*4.1. Sickness Absenteeism*

The average annual rate of days of absence of civil servants of the three Legislative Houses in this study was 8.73 d/s/yr, which is relatively moderate when compared to other national and international cases of work absences in the public service due to health problems. The sickness absenteeism of civil servants of the City Halls of Goiania, between 2005 and 2010, and Coritiba, between 2010 and 2015, had an average of working days lost of 12.07 d/s/yr [4] and 23.04 d/s/yr, respectively [6]. Similar studies with civil servants in Canada and Australia indicated an average civil servant absence of 11.6 d/s/yr [5]. However, much lower values of days away from work in the public sector have been reported, as in the case of the UK countries that achieved an average annual working day loss of 4.4 d/s/yr in 2018.

The poorer results of sick leave in the legislative houses of the present study may be related to the less adverse general working conditions in the executive branch, mainly in the areas of education, health, and public security. These areas present frequent occurrences of absence from work due to health problems, considering the pathological potential of these sectors, due to direct contact with the public, high social demand for such services, or operational difficulties faced by educators and health and public safety professionals. Moreover, the legislative servants participating in this study perform only internal administrative activities of support to parliamentarians and have their own health service that provides care, including basic health promotion actions.


**LYMPHOCYTE UPDATES - CANCER, AUTOIMMUNITY AND INFECTION**

Edited by **Gheorghita Isvoranu**

#### **Lymphocyte Updates - Cancer, Autoimmunity and Infection**

http://dx.doi.org/10.5772/66613 Edited by Gheorghita Isvoranu

#### **Contributors**

Simone Bürgler, Won Fen Wong, Hamiyet Donmez-Altuntas, Ahmet Eken, Tatsuaki Tsuruyama, Teodora Karan-Djurasevic, Sonja Pavlovic, Cadiele Oliana Reichert, Joel Da Cunha, Débora Levy, , Luciana Morganti Ferreira Maselli, Sérgio Bydlowski, Celso Spada, Luciana Marti, Julieta Luna-Herrera, Jorge Ismael Castañeda-Sánchez, Ana Rosa Muñóz-Duarte, María Lilia Domínguez-López, Juan José De La Cruz-López

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**Antiproliferative Effect of Colonic Fermented Phenolic Compounds from Jaboticaba (***Myrciaria trunciflora***) Fruit Peel in a 3D Cell Model of Colorectal Cancer**

**Paula Rossini Augusti 1,\*, Andréia Quatrin 2 , Renius Mello 2 , Vivian Caetano Bochi 3 , Eliseu Rodrigues 1 , Inês D. Prazeres 4,5, Ana Catarina Macedo 4,5, Sheila Cristina Oliveira-Alves 4,5, Tatiana Emanuelli 2 , Maria Rosário Bronze 4,5,6 and Ana Teresa Serra 4,5**

**Abstract:** Jaboticaba is a Brazilian native berry described as a rich source of phenolic compounds (PC) with health promoting effects. PC from jaboticaba peel powder (JPP) have low intestinal bio-accessibility and are catabolized by gut microbiota. However, the biological implication of PCderived metabolites produced during JPP digestion remains unclear. This study aimed to evaluate the antiproliferative effects of colonic fermented JPP (FJPP) in a 3D model of colorectal cancer (CRC) composed by HT29 spheroids. JPP samples fermented with human feces during 0, 2, 8, 24 or 48 h were incubated (10,000 µg mL−1 ) with spheroids, and cell viability was assessed after 72 h. Chemometric analyses (cluster and principal component analyses) were used to identify the main compounds responsible for the bioactive effect. The antiproliferative effect of FJPP in the CRC 3D model was increased between 8 h and 24 h of incubation, and this effect was associated with HHDP-digalloylglucose isomer and dihydroxyphenyl-γ-valerolactone. At 48 h of fermentation, the antiproliferative effect of FJPP was negligible, indicating that the presence of urolithins did not improve the bioactivity of JPP. These findings provide relevant knowledge on the role of colonic microbiota fermentation to generate active phenolic metabolites from JPP with positive impact on CRC.

**Keywords:** spheroids; HT29 cells; cluster analysis; principal component analysis; hydrolysable tannins; HHDP-digalloylglucose isomer; dihydroxyphenyl-*γ*-valerolactone

### **1. Introduction**

Colorectal cancer (CRC) is the third most common type of cancer and the worldwide incidence in the year 2018 was nearly two million people [1]. Almost 75% of all sporadic cases of CRC are directly affected by dietary intake, and the consumption of plant phytochemicals has been reported to be beneficial in CRC management [2]. Phytochemicals, such as phenolic compounds (PC), have chemopreventive properties mostly due to their

**Citation:** Augusti, P.R.; Quatrin, A.; Mello, R.; Bochi, V.C.; Rodrigues, E.; Prazeres, I.D.; Macedo, A.C.; Oliveira-Alves, S.C.; Emanuelli, T.; Bronze, M.R.; et al. Antiproliferative Effect of Colonic Fermented Phenolic Compounds from Jaboticaba (*Myrciaria trunciflora*) Fruit Peel in a 3D Cell Model of Colorectal Cancer. *Molecules* **2021**, *26*, 4469. https:// doi.org/10.3390/molecules26154469

Academic Editors: Višnja Stepani´c and Marta Kuˇcerová-Chlupáˇcová

Received: 14 June 2021 Accepted: 21 July 2021 Published: 24 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

capacity to inactivate reactive oxygen species, which plays a vital role in the initiation and progression of CRC [2]. Besides, PC may also modulate cell gene expression, apoptosis or differentiation [3]. Studies regarding CRC are usually carried out using monolayer cells. In vitro 3D cell culture models using human and patient-derived CRC cell lines have been described as to be promising tools to evaluate the role of many antitumoral compounds, since these models provide cell–cell and cell–matrix interactions and, in this way, a cellular context similar to the cancer microenvironment. In particular, spheroid cultures of human CRC cells produced in stirred culture systems have been shown to be enriched in cancer stem cells and better recapitulate the in vivo tumor behavior [4,5]. Of note, these models have been used to ensure an accurate evaluation of the anticancer potential of phytochemicals derived from brassicas, nuts and citrus fruits [4,6–8].

Jaboticaba fruit (*Myrciaria trunciflora*) is a Brazilian berry with great potential for the food industry, being consumed naturally or as juices, jams, wines and liqueurs [9]. This fruit is rich in PC, in particular anthocyanins, flavonols, and ellagitannins, which are mostly concentrated in the peel [10]. Following jaboticaba processing for juice production, the peel is discarded and becomes a large food-waste. Thus, studies regarding the bioactivity of jaboticaba peel compounds will contribute to the sustainability of this agri-food system, supporting waste reuse as a nutraceutical or pharma chemical source. Accordingly, some PC extracted from jaboticaba exhibited antiproliferative effects against HT29 and HCT116 colon cell lines [11]. However, PC from jaboticaba peel powder (JPP) have low intestinal bio-accessibility and undergo extensive catabolism by gut microbiota [12].

The biological implication of the gut-derived PC metabolites of JPP remains unknown as previous studies on JPP fermentation were limited to the description of PC transformation during simulated digestion [12]. Although the antiproliferative effect of JPP extract in HT29 monolayer cells has been recently reported [13], no studies were found regarding the antiproliferative effects in CRC 3D cell models or the effects of colonic fermented JPP (FJPP).

This study aimed to evaluate the antiproliferative effect of FJPP in a 3D model of CRC (HT29 spheroids). Moreover, PC composition data of FJPP were processed by multivariate analyses, such as cluster analysis (CA) and principal component analysis, to determine the relationship between PC generated during fermentation and the antiproliferative effect of FJPP.

### **2. Results and Discussion**

The inhibition of colon cancer cell line growth by PC has gained attention as having potential for a candidate compound for cancer therapeutics [3]. However, some caution in data interpretation is needed, since PC have low bioavailability. Given that a large portion of PC are eliminated in feces, the transformation of PC by gut microbiota may influence the therapeutic potential of these compounds [14]. Besides being substrates for colonic microbiota, PC fermentation may also generate products that benefit the intestinal environment [15].

PC from JPP are poorly absorbed in the small intestine, and most of these compounds reach the colon and suffer fermentation by gut microbiota [12]. After simulated salivary, gastric and intestinal digestion, the fraction of JPP that was not bio-accessible (JPP-IN) was used for the colonic fermentation assay. In vitro colonic fermentation is a simple model to simulate the catabolism of compounds by colonic microbiota, and several reports have demonstrated the catabolism of PC during colonic fermentation using this model [16,17].

In vitro gut fermentation assays are relatively simple and fast procedures that present an unmatched opportunity for performing studies frequently challenged in humans and animals owing to ethical concerns. Fresh feces are the usual source of gut microbiota, but the large inter-individual variability of the gut microbiota poses a great challenge for biological replications [18]. This issue can be partially overcome by pooling fecal samples from different donors, as conducted in the present study.

### *2.1. Antiproliferative Effect of FJPP in Monolayer Cultures and Spheroids of HT29 Cell Line* nate the interference of PC already present in the feces. Before antiproliferative assays, the

the large inter-individual variability of the gut microbiota poses a great challenge for biological replications [18]. This issue can be partially overcome by pooling fecal samples

*2.1. Antiproliferative Effect of FJPP in Monolayer Cultures and Spheroids of HT29 Cell Line*

suspension alone (no JPP-IN) that was fermented during the same time as JPP, to elimi-

*Molecules* **2021**, *26*, x FOR PEER REVIEW 3 of 14

from different donors, as conducted in the present study.

All antiproliferative assays were carried out using a feces control group, i.e., a fecal suspension alone (no JPP-IN) that was fermented for the same time as JPP, to eliminate the interference of PC already present in the feces. Before antiproliferative assays, the cytotoxicity of FJPP was tested in confluent Caco-2 cells at concentrations ranging from 125 to 10,000 µg mL−1 . None of the evaluated concentrations caused toxicity to these cells after exposure for 72 h (Figure S1, supplementary material), indicating their safety for intestinal epithelial cells. The antiproliferative effect of FJPP (125 to 2000 µg mL−1 ) was firstly screened in monolayer cultures of HT29 cells, and all samples inhibited cell proliferation with EC50 values ranging from 769 to >2000 µg mL−1 (Figure 1). However, at the end of fermentation, (24 h and 48 h) the antiproliferative effect decreased when compared with time 0 (non-fermented samples) (Figure 1, *p* < 0.05). cytotoxicity of FJPP was tested in confluent Caco-2 cells at concentrations ranging from 125 to 10,000 µg mL−1. None of the evaluated concentrations caused toxicity to these cells after exposition for 72 h (Figure S1, supplementary material), indicating their safety for intestinal epithelial cells. The antiproliferative effect of FJPP (125 to 2000 µg mL−1) was firstly screened in monolayer cultures of HT29 cells, and all samples inhibited cell proliferation with EC50 values ranging from 769 to >2000 µg mL−1 (Figure 1). However, at the end of fermentation, (24 h and 48 h) the antiproliferative effect was decreased when compared with time 0 (non-fermented samples) (Figure 1, *p* < 0.05).


**9. Estimation results**

Within this section, we report the cycle estimates and other parameters of interest under the multivariate unobserved component model by the maximum likelihood method using the Kalman filter. In a second step, we proceed to calculate the c and Beta parameters for the trend, cycle, and seasonal parameters using LLT and State Space model using as instruments the gold, energy, and the financial cycle.

The parameters' estimates for the likelihood estimator are shown in **Table 1**. All the parameters are statistically significant. Global consumption and global inflation are significantly negatively determined by the oil, the gold and the financial cycle, which is confirming the expected result compatible with the theory, as a higher price of commodities have a negative impact on the consumption and enhances the inflation which leads to affecting the global economy negatively. In opposite global investment is significantly positively determined by the oil, the gold, and the financial cycle as the oil, the gold are used as hedge funds by the investors as well as the trading of the stock market which promotes the global economy and affects the cyclicity of the global business cycle within the peak. The long-run global investment elasticity of the global output is the highest equal to 0.119. This is confirming that the three determinants are used as a hedge fund for the investors and the payment system and are consistent with the capital channel of the global system, which means that the commodities are distributed mainly for investment other than consumption. **Figure 6**a–d shows the trend of the global business cycle within the Multivariate Unobserved Components model. The time lag of one period for the global GDP has not pronounced an impact on the cyclicity of the global business cycle, which might be explained as the

*The estimate of the generalized UC model for the global GDP. The estimation result of the generalized UC model is reported. The sample period is 1984:Q 1–2020:Q4. Within the estimation, we are limiting to the standard format of the multivariate unobserved model using the maximum likelihood estimator with the Kalman filter. Source: Own study.*

### **Table 1.**

*Result maximum likelihood estimator with Kalman filter for multivariate unobserved model of the global GDP.*

### **Figure 6**

*a. the residual, actual, and fitted estimation of global GDP with maximum likelihood estimator. b. the residual, actual, and fitted estimation of global consumption with maximum likelihood estimator. c. the residual, actual and fitted estimation of global investment with maximum likelihood estimator. d. the residual, actual and fitted estimation of global inflation with maximum likelihood estimator. Source: Own study.*

*The Global Business Cycle within the New Commodities and the Financial Cycle… DOI: http://dx.doi.org/10.5772/intechopen.111482*

deterministic commodities influence the upswing and downswing of the business cycle. However, basic on the filtering data, it is confirmed that oil has a lag impact on the fluctuation of the business cycle, it reacts as an impulsion for the crisis, compared to the gold and the global financial cycle which react simultaneously with the trend of the global business cycle.

Related to the investment, the inflation, and the consumption the related graph will show the univariate unobserved component model for each variable determined by the oil, the gold, and the financial cycle. According to the main result, we can consider that the determinant oil, gold, and financial cycle was a passive influencer for the global cycle of investment, consumption, and inflation. It is in opposite to the theory; however, it can be explained economically that the macroeconomic aggregate is an endogenous aggregate of the fluctuation of the commodities and not the opposite. However, related to the global business cycle, the commodities are reacting as an intermediary between the global business cycle and the investment, consumption, and inflation. Our result confirms that the long-run relationship between commodities and the business cycle is influenced by the economic condition. The relationship between gold and the financial cycle is stable with significant shifts during peak and recession.


**Preparation, Characterization and Application of Ultra-Fine Modified Pigment in Textile Dyeing**

Chaoxia Wang and Yunjie Yin

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53489


**Acknowledgements**

Based on this study, some simple ratio algorithms using spectral bands in the red and rededge ranges were adequate for quantifying chlorophyll-*a* in coastal waters. These algorithms were validated in other environments such as Nebraska with some modification to the coefficients [160]. Therefore, there is a high potential that chlorophyll-*a* in coastal waters could be estimated using hyperspectral remote sensing without the interference from TSS. NIR/ red ratios covering red-edge bands from 692 to 705 nm were found to be useful for retrieving chlorophyll-*a* in Delaware coastal waters when using AISA images and they could work in similar situations in other coastal areas. This suggests the advantage of using ratio-based

The impacts of land use on aquatic health and marsh habitat have been a constant battle, and the rate of our wetlands lost is far more than they can be replaced. Tidal marsh ecosystems serve as great examples of dynamic ecosystems that can provide numerous lessons in restoration and management strategies. The ubiquitous incidence of aquatic species as both temporal and spatial perspectives indicates the importance of these types of systems to sustain the health of aquatic species and other important species in the face of anthropogenic and environmental stresses. Thus, it would be pertinent for managers to maintain the health of these areas by preventing any further alterations and damage, especially from nutrient runoff and

The Blackbird Creek Watershed is composed of only 4% urban development. This provided a great opportunity to study the area that has very little anthropogenic impact, relative to other watersheds. How trophic dynamics can be affected as a result of various land uses is one of the concerns. There are several plots of land designated as cropland in Blackbird Creek. An understanding of crop rotation in these plots both within and between seasons can provide insight on whether changes in crops across years can affect trophic interactions and food web dynamics. Additionally, effectiveness of riparian buffers as blockades for fertilizer runoff is a significant impact on water quality. With sites associated with buffers and sites without buffers, different food web characteristics can be identified, especially if there are inconsistencies in the nutrient concentrations. While this is intriguing, it would be more beneficial if a second watershed with more urban areas and anthropogenic effects was used. The St. Jones Watershed is composed of over 21% urban development. If similar work can be done in each of these watersheds, we may be able to discern the effects of different land uses on Delaware's

The Chesapeake Bay is under increasing pressures from anthropogenic disturbances at various temporal and spatial scales. Water quality monitoring is vital for assessing such impacts, and further provides important information for sustainable water resource managements. The research in this chapter demonstrates the applications of hyperspectral remote sensing in retrieval of the water quality parameters in such an optically complex system. Further development of retrieval algorithms is still needed in order for the remote sensing to be routinely

indices in estimating coastal chlorophyll-*a* concentrations by remote sensing.

abusive use of our coastal lands, and the invasion of *P. australis*.

**3. Final remarks**

208 Water Quality

coastal waterways.

used in the water quality monitoring.

We would like to thank the following individuals for their assistance and support for research discussed and in preparation of this chapter: Matthew Stone, Kris Roeske, Laurieann Phalen, Morgan State University Patuxent Environmental and Aquatic Environmental Laboratory (PEARL) and Delaware State University Aquatic Sciences Laboratory team members for field assistance and the early comments and discussions on the research papers. The authors also thank Dr. Shobha Sriharan at the Virginia State University for her support and Delaware State University for providing support and resources to conduct the research in this chapter. Case studies provided in this chapter are funded in the following order:

Case Study 2.1 is funded by NSF EPSCoR Grant Award# EPS-1301765 and the USDA-NIFA CBG Grant Award# 2013-38821-21246 to the Virginia State University.

Case Study 2.2 is funded by USDA-NIFA CBG Grant Award# 2010-38821-21454 to the Delaware State University and USDA Evans-Allen Grant Award# DELXDSUGO2015.

Case Study 2.3 is partly supported by NOAA-ECSC Grant Award# NA11SEC4810001, the NSF Grant Award# GEO0914546 to the Morgan State University, and the NSF Grant Award# 1036586 to the University of Maryland Eastern Shore and the USDA-NIFA CBG Grant Award # 2011-38821-30892 to the Virginia State University.

Case Study 2.4 is partly supported by NOAA-ECSC Grant Award# NA11SEC4810001, the NSF Grant Award# 1036586 to the University of Maryland Eastern Shore, and the USDA-NIFA Grant Award# 2013-38821-21246 to the Virginia State University.

Case Study 2.5 is funded by NOAA-ECSC Grant Award# NA11SEC4810001.


**Curtise K. C. Ng**

Dr. Curtise K. C. Ng received his Bachelor of Science (Honours) Radiography degree in 2002 and Doctor of Philosophy degree in imaging informatics in 2007 from The Hong Kong Polytechnic University. In 2008, he decided to broaden his horizon and took up the Medical Imaging Lecturer position at Curtin University in Australia and has become a tenured Senior Lecturer since 2011. He has been awarded United Kingdom Higher Education Academy Senior Fellow status since 2021. Currently, he is a tenured Senior Lecturer of Curtin Medical School.

Dr. Ng is an active researcher in Radiology, Radiation Oncology, and Medical Radiation Science focussing on imaging informatics (including artificial intelligence) and education research. He was appointed as the Associate Editor of the *Journal of Medical Imaging and Radiation Sciences* (official journal of Canadian Association of Medical Radiation Technologists, Hong Kong Radiographers Association, Portuguese Association of Nuclear Medicine Technologists, Alliance in Nuclear Scintigraphy in Australia and Society of Medical Radiographers—Malta) between 2013 and 2019. Currently, he is the Guest Editor of a Special Issue of *Children* titled "Advanced Research in Pediatric Radiology and Nuclear Medicine (Volume II)" and is an Editorial Board Member of *BMC Medical Education* journal. He also serves as a reviewer for 30 journals such as *BMJ Open*, *International Journal of Medical Informatics*, *Knowledge-based Systems*, *Medicine*, *PLOS ONE* and *Scientific Reports*.


*Article* **Robust Inference in the Capital Asset Pricing Model Using the Multivariate** *t***-Distribution**

**Manuel Galea 1,\*, David Cademartori 2, Roberto Curci 3 and Alonso Molina 1**

Received: 1 May 2020; Accepted: 9 June 2020; Published: 13 June 2020

**Abstract:** In this paper, we consider asset pricing models under the multivariate *t*-distribution with finite second moment. Such a distribution, which contains the normal distribution, offers a more flexible framework for modeling asset returns. The main objective of this work is to develop statistical inference tools, such as parameter estimation and linear hypothesis tests in asset pricing models, with an emphasis on the Capital Asset Pricing Model (CAPM). An extension of the CAPM, the Multifactor Asset Pricing Model (MAPM), is also discussed. A simple algorithm to estimate the model parameters, including the kurtosis parameter, is implemented. Analytical expressions for the Score function and Fisher information matrix are provided. For linear hypothesis tests, the four most widely used tests (likelihood-ratio, Wald, score, and gradient statistics) are considered. In order to test the mean-variance efficiency, explicit expressions for these four statistical tests are also presented. The results are illustrated using two real data sets: the Chilean Stock Market data set and another from the New York Stock Exchange. The asset pricing model under the multivariate *t*-distribution presents a good fit, clearly better than the asset pricing model under the assumption of normality, in both data sets.

**Keywords:** capital asset pricing model; estimation of systematic risk; tests of mean-variance efficiency; *t*-distribution; generalized method of moments; multifactor asset pricing model