Molecular Cell
Article
miR-182-Mediated Downregulation
of BRCA1Impacts DNA Repair
and Sensitivity to PARP Inhibitors
Patryk Moskwa,1Francesca M.Buffa,3Yunfeng Pan,1Rohit Panchakshari,1Ponnari Gottipati,4Ruth J.Muschel,4
John Beech,4Ritu Kulshrestha,1Kotb Abdelmohsen,5David M.Weinstock,2Myriam Gorospe,5Adrian L.Harris,3 Thomas Helleday,4,6,7,*and Dipanjan Chowdhury1,*
1Department of Radiation Oncology
2Department of Medical Oncology
Dana Farber Cancer Institute,Harvard Medical School,Boston,MA02115,USA
3Cancer Research UK Department of Medical Oncology,University of Oxford,Weatherall Institute of Molecular Medicine,
John Radcliffe Hospital,Headington,Oxford OX39DS,UK
4Cancer Research UK-Medical Research Council Gray Institute for Radiation Oncology and Biology,University of Oxford,
Oxford OX37DQ,UK
5Laboratory of Cellular and Molecular Biology,NIA-IRP,NIH,Baltimore,MD21224,USA
6Department of Genetics Microbiology and Toxicology,Stockholm University,S-10691Stockholm,Sweden
7Science for Life Laboratory,Stockholm University,Box1031,SE-17121Solna,Sweden
*Correspondence:helleday@gmt.su.se(T.H.),dipanjan_chowdhury@https://www.doczj.com/doc/518629244.html,(D.C.)
DOI10.1016/j.molcel.2010.12.005
SUMMARY
Expression of BRCA1is commonly decreased in sporadic breast tumors,and this correlates with poor prognosis of breast cancer patients.Here we show that BRCA1transcripts are selectively enriched in the Argonaute/miR-182complex and miR-182 downregulates BRCA1expression.Antagonizing miR-182enhances BRCA1protein levels and protects them from IR-induced cell death,while overexpressing miR-182reduces BRCA1protein, impairs homologous recombination-mediated repair,and render cells hypersensitive to IR.The impaired DNA repair phenotype induced by miR-182overexpression can be fully rescued by overex-pressing miR-182-insensitive BRCA1.Consistent with a BRCA1-de?ciency phenotype,miR-182-over-expressing breast tumor cells are hypersensitive to inhibitors of poly(ADP-ribose)polymerase1 (PARP1).Conversely,antagonizing miR-182 enhances BRCA1levels and induces resistance to PARP1inhibitor.Finally,a clinical-grade PARP1 inhibitor impacts outgrowth of miR-182-expressing tumors in animal models.Together these results suggest that miR-182-mediated downregulation of BRCA1impedes DNA repair and may impact breast cancer therapy.
INTRODUCTION
Germline mutations or deletions in breast cancer susceptibility gene BRCA1contribute to familial breast tumor formation,but there is limited evidence for direct mutation of the BRCA1 gene in the sporadic form of the disease.However,decreased expression of the BRCA1gene has been shown to be common (30%–65%)in sporadic basal-like breast cancer,and the magni-tude of the decrease correlates with disease progression(Mueller and Roskelley,2003;Thompson et al.,1995;Turner et al.,2004, 2007;Wilson et al.,1999).Because sporadic tumors account for $90%of the total breast cancer burden,a key question that emerges is how BRCA1expression is suppressed in these tumors.
DNA methylation,which can be permanent and heritable,is associated with decreased tumor suppressor gene expression in a number of disease contexts(Herman and Baylin,2000). Although promoter methylation may result in very low levels of BRCA1,aberrant methylation of the BRCA1promoter is found only in a relatively moderate percentage(10%–15%)of sporadic breast tumors(Catteau et al.,1999;Esteller et al.,2000;Matros et al.,2005;Rice et al.,2000),and there is no signi?cant correla-tion with clinical or pathological parameters of the disease (Matros et al.,2005).Other factors which have been reported to potentially contribute to diminished BRCA1expression are the transcriptional suppressors ID4(Turner et al.,2007)and HMGA1(Baldassarre et al.,2003).However,it is still unclear how BRCA1silencing occurs in the majority of sporadic basal-like breast tumors.
From a therapeutic perspective,the expression level of BRCA1is a major determinant of response to different classes of chemotherapy(Mullan et al.,2006).BRCA1-de?cient tumors are hypersensitive to DNA damaging chemotherapeutic agents (such as cisplatin,mitomycin C,etc.)(Bhattacharyya et al., 2000;Fedier et al.,2003;Moynahan et al.,2001).Based on the principle of‘‘synthetic lethality,’’BRCA mutation-associated cancers with impaired homologous recombination(HR)-medi-ated repair of DNA double-strand breaks(DSBs),are being selectively targeted by inhibitors of the DNA repair protein
PARP1(Bryant et al.,2005;Farmer et al.,2005).Conversely,the presence of functional BRCA1sensitizes tumor cells to antimi-crotubule agents(such as vincristine,paclitaxel)(Fedier et al., 2003;Mullan et al.,2001).Therefore,the cellular level of BRCA1can directly impact malignant transformation and thera-peutic response.
Because other DNA repair factors have recently been reported to be regulated by microRNAs(miRNAs)(Crosby et al.,2009;Lal et al.,2009),we hypothesized that speci?c miRNAs may suppress BRCA1expression in breast tumors.miRNAs are small ($22nt)noncoding RNAs that regulate posttranscriptional gene expression by blocking translation of target mRNAs or by accel-erating their degradation(Bartel,2009;Fabian et al.,2010). Although studies addressing their role in cancer pathogenesis are at an early stage,it is apparent that loss-or gain-of-function of speci?c miRNAs contributes to cellular transformation and tumorigenesis(Chang and Mendell,2007;Garzon et al.,2009; Ventura and Jacks,2009).Using the experimental system of in vitro hematopoietic cell differentiation and miRNA expression analysis,we have recently discovered that miRNAs downregu-late DSB repair factors and suppress DNA repair in terminally differentiated blood cells(Lal et al.,2009).Our goal was to iden-tify miRNAs targeting BRCA1and other DSB factors using this same strategy.
RESULTS
Radiation Response of MicroRNA Cluster-183
in Dividing and Postmitotic Cells
In order to identify the differentiation-induced miRNAs that play a role in the DNA damage response,proliferating progenitor K562cells and postmitotic differentiated K562cells(cells were treated with12-O-tetradecanoylphorbol-13-acetate[TPA]to produce terminally differentiated megakaryocytes)were exposed to IR,and the expression of miRNAs was studied by microarray analysis.We were particularly interested in the IR response of a subset of miRNAs that have previously been shown to be upregulated in multiple blood lineages(Lal et al., 2009).We hypothesized that in postmitotic blood cells DNA damage induces apoptosis and miRNAs attenuate the DSB repair machinery promoting cell death.Therefore,the expression of these miRNAs would be indifferent to IR in postmitotic cells. However,upon irradiation of proliferating progenitor cells,we postulated that these miRNAs are downmodulated to allow increased production of DNA repair proteins and boost the DNA damage response.Following exposure to IR,only?ve miRNAs were downregulated>2-fold in the proliferating K562 cells,but not in the postmitotic cells(see Figure S1A available online).Three of the miRNAs in this set,miR-96,miR-183,and miR-182,are encoded in an$5kb gene segment.They are pro-cessed from a well-conserved polycistronic transcript on human chromosome7q32.2(Figure S1B).These miRNAs (termed miRNA cluster-183)have a sensory role(Lewis et al., 2009;Mencia et al.,2009)and are expressed at high levels in mouse retina and sensory hair cells of the ear(Friedman et al., 2009;Jin et al.,2009;Pierce et al.,2008;Xu et al.,2007).miRNA cluster-183is aberrantly expressed in a variety of tumors(Ban-dres et al.,2006;Gaur et al.,2007;Hanke et al.,2009;Lowery et al.,2010;Segura et al.,2009;Watkins et al.,2010;Zhang et al.,2006)and is potentially useful for tumor classi?cation (Gaur et al.,2007).We veri?ed the microarray results by qRT-PCR.miR-183,miR-96,and miR-182were signi?cantly up-regulated during terminal differentiation of HL60and K562,(Fig-ure1A).As expected,there was no signi?cant change in expres-sion of miRNA cluster-183in differentiated cells exposed to IR (Figure S1C).However,there was a sharp decrease in expres-sion of miR-96,miR-183,and miR-182in undifferentiated HL60 cells(Figure1B)within30min of IR exposure.Importantly, the expression levels diminished in an IR dosage-dependent manner.The rapid and dramatic change in expression of these miRNAs in response to IR suggests a possible involvement in the DNA damage response.
miR-182Targets BRCA1
We had postulated that miRNA cluster-183was rapidly downre-gulated in response to IR in dividing cells to allow increased production of DNA repair factors.In order to identify DNA repair factors targeted by these miRNAs we adopted a computational approach.Several of the available prediction algorithms(such as TargetScan and Pictar)are largely based on evolutionary conservation of target sites across species(Bartel,2009;Sethu-pathy et al.,2006).However several critical DSB repair factors (such as MDC1,53BP1,DNA-PK,BRCA1,and BRCA2)are not found in lower eukaryotes,potentially making these algorithms less effective in identifying DNA repair targets.We used RNA22,which is distinct from other methods in that it obviates the use of a cross-species sequence conservation?lter,thus allowing the discovery of miRNA binding sites that may not be present in closely related species(http://cbcsrv.watson.ibm. com/rna22.html)(Miranda et al.,2006).One limitation of this method is that it predicts hundreds or even thousands of poten-tial targets for each miRNA,making it dif?cult to identify the most important targets.The problem was further compounded by the fact that we had three miRNAs,and there was a cumulative list of targets.We therefore focused on miR-182,which was predicted to target BRCA1.
miR-182is predicted to target several DSB repair proteins (Figure S1D),which include BRCA1,NHEJ1/XLF,etc.However, bioinformatic algorithms have a high margin of error,and the majority of predicted genes may not be real targets(Sethupathy et al.,2006).To screen predicted targets,we adapted a recently described(Easow et al.,2007;Hendrickson et al.,2008;Kargi-nov et al.,2007)biochemical approach(Figure1C).miRNAs target their corresponding mRNAs in association with a protein complex that includes the Argonaute proteins,AGO1and AGO2.This interaction allows for the identi?cation of miRNA-target interactions that occur in vivo.Immunoprecipitation(IP) of a hemagglutinin(HA)-tagged AGO1can recover miRNA/ mRNA complexes.In cells that overexpress a speci?c miRNA, IP of HA-AGO1selectively enriches for the overexpressed miRNA and its corresponding target https://www.doczj.com/doc/518629244.html,ing this strategy, we found that the miR-182/AGO1complex associates selectively with the BRCA1transcript(Figure1C)at levels comparable with a validated miR-182target,FOXO3mRNA (Segura et al.,2009).Levels of BRCA1mRNA were signi?cantly higher than control transcripts(5S rRNA and GAPDH mRNA)and
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miR-182Targets BRCA1
other predicted targets (NHEJ1mRNA).The BRCA130UTR is ($1400nt)long and has four potential miR-182miRNA recogni-tion elements (MREs)(Figure S1E),and MRE #3is the most conserved.It is important to note that none of these four sites have perfect complementarity to the ‘‘seed’’sequence.To verify that BRCA1is regulated by miR-182,we used the luciferase reporter assay.Luciferase activity was reduced $2-fold by miR-182expression in cells transfected with wild-type (WT)BRCA1-reporter.Mutation of the predicted MREs completely restored luciferase expression (Figure 1D).Furthermore,we tested the ef?cacy of each individual MRE and luciferase activity was suppressed to varying degrees by MREs #1,#3,and #4,but MRE#2had no impact on luciferase activity (Figure 1E).Together these results suggest that miR-182targets the BRCA1transcript directly by interaction with its 30UTR.
miR-182-Mediated Suppression of BRCA1Impedes DNA Repair
Next,we looked at BRCA1expression levels after ectopic over-expression of miR-182and observed a signi?cant decrease in BRCA1protein (Figure 2A).Importantly,overexpression of miR-183and miR-96,which are coexpressed with miR-182,
did not affect BRCA1protein levels.The physiological relevance of the BRCA1/miR-182interaction was further established by evaluating the endogenous expression pattern of miR-182and BRCA1.miR-182is overexpressed in the course of hematopoi-etic differentiation.If miR-182is regulating BRCA1levels,then the prediction would be that BRCA1levels diminish in parallel with increasing expression of miR-182.We differentiated HL60cells to granulocytes using DMSO and monitored the expression levels of miR-182and BRCA1over 8days.Consistent with our prediction,we observed a striking inverse correlation of BRCA1protein levels,with miR-182expression (Figure 2B).Together these results suggest that BRCA1is a physiologically relevant target of miR-182.
BRCA1is an integral component of the cellular DNA damage response (Boulton,2006;Huen et al.,2010).To determine whether miR-182-mediated BRCA1downregulation affects DNA repair,we measured the persistence of DSBs after IR,as an indicator of unrepaired damaged DNA,by single-cell gel elec-trophoresis (neutral comet assay)(Figure 2C).HL60cells with ectopic overexpression of miR-182had lower levels of BRCA1protein and signi?cantly higher residual DNA damage relative to control cells (Figure 2C,left panel).Conversely,HL60
cells
Figure 1.Expression of miRNA Cluster-183Is Induced during Differentiation and Sup-pressed by g -Radiation,and BRCA1Is a Target of miR-182
(A and B)(A)Expression of miR-183,miR-96,and miR-182during differentiation of HL60cells to neutrophils (DMSO,8days,p <0.001)and macro-phages (TPA,3days,p <0.001);K562cells to megakaryocytes (TPA,3days,p <0.002)and to erythrocytes (Hemin,4days,p <0.003).(B)miR-183,miR-96,and miR-182are rapidly down-regulated with g -radiation in proliferating cells.HL60and K562cells (data not shown)were exposed to indicate doses of g -radiation and RNA isolated 30min after exposure.There was signi?cant reduction in miR-183(p <0.002),miR-96(*p <0.003),and miR-182(*p <0.002)at 3Gy.In all the above experiments (A and B),the expression of miR-183,miR-96,and miR-182was analyzed with qRT-PCR and normalized to RNU6B,mean ±SD,n =3–6independent experi-ments,are shown.
(C)Isolation of target transcripts associated with miR-182.HeLa cells were cotransfected with expression vectors for HA-tagged AGO1and miR-182/miR-scr.The immunoprecipitated RNA was analyzed by qRT-PCR using gene-speci?c primers and normalized to 5S rRNA.FOXO3and GAPDH served as positive and negative controls,respectively.BRCA1,and not NHEJ1,was signi?-cantly (p <0.002)enriched in the pull-down.
(D and E)miR-182targets the 30UTR of BRCA1mRNA in a luciferase reporter assay.HeLa cells were cotransfected with BRCA130UTR-luciferase reporter,wild-type (WT,stripped),or mutant (M,gray)(D),or with the four predicted miR-182MREs in the BRCA130UTR (E)and control mimic
(miR-scr,black)or miR-182mimic (stripped and gray)for 48hr before analysis.Fire?y luciferase activity of the reporter was normalized to an internal Renilla lucif-erase control.Mean ±SD of three independent experiments is shown.miR-182signi?cantly (p <0.001)suppresses lucifererase activity from BRCA1-WT reporter,but mutation in the miR-182recognition sites in BRCA1-M rescues this suppression (p <0.001).
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miR-182Targets BRCA1
differentiated with DMSO,and transfected with miR-182anti-sense oligonucleotides (termed antagomirs,ASO)had signi?-cantly lower amounts of DNA breaks and higher levels of BRCA1protein (Figure 2C,right panel).
miR-182Expression Impacts HR-Mediated Repair
DSBs are one of the most deleterious types of damage caused by radiation (Jackson and Bartek,2009).Two major pathways,HR and nonhomologous end-joining (NHEJ),have evolved to deal with DSBs,and the critical components of these pathways are conserved from yeast to mammals (Shrivastav et al.,2008).BRCA1is involved in the HR-mediated DSB repair pathway (Moynahan et al.,1999),and recent reports suggest that it may impact the pathway choice (Bouwman et al.,2010;Bunting et al.,2010;Cao et al.,2009).To test whether expression of miR-182impacts HR repair,we assayed for HR-mediated repair of an I-SceI-induced DSB,in U2OS cells,using a recombination substrate DR-GFP (Nakanishi et al.,2005).Consistent with the
role of BRCA1in HR (Moynahan et al.,1999;San Filippo et al.,2008),cells overexpressing miR-182had signi?cantly reduced HR ef?ciency (Figure 2D).The cumulative impact of BRCA1de?-ciency on cellular DNA damage response is increased sensitivity to IR and other DNA damaging agents (Moynahan et al.,2001;Scully et al.,1999).Manipulating miR-182levels (via mimics or antagomirs)in HeLa cells impacts sensitivity to IR (Figure 2E).Importantly,the effect of miR-182on IR sensitivity was fully rescued by overexpressing miR-182-insensitive BRCA1.Together these results suggest that miR-182-mediated downre-gulation of BRCA1impedes the DNA damage response.miR-182Levels Correlate with BRCA1Expression in Breast Tumor Lines
BRCA1expression affects both breast tumor development and therapy (Mullan et al.,2006;Narod and Foulkes,2004;Palacios et al.,2008).Based on molecular pro?ling of tumors,breast cancer types have been divided into those with high
expression
Figure 2.Reduction of BRCA1Protein Levels by miR-182Impacts DNA Repair
(A)miR-182suppresses BRCA1expression.HL60cells transiently transfected with expression vectors for miR-183,miR-96,miR-182,and control (miR-scr)were harvested after 3days and cell lysates analyzed by immunoblot after normalization for total protein.The indicated nonspeci?c band served as a visual representa-tion for loading control.
(B)miR-182and BRCA1protein inversely correlate in DMSO-treated HL60cells.miR-182expression and BRCA1protein during DMSO-induced differentiation of HL60cells have been shown.miR-182was quanti?ed by qRT-PCR normalized to RNU6B.Tubulin served as a loading control for the immunoblot.
(C)Altering miR-182levels impacts the amount of unrepaired DSB by comet assay.Proliferating HL60cells (left panel)were transfected with control or miR-182mimic,and differentiated HL60cells (right panel)were transfected with control or miR-182antagomir (ASO).Transfected cells were irradiated,allowed to repair for 18hr,and analyzed by single-cell gel electrophoresis (comet assay).BRCA1protein is compared to tubulin levels in the immunoblots.Representative images are shown in the upper panel and the mean ±SD for each condition below.Residual DNA damage after irradiation is signi?cantly altered in miR-182mimic (p <0.001)or miR-182ASO (p <0.001)transfected cells.
(D)Overexpression of miR-182impedes HR-medi-ated repair of DSBs.U2OS cells carrying the recombination substrate (DR-GFP)were stably transfected with expression vectors for miR-182or control.I-SceI expression plasmid was tran-siently transfected and the GFP-positive cells analyzed 48hr later by ?ow cytometry (FACS).miR-182expression and representative FACS
pro?les are shown.HR repair was signi?cantly (p <0.002)impaired (lower panel).Mean ±SD of three independent experiments is shown.
(E)BRCA1mediates the DNA damage sensitivity induced by miR-182.Cells were mock transfected,transfected with miR-182antagomir (ASO),transfected with miR-182mimic or BRCA1cDNA lacking the 30UTR or both.Cell viability was assayed after indicated doses of g -radiation by clonogenic cell survival assay.Curves were generated from three independent experiments.miR-182mimic signi?cantly (p <0.003)enhanced sensitivity to IR,whereas miR-182ASO reduced (p <0.001)IR sensitivity.Representative immunoblots for (D)and (E)are shown.
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miR-182Targets BRCA1
of the ER gene (luminal)and those that do not express ER (basal)(Perou et al.,2000).ER-negative status is an intrinsic feature of BRCA1-related breast cancer (Atchley et al.,2008;Foulkes et al.,2004).We investigated BRCA1protein and miR-182expression in a panel of breast cancer lines derived from ER-positive and -negative sporadic tumors (Figure 3A).Consistent with clinical data,the basal-like ER-negative cell lines 21NT,BT549,HS578T,and HCC38had relatively low levels of BRCA1protein (Neve et al.,2006).Interestingly,in ?ve of the six ER-negative cell lines there was inverse correlation of BRCA1protein and miR-182expression (Figure 3B).To test whether the cell cycle pro?le contributes to this correlation,we compared the cell cycle pro?le of HS578T (no correlation of BRCA1protein and miR-182)with three other ER-negative cell lines (Figure 3C).Interestingly,HS578T has a very distinct pro?le with a mixed diploid and polyploid cell population (Zajac et al.,2008).Finally,in synchronized 21NT cells we examined the
rela-
Figure 3.Cell Cycle-Dependent Correlation of miR-182and BRCA1in Breast Tumor Lines
(A and B)miR-182and BRCA1protein inversely correlate in breast tumor cell lines.miR-182quan-ti?ed by qRT-PCR (normalized to RNU6)and rela-tive to nontumorigenic breast epithelial cell,HMEC,is shown on the upper panel.Mean ±SD,n =3independent experiments,is shown.A repre-sentative immunoblot for BRCA1(tubulin as control)from the indicated cells is shown in the lower panel.Relative BRCA1expression was quanti?ed by densitometry using tubulin as control and normalized to expression in HMEC.The estrogen receptor (ER)expression status of the different tumor lines has been indicated.
(C)Cell cycle pro?le of the indicated ER-negative cell lines.Asynchronously growing cells were ?xed,stained with PI,and analyzed by ?ow cytom-etry.The cell cycle distribution was assessed using FloJo.Representative images from three independent experiments are shown.
(D)Expression of miR-182and BRCA1in synchro-nized cells.21NT cells were synchronized using a combination of double thymidine block and no-cadozole and the relative amount of miR-182and BRCA1mRNA determined by qRT-PCR (normalized to 5S rRNA).Representative images of the cell cycle pro?le at indicated times after release are shown on the left panel.Mean ±SD,n =3independent experiments,is shown on the right panel.
tive expression of miR-182and BRCA1in the course of the cell cycle (Figure 3D).As described previously (Gudas et al.,1996;Shrivastav et al.,2008),BRCA1expres-sion increases with entry into S phase.Interestingly,miR-182is coexpressed with BRCA1during the cell cycle.This is consistent with a report by Bartel and colleagues (Shkumatava et al.,2009)in which they show that miRNAs and their
biologically important target transcripts are coexpressed.Together these results suggest that miR-182may regulate BRCA1expression during the cell cycle and that their correlation may depend on cellular DNA content.
BRCA1Is Targeted by miR-182in Breast Tumor Lines The physiological relevance of the BRCA1/miR-182interaction was further established by evaluating the endogenous expres-sion pattern of miR-182and BRCA1in postmitotic breast epithe-lial cells.It has been reported that TPA treatment of breast cancer lines leads to a postmitotic state (Cunliffe et al.,2003),and we con?rmed this result (Figure S2).We treated MCF7cells with TPA and monitored the expression levels of miR-182and BRCA1over 3days.Consistent with our expectation,we observed a striking inverse correlation of BRCA1protein levels,with miR-182expression (Figure 4A).Like blood cells,upon IR treatment there was rapid and dosage-dependent decrease of
Molecular Cell
miR-182Targets BRCA1
miR-182expression in proliferating MCF7cells,but not postmi-totic MCF7cells (Figure 4B).Relative decrease in miR-182levels with IR exposure in MCF7cells is comparable to levels in primary human mammary epithelial cells (HMECs,data not shown).Inter-estingly,the decrease in miR-182is speci?c to IR and exposure of MCF7cells to other DNA damaging agents,UV (single strand breaks/crosslinks)and camptothecin (replication stress),does not impact miR-182levels (Figure 4C).
miR-182In?uences the DNA Damage Response in Breast Tumor Lines
To determine whether miR-182-mediated BRCA1downregula-tion in breast cancer lines affects DNA repair,we overexpressed miR-182in MDA-MB231cells (low endogenous miR-182,high BRCA1protein)and reduced miR-182expression in 21NT cells (high endogenous miR-182,low BRCA1protein).MDA-MB231cells with ectopic overexpression of miR-182had lower levels of BRCA1protein and signi?cantly higher residual DNA damage relative to control cells (Figure 4D,left panel).Conversely,21NT cells transfected with miR-182-(ASO)had higher levels of BRCA1protein and signi?cantly lower amounts of DNA breaks (Figure 4D,right panel).Together these results suggest that
BRCA1is a physiologically relevant target of miR-182in breast cancer cells.
To determine the therapeutic impact of miR-182-mediated regulation of BRCA1in breast cancer,we overexpressed miR-182in MDA-MB231cells and reduced miR-182expression in 21NT cells and assessed their sensitivity to IR.Consis-tent with the DNA repair assays,MDA-MB231cells overexpressing miR-182were signi?cantly more sensitive to different doses of IR (Figure 5A,left panel).Importantly,the effect of miR-182on IR sensitivity was fully rescued by overexpressing miR-182-insensitive BRCA1,further con?rming that miR-182impacts the radiosensitivity of breast tumor cells via BRCA1.Furthermore,antagonizing miR-182expression in 21NT by miR-182-(ASO)induced signi?cant radio resistance in these cells (Figure 5A,right panel).Together these results strongly suggest that miR-182-mediated downregulation of BRCA1signi?cantly impacts the radiation response of breast cancer cells.
miR-182Impacts PARP Inhibitor Sensitivity
HR de?ciency of BRCA mutation-associated breast tumors selectively sensitizes them to PARP1inhibitors (Bryant et al.,2005;Farmer et al.,2005),which is a working
therapeutic
Figure 4.miR-182Targets BRCA1in Breast Cancer Cell Lines and Impacts Function
(A)miR-182and BRCA1protein inversely correlate in TPA-treated MCF7cells.miR-182expression and BRCA1protein levels during TPA-induced differentiation of MCF7cells have been shown.miR-182was quanti?ed by qRT-PCR normalized to RNU6B.Mean ±SD,n =3independent experi-ments,p <0.0091,are shown.Tubulin served as a loading control for the immunoblot.
(B and C)miR-182is rapidly downregulated with IR,and not other DNA damaging agents,in prolif-erating breast epithelial cells.Proliferating MCF7cells (B,left panel,and C,right panel)and TPA-treated postmitotic MCF7cells (B,right panel)were exposed to indicated doses of IR,UV (C,middle panel)and camptothecin (C,left panel)for 1hr.RNA isolated 30min after exposure.There was signi?cant reduction of miR-182in MCF7cells (*p <0.007).The expression of miR-182was analyzed with qRT-PCR and normalized to RNU6B,and in all panels,mean ±SD,n =3–6independent experiment,are shown.
(D)Altering miR-182levels impacts the amount of unrepaired DSB by comet assay in ER-negative tumor cells.MDA-MB231cells (left panel)were transfected with either control mimic (miR-scr)or 182mimic (miR-182).Conversely,21NT cells (right panel)were transfected with antagomirs (ASO),either control (AS0-scr)or ASO-182.Transfected cells were irradiated at indicated doses,allowed to repair for 18hr,and analyzed by single-cell gel electrophoresis (comet assay).BRCA1protein is compared to tubulin levels in the immunoblots.Representative images are shown in the upper panel,and the mean ±SD,n =3independent experiments,are shown below.Residual DNA damage after irradiation is signi?cantly altered in 182mimic (miR-182,p <0.001)or ASO-182(p <0.001)transfected cells.
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miR-182Targets BRCA1
strategy to eliminate these tumors (Fong et al.,2009;Tutt et al.,2010).Since miR-182in?uences HR-mediated repair (Figure 2D)we speculated that miR-182expression will impact cellular sensitivity to PARP1inhibitors.Overexpressing miR-182dimin-ished BRCA1levels and sensitized MDA-MB231cells to PARP1inhibition (Figure 5B,left panel)using the PARP1inhibi-tors 4-amino-1,8-naphthalimide (ANI)and ABT-888(data not shown).This effect is reverted by expressing miR-182-insensi-tive BRCA1transcripts.Conversely,reducing miR-182enhanced BRCA1expression and induced resistance to ANI (Figure 5B,right panel)and ABT-888(data not shown)in 21NT cells.Next we tested if the clinical PARP inhibitor olaparib can retard outgrowth of miR-182-expressing tumors.We treated mice 2days after injection or those bearing xenografts,derived from MDA-MB-231cells either stably expressing miR-182or scramble control (Figure S3),with olaparib or vehicle
control
Figure 5.miR-182-Mediated Regulation of BRCA1Impacts Sensitivity to Radiation and PARP1Inhibitor in Breast Cancer Cells
(A and B)BRCA1mediates the radiosensitivity,and sensitivity to PARP1inhibitor,induced by miR-182in breast cancer cell lines.MDA-MB231cells (left panels)were transfected with either control mimic or 182mimic or BRCA1cDNA lack-ing the 30UTR or both.Conversely,21NT cells (right panels)were transfected with either control ASO or 182ASO.Cell viability was assayed by clonogenic cell survival assay after indicated doses of g -radiation (A)or in the presence of PARP1inhibitor (ANI)at indicated concentrations (B).Curves were generated from three indepen-dent experiments.miR-182mimic signi?cantly enhanced sensitivity to IR (p <0.004)and to ANI (p <0.001),whereas miR-182ASO reduced sensi-tivity to IR (p <0.001)and ANI (p <0.002).Repre-sentative immunoblots for (A)and (B)are shown.(C)miR-182impacts PARP inhibitor sensitivity in xenograft mouse models.MDA-MB231cells stably expressing miR-scr or miR-182were implanted in each thigh of nude mice (n =5),and olaparib treatment was initiated either 2days postimplantation (left panel)or tumors outgrown (4–7weeks)were treated (right panel).Tumor volume was determined in 7days intervals and the median fold differences after olaparib treat-ment represented graphically.
for 10days.As anticipated,a 10day treatment with olaparib retarded tumor outgrowth in animals treated both pre-or posttumor formation (Figure 5C).In untreated animals the tumors grew at a comparable rate in the presence or absence of miR-182(Figure S4).We har-vested the tumors and con?rmed the expression of miR-182(data not shown).Altogether,these results convincingly demonstrate that miR-182is a mediator of the cellular response to PARP inhibi-tors.Importantly,the observation that the DNA repair-de?cient phenotype induced by miR-182was largely rescued by miR-182-resistant BRCA1transcripts suggests that the key target of miR-182in DNA repair is BRCA1.DISCUSSION
We have previously used the experimental system of in vitro hematopoietic cell differentiation to ?nd miRNAs involved in DSB repair (Lal et al.,2009),and the same strategy was applied to identify miR-182.A rapid,IR dosage-dependent decrease in miR-182expression further implicated a role in the DNA damage response.This IR-induced change in miR-182is independent of p53,as it occurs in both p53-pro?cient (MCF7,HMEC)as well as p53-de?cient (K562,HL60)https://www.doczj.com/doc/518629244.html,ing a combination of compu-tational and biochemical methods,we found that BRCA1was
Molecular Cell
miR-182Targets BRCA1
targeted by miR-182.It is feasible that other factors in the DNA damage response are affected by miR-182expression. However,the observation that the DNA repair-de?cient pheno-type induced by miR-182was largely rescued by miR-182-resis-tant BRCA1transcripts suggests that the key target of miR-182 in DNA repair is BRCA1.
Decreased expression of the BRCA1has been shown to be common in sporadic basal-like breast cancer(Mueller and Roskelley,2003;Turner et al.,2004).Although the magnitude of the decrease correlates with disease progression,the molec-ular mechanism of BRCA1suppression in sporadic tumors is unclear.We anticipate that overexpression of miRNAs such as miR-182may play a role in BRCA1downregulation in sporadic breast tumors.Manipulation of miR-182expression in multiple breast tumor lines impacts BRCA1levels and sensitivity to PARP1inhibition,either in cultured cells or in xenograft models. This observation has potential clinical relevance.Although familial breast cancer patients with BRCA mutations are currently being treated with PARP inhibitors,the majority of patients with breast cancer($90%)have the sporadic form of the disease and do not have BRCA mutations.It is currently unclear if patients with sporadic breast cancer bene?t from the therapeutic approach with PARP inhibitors.Our results suggest that sporadic tumors overexpressing miRNAs which target BRCA proteins(such as miR-182)may also be susceptible to PARP inhibition or other strategies based on synthetic lethality with BRCA.Future studies will determine whether expression levels of miR-182and possibly other miRNAs that regulate BRCA1can serve as determinants of therapeutic strategy and of clinical outcome for patients with breast cancer. EXPERIMENTAL PROCEDURES
Cell Culture and Differentiation
HL60cells and K562cells were grown and differentiated into different lineages as previously described(Lal et al.,2009).MCF-7cells(106cells/6cm dish) were differentiated with100nM TPA for3days in DMEM with10%(v/v)FBS (Cunliffe et al.,2003).The breast cancer lines and human mammary epithelial cells used in Figure3were cultured in media according to protocols from ATCC(https://www.doczj.com/doc/518629244.html,/).
miRNA Microarray,RNA Isolation,and Quantitative PCR Undifferentiated and differentiated K562cells were untreated or exposed to 2Gy of g-radiation.Total RNA was extracted after2hr with Trizol(Invitrogen) according to the manufacturer’s manual and treated with10U DNase I for 30min at37 C in50m l.RNase-free water was added and DNase I removed with phenol-choloroform(Ambion).The TaqMan Human MicroRNA Array v 1.0(Early Access)platform was used for quantitative miRNA analysis and was carried out in the Dana-Farber Cancer Institute core facility.In subsequent experiments,TaqMan MicroRNA Assay from Applied Biosystems was used as per the manufacturer’s instructions.mRNA expression was anlayzed by qPCR using SYBR Green master mix(Applied Biosystems)according to the manu-facturer’s manual and the Bio-Rad iCycler.Gene-speci?c primers were as follows:BRCA1F,CAACATGCCCACAGATCAAC,R,ATGGAAGCCATTGTCC TCTG;NHEJ1F,AGTGCCAAGTGAGGGAGCTA,R,CCACTTGGACCTCTTG TGT;FOXO3F,GATAAGGGCGACAGCAACAG;R,CCAGTTCCCTCATTCTG GAC;5S rRNA F,GCCCGATCTCGTCTGATCT;R,AGCCTACAGCACCCGG TATT;GAPDH F,TGCACCACCAACTGCTTAGC;R,GGCATGGACTGTGGT CATGAG;and FNDC3A F,CTTGGAGCTGGTCCTTTCAG;R,CCTTCCCCAG CTTCATTACA.Algorithm Tools to Predict Targets of miR-183Cluster
We applied RNA22algorithm(https://www.doczj.com/doc/518629244.html,/rna22_tar gets.html)to?nd targets of miR-182.30UTR of BRCA1was further analyzed by RNAhybrid(http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/)and PITA algorithm(http://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.html).
Immunoprecipitation of miR-182Targets
miR-182was cloned from HL60cDNA in pcDNA3.1-Puro expression vector (Invitrogen)using the following primers:miR-182,F,CGGCGGCCGCGATAT GAGGGGAAGGGAGGA;R,CGGCGGCCGCGAGAAGGTTCACCACCCAGA. Cells were cotranfected with HA-AGO1(Addgene)and miR-182or miR-scr (expression vectors).After2days,cells were harvested in400m l lyses buffer (100mM KCl,5mM MgCl2,10mM HEPES[pH7.0],0.5%Nonidet P-40)con-taining freshly added RNaseOUT(Invitrogen)and Protease Inhibitor Cocktail (Roche).After centrifugation,a50m l aliquot was taken as input for subsequent RNA extraction.The remaining supernatant was gently shaken with HA beads (HA-probe Santa Cruz sc-7392)for4hr at4 C in spin columns(Pierce Spin Columns-Screw Cap).The columns were drained and washed,and the retained beads were treated with5U DNaseI in NT2buffer(50mM Tris[pH 7.4],150mM NaCl,1mM MgCl2,0.05%Nonidet P-40)for10min at37 C, washed,and treated with Proteinase K.Finally the beads were resuspended and RNA extracted using acid phenol-choloroform(Ambion).The analysis was done as follows:scrambled control,pull-down/input(say=A);miR-182, pull-down/input(say=B);fold enrichment=B/A.
Luciferase Assay
The WT and mutated(M)miR-182recognition elements of30UTR-BRCA1were annealed(for sequence,see below)and cloned in pMIR-REPORT(Ambion) downstream to Fire?y Luciferase.The luciferase assays were done in HeLa cells as described previously(Lal et al.,2009).
The primer sequences are as follows:WT F,CTAGTAGAAGAGATTTCTA AAAGTCTGAGATATATTTGCTAGATTTCTAAAGAATGTGTTCTAAAACAGCA GAAGATTTTCAAGAACCGGTTTCCAAAGACAG;WT R,AGCTCTGTCTTTG GAAACCGGTTCTTGAAAATCTTCTGCTGTTTTAGAACACATTCTTTAGAAAT CTAGCAAATATATCTCAGACTTTTAGAAATCTCTT;M F,CTAGAAGAGA C GATACCC GTCTGAGATATATTTGCTAG GCGATACCC G GG TGTGTTCTAAA AGCAGAAG CCGATACCCGG CCG GCGATACCC GACAG;and M R,AGC TCTGTCGGGTATCGCCGGCCGGGTATCGCTTCTGCTGTTTTAGAACACAC CCGGGTATCGCCTAGCAAATATATCTCAGACGGGTATCGCTCTCTTCTAG. The mutant residues have been indicated.
Primers for the individual sites are as follows:(site I)F,CTAGGGAAAATGA AACTAGAAGAGATTTCTAAAAGTCT;R,AGCTAGACTTTTAGAAATCTCTTCT AGTTTCATTTTCC;(site II)F,CTAGAAAGTCTGAGATATATTTGCTAGATTTCT AAAGAATG;R,AGCTCATTCTTTAGAAATCTAGCAAATATATCTCAGACTTT; (site III)F,CTAGAGAATGTGTTCTAAAACAGCAGAAGATTTTCAAGAACCG; R,AGCTCGGTTCTTGAAAATCTTCTGCTGTTTTAGAACACATTCT;(site IV)F, CTAGAAAACAGCAGAAGATTTTCAAGAACCGGTTTCCAAAGACAG;R,AGC TCTGTCTTTGGAAACCGGTTCTTGAAAATCTTCTGCTGTTTT.
Immunoblots
The immunoblots were done as described previously(Lal et al.,2009;Lee et al.,2010)with BRCA1antibodies1:500dilution(CALBIOCHEM,MS110) and tubulin antibodies1:10000dilution(Sigma,Clone B-5-1-2).
Single-Cell Gel Electrophoresis,Comet,Assay
21NT cells were transfected with100nM,control or miR-182antagomir(Am-bion),whereas MDA-MB231cells were transfected with10nM,control or miR-182mimic(Ambion).After3days,cells were irradiated and allowed to recover for18hr prior to analysis.The single cell comet assays(Trevigen)were carried out as previously described(Lal et al.,2009;Lee et al.,2010).
Homologous Recombination Reporter Assay
HR assay was done as described(Chowdhury et al.,2008;Lee et al.,2010). Brie?y,23105cells plated overnight in24-well plates were transfected with 0.8m g of I-SceI expression plasmid(pCBA Sce)using Lipofectamine2000. Two days later,GFP-positive cells were assayed by FACScan.
Molecular Cell
miR-182Targets BRCA1
Clonogenic Assay
HeLa cells(0.353106cells/well),MDA-MB231cells(0.43106cells/well),and 21NT cells(0.53106)were seeded overnight and transfected with10nM of miRNA mimics or antagomirs(Ambion).In rescue experiments,miR-182or control mimics were cotransfected with1m g/ml BRCA1(pcDNA3.1vector). After2days,1000cells in4ml DMEM media(10%FBS v/v)were seeded on 6cm dishes in four replicates and incubated overnight before treatment. PARP inhibitors(4-amino-1,8-naphthalimide[Sigma]or ABT-888or olaparib [(ChemieTek]in DMSO)were added to the growth media at indicated concen-trations.Irradiated cells as well as cells in the presence of PARP inhibitor were allowed to form colonies for14days.For evaluation,formed colonies were stained with crystal violet and surviving colonies containing>50cells were counted.Plating ef?ciency was30%à50%.
Cell Cycle Synchronization
21NT cells were seeded on a6-well plate(2.53105/well)in DMEM10%FBS media and treated twice with2mM thymidine for16hr with a9hr interval between treatments.Subsequently the cell were released into media contain-ing100ng/ml nocadozole and incubated for12hr.The cells were washed and released into DMEM20%FBS media and were collected for FACS analysis and RNA extraction after indicated time intervals.For FACS the cell were?xed in70%ethanol,washed twice with PBS buffer,and analyzed in PI/RNase staining buffer(BD PharMingen).
Xenograft Experiments
MDA-MB231cells(2.53106/injection)stably expressing miR-scr or miR-182 from pcDNA3.1(+)Neo were injected subcutaneously into CD1nude mice.Ola-parib(50mg/kg,obtained in collaboration with AstraZeneca)or vehicle(PBS+ 10%DMSO+10%HPCB)treatment was initiated either at2days postinjec-tion or at detectable tumor volume($70mm3)between4and7weeks later. Each animal obtained ten intraperitoneal injections on10consecutive days. Measurement of tumor volume by calipers was commenced7days post the subcutaneous injection and continued in7day intervals.
SUPPLEMENTAL INFORMATION
Supplemental Information includes three?gures and Supplemental Refer-ences and can be found with this article at doi:10.1016/j.molcel.2010.12.005.
ACKNOWLEDGMENTS
This work was supported by R01CA142698(NCI),JCRT and a Barr Award (D.C.);by the Medical Research Council(T.H.);and by the NIHR Biomedical Research Centre,Oxford(A.L.H.).M.G.and K.A.were supported by the NIA-IRP,NIH.Dr.Mark O’Connor(AstraZeneca)kindly provided olaparib. We thank members of the Harris,Helleday,and Chowdhury laboratories for useful discussions.
Received:August24,2010
Revised:October20,2010
Accepted:November11,2010
Published online:December30,2010
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Molecular Cell
miR-182Targets BRCA1
bt建设项目合同范本2篇Model contract for BT Construction Project 甲方： 乙方： 签订日期：年月日
bt建设项目合同范本2篇 小泰温馨提示：建设工程合同是承包人进行工程建设，发包人支付价款的合同。建设工程合同包括即建设工程勘察合同、建设工程设计合同和建设工程施工合同。本文档根据建设工程合同内容要求和特点展开说明，具有实践指导意义，便于学习和使用，本文下载后内容可随意修改调整及打印。 本文简要目录如下：【下载该文档后使用Word打开，按住键盘Ctrl键且鼠标单击目录内容即可跳转到对应篇章】 1、篇章1：bt建设项目合同范本 2、篇章2：bt建设项目合同范本 篇章1:bt建设项目合同范本 甲方: **×××××公司 鉴于: 1.甲方经**区人民政府授权（授权文件见附件 1） ,是** 区人民政府确定的**×××××工程的最终项目业主;
2.甲方就**×××××工程（详后附（工程项目一览表）进行"BT" Build-Transfer）模式招标（或竞争性谈判） （,×××× ×公司投标并中标（或参与竞争性谈判并中 标） . 根据《中华人民共和国合同法》《中华人民共和国建筑 , 法》《建设工程质量管理条例》《国家计委关于印发促进 和 , , 引导民间投资的若干意见的通知》 ,建设部《关于加 快市政公用行业市场化进程的意见》《重庆市人民政府关于 加强和 , 规范政府投资项目 BT 融资建设管理的通知》渝府发（[2019]73 号）等法律法规,及《**区政府投资项目BT 融资建设管理实施意见》（合川府发〔2019〕17 号）和 合川区政府投资项目相关规定,经双方充分协商一致,订立本 合同, 以此共同恪守. 第一条定义 除合同条款另有约定外,本合同用语以下列定义为准: 1.1 本工程（或本项目） :指**工业园区××××× （详后附（工程项目一览表）工程. 1.2 本工程范围: [以下为参考示例,应根据不同工程明确详尽:
合伙企业变更决定书 合伙企业变更决定书1 根据《合伙企业法》和本企业合伙协议的有关规定，召开此次会议。参加本次会议的合伙人共2人，决定通过的事项如下: 同意本企业合伙人变更为XXX、XXX 同意本企业各合伙人出资额变更为:XXX认缴出资50万元、XXX认缴出资950万元 同意修改合伙协议 全体合伙人签字或盖章: XXX 盖章: XXX 签字: xxxx年3月8日 合伙企业变更决定书2 根据《中华人民共和国合伙企业法》、《中华人民共和国合伙企业登记管理办法》等相关法律法规的规定，(以下简称本合伙企业)于年月日召开合伙人会议，经全体合伙人决定，一致同意本合伙企业的以下变更： 1、同意合伙人将其在本合伙企业的万元出资额(占合伙企业出资额的%)转让给，转让价格为人民币万元。其他合伙人同意放弃优先购买权。 2、上述转让后，本合伙企业合伙人出资变更为：
3、其他企业登记内容不变。 4、同意对合伙协议作出相应修改，并同意修改后的合伙协议。 此页无正文，为《合伙企业变更决定书》签字页： 年月日 合伙企业变更决定书3 参加表决合伙人: 、、。 新增合伙人: 、。 根据《合伙企业法》和本企业合伙协议的有关规定，参加本次表决的合伙人共人，代表本企业合伙人 %的表决权，所作出的表决经本企业合伙人表决权的 %通过。决议事项如下: 1.同意改变合伙企业的名称为: 。 2.同意(原合伙人)将占本合伙企业的出资财产份额 %共万元转让给(其他/新合伙人) 。 3.同意改变合伙企业的经营场所为: 具体地址。 4.????????。(其它需要表决的事项请逐项列明) 5. 同意就上述改变事项修改合伙协议相关条款。 原合伙人:(签名或盖章) 新增合伙人:(签名或盖章) 年月日 注:1、本范本适用于各类型合伙企业; 2.合伙人对合伙企业有关事项作出决议，按照合伙协议约定的表决办法办理。合伙协议未约定或者约定不明确的，实行合伙
生产计划与物料管理 课程背景： 准时化生产是精益生产的一个重要内容，随着时代节奏加快的变化。客户对企业的交货期要求越来越苛刻，能否快速、准确地交货将成为企业未来生存重要考验。同时准时化生产也是企业降低成本，降低库存，增加现金流的制胜法宝。为什么我们很多企业生产周期很长，交货期一拖再拖，在制品堆满车间，产成品库存居高不下，而且有的产成品库龄很高。其实都与生产计划的编制不科学有着很大的关系。因此，学习准时化生产下的生产计划科学编制是企业迫在眉睫的课题，也是当务之急。 课程模型： 课程收益： 本课程从精益生产的角度出发，深入分析企业的生产形态、产品特点、工艺流程、标准工时、生产节拍、产能负荷关系和客户需求，如何在低的在制品和产成品库存的情况下，做到快速准确地交货。通过学习学员将得到以下收益： 1.了解“零库存”是精益生产的核心； 2.学会分析自己企业的生产形态和产品特点；
3.学会标准工时的制定方法； 4.学会产能和负荷的正确评估； 5.学会如何确定订单的生产节拍； 6.学会如何计算产品的生产周期； 7.学会如何进行生产线的平衡技巧； 8.学会在低在制品和低产成品库存下的快速交货的生产计划编制方法。 课程工具： 1.精益生产的确切定义： 2.“零库存”的三个重要环节：准时化生产的三个标准： 3.企业常见的2种生产形态： 4.标准工时构成三要素： 5.作业要素划分三原则： 6.生产排序的四大原则： 7.确定各工序投入量和产出量的方法： 8.确定生产线生产能力方法： 9.确定生产周期两个重要指标： 10.装配型生产排产技巧： 11.确定各工序的投入期和产出期的方法： 12.生产线平衡的四大方法：订单来源两个方向： 13.产品ABC分析的计算流程： 14.预测型产品计划编制原则： 15.AB类产品双堆法排产技巧： 16.物料ABC判断标准： 17.ABC物料分析计算流程 课程时间：2天，6小时/天 授课方式：理论讲授(50%)+案例分析、小组研讨(30%)+实操练习(20%)
XXXXX工程 投资建设-移交-回购合同 1.总则 为加快城市基础设施建设，改善XX城区交通状况，经XXX 人民政府批准，XXXXX工程项目已经进入建设阶段。根据中华人民共和国法律、法规和规范性文件的相关规定，依循平等互利、诚实信用的原则，经XX人民政府（以下简称“甲方”）、通号创新投资有限公司公司（以下简称“乙方”）和XXX平台公司（以下简称“丙方”）友好协商，就XXX工程投资建设－移交－回购项目，双方于XX年XX月XX日在XXX签署本合同，以资共同恪守。 1.1 鉴于条款 1.1.1甲方依照职权并根据授权，采取投资建设－移交－回购模式建设XXX工程。甲方授权XXX平台公司为本项目的日常管理机构。乙方具有相应的投融资能力。经甲方对乙方进行综合考察评估（或公开招投标或竞争性谈判），最终确定乙方为本项目的投资建设方。 1.1.2甲方同意按本合同约定向乙方支付回购价款，乙方同意按照本合同约定投资、通过合法程序将工程发包给具有相应资质的施工单位施工并最终向甲方移交工程项目。丙方同意以XXX 地块对本项目的回购提供抵押担保（或银行保函）。 1.2词语定义 在本合同中，下文定义的措词和用语，应具有本款赋予的含
义。 1.2.1 “项目名称”（下称“本项目”）： 1.2.2“工程内容”： 1.2.3“甲方”：指XXX人民政府，系本合同项下项目回购方。 1.2.4“乙方”：指通号创新投资有限公司，系本合同项下项目投资建设方。 1.2.5“丙方”：指XXX平台公司，系本合同项下项目回购担保方。 1.2.6 “项目管理机构”：指乙方为投资、建设、管理本项目而在项目所在地设立的管理机构。 1.2.7 “回购基数”：是经甲方和乙方最终确认的项目建安投资。回购基数=建安投资。 1.2.8“建安投资”：指由建筑安装工程费、工程变更、价格调整和其它费用等组成。 1.2.9“回购价款”：回购价款为回购基数与投资收益之和。 1.2.10“投资收益”：是指甲方应支付给乙方，乙方在本合同项下投资建设应得的投资收益，包括建设期投资收益和回购期投资收益。计算公式为：“建设期投资收益额”=∑（按每月计量确认的月建安投资×年投资收益率×资金占用天数/365），其中：资金占用天数为资金注入之日至工程交工验收合格签字之日；“回购期投资收益额”=∑（未回购部分的建安投资×年投资收益率）。 1.2.11“投资收益率”：指对本项目进行投资建设并移交给甲方，甲方给予乙方投资收益的比率。本项目的年投资收益率为同期中国人民银行3-5年期贷款基准利率计算利息的基础上+5%。
公共关系学-公共关系传播模式与媒介 (总分：100.00，做题时间：90分钟) 一、{{B}}单项选择题{{/B}}(总题数：26，分数：26.00) 1.传播学中，总体研究范畴的规划者是美国人______ ?A.卢因 ?B.哈罗德·拉斯韦尔 ?C.拉扎斯菲尔德 ?D.麦库姆斯 （分数：1.00） A. B. √ C. D. 解析：[解析] 本题考查考生对学科发展史的了解。传播学中，美国人哈罗德·拉斯韦尔是总体研究范畴的规划者。2.发表了《社会传播的结构与功能》一文，成为传播学的创始人之一的是______ ?A.拉斯韦尔 ?B.卢因 ?C.拉扎斯菲尔德 ?D.麦库姆斯 （分数：1.00） A. √ B. C. D. 解析：[解析] 传播学中，拉斯韦尔是总体研究范畴的规划者，他在1948年发表的《社会传播的结构与功能》一文，使其成为传播学的创始人之一。 3.拉斯韦尔提出的界定传播研究范畴的经典模式是______ ?A.香农模式 ?B.5W模式 ?C.两级传播模式 ?D.议题设置论 （分数：1.00） A. B. √ C. D. 解析：[解析] 本题属于识记内容，考生需认真掌握。5W模式是拉斯韦尔在《社会传播的结构与功能》一文中提出的界
定传播研究范畴的经典模式。 4.“把关人”这一概念出自______ ?A.《原则宣言》 ?B.《修辞学》 ?C.《社会传播的结构与功能》 ?D.《群体生活的渠道》 （分数：1.00） A. B. C. D. √ 解析：[解析] 本题属于识记内容，考生需牢记。1947年，德国著名社会心理学家卢因在《群体生活的渠道》一文中最早提出了“把关人”这一概念。 5.传播学学者十分重视把关人的作用，并认为这是一种信息传播的______ ?A.特殊现象 ?B.简单现象 ?C.普遍现象 ?D.复杂现象 （分数：1.00） A. B. C. √ D. 解析：[解析] 本题考查考生对“把关人”这一知识点掌握的熟练程度，属于识记内容。传播学学者十分重视把关人在信息传播过程中的枢纽作用，认为把关人是一种信息传播的普遍现象。 6.在信息传播过程中，对信息的提供、制作、编辑和报道能够采取“疏导”与“抑制”行为的关键人物是______ ?A.守门人 ?B.媒介 ?C.舆论指导者 ?D.监督人 （分数：1.00） A. √ B. C. D. 解析：[解析] “把关人”又称“守门人”，它是指在信息传播过程中，对信息的提供、制作、编辑和报道能够采取“疏导”与“抑制”行为的关键人物。 ______ 把关人原有的意见、经验、兴趣和精神状态的总和指的是7. ?A.知识资源 ?B.预存立场
智慧社区建设合作协议 编号：WYR-A-2015001 甲方： 地址： 法定代表人或负责人： 乙方： 地址： 法定代表人或负责人： 为了协助提高小区的治安综合治理能力，最大限度降低小区业主人身及财产安全风险，更便捷、更科技、更智能的服务于业主，进一步提升小区物业的服务质量，增加业主的满意度。经甲方与小区业主委员会协商同意，一致决定将乙方的“民生驿站”智慧社区云平台引入本小区，服务于小区业主。 根据《中华人民共和国合同法》及相关法律法规，甲乙双方本着诚信、平等、自愿、双赢的原则，经双方友好协商达成如下协议，双方共同信守。 （以下简称“甲方”）是“民生驿站”智慧社区云平台的使用者。 （以下简称“乙方”）是“民生驿站”智慧社区云平台的提供者。 第一条项目情况 一、小区基本情况
项目地址：。 小区业主：栋单元户人（约）。 小区门禁：扇大门、扇单元门、车库单元门。 二、云平台介绍 “民生驿站”智慧社区云平台（以下简称“云平台”）是重庆百金穗电子商务股份有限公司自主研发的基于智能安防、智能家居、政务民生、公共服务等的移动终端智慧民生项目。主要功能如下：（一）经授权的业主可通过门禁卡、智能手机客户端方便出入小区大门及单元门。 （二）来访客人可通过小区及单元门口机与业主智能手机客户端可视对讲，业主可实现远程开门。 （三）物业公司可利用“云平台”提供物业服务、发送公告、征集民意、传递关怀等。 （四）经授权业主可通过智能手机客户端、缴纳生活日常费用、采购生活用品、各类远程报修等。 （五）业主可享受智能家居及远程报警的各类服务(智能家居及报警设备需业主购买)。 第二条合作内容与方式 一、合作内容： （一）硬件改造：根据甲方提供的小区的实际情况，小区的硬件改造费用共计元（大写：）（报价清单见附表）；该改造项目由方投资建设，则该设备的所有权及处置权
合伙人变更决定书样本 怎样写合伙人变更决定书?合伙人变更需要什么材料?那么，下面请看我给大家整理收集的合伙人变更决定书样本，供大家阅读参考。 合伙人变更决定书样本1 根据《合伙企业法》及合伙协议的约定，经全体合伙人一致同意，于年月日就合伙企业以下事宜作出变更: 1.(1)同意将合伙企业名称变更为 (名称变更); (2)同意将公司住所变更至 (住所变更); (3)撤销为执行事务合伙人的委托，重新委托为执行事务合伙人(执行事务合伙人变更); (4)同意合伙企业类型变更为 (合伙企业类型变更，同时需变更名称); (5)同意吸收为新合伙人，新合伙人以货币出资，出资额为万元，于年月日缴付，本企业出资总额增至万元(新合伙人入伙)。 (6)同意××××出资额由万元增加(或减少)至万元，其中万元的出资额，出资方式变更为 (出资额、出资方式变更)。 2.相应修改合伙协议。 全体合伙人签或或者合伙协议约定的人员签名(或盖章)： (注：自然人签名，法人、其他组织加盖公章) 年月日 合伙人变更决定书样本2 会议时间：xx 年xx 月xx 日 会议地点：xxx 参加会议人员：(全体合伙人)xx 、xx 、 xx， 会议议题：协商表决变更合伙企业xxx 事宜。 根据《中华人民共和国合伙企业法》及合伙协议， (合伙企业)经全体合伙人协商一致通过如下决议： 一、同意将xxx变更为xxxx 。 二、同意将合伙企业经营范围由xxxx变更为xxxxx
(以上经营范围以登记机关核发的营业执照记载项目为准;涉及许可审批的经营范围及期限以许可审批机关审批的为准)。 同意就上述变更事项修改合伙协议相关条款，同意年月日新签订的合伙协议，新签订的合伙协议自本决议通过之日起生效。 全体合伙人签字：xxx xx 年xx 月xx 日 相关知识 一.合伙企业变更登记应提交文件材料 1. 执行事务合伙人或者委派代表签署的《合伙企业变更登记申请书》。 2. 原全体合伙人签署的《变更决定书》，或者合伙协议约定的人员签署的《变更决定书》。 3. 《指定代表或者委托代理人的委托书》及指定代表或委托代理人的身份证件复印件(本人签署"此件与原件一致"并本人签字)。 4. 合伙企业变更经营场所的，应当在迁入新经营场所前申请变更登记，并提交新经营场所使用证明。 合伙企业变更经营场所跨企业登记机关辖区的，应当在迁入新经营场所前向迁入地企业登记机关申请变更登记。 5. 法人、其他组织委派的执行合伙事务的授权代表发生变化的，提交其继任代表的自然人身份证明复印件(本人签署"此件与原件一致"并本人签字)和继任委派书。 6. 合伙企业变更企业类型的，应当办理企业名称变更;普通合伙企业变更为特殊的普通合伙企业的，提交由国家有关部门或授权机构颁发的各合伙人的职业资格证明。 7 .合伙企业修改合伙协议的，应当提交依据设立登记时合伙协议的约定作出的《合伙协议修正案》,由全体合伙人签名、盖章。 8. 新合伙人入伙的，提交新合伙人的主体资格证明(盖公章)或自然人身份证明(本人签署"此件与原件一致"并本人签字)、《入伙协议》以及全体合伙人对新合伙人认缴或者实际缴付出资的《出资确认书》。 9.合伙人增加或减少对合伙企业出资的，提交全体合伙人对该合伙人认缴
第一讲物料控制管理概述 生产计划与物料控制重要性 对于一个现代化的公司来说，生产计划与物料控制非常重要。科学的管理理论认为，在生产计划与物料控制层面上，所有的PMC人员（即生产计划与物料控制人员）都是生产部门的上司。在一个建立了现代化管理制度的企业中，生产计划与物料控制应该是一个统筹部门；生产计划与物料控制层面的工作是所有生产进程的前提，如果生产计划与物料控制层面的工作进行得不够顺利，那么所有的生产流程都会受到延误。因此，现代企业要充分重视生产计划与物料控制工作的重要性，公司的组织架构、岗位职责、制度和流程都要围绕生产计划与物料控制来制定。 物料管理部门的统筹作用具体体现在两个方面： 其一，在产品研发过程中，物料管理部门要与研发部门相协调，同步参与研发过程。物料部门参与了研发过程，就会更明白产品的指标标准范例，更有利于采购工作； 其二，物料部门可以督促研发部门的进度以便符合其排期要求。物料部门的统筹作用还体现在工程更改的时候，如果出现了工程更改的情况，物料部门有必要与研发部门沟通，譬如是否可以把呆料改作它料。 物料管理范围和意义 1.物料管理的范围 物料管理包括三方面的内容：物料计划及物料控制、采购和仓储。传统管理理论认为，企业的生产计划同物料控制工作与采购工作存在巨大差别，因此应该由不同部门进行管理。但在实际工作中，企业管理人员会经常发现，生产计划与物料控制工作往往同采购工作存在冲突，采购部门的工作往往令生产计划与物料控制部门无法满意。要解决这个问题，需要对企业的组织架构进行调整。 采购部门的工作一般分为两个部分：寻找供应商议价以及跟进采购工作。按照生产计划与物料控制理论，跟进采购单等采购工作应该从采购部门剥离出来，划入生产计划与物料控制部门管理。这样进行调整之后，就能够较好地解决物料控制工作与采购工作之间的冲突。传统企业组织架构的依据是部门的功能，而现代企业组织机构的依据是企业的生产流程。 2.物料管理的意义 物料控制的首要意义在于降低企业的物料成本，确保企业获得比较丰厚的利润。在市场经济条件下，一个生产新产品的企业刚开始可能获取很高的利润，但随着竞争对手的增多，
YOUR LOGO 公路BT项目合同新范本正式版 After The Contract Is Signed, There Will Be Legal Reliance And Binding On All Parties. And During The Period Of Cooperation, There Are Laws To Follow And Evidence To Find 专业合同范本系列，下载即可用
公路BT项目合同新范本正式版 使用说明：当事人在信任或者不信任的状态下,使用合同文本签订完毕，就有了法律依靠,对当事人多方皆有约束力。且在履行合作期间,有法可依,有据可寻，材料内容可根据实际情况作相应修改，请在使用时认真阅读。 建设单位:____,以下简称甲方; 订立条约双方: 设计单位:____,以下简称乙方。 为了明白责任,分工协作,共同完成国度建设项目标设计任务,根据《建设工程勘探设计条约条例》的规定和__答应的计划任务书,经甲乙双方充分协商,特签订本条约,以便共同遵守。 第一条 工程名称、规模、投资额、建设地点 甲主委托乙方承担____工程的设计项目,建筑安装面积为____平方米,答应总投资为____万元,建设地点在____。 第二条 甲方的义务 一.甲方应在__年__月__日以前,向乙方提交业经上报答应的设计任务书、工程选址报告,以及原料(或经过答应的资源报告)、燃料、水、电、运输等方面的协议文件和能满足初步设计要求的勘探材料、需要经过科研取得的技能材料。甲方在__年__月__日施工图设计前,应提供经过答应的初步设计文件和能满足施工图设计要求的勘探材料、施工的条件,以及有关设备的技能材料。
变更决定书 出席会议合伙人：、、。 新增合伙人：、。（注：如没有新增合伙人，该项可删除）根据《合伙企业法》和本企业合伙协议的有关规定，本次合伙人会议于年月日在（注：地点）召开。出席本次会议的合伙人共人，代表本企业合伙人%的表决权，所作出的表决经本企业合伙人表决权的%通过。决议事项如下： 1、同意。 2、同意。 …… （其它需要表决的事项请逐项列明） 同意就上述决议事项修改合伙协议相关条款。 或：同意就上述决议事项重新制定合伙协议并启用新合伙协议。 原合伙人：（签名或盖章）新增合伙人：（签名或盖章） （注：如没有新增合伙人，该项可删除。） 注：合伙人为自然人的由本人签字，合伙人为法人或其他组织的由其法定代表人或负责人（有权签字人）签字，并加盖公章。 年月日（企业盖章）
注：决议内容参考 第一条变更或备案的情形： 1、名称变更 同意合伙企业名称变更为：。 2、经营场所变更/备案 同意经营场所由变更为： （注：具体地址）。 3、合伙企业类型变更 同意变更合伙企业类型，由变更为。 （注：此项变更指因合伙人类型变更导致的由有限合伙企业变更为普通合伙企业，与合伙人类型变更同时办理） 4、执行事务合伙人（委派代表）变更 同意变更执行事务合伙人，执行事务合伙人由变更 为。 （注：执行事务合伙人必须由普通合伙人担任） 5、执行事务合伙人的委派代表变更 同意变更执行事务合伙人的委派代表，委派代表由变更 为。 （注：此项变更适用于法人或其他组织担任执行事务合伙人的情形） 6、合伙企业出资额变更 同意合伙企业出资额由人民币万元变更为万元。 本次新增出资人民币万元，其中（合伙人姓名、名称）以
《茂名市政府投资项目BT合同范本》业经市人民政府十一届六次常务会议通过，现印发给你们，请认真贯彻执行。执行中遇到的问题，请直接向市进展和改革局反映。 二О一二年六月十九日 投资建设——回购合同 项目名称： 项目地点： BT发包人： BT投资人： 目录 第一部分协议书
一、项目概况 (1) 二、工程承包范围和承包方式 (1) 三、合同工期 (2) 四、质量标准 (2) 五、合同价款 (2) 六、有关费率 (2) 七、BT投资人承诺 (2) 八、BT发包人承诺 (3) 九、合同份数 (3) 十、合同生效 (3)
第二部分合同条款 一、总则 (4) 1、定义与解释 (4) 2、项目公司 (5) 3、组成合同的文件及解释顺序 (6) 二、项目前期工作 (6) 4、项目许可文件 (6) 5、前期工作 (7) 三、工程相关单位的选择 (7) 6、监理、勘察设计单位的选择 (7)
7、施工单位的选择 (7) 8、BT投资人建设治理责任 (7) 四、项目建设 (7) 9、设计修改和设计变更 (8) 10、建设工程质量 (8) 11、安全文明施工 (12) 12、项目建设进度 (13) 五、验收和移交 (17) 13、工程竣工和验收 (17) 14、单项工程移交 (18)
15、项目整体移交 (19) 六、缺陷责任与质量保修 (19) 16、缺陷责任与质量保修 (19) 七、项目监管 (20) 17、项目监管 (20) 八、回购价款 (21) 18、建安工程费计算 (21) 19、预算、结算的编制、审核、备案 (23) 20、回购价款的计算 (23) 21、回购价款的调整 (25)
合伙企业变更登记申请书 一、申请变更登记事项 执行事务合伙人或委派代表签字：申请日期：
执行事务合伙人或委派代表委托代理人的委托书 作为合伙企业（名称）的执行事务合伙人或委派代表，现委托代理人向登记机关申请办理合伙企业的变更登记事宜。 执行事务合伙人或委派代表：受托人： 年月日年月日
. 整理范本编辑word！二、变更后的全体合伙人名录及出资情况 执行事务合伙人或委派代表签字：申请日期：
新合伙人的主体资格证明或者自然人的身份证明复印件
合伙企业变更登记应提交文件材料及填表说明 一.合伙企业变更登记应提交文件材料 1.执行事务合伙人或者委派代表签署的《合伙企业变更登记申请书》。 2.全体合伙人签署的变更决定书，或者合伙协议约定的人员签署的变更决定书。 3.执行事务合伙人或者委派代表指定的代表或者委托的代理人的委托书。 4. 合伙企业变更经营场所的，应当在迁入新经营场所前申请变更登记，并提交新经营场所使用证明。 合伙企业变更经营场所跨企业登记机关辖区的，应当在迁入新经营场所前向迁入地企业登记机关申请变更登记。 5.法人、其他组织委派的执行合伙事务的代表发生变化的，提交其继任代表的自然人身份证明复印件和继任委派书。 6.合伙企业变更企业类型的，应当办理企业名称变更；普通合伙企业变更为特殊的普通合伙企业的，提交由国家有关部门或授权机构颁发的各合伙人的职业资格证明。 7．合伙企业修改合伙协议的，应当提交由全体合伙人签名、盖章的新修改的合伙协议或者依据设立登记时合伙协议的约定作出的修改合伙协议的决议。 8．新合伙人入伙的，提交新合伙人的主体资格证明或自然人身份证明、入伙协议以及全体合伙人对新合伙人认缴或者实际缴付出资的确认书。 9．合伙人增加或减少对合伙企业出资的，提交全体合伙人对该合伙人认缴或者实际缴付出资的确认书。 10．法律、行政法规或者国务院规定在登记前须经批准的项目的，提交有关批准文件。 11．国家工商行政管理总局规定提交的其他文件。 二.填表说明： 1．经办人是指执行事务合伙人或委派代表指定的代表或者委托的代理人。 2. 经办人提交的文件、证明应当是原件，不能提交原件的，其复制件应当由登记机关核对。 3. 经办人应当使用钢笔、毛笔认真填写表格或签字。 4．经办人只填写申请书中登记事项变更的栏目，登记事项未变的不填。 5．需要由全体合伙人签署的文件，合伙人是自然人的，由本人签字；合伙人是法人或其他组织的，由其法定代表人（负责人）签字并加盖公章。
合作框架协议 甲方： 乙方： 二?一四年二月
合作框架协议 甲方: 乙方: 为加快 _______________ 市政基础设施的建设步伐，甲乙双方根据《中华人 民共和国政府采购法》和《中华人民共和国合同法》等法律规定，本着平等互利的原则，经过友好协商，一致同意建立合作关系，特订立本合作框架协议。 一、合作内容 甲乙双方拟就 _________________________ 项目展开合作。拟合作具体项目 符合本合作框架协议BT合作模式原则性条件的由双方按法定程序推进实施。 二、合作方式 _______________________ 项目以BT方式投资、建设和移交，即乙方负责筹措大部分建设资金并作为工程总承包单位组织项目建设，建设完毕后将项目移交给甲方，甲方在约定期限内对项目进行回购。 本项目根据前期工作完成情况逐个实施、逐个签订BT合同并逐个回购。甲乙双方根据具体项目签订BT合同。 三、合作条件 （一）B T项目投资 1._______________________ BT项目的投资总额约元人民币，其中包括工程建设费用约亿 元人民币，前期费用约—亿元人民币。乙方负责工程建设费用的投资，甲方负责前期费用的投入。最终投资额根据BT合同约定确定。 2 ?乙方投资本金的到账资金以专用共管账户开户银行出具的到账凭证为准。乙方投资本金总额最终为乙方实际投入专用共管账户的经双方确认的金额。 3?项目的实际工程结算金额超出BT合同约定的乙方总投资额部分由甲方筹集支付，乙方不再追加投资。 （二）工程造价确定原则 1?工程预算编制原则
项目工程建设费用按照施工图设计及工程量清单，以《____ 工程预算定额》及其取费标准和相关配套文件、当地《______ 工程造价信息》为依据编制，工程 预算不下浮。工程预算金额由双方协商确定后报_______ 审计局确认，将作为工程进度款支付和工程结算的依据。 2?工程建设费用调整原则 施工建设中若发生设计变更、技措洽商、增项增（减）量、工料机价格涨跌等，由BT合同甲方、乙方与监理共同予以书面确认，并计入相应工程付款证书中。调整后的增加费用，从不可预见费中优先支付，不可预见费不足支付部分由甲方筹集支付。 3?工、料、机调价原则 在建设期内发生人工、材料、机械台班价格涨跌时，工程建设费用应按该部分工程施工期当期《__________ 工程造价信息》中的工、料、机指导价作相应调整，每月调整一次。人工、材料、机械据实调整，材料调整范围根据各项目具体情况在BT合同中确定。 各项目调价的工程材料数量，根据同期经监理工程师确认的实际数量，按照批准的配合比或定额计算材料用量确定。工程交工后，施工方按实际施工完成工作内容，编制工程造价结算书，由监理预审后，送审计部门审核，并以双方确认的审核结果作为工程结算的依据。 （三）B T项目周期 1.BT项目周期分建设期和回购期。 2.__________________________ BT项目的建设期预计为：年。各项目的开工日期根据项目情况由双 方根据各项工作进度协商确定，实际开工时间以BT合同约定为准。 3.____________ 回购期：年。回购时点在BT合同中一经确定则固定不变。甲方应 向乙方支付第一笔投资本金回购款的日期为乙方首笔投资投入BT合同双方专用 共管账户之日起第____ 年的同一日。 （四）B T项目回购原则 1.项目回购款组成
变更决定书 出席会议合伙人：、、。新增合伙人：、。（注：如没有新增合伙人，该项可删除） 根据《合伙企业法》和本企业合伙协议的有关规定，本次合伙人会议于年月日在 （注：地点）召开。出席本次会议的合伙人共人，代表本企业合伙人 %的表决权，所作出的表决经本企业合伙人表决权的 %通过。决议事项如下： 一、同意。 二、同意。 …… 同意就上述决议事项重新制定合伙协议并启用新合伙协议。
原合伙人签署： 新增合伙人签署：（注：如没有新增合伙人，该项可删除。） 注：合伙人为自然人的由本人签字，合伙人为法人或其他组织的由其法定代表人或负责人（有权签字人）签字，并加盖公章。 年月日 注：决定内容参考 （一）变更或备案的情形： 1、名称变更 同意合伙企业名称变更 为：。 2、经营场所变更/生产经营地备案 同意经营场所变更为：。（注：新的具体地址）。 3、合伙企业类型变更 同意变更合伙企业类型，由变更 为。 （注：此项变更指因合伙人类型变更导致的有限合伙
企业、普通合伙企业和特殊普通合伙企业类型之间的转换，与合伙人类型变更同时办理） 4、执行事务合伙人（委派代表）变更 同意变更执行事务合伙人， 执行事务合伙人由变更为。 （注：执行事务合伙人必须由普通合伙人担任） 5、执行事务合伙人的委派代表变更 同意变更执行事务合伙人的委派代表， 委派代表由变更为。 （注：此项变更适用于法人或其他组织担任执行事务合伙人的情形，同时出具法人或其他组织委派代表的委托书） 6、合伙企业出资额变更 同意合伙企业的出资额由人民币万元变更为 万元。 本次新增出资人民币万元，其中有限合伙人/普通合伙人（注：合伙人姓名或名称）以方式认缴出资人民币万元，于年月日前缴足……（注：增资格式） 本次减少出资额人民币万元，其中有限合伙人/普通合伙人（注：合伙人姓名或名称）减少出资人民币万元……（注：减资格式）
生产计划与物料控制（PMC） 讲师：赵又德 【课程背景】 生产计划与物料控制是企业生产运作的核心，本培训项目根据制造业面临着销售预测不准、客户需求多变、产品寿命周期短、技术更新快、产销协调困难、生产计划执行不到位、物料控制难等问题，通过培训、案例分析和互动游戏，使学员了解国内外制造业生产计划与物料控制的最新理念和成功经验。 【课程收益】 ◆掌握如何构建适应市场需求变化的、协调性强的PMC组织架构 ◆掌握物料全过程管理的最新管理模式，从根本上解决产销协调不良、控制不力及信息沟通不畅的问 题 ◆掌握不断创新和探索新的管理模式，提升PMC对市场需求的快速反应 ◆掌握对生产运作和物流过程的控制力度及降低生产运作成本之能力 ◆掌握MRP运行机理及如何与生产计划、库存的有效配合 【课程对象】 制造业企业总经理、副总经理、PMC、采购、财务、仓储、资材及库存管理等部门经理（本课程尤其适合制造业企业全体干部集体参会、思考、研讨生产计划、物料控制、如何建立PMC组织架构、缩短生产周期等相关问题，召开会后会，效果极佳）。 【课时】 1天 【课程大纲】 第一部分如何构建高效运行的生产计划与物流控制运作体系？ 1.目前制造企业生产与物料控制面临机遇与挑战 １.１现代生产管理的发展趋势
１.２最有效的利用资源和经济效益最大化 2. PMC在生产运作管理中扮演的角色分析？ 2.1何为生产计划与物料控制（PMC）？ 2.2什么说ＰＭＣ是生产运作管理的总导演？ 【案例分析】：某企业因ＰＭＣ执行力欠缺导致生产瘫痪的教训 【案例分析】：某公司生管组织及工作职能实例分析 【案例分析】：某公司高效的销售计划/生产计划/物料计划组织架构实例分析 第二部分如何处理订单并构建高效运行的产销链接运作体系？ 1.如何及时高效处理订单与搞好产销协调？ 【案例分析】：某公司订单评审的相关部门及职责、流程、评审表分析 【实例分析】：某公司有效的产销链接方式分析 【实例分析】：某香精公司的高效客户订单处理程序实例分析 2.如何对插单与紧急订单进行处理？ 3.１如何确定插单与紧急订单的类型 【案例分析】：某公司决定接收订单的要素分析模型分析 3.2 插单与紧急订单难以处理原因分析 【案例分析】：某公司各部门联合处理插单与紧急订单的有效措施（业务部、生产部、生产车间及班组等） 【案例分析】：某厂正确处理紧急订单的措施分析 第三部分物料需求计划（MRP）与生产计划的有效配合 1. 物料需求计划编制程序 1.1 常备性物料计划制定 1.2 专用性物料计划制定 2. 如何配合生产计划，有效实施物料需求计划（MRP）？ 2.1 生产计划的日程基准概念及构成分析 【案例分析】：某公司日程基准表的计算
文件编号：RHD-QB-K5790 （合同范本系列） 甲方：XXXXXX 乙方：XXXXXX 签订日期：XXXXXX 公路BT项目合同范本 新标准版本
公路BT项目合同范本新标准版本操作指导：该合同文件为经过平等协商和在真实、充分表达各自意愿的基础上，本着诚实守信、互惠互利的原则，根据有关法律法规的规定，达成如下条款，并由双方共同恪守。，其中条款可根据自己现实基础上调整，请仔细浏览后进行编辑与保存。 建设单位:____,以下简称甲方; 订立条约双方: 设计单位:____,以下简称乙方。 为了明白责任,分工协作,共同完成国度建设项目标设计任务,根据《建设工程勘探设计条约条例》的规定和__答应的计划任务书,经甲乙双方充分协商,特签订本条约,以便共同遵守。 第一条 工程名称、规模、投资额、建设地点 甲主委托乙方承担____工程的设计项目,建筑安装面积为____平方米,答应总投资为____万元,建设地点在
____。 第二条 甲方的义务 一.甲方应在__年__月__日以前,向乙方提交业经上报答应的设计任务书、工程选址报告,以及原料(或经过答应的资源报告)、燃料、水、电、运输等方面的协议文件和能满足初步设计要求的勘探材料、需要经过科研取得的技能材料。甲方在__年__月__日施工图设计前,应提供经过答应的初步设计文件和能满足施工图设计要求的勘探材料、施工的条件,以及有关设备的技能材料。 甲方对上述材料必须保证质量,不得随意变更。 二.及时办理各设计阶段的设计文件审批工作。 三.在工程开工前,甲方应组织有关施工单位,由乙方进行设计技能交底;工程竣工后,甲方应通知乙方参
合伙企业变更决定书范本 如何起草合伙企业变更决定书?下面请看第一范文网小编给大家整理收集的合伙企业变更决定书范本，供大家阅读参考。 合伙企业变更决定书范本1 根据《合伙企业法》及合伙协议的约定，经全体合伙人一致同意，于年月日就合伙企业以下事宜作出变更: 1.(1)同意将合伙企业名称变更为(名称变更); (2)同意将公司住所变更至(住所变更); (3)撤销为执行事务合伙人的委托，重新委托为执行事务合伙人(执行事务合伙人变更); (4)同意合伙企业类型变更为(合伙企业类型变更，同时需变更名称); (5)同意吸收为新合伙人，新合伙人以货币出资，出资额为万元，于年月日缴付，本企业出资总额增至万元(新合伙人入伙)。 (6)同意××××出资额由万元增加(或减少)至万元，其中万元的出资额，出资方式变更为(出资额、出资方式变更)。 2.相应修改合伙协议。 全体合伙人签或或者合伙协议约定的人员签名(或盖章)： (注：自然人签名，法人、其他组织加盖公章) 年月日 合伙企业变更决定书范本2 根据《合伙企业法》和本企业合伙协议的有关规定，召开此次会议。参加本次会议的合伙人共2人，决定通过的事项如下： 同意本企业合伙人变更为XXX、XXX 同意本企业各合伙人出资额变更为：XXX认缴出资50万元、XXX认缴出资950万元 同意修改合伙协议 全体合伙人签字或盖章： XXX 盖章： XXX 签字：
xxxx年3月8日 合伙企业变更决定书范本3 经本企业全体合伙人协商决定赣州市XXX厂(服务中心)变更如下登记事项： 1、同意执行事务的合伙人变更为： 2、同意合伙企业类型变更为： 3、同意经营范围变更为： 4、同意主要经营场所变更为： 5、就上述变更事项修改合伙协议 全体合伙人签字： xxxx 年月日
合同号： 项目名称： 投资建设-移交 （BT）合同 XXXX人民政府 二〇一〇年月日印制
目录 一、总则 (2) 二、合同文件的组成与解释顺序 (3) 三、项目 (4) 四、项目管理机构 (4) 五、投资 (5) 六、建设 (7) 七、项目的设计、监理、造价咨询与审计 (7) 八、变更 (8) 九、工程造价及投资确认 (9) 十、验收保修 (10) 十一、项目移交 (11) 十二、项目移交时的状态 (11) 十三、风险转移 (11) 十四、质量保证金 (11) 十五、回购 (12) 十六、双方的权利与义务 (15) 十七、不可抗力、风险责任与保险 (17) 十八、违约 (18) 十九、争议 (22) 二十、通知事项 (22) 二十一、特别约定事项 (24) 二十二、合同的份数、生效 (25) 二十三、合同附件 (25)
项目名称： 项目投资建设—移交（BT）合同 回购方：XXXX人民政府（以下简称甲方） 投资建设方：（以下简称乙方） 甲、乙双方依据《中华人民共和国合同法》、《中华人民共和国建筑法》、《中华人民共和国招标投标法》及国家有关法律、法规和规范性文件，遵循平等、自愿、公平和诚实信用的原则，订立本合同。 一、总则 1．词语定义 1.1“本项目”: 1.2“甲方”包括“回购方”、“业主方”：均指XXXX人民政府。 1.3“乙方”、“投资建设方”：是指 1.4“项目部”：是指由乙方根据项目管理及建设要求按照招投标文件及有关法律规定而组建的机构。 1.5“回购款”：回购款＝回购基数+投资利息+投资回报。 1.6“回购基数”：是指经回购方确认并经审计单位审定后的投资资金总额，包括全部建安工程费及与项目有关的公共基础设施建设费用、工程建设其它费（含征地拆迁费、管线迁改费、勘察设计费、监理费、招标代理服务费等），以及与项目有关的其他各项税费及向政府各管理部门缴纳的各项费用及其他支出。
合伙人变更决定书 导读：本文是关于合伙人变更决定书，希望能帮助到您！ 合伙人变更决定书范文篇一 变更决定书出席会议合伙人： 新增合伙人： 根据《合伙企业法》和本企业合伙协议的有关规定，本次合伙人会议于年月日在 (注：地点)召开。出席本次会议的合伙人共人，代表本企业合伙人 %的表决权，所作出的表决经本企业合伙人表决权的 %通过。决议事项如下：1、同意。2、同意。……(其它需要表决的事项请逐项列明)同意就上述决议事项修改合伙协议相关条款。或：同意就上述决议事项重新制定合伙协议并启用新合伙协议。 原合伙人：(签名或盖章) 新增合伙人：(签名或盖章) 合伙人变更决定书范文篇二 根据《合伙企业法》及合伙协议的约定，经全体合伙人一致同意，于年月日就合伙企业以下事宜作出变更: 1.(1)同意将合伙企业名称变更为 (名称变更); (2)同意将公司住所变更至 (住所变更); (3)撤销为执行事务合伙人的委托，重新委托为执行事务合伙人(执行事务合伙人变更); (4)同意合伙企业类型变更为 (合伙企业类型变更，同时需变更名称); (5)同意吸收为新合伙人，新合伙人以货币出资，出资额为万元，于
年月日缴付，本企业出资总额增至万元(新合伙人入伙)。 (6)同意××××出资额由万元增加(或减少)至万元，其中万元的出资额，出资方式变更为 (出资额、出资方式变更)。 2.相应修改合伙协议。 原合伙人：(签名或盖章) 新增合伙人：(签名或盖章) 合伙人变更决定书范文篇三 会议时间：年月日 会议地点： 参加会议人员：(全体合伙人) 、、， 会议议题：协商表决变更合伙企业事宜。 根据《中华人民共和国合伙企业法》及合伙协议， (合伙企业)经全体合伙人协商一致通过如下决议： 一、同意将名称变更为。 二、同意将合伙企业经营范围由 变更为 (以上经营范围以登记机关核发的营业执照记载项目为准;涉及许可审批的经营范围及期限以许可审批机关审批的为准)。 同意就上述变更事项修改合伙协议相关条款，同意年月日新签订的合伙协议，新签订的合伙协议自本决议通过之日起生效。 全体合伙人签字： 年月日 合伙企业变更登记应提交文件材料 1. 执行事务合伙人或者委派代表签署的《合伙企业变更登记申请书》。
云南省基础设施项目 投资建设-回购（BT）合同范本
回购人：______________________________投资人：______________________________签约地点：_____________________________签约日期：_____________________________
目录 陈述_______________________________________________ 5第一章特许权_______________________________________ 5第1条定义和解释_______________________________ 5 1.1 定义____________________________________ 5 1.2 释义____________________________________ 8 第2条双方义务_________________________________ 9 2.1 特许权的授予____________________________ 9 2.2 担保权__________________________________ 9 2.3 投资人的要紧义务________________________ 9 2.4 回购人的要紧义务_______________________ 10 2.5 建设期_________________________________ 10 2.6 建设期的延长___________________________ 10 第3条合作前提条件____________________________ 10 3.1 投资人条件_____________________________ 10 3.2 回购人条件_____________________________ 10 3.3 回购承诺函和回购担保文件_______________ 11