*2.1. Purification and Identification of the Alkene Cleavage Activity*

Within a screening of 17 basidiomycetes for alkene cleavage activity using the substrate *trans*-anethole (Table S1) *P. sapidus* turned out to be a promising candidate for the production of the desired activity. The lyophilized mycelium as well as the culture supernatant was examined for the ability to cleave *trans*-anethole after submerged cultivation. The culture supernatant showed no activity, whereas the incubation in the presence of the mycelium resulted in formation of 5.36 mM *p*-anisaldehyde (molar yield of 79.03%; Figure 1).

**Figure 1.** Bioconversion of *trans*-anethole by *P. sapidus*. (**a**) Alkene cleavage of *trans*-anethole resulted in the formation of *p*-anisaldehyde. (**b**) GC-FID chromatogram of an *n*-hexane extract of the conversion of the blank sample (blue) and after incubation with lyophilized mycelium of *P. sapidus* (orange). Retention times: *trans*-anethole (11.53 min) and *p*-anisaldehyde (13.45 min).

For the identification of the enzyme catalyzing the *trans*-anethole cleavage, it was semi-purified from the rehydrated mycelium by hydrophobic interaction and anion exchange chromatography (IEX). During the purification, a high activity loss occurred, which resulted in low product concentrations after conversion (Table S2). This was most likely a result of protein loss, enzyme degradation or denaturation as described for other enzymes [22–24]. Another possibility is the loss of cofactors or –substrates, such as metal ions or peroxides during the purification steps [6]. Addition of Mn2+ led to a 15-fold increase in *p*-anisaldehyde concentration, which was further increased by addition of hydrogen peroxide, indicating a cosubstrate dependency (Table S2). Chemical conversion by Mn2+ alone was excluded, while product formation was observed with hydrogen peroxide (18 μM), but with a yield around 30-fold lower than the one for the enzymatic reaction (Table S2). Thus, the improved bioconversion in the presence of Mn2+ and hydrogen peroxide was verified to be the result of an increased enzyme activity.

A class of fungal enzymes that requires hydrogen peroxide and some of which need Mn2+ for catalysis are peroxidases [12,25,26]. The anion exchange fractions, which showed alkene cleavage activity, also exhibited peroxidase activity, thus verifying the presence of a peroxidase. For visualization of the activity, a semi-native PAGE was performed and stained with ABTS in the presence of hydrogen peroxide (Figure 2). Two peroxidases running at 45 and 52 kDa were detected. The respective protein bands stained with Coomassie Brilliant Blue were excised for electrospray ionization tandem mass spectrometry. Due to the low protein concentration of the 52 kDa band, no meaningful peptides were found (Figure 2, lane 1). This paper presents the data obtained for the protein running at 45 kDa (Figure 2, arrow).

Three tryptic peptides (EGSELLGAR, DGSFLTFR, and SGAPIEITPLKDDPK) were identified by ESI-MS/MS. Homology searches against the public database NCBI using the mascot search engine (Matrix Science, London, UK) identified a DyP-type peroxidase of *P. ostreatus* PC15 (*Pleos*-DyP4 [27]; GenBank accession no. KDQ22873.1), a close relative of *P. sapidus*, as the best hit.

**Figure 2.** Semi-native PAGE of the active fraction after purification of the alkene cleavage enzyme from *P. sapidus* by IEX. 1: gel stained with Coomassie Brilliant Blue; 2: gel stained with ABTS in the presence of hydrogen peroxide, M: pre-stained molecular mass marker. An arrow and a box marks the protein that was successfully identified by sequencing.

#### *2.2. Amplification and Expression of PsaPOX*

Specific primers successfully amplified the 1512 bp coding region of the gene from *P. sapidus*. The translated amino acid sequence of 504 aa contained the peptide fragments that were obtained by ESI-MS/MS and showed highest identity (94%) to the sequence of *Pleos*-DyP4 (Figure 3), which has not been investigated for its alkene cleavage activity against *trans*-anethole or other substrates before [27,28]. In our hands, *P. ostreatus* showed a weaker *trans*-anethole cleavage activity, too (Table S1). Identity of the *P. sapidus* peroxidase (PsaPOX) to other DyPs and proteins was lower than 60%. A sequence alignment with other DyPs (Figure 3) confirmed that PsaPOX exhibited the typical GXXGD motif and all important residues known for the catalytic activity of DyPs, such as the proximal histidine (His-334) (fifth ligand of heme iron) and distal Asp-196 and Arg-360 involved in the activation of the enzyme (formation of compound I) by H2O2 cleavage [14,15]. Furthermore, Trp-405 was identified as a homolog to the surface exposed Trp-377 of AauDyP of *Auricularia auricula-judae*, which serves as an oxidation site for bulky substrates such as Reactive blue 19 (RBB19) using a long-range electron transfer [29]. Comparison of the PsaPOX and *Pleos*-DyP4 sequence indicated that PsaPOX exhibited a non-canonical Mn2+-oxidation site on its surface (Asp-215, Glu-345, Asp-352 and Asp-354; Trp-339 participates in the electron transport from the oxidation site to the heme) like *Pleos*-DyP4 [28] and can oxidize Mn2+ to Mn3+, which is known for a few fungal DyPs only [24,27,30,31].

A structural homology model of PsaPOX (Figure S1), which was generated using the X-ray crystal structure of *Pleos*-DyP4 (PDB-ID 6fsk) on the SWISS-MODEL server, possessed typical characteristics of the DyP-type peroxidase family (N- and C-terminal ferredoxin-like domain, each formed by four-stranded antiparallel β-sheets and several α-helices) [15] and supported the classification of PsaPOX as DyP. Furthermore, the analysis of the amino acid sequence using PeroxiBase [32] related the *P. sapidus* peroxidase (PsaPOX) to the class "DyP-type peroxidase D". As known from literature, DyPs differ significantly in amino acid sequence, tertiary structure, and catalytic residues from other representatives of the heme peroxidases, such as HRP, human myeloperoxidase, lignin peroxidase, or *Coprinus cinereus* peroxidase [12,15,33], all of which are able to cleave *trans*-anethole or structurally related alkenes [10,11]. So far no DyP is known to catalyze the mentioned reaction.

**Figure 3.** Alignment of alkene cleaving peroxidase from *P. sapidus* (PsaPOX) with the *Pleos*-DyP4 of *P. ostreatus* (PosDyP; KDQ22873.1) and other characterized DyPs. AgaDyP: *Armillaria gallica* (PBK80505.1), VvoDyP: *Volvariella volvacea* (AKU04643.1), CtrDyP: *Coriolopsis trogii* (AUW34346.1), GluDyP: *Ganoderma lucidum* (ADN05763.1), and TveDyP: *Trametes versicolor* (XP\_008039377.1). Inverted triangles show amino acids important for heme binding (histidine (magenta) functions as ligand for heme and the four other amino acid residues form a hydrogen peroxide binding pocket). Aspartic acid, which forms a hydrogen bond with histidine to stabilize compound I (oxidized heme after transfer of two electrons to H2O2) is shown in grey. The black box indicates the GXXDG motif containing the catalytic aspartic acid residue (yellow), which cleaves H2O2 heterolytically with the help of the neighboring arginine (green) to form compound I, and the circle presents an exposed tryptophan potentially involved in an LRET (long range electron transfer). Important amino acids for Mn2+-oxidation are highlighted in cyan; asterisks indicate conserved residues, colons equivalent residues and dots partial residue conservation. Peptides identified by protein sequencing are underlined. Alignment was performed with Clustal Omega (European Bioinformatics Institute, Hinxton, UK).

## *2.3. Production and Purification of the Recombinant PsaPOX*

The *PsaPOX* gene was amplified and cloned into the *K. pfa*ffi*i* expression vector pPIC9. The initial expression of the gene yielded average peroxidase activities of 65 U/L after 72 h of cultivation. The best performing colonies produced activities up to 142 U/L, indicating a multiple insertion of the expression construct [34]. Similar results were obtained for the heterologous production of a DyP from *Funalia trogii* in *K. pfa*ffi*i* previously [24]. Further experiments were performed using the clone with the highest peroxidase activity for maximum protein production.

The recombinant peroxidase was purified by Ni-NTA affinity. Using SDS-PAGE, a molecular mass of around 61 kDa was determined (Figure 4a), which is slightly higher than the calculated molecular mass of 54.9 kDa (ExPASy). In addition, the native recombinant enzyme was detected at 52 kDa after semi-native PAGE, while the native wild-type enzyme showed a band at 45 kDa (Figures 2 and 4b). Deglycosylation by endoglycosidase H (EndoH) showed that the higher molecular mass was attributed to post-translational modifications by *K. pfa*ffi*i*, as has been described for other proteins [35,36]. The wild-type peroxidase, on the contrary, was not glycosylated (see Figures 2 and 4b). That is uncommon for DyPs, which usually exhibit a carbohydrate content of 9 to 30% [12]. The molecular mass of the monomeric PsaPOX was similar to other DyP-type peroxidases [12].

**Figure 4.** Purification of the recombinant PsaPOX by Ni-IMAC. (**a**) SDS-PAGE stained with Coomassie Brilliant Blue. 1: flow through, 2: elution fraction, 3: elution fraction incubated with EndoH, 4: EndoH, M1: molecular mass marker. (**b**) Semi-native PAGE stained with ABTS in the presence of hydrogen peroxide. 5: elution fraction, 6: elution fraction incubated with EndoH, M2: pre-stained molecular mass marker. (**c**) Isoelectric focusing gel. 7: elution fraction stained with phenylendiamine in the presence of urea peroxide. M3: standard protein marker for isoelectric focusing stained with Coomassie Brilliant Blue.

Analysis of the purified recombinant peroxidase by isoelectric focusing indicated an isoelectric point around pH 6.7 (Figure 4c), which differs slightly from the calculated value of 6.28 (ExPASy), but was similar to the isoelectric point of another DyP-type peroxidase from *P. sapidus* [19]. Most other proteins belonging to the DyP-type peroxidase family showed lower values (pI 3.5-4.3, [12]).

#### *2.4. Biochemical Characterization of PsaPOX*

The influence of pH and temperature on PsaPOX activity and stability was determined using ABTS in the presence of hydrogen peroxide as substrate (Figure 5). The enzyme showed a pH optimum of 3.5 while more than 50% of activity was conserved between pH 3 and 5 (Figure 5a). At lower or higher pH values of ≤ 25% of activity remained, most likely due to conformational changes of the enzyme. The results were consistent with the findings for other fungal DyPs, which had pH optima in the range between pH 2 and 5 [13,19,27]. PsaPOX showed the highest pH stability with a residual peroxidase activity of ≥90% between pH 2.0 and 5.5 after 1 h of incubation (Figure 5c). At pH values higher than six, near the isoelectric point, the stability decreased drastically, probably due to a reduced solubility and changes of the protein structure, which may have resulted in protein aggregation.

**Figure 5.** Influence of pH and temperature on activity and stability of PsaPOX. The pH optimum (**a**) was determined to be 3.5 and the temperature optimum (**b**) 40 ◦C. Relative peroxidase activity [%] was defined as the percentage of activity detected with respect to the highest activity in each experiment. pH stability (**c**) was determined after incubation of PsaPOX in Britton Robinson buffer ranging from pH 2.0 to 9.5 for 1 h at RT and temperature stability (**d**) after incubation at 20 to 90 ◦C and pH 3.5 for 1 h. Residual activities were determined at pH 3.5 and 40 ◦C. Values are the average of triplicate experiments with standard deviations shown as error bars.

Peroxidase activity of PsaPOX increased with rising temperature, reaching its maximum at 40 ◦C (Figure 5b), which was similar to the optimum (30–40 ◦C) of a recombinant DyP from *P. ostreatus* [13], but higher than the optimum (RT) of another DyP-type peroxidase of *P. sapidus* produced heterologously in *Escherichia coli* [37]. With further temperature increase, the peroxidase activity of PsaPOX decreased continuously. The temperature stability of PsaPOX was determined after an incubation for 1 h at different temperatures (Figure 5d). The enzyme was relatively stable at temperatures from 20 to 60 ◦C with a residual activity ≥80%. At higher temperatures, a high loss of activity was observed due to protein denaturation, resulting in residual activities <5%. The temperature stability of PsaPOX was higher than the stabilities of DyPs from *Bjerkandara adusta* and *Auricularia auricular-judae*, which were produced heterologously in *E. coli* (residual activity ≥80% and <5% after 1 h at 20–50 ◦C and 60 ◦C, respectively [13]), and of a DyP from *P. sapidus* produced in *Trichoderma reesei* (residual activity ≥80% and <65% after 5 min at 15-45 ◦C and 50 ◦C, respectively [19]).

As mentioned above, the addition of hydrogen peroxide as well as Mn2+ led to an increase of the product concentration for the biotransformation of *trans*-anethole using the lyophilized mycelium of *P. sapidus* containing the wild-type PsaPOX (Table S2). For this reason, the hydrogen peroxide and Mn2+ dependencies were examined for the recombinant enzyme using ABTS as substrate at optimal pH and temperature (Figure 6). As expected, no peroxidase activity was detectable without hydrogen peroxide. The activity rose with increasing peroxide concentration and reached its optimum in the presence of 100 μM H2O2 (Figure 6a). An increase of the hydrogen peroxide concentration led to a continuous activity decrease. Suicide inhibition in the presence of excess hydrogen peroxide is well known for classical peroxidases as a result of the formation of an inactive oxidative state (Compound III) by reaction of H2O2 and Compound II [20,21], even if the existence of Compound II has not been confirmed for DyP-type peroxidases universally [17]. However, inhibition of other DyPs in the presence of higher hydrogen peroxide concentrations has been reported [18,19].

**Figure 6.** Effect of hydrogen peroxide (**a**) and Mn2+ concentration (**b**) on the activity of PsaPOX. Relative peroxidase activity [%] was defined as the percentage of activity detected with respect to the highest activity obtained in each experiment. Values are the average of triplicate experiments with standard deviations shown as error bars.

Investigation of the Mn2+ dependency (Figure 6b) showed that PsaPOX activity rose with increasing Mn2+ concentration, but was not completely dependent on the addition of Mn2+. 30% of peroxidase activity were detected without addition of Mn2+. PsaPOX reached the maximal activity in the presence of 25 mM Mn2+. Evaluation of Mn3+ formation by Mn2+ oxidation revealed a manganese peroxidase activity of 0.4 U compared to 1 U of peroxidase activity using ABTS as substrate. This result fits the prediction of a Mn2+ oxidation site. Only a few fungal DyPs are known to catalyze the oxidation of Mn2+ [24,27,30,31]. Calculation of kinetic constants (Table 1) showed that the catalytic efficiency of PsaPOX towards Mn2+ was similar to the one of *Pleos*-DyP1 from *P. ostreatus* and Ftr-DyP from *Funalia trogii* [24,27]. However, the catalytic efficiency of *Pleos*-DyP4 was higher [27].

**Table 1.** Michaelis constants (*K*m), catalytic constants (*k*cat), and catalytic efficiencies (*k*cat/*K*m) for PsaPOX using ABTS, Mn2+, Reactive blue 19 (RB19), and Reactive black 5 (RB5) as substrate. Values are the average of triplicate experiments with indication of standard deviations.

n. d.: no activity was detected.

Kinetic parameters were also calculated for the oxidation of ABTS at optimal conditions (Table 1). The affinity of PsaPOX to ABTS (37 μM) was similar to a DyP from *Irpex lacteus* (28 μM), but higher in comparison to the FtrDyP from *F. trogii* (182 μM) and the *Pleos*-DyP2 from *P. ostreatus* (787 μM) [18,24,27]. In contrast, the catalytic efficiency of PsaPOX (184 s−1 mM−1) was lower than the efficiency of the DyP from *Irpex lacteus* (8000 s−1 mM−1) and *Pleos*-DyP4 (352 s−1 mM−1), but higher than the efficiency of the FtrDyP (54 s−1 mM−1).

It is known that DyP-type peroxidases typically oxidize anthraquinones and other dyes. Exemplary, decolorization of Reactive blue 19 (anthraquinone dye) and Reactive black 5 (recalcitrant azo dye) by recombinant PsaPOX (1 U/L) was tested. Unexpectedly, PsaPOX showed activity for neither of the substrates (Table 1), although the protein sequence and tertiary structure as well as the presence of typical catalytic residues and the GXXDG motif identified the enzyme as a DyP-type peroxidase. However, *Pleos*-DyP1 from *P. ostreatus* and *Tv*DyP1 from *Trametes versicolor* also did not oxidize the

high redox-potential Reactive black 5, although they degraded Reactive blue 19 [27,30]. A missing activity against Reactive blue 19 or another anthraquinone has not been described for a fungal or class D type DyP before, but one bacterial DyP of *Pseudomonas fluorescens* (DyP2B, DyP typ class B) is known not to oxidize the anthraquinone dye Reactive blue 4 [38].


To accomplish RT 1, we performed the first interdisciplinary literature review on media bias and approaches to analyze it as devised in the social sciences, computer science, and related disciplines. The review includes almost 200 research publications and related approaches. We found that automated bias identification approaches proposed so far often yield inconclusive or superficial results, especially compared to the results of decade-long research on the topic in the social sciences.

To facilitate interdisciplinary research on media bias, we established a shared conceptual understanding by mapping the state of the art from the social sciences to a framework, which approaches from computer science can target.

#### **Research Task RT 2**

*Devise a bias identification approach that addresses the identified weaknesses of current bias identification approaches.*

To overcome the deficiency of the current automated approaches for bias identification and communication, we introduced a novel approach named personoriented framing analysis (PFA). Compared to prior automated approaches, PFA does not treat media bias as a single, broadly defined concept but analyzes intext features representing specific person-oriented bias forms. To achieve this, PFA roughly resembles the manual process frame analysis as conducted by researchers in the social sciences. In contrast to these analyses, PFA does not require reading or annotating news articles but eliminates these tasks.

As a practical side contribution, we devised a system for crawling and extracting news articles from online outlets. The system can be used before PFA in order to gather news articles of interest. Additionally, the system has proven helpful throughout the research described in the thesis, e.g., to create training and test datasets.1

#### **Research Task RT 3**

*Develop methods for the devised approach and evaluate their technical performance.*

To detect person-oriented framing, we devised two analysis components. Our first component aims to identify persons and their mentions across a given set of news articles reporting on an event. Subsequently, our second component aims to determine how the individual persons are portrayed and then groups articles that portray the persons similarly, i.e., that frame the event similarly.

For the first component, we devised a method for context-driven cross-document coreference resolution that is the first to resolve also highly event-specific coreferences as they occur across slanted news articles, e.g., "Mr. Tough Guy" and "John Bolton" [230]. To evaluate this method, we created a test dataset of 50 news articles and 10 events. Each event contains articles by five outlets covering the political spectrum, including left-wing, center, and right-wing. When creating the dataset, we aimed to enable the annotation of also subtle and complex concept types, which the coreference resolution should resolve. To ensure reliable annotation despite the concept types' complexity, we conducted a manual content analysis as established in the social sciences. Our evaluation showed that our method reliably extracts and resolves individual persons (*F*1*m* = 88*.*7 compared to *F*1*m* = 81*.*9 achieved by the best baseline).

For the second component, we devised a method for target-dependent sentiment classification (TSC) in the domain of news articles. To evaluate this method, we created the first large-scale dataset for TSC in news articles reporting on policy issues. Our dataset consists of over 11k sentences. Each sentence includes at least one person mention and a sentiment label. Using an additional expert annotation on a random subset of the dataset, we confirmed the high quality of our ground truth. Not only increases our TSC method the state-of-the-art classification performance (*F*1*m* = 83*.*1 compared to *F*1*m* = 81*.*7), but our dataset is also the first to

1 Our system is also used by numerous researchers to create pre-training datasets for deep learning language models [163, 387, 404], including the RoBERTa language model [214]. The tool has also shown helpful in COVID-19-related research [186, 301].

even enable TSC on the news domain under real-world conditions. News TSC was previously practically impossible due to the implicit character of sentiment in news articles compared to the established TSC domains.

Side contributions of our initial research to address RT 3 include a system that extracts 5W1H phrases describing the main event of an article, i.e., who did what, when, where, why, and how. Further, we devised and annotated socalled frame properties, which we had planned to use to categorize how sentences portray persons. Afterward, our exploratory research early showed a vital issue of automatically detecting substantial frames or derivatives: very high, initial annotation cost required to create a sufficiently large training dataset. Consequently, we devised the previously mentioned TSC approach to capture the fundamental effects of person-oriented framing, i.e., change of a person's sentiment polarity.

#### **Research Task RT 4**

*Implement a prototype of a bias identification and communication system that employs the developed methods to reveal biases in real-world news coverage to non-expert news consumers.*

To evaluate PFA in a setting that resembles real-world news consumption, we developed Newsalyze, a system for bias identification and communication. Besides integrating the previously devised bias identification methods into the system, we developed visualizations for non-expert news consumers. First, an overview allows for getting a synopsis of current events quickly. Second, an article view shows a single news article. We designed the visualizations to suit typical news consumption while complementarily showing bias-revealing information, such as the identified person-oriented frames present in the news coverage on a given event.

#### **Research Task RT 5**

*Evaluate the approach's effectiveness in revealing biases by testing the implemented prototype in a user study.*

To validate our approach's holistic and practical effectiveness in revealing biases, we conducted a large-scale user study (*n* =160) on 30 news articles. Our studies measure the change in respondents' bias-awareness after exposure to one of our visualizations or baselines. We designed the studies to approximate real-world news consumption. Not only showed the study results that Newsalyze and the PFA approach significantly, consistently, and strongly increased bias-awareness in nonexpert news consumers. By employing a conjoint design in our experiments, we were also able to pinpoint the effects of individual components in the visualizations. We used the conjoint design in the pre-studies, among others, to make a founded selection of strong visualization variants for the main study.

In addition to demonstrating the high effectiveness of PFA in a setting that resembles real-world news coverage, the evaluation led to the following conclusions:

## **7.3 Future Work**

We intentionally formulated our research question rather openly and broadly to reflect the young state of the art in computer science. This way, our research question expresses the need to investigate how other disciplines define and analyze bias. Such disciplines traditionally include the social sciences, where media bias has been subject to research for decades resulting in comprehensive models to describe and effective methods to analyze it.

How can an automated approach identify relevant frames in news articles reporting on a political event and then communicate the identified frames to non-expert news consumers to effectively reveal biases?

As part of the literature review, we established a shared conceptual understanding by mapping the state of the art from the social sciences to a framework, which computer science approaches can target. Using this framework and the identified weaknesses of prior bias identification and communication approaches, we narrowed our open research question down to a specific research objective (Chap. 3). Our objective focuses on the identification of person-oriented framing:

Devise an approach to reveal substantial biases in English news articles reporting on a given political event by automatically identifying text-based, person-oriented frames and then communicating them to non-expert news consumers. Implement and evaluate the approach and its methods.

Concerning both the broad research question and the specific objective, this section discusses the most important limitations of our research and derives future research ideas to address these limitations.

#### *7.3.1 Context-Driven Cross-Document Coreference Resolution*

We devised the first method for context-driven cross-document coreference resolution. Naturally, our method can only serve as the first step in this novel task. Albeit the method's design in principle allows for using the method outside the scope of PFA, we focused on our research objective when devising and evaluating the method. Our evaluation was not intended to and cannot elucidate how effective our method is when applied to other domains and in other use cases. Before applying our method in other use cases, we propose to conduct a more sophisticated evaluation, following all standards of coreference resolution evaluation. Specifically, we propose to test the method on established datasets for coreference resolution and compare it to a larger set of related methods. We also propose creating a larger annotated dataset and thereby address various minor findings of the current annotations.

A larger dataset would also enable the training of deep learning models for crossdocument coreference resolution. Recent models for single-document coreference resolution achieve strongly increased performance compared to earlier traditional methods [390]. We thus expect that such models could achieve higher performance than our method employing hand-crafted rules. However, as described in Sect. 4.3, the creation of a sufficiently large training dataset would cause high annotation cost.

#### *7.3.2 Political Framing and Person-Independent Biases*

Detecting political framing directly as opposed to detecting its effects on the framing of persons (in PFA expressed through polarity) is beyond the scope of our objective but relevant to the overall research question. While PFA represents an effective and cost-efficient approach to identify substantial frames, its focus on persons implies that the approach may fail if large parts of the coverage on an event are not person-

**Table 7.2** Two news excerpts with opposing perspectives on the same event. The perspectives result from highlighting specific aspects of the event, here the negative consequences of loosening versus continuing COVID-19-related restrictions in Germany, March 2020. Free translation from [315, 354]

centric. Because of this expected shortcoming, we had initially attempted to identify topic-independent framing categories. Similar to other researchers' attempts to define or identify such categories directly, the approach turned out to be impractical. Reasons included the high cost of annotating a large dataset, which is required for training models to classify also nuanced and subtle framing categories.

Table 7.2 shows an example of framing that PFA cannot detect. Our approach would miss the two content-based frames, e.g., "restrictions are necessary" (first article) and "restrictions damage economy" (second article), because they are not directly related to individual persons. Instead, the articles focus on health or social implications versus economic consequences. To identify framing effects in nonperson-oriented topics, we propose extending our approach to additional concept types, such as actions, person groups, countries, and objects. Our method for cross-document coreference resolution already resolves these types (Sect. 4.3.4). To classify the sentiment of these new types using target-dependent sentiment classification, we propose to extend the NewsMTSC dataset.

More closely resembling frame analyses as conducted in the social sciences, another idea is to determine frames directly rather than their effects. Key differences of person-oriented frames identified by PFA and political frames as defined by Entman [79] include that PFA defines person-oriented frames implicitly (each frame is defined by its articles) and inductively (frames are derived during the analysis). In contrast, social science researchers define frame for a specific research question before quantifying them in news coverage. In our view, this line of research represents the most refined approach, relying directly on decade-long, established social science research. Recent language models yielded a decisive performance leap in many natural language understanding tasks. However, training these models would cause high cost before they can classify subtle, analysis question-specific, and—viewed from a technical perspective—in part (intentionally) subjective frames (Sect. 6.10).

A pragmatic alternative to political frames might be topic-independent frame derivatives, such as microframes [193], our frame properties (see Sect. 5.2), or frame types [45, 46]. Each of these, however, has their limitations or challenges. Typically, they require manual validation or cause high annotation effort. Finally, active learning might be a suitable method to reduce the annotation cost. In active learning, human annotators need to label only a subset of examples. Typically, this subset consists of those examples the model to be trained is least sure how to classify. An iterative process of (re)training the model, selecting uncertain examples, and manually labeling them is repeated until the trained model achieves a sufficiently high classification performance.

Automated identification of framing effects or political frames directly could also be helpful in social science research. While frame analysis is an established means for analyzing how the media reports events and topics, the manual effort prevents conducting such analysis at scale. An automated approach for frame identification could assist researchers, especially in the early phases of their research. For example, PFA could serve as a tool to enable low-effort inductive exploration of news coverage by revealing which person-oriented frames are present in the data to be analyzed. Once extended to identify frames independent of persons or generally political frames, our approach could be helpful also in later phases of frame analyses.

Lastly, we propose to inspect the devised methods for biases they may have inherited from their training or fine-tuning data. We inspected both the annotated data and the model's predictions for structural biases indirectly, e.g., as part of the expert validation and manual error analysis. However, we did not directly probe the models and results for biases. This is advised, for example, because language models can be prone to gender-induced or other bias-related prediction errors due to their pre-training data [320]. Likewise, our fine-tuning data and the other datasets may contain structural biases despite the implemented quality measures. For example, the expert validation may entail biases since all experts were influenced by Western culture. Means to probe for biases in language models are already discussed in the literature (cf. [223]).

#### *7.3.3 Bias Identification and Communication*

This section uses the findings of our PFA evaluation to discuss both conceptual and technical means to address these limitations of the PFA approach and its evaluation.

In our view, an important limitation of the experiments and results is their generalizability, e.g., due to the study's design, its deployment, and the samples of respondents and events. For example, we measured respondents' bias-awareness using a set of questions. A future study could measure bias-awareness more directly, e.g., by observing respondents' news consumption behavior over a longer time frame. Like prior work, such a study would assume that effective bias communication encourages news consumers to view and compare more articles than bias-agnostic visualizations.

We also propose to sample respondents from other countries than the USA alone. Currently, we cannot generalize the study's findings to other countries' populations since political and media landscapes differ across countries. For example, while in the USA, the two-party system may lead to more polarizing news coverage, countries with multi-party systems typically have more diversified media landscapes [395]. We expect that these differences affect bias-awareness in general and thus also the effectiveness of our approach. Enabling studies in other countries requires extending the PFA approach to analyze news articles written in other languages. Ideas range from devising language-specific methods, where we expect overall high research effort and high-quality analysis results, to using neural machine translation before the PFA approach, which would require investigating the stability of taskspecific linguistic properties, e.g., whether the subtle nuances of word choices are translated well.

The generalizability could also be improved by diversifying the event sample, which consisted in our study of 3 events and 30 articles. Besides systematically adding more events and articles, another future work idea is to randomly sample events and articles. While a random sample would reduce data selection biases, it would also require a larger respondent sample to compensate the noise introduced by the increased content diversity.

To further diversify the event sample, we propose investigating which technical changes are required to enable the PFA approach to analyze news from other sources than online newspapers. While we already included a few alternative outlets in our study, such as Breitbart, especially the news published on the increasingly consumed social media channels is different from news articles, such as concerning the texts' lengths and writing styles. We expect that the domains' differences require adapting the PFA approach. As a visionary outlook to broaden the generalizability of bias-awareness studies further, future approaches could aim to identify and reveal biases targeting not only persons. Ideas for extending the PFA approach range from analyzing other target concept types or political framing (as outlined previously in Sect. 7.3.2) to analyzing other forms of biases, such as through picture selection (Sect. 2.2.3).

We also propose to create a framing ground truth dataset using manual frame analysis. Such a dataset would enable measuring the influence of the articles' content and frames on respondents' bias-awareness. For example, by relating articles' content and frames with respondents' attitude toward these frames, the so-called hostile media effect could be investigated and how it affects the visualizations' effectiveness. Additionally, a framing ground truth would enable technical evaluation of the frame classification performance of approaches.

Our study showed effects of the PFA approach and the bias-sensitive overview but overall no effects of the article visualization and respondents' demographic factors. In the article view, only one visual clue conditionally increased biasawareness.2 Besides, the article view suffered from minor user experience (UX) issues and a conceptual issue, which lies in the relativity of bias. Specifically, to reveal bias, our article view should more prominently communicate frames of articles other than the viewed article. A method that could directly be integrated into our approach is to show summaries of other articles or their frames. Another future work idea is to enable users to contrast "facts" across articles, e.g., whether and how the facts of the currently viewed article are stated in other articles. To enable the mapping of such facts, methods for semantic textual similarity (STS) could be used. We already proposed exploratory systems for this line of research, but preliminary results indicate the difficulty of this approach [77, 139].

The respondent sample was too small to yield significant effects in the article visualization and respondents' attributes. On the one hand, the distributions of respondents' attributes, such as gender, political orientation, and education level, roughly approximated the US distribution. However, on the other hand, some distributions, such as of the education level, were thus imbalanced and contained too few observations in individual attribute levels, which prevented statistical analysis of these. Increasing the respondent sample would enable statistical analysis of the respondents' attributes. As discussed previously in this section, a larger respondent sample would also facilitate statistical soundness regarding the effect of the article view by compensating the article view's high content diversity.

#### *7.3.4 Societal Implications*

I conclude this thesis with an open outlook on the societal implications of using PFA and other automated approaches as part of daily news consumption. Understanding such societal implications is traditionally beyond the scope of computer science, and this dissertation cannot elucidate these implications. However, I intend to raise essential questions that could serve as a foundation for further, in-depth research across the topic's involved disciplines, especially in the social sciences.

2 The visual clue is highlighting persons' mentions in sentences of a given article. These in-text highlights use a different color depending on the sentences' sentiment toward the person; see Sect. 6.7.2.

subsequent visualization to news consumers? Which criteria are important for such selection?

The research described in this thesis highlights the effectiveness of taking an interdisciplinary approach to tackle the adverse issues caused by media bias. At the same time, the previously raised questions highlight the need for further interdisciplinary research on media bias. Not only is there a pressing need to do so, but now is also a perfect point in time. For example, news articles, representing the base of much research on media bias, are readily available in large quantities since much news is published online. Additionally, the rise of deep learning and recent language models has led to unprecedented advancements in natural language understanding. These advancements can and have enabled automated analysis of tasks that were previously difficult or nearly impossible to fulfill. This thesis, for example, devised the first model capable of classifying the subtle and implicit sentiment connotations common to news articles. I hope that with the shared conceptual understanding devised in the literature review and the proposed PFA approach, this thesis facilitates further interdisciplinary research on media bias.

My vision is that bias identification approaches, such as PFA, are integrated into popular news aggregators and news applications to enable bias-sensitive news consumption at scale. Enabling news consumers to critically assess and contrast news coverage can help mitigate the severe effects of systematically biased news coverage. Supporting individuals in making more informed choices is crucial in collective decision-making, such as democratic elections. In times of misinformation campaigns, "fake news," and other means to intentionally alter the public discourse, bias-sensitive news consumption is of unprecedented relevance. The PFA approach contributes to the required media literacy during daily news consumption by revealing substantial biases without tedious effort.

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

## **Appendix A**

The Appendix includes further material related to the methods and prototype, an overview of publications receiving awards, and other resources.

#### **A.1 Real-World Example**

Table A.1.

**Table A.1** Articles and their URLs used in the real-world example

#### **A.2 Context-Driven Cross-Document Coreference Resolution**

This section contains additional information that complements the tables and numbers presented in the evaluation of our method for context-driven cross-document coreference resolution (Sect. <IP_ADDRESS>) (Table A.2).

**Table A.2** Overview of the normalized composition of the concept types across the events in NewsWCL50. Column "ID" refers to the events' IDs

#### **A.3 Prototype Evaluation**

Figures A.1, A.2, A.3, A.4, and A.5.

**Fig. A.1** Gender distribution of the respondent sample in the main study

**Fig. A.2** Age distribution of the respondent sample in the main study

**Fig. A.3** Education distribution of the respondent sample in the main study

**Fig. A.4** Political orientation distribution of the respondent sample in the main study, very liberal (1)–very conservative (10)

**Fig. A.5** Distribution of the respondent sample in the main study on the attribute: news consumption

## **A.4 Data and Source Code Downloads**

Following the principles of open access research, the author publishes all methods and materials after they were reviewed in a peer review process to ensure easy availability and high quality. Thus, the methods presented in this thesis and further resources are available on GitHub: https://github.com/fhamborg.

The prototype system Newsalyze is available at https://newsalyze.org/.

#### **A.5 Publication Awards**

In the course of the research summarized in this dissertation, several of the author's publications were honored with an award or nominated for one. From the following four publications, the first two represent work that the author conducted independent of this dissertation. The third and fourth publications are directly relevant for this thesis.

#### **Best Student Paper Award**

*F. Hamborg, N. Meuschke, and B. Gipp, "Matrix-based News Aggregation: Exploring Different News Perspectives," in Proceedings of the ACM/IEEE Joint Conference on Digital Libraries (JCDL), 2017.*

This full paper proposes an approach to tackle media bias that the author devised before his doctoral research.

http://jcdl.org/awards.php

#### **Outstanding Paper Award**

*M. Schubotz, P. Scharpf, K. Dudhat, Y. Nagar, F. Hamborg, and B. Gipp, "Introducing MathQA: a Math-Aware question answering system," Information Discovery and Delivery, 2018.*

Related to the overall field of information retrieval, this short paper introduces a question answering system for mathematical knowledge.

https://emeraldgrouppublishing.com/about/our-awards/emerald-literati-awards

#### **Best Short Paper Award Nominee**

*F. Hamborg, A. Zhukova, and B. Gipp, "Illegal Aliens or Undocumented Immigrants? Towards the Automated Identification of Bias by Word Choice and Labeling," in Proceedings of the iConference 2019, 2019.*

This short paper proposes an earlier variant of the person-oriented analysis approach that is summarized in Chap. 3.

https://ischools.org/Awards-2019

#### **Best Full Paper Award Nominee**

*F. Hamborg, S. Lachnit, M. Schubotz, T. Hepp, and B. Gipp, "Giveme5W: Main Event Retrieval from News Articles by Extraction of the Five Journalistic W Questions," in Proceedings of the iConference 2018, 2018.*

This full paper proposes an earlier variant of the event extraction system that is summarized in Sect. 4.2.

https://ischools.org/iConference-2018-Summary

## **Glossary**

## **References**

USA: Association for Computational Linguistics, 2004, pp. 1–4. DOI: https://doi.org/10.3115/ 1614025.1614026. URL: http://portal.acm.org/citation.cfm?doid=1614025.1614026.

D'Informatique Documentaire, 2004, pp. 186–194. URL: https://dl.acm.org/doi/abs/10.5555/ 2816272.2816290.

Linguistics, 2013, pp. 1650–1659. ISBN:<PHONE_NUMBER>503. URL: https://www.aclweb.org/ anthology/P13-1162.pdf.

https://doi.org/10.1007/978-3-540-69858-6\_21. URL: http://link.springer.com/10.1007/978- 3-540-69858-6\_21.

115. ISBN: 978-1-7281-4941-7. DOI: https://doi.org/10.1109/VISUAL.2019.8933611. URL: https://ieeexplore.ieee.org/document/8933611/.

tics, 2020, pp. 3188–3197. DOI: https://doi.org/10.18653/v1/2020.acl-main.291. URL: https:// www.aclweb.org/anthology/2020.acl-main.291.


**3. Food Safety in H. alal Assurance ¯**

Nowadays food safety is a responsibility of government agencies and organizations. European Community (EC) legislation (see Table 1) is primarily geared towards ensuring the production of safe foods for human health but also for ensuring free competition in the food market.

For foodstuffs of animal origin, further specific hygiene requirements are necessary (prescribed by Reg. 853/2004) [22], as these products may present macrobiotic and chemical risks for the human health and therefore, they require the application of specific rules. The rules dictated by Reg. 853/2004 are added to those related to animal welfare [72], without posing particular issues to ritual slaughter operators.

In addition to the above mentioned laws, Regulation (EC) N. 1935/2004 of the European Parliament and of the Council of 27 October 2004 on the regulation of materials and articles intended to come into contact with foodstuffs [23] should also be taken in account. In general, once these materials come into contact with food, they must not cause unacceptable changes in foodstuffs; nor should these provisions pose particular problems to h. alal slaughter operators. ¯

Hazard Analysis and Critical Control point (HACCP) system is considered to be effective for enhancing food safety; furthermore, other standards, such as ISO series, Approved Quality Assurance (AQA), Good Agricultural Practices (GAP), Good Manufacturing Practices (GMP) and the Food Safety Management System (FSMS) could be considered for food quality and safety. These standards could be contemplated by more than one hundred active certifying bodies, governmental or non-governmental organizations, for h. alal compliance [ ¯ 73]. In a recent study, many areas for potential research in halal¯ assurance in the food industry have been identified, and critical issues have been highlighted [74]. Incorporating halal features into the HACCP system could be a plausible tool for hal ¯ al assurance. ¯ As the HACCP and H. alal certification processes are similar, the integration of the hal ¯ al assurance ¯ scheme into the HACCP system could be feasible [75].

Although the knowledge of all factors influencing the h. alal assurance systems is a determinant to ¯ help companies to identify intervention strategies to improve their performance, limited literature is available on this issue [76]. Recently, Malaysian researchers have applied an interesting study design to explore critical factors affecting the h. alal assurance systems: di ¯ fferent factors in every country, region or food chain can be found, with differences resulting between food sectors and subsectors and among small or medium (SME) and large-sized enterprises [77]. These studies are particularly important for SMEs that have limited resources.


**6. Future**

**Figure 12.** Add caption

116 Telemedicine

**Figure 13.** Add caption

To our knowledge, no study has evaluated the child and non-offending caregivers' satisfaction with telemedicine for child abuse evaluations. However, child and parent satisfaction with telemedicineutilizedbyothermedicalsubspecialtieshasbeenpromising.Aself-reportquestion‐ naireadministeredtopatientsreceivingpsychologicalinterventionforchildhooddepressionvia telemedicine over an 8 week period revealed that the participants and their caregivers had very high satisfaction with the services received and had similar rates of attendance when compared to the control group receiving the same services face-to-face [7]. The authors also noted that due to the children's previous experiences with other forms of technology, they appeared to adapt easily to the use of the equipment used in the study [7]. Satisfaction surveys distributed to burn centerphysicians,referringMDorRNproviders,andpatients/familymemberswhereteleconsul‐ tationwasutilized,alsorevealedthatallinvolvedintheprocess,includingthepatientandfamily, felt the encounter was helpful and that they were comfortable with the technology [8]. During an initial review of Florida's program between1999 and 2000, medical personnel reported that the children had a very high comfort level with the equipment involved [9]. With the ever growing technology present in children and parent's everyday lives, it is likely that there will be both a relatively high comfort level with the equipment and high satisfaction with the overall assess‐ ment conducted via telemedicine.

## **7. Conclusions**

Telemedicine has proved to be a very useful method in conducting child abuse assessments to rural areas in the absence of local child abuse experts. The increases in child abuse reporting and lack of experts qualified to medically assess abuse cases further support the use of telemedicine. A variety of applications and uses provides a wide range of possibilities at varying costs that can be adapted by other child welfare programs throughout the world and have been demonstrated nationwide in the U.S. Telemedicine assessments and evidence obtained during the examinations have been successfully used in criminal and civil court without legal challenge. The use of telemedicine in child abuse will ensure accurate diagnosis by an expert, appropriate treatment and follow up, and reduce risk of future maltreatment.

[6] Maguire, S, Mann, M. K, Sibert, J, & Kemp, A. Are there patterns of bruising in child‐ hood which are diagnostic or suggestive of abuse? A systematic review. Arch Dis

Telemedicine: Reducing Trauma in Evaluating Abuse

http://dx.doi.org/10.5772/55070

119

[7] Nelson, E. L, Barnard, M, & Cain, S. Treating childhood depression over videoconfer‐

[8] Saffle, J. R, Edelman, L, Theurer, B. S, Morris, S. E, & Cochran, A. C. Telemedicine evaluation of acute burns is accurate and cost-effective. The Journal of Trauma

[9] Pammer, w, Haney, M, Wood, B. M, Brooks, R. G, Morse, K, Hicks, P, Handler, E. G, Rogers, H, & Jennett, P. Use of telehealth technology to extend child protection team

encing. Telemedicine Journal and e-health (2003). , 9(1), 49-55.

Child (2005). , 90, 182-186.

(2009). , 67(2), 358-365.

services. Pediatrics (2001). , 108(3), 584-590.


**1. Introduction**

In several plant groups, especially those with polyploid complexes as *Triticum* (the wheat genus, Poaceae), related species can be used as important sources of genes. In the tribe Triti‐ ceae as a whole, which comprises other important cereals as barley (*Hordeum vulgare*) and rye (*Secale cereale*), there are high rates of successful interspecific hybridization [1-2]. Due to the ease in obtaining these hybrids, plus the high amount of available information on the genomes of the species, the interspecific hybrids are potentially useful for the genetic im‐ provement of these crops [3-4]. Thus, the hybrids and their derivatives from breeding pro‐ grams can be analyzed by means of different approaches, aiming the full knowledge on the phenotypic constitution of the plant material for its subsequent utilization.

Many cytogenetic methods can be applied during the process of crop improvement, mainly regarding the characterization of chromosome types among accessions of a germplasm col‐ lection [5-6]. Since the discovery of the nucleic acid hybridization reaction by Hall & Spiegel‐ mann [7], and later by using fluorescent detection rather than radioactive isotopes [8], the fluorescent *in situ* hybridization (FISH) and its variations have been largely employed in karyotype characterization of plants [9]. The technique basically consists on the pairing of a given probe (a DNA or RNA fragment) with a specific sequence on the target genome, aim‐ ing to indicate its exact location in a chromosome.

When the objective is to distinguish parental chromosomes (or chromosome segments) in an interspecific hybridization or the distinct genomes of an allopolyploid, the entire genome of one parent should be labeled and used as probe [10]. In this case, the technique is called genom‐

© 2013 Patussi Brammer et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Patussi Brammer et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ic *in situ* hybridization (GISH). On the other hand, the genome of the second parent (unlabeled) is used as blocking DNA, aiming to avoid non-specific hybridizations due to the similarity of the two parental genomes. Thus, both parental genomes (the probe and the blocking DNA) must be used together in the same hybridization mixture. The proportion probe:blocking DNA should be adequate to avoid the detection of the second parent (see Fig. 1).

**Figure 1.** Main steps of the genomic *in situ* hybridization (GISH). (A) Direct and indirect probe labeling. (B) Fragmenta‐ tion of the blocking DNA. (C) Slide preparation. (D) Probe and blocking DNA denaturation in a hybridization mixture. (E) Addition of the hybridization mixture with the probe and the blocking DNA. (F) Denaturation of the chromosome DNA. (G) *In situ* hybridization of probe and blocking DNA in the target sequence of the chromosome. (H) Detection of the probe in the chromosome DNA of one parent, in an indirect labeling. (I) Chromosome DNA molecule of the sec‐ ond parent associated to the unlabeled blocking DNA. (J) Visualization of hybridization signals associated to a probe (green) in a fluorescence microscope. Unmarked chromosomes are visualized with a counter-staining (blue). When the probe labeling is direct, the detection step of the GISH can be excluded. The fluorochromes are the signaling mol‐ ecules and can be directly visualized in a fluorescence microscope with the appropriate filter. Santelmo Vasconcelos & Ana C. Brasileiro-Vidal.

The probe labeling can be either direct or indirect (Fig. 1A). In the direct labeling, the marked nucleotides are associated to fluorochromes, which can be directly visualized in a fluorescence microscope with the proper filter, after *in situ* hybridization procedures. On the other hand, in the indirect labeling, the marked nucleotides are associated to marker molecules (Fig. 1A), which cannot be visualized in microscopes. Thus, after *in situ* hybridization procedures, the la‐ beled probes are recognized by antibodies conjugated to fluorochromes, allowing for the probe detection and visualization (Fig. 1J).

The GISH has direct applications on the understanding of the genome evolution of poly‐ ploid hybrids, partial allopolyploids and recombinant inbred lines, as well as in detecting the amount of introgressed chromatin during the production of new lineages [11-13]. There‐ fore, the GISH has efficiently contributed for the analysis on the karyotypic stability of plant materials, indicating the best genotypes, and helping the assisted selection in different phas‐ es of crop improvement [9, 14].

Here we describe and discuss the main methodological steps of the GISH process as well as the importance of such an approach for the establishment of successful inbred lines, using as example hybrids between common wheat (*T. aestivum*, 2*n* = 6*x* = 42, AABBDD genome) and rye (*S. cereale*, 2*n* = 2*x* = 14, RR genome), hexaploid (2*n* = 6*x* = 42, AABBRR genome) and octo‐ ploid (2*n* = 8*x* = 56, AABBDDRR genomes) triticale lines and their derivatives. For this analy‐ sis, genomic DNA from rye was used as probe and wheat genomic DNA was used as blocking agent in a proportion of 1:10 (probe:blocking agent). Variations of the technique for other Triticeae species will also be discussed.


**5. Postharvest treatments**

### **5.1 Physical treatments**

Physical methods include high or low temperature treatments**,** irradiation and use of modified or controlled atmospheres.

#### *5.1.1 High-temperature treatments*

High temperature treatments can control insect pests, prevent pathogen infection, induce resistance to chilling injury, slow fruit ripening, and extend postharvest shelf life [34, 35]. Application of thermal treatments reduced the fungal development, ripening rate and extended the shelf life in strawberry [36]. Strawberry shelf life may be improved by an appropriate thermal treatment that could be used instead of fumigation to allow a more advantageous usage of this fruit in the commercial chain.

#### *5.1.2 Low temperature*

Freezing of fruits and vegetable is one of the most common ways for maintaining the quality of these products. Frozen storage of strawberry, at 18°C after 7 months, had a specific effect on color but no significant different in total anthocyanin was observed [37]. Decrease of anthocyanin content in frozen storage strawberry, at 20°C after 6 months, depending on variety was 11–27.5% [38]. The storage temperatures of 18 and 24°C were best for preserving the qualitative characteristics (color, texture, flavor and wholeness) of the strawberries [39].

#### *5.1.3 Irradiation*

Alternative control methods that do not leave residues, such as postharvest UV-C radiation, have been shown to prevent decay and improve fruit quality [40–44]. Ultraviolet C (UV-C) radiation is known for preventing fungal decay and enhancing phytochemical content in fruit when applied postharvest. Additionally, it has been reported that postharvest UV-C radiation induces secondary metabolites production that protect fruit against abiotic and biotic stresses [45]. Furthermore, these metabolites (phenolic compounds, anthocyanins, carotenoids) also play an important role in fruit quality with impact on human health [46]. UV-treated fruits had a lower respiration rate, higher titratable acidity and anthocyanin content, and were firmer than the untreated fruits. The percentage of free sugars increased faster in UV treated fruits at the beginning of the storage period [40]. Freshly harvested strawberries of cv. Kent, at 25–50% red were exposed to UV-C at doses of 0.25 and 1.0 kJ/m2 and stored at 4 or 13°C after exposure which has resulted in controlling the decay caused by *Botrytis cinerea* at both storage temperatures and extended the shelf-life of the fruits by 4–5 days [40].

#### **5.2 Chemical treatments**

#### *5.2.1 Fumigation*

Methyl bromide fumigation is the current treatment for postharvest strawberry disinfestation of pests such as western flower thrips [*Frankliniella occidentalis* (Pergande)] and two-spotted spider mite (*Tetranychus urticae* Koch). Due to the reduced availability and increased cost of methyl bromide (as a result of its phase out in 2005 for all uses except quarantine treatments), an alternative treatment is

**29**

*Fruit Physiology and Postharvest Management of Strawberry*

desirable. Low molecular weight volatile compounds such as ethyl formate (EF) are produced by several fruits and vegetables which are important components for flavor and aroma and also have been revealed to have insecticidal and fungicidal properties [47]. Before the product reaches the market, these low molecular weight volatile compounds can potentially undergo degradation to biogenic levels in the tissues of treated commodities which is an advantage over conventional chemicals,

Ethyl formate is currently in the process of being formulated with CO2 for commercial use in Australia and New Zealand. Simpson et al. [48] showed that CO2 in combination with Ethyl formate significantly reduced pest population (Western flower thrips and Red spider mite) without causing any damage to the fruit quality. Nitrous oxide (NO) has been found to be ubiquitous in postharvest climacteric and nonclimacteric fruit, vegetables and flowers, with higher levels present in unripe than in ripe tissues [49, 50]. Since ethylene accumulation initiates ripening of climacteric produce and enhances senescence of nonclimacteric produce, it was speculated that application of NO might retard ripening and senescence in postharvest tissues [51].  The postharvest life can, however, be extended by minimiz-

ing the concentration of ethylene in the atmosphere around fruit [51, 52]. Wills et al. [53] performed fumigation in strawberry in an atmosphere of anaerobic nitrogen for up to 2 h at 20°C with nitric oxide concentrations ranging

dative role in enhancing postharvest shelf life of strawberry fruits [54].

weight loss and decay, higher firmness and hue angle than control [57].

Salicylic acid (SA) is a simple phenolic compound. It is recognized as a plant growth regulator, because of its external application effect on many plant growth physiological processes [55].  They also studied the reversible effect of SA and recommended plant SA treatment in all different growth stages like vegetative, fruit development and postharvest stage. Fruits of the plants of strawberry cv. Camarosa which received SA (0.03 mM) after 7 days at 28°C in their nutrient solution had less

Among secondary nutrients, calcium acts a major role in maintaining the quality of fruit and vegetables. Increasing the "Ca" content in the cell wall of fruit tissue

then held at 20 and 5°C in air containing 0.1 ml l−1

Hydrogen sulfide acts as an important gaseous regulator in plants like nitrous oxide. Fumigation with hydrogen sulfide (H2S) gas released from the H2S donor NaHS increased the postharvest shelf life of strawberry fruits depending on dose used [54]. Strawberry fruits fumigated with various doses of H2S has resulted in significantly lower rot index, maximum fruit firmness, and minimum respiration intensity and polygalacturonase activities than controls. Treatment with H2S maintained higher activity levels of enzymes catalase, ascorbate peroxidase, guaiacol peroxidase, and glutathione reductase and lowers the activities of lipoxygenase relative to untreated (controls). It also reduced hydrogen peroxide, malondialdehyde, and superoxide anion to levels below control fruits during storage. Furthermore, H2S treatment maintained higher contents of soluble proteins, reducing sugars, free amino acid, and endogenous H2S in fruits. This interprets that H2S plays an antioxi-

ethylene

*DOI: http://dx.doi.org/10.5772/intechopen.84205*

which can persist as residues in food products.

which had resulted in extension of postharvest life.

from 1.0 to 4000 ml l−1

*5.2.2 Salicylic acid*

*5.2.3 Calcium dips*

#### *Fruit Physiology and Postharvest Management of Strawberry DOI: http://dx.doi.org/10.5772/intechopen.84205*

*Strawberry - Pre- and Post-Harvest Management Techniques for Higher Fruit Quality*

Physical methods include high or low temperature treatments**,** irradiation and

High temperature treatments can control insect pests, prevent pathogen infection, induce resistance to chilling injury, slow fruit ripening, and extend postharvest shelf life [34, 35]. Application of thermal treatments reduced the fungal development, ripening rate and extended the shelf life in strawberry [36]. Strawberry shelf life may be improved by an appropriate thermal treatment that could be used instead of fumigation to allow a more advantageous usage of this fruit in the commercial chain.

Freezing of fruits and vegetable is one of the most common ways for maintaining the quality of these products. Frozen storage of strawberry, at 18°C after 7 months, had a specific effect on color but no significant different in total anthocyanin was observed [37]. Decrease of anthocyanin content in frozen storage strawberry, at 20°C after 6 months, depending on variety was 11–27.5% [38]. The storage temperatures of 18 and 24°C were best for preserving the qualitative characteristics

Alternative control methods that do not leave residues, such as postharvest UV-C

and stored at 4 or 13°C after exposure which has resulted in controlling

the decay caused by *Botrytis cinerea* at both storage temperatures and extended the

Methyl bromide fumigation is the current treatment for postharvest strawberry

disinfestation of pests such as western flower thrips [*Frankliniella occidentalis* (Pergande)] and two-spotted spider mite (*Tetranychus urticae* Koch). Due to the reduced availability and increased cost of methyl bromide (as a result of its phase out in 2005 for all uses except quarantine treatments), an alternative treatment is

radiation, have been shown to prevent decay and improve fruit quality [40–44]. Ultraviolet C (UV-C) radiation is known for preventing fungal decay and enhancing phytochemical content in fruit when applied postharvest. Additionally, it has been reported that postharvest UV-C radiation induces secondary metabolites production that protect fruit against abiotic and biotic stresses [45]. Furthermore, these metabolites (phenolic compounds, anthocyanins, carotenoids) also play an important role in fruit quality with impact on human health [46]. UV-treated fruits had a lower respiration rate, higher titratable acidity and anthocyanin content, and were firmer than the untreated fruits. The percentage of free sugars increased faster in UV treated fruits at the beginning of the storage period [40]. Freshly harvested strawberries of cv. Kent, at 25–50% red were exposed to UV-C at doses of 0.25 and

(color, texture, flavor and wholeness) of the strawberries [39].

**5. Postharvest treatments**

*5.1.1 High-temperature treatments*

use of modified or controlled atmospheres.

**5.1 Physical treatments**

*5.1.2 Low temperature*

*5.1.3 Irradiation*

1.0 kJ/m2

shelf-life of the fruits by 4–5 days [40].

**5.2 Chemical treatments**

*5.2.1 Fumigation*

**28**

desirable. Low molecular weight volatile compounds such as ethyl formate (EF) are produced by several fruits and vegetables which are important components for flavor and aroma and also have been revealed to have insecticidal and fungicidal properties [47]. Before the product reaches the market, these low molecular weight volatile compounds can potentially undergo degradation to biogenic levels in the tissues of treated commodities which is an advantage over conventional chemicals, which can persist as residues in food products.

Ethyl formate is currently in the process of being formulated with CO2 for commercial use in Australia and New Zealand. Simpson et al. [48] showed that CO2 in combination with Ethyl formate significantly reduced pest population (Western flower thrips and Red spider mite) without causing any damage to the fruit quality.

Nitrous oxide (NO) has been found to be ubiquitous in postharvest climacteric and nonclimacteric fruit, vegetables and flowers, with higher levels present in unripe than in ripe tissues [49, 50]. Since ethylene accumulation initiates ripening of climacteric produce and enhances senescence of nonclimacteric produce, it was speculated that application of NO might retard ripening and senescence in postharvest tissues [51].  The postharvest life can, however, be extended by minimizing the concentration of ethylene in the atmosphere around fruit [51, 52].

Wills et al. [53] performed fumigation in strawberry in an atmosphere of anaerobic nitrogen for up to 2 h at 20°C with nitric oxide concentrations ranging from 1.0 to 4000 ml l−1 then held at 20 and 5°C in air containing 0.1 ml l−1 ethylene which had resulted in extension of postharvest life.

Hydrogen sulfide acts as an important gaseous regulator in plants like nitrous oxide. Fumigation with hydrogen sulfide (H2S) gas released from the H2S donor NaHS increased the postharvest shelf life of strawberry fruits depending on dose used [54]. Strawberry fruits fumigated with various doses of H2S has resulted in significantly lower rot index, maximum fruit firmness, and minimum respiration intensity and polygalacturonase activities than controls. Treatment with H2S maintained higher activity levels of enzymes catalase, ascorbate peroxidase, guaiacol peroxidase, and glutathione reductase and lowers the activities of lipoxygenase relative to untreated (controls). It also reduced hydrogen peroxide, malondialdehyde, and superoxide anion to levels below control fruits during storage. Furthermore, H2S treatment maintained higher contents of soluble proteins, reducing sugars, free amino acid, and endogenous H2S in fruits. This interprets that H2S plays an antioxidative role in enhancing postharvest shelf life of strawberry fruits [54].

### *5.2.2 Salicylic acid*

Salicylic acid (SA) is a simple phenolic compound. It is recognized as a plant growth regulator, because of its external application effect on many plant growth physiological processes [55].  They also studied the reversible effect of SA and recommended plant SA treatment in all different growth stages like vegetative, fruit development and postharvest stage. Fruits of the plants of strawberry cv. Camarosa which received SA (0.03 mM) after 7 days at 28°C in their nutrient solution had less weight loss and decay, higher firmness and hue angle than control [57].

#### *5.2.3 Calcium dips*

Among secondary nutrients, calcium acts a major role in maintaining the quality of fruit and vegetables. Increasing the "Ca" content in the cell wall of fruit tissue

### *Strawberry - Pre- and Post-Harvest Management Techniques for Higher Fruit Quality*

can aid to delay softening and mold growth and decrease the occurrence of physiological disorders [58]. Common techniques like dipping and vacuum or pressure infiltrations are used to increase cell wall Ca content of fruit tissue after harvest. The firming effect can be explained by the crosslinks formation between the carboxyl groups of polyuronide chains found in the middle lamella of the cell wall; Ca also increases cell turgor pressure [59, 60] and stabilizes the cell membrane [61]. Calcium dips have been employed to improve firmness and extend the postharvest shelf-life of a wide range of fruit and vegetables. In strawberries, CaCl2 dips in combination with heat treatment or modified atmosphere storage and refrigeration increase calcium content and fruit firmness and delay postharvest decay [62, 63]. Calcium dips were effective in decreasing surface damage and delaying both fungal decay and loss of firmness in strawberries, compared to untreated fruit [64].


3.5.3. Changes in Endogenous ZR

The ZR content presented a trend of decreasing before increasing (Figure 4C). After the initiation period, the ZR content of the treatments and control exhibited a decreasing trend, with a more obvious decrease exhibited in the treatment group. After callus formation, the ZR content in the control group was higher than that in the treatment groups, indicating that high ZR concentration has an inhibitory impact on adventitious root formation.


*2.6. Tape-Stripping Studies. Drug Penetration through the Skin and Stratum Corneum Depth*

The assay was performed using porcine skin samples. Skin was removed from the connective tissues and placed onto a glass slide, with the outside layer facing upwards. An aluminium mask was placed over it, leaving the application area uncovered. One hundred microliters of each formulation containing lipid vesicles was applied to the delimited application area of skin. The system was then incubated at 32 ◦C for 2, 4, and 6 h. Each test was performed in triplicate (*n* = 3). After incubation, 20 strips of adhesive tape were applied sequentially to the skin using a roller, according to the standardized procedure in our lab, and then removed with a forceps [65]. The amount of stratum corneum removed was determined by infrared densitometry (SquameScan™ 850A device, Wetzlar, Germany), and strips were grouped in different pools, following the sequence: 1, 2, 3–5, 6–10, 11–15, 16–20 [66]. The B12 amount present in each sample was extracted overnight from the strips using a methanol:water mixture (50:50 *v/v*) as extraction solvent, since it afforded an optimal B12 recovery ratio within the established range (100 ± 20%) [67]. Subsequently, B12 was quantified by HPLC with no interferences with the analytical method.


**5. Granuloma: definition**

Granuloma is a foreign body reaction against foreign substances that enter the skin. Granuloma occurs due to continuous or chronic inflammation against foreign substances. Silicone is a foreign substance in the body, which will be encapsulated by the body. Datia cells (giant cells) will encapsulate silicone material, and therefore inflammatory mediators cannot perform phagocytosis, which results in continuous inflammation and causes side effect. The encapsulated material has poor vascularization; therefore, it may potentially induce infections [6].