Cryopreservation and revival of cell lines

Phenol solution saturated with 0.1 M citrate buffer (pH 4.3 ± 0.2)was procured from Sigma (P4682)

Buffer C100 mM Tris-HCl (pH 7.5)10 mM EDTA10% SDSPhenol:Chloroform:Isoamyl alcohol (25:24:1) solution25 ml Tris-equilibrated phenol (pH 8.0)24 ml Chloroform1 ml Isoamyl alcoholDNA sample loading buffer0.25% Bromophenol blue0.25% Xylene cyanol15% FicollStock solution of the loading buffer was prepared in water as a 6 X concentrate and was added to the sample DNA to the final concentration of 1 X.RNA isolation bufferAE buffer3 M sodium acetate0.5 M EDTA (pH 8.0)Reagents used for RNA isolation were prepared in DEPC-treatedwater and stored at 4°C. For preparationof DEPC-treated water,0.1 ml DEPC was added to 100 ml waterand kept overnight onamagnetic stirrer. Followingincubation,the solution was autoclaved to remove any traces of DEPC.Acid phenol solution

Genomic DNA isolation buffersBuffer A50 mM Tris-HCl10 mM EDTA150 mM NaCl1% Triton-X1% SDSBuffer B50 mM Tris-HCl (pH 7.5)10 mM EDTA1.1 M Sorbitol50 mM β-mercaptoethanol (Added freshbefore use)

Buffers used for nucleicacid extraction

Casamino acid (CAA)0.67% Yeast Nitrogen Base2% Dextrose0.6% Casamino acid

Yeast Extract-Peptone-Dextrose (YPD)1% Yeast Extract2% Peptone2% DextroseYeast Nitrogen Base (YNB)0.67% Yeast Nitrogen Base2% DextroseFor alternate carbon source utilization experiments, dextrose was replacedwith other carbon sources viz.,ethanol, glycerol, oleic acid and sodium acetate.Ethanol, oleic acid and sodium acetate were used at afinal concentration of 2%and glycerol was used at a final concentration of 3%

Yeast media

Oligonucleotides used for generation of C. glabratadeletion strains, for cloning and for quantitative Real time Polymerase Chain Reaction (qPCR)were commercially synthesized either at MWG Biotech Pvt. Limited, Bangalore, India or at Xcelris genomics Pvt. Limited, Ahemdabad, India. All the oligonucleotides used were designed by using freely available online tool Primer 3 plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/) and are listed in Table 2.3

Oligonucleotides

the selection marker.Knockdown was confirmed by immunoblot analysis with an IP6K1 specific antibody (Table 2.3) as described in Section 2.2.10

lines were used for stable knockdown of IP6K1 expression. Viral particles harboring either non-targeting control or IP6K1directed shRNA were used to infect HeLa or HCT116 cell lines at 0.5 MOI, following treatment with polybrene (8 μg/mL)for 2 h.After 48 h, transduced cells were selected with 2 μg/mL puromycin. Medium was changed twice a week and observed for colony formation. After reaching the optimum growth, selected cells were maintained in DMEM supplemented with 10% FBS and 1 μg/mL puromycin as

Generation of stable cell lines expressing shIP6K1-HeLa and HCT116 cell

Cells were plated at a density of 30,000 cells per well in triplicates in a 24-well plate. After 24 h, cells at a confluency of 30-40%, were treated with different genotoxic agents such as hydroxyurea (0.5 mM), neocarzinostatin (0.25 μg/mL) and mitomycin-C (1 μg/mL) for 12 h. Spent media containing drug was removed and cells were washed gently twice with PBS. Cells were then incubated for different lengths of time ranging from 24 h to 120 h, in fresh complete DMEMto allow them to recover from genotoxic stress. At each time point cell survival was analyzed by MTT assay as described in 2.2.1. Cell survival was expressed as a fold increase in cell population relative to cells treated with drug for 12 h

DNA damage and recovery assay

Celllines

The cell lines used in the study are mouse embryonic fibroblasts (MEFs) derived from wild type (WT) and Ip6k1knockout mouse embryos. The MEFs were immortalized with SV40 large T antigen (Bhandariet al., 2008)and single cell derived lines were generated in the lab. Ip6k1knockout MEFs display 70% lower levels of IP7compared with wild type MEFs (Bhandariet al., 2008). Ip6k1-/-MEFs expressing kinase active or inactive forms of IP6K1 were generated in the lab (Rescue MEFs). MEFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 1 mM L-Glutamine (Life Technologies), 100U/mL penicillin, and 100 μg/mL streptomycin (Life Technologies).Rescue MEFs were cultured in complete DMEM supplemented with G418 (200 μg/mL) as selection marker. HCT116 (colon cancer cells, a gift from Dr. Sagar Sengupta, NII, New Delhi) or HeLa (cervical cancer cells) expressing non-targeting control and shRNA against human IP6K1were cultured in complete DMEM containing puromycin (2μg/mL). The amphotropic Phoenix cells (a gift from Dr. Shweta Tyagi, CDFD, Hyderabad) and HEK293T packaging cells were usedfor generating lentiviral particles containing shRNA against human IP6K1or mouse Ip6k2and were maintained in complete DMEM

The slides for microscopy were prepared as described in Dajkovic et al.,(2008)with slight modifications. After wiping the glass slide with ethanol, 200μL of 1% molten agarose was layered on it between two strips of tape and clean cover-slip placed on it to obtain levelled surface. The agarose was allowed to solidify and the cover-slip was carefully removed and5μlof sample was put on top of the agarose and carefully covered with a cover-slip

Preparation of microscopic slides

Semi-dry transfer apparatus (Bio-Rad trans blot semi dry transfer cell)was used for the transfer of RNA from the gel to the membrane. The Hybond-N+ membrane from Amersham biosciences was used which was cut as per dimensions of the gel containing the RNA samples. For each transfer 6 pieces of Whatman3mmsheets of the size of the membrane were used. The membrane was soaked for 30-60 minutes in 0.5XTBE before transfer. The transferapparatus was set up as describedby the manufacturer. Transfer was done in 0.5XTBE buffer at 20V, 400mA and 100W for 1.15 hours

Transfer of RNA to the membrane

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

Recombineering was performed as described in(Yuet al., 2000)for engineering the linear DNA on the chromosome. The oligonucleotide primers were designed to amplify the DNA cassette to be engineered. Oligonucleotidesused for recombination contained30–50nt homology at the 5ʹ endtothesequences at the target siteand 20nt homology tothe DNA cassette at the 3ʹ end. The DNA cassettefor recombinationwas generated by PCR and would contain30-50 bp homologiesto the target site. A strain with the target DNA and carrying a defective λ-prophage with gam,betaand exo genes (thatfacilitate homologous recombination)under the control of a temperature-sensitive λ cI-repressorwas grown at 30oC. At an A600of 0.4, the culture was shifted to 42oC for 15 minutes to express gam,betaand exo genes. Cells becomecapable ofrecombining linear DNA introduced into the cell by electroporation. 50-100ng ofamplified DNA cassettewas used for electroporation whichwas performed using theBio-Rad Gene Pulser set at 1.8 kV, 25 μF with Pulse controller of 200 ohms

Recombineering

Typically 400-500ng of DNA was used in each ligation reaction. The ratio of vectorto insert was maintained between 1:3 and 1:5 for cohesive end ligation. The reaction was generally performed in 15μl volume containing ligation buffer (provided by the manufacturer) and 0.075 Weiss unit of T4 DNA ligase at 16ºC overnight (14-16 hours)

Ligation of DNA

DNA fragments to be used for specific purposes like ligation or radioactive labellingwere eluted from the agarose gel after electrophoresis. The gel piece containing the desired band was sliced out from the gel and the DNA was purified using commercially available purification kit (Qiagen)for this purpose. The efficiency of elution was determined by checking a small aliquot of DNA sample on the gel

Purification of DNA by gel elution

Around 0.5-1μg DNA was regularly used for each restriction digestion. 2 to 5 units of restriction enzyme were used in the total reaction volume of 20μl containing 2μl of the corresponding buffer supplied at 10X concentration by the manufacturer. The reaction was incubated for 3hours at the temperaturerecommended by the manufacturer. The DNA fragments were visualized after electrophoresis on 0.8 to 1.5% agarose gels. Commercially available DNA size markers were run along with the digestion samples to compare with and to estimate the sizes of the restriction fragments

Restriction enzyme digestion and analysis

The DNA samples were mixed with appropriate volumes of 6X loading dye (0.25%bromophenol blue and 0.25% xylene cyanol and 30% glycerol in water) and subjected to electrophoresis through 0.8 to 1.5 % agarose gel in TAE buffer. The Goodview nucleic acid stain(supplied as 20000X; Beijing SBS Genetech Co. Ltd.) was added to the gel at the time of casting or 6X EZ-Vision One DNA dye(Amresco) was used as loading buffer, both being commercially available non-carcinogenic dyes to aid visualization of bands. The visualization was doneby fluorescence under UV light in a UV transilluminator

Agarose gel electrophoresis

following the manufacturer’s instructions. For genomic DNA, 1ml culture was used for DNA isolationusing Qiagen or Invitrogen kits. The quality of plasmid/genomic DNApreparations was assessed following electrophoresis on 0.8% agarose gels

3ml (for high copy number)or 10 ml (for low-copy number) of cells from an overnight culture were pelleted by centrifuging for 5 minutes at 6000rpm forthe plasmid isolation which was carried out with the commercially available kits (Qiagen or Invitrogen)

Isolation of plasmid and chromosomal DNA

Recombinant DNA techniques

To 2 ml of fresh overnight culture of recipient strain, 108 pfu equivalent of phage lysate was added and incubated at 37ºC without shaking for 30 minutes to facilitate phage adsorption. The unadsorbed phage particles were removed by centrifugation at 6000 rpm for 5 minutes and the pellet ofbacterial cells was resuspended in 5 ml of LB broth containing 20 mM sodium citrate to prevent further phage adsorption. This was incubated for 25-60 minutes at desired temperaturewithout shaking to allow the phenotypic expression of the antibiotic resistance gene. The mixture was then centrifuged and the pellet was resuspended in 300 μl of 0.1M citrate buffer. 100 μl aliquots were spreadon appropriate antibiotic containing plates supplemented with 2.5 mM sodium citrate. A control tube without addition of P1 lysate was also processed in the same way. In the case of selection of nutritional requirement, the infection mixture was centrifuged, resuspended in 300 μl of 0.1M citrate buffer and plated without phenotypic expression

Phage P1 transduction

Table 2.2 List of plasmids used in this study

Plasmids

She then handed over a platinum card which bore the name of her father, the Emperor of Japan.

As the manageress tapped in the magic numbers from the card,7 Sayako tried on a soft green-coloured suede coat which was also be­ing worn by a red-haired mannequin. The suede coat cost one penny less than a thousand pounds.

She then handed over a platinum card which bore the name of her father, the Emperor of Japan.As the manageress tapped in the magic numbers from the card,7 Sayako tried on a soft green-coloured suede coat which was also be­ing worn by a red-haired mannequin. The suede coat cost one penny less than a thousand pounds.'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.

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reviewed negatively

This sectional combination for the submersion of the Constitution, has been aided in some of the States by elevating to citizenship, persons who, by the supreme law of the land, are incapable of becoming citizens; and their votes have been used to inaugurate a new policy, hostile to the South, and destructive of its beliefs and safety.

The States of Maine, New Hampshire, Vermont, Massachusetts, Connecticut, Rhode Island, New York, Pennsylvania, Illinois, Indiana, Michigan, Wisconsin and Iowa, have enacted laws which either nullify the Acts of Congress or render useless any attempt to execute them. In many of these States the fugitive is discharged from service or labor claimed, and in none of them has the State Government complied with the stipulation made in the Constitution.

But an increasing hostility on the part of the non-slaveholding States to the institution of slavery, has led to a disregard of their obligations, and the laws of the General Government have ceased to effect the objects of the Constitution.

We assert that fourteen of the States have deliberately refused, for years past, to fulfill their constitutional obligations, and we refer to their own Statutes for the proof.

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Pharmacologic Therapy for Type 2 Diabetes

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It appears therefore that the only alternative which now offers itself to the inhabitants of Lower Canada is a choice between dissolution pure and simple, or Confederation on one side, and representation by population on the other. And however opposed Lower Canada may be to representation by population, is there not imminent danger that it may be finally imposed upon it, if it resist all measures of reform, the object of which is to leave to the local authorities of each section the control of its own interests and institutions. We should not forget that the same authority which imposed on us the Act of Union, or which altered it without our consent, by repealing the clause which required the concurrence of two thirds of the members of both Houses in order to change the representation respecting the two sections, may again intervene to impose upon us this new change.

Her Majesty’s Government cannot but express the earnest hope, that the arrangements which may be adopted in this respect may not be of such a nature as to increase—at least in any considerable degree—the whole expenditure, or to make any material addition to the taxation, and thereby retard the internal industry, or tend to impose new burdens on the commerce of the country.

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.

Let us then have direct taxation, and what will be the result ? If there is a large expenditure on the part of the General Government, in addition to this taxation, political agitators will arise, who will cry out that the public burdens are unequally borne —(hear)—that two-thirds of the revenue is borne by the people living west of Quebec— that is, the population west of this city will, man for man, pay twice as much to the public exchequer as the population east of it.

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.

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Smart Cities: Stockholm Royal Seaport Project Update

Urban Green Space

Last week Stefka Wiese from GreenGasDrive was with us. The startup wants to make GreenCNG available nationwide for public and private fleet customers in the CNG filling station network. It should be made climate neutral, especially from waste biogas. In addition, GreenGasDrive advises on upcoming investment decisions and utilization optimization

A fully integrated logistics concept to establish environment and climate friendly supply chains in urban districts is going to evolve from the project. Co-creation processes, which are supported by online tools, are being used to develop the concept. The development and testing of sustainable business and deployment models is the goal of Distribut(e) and this will be addressed through the development of a shared e-logistics system in Klausenerplatz and at Mierendorff-Insel. The system will relate to a specific district (Kiez) in Berlin. A digital dialogue platform will be set up for the purpose of ordering local goods and for the booking of cargo bikes. Other low-threshold service offerings will be developed using Urban Design Thinking and will be integrated into the operating model.Focus Areas Integrated shared e-logistics at Mierendorff-Insel and at KlausenerplatzUrban Design Thinking: Formats for the co-creation process for sustainable urban district logisticsEfficiency enhancement in the delivery of goods in the ‘last mile’Mobile and online participation: Promotion of local production chains and activation of local stakeholders such as small to medium sized companies and local residentsIdentification of new sharing and value-in-use conceptsHigher standards in terms of the transparency, sovereignty, security and efficiency of data on the online platform

system of taxation, my honorable friends opposite would have had a much better chance of success in blowing the bellows of agitation than they now have. (Laughter, and cheers.) The objection, moreover, was not confined to Lower Canada—all the Lower Provinces stood in exactly the same position. They have not a municipal system such as we have, discharging many of the functions of government; but their General Government performs all the duties which in Upper Canada devolve upon our municipal councils, as well as upon Parliament. If then the Lower Provinces had been asked to maintain their customs duties for federal purposes, and to impose on themselves by the same act direct taxation for all their local purposes, the chances of carrying the scheme of union would have been greatly lessened. (Hear, hear.) But I apprehend that if we did not succeed in putting this matter on the footing that would have been the best, at least we did the next best thing. Two courses were open to us—either to surrender to the local governments some source of indirect revenue, some tax which the General Government proposed to retain,—or collect the money by the federal machinery, and distribute it to the local governments for local purposes. And we decided in favor of the latter. We asked the representatives of the different, governments to estimate how much they would require after the inauguration of the federal system to carry on their local machinery.

I trust I commit no breach of discretion in stating that in Conference I was one of the strongest advocates for defraying the whole of the local expenditures of the local governments by means of direct taxation, and that there were liberal men in all sections of the provinces who would gladly have had it so arranged. But, Mr. SPEAKER, there was one difficulty in the way—a difficulty which has often before been encountered in this world—and that difficulty was simply this, it could not be done. (Hear, and laughter.) We could neither have carried it in Conference nor yet in any one of the existing provincial legislatures. Our friends in Lower Canada, I am afraid, have a constitutional disinclination to direct taxation, and it was obvious that if the Confederation scheme had had attached to it a provision for the imposition of such a

the second feature of this scheme as a remedial measure is, that it removes, to a large extent, the injustice of which Upper Canada has complained in financial matters. We in Upper Canada have complained that though we paid into the public treasury more than three fourths of the whole revenue, we had less control over the system of taxation and the expenditure of the public moneys than the people of Lower Canada.

But, sir, I am told that though true it is that local matters are to be separated and the burden of local expenditure placed upon local shoulders, we have made an exception from that principle in providing that a subsidy of eighty cents per head shall be taken from the federal chest and granted to the local governments for local purposes.

constitution of the Legislative Council, and insisted not only that it should have remained elective, but that the principle of representation according to population should also have prevailed. But who ever heard that in a Federal Constitution the Upper House should be arranged on that principle? If that view be the sound one, the better way would be to have but one House, for the only effect of having two Houses, both elected on the basis of population, would be that one would constantly be combating the other, and the wheels of government would unavoidably be brought to a stand-still. In such a case the more powerful members of the Confederacy would be wholly unrestrained, and would completely overwhelm the weaker. This was fully considered on the adoption of a Constitution for the United States, according to which it is well known that the smaller States are represented in the Senate by the same number of senators as the larger ones—there being two members for each. The same principle has been adopted in arranging the terms of this proposed union, and for the same reason; viz., to protect the weaker parties to the compact.

It soon became apparent that, while they protected the sovereignty of the states, the Articles had created a central government too weak to function effectively.

The central government had authority to act in only a few areas, such as national defense, in which the states were assumed to have a common interest (and would, indeed, have to supply militias). This arrangement was meant to prevent the national government from becoming too powerful or abusing the rights of individual citizens. In the careful balance between power for the national government and liberty for the states, the Articles of Confederation favored the states.

Fearful of replacing one oppressive national government with another, however, the framers of the Articles of Confederation created an alliance of sovereign states held together by a weak central government.

Laws were made by Parliament, and property-owning males were allowed to vote for representatives to Parliament. Thus, Americans were accustomed to the idea of representative government from the beginning.

This document would profoundly influence the U.S. Constitution and Bill of Rights.

4

The Standards set grade-specific standards but do not define the intervention methods or materials necessary to support students who are well below or well above grade-level expectations.

they do not describe all that can or shouldbe taught

6 | iNTrOduCTiONCommon Core State StandardS for enGLISH LanGUaGeartS & LIteraCy In HIStory/SoCIaL StUdIeS, SCIenCe, and teCHnICaL SUbjeCtS1.The Standards define what all students are expected to know and be able to do, not how teachers should teach.

The Common Core State Standards for English Language Arts & Literacy in History/Social Studies, Science, and Technical Subjects (“the Standards”) are the culmination of an extended, broad-based effort to fulfill the charge issued by the states to create the next generation of K–12 standards in order to help ensure that all students are college and career ready in literacy no later than the

students must be able to understand and describe the text before they are able to analyze its elements.

Important hints that an author drops to prepare the reader for what is to come, and help the reader anticipate the outcome.

If you need more information contact the Documentation Centre on the Bombing of Gernika dokumentaziozentrua@gernika-lumo.net.

Acknowledgement of German involvement in the bombing of Gernika did not come until 1997, when President Herzog sent a letter to the survivors in which he admitted that Germany had been involved in the attack in 1937.

Today Gernika is an advanced example of reconciliation; the process has not yet been completed, but the journey embarked upon is a long one. Initially it proved necessary to cover serious deficits, since Germany delayed the first steps to repair the damage caused.

The atmosphere in Gernika before the war was tense for a long time. Franco introduced tough repression of both ideology and culture. There were new regulations and codes of conduct; people were reported to the authorities and searches were carried out on a daily basis, and nothing escaped scrupulous examination by the Civil Guard. The people of Gernika were forced to confront fear, distrust and mutual suspicion. Nobody gave them back the happy, open town they had once known.

Reconstruction took five long years and, paradoxically, the person who was ultimately responsible for the destruction of Gernika, Francisco Franco, was made an adopted son of the town.

"The statement released by Salamanca according to which Guernica was destroyed by the reds is completely untrue.

Franco's army never acknowledged responsibility - on the contrary, evidence was twisted, and his press service accused the Basque republicans (referred to as reds and separatists) of having set fire to the town during their retreat towards Bilbao.

The news was published in the main European organs thanks to the rapid action taken by a number of journalists, of which the best-known is George Steer.

"Before God and History, which will eventually judge us all, I hereby state that for three and a half hours German planes bombed the defenceless civilian population of Gernika with unprecedented viciousness. They reduced the town to ashes and machine-gunned women and children, many of whom were killed, while the rest fled in terror." Jose Antonio Aguirre. President of the Basque Government. "Aguirre is lying. We respected Gernika in the same way as we respect everything Spanish." Francisco Franco.

direct taxation, all the means whereby the industry of the people may be made to contribute to the wants of the state, it must be evident to every one that some portion of the resources thus placed at the disposal of the General Government must in some form or other be available to supply the hiatus that would otherwise take place between the sources of local revenue and the demands of local expenditure. The members of the Conference considered this question with the most earnest desire to reduce to the lowest possible limits the sum that was thus required, and I think the figures that I have already given to the House afford the best possible evidence that no disposition existed, at any rate on the part of our friends from the Lower Provinces

If, nevertheless, the local revenues become inadequate, it will be necessary for the local governments to resort to direct taxation ; and I do not hesitate to say that one of the wisest provisions in the proposed Constitution, and that which affords the surest guarantee that the people will take a healthy interest in their own affairs and see that no extravagance is committed by those placed in power over them, is to be found in the fact that those who are called upon to administer public affairs will feel, when they resort to direct taxation, that a solemn responsibility rests upon them, and that that responsibility will be exacted by the people in the most peremptory manner. (Hear, hear.) If the men in power find that they are required, by means of direct taxation, to procure the funds necessary to administer the local affairs, for which abundant provision is made in the scheme, they will pause before they enter upon any career of extravagance.

But this precedent could not be urged as an objection to Federation, inasmuch as it would be for the General Government to deal with our commercial matters. There could be no reason for well-grounded fear that the minority could be made to suffer by means of any laws affecting the rights of property.

It was desirable the General Government should have the control of the medium through which the trade and commerce of the country was carried on, and that in the establishment of banks, the issue of paper money and in offering to the public the paper representative of their labor, in whatever part of the country, there should be the same legislative security for the people

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 would have the regulation of all the trade and commerce of the country, for besides that these were subjects in reference to which no local interest could exisit, it was desirable that they should be dealt with throughout the Confederation on the same principles.

$61,099.60

Que, hermanas

n roble, sin alma 120 de discurso y

Que,

sin y

vocês se tornarão merecedores do seu Reino

This likely means that Facebook will become embroiled in messy political debates.

There are many myths about Internet search engines that proliferate, including the notion that what rises to the top of the information pile is strictly what is most popular as indicated by hyperlinking. Indeed, what is most popular on the web is not necessarily what is most trustworthy or truthful.

FBI statistics show that most violence against white Americans is committed by other white Americans, and that most violence against African-Americans is committed by other African-Americans.

At a White House dinner in 2015, Mr. Zuckerberg had even asked the Chinese president, Xi Jinping, whether Mr. Xi might offer a Chinese name for his soon-to-be-born first child — usually a privilege reserved for older relatives, or sometimes a fortune te

en retirar la vista y el oído,

Se trata de palabras o frases tachadas y sustituidas por otras o reorganizadas dentro de la estrofa. Es conocida la tendencia de Lope a la intervención in itinere[31]:

machine learning

open access

The graph below shows attitudes of homeowners towards owning their home. In this case, since the statistic of interest is what percentage of homeowners agree with each statement, it is probably best to keep the graph as is, with the total exceeding 100%.

As you decide how much data to illustrate, keep in mind the idea that more is not always synonymous with better unless it’s meaningful and presented in support of your key message.

Another way to do this would be to publish interactive versions of your visualizations that allow the viewers to dive in and explore the information themselves.

estab-lish their authority

A qualitative explorationin South Africa found that women who describednon-consensual, coerced, or violent sexual experi-ences with intimate partners would frequentlydescribe these experiences as disappointing, emo-tionally hurtful, or traumatic, but rarely categorizedthem as rape and often attributed them to men’s‘‘natural’’sexual drives and entitlement.34

EN knock-out mouseembryonic fibroblasts (PTEN KO MEF) have 2–3-fold higherconcentrations of F2,6P2, the most potent allosteric activator ofthe glycolytic enzyme phosphofructokinase-1 (PFK-1)

This has beentraditionally attributed to the hyperactivation of PI3K/Akt sig-naling that results from PTEN loss. Here, we propose a novelmechanism whereby the loss of PTEN negatively affects theactivity of the E3 ligase APC/C-Cdh1, resulting in the stabiliza-tion of the enzyme PFKFB3 and increased synthesis of its prod-uct fructose 2,6-bisphosphate (F2,6P2)

Unlike normal differentiated cells, tumor cells metabolizeglucose via glycolysis under aerobic conditions, a hallmark ofcancer known as the Warburg effect

a flexible feel inviting improvisation

Cindy A. Buckmaster: Animal research Is a labor of love for animals and people

simply

name

basic

member

how it uses zones

In general, add providers to the root module so that the same instance of a service is available everywhere.

you get a new instance of the service with each new instance of that component

Component classes should be lean. They don't fetch data from the server, validate user input, or log directly to the console. They delegate such tasks to services.

While a component is technically a directive, components are so distinctive and central to Angular applications that this architectural overview separates components from directives.

other metadata decorators

The very last person to see Hae at school that day, we think, is Inez Butler Hendrix.

Opponents concentrate on the 20,000 or so oaks and other trees to be removed on the Walt Ranch. The county stresses the 221,429 trees that are to remain.

But the county disputes Circle Oaks claims that environmental impact report’s groundwater section has numerous errors and requires vague and unenforceable safeguards for Circle Oaks water.

the county holds to the environmental impact report’s assertion that vineyard project erosion control steps would reduce erosion from the property by 44 percent.

The county responds that the environmental impact report includes the necessary analysis for species found on the Walt Ranch. For example, it says, the California longhorn elderberry beetle and not the valley elderberry longhorn beetle live there.

The project has shrunk since the Halls proposed it in 2007. The latest version calls for 209 acres of vineyards within 316 acres of graded areas—including roads and other features — scattered across the 2,300-acre Walt Ranch. No winery is proposed. The original project was for 397 acres of vineyards within a 538-acre development area.

Notice is given that thePBES Directorhas approvedAgriculturalErosionControl Plan No. P11-00205-ECPA which generally conforms to theReduced Intensity Alternative described in the Final EIR and consists ofapproximately 209 netacresof vineyard (±316grossacres).