Neuronal networks are the central functional units of the nervous system. Knowledge about the identity of participating neurons and the assembly of these during development is crucial for the understanding of CNS function. A promising system to dissect the development and functionalities of a neuronal network is the central pattern generator (CPG) for locomotion. We used screening approaches to identify spinal neuronal subpopulations by their specific gene expression, potentially involved in CPG function. Amongst others we found paired-like homeodomain transcription factor 2 (Pitx2) as a cholinergic interneuron marker for partition cells, with a possible role in the spinal network for locomotion. In addition, we present two genes, Chondrolectin (Chodl) and Estrogen-related receptor beta (ERRβ) as novel markers for fast and slow motor neurons, respectively. The neuronal components of the CPG integrate three key functions; rhythm generation, ipsilateral flexors/extensors coordination and bilateral coordination over the midline. Commissural interneurons (CINs) are considered to participate in the latter. During development axons are guided to their targets by the help of axon guidance molecules. Netrin-1 and its receptor DCC (Deleted in Colorectal Cancer) have been shown to play an important role for spinal cord neurons in axon-pathfinding and migration towards the midline. We show that loss of netrin-1 functionally results in a switch from alternating to synchronous left-right locomotor activity and deletion of DCC surprisingly leads to a different phenotype, best described as uncoordination. Thus, during development, netrin-1 and DCC play a critical role for the establishment of a functional balanced CPG. Further we show a selective loss of CINs, predominantly from dorsally originating subtypes, not affecting the ventral-most V3 subtype in netrin-1 mutant mice, but a loss of CINs from all progenitor domains in Dcc mutant mice. Together, our data suggest a netrin-1-independent mechanism for DCC in axon guidance and a role of the most ventral originating CINs as part of the neuronal network controlling synchronous activities over the midline. Another pair of axon guidance molecules, EphA4 and ephrinB3, has been shown to cooperate in preventing ipsilateral interneurons from crossing the spinal midline and if either molecule is deleted in mice, this will result in a defect in left-right coordination of locomotion. We provide in vivo and in vitro evidence that the GTPase-activating protein α2-chimerin, as a downstream molecule of EphA4 signaling, is essential in axon guidance decisions involved in the correct formation of the spinal circuitry for locomotion.

本研究目的，在探討以行為症狀區分的ADHD次分類型，是否也能在認知功能上有其相對映的區辨？本研究以行為症狀量表DBRS，測量受試者在不專心注意與過動/易衝動向度上的行為症狀表現，以神經心理測驗GDS，測量受試者在不專心注意與過動/易衝動向度上的認知功能表現。 本研究假設認為，以行為症狀量表DBRS所區辨出的不同ADHD次分類型，在神經心理測驗GDS上，應具有不同的認知功能表現模式。透過對63名6~9歲的男性學齡受試者，由行為症狀量表DBRS區分ADHD次分類型，並以單因子變異數分析及Pearson積差相關等方式，檢驗不同次分類型間在神經心理測驗GDS上的認知功能差異與關係。 研究結果顯示，以DBRS所區分的次分類型，在GDS各相關認知功能指標上，都無法有效獲得顯著的差異與關係；大部分的測驗指標，皆未能有效支持本研究假設。研究結果顯示，在本研究樣本中，受試者母親在DBRS上填答的行為症狀表現，與受試者在神經心理測驗GDS上的認知功能表現，未能展現出一致及可茲對映的區辨能力。並在討論中針對此結果，提出可能的原因及相關的建議。 / The purpose of this study is to explore that if ADHD subtypes which are discriminated by behavioral symptoms could reflect the same discriminance on cognitive functions. In this study, the performance of inattention and hyperactive/impulsive behavioral symptoms are measured by the behavioral symptom scale, DBRS; and the performance of inattention and hyperactive/impulsive cognitive functions are measured by the neuropsychological testing, GDS. The hypotheses of this study are that the different ADHD subtypes which are measured by behavioral symptom scale, DBRS, should have different performance pattern of cognitive functions in GDS. This study sorts 63 male subjects, aged from 6 to 9 years old, into 4 subtypes and compares the differences by one way ANOVA and explores the relationships by Pearson product-moment correlation, so as to exam the cognitive differences and the relationships between subtypes on neuropsychological test, GDS. The results show that the subtypes which is discriminated by DBRS have no significant differences on most related cognitive-functioning indexes of GDS. Most testing indexes do not support the hypothese in this study. From the samples of this study the results show that the performance of behavioral symptoms on DBRS, based on mother's evaluation of subject's behavior, and subjects’ performance of cognitive functions on GDS do not have consistent or matched patern. The probabilistic explanation and related suggestion will be proposed in discussion.

Dendritic cells (DC) are antigen-presenting cells that are critical for the initiation and regulation of the immune response. Several DC subtypes within mouse spleen have previously been characterised and these include the plasmacytoid (pDC), and conventional DC (cDC) of the CD8+ and CD8- subtypes. Each subtype appears to have a specialised role in the various arms of immunity and tolerance. Less clear is the process by which these DC develop from haematopoietic precursors, of the precursor stages and branch points from bone marrow (BM) stem cells to each of the peripheral DC subtypes. The research described herein had the aim of identifying and isolating some of the intermediate precursors of DC, downstream of stem cells, and determining whether these differed in the steady-state versus inflammation. Particular was given to DC of the spleen. Experiments that sought the identity of such precursors involved both i) transfer of cell fractions that contained DC precursors into steady-state or inflamed recipient mice to assess their in vivo development at later times, and ii) analysis of an in vitro culture system to question whether it reflected development of the steady-state DC subtypes.

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2018-05-11T13:03:15Z No. of bitstreams: 1 PDF - Aline Ferreira de Araújo Jerônimo.pdf: 26670711 bytes, checksum: 92864afd3e086a9cb19dbb47d53bcc6a (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2018-05-22T20:48:51Z (GMT) No. of bitstreams: 1 PDF - Aline Ferreira de Araújo Jerônimo.pdf: 26670711 bytes, checksum: 92864afd3e086a9cb19dbb47d53bcc6a (MD5) / Made available in DSpace on 2018-05-22T20:48:51Z (GMT). No. of bitstreams: 1 PDF - Aline Ferreira de Araújo Jerônimo.pdf: 26670711 bytes, checksum: 92864afd3e086a9cb19dbb47d53bcc6a (MD5) Previous issue date: 2017-03-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Background: Breast cancer is the type of cancer that most causes deaths among women in developing countries. In Brazil, an incidence of the disease increases and the number of new cases of breast cancer can be attributed to the increase in life expectation of women and changes related to reproductive factors and lifestyle. Objectives: The aim of this study was to analyze the literature on breast cancer risk factors and knowledge about the disease and to identify associations between risk factors and triple-negative breast cancer. Methods: Two scientific articles were developed for publication. The first article deals with an integrative review on the knowledge of breast cancer and its risk factors in Latin American studies. The second article deals with a cross-sectional study on lifestyle risk factors and triple-negative breast cancer, performed with 236 women diagnosed with invasive breast cancer in the state of Paraiba. Results: In the integrative review, of 47 studies selected, 27 were about risk of breast cancer and 20 were about knowledge or awareness. The main risk factors identified by the authors were obesity and overweight, old age, family history of breast cancer, nulliparity and menopause. The articles on knowledge focused in early diagnosis of breast cancer and breast self-examination was the most investigated method of detection. The cross - sectional study showed that smoking and obesity had associations with the triple- negative subtype of breast cancer. Women who smoked sometime in their lives had 3.70 (OR = 0.270, 95% CI: 0.10-0.68, p = 0.006) times less likely to have a triple-negative subtype. Patients with obesity had 3,514 (95% CI: 1.23 - 10.03; p = 0.019) more likely to have the triple-negative subtype when compared to the Luminal A subtype. Conclusions: The literature on breast cancer risk factors presents a better methodological outline and theoretical basis than the literature on the knowledge of the disease. Smoking is associated with lower chances of having triple-negative breast cancer, while obesity is associated with a higher chance of developing this type of breast cancer. / Introdução: O câncer de mama é o tipo de neoplasia que mais causa mortes entre as mulheres nos países em desenvolvimento. No Brasil, a incidência da doença é elevada no Nordeste do país e o aumento no número de casos novos pode ser atribuído às mudanças relacionadas aos fatores reprodutivos e do estilo de vida. Objetivos: Esta dissertação teve como objetivo analisar a literatura sobre os fatores de risco do câncer de mama e o conhecimento sobre a doença e identificar associações entre fatores de risco e o câncer de mama do tipo triplo-negativo. Métodos: Foram desenvolvidos dois artigos científicos para publicação. O primeiro artigo trata-se de uma revisão integrativa sobre o conhecimento da neoplasia mamária e seus fatores de risco em estudos Latino americanos. O segundo artigo trata-se de um estudo transversal sobre fatores de risco do estilo de vida e o câncer de mama triplo-negativo, realizado com 236 mulheres diagnosticadas com câncer de mama invasivo no estado da Paraíba. Resultados: Na revisão integrativa da literatura foram selecionados 47 artigos, dos quais 27 eram sobre fatores de risco do câncer de mama e 20 eram sobre conhecimento ou consciência sobre a doença. Os principais fatores de risco identificados pelos autores foram obesidade e sobrepeso, idade avançada, historia familiar de câncer de mama, nuliparidade e menopausa. Os artigos sobre conhecimento destacaram o diagnóstico precoce da neoplasia mamária e o autoexame foi o método de detecção mais investigado. O estudo transversal mostrou que tabagismo e obesidade tiveram associações com o subtipo triplo-negativo do câncer de mama. As mulheres que já fumaram alguma vez na vida tiveram 3.70 (OR= 0.270; 95% CI: 0.10-0.68; p= 0.006) vezes menos chances de ter o subtipo triplo-negativo. Pacientes com obesidade tiveram 3.514 (95% CI: 1.23- 10.03; p= 0.019) mais chances de ter o subtipo triplo-negativo quando comparadas com o subtipo Luminal A. Conclusões: A literatura sobre fatores de risco do câncer de mama apresenta melhor delineamento metodológico e fundamentação teórica do que a literatura sobre o conhecimento da doença. O tabagismo está associado a menores chances de ter o câncer de mama triplo-negativo, enquanto que a obesidade está associada a maiores chances de desenvolver esse tipo de câncer de mama.

Made available in DSpace on 2014-07-29T15:29:09Z (GMT). No. of bitstreams: 1 dissertacao adriana santarem ferreira.pdf: 1923903 bytes, checksum: fe2d53ac48a0f70b931e84fbecdbf60c (MD5) Previous issue date: 2011-03-11 / The antiretroviral therapy (ART) has reduced morbidity and mortality related to human immunodeficiency virus (HIV) infection. In spite of this advance, the antiretroviral resistance mutations and viral genetic diversity remain the main obstacles in the fight against AIDS. The resistance pattern of HIV-1 to antiretrovirals can be evaluated by resistance tests, specially the genotype testing that allows detection of mutations in the viral genome. This study describes the prevalence of primary HIV-1 drug resistance and subtypes circulating in Mato Grosso, Central West Brazil. Plasma samples from 105 naive patients were colleted during the years 2008 and 2009 to perform the viral resistance genotyping at Tropical Pathology and Public Health Institute, Federal University of Goiás. Protease (PR) and partial reverse transcriptase (RT) were amplified and sequenced from plasma RNA. HIV-1 pol subtypes were assigned by phylogenetic analysis through Los Alamos Database. ARV resistance mutations were analyzed by Stanford University Database and International AIDS Society. Ninety two of the 105 samples had their RNA amplified, 5 (5,43%) of them harboring a resistant strain. Nucleoside reverse-transcriptase inhibitors, nonnucleoside reverse-transcriptase inhibitors and protease inhibitors associed mutations were present in 3 (3,26%), 1(1,08%) and 1(1,08%) samples respectively. Reverse transcriptase gene mutations were observed at códons 219 (K219E), 67 (D67N) e 103 (K103N). Protease gene mutation was observed at códon 90 (L90M). This study revealed that the main mutations are related to reversetranscriptase inhibitors, mainly INTR, reflecting the widespread use of reversetranscriptase inhibitors in the initiation of antiretroviral therapy. Moreover, shows the wide genetic diversity with a significant proportion of distinct BF1 recombinants and the co-circulation of subtypes B, F1 and C in Central West Brazil. / O uso de antirretrovirais (ARVs) reduziu a morbi-mortalidade relacionada à infecção pelo vírus da imunodeficiência humana (HIV). Apesar deste avanço, as mutações de resistência aos antirretrovirais e a diversidade genética do HIV-1 permanecem os principais obstáculos na luta contra a AIDS. O padrão de resistência do HIV-1 aos ARVs pode ser determinado através de testes de resistência, especialmente a genotipagem que permite a detecção de mutações do genoma viral. Este estudo descreve a prevalência de resistência primária às drogas antirretrovirais e subtipos circulantes no Estado de Mato Grosso, no centro-oeste brasileiro. Foram colhidas 105 amostras de plasma de pacientes infectados com HIV-1, virgens de tratamento, durante o período de outubro de 2008 a setembro de 2009 para a realização do teste de resistência genotípica no Instituto de Patologia Tropical e Saúde Pública da Universidade Federal de Goiás. A protease e fragmento da transcriptase reversa do HIV-1 foram amplificados e seqüenciados a partir do RNA plasmático. Os subtipos do gene pol do HIV-1 foram avaliados por análise filogenética por meio do banco de dados de Los Alamos. As mutações de resistência aos ARVs foram analisadas pelo banco de dados da Universidade de Stanford e International AIDS Society. Dos 105 pacientes, 92 tiveram o RNA do HIV-1 amplificado e seqüenciado, sendo que 5 (5,43%) apresentaram cepas resistentes. Entre os 5 pacientes com resistência, 3 (3,26%) apresentavam mutações para os inibidores da transcriptase reversa análogos ao nucleosideo, 1(1,08%) para os inibidores da transcriptase reversa não análogos ao nucleosideo e 1 (1,08%) para os inibidores da protease. As mutações no gene da transcriptase reversa foram observadas nos códons 219 (K219E), 67 (D67N) e 103 (K103N). A mutação no gene da protease foi observada no códon 90 (L90M). Este estudo revelou que as principais mutações encontradas estão relacionadas aos inibidores da transcriptase reversa, principalmente INTR, refletindo o amplo uso de inibidores da transcriptase reversa no início da terapia antirretroviral. Além disso, mostra a grande diversidade genética com significativa proporção de recombinantes BF1 e a co-circulação de subtipos B, F1 e C no centro-oeste brasileiro.

nÃo hà / A variabilidade genÃtica do HIV-1 à reconhecida como um problema em potencial para o diagnÃstico e tratamento do HIV/AIDS, assim como para a transmissÃo, progressÃo da doenÃa e desenvolvimento de vacinas globalmente efetivas, o que torna importante o monitoramento da distribuiÃÃo global dos diferentes subtipos de HIV-1 e formas recombinantes circulantes (CRFs) no Brasil. O presente trabalho teve como objetivo descrever a prevalÃncia dos subtipos do HIV-1 circulantes no Estado do Piauà e o perfil de resistÃncia das cepas identificadas aos ARV, assim como verificar possÃveis associaÃÃes entre os subtipos virais e as informaÃÃes epidemiolÃgicas e laboratoriais da populaÃÃo estudada. As amostras de sangue de 60 pacientes portadores do HIV-1/AIDS foram coletadas no LaboratÃrio Central de SaÃde PÃblica da cidade de Teresina/PI, no perÃodo de maio a abril de 2009. ApÃs a extraÃÃo do DNA proviral, foi realizada a tÃcnica de Nested PCR, para amplificaÃÃo de duas regiÃes genÃmicas (pro e tr), sendo 37 amostras seqÃenciadas posteriormente. Em relaÃÃo à anÃlise do segmento do gene pro, 32 (86,5%) foram do subtipo B e 2 (5,4%) do subtipo D, jà em relaÃÃo ao segmento do gene da tr, todas amostras pertenceram ao subtipo B (20). Na anÃlise estatÃstica foram encontradas associaÃÃes significantes (p < 0,05) entre os subtipos virais com usuÃrios de drogas endovenosas, transfusÃo sanguÃnea e DST. AlÃm disso, verificou-se uma baixa prevalÃncia de cepas com mutaÃÃes resistentes aos inibidores de protease (IP) (2,9%; 1/34), inibidores de transcriptase reversa nucleosÃdicos (ITRN) (15%; 3/20) e nÃo nucleosÃdicos (ITRNN) (10%; 2/20), sugerindo que hà baixa circulaÃÃo de cepas do HIV-1 resistentes aos ARV no estado do PiauÃ. / HIV-1 genetic variability is a wellkown problem that makes diagnosis and treatment difficult to manage. Also, this variability is a problem for trasmission, disease progression and for the development of vaccines, being important to know the subtypes presented in each population worldwilde and in Brazil to provide more effective treatment. The present study have the aim to describe the subtype prevalent in Piauà state, the resistance profile of thouse to antiviral treatment, laboratory exams and the epidemiologic point of view of HIV-1 in PiauÃ. Samples of blood from 60 patients (HIV-1 positive) were colect at Central Public Laboratory in Teresina/PI, from maio to april 2009. Proviral DNA were isolated and Nested PCR were performed for two genomic regions (pro e tr), being sequenced 37 samples. Analysis to the segment for gene pro, 32 (86,5%) were subtype B and 2 (5,4%) subtype D. For tr segment all samples were subtype B (20). Statistics analysis found a significant association between vÃrus subtype and drug users, blood transfusion and STD (p < 0,05). Furthermore, the study reveal that even with two subtypes of HIV-1 detected (B e D), a low prevalence of drug resistance was observed to the protease inhibitors (PI) (2,9%; 1/34), nucleosidic reverse transcriptase inhibitor (NRTI) (15%; 3/20) and non nucleosidic (NNRTI) (10%; 2/20).

abstract: Eosinophils are innate immune cells that are most commonly associated with parasite infection and allergic responses. Recent studies, though, have identified eosinophils as cells with diverse effector functions at baseline and in disease. Eosinophils in specific tissue immune environments are proposed to promote unique and specific effector functions, suggesting these cells have the capacity to differentiate into unique subtypes. The studies here focus on defining these subtypes using functional, molecular, and genetic analysis as well as using novel techniques to image these subtypes in situ. To characterized these subtypes, an in vitro cytokine induced type 1 (E1) and type 2 (E2) eosinophil model was developed that display features and functions of eosinophils found in vivo. For example, E1 eosinophils secrete type 1 mediators (e.g., IL-12, CXCL9 and CXCL10), express iNOS and express increased levels of the surface molecules PDL1 and MHC-I. Conversely, E2 eosinophils release type 2 mediators (e.g., IL4, IL13, CCL17, and CCL22), degranulate and express increased surface molecules CD11b, ST2 and Siglec-F. Completion of differential expression analysis of RNAseq on these subtypes revealed 500 and 655 unique genes were upregulated in E1 and E2 eosinophils, respectively. Functional enrichment studies showed interferon regulatory factor (IRF) transcription factors were uniquely regulated in both mouse and human E1 and E2 eosinophils. These subtypes are sensitive to their environment, modulating their IRF and cell surface expression when stimulated with opposing cytokines, suggesting plasticity. To identify and study these subtypes in situ, chromogenic and fluorescent eosinophil-specific immunostaining protocols were developed. Methods were created and optimized, here, to identify eosinophils by their granule proteins in formalin fixed mouse tissues. Yet, eosinophil-specific antibodies alone are not enough to identify and study the complex interactions eosinophil subtypes perform within a tissue. Therefore, as part of this thesis, a novel highly-multiplexed immunohistochemistry technique was developed utilizing cleavable linkers to address these concerns. This technique is capable of analyzing up to 22 markers within a single biopsy with single-cell resolution. With this approach, eosinophil subtypes can be studied in situ in routine patient biopsies. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

Response to Intervention (RTI) has become the gateway to identification for many students with specific learning disabilities. Those include students in the 17 states that require RTI as the source of eligibility data as well as many students in the 33 states that allow districts to choose RTI among other options (e.g., discrepancy). There is concern that the use of RTI may infringe on due-process protections and child-find responsibilities. Specifically, the number of days students must remain in RTI may delay their referral for comprehensive evaluation and, hence, potential eligibility for special education. In the present study, the authors surveyed District Directors of Special Education in selected states to determine whether guidelines or policies were present to govern referrals for a comprehensive evaluation and how long a student may remain in RTI tiers before referral or eligibility determination. The extent to which districts permit individual schools to enact their own policies was also investigated. Results indicate that (a) RTI is often used to identify specific learning disabilities without clear guidelines, (b) many aspects of RTI are implemented without being communicated within state and district administrative levels, and (c) RTI is the required specific learning disability assessment determinant in a significant percentage of districts in states that allow that choice. Implications for future research and practice are discussed.

Previous studies have used new angiotensin II (AII) receptor subtype selective compounds to localize AII receptor subtypes within discrete rat brain nuclei. The purpose of this autoradiographic study was to extend these preliminary findings and provide a comprehensive analysis of AII binding sites in 22 rat brain nuclei and the anterior pituitary, to include estimates of the binding affinity for 125I sar1 ile8 AII (125I SIAII) at each nucleus, and determine the fractional distribution of each subtype at each nucleus. Estimates of K(D), in separate experiments revealed that AT1 nuclei had a consistently higher affinity for 125I SIAII than AT2 nuclei (0.66 vs. 2.55 nM). Displacement of subsaturating concentrations of 125I SIAII by 10-8-10-4 M DuP753 (selective for the AT1 subtype) or PD123177 (selective for the AT2 subtype) indicated that approximately half of the brain regions surveyed contained predominantly AT1 sites and half contained predominantly AT2 sites. Binding was partially displaced by both compounds in several regions and two site analyses were performed to estimate the distribution of subtypes within each nucleus. The data were then corrected for differential occupancy by 125I SIAII. Brain nuclei associated with cardiovascular or dipsogenic actions of AII, e.g., subfornical organ, organum vasculosum of the lamina terminalis, median preoptic nucleus, nucleus of the solitary tract and area postrema, contained pure, or almost pure, populations of AT1 receptors. The functions of AII in brain regions containing predominantly AT2 binding sites, e.g., thalamus, colliculi, inferior olive and locus ceruleus, remain undefined. Thus, AII binding sites in the rat brain have been differentiated into two subtypes with similar characteristics to those reported in peripheral tissues. However, the unexpected finding that they can be differentiated on the basis of their affinity for 125I SIAII raises questions concerning their coidentity with peripheral receptor subtypes.

Objective: This study aimed to identify the programmed death ligand-1 (PDL1, also termed as CD274) and its positively correlated immune checkpoint genes (ICGs) and to determine the immune subtypes of CD274-centered ICG combinations in oral and squamous cell carcinoma (OSCC). Materials and Methods: Firstly, the 95 ICGs obtained via literature reviews were identified in the Cancer Genome Atlas (TCGA) database in relation to OSCC, and such 88 ICG expression profiles were extracted. ICGs positively correlated with CD274 were utilized for subsequent analysis. The relationship between ICGs positively correlated with CD274 and immunotherapy biomarkers (tumor mutation burden (TMB), and adaptive immune resistance pathway genes) was investigated, and the relationships of these genes with OSCC clinical features were explored. The prognostic values of CD274 and its positively correlated ICGs and also their associated gene pairs were revealed using the survival analysis. Results: Eight ICGs, including CTLA4, ICOS, TNFRSF4, CD27, B- and T-lymphocyte attenuator (BTLA), ADORA2A, CD40LG, and CD28, were found to be positively correlated with CD274. Among the eight ICGs, seven ICGs (CTLA4, ICOS, TNFRSF4, CD27, BTLA, CD40LG, and CD28) were significantly negatively correlated with TMB. The majority of the adaptive immune resistance pathway genes were positively correlated with ICGs positively correlated with CD274. The survival analysis utilizing the TCGA-OSCC data showed that, although CD274 was not significantly associated with overall survival (OS), the majority of ICGs positively correlated with CD274 (BTLA, CD27, CTLA4, CD40LG, CD28, ICOS, and TNFRSF4) were significantly correlated with OS, whereby their low-expression predicted a favorable prognosis. The survival analysis based on the gene pair subtypes showed that the combination subtypes of CD274_low/BTLA_low, CD274_low/CD27_low, CD274_low/CTLA4_low, CD8A_high/BTLA_low, CD8A_high/CD27_low, and CD8A_high/CTLA4_low predicted favorable OS. Conclusion: The results in this study provide a theoretical basis for prognostic immune subtyping of OSCC and highlight the importance of developing future immunotherapeutic strategies for treating oral cancer.