All reagents required for RNA extraction were preparedin DEPC-treated water. RNasecontamination from non-autoclavable items wasremoved by wiping them with RNaseZap® (Ambion). Total RNA from yeast cells was extractedusing acid phenolextractionmethod. Briefly, yeast cells were grown underappropriate conditions and at suitabletime points,cells were harvested by centrifugation at 4,000 rpm for 5 min. The cell pellet was washed twice with ice-cold DEPC-treated water, resuspended in 350 μl of AE buffer and transferred toa1.5 ml microcentrifuge tube. To this,40 μl of 10% SDS and 400 μl of acid phenol (pH 4.3) was added. The cell suspension was mixed well by vortexing thrice, short pulsesof10 seconds each,and incubated at 65°C for 15 min with continuous agitation at 800 rpm. Post incubation, cells were kepton ice for 5 min and centrifuged at 13,000 rpm in a refrigerated centrifuge set at 4°C for 10 min. After centrifugation, aqueous layer was transferred to a new1.5 ml microcentrifuge tube and 400 μl of chloroform was added. Tubes were mixed well by gentlyinverting them 4-5 times and centrifuged at 13,000 rpm for 10 min. The aqueous layer was separated and transferred to a new1.5 ml

Total RNA isolation

This method was used to isolate highly pure genomic DNA. Briefly, 10 ml overnight grownC. glabratacultures were spun downandwashed with 10 ml sterile water. Washed cells wereresuspended in500 μl sterile water and transferred toa1.5 ml microcentrifuge tube. Tubes were spundownat 4,000 rpm for 5 min, supernatant was discarded andcell pellet was resuspended in 500 μl of buffer containing 100 mM EDTA and 5% β-mercaptoethanol and incubatedat 42°C for 10 min. Post incubation, cells were spun down at 4,000 rpm for 5 min and resuspended in freshly prepared Buffer B. To this, one tip-full of lyticase (Sigma, L4025) was added and incubated at 37°C for 1 h.After incubation, spheroplasts were collected by spinning downtubes at 6,000 rpm for 5 min, supernatant was discarded and the pellet was resuspended in 500 μl of Buffer C. DNA was extracted twice with 500 μl of PCI (25:24:1) solution and the aqueous layer was transferred toa new1.5 ml microcentrifuge tube. To this, 2.5 volume of absolute ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.3) wereadded. Tubes were spundownat 13,000 rpm for 10 min, DNA pellet was resuspended in 200 μl of 1X TE buffer containing0.3 μl of RNase cocktail (Ambion) and incubated at 37°C for30 min. DNA was precipitated again by adding absolute ethanol and sodium acetate as mentioned above. DNA pellet was washed once with 70% ethanol, centrifuged at 13,000 rpm for 10 min, air-dried at room temperature and was resuspended in 100-200 μl of 1X TE buffer by gently tapping the tube. DNAwas stored at -20°C until use

Spheroplast lysis method

Yeast genomic DNA was isolated by mechanically lysing the yeast cells. Briefly, 10 ml of overnight grown yeast culture was transferred toa 15 ml centrifuge tube andcells were spun down at 4,000 rpm for 5 min. Media was decanted and cells were washed with 10 ml sterile water. Washedcells were resuspended in 500 μl of Buffer A and transferred to a 1.5 ml microcentrifuge tube. Tubes were incubated at 65°C for 15 min. Post incubation,500 μl of PCI (25:24:1) solution was added. To this, 0.5 g of 0.5 mm glass beads were added and cells were lysed mechanically in a bead-beatinghomogenizer (MP Biomedicals,FastPrep®-24) thrice, 45 seceach, with intermittent cooling on ice. Tubes were spun at 12,000 rpm for 5 min and the aqueous layer was transferred to a new 1.5 ml microcentrifuge tube. To this, 500 μl of PCI solution was addedand mixed gently by inverting the tubes.Tubes were centrifuged again at 12,000 rpm for 5 min and aqueous layer was transferred to another 1.5 ml microcentrifuge tube. Next, 2.5 volume of absolute ethanol was added to the aqueous layer, mixed well and centrifuged at 13,000 rpm for 10 min. Supernatant was decanted and the DNA pellet was washed once with 70% ethanol and centrifuged at 13,000 rpm for 10min. Washed DNA pellet was air-dried and dissolved in 100-200 μl of 1X TE buffer by gently tapping the tubes

Glass bead lysis method

Yeast genomic DNA isolation

Molecular biology methods

To phenotypically characterize C. glabratamutants,serial dilution spot growth assays were performed. Briefly, the optical density of overnight-grown C. glabratacultures wasnormalized to OD600of 1.0andnormalized cultures were further diluted 10-fold in 1X sterile PBS five times. 3 μl of serially diluted culture were spotted on test plates. Plates were incubated at 30°C (unless mentioned otherwise) for 24-48hand growth was recorded by capturing plate images. For experiments involvingchecking theability of mutants to utilize non-fermentable carbon sources,growth was scoredafter 6-7 days of incubation

Serial dilution spot growth assay

phorbol myristateacetate (PMA) (Tsuchiya et al., 1982). For PMA treatment, THP-1 cells were allowed to grow till 70-80% confluence and were collectedin a centrifuge tube by centrifugationat 1,000 rpm for 3 min. THP-1 cell pelletswere resuspended in 4-5 ml of pre-warmed complete RPMI-1640 medium, 100 μl of this cell suspension was appropriately diluted in PBS (1X) and viability was determined by counting trypan blue stained cellsusing hemocytometer. THP-1 cell suspension was diluted appropriately to obtainafinal cell density of 106cells/ml with pre-warmed complete RPMI-1640 medium. PMA was added totheTHP-1 cell suspension at a final concentration of 16 nM and mixed well by gently inverting the tubes. PMA-treated cells were seeded either in 24-well cell culture plates or in cell culture dishes and allowed to grow for 12 h under tissue culture conditions i.e. at 37°C and 5% CO2.After 12 h incubation, spent medium was replaced withfresh pre-warmed complete RPMI-1640 medium and cells were allowed to recover for another 12 h

THP-1 monocytes getdifferentiated intophagocytic macrophages upon treatment with

PMA (Phorbol myristateacetate) treatment of THP-1 monocytic cells

2% DTTThe stock solution of SDS loading buffer was made asa4 X concentrateand was added to the protein sample to the final concentration of 1 X.SDS-PAGE running buffer0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSThe stock solution was prepared as a 10 X concentrate and was diluted to 1 X concentration prior to use.Resolving gel mix (12%, 10 ml)3.3 ml H2O4 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix2.5 ml 1.5 M Tris-HCl (pH 8.8)100 μl 10% SDS100 μl 10% Ammonium persulfate (APS)4 μl N,N,N′,N′-Tetramethylethylenediamine (TEMED)Stacking gel mix (5%, 3 ml)2.1 ml H2O0.5 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix380 μl 1 M Tris-HCl (pH 6.8)30 μl 10% SDS30 μl 10% APS3 μl TEMED

2% DTTThe stock solution of SDS loading buffer was made asa4 X concentrateand was added to the protein sample to the final concentration of 1 X.SDS-PAGE running buffer0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSThe stock solution was prepared as a 10 X concentrate and was diluted to 1 X concentration prior to use.Resolving gel mix (12%, 10 ml)3.3 ml H2O4 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix2.5 ml 1.5 M Tris-HCl (pH 8.8)100 μl 10% SDS100 μl 10% Ammonium persulfate (APS)4 μl N,N,N′,N′-Tetramethylethylenediamine (TEMED)Stacking gel mix (5%, 3 ml)2.1 ml H2O0.5 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix380 μl 1 M Tris-HCl (pH 6.8)30 μl 10% SDS30 μl 10% APS3 μl TEMED

Total cell lysis buffer (Homogenization buffer)50 mM Tris-HCl (pH 7.5)2 mM EDTA10 mM Sodium fluoride*1 mM Sodium orthovanadate*1 X protease inhibitor cocktail (Sigma, P 8215)** Were added fresh before use.SDS-PAGE30% acrylamide solution29 g Acrylamide1 g N,N’-MethylenebisacrylamideDissolved in 100 ml H2O10% Sodium Dodecyl Sulfate (SDS)10 g SDS in 100 ml H2OSDS loading buffer130 mM Tris-HCl (pH 8.0)20% (v/v) Glycerol4.6% (w/v) SDS0.02% Bromophenol blue

Buffers for protein extraction and separation by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis)

Table 2.4: List of antibodies used in thisstudy

All antibodies used in thisstudy, their clonality and dilutions used,Manufacturers’ details,and catalogue numbersare listed in Table 2.4

Antibodies

Cells were grown in 35 mm dishes at 20% initial confluence. At 40-50% confluence, cells were treated with different genotoxic agents as described in Section 2.2.2. Post treatment, cells were washed twice with PBS and replaced with fresh media to allow recovery for different lengths of time. At each time point, cells were harvested and fixed with 70% ethanol at -20°C overnight. Fixation was carried out by adding 70% ethanol drop by drop, while the cells were being vortexed at a low speed.The fixed samples were brought to room temperature, pelleted down at 2000 rpm for 3 min and washed twice with PBS. Cells were stained with PI solution containing 0.1% Triton X-100, 0.2 mg RNase and 20μg propidium iodide and incubated at 37ºC for 30 min in the dark. Samples were analyzed by flow cytometry (FACS ARIA, BD). Data was analyzed usingFACS DIVA (BD) and FlowJo (FLOWJO, LLC) softwares to identify different stages of the cell cycle

Cell cycle analysis by PI staining

All animal experiments were conducted as per guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment, Forest, and Climate Change, Government of India,and these experiments were approved by the Institutional Animal Ethics Committee (Protocol numbers PCD/CDFD/02-version 2 and PCD/CDFD/08). Mice used for this study were housed in the Centre for DNA Fingerprinting and Diagnostics animal facility located within the premises of Vimta Labs, Hyderbad.Ip6k1+/-heterozygous mice were bred to generate age and sex matched Ip6k1+/+and Ip6k1-/-littermates for experiments. Foxn1numice were generated by breeding homozygous males with heterozygous females.These mice were used for in vivotumourigenic assays

Mice

Viewing slides under microscope

A drop of immersion oil was put on top of the cover-slip before viewing it under microscope. The cells were viewed at 100X resolution of Nikon Eclipse 80i microscope.Thedifferential interference contrast images of the cells were captured using NIS-Elements D3.0 software also used to find out mean cell size using at least 100 randomly selected cells.Fluorescence images were captured on Zeiss LSM 710 Meta inverted confocal microscope

The RNA was cross-linked onto the membrane after transfer by exposing it to the UV light of 200KJ/cm2 energy in a UV-crosslinker

UV-crosslinking of the RNA

Band intensities in gel autoradiogramswere determined by densitometry with the aid of the Fujifilm Multi Gauge V3.0 imaging system. Equal areas of radioactive bands were boxed and the PSL (Photo stimulated luminescence) values were further considered. Background signal (obtained from equal area as that of the radioactive band but from other part of the gel/blot) is subtracted from the signal intensities obtained from radioactive bands to get the final values

Densitometry

Oligonucleotides and PCR products were end-labelled using phage T4-polynucleotidekinase (PNK, New England Biolabs or Fermentas or Sigma) with 32P-γ-ATP. The radiolabelling reaction mixture (20μl) contained 1X of buffer provided by the company, 10 units of T4-PNK and 40μCi of 32P-γ-ATP. The reaction mix was incubated for 1 hrat 37ºC and the reaction was heat-inactivated at 65oC for 20 minutes. The labelled oligonucleotides and DNA fragments were purifiedby the Qiagen nucleotide removal kit. Labelling efficiency was checkedeither by using Geiger-Muller (GM) counter orusing liquid scintillation counter.For scintillation counting, 1μl of radioactive sample wasadded to the 5ml scintillation cocktail, and radioactivity count was determined in the 32P channel of scintillation counter (Perkin Elmer, Liquid Scintillation analyzer, Tri-Carb 2910 TR, USA). Liquid scintillation cocktail consists of 5g PPO (2,5-diphenyloxazol) and 0.3g POPOP (1,4-bis (5 phenyl 1,2-oxazole) Benzene, adjusted to a volume of 1L in toluene

Radiolabelling of oligonucleotides

DNA sequencing

Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits on an automated sequencer following the manufacturer's instructions byan outsourced sequencing facility

The semi-quantitative reverse transcription-PCR (RT-PCR) involves the synthesis ofcomplementary DNA (cDNA)from RNA. For this, 1μgof RNAwas treated with 1μl (1 unit) DNase I enzyme (Sigma, amplification grade) for20 min to remove DNA contamination. DNase I was inactivated by heating at 70oC for 10 min. Next, 5pmol reverse primer wasadded along with dNTPs and volume made to 10μlwith DEPC-treated water; the mix washeated at 65oC for 5 min and incubated on ice forat least 1 min. The reverse transcription reaction was set up with this mix using the Superscript III RT kit (Invitrogen) as per manufacturer’s protocolto obtain cDNA. The cDNA servedas the template for setting up a PCR for requirednumber of cycles. The samples were finally run on agarose gels

Reverse transcription(RT)-PCR

Concentrations of DNApreparations were estimated by nanodrop or by gel electrophoresis followed by densitometric analysis.Concentration of RNA preparations were estimated by nanodrop

Estimation of DNA and RNA concentrations

Total RNA extraction from E. colicells was doneusing Qiagen RNeasy minikit. Cells were grown to an A600of 0.6 and harvested(amaximumof107cells)at 6000rpm for 5min at room temperature to prevent cells for encountering any stress in cold. Rest of the steps were followed exactly as mentioned in the manufacturer’s protocol. The quality of RNA preparations was assessed following electrophoresison 1.4% agarose-formaldehyde-MOPS gels.Ingeneral,forawild-typestrainRNAyieldwouldbe~0.5-1μg

Isolation of total cellular RNA

For high fidelity PCR, Herculase II fusion DNA polymerase (AgilentTechnologies)was used. Approximately 0.5μg of chromosomal DNAwas used as a template in a 50μl reaction volume

The PCRs were normally performed using Taqpolymerasefrom Roche or Fermentas. Approximately 1-5ng of plasmid or 5-100ng of chromosomal DNA was used as a template in a50μlreaction volume containing 200μM of each dNTP, 20pM each of the forward and reverse primers and 1 unit of Taq DNA polymerase. For colony PCR E. coli cells from a freshly grown plate were resuspended in 10μl of sterile Milli-Q water to get a cell suspension and this was used as a template in a PCR reaction at a final volume of 50μl. The samples were typically subjected to 30 cycles of amplification with the following general conditions: Initial denaturation 95ºC5minutes Denaturation 95ºC 1 minute Annealing 55ºC 1 minute Extension 72ºC 1 minute/kb of DNA template to be amplified Final extension 72ºC 10 minutes

Polymerase Chain Reaction (PCR)

Molecular techniques

Restriction enzyme digestion and analysis

desired temperaturefor 45 minutes and plated on an appropriate selective medium at various dilutions. An aliquot of cell suspension to which plasmid DNA was not added served as a negative control. B. Inoue method i. Preparation of high efficiency competent cells Competent cells for high efficiency transformation were prepared by the method of (Inoueet al., 1990)with few modifications. An overnight culture of the strain (routinely DH5α) was subculturedinto fresh sterile LB broth in 1:100 dilutions and grown at 18ºC to anA600of 0.55. The cells were harvested by centrifugation at 2500rpm for 10 minutes at4ºC. Thesecells wereresuspended in0.4 volumes of INOUE buffer andincubated in ice for 10 minutes. The cells were recovered by centrifugation at 2500rpm at 4ºC for 10 minutes and finally resuspended in 0.01 volume of the same buffer. Sterile DMSO was added to a final concentration of 7%. After incubating for 10 minutes in ice, the cells were aliquoted in 100μl volumes, snap frozen in liquid nitrogen and stored at –80ºC. ii. Transformation protocolFor transformation, the required number of vials wasthawed on ice and the transformation protocol as described for CaCl2method was employed

A. Calcium chloride(CaCl2)method For routine plasmid transformation, following method which is a modification of that described by(Cohenet al.,1972)was used. An overnight culture of recipient strain was subcultured 1:100 in fresh LB medium and grown till mid-exponential phase. The culture was chilled on ice for 20 minutes, and the steps thereafter performed at 4ºC. 10 ml of culture was centrifuged and pelletwas resuspended in 5 ml of 0.1M CaCl2. After 5 minutes of incubation on ice, the cells were again centrifuged and resuspended in 1ml of 0.1M CaCl2. The suspension was incubated onice for 45 minutes. To the 100μl aliquot of the cellsuspension plasmid DNA (20-200ng in less than 10μl volumes) was added, incubated for 30-40 minutes on ice and given a heat shock for 90 seconds at 42ºC. The cultures were rapidly chilled for 1 minute, mixed with 0.9ml of LB broth and incubated at

Transformation

Table 2.3: Oligonucleotide primers

Primers

What other colours do you have this in?' asked Sayako of the assistants, who were packing her suits, loafers, bags and wig.'Just one other colour,' said an assistant (who thought, Jesus, we'll have a drink after work tonight).She hurried to the back of the shop and quickly returned with a toffee-brown version of the sumptuous coat.8'Yes,' said Sayako. 'I take both and, of course, boots to match, size four.' She pointed to the boots worn by the red-haired manne­quin.The pile on the counter grew. Her bodyguard standing inside the shop door shifted impatiently.When the Princess and her purchases had been driven away, the manageress and her assistants screamed and yelled and hugged each other for joy.

'What other colours do you have this in?' asked Sayako of the assistants, who were packing her suits, loafers, bags and wig.'Just one other colour,' said an assistant (who thought, Jesus, we'll have a drink after work tonight).She hurried to the back of the shop and quickly returned with a toffee-brown version of the sumptuous coat.8'Yes,' said Sayako. 'I take both and, of course, boots to match, size four.' She pointed to the boots worn by the red-haired manne­quin.The pile on the counter grew. Her bodyguard standing inside the shop door shifted impatiently.When the Princess and her purchases had been driven away, the manageress and her assistants screamed and yelled and hugged each other for joy.

ZFIN_ZDB-GENO-140423-3

ZFIN_ZDB-ALT-080321-3

ZFIN: ZDB-ALT-120409-3

ZFIN_ZDB-GENO-140423-3

ZFIN ID: ZDB-ALT-070612-3

involves a complex interaction of conscious and unconscious processes.

latent learning

Remember, the best way to teach a person or animal a behavior is to use positive reinforcement

Why is shaping needed?

In his operant conditioning experiments, Skinner often used an approach called shaping

In discussing operant conditioning, we use several everyday words—positive, negative, reinforcement, and punishment—in a specialized manner. In operant conditioning, positive and negative do not mean good and bad

There are specific steps in the process of modeling that must be followed if learning is to be successful.

It was then that Claire knew she wanted to discipline her children in a different manner.

PICKERING[zait/?enthusiasm]IcamefromIndiatomeetyou.HIGGINS.IwasgoingtoIndiatomeetyou.

Type hints cheat sheet (Python 3)¶ This document is a quick cheat sheet showing how the PEP 484 type annotation notation represents various common types in Python 3.

three types of gymnastics

DC3

Wherefore, after I have abridged the record of my father, then will I make an account of mine own life.

foolish imaginations of his heart

The evidence strongly suggests that the Canadiens adopted a casual attitude toward the clergy, which could (and did) sometimes express itself as contempt

accustom the people to direct taxation

however, have said that they were in favor of direct taxation for the support of the local governments, because it would lead those who have to pay the taxes to look more closely into what was going on, and the manner in which their money was expended. (Hear, hear.) There seems also to have been a feeling in the Lower Provinces in favor of a legislative union, and the Hon. Mr. GREY seems to be combatting that idea. He says that with a legislative union, municipal institutions, and direct taxation in every province, would be the only means of getting along. He expressed himself as opposed to that and in favor of a Federal union, which he thought would afford them all the advantage that could be attained, commercially, by union, and would allow each province to retain control over its own local affairs. The local legislatures, he said, were to be deprived of no power over their own affairs that they formerly possessed. But in Canada it was represented that the local legislatures were to be only the shadow of the General Legislature—that they were to have merely a shadow of power, as all their proceedings were to be controlled by the Federal Government. That is the position taken by the advocates of the measure on this floor. So it seems that those gentlemen who have represented to us that they acted in great harmony, and came to a common decision when they were in conference, take a widely different view of the questions supposed to have been agreed upon, and give very different accounts of what were the views of parties to the conference on the various subjects. (Hear, hear.) In the Lower Provinces they were strongly opposed to direct taxation, while here it was present end as one of the advantages to accrue from the Federation. (Cries of No, no.) Well, Mr. SPEAKER, I say yes. That view of the case has been taken. If the amount allowed for the expenses of local legislation—the 80 cents per head—was found insufficient, the local parliaments must resort to direct taxation to make up the deficiency, while in tile Lower Provinces, it seems, nothing of that kind was to follow.

Another question on which the hon. member has also called us to account, relates to the export duties on timber and coals. In clause 29, which relates to the powers of the Federal Parliament, the third section reads as follows : This imposition or regulation of duties of customs on imports or exports, except on exports of timber, logs, masts, spars, deals, and sawn lumber from New Brunswick, and of coal and other minerals from Nova Scotia. The fact that this power has been conferred on the Government does not imply that it will be exercised. The power was granted simply because it might be necessary in certain cases mentioned. Now this is the reason for the second part of the clause which I have just read to the House, and which I cannot better explain than by citing some expressions of a speech by the Hon. the Minister of Finance on the subject. Nevertheless, as there are several honorable members in the House who do not understand English, I think it will perhaps be better to explain them in French. Here then was the thought of the Convention : as in New Brunswick the Government had found that it was a great disadvantage to collect the duties on timber according to the system formerly adopted, and they had substituted an export duty which superseded all other dues on that product, it was no more than right that this source of revenue should remain in New Brunswick, to which province it was an object of absolute necessity to defray its local expenses. In Canada we retain, under the new Constitution, our own method of collecting similar duties. As to New Brunswick, the duty on the article in question is their principal revenue, as coal is almost the sole revenue of Nova Scotia ; and if they had been deprived of them, they would have peremptorily refused to join the Confederation. (Hear, hear.) Their demand was perfectly just, and could not therefore be refused. Moreover, we have no right to complain, for they leave us all our mines and our lands, and we shall now, as heretofore, collect the proceeds for our own use and profit. The honorable member for Hochelaga says that it will be impossible to administer the affairs of the local legislatures without having recourse to direct taxation ; but a man of his experience ought not to have made that assertion. Instead of attempting to trade on popular prejudice, he ought to have admitted at once that the right granted by the new Constitution of levying direct taxes, is the same that already exists in the present Constitution ; it is the same right that all our municipalities possess.

reference to the meaning of the 5th sub-section of the 29th clause, which commits to the General Parliament ” the raising of money by all or any other modes or system of taxation.” Am I to understand that he General Government are to have the power of imposing local taxation upon the lands of the provinces ? HON. MR. CAMPBELL—The general national power of taxation is to be in the General Government.

Stadtinfo Köln (City Info Cologne) is a research project financed by the German Federal Ministry of Research that centres around the collection of various traffic data to be distributed to diverse platforms including the Internet, portable devices such as PDAs and mobile telephones, in-car navigation systems and variable message signs throughout the city. The project was implemented over a four-year period from 1998 to October 2002 by 15 partners in co-operation with the city of Cologne at a cost of €16.1 million.

The Cologne-based TÜV Rheinland headquarters is revitalizing its approximately 100,000 square meter business park with ten buildings in Poll. The management of TÜV Rheinland Immobiliengesellschaft mbH & Co. KG has developed an innovative concept with the engineering experts from Drees & Sommer as energy designer, building physicist and TGA planner: In future, there should only be one energy center. All buildings in the property are supplied with heat and cooling via the power grid of the new energy center. For heat supply, hybrid energy sources are used. These consist of the renewable raw material wood, a wood pellet boiler plant, as well as the fossil energy natural gas, gas condensing boilers and an integrated combined heat and power plant. The cold is generated by free-cooling, high-efficiency compression machines and absorption chillers. This can save 30 percent of primary energy compared to today. In addition, CO2 emissions will be reduced by more than 30 percent. The overall concept is modular in design and adaptable for the future.

KVB cycle hire Smart mobility   Smart mobility is climate-friendly, sustainable, space-saving and networked. It relies on diversity and multimodality. The resident of a smart city does not remain loyal to one mode of transport. The result is a mobility patchwork that is tailored to the individual circumstances and that can be configured quickly and easily at any time. Energy-efficient and space-saving mobility has priority here. "Sharing" is smart! The sharing of things and information already establishes itself under the term "sharing economy" and places the function before the property, in order to use existing resources more efficiently. Smart mobility in urban areas is therefore primarily a matter of sharing a networked mobility offer from buses, trains, bicycles and cars. Smart mobility is not just a technological task. Especially in the inner cities, walking and cycling will provide space for quality of life and urban development through active mobility. This is where the bicycle rental system of the Cologne Transport Company (KVB) comes in by closing a gap in the combination of environmentally conscious and mobility-active mobility. The bicycle rental system of KVB stands for an open architecture. It is therefore not a system with only fixed station terminals after the well-known role models from other major cities, because a template for all cases, the complex events of a city can consider insufficient. The system offers users fully flexible rental and return in the street, but also stationary station terminals depending on the available options and needs. The rental terminals cover the entire span between conventional stations and purely virtual stations.  

n times of energy transition and scarce resources, the architectural concept of Concrete Apartments Cologne is based on the requirements of the future - it is designed as an energy-saving passive house. This contains • a 26 cm thick external insulation made of rock wool, • triple glazed windows, • optimum recovery of radiated heat from residents and household appliances, • a ventilation system with a constant base temperature of 20 ° C - summer and winter - as well as • a digital control system that directs the use of luminaires and large consumers. Only those who like it even warmer must turn on the heating controller. All rooms are equipped with presence detectors, which automatically switch off lamps, for example, when not in use - this also saves energy. Of course, residents can also make the scheme manually. The energy and heat for the Boarding House creates its own, energy-efficient combined heat and power plant. State-of-the-art technology is also used here: surplus electricity is optionally fed into the public grid or used for the charging station for electric vehicles in the courtyard.

The diesel exhaust gases of the Rhine ships pollute the Cologne air with pollutants and fine dust and the climate with a significant amount of CO 2 . A part of it does not arise during the journey, but while the ships are at anchor. Because their generators must also run to generate the necessary electricity. Here, "Landstrom" provides a remedy: Since 2015, RheinEnergie has gradually been equipping a large part of the moorings along the Rhine with uniform power connections. Consequence: During the lay times the ship diesels can be turned off.

As well as energy-saving lighting, Smart Home is an important building block for an energy-efficient and comfortable future. With smart homes and smart meters in the network, homeowners and store owners can reduce their electricity and heating costs by an average of 7%! Add to that the great comfort of making the apartment burglar-proof and controlling almost every aspect of heating, electricity or security in the building. So you can control from your smartphone whether the stove is still on at home, a window has been left open, the heating is running at full speed or the light is on. In addition, before the house is on fire, modern, networked smoke detectors report any alarm directly to the owner's smartphone. It can automatically be initiated various steps, such. B. that the fire department is called. In order to test some scenarios and saving opportunities in everyday life and to make known the possibilities offered by these modern technologies, Smart Home applications were installed on the Klimastraße in nine private apartments of the Nippes Tower and in the bookstore Neusser Straße. This was financed by the project Klimastraße or the company RocketHome . In addition, it is planned to equip the entire climate road with smart meters from RheinEnergie.

Neusser Straße in the district of Nippes shows what a future SmartCity could look like, because a section of the street becomes Cologne's climatic road. There, the most important energy projects are implemented. All facets of climate protection are taken into account: from optimal building insulation and maximum heat efficiency to charging stations for electric vehicles and low-energy street lighting. Klimastraße offers innovative companies the opportunity to test their new products and services in everyday life. If possible, companies finance their projects themselves, promising projects are funded from the project budget of RheinEnergie AG. Companies also gain additional value by exchanging valuable information and innovative ideas with other companies, including at climate road events. For all the enthusiasm for innovation, of course, only technology is used that meets the very strict German safety requirements. In addition, RheinEnergie and the City of Cologne make sure that the high Cologne supply standards are adhered to. For all new projects, safety comes first - technically as well as logistically. That is why not everything changes in the climate route - but certainly much better. The following section deals in more detail with the individual projects.

The energy transition presents network operators and energy providers with particular challenges. Both have to deal with an increasing share of electricity from renewable sources in the electricity grid. Wind turbines and photovoltaic systems produce electricity, however, depending on the weather and therefore fluctuating. For a secure supply, it is necessary that electricity production and consumption are always balanced as much as possible. In order for this to succeed, utilities and network operators must always know where and in what quantity energy is generated and consumed. Only then can production and consumption be optimally coordinated. However, continuous metering is not possible with today's metering technology.The solution to the problem is smart metering. In the future, so-called "smart metering systems" will transfer consumption data to grid operators and energy providers. This ensures that they can optimally control the network at any time. The technology is mainly used in households and businesses with high annual consumption.Consumers can access the data at any time. The additional transparency helps them to further increase their energy efficiency and thereby reduce costs. New services provided by energy suppliers are intended to reinforce the positive effects.

In the framework of the project "Celsius" we investigate which method leads to the best possible results in order to increase the chances of realization. For this purpose, demonstration plants were built at three different locations in the city. In Cologne-Wahn and Cologne-Mülheim, the heat is extracted directly from the sewer using so-called gutter heat exchangers. The heat exchangers with a length of 60 and 120 meters are installed at the bottom of the canal. The heat transfer medium transports the heat from there to the heat pumps with a capacity of 150 or 200 kW in the boiler rooms of the schools supplied. In Cologne-Nippes, a total of three schools and a sports hall are supplied by sewage heat. Here, the wastewater is pumped through a newly laid, 400-meter-long bypass to the boiler room of the Edith Stein-.Realschule. There, in the largest direct evaporator in Germany (400 kW), heat is transferred directly to the heating circuit of the schools. With the three demonstration plants, an environmental relief of a total of 500 t CO2 / year is achieved. The use of wastewater heat is technically mature and well developed. Nevertheless, this form of waste heat utilization has so far been a niche existence. This is partly because it is still little known, often the necessary information is not available locally, their implementation is relatively complex and requires high investment. Further reducing these barriers is the goal of the Cologne CELSIUS project.  

District heating is one of the key pillars of our sustainable energy action plan. This plan has been decided by the local parliament in 2008 and renewed in 2015. Our first priority is to cut in half the total energy demand of the city until 2050 and then cover the rest with renewable energy and/or waste heat. To use large amounts of waste heat (e.g. from a waste incineration plant, industry, datacentres …) you need a distribution system, because it is not useable only locally. This is why we want to increase the share of district heating in the city. For the future we see a district heating system which will be “open source technology” – everyone can use the heat and also be a prosumer, delivering surplus energy, e.g. from a solar – thermal plant, to the system. There will not be any longer central DH-Stations but smaller plants and the use of all waste heat sources we can get.

The Federal Government will have the right of imposing taxes on the provinces without the concurrence of the local governments. Under article five of the 29th resolution, the Federal Government may raise moneys by all modes or systems of taxation, and I look upon this power as most excessive. Thus, in case it should happen, as I said a moment ago, that the Lower Canada Government refused to undertake the payment of the debt contracted for the redemption of the Seigniorial Tenure, the Federal Government would have two methods of compelling it to do so. First, by retaining the amount out of the eighty cents per head indemnity to be accorded to the Local Government, and secondly, by imposing a local and direct tax. The Lieutenant Governor of the Local Government will be appointed by the Federal Government, and will be guided by its instructions. We are not told whether the Local Government will be responsible to the Local Legislature; whether there will be only one or two branches of the Legislature, nor how the Legislative Council will be composed, if there is to be one ; we are refused any information whatsoever on these points, which are nevertheless of some importance.

smart­PORT lo­gis­tics Thanks to in­tel­li­gen­t so­lu­tions for the flow of traf­fic and goods, the HPA is im­prov­ing the port's ef­fici­ency. smart­PORT lo­gis­tics com­bines eco­no­mic and eco­lo­gical as­pec­ts in three sub-sec­tors: traf­fic flows, in­fra­structure and the flow of goods. An in­ter­mo­da­l Port­Traf­fic cen­tre for sea, rail and road trans­port forms the ba­sis for net­work­ing the flow of traf­fic. In­tel­li­gen­t net­work­ing is a pre­req­ui­site for smooth, ef­fici­en­t trans­port in the port of Ham­bur­g and ul­ti­mately for the flow of goods: op­ti­mum da­ta cap­ture and rapid in­for­ma­ti­on shar­ing al­low lo­gis­tics man­agers, car­ri­ers and agen­ts to se­lect the most efficien­t means of trans­port for their goods.

The Green-Space Information System (GRIS) is an EDP procedure of the borough departments of green spaces and the Senate Department for Environment, Transport and Climate Protection, Commission III C, Open-Space Planning and Urban Green Spaces.

Further, in Lower Canada, each locality is told that it may rest satisfied it will not be overlooked, for each is to be represented in the Legislative Council by a gentleman residing or holding property in it

Crime increasing in the past 3 years47.75

3 Steps for Licensing Your 3d Printed Stuff by Michael Weinberg. CC BY-SA 3.0 A set of instructions for how to license 3d printed materials https://www.publicknowledge.org/assets/uploads/documents/3_Steps_for_Licensing_Your_3D_Printed_Stuff.pdf

Teach Source EvaluationSkillsIf you want to teach source evaluation skills, have small groups conduct research to answer a three-part problem such as this:1.How high is Mt. Fuji in feet?2.Find a different answer to this same question.3.Which answer do you trust and why do you trust it?

5

“I support a social transition for a kid who is in distress and needs to live in a different way. And I do so because I am very focused on what the child needs at that time,” said Johanna Olson-Kennedy, medical director of the Center for Transyouth Health and Development at Children’s Hospital Los Angeles, the largest transgender youth clinic in the United States with some 750 patients. A social transition to the other gender helps children learn, make friends, and participate in family activities. Some will decide later they are not transgender, but Olson-Kennedy says the potential harm in such cases may be overstated.

I finally want to express how much easier both my waking and my sleeping hours would be if there were one book in existence that would tell me something specific about my life. One book based in Black feminist and Black lesbian experience, fiction or non-fiction. Just one work to reflect the reality that I and the Black women whom I love are trying to create. When such a book exists then each of us will not only know better how to live, but how to dream.

they found in each other's eyes the intimacy they were looking for.

Nel imagines a "fiery prince" who never quite arrives while Sula gallops like a prince "on a gray

Thou wilt see (him) and [like a woman(?)] thou wilt rejoice.

the Central Government would have the power of raising money by all the other modes and systems of taxation—the power of taxation had been confided to the General Legislature—and there was only one method left to the Local Governments, if their own resources became exhausted, and this was direct taxation.

It was adopted by the Imperial Legislature with the view of remedying difficulties which then existed between the two Provinces. The inherent defect in the Imperial Act for the Union of the two Canadas was this : it attempted to combine the federal principle with unity of action. It endeavoured to give equal representation to the two sections of the Province, while it brought them together for the purpose of dealing as one with all subjects both (general and local,

Those claims were in themselves undoubtedly founded in justice— but at the same time there was great reason in the objections taken to them—they involved an interference with the Federal principle recognized in the Union Act, an interference which amounted to an entire change in the principles on which the Government of the country was to be administered, and could not be received otherwise than with dread by a large class, if not by the whole of the population of Lower Canada.

should have been a concession to Upper Canada, of additional members in proportion to its population, but that concession would, as be had already remarked, have been an invasion of the Federal principle, contained in the Union Act, and would unquestionably have been represented to the uttermost by a large proportion if not by the whole of Lower Canada.

. Should African American Vernacular English (AAVE) be ac- ceptable to use in the classroom? Is AAVE a dialect or a language? Can AAVE be recognized as a second lan- guage that is accepted and validated within my classroom communi

She asked if she might visit the classroom and talk to them the next day. Little did I know at the time how difficult it was for Aaron's mother to cross the threshold of the school. In future conversations, she revealed many of her own negative school experiences (including bully- ing and verbal abuse by both her peers and teachers) and how ner- vous it made her to step into a school building agai

$62,932.59

maginativeintervention

President Barack Obama, in his 2013 State of the Union address

3-D print your way to freedom and prosperity

utomatic interaction analysi

Social-media companies must take responsibility for preventing the spread of obvious falsehoods, while allowing users to argue for different interpretations of the truth.

Research shows that users typically use very few search terms when seeking information in a search engine and rarely use Advanced Search queries, as most queries are different from traditional offline information seeking behavior.7

Online search can oversimplify complex phenomena. The results, ranked by algorithms treated as trade secrets by Google, are divorced from context and lack guidance on their veracity or reliability.

One factor driving Mr. Zuckerberg may be the brisk ad business that Facebook does from its Hong Kong offices, where the company helps Chinese companies — and the government’s own propaganda organs — spread their messages.

mil hijos de padres sabios,

Joshua:                   The first thing I would say is, with respect to stuff, let it go. It’s just stuff and it has only the meaning that you give it. Things don’t have meaning unless we give a meaning. Second thing I would say is, it has to do with the people in our lives. You can’t change the people around you, but you can change the people around you. What I mean by that is, you can’t try to change someone. Make them, mold them into what you want them to be, but you can surround yourself by supportive people with similar interests and values. They may even have different beliefs. I look at me and Ryan, we have radically different personalities, we have different belief systems, different political leanings, but we have similar values. I surround myself with people who make me a better version of myself. The last thing I would say is try to love people and use things because the opposite never works.

I published a study on football players, NFL players who had been hit in the head thousands of times. What we did for them is put them on a multiple vitamin, high dose omega-3 fatty acids 5.6 grams, and blend ... I like higher EPA than DHA. A supplement I created calledBrain and Memory Power Boost that works in seven different ways, and 80% of our players showed improvement including, not just self-report, they showed improvement in blood flow to their prefrontal cortex zones back. That's not a placebo thing. A placebo thing is not actually going to improve blood flow to the focus, or thought judgment, impulse control part of your brain.

He's taking one of the big box stores fish oil. His omega-3 fatty acid probably was 5%, when it should be 8% or above. I had him stop and give him ours, Omega-3 Power, two capsules a day and his level three months later was 10; so quality matters. Plus, if you read a lot of label. Rancidity matters. You want to make sure the expiration date is there, and you want to make sure you have a high quality brand. I know, I really care about quality like you really care about quality. Then, you have to actually read the level on how much is it. Two of our capsules have 146 mg of omega-3 fatty acids where two capsules, one of the big box stores may have 120 mg. I recommend kids get at least 1,000 mg, adults 1,400 or often more. If you're on a repair situation, it's more like five grams.

These changes can help improve survey readability, flow, length, and overall response rates.

Would you spend time explaining algebra to a group of engineers?

A substantial minority of women reported only1 lifetime sexual partner in both ACASI and DHSdata, but no participants in the qualitative

User Package The Packages/User/ folder is the User package. It is unique in that it is loaded last by Sublime Text. This allows users to place changes to .sublime-settings and .sublime-keymap files in this folder. Sublime Text loads these files by name. Thus if a package has a file named Package Control.sublime-settings in the package, a file with the same name in the User package will override any of the settings in the original file. The same is true for key bindings.