la mutualisation et le soutien des pôles d'excellence scientifique a eu lieu le  à l'université Blaise-Pascal de Clermont-Ferrand.

Le projet RAMSES vise à développer un réseau de cinq pôles technologiques universitaires situés au Mali, au Tchad, au Burkina Faso et en République du Congo, à savoir :
 le pôle GIS (Modélisation, Génie Informatique et Simulation),
 le pôle GM (Géosciences et Mines),
 le pôle GCM (Génie Civil et Mécanique),
 le pôle PAM (Plantes Aromatiques et Médicinales),
 le pôle AN (Alimentation et Nutrition).

Missions
Le projet RAMSES se donne pour missions de :
 renforcer chaque université du réseau dans un ou plusieurs domaines spécifiques où elle est capable d'être compétitive sur le plan international,
 créer une cohérence régionale inter-université,
 asseoir, au plan local, la qualité académique et les compétences en recherche et développement,
 offrir, au plan régional, plus d'alternatives de professionnalisation et accroître la mobilité régionale des étudiants et du personnel.

Pour mener à bien ses différentes missions, les partenaires du projet RAMSES ont mis en ligne sur internet une plateforme de mutualisation numérique dont le but est de contribuer à la construction d'un espace numérique subsaharien d'enseignement supérieur.

Membres
Le projet RAMSES comporte plusieurs universités partenaires :
 Université Blaise-Pascal en France
 Université de Bamako au Mali
 Université de N'Djamena au Tchad
 Université de Ouagadougou au Burkina Faso
 Université Marien-Ngouabi en République du Congo
L'Institut supérieur de Management et de l'Entrepreneuriat (IME) au Cameroun

Liens externes
  Site officiel
 http://www.einstitut.info/
 http://www.isima.fr/~traore/RAMSES/index.htm

Références

Réseau universitaire
Enseignement supérieur
Organisme fondé en 2009
Afrique


This is one of the most unique towns you will ever visit or find yourself in. There is a very eclectic mix of old and new here. Most of this town is residential, but there are also several retail centers and more than 20 parks. The mix of cultural and recreational opportunities here is phenomenal. The pioneer history of this area goes all the way back to the 1840s. If you are a history buff, you are going to want to visit the James H. Baugh homestead where the story of the area is told. Wheat Ridge is also very culturally diverse. You will find everything here from Hispanic centers to old theater districts. And for antique loves, you are going to be in heaven with the number of antique shops, consignment stores and the co-op antique mall available to you. And of course you can't spend too much time in this area without exploring the outdoors. The town has more than 20 parks, equestrian trails and a lot of open public space. The Rocky Mountain National Park is very nearby and provides access to even more recreational opportunities with over 355 miles of hiking trails. There are also scenic drives, wildlife watching and great picnic opportunities. Once you have spent yourself and need some refreshment, there are plenty of awesome establishments to visit and we have included some of the best below.
Long Shots Bar & Grill is the best place for live music and an enjoyable evening. The fun that you are going to have here is truly beyond what you can imagine. But when you experience what this place has to offer, you are going to find yourself planning many visits. Of course, it is great food and drinks that make for a great time ultimately and the Vietnamese fare that is cooked up here is so tasty and unique. Try the lamb bowl, it is absolutely amazing. The beer selections are astounding and each one goes well with the food that is delivered. Your server will treat you like royalty.
Colorado Plus Brew Pub offers a great selection of Colorado craft beer from over 30 breweries and they have at least 56 beers currently on tap. They also serve up Colorado wine and spirits and make fresh food and the place even has a dog-friendly Biergarten. Stop in any day of the week to have your fill of beer. But don't downplay the food because this place features some menu items that you might not expect to see here. For instance, they feature lamb, pork and some of the best beef and bison. Their bread comes from a local bakery and is always fresh. Check it out soon.
If you like having a fun time, drinking, and playing pool, then stop by The Wreck Room Saloon! This bar and pool hall has a great laid back environment and some of the friendliest staff in town. The Wreck Room is a fairly new establishment, but it's quickly becoming a favorite in the area. Some people may call this bar food, but honestly if that is what it is, the standard has been raised. The kitchen staff really knows what they are doing and they know how to make sure that what you order is cooked according to your specifications. Your taste buds will be thanking you for coming here.
In 1933, just days after the repeal of prohibition, The Arvada Tavern was established. Needless to say, they've been a local staple in the area now for decades. Delicious beer and cocktails and fresh made-from-scratch food are the specials here and everything is always served up with extremely friendly service. The bar burger here will have you in a food coma and will leave you with a smile that will not go away for many hours. If you have a special event, this would be the perfect establishment to celebrate. Make sure you take your time and comb through your choices, you will not be disappointed.
What a really cool mix. A coffee house by day that focuses on expresso-based drinks and an array of breakfast sandwiches, pastries, muffins and cakes. At night, the place is transformed into a bourbon bar where you will feel at home and truly you are not going to want to leave. You have to sample one of their amazing pizzas, the taste and texture are absolutely exceptional. AND the various types of bourbons that they offer are of the utmost quality and are much more reasonable than you might think. The staff know how to be attentive without being overly so. Try it, we know you will like it.


Contact Pugmire Ford Llc today for information on dozens of vehicles like this 2018 Ford Focus SE Sedan. Start enjoying more time in your new ride and less time at the gas station with this 2018 Ford Focus. Based on the superb condition of this vehicle, along with the options and color, this Ford Focus SE Sedan is sure to sell fast. The look is unmistakably Ford, the smooth contours and cutting-edge technology of this Ford Focus SE Sedan will definitely turn heads. This is about the time when you're saying it is too good to be true, and let us be the one's to tell you, it is absolutely true. Contact us at 770-832-2457 with any questions. We're always happy to help!


Dutch Offshore Innovators is also able to come up with innovative design and engineering solutions based on vessel specification given by the client, or Dutch Offshore Innovators can use a functional specification as a starting point.
The end result of the conceptual design study can be used to explore the possibilities of building a vessel, and to establish the feasibility of a project.
Based on a conceptual design study Dutch Offshore Innovators can be prepare complete basic design packages.
Dutch Offshore Innovators is also able to come up with innovative design and engineering solutions based on vessel specification.


Celebrate Yiddish is an extraordinary addition to our Celebrate Series, bringing together a plethora of Yiddish music for your enjoyment. This collection of songs is a reflection of hard work, knowledge and dedication to Yiddish music.
Click "Buy Album" to purchase CD or Digital Album.


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Frontiers in Behavioral Neuroscience
Motivation and Reward
Front. Behav. Neurosci., 03 September 2014 | https://doi.org/10.3389/fnbeh.2014.00303
Functional characterization of dopamine transporter in vivo using Drosophila melanogaster behavioral assays
Taro Ueno1,2* and Kazuhiko Kume1,3*
1Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan
2Department of Sensory and Motor Systems, Tokyo Metropolitan Institute of Medical Science, Setagaya, Tokyo, Japan
3Department of Neuropharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Mizuho, Nagoya, Japan
Dopamine mediates diverse functions such as motivation, reward, attention, learning/memory and sleep/arousal. Recent studies using model organisms including the fruit fly, have elucidated various physiological functions of dopamine, and identified specific neural circuits for these functions. Flies with mutations in the Drosophila dopamine transporter (dDAT) gene show enhanced dopamine signaling, and short sleep and memory impairment phenotypes. However, understanding the mechanism by which dopamine signaling causes these phenotypes requires an understanding of the dynamics of dopamine release. Here we report the effects of dDAT expression on behavioral traits. We show that dDAT expression in a subset of dopaminergic neurons is sufficient for normal sleep. dDAT expression in other cell types such as Kenyon cells and glial cells can also rescue the short sleep phenotype of dDAT mutants. dDAT mutants also show a down-regulation of the D1-like dopamine receptor dDA1, and this phenotype is rescued when dDAT is expressed in the same cell types in which it rescues sleep. On the other hand, dDAT overexpression in mushroom bodies, which are the target of memory forming dopamine neurons, abolishes olfactory aversive memory. Our data demonstrate that expression of extrasynaptic dopamine transporters can rescue some aspects of dopamine signaling in dopamine transporter mutants. These results provide novel insights into regulatory systems that modulate dopamine signaling.
The neurotransmitter dopamine is crucial for arousal, motor function and memory. Further, accurate temporal and spatial regulation of dopamine signaling is required for these activities. The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft, and has a key role in limiting dopamine signaling. DAT is inhibited by therapeutic agents such as Ritalin (methylphenidate) or psychostimulant drugs of abuse such as amphetamine and cocaine, demonstrating its importance in regulating behaviors. Besides directly regulating dopamine signaling, DAT is thought to participate in gating the spillover of dopamine (Floresco et al., 2003; Cragg and Rice, 2004). This spillover of dopamine from a release site into the extrasynaptic space (i.e., volume transmission) has been reported in mammals, and is proposed to modulate brain function.
The Drosophila dDAT mutant, fumin (fmn), sleeps less than wild-type flies due to an increase in postsynaptic dopamine signaling (Kume et al., 2005). In vivo voltametric studies have shown that the fumin mutants are defective for dopamine clearance (Makos et al., 2009). Increased dopamine signaling in fumin mutants also alters their activity, metabolic rate, longevity, temperature sensitivity and sensitivity to dietary calories (Ueno et al., 2012a,b; Yamazaki et al., 2012). A similar effect is seen in mammals, where deletion of the dopamine transporter gene results in a decrease in sleep (Wisor et al., 2001). Recent studies have identified a dopamine pathway for controlling fly sleep (Liu et al., 2012b; Ueno et al., 2012c) which is distinct from other dopamine pathways responsible for memory formation (Aso et al., 2010), and the response to ethanol (Kong et al., 2010).
Due to its powerful genetics, Drosophila is an ideal model organism for identifying genes or neural circuits underlying specific behaviors. Currently, little is known about the dynamics and distribution of the released dopamine. For example, it is unknown how far dopamine, released from specific sites, diffuses, and how long the effects of released dopamine last. To investigate the effects of released neurotransmitters, genetic perturbation of plasma membrane transporters can be used to great advantage for controlling the intensity of extracellular signals.
In this report, we describe effects of altered dDAT expression on fly behavior. Using tissue specific knockdown and rescue experiments, we found that dDAT expression in a subset of dopaminergic neurons is sufficient for normal sleep. In addition, we also found that dDAT expression in glial cells could rescue the short sleep phenotype of fumin mutants. dDAT expression in postsynaptic sites for memory formation (the mushroom bodies), abolished olfactory memory formation. These results shed light on the physiological function of dDAT in dopamine signaling and in regulating extrasynaptic dopamine spillover in vivo.
Fly Stocks
Flies were reared on a standard corn meal, yeast, glucose agar medium at 24.5°C under a 12 h:12 h light:dark cycle. The fmn (fumin) mutant was described previously (Kume et al., 2005). TH-GAL4 was a gift from J. Hirsh. 104Y was a gift from D. Armstrong. NP lines were obtained from the Kyoto Drosophila Genetic Resource Center, To remove possible modifiers and allow comparisons in a common genetic background, we outcrossed all the alleles into the w1118 or fmn background over at least five consecutive generations. As controls we used w1118 or fmn flies. The transgenic RNA interference (RNAi) line for DAT (Transformant ID: 106961), UAS-Dicer-2 flies (Stock numbers: 60008) and the w1118 (60000) which is the genetic background of the RNAi line were obtained from the Vienna Drosophila RNAi Center (VDRC), Vienna, Austria (Dietzl et al., 2007).
Sleep Analysis
Flies were placed individually in glass tubes (length, 65 mm; inside diameter, 3 mm) containing 1% agar and 5% sucrose at 24.5°C. Male flies were used for sleep analysis. They were entrained for at least 3 d to LD conditions before being transferred to constant dark (DD) conditions. Locomotor activity was monitored by recording infrared beam crossings by individual flies in 1 min bins using the Drosophila activity monitoring system (Trikinetics, Waltham, MA, USA). Based on previous reports (Hendricks et al., 2000; Shaw et al., 2000), sleep was defined as periods of inactivity lasting 5 min or longer. For the pharmacological manipulations, 3-iodo-d-tyrosine and RU486 were obtained from Sigma-Aldrich. Flies were transferred to vehicle (1% agar + 5% sucrose) with 3 mM 3-iodo-d-tyrosine, 0.5 mM RU486. Daily sleep time was calculated with software written in Excel or R 2.11.1.
Measurements of Transcript Levels
The efficiency of dDAT RNAi were examined by qPCR. Total RNA was extracted from 10 heads by using RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized from the total RNA using oligo (dT)20 primer and ReverTra Ace reverse transcriptase (Toyobo). The cDNA was used for qPCR using THUNDERBIRD SYBR qPCR Mix (Toyobo). The primers were 5′-AACAATAGCA TCAGCGACGA-3′ and 5′- CAGGTTATGG CACCCTTACG-3′ for dDAT, 5′- TGGTACGACA ACGAGTTTGG-3′ and 5′- TTTCAGGCCG TTTCTGAAGT-3′ for GAPDH2. Before qPCR, all the primer sets were confirmed to yield one major band corresponding to each targeted mRNA on the gel when used in conventional RT-PCR. Each expression level was first normalized to GAPDH2. Then, the values were normalized to the average of independent control sample.
Brains of adult flies were dissected in cold PBS and fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. After three 20 min washes in 0.3% Triton X-100 in PBS, samples were blocked and penetrated in 5% normal sheep serum (NSS) for 30 min at room temperature. Samples were then incubated in a primary antibody solution in 5% NSS at 4°C overnight. The following primary antibodies were used in this study: chicken anti-GFP (1:1000; Abcam), rabbit anti-TH (1:25 Pel-Freez), rat anti-DAT (1:1000; Millipore) and rabbit anti-dDA1 (1:1250: gift from Dr. Fred W. Wolf). After three 20 min washes in 0.3% Triton X-100 in PBS, brains were incubated at 4°C overnight in a secondary-antibody solution in 5% NSS. The following secondary antibodies were used: Alexa Fluor 568 goat anti-rat IgG (1:200; Invitrogen), Alexa Fluor 488 goat anti-chicken IgG (1:200; Invitrogen) and Alexa Fluor 568 goat anti-rabbit IgG (1:200; Invitrogen). After three 20 min washes in PBS, brains were mounted using PermaFluor (Thermo Scientific) between two coverslips separated by electrical tape of ~200 μm thickness, so that the brain sample was not flattened. Immunolabeled adult brains were imaged under a Leica TCS SP2 AOBS confocal microscope using 20x magnification. Z-stack images were scanned at 2 μm section intervals with a resolution of 1024 × 1024 pixels.
For quantification of dDA1 signal, a confocal slice image containing the holizontal lobes of the mushroom body was selected from a stack. The average pixel intensity values in the horizontal lobes and the adjacent control region (aimpr: anterior inferior medial protocerebrum) was measured by NIH ImageJ. The signal of the dDA1 in horizontal lobes was normalized by the control region of an identical confocal slice.
Aversive Odor Memory
Olfactory conditioning with two odors (4-methylcyclohexanol and 3-octanol) was performed as described previously (Tully and Quinn, 1985). Briefly, approximately 100 flies were placed in a training chamber where they were exposed to odors and electric shocks. One of the aversive odors, OCT (3-octanol, Sigma) or MCH (4-methylcyclohexanol, Sigma), was paired with 12 pulses of electrical shocks (60 V DC) for 1 min, whereas the other was not. Testing was performed for 2 min after the training by placing flies at a choice point in a T-maze between the two odors for 2 min. If no flies moved to either arm of the T-maze or if a small number of flies were trapped in the middle compartment, it indicated that the flies did not choose either odor. A performance index (PI) was calculated so that a 50:50 distribution (no memory) yielded a PI of 0 and a 0:100 distribution away from the shock-paired odor yielded a PI of 100. Individual PIs were always the average of two experiments where the shock-paired odor was alternated.
The significance level in each experiment was set to 5%. Comparisons were made by using either Student's t-test (for two groups) or analysis of one-way ANOVA with Tukey-Kramer HSD post hoc test (for more than two groups). Bars and error bars represent means and SEM, respectively. Data were analyzed as described in the figure legends using R 2.11.1.
Short Sleep Phenotype in fumin and dDAT Knockdown Flies
As previously reported (Kume et al., 2005), the dDAT mutant fumin shows a short sleep phenotype (Figures 1A,B). To further investigate behavioral effects of dDAT expression, we examined the sleep phenotype of dDAT knockdown flies. Using GAL4/UAS system (Brand and Perrimon, 1993), we expressed dsRNA to induce RNAi in a cell specific manner. As shown in Figures 1A,B, flies with pan-neuronal knockdown of dDAT using Elav-GAL4 displayed a significant sleep reduction compared with control flies. The total daily sleep of dDAT RNAi flies decreased to about half that of control flies (Figure 1C). Next, we examined the effect of dDAT RNAi in specific cells or various brain regions using a series of GAL4 drivers. We performed this GAL4 driver screening in constant darkness since light has buffering effect against wake promoting effect of dopamine (Shang et al., 2011). It has been reported that dDAT gene expression is restricted to dopaminergic neurons (Porzgen et al., 2001). Consistent with the previous reports, dDAT knockdown in glial cells resulted in no significant sleep decrease. Flies subjected to dDAT knockdown in the dopamine neurons using Ddc-GAL4 showed a significantly short sleep time compared with control. On the other hand, dDAT knockdown in dopaminergic neurons using TH-GAL4 resulted in no significant sleep reduction. Ddc-GAL4 labels dopamine neurons including PAM cluster neurons, whereas this cluster neurons are sparcely labeled by the TH-GAL4 (Liu et al., 2012a). To check the efficiency of RNAi, we quantified dDAT mRNA level using qPCR. As shown in Figure 1D, pan-neuronal knockdown of dDAT using Elav-GAL4 decreased dDAT mRNA level to one-fifth of control. dDAT knockdown in dopaminergic neurons using TH-GAL4 or Ddc-GAL4 both showed decreased dDAT mRNA level to one-fourth of control, which is consistent with the previous report that showed dDAT expression is restricted to dopaminergic neurons. These results suggest that GAL4 expression pattern rather than GAL4 expression level contribute to the sleep phenotype. To further investigate the effect of dDAT knockdown in a subset of dopaminergic neurons, we crossed with HL5-, HL7-, HL9-GAL4 drivers. These drivers have GAL4 expression in PAM clusters whereas other clusters such as PPL1 neurons are sparcely labeled (Claridge-Chang et al., 2009; Figure 1E). Despite dense expression in PAM clusters, dDAT knockdown using these drivers showed no significant sleep reduction. We further knocked down dDAT in mushroom body and fan-shaped body. These neuropiles receive projection of dopaminergic neurons and contribute to arousal effect of dopamine (Andretic et al., 2008; Liu et al., 2012b; Ueno et al., 2012c). Consistent with Portzgen et al. that showed restricted dDAT expression in dopamine neurons, dDAT knockdown in these postsynaptic neurons has no effect on sleep phenotype. Taken these results together, we hypothesize that dDAT expression in a subset of dopamine neurons is sufficient for the reuptake of dopamine from synaptic cleft then shut down postsynaptic dopamine signaling for normal sleep.
Figure 1. Effect of dDAT knockdown on sleep. (A) Sleep profiles in 60 min intervals for each genotype (n = 32 flies). Sleep was monitored in 12 h light: 12 h dark condition (LD). Day and night are depicted by the white and black bars, respectively. (B) Sleep profiles in 60 min intervals for each genotype (n = 16–32 flies). Sleep was monitored in constant darkness condition (DD). Subjective day and night are depicted by the gray and black bars, respectively. (C) Total daily sleep amounts for flies with dDAT knockdown using various GAL4 drivers (n = 16–32 flies). Data are represented as the mean ± s.e.m. * P < 0.05; one-way ANOVA with Tukey-Kramer HSD post hoc test. (D) Efficiency of dDAT knockdown. The expression levels of dDAT gene in the head of flies expressing dDAT RNAi transgene by GAL4 drivers are expressed as the relative values to the control flies. n = 3 for each group. Data are represented as the mean ± s.e.m. * P < 0.05; one-way ANOVA with Tukey-Kramer HSD post hoc test. (E) Expression patterns of GAL4 drivers for dopaminergic neurons in the central brain. The GAL4 expression for each driver was visualized using UAS-mCD8::GFP (green). The brain was stained with antibody to nc82 to stain the neuropil (magenta). PAM, protocerebral anterior medial; PAL, protocerebral anterior lateral. Scale bars = 50 μm.
dDAT Expression in a Subset of Dopamine Neurons can Rescue the Short Sleep Phenotype in fumin Mutants
To further investigate the effect of dDAT expression on fly sleep, we rescued dDAT in fumin mutant. Using GAL4 drivers which labels a subset of dopamine neurons, we expressed dDAT in dopaminergic neurons in fumin mutant. Although we could not observe significant sleep reduction with dDAT knockdown using TH-GAL4 (Figure 1C), fumin with dDAT expression in TH-GAL4 labeled neurons showed significant increase of sleep (Figure 2A). As described above, dopaminergic neurons except for PAM cluster neurons are labeled by TH-GAL4 line. Other GAL4 drivers which labels a subset of dopaminergic neurons, HL5- and HL7-GAL4, could also rescue the short sleep phenotype of fumin by cell specific dDAT expression. HL5-GAL4 has dense expression in PAM cluster neurons whereas PPL1 and PPM3 cluster neurons which are one of the wake promoting dopaminergic neurons are not labeled by this GAL4 driver. HL7-GAL4 also labeles PAM cluster neurons but PPL1 and PPM3 clusters are sparcely labeled (Kong et al., 2010). Based on the discrepancy between dDAT knockdown and dDAT rescue experiments, we consider that dDAT expression in a subset of dopamine neurons is sufficient to rescue the short sleep phenotype of fumin mutant. On the other hand, fumin with dDAT expression by HL9-GAL4 showed no significant increase of sleep. It might be due to its low expression level since we observed lower expression in HL9-GAL4 compared with HL5- and HL7-GAL4 drivers.
Figure 2. Effect of dDAT expression on sleep. (A) The short sleep phenotype of fmn was rescued by expression of dDAT in various brain regions. Black bar indicates control fly without dDAT mutation. Blue bars indicate fmn GAL4 drivers without UAS-dDAT. Red bars indicate fmn GAL4 drivers with UAS-dDAT (n = 16–32 flies). Error bars represent s.e.m. * P < 0.05; two-sided Student's t-test. (B) Expression patterns of GAL4 drivers in the central brain. The GAL4 expression for each driver was visualized using UAS-mCD8::GFP (green). Dopamine neurons were visualized using anti-TH antibody counterstaining (magenta). PPM, protocerebral posterior medial; PPL, protocerebral posterior lateral. Scale bars = 50 μm. (C) dDAT expression in the fly brain detected with immunohistochemistry. dDAT expression in fmn flies and fmn 30Y > dDAT flies are shown. Left panel shows anterior view and right panel shows posterior view. Scale bars = 50 μm.
dDAT Expression in Non-Dopaminergic Cells can Rescue the Short Sleep Phenotype in fumin Mutants
Since we could rescue the short sleep phenotype of fumin by dDAT expression in different subset of dopaminergic neurons which were insensitive to dDAT knockdown, we further investigated the effect of ectopic dDAT expression in non-dopaminergic cells. We presumed if dDAT expression in a subset of dopamine neurons is sufficient for the shut down of postsynaptic dopamine signaling, it is possible to control dopamine signaling by dDAT expression in postsynaptic neurons or extrasynaptic cells such as glia.
In order to express dDAT in various regions of the brain in a fumin background, we outcrossed the GAL4 drivers to the fumin mutant. Next, we examined the effect of dDAT expression on the short sleep phenotype of the fmn mutant. We found that fmn flies with dDAT expression in non-dopaminergic neurons showed a significant sleep increase (Figure 2A). Mushroom body (Joiner et al., 2006; Pitman et al., 2006) and fan-shaped body (Donlea et al., 2011) receive dopaminergic projection and regulates sleep and arousal. Although we observed no significant changes in sleep by dDAT knockdown in these postsynaptic structure (Figure 1C), dDAT expression in these neuropile rescued short sleep of fumin mutant. In addition, dDAT expression in glial cells also rescued short sleep in fumin. To confirm that the drivers have no GAL4 expression in dopamine neurons, we expressed GFP using the GAL4/UAS system and then counter stained with anti-TH antibody. As the merged signal patterns shown in Figure 2B, TH-GAL4 drives GFP expression in most, but not all, dopamine neurons. Conversely, we could not detect any merged signals when expressing GFP using the other drivers, which suggests that these drivers only expressed dDAT in non-dopaminergic neurons. Next, we analyzed the localization of ectopically expressed dDAT using immunohistochemistry. Although we could not detect endogeneous dDAT expression due to high background with the antibody, ectopic expression of dDAT in the fmn mutant using the 30Y-GAL4 driver, which has strong expression in the mushroom body, showed dDAT immunostaining in all lobes and calyx of the mushroom body (Figure 2C). These lobes and calyx are composed of axon bundles and dendrites of Kenyon cells, thus ectopically expressed dDAT appears to be transported and localized to the synaptic area.
To further investigate if the effect of dDAT expression in non-dopaminergic neurons is due to developmental changes or not, we expressed dDAT in postsynaptic neurons or glial cells using spatiotemporal expression system, TARGET system (McGuire et al., 2003). In this system, dDAT expression is suppressed at 19°C by the temperature sensitive GAL4 inhibitor GAL80ts which is under the control of tubulin promotor. After shifting to 31°C, GAL80ts was inactivated and dDAT expression will be induced. All the flies were raised at 19°C to adulthood. At the permissive temperature, progeny from the experimental crosses showed similar sleep patterns to control flies. The temperature shift to 31°C significantly increased sleep time of flies which has temporal dDAT expression in mushroom body and glial cells (Figure 3). These results suggest that dDAT expression in non-dopaminergic neurons, such as the mushroom body or glial cells, can rescue the short sleep phenotype in fmn without developmental changes.
Figure 3. Effect of spatiotemporal dDAT expression on sleep. (A) Sleep profiles and daily sleep amounts for flies expressing dDAT in the mushroom body using the TARGET system. (B) Sleep profiles and daily sleep amounts for flies expressing dDAT in glial cells using the TARGET system. (C) Total daily sleep amounts for flies expressing dDAT in the mushroom body using the TARGET system. Daily sleep at 31°C is shown. (D) Total daily sleep amounts for flies expressing dDAT in glial cells using the TARGET system. Daily sleep at 31°C is shown. Data are represented as the mean ± s.e.m. * P < 0.05; one-way ANOVA with Tukey-Kramer HSD post hoc test (n = 32 flies).
Enhancement of Extracellular Dopamine Causes Downregulation of the D1-Like Receptor dDA1
The D1-like receptor dDA1 is a dopamine receptor expressed in the mushroom body and in central complex structures such as the ellipsoid body and fan-shaped body. dDA1 expression in the mushroom body mediates olfactory memory (Kim et al., 2007). As shown in Figure 4A, fmn mutants demonstrated decreased dDA1 expression in the mushroom body. Quantification of dDA1 expression based on image analysis of immunohistochemistry results (Knapek et al., 2011) also confirmed reduced dDA1 expression in the mushroom body (Figure 4B). Administration of a dopamine synthesis inhibitor (tyrosine hydroxylase inhibitor) rescued the short sleep phenotype of fmn mutants (Figure 4C) and increased dDA1 expression in the mushroom body (Figures 4D,E). Conditional rescue of dDAT in all neurons using the GeneSwitch system (Osterwalder et al., 2001) increased dDA1 expression in the mushroom body (Figure 4F). Without administration of RU486, there was also significant leakage of dDAT expression in the mushroom body as previously pointed out (Poirier et al., 2008). To investigate whether the decreased dDA1 expression was the result of short sleep or increased dopamine signaling, we checked the dDA1 expression in another sleep mutant. Both calcineurin knockout and knockdown result in the short sleep phenotype (Nakai et al., 2011; Tomita et al., 2011), but dDA1 expression in the calcineurin mutants was not reduced (Figure 4G). Taken these results together, increased dopamine signaling, rather than short sleep in fmn mutants, downregulates dDA1 expression in the mushroom body. Thus dDA1 expression level is applicable as an indicator of the dopamine signaling intensity.
Figure 4. The D1-like receptor dDA1 in the mushroom body is regulated by dopamine signaling. (A) dDA1 expression in the mushroom body. dDA1 expression in the mushroom body was visualized using the anti-dDA1 antibody. Robust dDA1 expression in the mushroom body was detected in the control, on the other hand, the fmn mutant showed decreased dDA1 expression in the mushroom body. Images using the maximum projection are shown. Scale bars = 50 μm. (B) Quantification of dDA1 expression. dDA1 expression was quantified by calculating the signal ratio of the horizontal lobe and the aimpr. The fmn mutant shows significantly decreased dDA1 expression compared with the control. * P < 0.05; two-sided Student's t-test. Scale bar = 50 μm. (C) The dopamine synthesis inhibitor 3IY rescues the short sleep phenotype of fmn mutants. fmn flies fed with vehicle (red line) show decreased sleep compared with control flies (black line), on the other hand, fmn flies fed with 3IY (blue line) show sleep levels similar to the control (n = 16 flies). (D) dDA1 expression was restored in fmn mutants fed with 3IY. dDA1 expression in the mushroom body was visualized using the anti-dDA1 antibody. Image using the maximum projection is shown. Scale bar = 50 μm. (E) Quantification of dDA1 expression in the mushroom body with drug administration. dDA1 expression in the mushroom body was calculated as shown in Figure 4B. * P < 0.05; two-sided Student's t-test. (F) Quantification of dDA1 expression in the mushroom body with temporal dDAT expression. dDAT expression was induced using the Elav-GeneSwitch. dDA1 expression of flies with or without RU486 0.5 mM administration for each genotype are shown. * P < 0.05; one-way ANOVA with Tukey-Kramer HSD post hoc test. (G) Quantification of dDA1 expression in the short sleep mutant. dDA1 expression of flies with either the CanA14F knockout or the CanB knockout are shown.
dDAT Expression Rescues Downregulation of dDA1
To examine the effect of dDAT expression on dopamine signaling, we verified the dDA1 level in fmn with dDAT expression. As shown in Figure 5, dDAT expression in a subset of dopaminergic neurons with TH-GAL4 rescued dDA1 expression in fmn. In addition, dDAT expression in the mushroom body (121Y-GAL4, 11Y-GAL4), glial cells (dEAAT1-GAL4) and pars intercerebralis (c767-GAL4) increased dDA1 expression in the mushroom body, indicating decrease of dopamine signaling. These results are consistent with the rescued sleep phenotype in fmn by ectopic dDAT expression (Figure 2A). On the other hand, npf-GAL4 driven dDAT expression in fmn flies could not rescue the short sleep phenotype (Figure 2A) and showed no significant changes in dDA1 expression in the mushroom body (Figure 5). These results indicate that the ectopic expression of dDAT suppresses the increased dopamine signaling in the fmn mutant.
Figure 5. dDAT expression rescues dDA1 down-regulation in the fmn mutant. dDA1 expression in the mushroom body was calculated as shown in Figure 4B. Error bars represent s.e.m. * P < 0.05; two-sided Student's t-test.
The Effect of dDAT Expression on Learning and Memory
Next, we investigated the effect of dDAT expression on aversive olfactory memory formation. Dopamine signaling mediates associative olfactory memory via dDA1 receptor in mushroom body (Kim et al., 2007). As previously reported (Zhang et al., 2008), fmn mutants have an impaired aversive olfactory memory due to excessive dopaminergic signaling (Figure 6A). dDAT expression in glial cells (using Repo-GAL4) as well as dopamine neurons (using TH-GAL4) promoted memory formation in fmn mutants although we could not find statistical significance. Surprisingly, however, when some GAL4 drivers which are expressed in the mushroom body (such as 30Y-GAL4 and OK107-GAL4) were used to drive dDAT, these flies did not demonstrate aversive olfactory memory (Figure 6A). 30Y-GAL4 and OK107-GAL4 have broad and strong GAL4 expression in the mushroom body (Aso et al., 2009), which suggest that post-synaptic strong dDAT expression abolished dopamine signaling after activation of dopamine neurons with electric punishment. Another possibility is that strong expression of dDAT in the mushroom body caused a developmental defect in the Kenyon cells since flies with chemical ablation of mushroom body are unable to perform in a classical conditioning paradigm (de Belle and Heisenberg, 1994). To exclude this possibility, we checked the memory performance of flies with temporal dDAT expression using Elav-GeneSwitch. Without administration of RU486, there was significant leakage of dDAT expression which may have affected memory performance (Figures 6B,C), since there was a slight improvement in the PI even in flies fed with vehicle (compare control of Figure 6A and vehicle of Figure 6B). However, administration of RU486 results in the impairment of memory performance in a dose dependent manner. Furthermore, the flies fed with 500 μM of RU486 did not demonstrate associative memory (Figure 6B). These results suggest that strong expression of dDAT at the post-synaptic site results in abolition of dopamine signaling after activation of dopamine neurons.
Figure 6. Effect of dDAT expression on memory. (A) The results of the 2 min memory (immediate memory) tests for each genotype are shown. Each genotype (n = 8–17 trials) was tested using an aversive olfactory conditioning test after training. Data are represented as the mean ± s.e.m. a: P < 0.05; one-way ANOVA with Tukey-Kramer HSD post hoc test. b: P < 0.05; two-sided Student's t- test compared with control. (B) Conditional expression of dDAT in all neurons abolishes memory formation. fmn Elav-GS UAS-dDAT or Elav-GS UAS-dDAT flies were fed with vehicle or RU486 and then tested using an aversive olfactory conditioning test after training. * P < 0.05; one-way ANOVA with Tukey-Kramer HSD post hoc test or two-sided Student's t-test. (C) Leakage and induction of gene expression with Elav-GS. mCD8::GFP was expressed using the Elav-GS driver. The central brains of flies fed with either vehicle or RU486 are shown. Scale bars = 50 μm.
In this study, we investigated the effect of dDAT expression on sleep and memory. We showed that dDAT expression in a subset of dopaminergic neurons is sufficient for normal sleep. We also demonstrated that dDAT expression in non-dopaminergic neurons rescue the short sleep phenotype of fmn mutant through the reduction of dopaminergic signaling. Finally, we also demonstrated that postsynaptic strong dDAT expression results in the abolishment of memory formation.
To investigate the role of dDAT on sleep, we set out to map the neural circuit in which the dDAT expression mediates sleep. Mushroom body receives projection of dopaminergic neurons and controls fly sleep (Joiner et al., 2006; Pitman et al., 2006). Previous study demonstrated that dDA1 expression in mushroom body mediates arousal effect of caffeine and methamphetamine (Andretic et al., 2008). Dopaminergic neurons also have projection to sleep promoting neuropile fan-shaped body and regulates sleep (Donlea et al., 2011; Liu et al., 2012b; Ueno et al., 2012c). Pan-neuronal knockdown of dDAT resulted in decrease of sleep similar to fmn mutant. On the other hand, flies with dDAT RNAi in glial cells or postsynaptic neurons showed no sleep changes which is consistent with the previous report that demonstrated restricted dDAT expression in dopaminergic neurons (Porzgen et al., 2001). We also demonstrated that dDAT knockdown in a subset of dopaminergic neurons using TH-, HL5-, HL7- and HL9-GAL4 is not sufficient to cause a short sleep phenotype although Ddc-GAL4 driven RNAi resulted in a sleep decrease. TH-GAL4 labels most dopaminergic neurons including fan-shaped body projecting PPL1 and PPM3 clusters but lacks GAL4 expression in PAM clusters. On the other hand, HL5-, HL7- and HL9-GAL4 drivers heavily labels PAM clusters. It is possible that GAL4 expression pattern in dopaminergic neurons is responsible for sleep regulation by dDAT since Ddc-GAL4 driven RNAi resulted in a sleep decrease. Another explanation is that dDAT expression in serotonin positive neurons contribute to sleep regulation since Ddc-GAL4 labels some serotonin-releasing neurons (Pech et al., 2013). In contrast to the results of knockdown experiments, we demonstrated that dDAT expression in a subset of dopaminergic neurons is sufficient to rescue the short sleep phenotype in fmn mutant. Taken these results together, we concluded that dDAT expression in a subset of dopaminergic neurons is sufficient for normal sleep. It is possible that diverse cells can redundantly fulfill the task of clearing the extracellular space from dopamine since dDAT expression in PAM-cluster neurons is not required but sufficient for proper sleep. We further investigated the effect of dDAT expression in non-dopaminergic neurons. dDAT expression rescued the short sleep phenotype of the fmn mutant not only in dopamine neurons but also in other cells such as Kenyon cells and glial cells. The effect of ectopic dDAT expression on dopamine signaling was confirmed by the rescue of dDA1 expression in the mushroom body which is down-regulated in the fmn mutant. The ectopic expression of dDAT in mushroom body resulted in a poor memory performance. These results may suggest the phenotype of dDAT mutant is not only the result of a simple enhancement of physiological dopamine pathway, but also the result of an extrasynaptic volume transmission, i.e., spillover. This should be taken into account when we interpret the phenotype of a neurotransmitter transporter mutant.
Dopamine needs to be cleared from synaptic cleft to maintain the proper signaling levels. dDAT reuptake dopamine from synaptic cleft into presynaptic dopamine neurons. In Drosophila, recycling of dopamine through glial cells also regulates dopamine signaling (Suh and Jackson, 2007). Ebony converts dopamine to N-β-alanyl dopamine which is passed from glial cells to presynaptic neurons and then converted back to dopamine. Fly homolog of Dysbindin, a human schizophrenia susceptibility gene, regulates dopamine signaling through upregulation of Ebony (Shao et al., 2011). Although we could not find any significant sleep changes with dDAT knockdown in glial cell, glial ebony might have contribution to sleep phenotype in fmn with dDAT expression in glial cells. Another mechanism to clear dopamine from synaptic cleft is degradation of dopamine by metabolic enzyme. In Drosophila, dopamine is metabolized primarily by arylalkylamine N-acetyltransferase (aaNAT). Flies with aaNAT mutation show defects in sleep homeostasis (Shaw et al., 2000).
Since synaptic and extrasynaptic glutamate uptake transporters are enriched on multiple presynaptic and postsynaptic cells (including glial cells), neurotransmitters such as glutamate are restricted to the synaptic cleft of the release site (Seal and Amara, 1999; Danbolt, 2001). In contrast, neurotransmitters such as gamma-aminobutyric acid (GABA) and monoamines show extrasynaptic (volume) transmission which is mediated by the diffusion of transmitters through the extracellular space (Cragg and Rice, 2004; Syková, 2004; Farrant and Nusser, 2005). Use of experimental methods such as microdialysis or voltammetry with DAT-knockout mice, or psychostimulants such as cocaine or methylphenidate, revealed that DAT inhibition increases the extracellular dopamine concentration (Benoit-Marand et al., 2000; Bradberry et al., 2000), although these invasive methods may affect the extracellular environment. On the other hand, using the fruit fly and its highly sophisticated genetic toolbox we can spatially and temporally control dDAT expression and analyze physiological function using behavioral assays. Park et al., studied the effect of ectopic Drosophila serotonin transporter (dSERT) expression in the fly brain and suggested additional factors may be needed for dSERT function (Park et al., 2006). By combining ectopic expression, a knockout mutant, a behavioral assay and immunohistochemistry, we revealed that ectopic dDAT expression affects the kinetics of dopamine elimination from the synaptic cleft under physiological conditions (i.e., without invasive experimental methods). Cell-type-specific ectopic expression of transporters for neurotransmitters in Drosophila will be a favorable tool by which the mechanisms underlying precise inter-cellular signaling in space and time will be elucidated.
A previous study explored the relative roles of the DAT on striatal extracellular dopamine during tonic vs. phasic activity of dopamine neurons in the substantia nigra (Floresco et al., 2003). They suggested that dopamine released during burst firing is normally rapidly removed by uptake before it can escape the synaptic cleft. Although the fly brain is tiny for electrophysiology, ectopic expression of dDAT in the fly brain might shed light on the relationship between physiological function and neural activity of dopamine neurons.
We thank Jun Tomita, Masako Yamazaki, Akiko Miyoshi, Yusei Kawahara, Wang Honghang for technical assistance, Fred W. Wolf for the kind gift of an antibody, Rob Jackson, Shoen Kume and the members of Kume lab for discussion and critical reading of the manuscript, Junjiro Horiuchi for improvement of English, and Paul Garrity, Doug Armstrong, Jay Hirsh, Kei Ito, Orie T. Shafer, the Kyoto Drosophila Genetic Resource Center, and the Bloomington Stock Center for fly stocks. This work was supported by a grant (22300132, 25290007, 26702037, 26560458) from the Japanese Society for the Promotion of Science (JSPS), Japan. This work was supported by the Grant-in-Aid for Scientific Research on Innovative Areas "Memory dynamism" (25115006) from the Ministry of Education, Culture, Sports, Science and Technology.Taro Ueno is a JSPS fellow.
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Keywords: sleep, dopamine, dopamine transporter, memory, Drosophila melanogaster, volume transmission
Citation: Ueno T and Kume K (2014) Functional characterization of dopamine transporter in vivo using Drosophila melanogaster behavioral assays. Front. Behav. Neurosci. 8:303. doi: 10.3389/fnbeh.2014.00303
Received: 23 June 2014; Accepted: 18 August 2014;
Published online: 03 September 2014.
Martin Giurfa, Centre National de la Recherche Scientifi que - Université Paul Sabatier-Toulouse III, France
Andre Fiala, Georg-August-Universität Göttingen, Germany
Laurent Seugnet, Lyon Neuroscience Researach Center (CRNL) INSERM U 1028 - Equipe Waking, France
Copyright © 2014 Ueno and Kume. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Taro Ueno, Department of Sensory and Motor Systems, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya, Tokyo 156-0057, Japan e-mail: ueno-tr@igakuken.or.jp;
Kazuhiko Kume, Department of Neuropharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe 3-1, Mizuho, Nagoya 467-8603, Japan e-mail: kume@phar.nagoya-cu.ac.jp


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Church Organist
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Carolyn Williams holds a Bachelor of Science in Music from Campbell College, a Master of Education in Guidance and Counseling from The University of North Carolina at Chapel Hill and a Doctor of Philosophy in Education Administration from the University of South Carolina at Columbia. She taught classroom music in the Department of Defense schools at Fort Bragg in Fayetteville. Carolyn retired from the Brunswick County Schools as Principal of Supply Elementary School in 2000. She has served as Music Director, Pianist, Organist, and Chair of the Music Committee at Trinity. Carolyn is a native of Brunswick County, having been born in Southport. She now lives in Southport . She enjoys singing in the Chancel Choir and playing handbells with the Celebration Ringers. She has raised Cocker Spaniel puppies in the past and hopes to soon have a new four-legged child.
Trinity United Methodist Church | 209 E. Nash St., Southport, NC 28461 | 910-457- 6633 | Church Annex Office | 300 N. Howe St., Southport, NC 28461 | 910-363-4409
Hours: Mon-Thurs 9 a.m. to 3 p.m., Fri 9 a.m. to noon | email: southporttrinityumc@gmail.com
Sunday services: Casual, 8:15 a.m., Sanctuary | Contemporary, 9:30 a.m., Murrow Hall | Traditional, 11 a.m., Sanctuary