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Hyperglycemia in critically ill patients was considered for many years an adaptive response to stress conditions being present in both patients with and without previous history of diabetes. Hyperglycemia is caused mainly by peripheral insulin resistance induced by the factors acting counteracting insulin signalling at the postreceptor level. Furthermore, hyperglycemia itself can then increase serum levels of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-6 (Il-6) and interleukin-8 (Il- 8) and others. On the contrary, peripheral insulin resistance induced by pro- inflammatory cytokines may further potentiate hyperglycemia. White adipose tissue represents in addition to its energy storage function also a very active endocrine active organ. In addition to regulation of a number of metabolic processes it also significantly modulates the inflammatory response. In critically ill patients, adipose tissue changes its morphology, i.e. the adipocytes are shrinking and adipose tissue is abundantly infiltrated by macrophages. Paradoxically, overweight and obese critically ill patients have lower mortality than underweight, lean and morbidly obese subjects. In our studies, we selected population of the patients undergoing elective major cardiac surgery with extracorporeal...

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Orientadores: Maria Salete Costa Gurgel, Sílvia de Barros-Mazon / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T03:12:53Z (GMT). No. of bitstreams: 1 Gimenez_RodrigoPinto_D.pdf: 2727620 bytes, checksum: 505f7780aca2f535e4882eb250f83b9d (MD5) Previous issue date: 2013 / Resumo: Introdução: A obesidade tem se mostrado responsável pelo aumento de 30% a 50% dos casos novos de câncer de mama, em particular na pós-menopausa. A mais recente hipótese para explicar tal fato situa os adipócitos e suas funções autócrina, parácrina e endócrina no centro do cenário, através da relação das adipocinas, por ele secretadas, com a obesidade e o câncer de mama. Objetivo: Artigo 1- Comparar o padrão de expressão imunoistoquímica da adiponectina (APN) e dos seus receptores tipos 1 e 2 (adipoR1/R2) no carcinoma invasor (CDI), carcinoma ductal in situ (CDIS) e lesões benignas da mama (BE) e correlacioná-los com parâmetros clínicos e histológicos. Artigo 2- Avaliar a expressão protéica da FABP4 nos tecidos epiteliais e adiposos mamário de portadoras de CDI, CDIS e lesões benignas da mama. Material e Métodos: Foram incluídos os blocos de parafina de 223 mulheres sendo 69 com CDI, 73 com CDIS e 81 com biópsias negativas para câncer de mama, tratadas no CAISM/UNICAMP de janeiro de 2008 a dezembro de 2011, e preparadas lâminas de Tissue Microarray (TMA). A expressão de APN e Adipo R1/R2 foi avaliada no tecido tumoral nos casos CDI e CDIS e no tecido epitelial e nos casos benignos. A expressão de FABP4 foi avaliada no tecido tumoral, na gordura peritumoral (GP) e na gordura mamária distante (GD) nos casos de CDI e CDIS, e no tecido epitelial e gorduras mamários nos casos benignos. Para avaliar uma possível relação entre a expressão dos marcadores entre si e com parâmetros antropométricos, clínicos e histopatológicos, foram utilizados os testes qui-quadrado ou exato de Fisher, Mann-Whitney, Kruskal-Wallis e correlação de Spearman. As determinações foram calculadas considerando o valor de ?=0,05 (p<0,05). Resultados: Artigo 1 - A APN mostrou-se expressa em 65% dos CDI, 48% dos CDIS e 12% das BE e AdipoR1 em 98%, 94% e 71%, respectivamente. Todos os casos de CDI e CDIS expressaram AdipoR2 contra 81% de BE. Nos CDI e CDIS observou-se associação entre maior expressão de APN e tumores RE negativo. No CDIS esta associação foi também observada com RP negativo. Artigo 2 - Observou-se expressão protéica da FABP4 no tecido epitelial em 90% dos CDI, 40% dos CDIS e 28% em BE. Considerando-se a GP e GD esta expressão foi maior nas BE que nos CDI, diferenças consideradas significativas. Nas pacientes com CDI a expressão da FABP4 foi maior quando o diagnóstico ocorreu até 50 anos de idade. A totalidade dos casos expressou moderada a intensamente este marcador no tecido gorduroso periepitelial e distante. Conclusões: As diferenças de expressões protéicas da adiponectina e dos seus receptores AdipoR1/R2 observadas em diferentes diagnósticos mamários sugerem sua participação no complexo mecanismo etiológico destas diferentes condições. Os resultados deste estudo indicam, ainda, que existe uma correlação direta entre expressão protéica da FABP4, câncer de mama e obesidade / Abstract: Introduction: Obesity has been shown to be responsible for a 30 to 50% increase in new breast cancer cases, in particular in the postmenopausal period. The most recent hypothesis that explains this fact places adipocytes and its autocrine, paracrine and endocrine functions at center stage, linking adipokines secreted by adipocytes to obesity and breast cancer. Objective: Article 1- to compare immunohistochemistry expression pattern of adiponectin (APN) and its receptors types 1 and 2 (adipoR1/R2) in invasive carcinoma (IDC), ductal carcinoma in situ (CDIS) and benign breast lesions (BE), correlated with clinical and histological parameters. Article 2- To assess FABP4 protein expression in epithelial and adipose breast tissue in women diagnosed with IDC, DCIS and benign breast lesions. Material and Methods: Paraffin-embedded blocks from 223 women were included. Of the total number of women, 69 had IDC CDI, 73 had CDIS and 81 had biopsies negative for breast cancer. The patients have been treated at CAISM/Unicamp from January 2008 to December 2011 and Tissue Microarray (TMA) slides were constructed. Expression of APN and Adipo R1/R2 was assessed in tumor tissue in cases of IDC and DCIS and in epithelial tissue in benign cases. FABP4 expression was evaluated in tumor tissue, peritumoral fat tissue (PF) and distant fat breast tissue (DF) in cases of IDC and DCIS and in the epithelial tissue and breast fat tissue in benign cases. To assess a possible relationship between marker expression and anthropometric, clinical and histopathological parameters, the chi-square test or Fisher's exact test, Mann-Whitney test, Kruskal-Wallis test and Spearman's correlation were used. Determinations were calculated, considering a value ?=0.05 (p<0.05) as significant. Results: Article 1 - APN was shown to be expressed in 65% of IDC, 48% of DCIS and 12% of BE and AdipoR1 in 98%, 94% and 71%, respectively. All IDC and DCIS cases expressed AdipoR2 versus 81% of BE. In IDC and DCIS, an association between a higher level of APN expression and ER-negative tumors was observed. In DCIS, this association was also observed with PR-negative tumors. Article 2 - FABP4 protein expression was observed in epithelial tissue in 90% of CDI, 40% of DCIS and 28% of BE. Considering PF and DF, FABP4 expression had a higher level in BE than in IDC, a difference that was considered significant. In patients with IDC, FABP4 expression was higher when diagnosis was made in patients aged up to 50 years. In all cases, this marker was moderately to intensely expressed in the peri-epithelial and distant fat tissue. Conclusions: Discrepancies in protein expression of adiponectin and its receptors AdipoR1/R2 observed in different breast diagnoses suggest its participation in the complex etiologic mechanism of these different conditions. Our results indicate that there is a direct correlation between FABP4 protein expression, breast cancer and obesity / Doutorado / Oncologia Ginecológica e Mamária / Doutor em Ciências da Saúde

BACKGROUND: Quantitative evidence of direct links between dietary intake during pregnancy and maternal and infant outcomes in the preterm population is lacking in the literature. However, studies are starting to investigate relationships between them, and data shows that many of the adverse effects of poor maternal diet are linked to inflammatory response and dysbiosis of the microbiome in both the mother and her offspring. OBJECTIVE: Our objective with this study was to analyze the relationships between sociodemographic factors, maternal diet, pH of breast milk, and infant inflammation. We aimed to assess the dietary pattern of our population of mothers who delivered preterm in comparison to other populations, as well as explore the possibility of using pH of mother’s own milk in future research. METHODS: We reviewed the Electronic Medical Records (EMRs) of participants to gather clinical and demographic characteristics (infants n = 53; mothers n = 45). Maternal participants also completed the Dietary Screener Questionnaire (DSQ). Spearman’s rank correlation and raw unadjusted linear regression analyses were used to investigate relationships between maternal diet and characteristics, mother’s own milk pH, and infant urinary intestinal fatty acid binding protein (I-FABP). Kruskal Wallis analysis was used to analyze between group differences of maternal comorbidities. RESULTS: None of our maternal participants met the guidelines for dietary recommendations by the United States Department of Agriculture (USDA) for pregnant women. This follows the trend in national data for women who are pregnant. The greatest number of women met the recommendation for fiber intake (n = 12). Maternal intake of fiber and whole grains was negatively correlated with pH of mother’s own milk (p <0.5). We did not find any significant correlations between maternal characteristics and maternal diet or pH of mother’s own milk. However, meeting the guidelines for added sugars differed by race (p = 0.03). We found no statistically significant correlations between urinary I-FABP and pH of mother’s own milk or maternal dietary intake. Urinary I-FABP values differed by infant sex (p = 0.03) and infant feeding status (> 50% formula or donor milk vs. < 50% formula or donor milk, p = 0.03). Analysis by groups showed statistically significant differences (p = 0.04). Preeclamptic participants had a higher intake of whole grains (0.97 oz) than women without preeclampsia (0.69 oz). Mothers with gestational diabetes had a lower intake of sugar (14.4 tsp) compared to women without diabetes (17.9 tsp) (p = 0.01). We found no other statistically significant results between groups for maternal diet, milk pH, or urinary I-FABP. CONCLUSION: Our findings suggest that additional research on mother’s own milk pH may be warranted, and that continued education on the importance of a healthy diet and its benefits during pregnancy is needed. Areas of planned future research include fat intake calculations and inflammatory measures of the maternal dietary data.

Background: Diagnosis of acute coronary syndromes (ACS) in the Emergency Department (ED) is a topical and contentious issue. Current diagnostic techniques rely on hospital admission for troponin testing. Only a minority of those admitted prove to have ACS while unacceptable proportions of those discharged have unrecognised ACS. Aims: We aimed to evaluate the diagnostic and prognostic value of individual clinical findings and novel biomarkers in ED patients with suspected cardiac chest pain. We then aimed to derive a clinical decision rule (CDR) to potentially enable safe, immediate discharge of a proportion of patients from the ED while risk stratifying others to facilitate triage to an appropriate level of in-patient care. Methods: We recruited patients who presented to the ED with suspected cardiac chest pain. Variables that have previously been shown to predict diagnosis of acute myocardial infarction (AMI) or to predict outcome were prospectively recorded. Blood was drawn at presentation for levels of eight biomarkers. Patients underwent 12-hour troponin testing and were followed up for the composite primary outcome of AMI, death or urgent coronary revascularisation for six months. Variables that were univariate predictors (p<0.05) of outcome were entered into a multivariate analysis using recursive partitioning. Results: While many clinical findings and levels of all eight novel biomarkers were found to be significant predictors of outcome, none could be used individually to confirm or exclude ACS in the ED. We derived a nine-point CDR that combined clinical findings with biomarker levels to effectively stratify patients into four risk groups. 14.2% of patients were identified as being at ‘no risk’ and had a 0.0% outcome rate. The rule performed significantly better than two commonly used risk scores and may improve on triage decisions made in actual clinical practice. Conclusion: ACS remains a difficult diagnosis to confidently confirm or refute in the ED. Our CDR may help to avoid unnecessary hospital admissions while improving on triage decisions made for the remaining in-patients. Prospective validation of our findings is warranted.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Nas abelhas sociais Apis mellifera a determinação de castas está relacionada à nutrição diferencial durante o desenvolvimento larval. Os indivíduos são alimentados com geléia real até o terceiro estágio larval, quando aqueles que são destinados a se tornarem operárias passam a receber uma mistura de secreções glandulares, mel e pólen. O conteúdo da dieta recebida após o terceiro estágio larval ativará respostas endócrinas diferenciais que resultarão no estímulo de vias distintas de expressão gênica que culminarão no desenvolvimento de rainhas e operárias. Vários modelos de determinação de castas foram propostos envolvendo diferentes fatores que atuam sobre o desenvolvimento de cada uma, em especial o Hormônio Juvenil (HJ), as vias de sinalização por insulina/IGF e TOR (target of rapamycin) a metilação diferencial e a proteína recentemente descoberta, royalactin, que favorecem o desenvolvimento de rainhas. Para o desenvolvimento de operárias foi sugerido estímulo de outras vias de sinalização, que possivelmente envolveria a participação dos genes ultraspiracle (usp), cryptocephal (crc) e retinoid- and fatty acid-binding protein (RfaBp). Utilizando diferentes abordagens avaliamos a participação destes genes no processo que culmina no desenvolvimento das castas. Através da análise de expressão gênica em larga escala utilizando microarrays, observamos a existência de genes diferencialmente expressos em rainhas e operárias, sendo a maior que parte deles apresentou expressão preferencial em operárias. Muitos destes genes, inclusive esterase do hormônio juvenil (jhe), failed axon connections (fax), activating transcription factor-3 (atf-3), cathepsin-D (cath-D) e peptidoglycan recognition protein-SC2 (pgrp-sc2), preferencialmente expressos em operárias, estão envolvidos, segundo análises de função por Gene Ontology, em processos essenciais no desenvolvimento das castas como crescimento, reprodução, apoptose, neurogênese, degradação do hormônio juvenil, entre outros. A partir destes resultados, incluímos o gene da esterase do hormônio juvenil (jhe) em nossas análises, como um possível candidato a determinante do desenvolvimento diferencial das operárias. Além disto, foi determinado o perfil de expressão de usp, crc, RfaBp e jhe, durante o desenvolvimento de rainhas e operárias. Observamos que os maiores níveis de expressão de cada um são encontrados em fases posteriores ao período crítico de determinação de castas e que em geral, os maiores níveis de expressão são encontrados em operárias, especialmente crc, RfaBp e jhe. Para usp, os níveis são distintos em rainhas e operária apenas em pontos específicos entre o quinto estágio larval e a fase pré-pupal. Adicionalmente avaliamos a influência da diminuição, através de interferência por RNA (RNAi), dos níveis de expressão de cada um destes genes sobre os níveis dos outros genes estudados, e também sua atuação no desenvolvimento. Vimos que mesmo pequenas modificações nestes níveis inibem ou estimulam a expressão de outros genes e, em alguns casos causam alterações no desenvolvimento das abelhas. Sabendo da importância dos microRNAs (miRNAs) na regulação da expressão gênica e do desenvolvimento, avaliamos os níveis de expressão dos miRNAs preditos como reguladores de jhe. Os resultados obtidos mostraram que alguns deles, como let-7, miR-2796 e miR-263b, por apresentarem correlação negativa com os níveis do gene alvo, são realmente fortes candidatos a seus reguladores. Além disto, alterações nos níveis do gene alvo, mostraram a capacidade de alterar os níveis de expressão da maioria dos miRNAs preditos. Este resultado foi corroborado por sequenciamento em larga escala das amostras tratadas com dsjhe e controle, que apontou também outros possíveis reguladores de jhe, entre eles miR-100, miR-306 e mi-13b. Analisando os resultados obtidos de forma conjunta podemos sugerir que o desenvolvimento de operárias está sob complexa regulação que envolve a participação dos genes aqui estudados, além de outros fatores como os miRNAs. Estes genes agem de maneira coordenada, inclusive com os miRNAs, em momentos específicos do desenvolvimento atuando sobre cascatas de expressão gênica de forma a ativar ou inibir a expressão uns dos outros e também de outros genes, o que culminará no desenvolvimento diferencial de rainhas e operárias em A. mellifera. / In Apis mellifera, a eusocial bee, caste determination during larval development is regulated by differential nutrition. All female larvae are fed royal jelly until the third larval stage, when the workerdestined ones have their diet switched to a gland secretion mix, honey and pollen, Queen-destined larvae, however, are provisioned with a rich diet throughout development. Changes in diet after the third developmental stage modulate larval endocrine responses and different nutritional regimes trigger distinct patterns of gene expression. Thus, nutritional regulation of specific signaling pathways controls development of worker and queen phenotypes. Several proposed models of this process involve caste-specific regulation of hormonal and nutritional factors and/or molecular processes including Juvenile Hormone (JH), insulin/IGF and TOR (target of rapamycin) signaling pathways, differential methylation and the newly discovered protein royalactin, which facilitates queen development. Previous research has also suggested the stimulus of other factors in signaling pathways that acts towards workers development, and they possibly involves the participation of some genes like ultraspiracle (usp), cryptocephal (crc), and retinoid- and fatty acid-binding protein (RfaBp). Using diverse molecular approaches, we evaluate the role of these genes in caste differentiation. We used microarrays to characterize global differences in gene expression between queen and worker larvae. Functional analysis of significantly, differentially expressed genes identified fundamental biological processes including growth, reproduction, apoptosis, neurogenesis and JH degradation that are involved in caste differentiation. Based on these findings, we selected several candidate genes including juvenile hormone esterase (jhe), failed axon connections (fax), activating transcription factor-3 (atf-3), cathepsin-D (cath-D), and peptidoglycan recognition protein-SC2 (pgrp-sc2) for investigation. Notably, these genes were preferentially upregulated in workers. In a separate experiment, we monitored expression of usp, crc, RfaBp and jhe, in queen and worker larval during stages critical to caste determination. In general, workers expressed crc, RfaBp and jhe at higher levels than queens. For usp, distinct expression levels between worker- and queen-destined larvae were observed only at specific points between the 5th larval stage and pre-pupal phase. Additionally we used RNA interference (RNAi) to monitor the impact of decreased levels of select candidate genes on larval development. We found that even small modification of gene expression levels inhibited or triggered expression of other genes, and, in some cases, caused developmental alterations. Furthermore, microRNAs (miRNAs) are also important regulators of gene expression during development and we identified miRNAs that were predicted jhe regulators and assessed their levels. Results determined that some miRNAs including let-7, miR-2796 e miR-263b were strong candidates for jhe regulation because they were significantly and negatively correlated with target gene expression levels. Furthermore, manipulation of target gene expression levels altered expression of most predicted miRNAs. These results were confirmed by deep sequencing of RNAi samples treated with double-stranded RNA for jhe gene (dsjhe) and controls (with no treatment) which also identified other candidate jhe regulators, like miR-100, miR-306 and mi-13b. Taken together, these results suggest that worker development is regulated by complex interactions that involve usp, crc, RfaBp and jhe in addition to other molecules, including miRNAs. These molecular participants coordinate development at specific time points by regulating activity of gene networks and each other, producing the differential development of workers and queens in A. mellifera.

Noncovalent interactions are involved in many biological processes in which biomolecules bind specifically and reversibly to a partner. Often, proteins do not have a biological activity without the presence of a partner, a ligand. Biological signals are produced when proteins interact with other proteins, peptides, oligonucleotides, nucleic acids, lipids, metal ions, polysaccharides or small organic molecules. Some key steps in the drug discovery process are based on noncovalent interactions. We have focused our research on the steps involving ligand screening, competitive binding and ‘off-target’ binding. The first paper in this thesis investigated the complicated electrospray ionization process with regards to noncovalent complexes. We have proposed a model that may explain how the equilibrium between a protein and ligand changes during the droplet evaporation/ionization process. The second paper describes an evaluation of an automated chip-based nano-ESI platform for ligand screening. The technique was compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation was obtained between the results obtained with the two methods. As a general conclusion we believe that the automated nano-ESI/MS should have a great potential to serve as a complementary screening method to conventional HTS. Alternatively, it could be used as a first screening method in an early phase of drug development programs when only small amounts of purified targets are available. In the third article, the advantage of using on-line microdialysis as a tool for enhanced resolution and sensitivity during detection of noncovalent interactions and competitive binding studies by ESI-MS was demonstrated. The microdialysis device was improved and a new approach for competitive binding studies was developed. The last article in the thesis reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II. / QC 20100705

Nas abelhas sociais Apis mellifera a determinação de castas está relacionada à nutrição diferencial durante o desenvolvimento larval. Os indivíduos são alimentados com geléia real até o terceiro estágio larval, quando aqueles que são destinados a se tornarem operárias passam a receber uma mistura de secreções glandulares, mel e pólen. O conteúdo da dieta recebida após o terceiro estágio larval ativará respostas endócrinas diferenciais que resultarão no estímulo de vias distintas de expressão gênica que culminarão no desenvolvimento de rainhas e operárias. Vários modelos de determinação de castas foram propostos envolvendo diferentes fatores que atuam sobre o desenvolvimento de cada uma, em especial o Hormônio Juvenil (HJ), as vias de sinalização por insulina/IGF e TOR (target of rapamycin) a metilação diferencial e a proteína recentemente descoberta, royalactin, que favorecem o desenvolvimento de rainhas. Para o desenvolvimento de operárias foi sugerido estímulo de outras vias de sinalização, que possivelmente envolveria a participação dos genes ultraspiracle (usp), cryptocephal (crc) e retinoid- and fatty acid-binding protein (RfaBp). Utilizando diferentes abordagens avaliamos a participação destes genes no processo que culmina no desenvolvimento das castas. Através da análise de expressão gênica em larga escala utilizando microarrays, observamos a existência de genes diferencialmente expressos em rainhas e operárias, sendo a maior que parte deles apresentou expressão preferencial em operárias. Muitos destes genes, inclusive esterase do hormônio juvenil (jhe), failed axon connections (fax), activating transcription factor-3 (atf-3), cathepsin-D (cath-D) e peptidoglycan recognition protein-SC2 (pgrp-sc2), preferencialmente expressos em operárias, estão envolvidos, segundo análises de função por Gene Ontology, em processos essenciais no desenvolvimento das castas como crescimento, reprodução, apoptose, neurogênese, degradação do hormônio juvenil, entre outros. A partir destes resultados, incluímos o gene da esterase do hormônio juvenil (jhe) em nossas análises, como um possível candidato a determinante do desenvolvimento diferencial das operárias. Além disto, foi determinado o perfil de expressão de usp, crc, RfaBp e jhe, durante o desenvolvimento de rainhas e operárias. Observamos que os maiores níveis de expressão de cada um são encontrados em fases posteriores ao período crítico de determinação de castas e que em geral, os maiores níveis de expressão são encontrados em operárias, especialmente crc, RfaBp e jhe. Para usp, os níveis são distintos em rainhas e operária apenas em pontos específicos entre o quinto estágio larval e a fase pré-pupal. Adicionalmente avaliamos a influência da diminuição, através de interferência por RNA (RNAi), dos níveis de expressão de cada um destes genes sobre os níveis dos outros genes estudados, e também sua atuação no desenvolvimento. Vimos que mesmo pequenas modificações nestes níveis inibem ou estimulam a expressão de outros genes e, em alguns casos causam alterações no desenvolvimento das abelhas. Sabendo da importância dos microRNAs (miRNAs) na regulação da expressão gênica e do desenvolvimento, avaliamos os níveis de expressão dos miRNAs preditos como reguladores de jhe. Os resultados obtidos mostraram que alguns deles, como let-7, miR-2796 e miR-263b, por apresentarem correlação negativa com os níveis do gene alvo, são realmente fortes candidatos a seus reguladores. Além disto, alterações nos níveis do gene alvo, mostraram a capacidade de alterar os níveis de expressão da maioria dos miRNAs preditos. Este resultado foi corroborado por sequenciamento em larga escala das amostras tratadas com dsjhe e controle, que apontou também outros possíveis reguladores de jhe, entre eles miR-100, miR-306 e mi-13b. Analisando os resultados obtidos de forma conjunta podemos sugerir que o desenvolvimento de operárias está sob complexa regulação que envolve a participação dos genes aqui estudados, além de outros fatores como os miRNAs. Estes genes agem de maneira coordenada, inclusive com os miRNAs, em momentos específicos do desenvolvimento atuando sobre cascatas de expressão gênica de forma a ativar ou inibir a expressão uns dos outros e também de outros genes, o que culminará no desenvolvimento diferencial de rainhas e operárias em A. mellifera. / In Apis mellifera, a eusocial bee, caste determination during larval development is regulated by differential nutrition. All female larvae are fed royal jelly until the third larval stage, when the workerdestined ones have their diet switched to a gland secretion mix, honey and pollen, Queen-destined larvae, however, are provisioned with a rich diet throughout development. Changes in diet after the third developmental stage modulate larval endocrine responses and different nutritional regimes trigger distinct patterns of gene expression. Thus, nutritional regulation of specific signaling pathways controls development of worker and queen phenotypes. Several proposed models of this process involve caste-specific regulation of hormonal and nutritional factors and/or molecular processes including Juvenile Hormone (JH), insulin/IGF and TOR (target of rapamycin) signaling pathways, differential methylation and the newly discovered protein royalactin, which facilitates queen development. Previous research has also suggested the stimulus of other factors in signaling pathways that acts towards workers development, and they possibly involves the participation of some genes like ultraspiracle (usp), cryptocephal (crc), and retinoid- and fatty acid-binding protein (RfaBp). Using diverse molecular approaches, we evaluate the role of these genes in caste differentiation. We used microarrays to characterize global differences in gene expression between queen and worker larvae. Functional analysis of significantly, differentially expressed genes identified fundamental biological processes including growth, reproduction, apoptosis, neurogenesis and JH degradation that are involved in caste differentiation. Based on these findings, we selected several candidate genes including juvenile hormone esterase (jhe), failed axon connections (fax), activating transcription factor-3 (atf-3), cathepsin-D (cath-D), and peptidoglycan recognition protein-SC2 (pgrp-sc2) for investigation. Notably, these genes were preferentially upregulated in workers. In a separate experiment, we monitored expression of usp, crc, RfaBp and jhe, in queen and worker larval during stages critical to caste determination. In general, workers expressed crc, RfaBp and jhe at higher levels than queens. For usp, distinct expression levels between worker- and queen-destined larvae were observed only at specific points between the 5th larval stage and pre-pupal phase. Additionally we used RNA interference (RNAi) to monitor the impact of decreased levels of select candidate genes on larval development. We found that even small modification of gene expression levels inhibited or triggered expression of other genes, and, in some cases, caused developmental alterations. Furthermore, microRNAs (miRNAs) are also important regulators of gene expression during development and we identified miRNAs that were predicted jhe regulators and assessed their levels. Results determined that some miRNAs including let-7, miR-2796 e miR-263b were strong candidates for jhe regulation because they were significantly and negatively correlated with target gene expression levels. Furthermore, manipulation of target gene expression levels altered expression of most predicted miRNAs. These results were confirmed by deep sequencing of RNAi samples treated with double-stranded RNA for jhe gene (dsjhe) and controls (with no treatment) which also identified other candidate jhe regulators, like miR-100, miR-306 and mi-13b. Taken together, these results suggest that worker development is regulated by complex interactions that involve usp, crc, RfaBp and jhe in addition to other molecules, including miRNAs. These molecular participants coordinate development at specific time points by regulating activity of gene networks and each other, producing the differential development of workers and queens in A. mellifera.