El gen K-ras, és molt important clínicament ja que es troba mutat en un 40% dels casos de carcinomes colorectals i en un 90% dels casos de carcinomes de pàncreas, que constitueixen la segona i quarta causa de mort per càncer en països desenvolupats i juga un paper important en el desenvolupament dels sarcomes. Degut a la importància clínica del gen K-ras, un model que millori el seu coneixement en l'àmbit molecular, ajudarà a predir el comportament biològic del tumor abans de decidir el tractament més adequat per un pacient concret. Aquests coneixements, poden portar a una millora substancial des del punt de vista terapèutic. Les mutacions activants de l'oncogen K-ras, s'han localitzat al codó 12 o 13 del mateix. Prèviament, s'ha associat la presència de mutacions al codó 13 amb un fenotip tumoral menys agressiu que quan aquestes es localitzen al codó 12. Concretament, la mutació al codó 13 s'associa amb menor capacitat invasiva local i metastàsica. L'objectiu d'aquesta tesi es trobar una explicació molecular al diferent comportament clínic descrit amb anterioritat en tumors humans segons si la mutació activant es troba al codó 12 o al codó 13 del gen K-ras. També es preten trobar una explicació molecular a la transformació produïda pel gen normal sobrexpressat, que prèviament s'havia descrit que en determinats models era tumorogènic. Aquest estudi s'ha realitzat en un model in vitro (fibroblasts NIH3T3 de ratolí), transfectats establement amb el gen K-ras humà amb les distintes mutacions i en un modelo in vivo, injectant subcutàniament els transfectants en ratolins atímics i estudiant els sarcomes generats.Les conclusions d'aquest treball són que en el nostre model d'estudi, la mutació al codó 12 de K-ras confereix un fenotip més transformant que la mutació al codó 13 o que la sobreexpresió del gen normal. La causa principal d'aquestes diferències en transformació és la major activació de la via de supervivència cel·lular PI3K/AKT als transfectants amb mutació al codó 12 que els confereix major resistència a l'apoptosi, tant en el model in vitro com in vivo. Per últim, la posició del codó mutat en K-ras confereix especifitat histològica als tumors generats que de manera coherent amb el seu origen mesenquimàtic (NIH3T3) són sarcomes. Els tumors generats amb mutació al codó 12 del gen K-ras constitueixen un possible model de fibrosarcoma i els generats amb mutació al codó 13 del gen K-ras constitueixen un possible model d'histioctioma fibrós maligne; dos tipus de sarcomes humans. / K-ras gene is the most frequently mutated ras gene in human tumors. It's mutated in 40% of cases of colorectal carcinomas and 90% of pancreatic adenocarcinomas. These are the second and fourth causes of cancer death in developed countries. K-ras play also an important role in development of sarcomas. A model that improve knowledge of K-ras in a molecular way will help to decide the right treatment to a specific patient.K-ras became oncogenic by single point mutations at codon 12 or 13. Several lines of evidence suggest that the malignant potencial of tumor cells may be influenced not only by the presence or absence of K-ras mutations, but by its molecular nature. Codon 13 mutations have been associated with less aggressive tumoral phenotype than codon 12 mutations in K-ras gene. Codon 13 mutation is associated with less invasive and metastasic capacity.In this thesis, is tested whether K-ras codon 12 mutation would confer upon the cell a more oncogenic phenotype than a K-ras codon 13 mutation and try to find a molecular explanation for these differences. It is also tested the transformation capacity of the K-ras proto-oncogene and molecular explanation of this effect.To this end, NIH3T3 cells (mouse fibroblasts) are transfected with a plasmid containing human K-ras with point mutations at codon 12 or at codon 13 or containing the K-ras proto-oncogene; selected stable transfectants; and evaluated the possible changes in different functions contributing to transformation. It's tested also in vivo model. We subcutaneously injected transfectants in nude mice and generated tumors were compared morphocally, functionally, and molecularly.The conclusion of this work is that K-ras with point mutations at codon 12 confer a more aggressive transforming phenotype, increasing the thresfold of apoptotic induction. In contrast, mutations at codon 13 or the overexpression of K-ras proto-oncogene reduce this thresfold. Increased activation of cellular survive pathway PI3K/AKT confer to K-ras with point mutations at codon 12 resistance to apoptosis, in vitro and in vivo model.And last; the position of mutated codon in K-ras gene confer histologic specificity to generated tumors.Tumors derived from transfectants with K-ras point mutations at codon 12 constitute a possible animal model of human fibrosarcoma. Tumors derived from transfectants with K-ras point mutations at codon 13 constitute a possible animal model of human malignant fibrous histiocytoma.

År 1961 publicerades en notis i tidningen DN, där Medicinalstyrelsen avrådde från internationell adoption i större omfattning. I synnerhet om det gällde från adoptivföräldrarna starkt skilda rasgrupper. Detta uttalande väckte stark mediedebatt i Sverige under de följande åren. Syftet med studien är att undersöka hur de olika tidningsdebattörerna ställde sig i adoptionsfrågan när det gällde utländska barn med annan ras eller etnicitet,samt få en ökad förståelse för varifrån dessa åsikter kan ha fått sin grund. Uppsatsen bygger på en kvalitativ innehållsanalys av artiklar som berör internationell adoption från 1961-1964. Syfte och frågeställningar diskuteras utifrån olika perspektiv på ras- och etnicitet, som kopplas till en socialkonstruvistisk bakgrund. Resultatet av studien visar att de parter som uttalade sig i artiklarna representerades av människor med olika förhållande till internationell adoption men att det ändå var journalisterna som dominerade antalet uttalande. Adoptivföräldrar kom först på tredje plats. De flesta ansåg att barnen hade goda möjligheter till anpassning i Sverige, det största hindret skulle vara om de var alltför märkta av sin tidigare miljö och att de riskerade att känna sig utanför på grund av sin avvikande hudfärg. De flesta debattörerna ville minska klyftan mellan "svarta" och "vita" men många fördomar fanns kvar främst genom att Sverige sedan 1920-talet varit världsledande inom rasbiologi. Arvet från den "vita" kolonialtiden samt den svenska afrikamissionen bidrog till den nedlåtande synen på främmlingar. Trots fördomar verkar de flesta debattörer positiva till internationell adoption, mycket pågrund av 1960-talets solidariska tänkande gentemot omvärlden.

<p>The genes encoding the Ras family of small GTPases are mutated to yield constitutively active GTP-bound oncoproteins in one-third of all human cancers. In many other cancers lacking Ras mutations, Ras is activated by other means. One common example of such activation is found in breast cancer, in which epidermal growth factor receptor (EGFR) family receptor tyrosine kinases, including EGFR and HER2 (ErbB-2/Neu), are frequently amplified and overexpressed, which in turn activates Ras. In human cells, activation of the Ral guanine nucleotide exchange factor, or RalGEF, effector pathway is necessary for Ras-mediated tumorigenesis and metastasis. RalGEFs activate the two highly similar Ral GTPases, RalA and RalB. While RalA has been shown to be required for Ras-mediated tumorigenesis, RalB is important for tumor metastasis. Activated Ral GTPases bind to and activate a limited number of effector proteins, including RalBP1, Sec5, and Exo84, to affect numerous diverse activities of the cell. This dissertation research sought to determine which of these well-characterized Ral effector proteins were required for oncogenic mutant Ras-induceded tumorigenesis and metastasis of human cells, as well as to examine the role of RalA in breast cancer cells that can activate Ras through EGFR and HER2 overexpression. </p><p> RNA interference-mediated loss-of-function analysis demonstrated that Sec5 and Exo84 are required for oncogenic Ras-mediated tumorigenesis, and, at least in part, metastasis. Additionally, both gain-of-function and inhibition studies showed that RalA activation is induced by EGFR and HER2 in breast cancer cell lines stimulated with EGF. Furthermore, stable suppression of RalA expression inhibited tumorigenic growth of breast cancer cells, and RalA activation was shown to be higher in a majority of mammary adenocarcinomas versus matched patient normal mammary tissue. These studies provide new insights into the importance of RalA activation in breast cancer, as well as the molecules downstream of RalA and RalB that may be responsible for mediating their effects on tumorigenesis and metastasis.</p> / Dissertation

Abstrate: Ras proto-oncogenes are members of the superfamily of GTP-binding proteins. Many tyrosine kinase receptors, including those for epidermal growth factor, insulin, and nerve growth factor, signal through RAS proteins. The product of members of this oncogene family (H-, K-, N-ras) is a 21 kD protein with nucleotide binding activity, involved in the transduction of information from the cell surface to the nucleus. The three RAS proteins exit in two states: a resting state in which they are bound to GDP and an active state in which they bind GTP. The most common form of mutational activation of Ras oncogenes in human tumors is through single base substations affecting either the GTP-binding of main (codons 12 and 13) or the GTPase domain (codon 61) of the protein. Thus, mutant RAS proteins result in constitutive activation of the downstream signaling cascade because their affinity for GTP is increased or their GTPase activity is decreased, so that the protein cannot return to the resting state. To investigate we have screened 89 thyroid tumor specimens, which include 8 follicular carcinomas (FC), 42 papillary carcinoma (PTC), 2 anaplastic carcinoma (AC),5 Hurthle cell adenoma (HA), 12 follicular adenoma (FA) and 20 nodular goiter (NG), for mutation in three Ras genes using PCR and automatic sequencing. Four tumors contained Ras gene mutation. Of these, three were identified among FC (37.5%), which mutation were in the codon 61 of each Ras genes. One mutation were at codon 61 of N-ras in FA specimens (8.3%). In addition, 33.7% (30/89) of specimens contain H-ras codon 27 polymorphism. In conclusion, our data indicated that the prevalence rates of Ras gene mutation were 5.8% and 2.7% in thyroid carcinoma and thyroid benign adenoma, respectively. Other environmental or genetic factors might also involved in the thyroid tumorigenesis and worth further investigation. The data were further confirmed using the combination of the PCR and denaturing gradient gel electrophoresis (DGGE). Four more cases of possible Ras mutation were detected which did not revealed by automatic sequencing , indicating that DGGE is a more sensitive method in detecting single nucleotide mutation. DGGE analysis should increase the detection rate of Ras gene mutation in our analysis.

[URE3] priono indukcija priklauso nuo Ras/PKA signalinio kelio aktyvumo. Galima šio kelio įtaka [PSI+] priono formavimuisi gali būti numanoma iš natyvaus Sup35 baltymo struktūros. Šio baltymo struktūroje yra nustatytos kelios menamos PKA fosforilinimo vietos. Siekiant patikrinti šią galimybę buvo atliktas trijų, Ras/PKA signalinio kelio aktyvumu besiskiriančių, izogeninių kamienų – 6-&#945;‘1-NB13-, 12-&#945;‘1-NB13-, 7-&#945;‘1-NB13 - [PSI+] priono indukcijos dažnio įvertinimas. Rezultatai parodė, kad terpėje esant turtingam azoto šaltiniui 6-&#945;‘1-NB13- kamieno ląstelėse padidinta CYR1, BCY1 ir Ras2Val19 genų raiška sumažina [PSI+] priono indukcijos dažnį. Tuo tarpu, terpėje esant neturtingam azoto šaltiniui 7-&#945;‘1-NB13 kamieno ląstelėse padidinta BCY1 geno raiška padidina [PSI+] priono indukcijos dažnį. Todėl galima daryti išvadą, kad [PSI+] priono indukcijos dažnis gali priklausyti nuo Ras/PKA signalinio kelio aktyvumo. / [URE3] prion induction depends on the activity of the Ras/PKA signal transduction pathway. Possible influence of this pathway on the formation of [PSI+] prion could be predicted from the structure of the native Sup35 protein. Several possible phosphorylation sites are known in the structure of this protein. In order to check this possibility analysis of prion induction frequency of three isogenic mutant strains – 6-&#945;‘1-NB13-, 12-&#945;‘1-NB13-, 7-&#945;‘1-NB13 – different in the activity of the Ras/PKA signal transduction pathway, was performed. Results showed that in the presence of rich nitrogen source in cells of the strain 6-&#945;‘1-NB13 the increased expression of CYR1, BCY1 and Ras2Val19 genes decreases the frequency of [PSI+] prion induction. Instead, in the presence of poor nitrogen source in cells of the strain 7-&#945;‘1-NB13 the increased expression of BCY1 gene increases the frequency of [PSI+] prion induction. So, it is possible to make a conclusion that the frequency of induction of the [PSI+] prion depends on the activity of the Ras/PKA signal transduction pathway.

Following starvation, Dictyostelium discoideum cells aggregate, a response that requires chemotaxis to cyclic AMP (cAMP) and the relay of the cAMP signal by the activation of adenylyl cyclase (ACA). Insertional inactivation of the rasG gene resulted in delayed aggregation and a partial inhibition of early gene expression, suggesting that RasG does have a role in early development. When the responses of rasG⁻ cells to cAMP were compared with the responses of rasC⁻ strain, these studies revealed that signal transduction through RasG is more important in chemotaxis and early gene expression, but that signal transduction through RasC is more important in ACA activation. Characterization of a rasC⁻/rasG⁻ mutant revealed that both cAMP chemotaxis and adenylyl cyclase (ACA) activation were negligible in this strain. The ectopic expression of carA from the actin 15 promoter restored early developmental gene expression to the rasC⁻/rasG⁻ strain, rendering it suitable for an analysis of cAMP signal transduction. Since there was negligible signaling through either the cAMP chemotactic pathway or the adenylyl cyclase activation pathway in this strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways. The mutational analysis of Switch I and Switch II regions also defined the key residues that generate functional differences between RasC and RasG. Rap1 is also activated in response to cAMP but its position in the signal transduction cascade was clarified by the finding that its activation was totally abolished in rasC⁻/rasG⁻/[act15]:carA and in rasG⁻ cells, but only slightly reduced in rasC⁻ cells. The finding that in vitro guanylyl cyclase activation is also abolished in the rasC/rasG⁻4act15]:carA strain identifies RasG⁻/RasC⁻ as the presumptive monomeric GTPases required for this activation. The phenotypes of the vegetative ras null mutants were also examined. The results indicate that RasG plays an important role in cytokinesis. The partial absence of chemotaxis to folate in rase cells compared to the total absence of chemotaxis to folate in rasC⁻/rasG⁻, and rasC⁻/rasG⁻/[act15]:carA cells suggests a compensatory role of RasC for RasG during this process, a similar phenomenon to that observed for cAMP chemotaxis by aggregating cells.

No description available.

Tumorigenesis is driven by the sequential accumulation of genetic lesions within a cell, each which confer the cell with traits that enable its abnormal growth. The result is a mass of dysregulated cells, or tumor, which, upon further mutation, may spread, or metastasize, to other organs of the body. The dissemination of tumor cells makes treatment difficult, and thus confers cancer with its associated lethality. Over the past 30 years, the RAS genes have been critical in teaching us the mechanisms underlying the molecular progression of cancer. RAS is mutated in 33% of all cancers and is often an early event in its stepwise progression. As a result, the RAS genes are widely accepted as ‘drivers’ or ‘initiators’ of human tumorigenesis. Unfortunately, efforts directed at targeting RAS in the clinic have as of yet been unsuccessful. This has triggered a need to identify genes that are required for RAS tumorigenesis that are therapeutically tractable. My research has focused on deciphering the potential role of the Rho GEF Arhgef2 in RAS-mediated tumorigenesis. I have found that Arhgef2 is a bona fide transcriptional target of RAS and is upregulated in human tumors harboring RAS mutations. Importantly, depletion of Arhgef2 in RAS-mutated cells inhibits their survival, proliferation, and tumor growth in murine models. In search of the mechanism underlying the requirement of Arhgef2 in RAS tumorigenesis, I have uncovered a novel function for Arhgef2 as a positive regulator of a central RAS pathway, the mitogen-activated protein kinase (MAPK) pathway. Thus, Arhgef2 is part of a positive feedback loop in which RAS-dependent increases in Arhgef2 expression results in the amplification of RAS signaling. Moreover, Arhgef2 confers tumor cells with properties favoring their malignant conversion, thereby implicating Arhgef2 in the formation of metastases. Together, these studies suggest that Arhgef2 plays an important role at multiple stages of tumorigenic progression and may therefore be a promising therapeutic target in RAS-mutated tumors.

Tumorigenesis is driven by the sequential accumulation of genetic lesions within a cell, each which confer the cell with traits that enable its abnormal growth. The result is a mass of dysregulated cells, or tumor, which, upon further mutation, may spread, or metastasize, to other organs of the body. The dissemination of tumor cells makes treatment difficult, and thus confers cancer with its associated lethality. Over the past 30 years, the RAS genes have been critical in teaching us the mechanisms underlying the molecular progression of cancer. RAS is mutated in 33% of all cancers and is often an early event in its stepwise progression. As a result, the RAS genes are widely accepted as ‘drivers’ or ‘initiators’ of human tumorigenesis. Unfortunately, efforts directed at targeting RAS in the clinic have as of yet been unsuccessful. This has triggered a need to identify genes that are required for RAS tumorigenesis that are therapeutically tractable. My research has focused on deciphering the potential role of the Rho GEF Arhgef2 in RAS-mediated tumorigenesis. I have found that Arhgef2 is a bona fide transcriptional target of RAS and is upregulated in human tumors harboring RAS mutations. Importantly, depletion of Arhgef2 in RAS-mutated cells inhibits their survival, proliferation, and tumor growth in murine models. In search of the mechanism underlying the requirement of Arhgef2 in RAS tumorigenesis, I have uncovered a novel function for Arhgef2 as a positive regulator of a central RAS pathway, the mitogen-activated protein kinase (MAPK) pathway. Thus, Arhgef2 is part of a positive feedback loop in which RAS-dependent increases in Arhgef2 expression results in the amplification of RAS signaling. Moreover, Arhgef2 confers tumor cells with properties favoring their malignant conversion, thereby implicating Arhgef2 in the formation of metastases. Together, these studies suggest that Arhgef2 plays an important role at multiple stages of tumorigenic progression and may therefore be a promising therapeutic target in RAS-mutated tumors.

No description available.