The Founders

*'GSD succeeded in its artistic aims but tended to falter philosophically because of a core weakness—it depended for its continued growth on the chance mix of individuals to create a living culture. Sometimes this worked and sometimes it didn't.'(p70)*

 **Figure 3.** Founders of Graphic Studio Dublin 1960.

Printmaking was also enjoying a resurgence in North America, in Britain, France, Russia and Scandinavia and many new print studios were being set up, often with the assistance of generous philanthropic donations. Hence GSD can be seen as part of a more widespread pattern on international development in fine-art printmaking. Many of the world's leading print studios were set up around this time. In the US: Pratt Contemporary Graphic Art Centre (1956), Tamarind Lithography Workshop (1960), Crown Point Press (1962) and Hollanders Workshop (1964). England saw the establishment of the Curwen Press (1958) and Scotland, Edinburgh Printmakers (1967). The seed funding of these various establishments tells its own story. In the US, big businesses were more established, and the germs of the concept destined to become corporate social responsibility were already visibly benefiting the arts. Pratt-Contemporaries received a donation of \$50,000 from the Rockefeller family, while Tamarind received \$135,000 from the Ford Motor Corporation. Figure 4 shows some of the printmaking techniques used in GSD.

**Figure 4.** A flavour of the work that takes place in the Graphic Studio Dublin.

However, raising money, especially for the Arts, in the cash-strapped Ireland of the 1960s, was a far trickier proposition. The Irish Export Board came forward with £120 and, while this was derisory when compared to US funding, in Ireland's low-wage and even lower-expectation economy, it was able to go a long way. However, one of the early successes of GSD was an imaginative business model they developed through which generous individual sponsors were encouraged to make a modest annual contribution in return for which they would receive as a gift a small portfolio of prints. This 'sponsors' portfolio' idea still persists to this day, some 50 years later. GSD became a thriving, successful, dynamic artistic community recognised by the Arts Council as part of the visual arts landscape in Ireland and recognised by artists as a commune where inexperienced and veteran printmakers; where master and pupil could work side by side.

Fast-forward, then, almost 50 years later. In 2007, experiencing growing pains and needing additional space to accommodate new equipment and to offer new, emerging printmaking techniques such as silk-screen, GSD now had its own CEO, its own Studio Director (a master-printmaker) and it had acquired its own gallery as a sales channel for artist members to both exhibit and sell their work. It decided to purchase new premises (see Figure 5) during the property boom in Ireland: a four-storey former granary close to the heart of the city on the northside of Dublin. To make the purchase, the organisation borrowed heavily. It had not owned premises before this one, it had been renting for the first 50 years, and the Board thought it was time to get a place they could consider home and call their own.

While the idea might have been laudable, the timing was catastrophic. It coincided with Ireland's first ever property 'bust', where property prices went into freefall, precipitating the deepest and longest recession in Ireland's history. The value of the property plummeted and the Board of GSD managed to negotiate a temporary 'interest only' payment arrangement on the property mortgage. However, the bank that owned the mortgage quickly sold it to a hard-nosed US vulture-fund who wanted to foreclose on the premises and to cast the artists out on the street. This would have left a generation of Irish printmakers without a place to work and without the facilities to make their art. The problems with the unsustainable mortgage were compounded by the fact that art purchases are among the first discretionary spends to get cut during a recession. People buy less art when their earnings are reduced, and hence, just when the organisation needed to show healthier revenues, its sales were drying up. This is where the VUCA environment loomed threateningly over the enterprise, with few reasons for optimism and every avenue for revenue generation apparently drying up.

**Figure 5.** The four-storey converted granary, new printmaking studio for GSD.

Innovation in cultural organisations is considered generally to fall into one of four broad typologies [60,61]:

However, sometimes, these conventional approaches to innovation are unlikely to deliver the sort of turnaround performance that was needed in this VUCA environment.

A new Board of directors was empanelled in GSD to take up this daunting challenge and this is where the Art Thinking strategy took root. To save costs, the services of the CEO were dispensed with. The Board has nine members, of which the majority (5) are artists and the rest are volunteers, generally those interested in Art from the professions. In this instance, there is a chairman, a director of marketing and strategy (the author), a head of Legal (a bank property lawyer), and a head of finance (a chartered accountant in a large practice). The challenge for the new board was to restore GSD's finances to the extent to which it could afford to service the full payments on a mortgage—a reduced mortgage from the one originally taken out. This meant not only getting the business back into a healthier shape, it also required that the mortgage be renegotiated with, ideally, some debt forgiveness.

The situation was so dire that the Board really had to embrace the principles of Art Thinking: it did not seem to them as if it would be enough to plot a route from current position A to an incrementally better position of A+. The Board just had to start with inventing a vision of what a desirable point B might look like in terms of revenues, audience numbers and costs. The Board decided to get all the artists on board to co-create a new, commercial strategy to pull the organisation back from the edge. Several artists (roughly 12) attended a Strategy Day in which a strategic North Star and set of guiding principles for the organisation were developed and several key effort priorities were agreed. The North Star was the organisation's guiding purpose and the effort priorities represented the key deliverables underpinning it. After that, the entire membership was invited to a brainstorming session which was run in a world-cafe format; roughly 40 members attended, which represented around 75% of members. The delegates were given a briefing on Art Thinking and given problem statements, such as: *wouldn't it be great if* ... *, or how might we* ... *?* Each table had to work through the effort priorities and give them some dimensions and suggest revenue generating projects that, while being imaginative enough to be exciting needed to be capable of being actually implemented. The brainstorming session was professionally facilitated by a consultant.

Inviting artists into the business planning process was also an unconventional (and courageous) step as artists invariably have a strong vision and point of view and they rarely pander to an audience's tastes; rather they like to explore new territories and bring the audience on the journey with them. Nevertheless, this artist collaboration yielded a large amount of good and useable concepts, which the Board started to implement straight away. Many were great sales ideas. One prompted a themed artist-led and very profitable promotion called *100 by 100*, in which 100 members produced one piece (an etching or print) which had to be inspired by something in Ireland's National Botanical Gardens, and they each made 100 prints of it. This was themed *the Botanicals*, as Dublin's Botanical Gardens allowed the show to be exhibited there during their busy season, thus providing GSD not only with a new audience for original art but with a new exhibition venue.

Another initiative was the selection of a street graffiti artist (Dublin's equivalent of Banksy) to come and make a series of prints in GSD (see Figure 6). This was a groundbreaking partnership, the artist, Maser, was well known for urban street art, and was an iconic figure in the youth market with a cult following on social media. To get Maser to make a series of fine-art prints was quite a coup for the studio, and the prints he produced sold so well and so fast that they broke all prior records.

**Figure 6.** A sample fine art print by street artist Maser—leading to record sales in GSD.

Others were that some of the artists' larger works were showcased in pop-up retail stores in some of Dublin's busiest shopping thoroughfares, such as the Powerscourt Townhouse Shopping Centre. New markets, new spaces and new audiences were being opened up through this strategy, and the sales were really feeling a dramatic lift. In searching to open up new and larger markets for Irish printmaking, the sponsor's portfolio was revived, and sales were made to institutional buyers like Yale University, Oxford University and in Ireland to the National Gallery and Trinity College.

The organisation also strengthened its Business to Arts partnerships and forged a deal with one of Ireland's biggest law firms to produce an original piece of art to go to their 750 best clients. This was a high-ticket, prestigious and profitable programme, putting original art into the homes of hundreds of well-paid business executives. Figure 7 shows the classification and the range of innovation activity that was undertaken at this time for the organisation. A lot of the activity focused on bringing the work to the attention of new and different audiences, especially people to whom printmaking was relatively unknown. A counter-intuitive feature of this case is that when threatened with the harsh financial crisis, rather than drawing in their horns: cutting costs and commissioning less work, the Board chose to do the opposite. They elected to take a different approach, to ignore the bleak reality and, instead, to imagine a different and better reality for the organisation. They began with plotting a strategic 'north star' and navigated towards this desired outcome.

**Figure 7.** Classification of the Art Thinking projects developed and delivered by the Board.


**7. Attractive lures and toxic lures**

Another aspect of behavioral control is the use of secondary plant substances that play an important role in the feeding of monophagous or oligophagous insects (Cohen 2003). These substances are used in insect behavioral and biology studies, such as attraction / repellence, feeding and oviposition deterrence etc (Martinez & Van Emden 2001). Such chemicals that indicate the presence of food are in many cases, secondary compounds that stimulate chemo‐ receptor cells located in taste sensilla across tarsi, antennas and mouth parts of insects, inducing feeding and oviposition. When these compounds induce feeding are called phagostimulants or feeding stimulants (Nation 2002).

Attractive food lures containing feeding stimulants make pest management by behavior ma‐ nipulation an effective tool, helping to reduce the amount and improve pesticides efficiency applied in agricultural production systems. These attractive lures have been used for the fol‐ lowing purposes: (i) identification and distribution of insect species, (ii) certification of a region or country for the absence of a particular pest species (free area), (iii) pest eradication pro‐ grams, (iv) Integrated Management Program, (v) pest monitoring (Nascimento *et al.* 2000).

these cases, adding a synergist, enhances the capture of both males and females. When using only the sexual pheromones in this technique, generally, only males are caught. The male removal from the area may not have a major impact on the size of the next generation

The results achieved with mass collection may not be suitable for pest control as, depending on the mating ability of the species, a single male can fertilize many females, thus preventing the elimination or reduction of the number of individuals of the next generation. To work around this, a combination of pheromone with a food attractant could lead to both males and females catch. The initial size of population should then be estimated by monitoring, before the use of mass collection technique. El-Sayed *et al.* (2006) indicate the technique for controlling populations at low density or sparsely isolated. In the absence of these conditions, it should not be used alone for pest control. When adults of both sexes are considered pests and in the presence of aggregation pheromone, the technique proves to be effective. Several examples can be found using this technique in agricultural pest control, as in the case of *C. pomonella* in Europe and America (Hagley, 1978, Willson & Trammel 1980, Emel'yanov & Bulyginskaya 1999, Beers *et al.* 2003), borer of the genus *Rhynchophorus* in Asia, Middle Eastern and South America; fruit flies in orchards; *A. grandis* in the United States of America and Mexico;

The results indicate that the success in suppressing pest population and damage reduction is not always ensured, especially when the pest is found in high population density. Some requirements must be observed according El-Sayed *et al.* (2006) to succeed in using the mass collection. The first is the arrangement of traps in the area, so that a large part of the population has contact with the attractive. The second requirement is that the attractive has to be more effective than the natural source of attraction, as virgin females, foods, among others. This attractive must have an effective release, ranging from emergency to the insect mating season. Traps effectiveness in capture and retention of insects must be observed as well as the cost of

The mass trapping, when is not enough by itself to control pest population, can be used in combination with other methods of pest control within the philosophy of Integrated Pest

Another aspect of behavioral control is the use of secondary plant substances that play an important role in the feeding of monophagous or oligophagous insects (Cohen 2003). These substances are used in insect behavioral and biology studies, such as attraction / repellence, feeding and oviposition deterrence etc (Martinez & Van Emden 2001). Such chemicals that indicate the presence of food are in many cases, secondary compounds that stimulate chemo‐ receptor cells located in taste sensilla across tarsi, antennas and mouth parts of insects, inducing feeding and oviposition. When these compounds induce feeding are called phagostimulants

depending on the reproductive biology of the species.

188 Insecticides - Development of Safer and More Effective Technologies

*Lymantria dispar* in USA, among others.

**7. Attractive lures and toxic lures**

or feeding stimulants (Nation 2002).

the technique.

Management.

For a higher persistence of phagostimulants at the lures, the starch can be used as a vehicle of feeding stimulant, for example, in combination with insecticides and dry bran. Several attractive, such as brown sugar, sucrose, hydrolyzed corn protein, fruit juice and wine vinegar in McPhail, Jackson and PET bottles traps are used for monitoring, and when added to insecticides are recommended for pest control, as example the fruit flies (Nascimento *et al.* 2000), contributing to the reduction of pest population in the field. Orange and grape juices at a concentration of 25% have been used in monitoring *A. fraterculus* in peach orchards (Guerra *et al.* 2007).

The attraction of *Mocis latipes* for sweet baits in traps has been studied by Landolt (1995) showing that honey solutions in water (20%) or unrefined sugar in water (5, 10 or 20%) caught a significant number of moths. This author also found that these baits prepared with three days in advance were more effective than baits made with fresh solutions.

Lures prepared with different concentrations of spinosad insecticide have been effective in fruit flies control, *C. capitata* and *A. fraterculus* (Raga & Sato 2005). Toxic lures formulated with hydrolyzed protein, consisting of Biofruit 3% plus malathion (500 Malathion EC) applied on plants located at the orchard edges has been effective in controlling *A. fraterculus* on peach trees. However, this practice is not employed by peaches producers, as it requires changes in the pest management system, replacing scheduled insecticides applications for monitoring implementation (Härter *et al.* 2010).

Toxic lures has also been shown important in the management of lepidopteran pests. Accord‐ ing to França *et al.* (2009) the mortality caused by lufenuron and deltamethrin, associated with 10% honey, increased directly with the evaluation periods (0, 0.5, 1, 2, 12 and 24) for males, females and adults of *N. elegantalis* after exposure to 10% honey attractive solution, achieving 100% of mortality in two hours.

One of the advantages of toxic lures is the environmental benefit, since they are not used in the total field area, compared with synthetic insecticides. So, it would provide reduced risk due to the decreased presence of toxic residues in fruits, decreased resurgence of secondary pests, re‐ duction in the amount of insecticides and higher preservation of natural enemies. The use of toxic lure in guava orchards, *Psidium guajava,* had lower influence on adults of Chrysopidae family, compared to synthetic insecticides application (Galli *et al.* 2004).

The great success of behavioral management is the ability to associate technologies to control pests, rationing insecticides use, due to applications based on monitoring. The techniques of behavioral control consort with other control methods such as conventional, and benefit not only the consumers but the ecosystem and all involved in the productive chain of agricultural products.


*Article* **Spatial and Temporal Trends of Burnt Area in Angola: Implications for Natural Vegetation and Protected Area Management**

#### **Silvia Catarino 1,2,\*, Maria Manuel Romeiras 1,3,\*, Rui Figueira 1,4, Valentine Aubard 2, João M. N. Silva 2 and José M. C. Pereira 2**

Received: 18 June 2020; Accepted: 5 August 2020; Published: 9 August 2020

**Abstract:** Fire is a key driver of natural ecosystems in Africa. However, human activity and climate change have altered fire frequency and severity, with negative consequences for biodiversity conservation. Angola ranks among the countries with the highest fire activity in sub-Saharan Africa. In this study, we investigated the spatial and temporal trends of the annual burnt area in Angola, from 2001 to 2019, and their association with terrestrial ecoregions, land cover, and protected areas. Based on satellite imagery, we analyzed the presence of significant trends in burnt area, applying the contextual Mann–Kendall test and the Theil–Sen slope estimator. Data on burnt areas were obtained from the moderate-resolution imaging spectroradiometer (MODIS) burnt area product and the analyses were processed in TerrSet. Our results showed that ca. 30% of the country's area burned every year. The highest percentage of annual burnt area was found in northeast and southeast Angola, which showed large clusters of decreasing trends of burnt area. The clusters of increasing trends were found mainly in central Angola, associated with savannas and grasslands of Angolan Miombo woodlands. The protected areas of Cameia, Luengue-Luiana, and Mavinga exhibited large areas of decreasing trends of burnt area. Conversely, 23% of the Bicuar National Park was included in clusters of increasing trends. Distinct patterns of land cover were found in areas of significant trends, where the clusters of increasing trends showed a higher fraction of forest cover (80%) than the clusters of decreasing trends (55%). The documentation of burnt area trends was very important in tropical regions, since it helped define conservation priorities and management strategies, allowing more effective management of forests and fires in countries with few human and financial resources.

**Keywords:** wildfires; MODIS burnt area product; WWF ecoregions; land cover; Miombo woodlands; biodiversity conservation; sub-Saharan Africa

#### **1. Introduction**

Fire is considered a key driver of biodiversity [1], playing an important role in the structure and distribution of ecosystems and biochemical cycles [2]. It determines the distribution, ecology, and maintenance of African savannas and grasslands [3–5]. Over the last million years, the frequent

occurrence of fires in Africa promoted the evolution of a more fire-tolerant and dependent flora [6]; without fire, large areas of savannas could develop into closed woodlands [4].

In southern Africa, however, the severity of fires caused by human activity is increasing [7], with negative consequences for biodiversity conservation and climate change. Approximately 70% of the global burnt area and 50% of fire-related carbon emissions are produced in Africa [8,9]. Uncontrolled fires can greatly damage residential zones, agricultural areas, and forests that are not adapted to fire, contributing to deforestation in many African countries, and producing gas emissions with harmful effects on human health and global climate [10–12]. In Africa, wildfires were identified as the main driver of forest degradation in remote areas of forests, which are more likely affected by recurring fires than by timber extraction [13]. On the other hand, fires are also used in the traditional slash-and-burn agriculture, which consists of cutting trees and woody plants from an area and then burning biomass, resulting in nutrient-rich ash that makes the soil more fertile for agriculture and grazing [13,14]. Savanna fires start by natural causes, mainly lightning, and human intervention, but the relative share of anthropogenic fires is quickly increasing in Africa. In savannas of West Africa, human activity is the most important factor causing fires [11].

The size of burnt areas varies greatly across countries and depends on rainfall seasonality, human activity, and available fuel [15,16]. The fire regime is strongly related to the land use practices and rural populations greatly influence the extent of fires and the annual area burnt [16,17].

Historically, humans have used fire to clean the land [16]. The rural population, which continues to grow in many African countries, frequently uses fire to promote grass growth for cattle to eliminate agricultural residues after harvests and to clear agricultural lands [16]. Moreover, wood and charcoal are the main sources of energy for cooking and heating in rural communities, their use enhancing the exploitation of natural resources and deforestation [11]. An increase in biomass burning causes local drying of the atmosphere, reducing rainfall in the region [12].

According to Archibald et al. [18], Angola, Zambia, and Mozambique are the countries with the most fire activity in southern Africa. In these countries, where the fires are mainly grass-fueled surface and closely related with areas of grass vegetation, such as savannas, grassland, forest and transitions, approximately 50% of the land area was affected by fire between 2001 and 2008, and a large part of it was burnt more than four times during this period [18].

Angola encompasses a great diversity of habitats from desert to tropical rain forests but is dominated by grasslands, savannas, and scrublands [19]. In total, 15 different ecoregions of the global ecoregions map of World Wildlife Fund (WWF) are present in Angola [20], of which the most widespread are the Miombo woodlands of the central plateau, and the western Congolian forest-savanna mosaics in the north [21]. Other important but less extensive ecoregions found in this country are the Atlantic Equatorial coastal forests in Cabinda and the mopane woodlands and the Namibian savanna woodlands in the southwest [20]. In this country, fires are generally classified as surface fires, occurring mainly during the dry season between late April and November [22,23]. Northern regions and central highlands burn considerably earlier than southern zones, when the grass fuel contains higher moisture, resulting in cooler, less intense fires that are less damaging to vegetation than the later and hotter fires in southern Angola [15,23].

Over the last years, intense fires are becoming more frequent in Angola [23]. Very dry seasons and consecutive years of drought with low-humidity conditions promote an increase in the occurrence and intensity of fire [24]. Moreover, droughts have led to significant food insecurity, reducing many populations to subsistence conditions and increasing use of fire to clear hunting zones and preparing new ones for cultivation [14]. Recent studies [23,25] show that Angolan forests have been strongly affected by severe and recurrent fires. Although a single fire has no strong effect on tree composition, the damage caused by recurrent fires in the late dry season can cause high tree mortality [26]. For woodlands in south Africa, a period of at least 5 years without fire is necessary for the successful reestablishment of some tree species [27,28], but such an interval rarely happens in Angolan savannas [17,29].

Currently, deforestation continues to increase in Angola, where one of the highest rates of tree cover loss in sub-Saharan Africa occurs [30–32]. Most of the native trees are under severe pressure due to logging, wood harvesting and charcoal production [21,33,34], as well as fire-related human and climate factors [35–37]. With southern Angola expected to become drier and warmer as a consequence of climate changes, fire frequency is likely to increase and the conservation of Angolan forests and savannas is urgently needed [38,39]. Effective biodiversity conservation requires that fire plays a natural role in ecosystems without representing a threat to ecosystems or human well-being [40].

The current forest degradation caused by fires is difficult to quantify, requiring a comparison of repeated measurements over time [26]. Over the last two decades, remote sensing has become a very useful technique to map burnt areas at regional and global scales, providing a comprehensive view of landscape patterns of fire (e.g., [10,18,29,41,42]). Mapping burnt areas over the years is crucial for resource managers to understand fire impacts on habitats, wildlife, and human settlements, particularly in the context of global climate change [11]. The moderate-resolution imaging spectroradiometer (MODIS) has been extensively used for the study of fire and burnt areas, including in southern Africa [18,24,25].  Mishra et al. [24] examined how variations in burned area and fire frequency were determined by rainfall, vegetation morphology, and land use in the semiarid savanna of Botswana. Schneibel et al. [25] studied the role of fire in forest degradation processes in south-central Angola. However, the study of spatial and temporal trends of burnt area and the determination of its magnitude have never been carried out in Angola. MODIS derived burnt-area product (MCD64A1) is based on changes in surface reflectance over time and includes per-pixel burnt area and quality information about recent fires [43,44]. It is available as monthly global maps, with good detection of small burns, low temporal uncertainty, and a small extent of unmapped areas [18,44,45].

The main goal of this study is to analyze spatial and temporal trends of the area annually burnt in Angola between 2001 and 2019, and how these trends affected the conservation goals of the country, i.e., the sustainable use of natural resources, the recovery of its biodiversity, and the rehabilitation of its protected areas after almost 30 year of war [14]. Specifically, we aimed to: (i) map fire incidence in Angola between 2001 and 2019; (ii) detect and analyze positive and negative trends in the annual burnt area; and (iii) analyze the association between the detected trends and the Angolan ecoregions, protected areas, and land cover classes. Our results will provide new data and useful information for the sustainable fire management and conservation of Angolan ecosystems.

#### **2. Materials and Methods**

#### *2.1. Study Area*

With ca. 1,246,700 km2, the Republic of Angola is the largest country in southern Africa. It is located between 4◦22 S and 18◦02 S, and 11◦41 E and 24◦05 E, and is organized into 18 provinces (Figure 1). The mean annual rainfall varies between ca. 50 mm in Namib and more than 1500 mm in Lunda Norte [46]. A very diverse climate and topography originated high species richness and habitat heterogeneity. In total, 15 ecoregions are recognized in Angola [20] (Figure S1), including deserts, savannas, grasslands, tropical forests, and mangroves. The current system of protected areas includes 14 areas (Figure 1), covering about 13% of the country [47,48].

With a population of over 26 million people and an annual growth rate of 3.3% according to the 2014 population census [49], this country struggles with food scarcity, poor medical support, and lack of infrastructures [25]. Approximately 37% of the population lives in rural areas and depends on agriculture for food and financial income [49,50]. The expansion of agricultural and urban areas and the demand for agricultural land are important causes of anthropogenic fire and deforestation [34,51].

**Figure 1.** Study area, with the 18 provinces of Angola and the protected areas system: BI, Bicuar; BU, Búfalo; KA, Cangandala; CM, Chimalavera; IO, Iona; IP, Ilheu dos Pássaros; KM, Cameia; KI, Quiçama; LL, Luengue-Luiana; LU, Luando; MV, Mavinga; MU, Mupa; MY, Maiombe; NA, Namibe.

#### *2.2. Burnt Area Data, Trend Estimation, and Testing*

Data on burnt areas in Angola were obtained from the MODIS burnt area product MCD64A1 v.006 [52] and downloaded using the Google Earth Engine platform. These data are produced from MODIS Terra and Aqua daily surface reflectance products at 500 m resolution [52–54]. Based on the daily surface reflectance, the algorithm identifies rapid changes to detect burning events and maps their spatial extent [52,54]. The algorithm uses a burn sensitive vegetation index derived from MODIS shortwave infrared atmospherically corrected surface reflectance to create thresholds that are applied to composite imagery data and then spatial and temporal active-fire information is used to estimate probabilistic thresholds suitable to classify individual grid cells as burnt or unburnt [44,52].

MODIS Terra and Aqua satellites were launched at different times. While the Terra sensor has been collecting data since February 2000, the Aqua sensor has been collecting data since June 2002 [55]. As the post-fire signal is persistent and the product of the burnt area is not very sensitive to the time of satellite passage [56], losing little information with just one sensor, we used monthly data from January 2001 to December 2019.

To analyze the mean annual fraction of the burnt area and the number of years of burning, we summed the data for the 12 months of each year (January to December) and resizes the resulting maps to a 5 km resolution grid using QGIS v.3.4.4 software [57]. Each cell was reclassified with the percentage of area burned and the number of years in which it was burned.

The analysis of the spatial and temporal trends of the burnt area was processed in TerrSet v.18.31 [58]. To avoid sequential correlation in time series (i.e., the lack of independence between observations), we applied the Durbin-Watson serial correlation test [59–61] and the pre-whitening procedure proposed by Wang and Swail [62], which maintains the same trend as the original series, but without serial correlation.

To identify significant trends, we applied the Mann–Kendall test [63,64] and the contextual Mann–Kendall test [65]. The Mann–Kendall test is a nonparametric test of trend monotonicity and a common approach in studies of environmental time series [61,65,66]. The contextual Mann–Kendall test, proposed by Neeti and Eastman [65], is a modified version of the Mann–Kendall test that includes geographical contextual information, i.e., neighboring cells are involved in the determination of trends, allowing the detection of homogeneous regions with similar trends. The contextual analysis imparts greater confidence to the identified trends [61].

Then, the Theil–Sen slope estimator was applied to determine the rate of burnt area change in each cell with significant trends [67,68]. This non-parametric technique is robust against outliers and provides an indicator of change over time [66]. It is defined as the median of the slopes calculated between observations in all pair-wise time steps and is suitable for application to time series with large annual variations, such as those concerning annual burnt area [61]. An increasing trend is indicated by a positive Z value, while a decreasing one is indicated by a negative Z value.

#### *2.3. WWF Ecoregions, Protected Areas, and Land Cover Data*

Ecological conditions, changes in land use, and population dynamics are important drivers of fire occurrence over time. To analyze the association between these factors and the trends of the burnt areas, we compared the maps of burnt area trends with the WWF ecoregions (Figure 2a) [20], the Angolan system of protected areas [47], and land cover time-series maps (Figure 2b) [69,70].

WWF terrestrial ecoregions represent the high variability of biogeographic diversity and ecological conditions (see Figure 2a) [20,71–73]. This map was produced at a global scale and reflects the regionalization of the Earth's terrestrial biodiversity, defining units that contain a distinct assemblage of natural communities sharing a large majority of species, dynamics, and environmental conditions [20]. WWF ecoregions has been used as a basis for studies on biotic diversity and conservation in Angola [19,21,74].

Maps concerning the Angolan system of protected areas were downloaded from the World Database of Protected Areas [75]. The present extent of Luengue-Luiana and Mavinga national parks was not updated in WDPA, so it was vectorized based on published Angolan legislation (Diário da República de Angola, law 38/11 29 December 2011, p. 6340).

Land cover data were obtained from the European Space Agency (ESA) Climate Change Initiative Land Cover (CCI-LC) project [76] which produced annual land cover maps for 1992–2015 (version 2.0.7) and 2016–2018 (version 2.1.1), combining remote sensing products and ground observations [69,70]. These maps provide data describing the land surface at 300 m resolution, divided into 22 classes (see Figure 2b). To understand changes in land cover over time, we used the most recent land cover data available (2018) and data collected every four years (2002, 2006, 2010, and 2014). Finally, the maps of burnt area and significant trends were spatially intersected with the WWF ecoregions, protected areas, and land cover maps using QGIS v.3.4.4 software [57].

**Figure 2.** World Wildlife Fund (WWF) ecoregions and land cover map of Angola: (**a**) WWF ecoregions occurring in Angola [20]. The black lines represent the boundaries of the 18 provinces of Angola; (**b**) land cover map of Angola in 2018 (300 m resolution) adapted the European Space Agency (ESA) Climate Change Initiative Land Cover (CCI-LC) project [70].

#### **3. Results**

#### *3.1. Area Burnt in Angola since 2001*

Our results revealed that Angola has been extensively affected by fires every year. On average, ca. 368,300 km2 burn every year since 2001, corresponding to 30% of the country's area. The highest values of burnt area were recorded between 2003 and 2005, with more than 400,000 km2 burnt each year, and the lowest value was 315,580 km2 in 2018 (Figure 3a).

**Figure 3.** Burnt area in Angola, from 2001 to 2019: (**a**) Absolute burnt area (km2) per year; (**b**) map of the mean annual fraction of burnt area in each cell (5 km resolution); (**c**) percentage of the area burnt in the four provinces with the highest values; (**d**) map of the number of years when each cell was completely or partially burnt since 2001 (5 km resolution).

The northeast and southeast regions of Angola presented the highest percentage of annual burnt area per cell (Figure 3b). The provinces with the largest annual burnt areas were Lunda Norte (54%, ca. 58,840 km2), Lunda Sul (53%, ca. 41,375 km2), Cuando Cubango (42%, ca. 84,455 km2), and Malanje (41%, ca. 33,586 km2) (Figure 3c, Table S1). Among them, Cuando Cubango showed the highest variations over consecutive years. The most affected municipalities were Lubalo, Cuilo, and Lucapa in Lunda Norte; Saurimo and Muconda in Lunda Sul; Dirico and Rivungo in Cuando Cubango; and Luquenbo and Cangandala in Malanje. Intermediate values were found in Cuanza Sul, Moxico, Bié, Cunene, Cuanza Norte, Huambo, Zaire, Huíla, Uíge, and Benguela, with 12–30% of area burnt every year. The provinces of Bengo, Cabinda, Luanda, and Namibe had the lowest values, under 10%. Fire events are recurrent in Angola, except in the arid southwestern region. Figure 3d shows the number of years when each cell burnt totally or partially between 2001 and 2019. Since 2001, 64% of the cells burnt 15 years or more.

#### *3.2. Spatial Trends of Burnt Area in Angola since 2001*

The results of the contextual Mann–Kendall test highlights large areas of significant increasing and decreasing trends in the burnt area of Angola. Figure 4 shows the results of this test, where the red cells represent increasing trends (positive Z value) and green cells represent decreasing trends (negative Z values). The significant areas with a confidence level of 95% are outlined with a black line. The Mann–Kendall test was also performed (Figure S1), but the contextual Mann–Kendall test (Figure 4) performed better in creating larger and more homogenous clusters of cells corresponding to significant areas.

**Figure 4.** Trends of the annual burnt area in Angola for the 2001–2019 period, based on the contextual Mann–Kendall test. The positive z-scores correspond to areas of increasing trends of burnt area, while the negative z-scores correspond to decreasing trends. The significant areas (with a confidence level of 95%) were outlined with black lines. Protected areas are indicated with blue outlines: BI, Bicuar; BU, Búfalo; KA, Cangandala; CM, Chimalavera; IO, Iona; IP, Ilheu dos Pássaros; KM, Cameia; KI, Quiçama; LL, Luengue-Luiana; LU, Luando; MV, Mavinga; MU, Mupa; MY, Maiombe; NA, Namibe.

We detected significant areas of increasing trends in ca. 119,410 km2 (10% of the country's area), mainly in the central region of the country. With more than 9400 km2, the single largest area is located in the north of the Cuando Cubango province, and the second largest area, with ca. 4330 km2, is found in Bié. The provinces with the most extensive areas of significant positive trends are Moxico (34,731 km2), Cuando Cubango (18,628 km2), Bié (15,830 km2), and Huíla (12,658 km2) (Figure 5, Table 1). Significant areas of decreasing trends was detected in ca. 154,240 km2 (12% of the country's area), spread all across Angola, except in its central region. The single largest area has more than 21,800 km2 and covers part of the Cuando Cubango and Cunene provinces; the second largest single area has approximately 14,000 km2 and is located in Lunda Sul. The provinces with the most extensive areas of significant negative trends are Cuando Cubango (34,584 km2), Lunda Sul (21,049 km2), and Lunda Norte (19,353 km2) (Figure 5, Table 1).

**Figure 5.** Areas of significant increasing and decreasing trends in Angolan provinces.

**Table 1.** Area and proportion of increasing and decreasing significant trends of annual burnt area in Angolan provinces, WWF ecoregions, and protected areas, from 2001 to 2019.

**Table 1.** *Cont*.

#### *3.3. Burnt Area Trends According to WWF Ecoregions, Protected Areas, and Land Cover*

Our results revealed a very uneven distribution of burnt area trends across WWF ecoregions (Figure 6a, Figure S2, and Table 1). Central African mangroves and Zambezian Cryptosepalum dry forests are among the smallest ecoregions occurring in Angola, but they displayed the highest percentage of increasing trends, and no significant decreasing trends. Western Zambezian grasslands, Central Zambezian Miombo woodlands, and Angolan Miombo woodlands also showed very high percentages of increasing trends (25%, 16%, and 14%, respectively) and much lower percentages of decreasing trends (3%, 4%, and 10%, respectively). The Angolan Miombo woodlands is the largest ecoregion (more than 628,000 km2), covering the central region of Angola.

The high percentage of decreasing trends was found in the Angolan Montane forest-grassland mosaic (32%), the Southern Congolian forest-savanna mosaic (32%), the Zambezian Baikiaea woodlands (25%), the Zambezian flooded grasslands (21%), and the Western Congolian forest-savanna mosaic (14%). The Namibian Savanna woodlands, Kaokoveld desert, and Atlantic Equatorial coastal forests exhibited no significant trends of burnt area.

In protected areas, burning trends are mainly negative. Our results revealed areas of decreasing trends in five national parks, totaling ca. 19,560 km2, which represents 12% of the national protected area (Figure 4, Table 1). Cameia has the highest percentage of decreasing trends (28%, 3918 km2), followed by Luengue-Luiana (18%, 7811 km2) and Mavinga (17%, 7466 km2) national parks (Figure 6b). The area of increasing trends was ca. 5710 km2, corresponding to 4% of the global protected area. It was detected in eight protected areas and the highest proportion was found in Bicuar National Park (23% of the park's area, 1809 km2) (Figure 6b, Table 1). Luando reserve (9%, 769 km2), Cangandala (5%, 33 km2), Mupa (5%, 240 km2), and Luengue-Luiana (4%, 1949 km2) national parks also have important fractions of increasing trends. Some national parks, such as Namibe, Iona, and Maiombe, showed no significant trend.

Concerning the land cover, Angola is covered mainly by natural vegetation as forest, shrubland, and grassland. In 2018, forest (including broadleaved, deciduous, evergreen, and flooded tree classes) was found in 59% of the country's area, shrubland in 18% and grassland in 12% (Figure 7a). Cropland was identified in ca. 48,900 km2, representing only 4% of the territory. Comparing the composition of the areas included in the significant clusters, clusters with increasing trends presented a much higher fraction of forest classes (80%) than clusters with decreasing trends (55%). In clusters with decreasing trends, the proportions of grassland and shrubland were much higher (20% and 19%, respectively) than in the increasing ones (4% and 10%, respectively) (Figure 7a and Figure S3).

**Figure 6.** Areas (%) of significant increasing and decreasing trends: (**a**) in WWF ecoregions; (**b**) in protected areas.

Areas with significant increasing and decreasing trends show similar patterns of land cover changes since 2002 (Table 2), occurring a global decrease in natural vegetation extent, i.e., grassland, shrubland, and forest, as well as an increase in cropland and urban areas (Figure 7). In significant clusters, forest shows a small increase in area between 2002 and 2006, but after 2006 it decreases and reaches the lowest value in 2018. Shrubland has maintained almost stable since 2010, with a small increase during the last four years in clusters with decreasing trends. Between 2002 and 2018, clusters with increasing trends have lost of 0.04% of forest, 0.38% of shrubland, and 1.26% of grassland, but displayed an increase of cropland (8.84%). Decreasing trend clusters lost 0.13% of forest area, 0.40% of shrubland and 1.19% of grassland, and has a great increase of cropland by 15.87% (Figure 8). Urban areas represented a small fraction inside the clusters, but they have a great increase, mainly in clusters with decreasing trends.

**Figure 7.** Land cover and the significant trends of burnt areas from 2002 to 2018, in increasing and decreasing trends clusters: (**a**) Proportions of the main land cover classes in 2018 (the last year for which information is available); (**b**) area of forest; (**c**) area of grassland; (**d**) area of shrubland; (**e**) area of cropland; (**f**) area of urban areas.

**Figure 8.** Changes in area of natural land cover classes (grassland, shrubland and forest) since 2002 to 2018, (**a**) in increasing trends clusters, and (**b**) in decreasing trend clusters.

**Table 2.** Area of land cover classes within the significant clusters of burnt area trends, in 2002, 2006, 2010, 2014, and 2018.

#### *Diversity* **2020**, *12*, 307

#### **4. Discussion**

#### *4.1. Trends of Annual Burnt Area in Angolan Provinces and Ecoregions*

This study presents a spatial and temporal analysis of the area burnt in Angola based on MODIS satellite images collected from 2001 to 2019, which allows us to assess the main trends over the years. Our results unveiled new information about fire in Angola, revealing the main areas of increasing and decreasing trends of the annual burnt area. There are several studies on fire regimes and dynamics at the global scale or focused in southern Africa (e.g., [17,18,42,77,78]); however, this is the first study on fire trends carried out specifically in Angola and focused on its ecosystems and protected areas. This country encompasses unique habitats and species, and is possibly one of the least documented biodiversity hotspot areas in the world [79]. However, with continued population growth and lack of financial support, subsistence agriculture and human pressure on the land will continue to increase, putting high pressure on natural resources [19,34,51] and endangering the conservation of the Angolan natural heritage [37,47].

The highest values of burnt area were recorded between 2003 and 2005, which may be a consequence of the end of the civil war. During the civil war, ca. one million people were killed and over four million people displaced. Moreover, there was a massive migration from rural communities to the cities and fields were abandoned [80,81]. When the war ended, some displaced people returned to rural areas and burnt natural vegetation to clear land for agriculture.

The largest burnt areas and the highest frequency of fire were found in grasslands and savannas of Lunda Norte, Lunda Sul, and Malanje (northeast Angola), as well as in open savannas of Cuando Cubango (southeast Angola). This agrees with previous studies that recognized these regions as the most affected by fires in Angola [15,17,23,77]. However, these provinces presented small areas of increasing trends and the largest clusters of decreasing trends of burnt area, suggesting a possible decrease in fire extent and/or frequency in the coming years, if the current trends are maintained. Lunda Norte and Lunda Sul are formed mainly by vast plains drained by the tributaries of the Cassai–Congo Basin [46]. According to the Köppen–Geiger climatic classification, the climate is humid tropical and tropical savanna, with hot and humid summers, and warm and dry winters [82]. The population density and the practice of agriculture are very low, but the wind plays an important role in the occurrence of fires, drying the grasslands [49,83]. Cuando Cubango is composed mainly of Kalahari sands, as it is a vast plain with slow flowing rivers. The climate is humid subtropical in the north and hot semi-arid in the south, with a low average annual rainfall, ranging from 1000 to 600 mm [46,82]. The population density is low in this region but more than half of the people work in agricultural activities [49]. Malanje has a similar pattern, with low population density but high intensity of agricultural activities [4]. This province includes a plateau in its central region, with elevation from 1000 to 1250 m, and the climate is tropical savanna [46,82].

The main areas of increasing trends were found in central Angola. This region includes Cuanza Sul, Bié, Huambo, Moxico, and Huíla provinces, and consists mainly of Angolan Miombo woodlands. Moxico is located in central-eastern Angola, the mean annual rainfall ranges from 900 to 1300 mm and is dominated by a humid subtropical climate. Cuanza Sul, Bié, Huambo, and Huíla are located in the extensive region of the ancient plateau, characterized by high levels of annual precipitation in the central zone, reaching 1300 mm in the province of Huambo. [46,49,82]. These provinces are dominated by tropical savanna, humid subtropical, and oceanic climates [82]. Currently, they have a very high population density and a high fraction of families working in agriculture. The Angolan Miombo woodlands is an ecoregion almost exclusive to Angola, differing from other savannas and forests in its high abundance of trees of the Leguminosae family, specifically, *Brachystegia, Julbernardia,* and *Isoberlinia* [72,84]. Several authors recognized fire as one of the most important ecological factors in Miombo, preserving the balance between grasses and trees [11,85]. Most plant species of Miombo show some degree of fire resistance, yet they cannot survive intense and recurrent fires [11]. The natural cause of Miombo fires is mainly thunderstorms in the early rainy season, but most fires are caused by

human action and occur during the dry and hot season [11], when the grass fuel is drier, resulting in more intense, damaging fires. A continuous increase in burnt area for a long period could represent a serious threat for tree species, transforming Miombo woodlands into open savannas [23].

The Central Zambezian Miombo woodlands should also be a priority for future studies and fire management measures. The ecoregion is found in the eastern province of Moxico and show more than 16% of the area with significant increasing trends. Both ecoregions, the Angolan Miombo woodlands and the Central Zambezian Miombo woodlands, are included in the regional center of Zambezian endemism (defined by White [86]), housing a high richness and endemism of plant and animal species [72]. Fire is an integral part of the Miombo ecology, but changes in fire regimes, such as the increase in frequency or severity, can result in drastic landscape changes, reducing or removing some species. Zambezian Cryptosepalum dry forests, Western Zambezian grasslands, and Central African mangroves showed the highest percentage of increasing trends. However, these ecoregions are very little represented in the country, with less than 5000 km2 each, and these results cannot be considered representative. Mangroves are present in a small area on the bank of the Congo River, surrounded by extensive areas of Angolan scrap savanna and woodlands. The cluster of growing trends detected in the mangrove area is shared with an extensive area of scrap savanna, suggesting that it may correspond to a commission error, resulting from the effect of the neighboring cells involved in the determination of trends. The same may be occurring with Zambezian Cryptosepalum dry forests and Western Zambezian grasslands, these ecoregions are surrounding by Angolan and Central Zambezian Miombo woodlands, which has large clusters of significant increasing trends of burnt area in transitional zones.

The WWF ecoregions show very clear patterns of increasing and decreasing trends in the area burnt. The ecoregions represent the original distribution of distinct assemblages of species and communities [20], better distinguishing the structural organization of the landscape than the generic vegetation classes. The definition of fire management measures and laws must be adapted to each ecoregion's characteristics, addressing socioeconomic and ecological problems that restore, protect, and maintain natural habitats.

#### *4.2. Implications for Vegetation and Land Cover*

The largest clusters of decreasing trends in burnt area are found in the regions most affected by fire events every year, such as Lunda Norte, Lunda Sul, and Cuando-Cubango. These regions have a lower percentage of forest (ca. 55%) and a large percentage of shrubland (19%) and grassland (20%), a pattern probably resulting from the recurrent fires identified in these provinces that limit the growth of trees and shrubs. In these clusters, there was also a strong increase in the extent of urban areas, which more than doubled from 2002 to 2018, and of cropland areas, which slowly but continuously has been growing over the years.

On the other hand, the areas of increasing trends are characterized by a large proportion of forest (ca. 80%) and small fractions of shrubland (10%) and grassland (4%). These areas also have a relatively high proportion of cropland (4%), which substantially increased since 2004. In these regions, the continuous increasing trend of the annual burnt area may lead to the conversion of forests and woodlands into grasslands and shrublands. Interestingly, Cuando Cubango is among the provinces that displays more areas of significant positive trends but also more areas of negative trends. This reveals high turnover of burnt areas in the province, possibly resulting in high levels of land cover changes.

Our findings show a reduction in forest cover over the years, which is in agreement with other studies [23,42], confirming that land cover changes in Africa have been dominated by woodland and forest losses. The fires have had severe effects on Angolan woodlands, converting large areas of forest and woodlands into open land [25]. For instance, a study by Palacios et al. [87] reported that 78.4% of Huambo province was covered by Miombo woodland in 2002, but 13 years later it had dropped to 48.3%. Similar losses in western Cuando Cubango, eastern Huíla, and eastern Huambo were reported by Schneibel et al. [88], and Mendelsohn and Mendelsohn [89]. However, the rate of forest loss detected in this study appears to be much lower than the rate reported by Hansen et al. [30], as reported by Food and Agriculture Organization of the United Nations (FAO) [31,90] and Global Forest Watch [32]. Other studies of land cover change [91,92] have also detected a much lower estimate of total forest area and forest loss when using ESA CCI-LC maps. One possible explanation is the different data sources. FAO collected forest resource data from the member countries [31], using satellite imagery and field surveys in different countries, while Hansen et al. [30] used high spatial resolution satellite data (30 m) obtained from NASA's Landsat, which allows for the detection of small-scale forest clear cuts. The different fraction of canopy considered in the forest definitions is also a possible cause of the disparity in the results. FAO [31] adopted a definition >10% canopy cover, while Global Forest Watch defined >30% canopy cover as forest. In our study, forest is defined as areas with >15% canopy cover, which does not reflect the decrease in the percentage of tree cover within each area, unless it falls below 15% of coverage.

The expansion of shifting cultivation practiced by smallholders and the overexploitation for timber, fuelwood, and charcoal are possibly the most important factors driving the rapid loss of Angolan woodlands and forests, as well as their very slow recovery rate [23]. The relative lack of nutrients and moisture in soils forces the farmers to leave their fields and use fire to clear new fields and enrich the soil with nutrients from the ash of the woody biomass [93]. The rate at which the woodlands are cleared and converted into cropland is dependent on the need to feed a growing human population. To reduce the impacts of human activity on fire processes, management strategies should be urgently implemented. These strategies must incorporate the ecological and socio-economic roles of fire, as well as the conservation of Angolan ecosystems [16].

#### *4.3. Conservation and Management of Protected Areas*

The protected areas system shows a strong impact on the burnt areas, presenting larger clusters of decreasing trends than of increasing trends. For instance, Cameia, Luengue-Luiana, and Mavinga national parks are located in eastern Angola (Moxico and Cuando Cubango provinces) and have the largest areas of decreasing annual burnt area. The decreasing trends detected in these areas may be related to the increase in the number of herbivores after the civil war. For instance, a study carried out in Luiana National Park showed a rapid increase in the numbers of African elephants between 2004 and 2005 [94]. According to Goldammer and Ronde [11], the abundance of mammalian herbivores is an important determinant of fire intensity. When the number of herbivores is high, less fuel is available and fires can affect smaller areas, with lower intensities [11,95].

Bicuar National Park is the most worrying protected areas, with increasing trends in more than 23% of its area. It is the only protected area of Huíla, located in a transition zone between the Angolan Miombo woodlands and the Zambezian Baikiaea woodlands ecoregions. With an area of almost 8000 km2, it consists mainly of riverbanks of the Cunene River, sandy hills, and river valleys with savannah grasslands. It was created in 1938 as a hunting reserve due to the abundance of game species, such as African bush elephant (*Loxodonta africana*), blue wildebeest (*Connochaetes taurinus* subsp. *taurinus*), and eland (*Tragelaphus oryx*) [96]. The analysis of Luando Integral Nature Reserve also reveals significant increasing trends in ca. 9% of this area. The status of protected area was acquired in 1955, to conserve the giant sable antelope (*Hippotragus niger* subsp. *variani*) [97], including more than 8000 km2 of the Angolan Miombo woodlands.

Angolan protected areas need a real and efficient administration, in addition to integrated biodiversity management plans. In 2006, the National Parks of Quissama, Mupa, Cangandala, Iona, Cameia, and Bicuar were almost completely abandoned, without equipment or supervision [98]. Parts of these protected areas are occupied by human populations that hunt and burn without control, leading to the disappearance of large mammals and converting large areas into agricultural land [78]. However, the Protected Areas system was greatly expanded in 2011 and more resources are being made available for more effective management [46]. The difficulties imposed by limited budgets, scarce

human resources, and weak technical capacity impose the need to identify priority areas, where the government's conservation resources must be allocated first.

Fires have been the main management tool used in protected areas of the African savanna [95]. During the 20th century, managers of protected areas have considered both the suppression of unwanted fires and ignition of prescribed fires to achieve different goals, such as reducing the putative negative effects of fire on ecosystems, reducing the risk of unplanned fires, improving visibility for game viewing and policing, improving forage quality, and fighting invasive alien species [95,99–101]. Fire managers must decide how, where, and when to apply fires to achieve their goals of management, while also respecting legal constraints. During the 21st century, fire management in Angola will face new challenges imposed by the effects of climate change and a growing human population.

#### **5. Conclusions**

In this study, we detected spatial and temporal patterns of burnt areas across Angola, which is a crucial step for the definition of conservation priorities and management strategies. The identification of the areas with significant trends is extremely important in tropical areas with low field data available and few human and financial resources, as is the case of Angola. This will allow for efficient distribution of fire management resources and public investment, resulting in improved management of Angolan vegetation.

Understanding the role of climatic and anthropogenic factors on these trends is very important. The overexploitation of forests for the production of timber, firewood, and charcoal also enhances grass development, which in turn promotes more intense fires in the dry season. Effective actions to limit fire events are urgently needed to protect fire-sensitive resources, natural ecosystems, and to reduce carbon emissions to the atmosphere. While most southern African countries have legislation and regulations to use and control fire, these are rarely applied due to enforcement difficulties [11]. Fire control is also difficult because, in Africa, fires occur in thousands of small, dispersed events. Moreover, climate change will bring more frequent and intense droughts to Angola, with strong impacts on water resources, agricultural productivity, and wildfire potential. All of these factors are expected to play an important-negative-role on the trends of burnt areas, land cover change, and biodiversity conservation.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1424-2818/12/8/307/s1, Figure S1: Trends of annual burnt area in Angola for the 2001–2019 period, based on Mann–Kendall single-cell analysis. The positive z-scores correspond to areas of increasing trends of burnt area, while the negative z-scores correspond to decreasing trends. The significant areas (with a confidence level of 95%) were overlaid with black lines. Protected areas are limited with blue lines: BI, Bicuar; BU, Búfalo; KA, Cangandala; CM, Chimalavera; IO, Iona; IP, Ilheu dos pássaros; KM, Cameia; KI, Quiçama; LL, Luengue-Luiana; LU, Luando; MV, Mavinga; MU, Mupa; MY, Maiombe; NA, Namibe. Figure S2: WWF ecoregions occurring in Angola and the significant cluster of increasing and decreasing trends of annual burnt area detected in 2001– 2019 period, based on the Contextual Mann–Kendall test. Figure S3: Land cover map of Angola in 2018 (300 m resolution), adapted the European Space Agency (ESA) Climate Change Initiative Land Cover (CCI-LC), and the significant cluster of increasing and decreasing trends of annual burnt area detected in 2001–2019 period, based on the contextual Mann–Kendall test. Table S1: Estimated fraction of burnt area per year and the average of the burnt area from 2001 to 2019 in each province.

**Author Contributions:** Conceptualization, S.C., M.M.R. and J.M.C.P.; methodology, S.C., J.M.N.S. and J.M.C.P.; formal analysis, S.C., V.A., J.M.N.S. and J.M.C.P.; writing—original draft preparation, S.C.; writing—review and editing, M.M.R., R.F., V.A., J.M.N.S. and J.M.C.P. All authors have read and agreed this version of the manuscript.

**Funding:** This research was funded by Foundation for Science and Technology (FCT) of the Portuguese Government through the grants SFRH/BD/120054/2016 to S.C., UID/AGR/04129/2019 to Linking Landscape, Environment, Agriculture and Food (LEAF), UID/BIA/00329/2019 to Centre for Ecology, Evolution and Environmental Changes (cE3c), UIDB/00239/2020 to Forest Research Centre (CEF).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

© 2020 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 (http://creativecommons.org/licenses/by/4.0/).

MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel. +41 61 683 77 34 Fax +41 61 302 89 18 www.mdpi.com

*Diversity* Editorial Office E-mail<EMAIL_ADDRESS>www.mdpi.com/journal/diversity

MDPI St. Alban-Anlage 66 4052 Basel Switzerland

Tel: +41 61 683 77 34 Fax: +41 61 302 89 18


*Water Chemistry*

*Pollution of Water Sources from Agricultural and Industrial Effluents: Special Attention… DOI: http://dx.doi.org/10.5772/intechopen.86921*

**Figure 5.** *Solubility curves for common metals in freshwater with pH [60].*

of metal speciation and redox conditions is hexavalent chromium (Cr(VI)). Many of metals are transition metals (such as cadmium and Cu) or heavy metals (such as lead and silver) that can form metalloids which bond to organic compounds to form lipophilic substances that are often highly toxic. Metals are also lost from solution by precipitation as the pH changes [7, 60, 61]. **Table 4** shows the ranking of these metals according to their toxicity through biological and carcinogenicity tests [62].

#### *<IP_ADDRESS>.1 Heavy metal pollutants*

Any metallic element with relatively high density as compared to water and toxic even at low concentrations is termed as "heavy metal" [63, 64]. Among these metals, Cr is one of the top 16 major toxic contaminants that have detrimental effects on human health [64, 65]. Besides Cr, Cu is generally considered as a highly harmful metal at high concentration [66]. Accordingly, the following section discusses and summarizes relevant information about Cr and Cu.

Cr as a metallic element was first discovered and isolated in 1797 by the French chemist Nicolas-Louis Vauquelin [35, 67]. The world resources of Cr exceed 10.9 billion metric tons of shipping-grade chromite [67]. Ferric chromite (FeCr2O4) is the principal Cr ore, found mostly (with 96% of the world's reserves) in South Africa. Minor common sources include chrome ochre (Cr2O3), and crocoite (PbCrO4) are also present [58]. Cr is widely used in engineering and chemical industries because of its durability and esthetic quality. The principal uses for Cr are metallurgical (67%), refractories (18%), and chemical (15%). In addition, Cr exits in three stable forms in the environment with different oxidation states and ionic nature [65]. The physicochemical properties of Cr are presented in **Table 5** [67].

*Water Chemistry*

**50**

**Nanomaterial**

**Type** Nanocomposite

Chitosan/Zeolite/nano ZrO2

**Table 3.**

*Comparison of NO3ˉ removal by different nanomaterials [53].*

—

20 mg/L

3

0.02 g

35 °C

60 min

23.58 mg/g

Doped composite

20 mg/L

6–8

50 mg

293–

30 min

83%

313 K

**Modification**

**Initial NO3ˉ**

**pH**

**Nanomaterial**

**T°**

**Contact**

**NO3ˉ removal**

**efficiency**

**Time**

**dose**

**concentration**

#### **Table 4.**

*Classification of metals according to their toxicity [62].*

#### **Table 5.**

*Physical and chemical properties of Cr [67].*

The oxidation states of Cr can go from −2 to +6; however, only the +6 and +3 oxidation states are commonly encountered in the environment. Cr(III) is highly insoluble, relatively immobile, and most thermodynamically stable [58], while Cr(VI) has high water mobility and solubility and can be easily reduced. Cr(VI) is recognized to be more poisonous (100 times more toxic than Cr(III)), mutagenic, and carcinogenic in nature [68].

#### *<IP_ADDRESS>.1.1 Chromium VI sources*

To date, naturally occurring Cr(VI) in groundwater has been detected in the following geologic environments: (i) chromite ore bodies, (ii) arid alluvial basins in the southwest United States, (iii) saline brines in evaporate basins, and (iv)

**53**

*Pollution of Water Sources from Agricultural and Industrial Effluents: Special Attention…*

serpentinite ultramafic terrains [67], while the major industrial source of Cr(VI) emissions are (i) chemical manufacturing industry, (ii) metal finishing industry, (iii) manufacturers of pharmaceuticals, (iv) electrical and aircraft manufacturers, (v) cement-producing plants (as cement contains Cr), and (vi) production of wood, stone, glass, and clay products. Owlad et al. [69] reported the concentration of Cr(VI) in the wastewater of several industries (as in **Table 6**) and estimated the concentration of Cr(VI) in wastewater caused by industries between 0.1 and

In aqueous solution, Cr(VI) may exist in a variety of oxospecies (viz., dichro-

depending on pH of the solution, total Cr concentration, the presence of oxidizing and reducing compounds, the redox potential, and kinetics of the redox reactions [64, 70]. At pH < 1, the predominant species is H2CrO4, while as the pH is raised

concentrations are greater than 1 mM, or it may even dominate when the total Cr(Vl) concentrations are greater than 30 mM. At a pH > 8 only the yellow ion

centration, and H2CrO4 is a very strong acid [70]. The chemical equilibria which are

↔ H+ + CrO4

↔ Cr2 O7

Ingesting large quantity of Cr(VI), either by human or animal, can be corrosive to the skin and eyes and causes stomach upsets and ulcers, convulsions, kidney and liver damage [72, 73]. Allergic reactions consisting of severe redness and swelling of the skin have been noted. Some evidence on animal studies show that Cr(VI) compounds can cause cancer in various tissues due to the low water insolubility [35, 67, 72]. Indeed, due to the fact that Cr(VI) is unstable in the body, easy to diffuse across the membrane, and is reduced intracellularly, it can provide very reactive pentavalent Cr and trivalent Cr which can alter DNA. Ingestion of 1.0–5.0 g of Cr(VI) as chromate results in severe acute gastrointestinal disorders, hemorrhagic diathesis, and convulsions [74]. In 1998, Dartsch et al. [75] noticed that 5 mmol/L

Furthermore, Cr(VI) compounds at high concentration are extremely toxic to plants and retard their growth. Cr and Cr(VI) compounds can cause severe phytotoxicity which may result in reduction of seed germination, nutrient imbalance, degradation of pigments, decrease of antioxidant and enzyme concentration, and oxidative stress [76]. Indeed, Rout et al. [77] demonstrated a 25% reduction in seed germination in the presence of 200 μM Cr(VI). According to Davies et al. [78] Cr

2− anions prevail.

2− dominates in acidic solution at Cr(VI) concentration above

2− dominates in basic solutions independently of Cr(VI) con-

2−), H2CrO4, and acid chromates (HCrO4

−))

− dominates when the

2− becomes significant when

2− (5)

2− + H2O (6)

↔ H+ + HCrO4 (4)

*DOI: http://dx.doi.org/10.5772/intechopen.86921*

200 mg/L.

mates (Cr2O7

CrO2

*<IP_ADDRESS>.1.2 Speciation of Cr(VI)*

from pH 2 to 6, the HCrO4

− exists. Cr2O7

0.01 M, while CrO4

2−), chromates (CrO4

− and Cr2O7

H2 CrO4 +

> H CrO4 −

> > −

According to Palmer and Puls [71] under pH 6.5, HCrO4

Cr(VI) concentrations are low (<30 mM), but Cr2O7

involved for Cr(VI) dissociation are as in Eqs. (4)–(6):

*<IP_ADDRESS>.1.3 Cr(VI) effects on human health and environment*

2 HCrO4

Cr(VI) resulted in 50% cell death.

*Pollution of Water Sources from Agricultural and Industrial Effluents: Special Attention… DOI: http://dx.doi.org/10.5772/intechopen.86921*

serpentinite ultramafic terrains [67], while the major industrial source of Cr(VI) emissions are (i) chemical manufacturing industry, (ii) metal finishing industry, (iii) manufacturers of pharmaceuticals, (iv) electrical and aircraft manufacturers, (v) cement-producing plants (as cement contains Cr), and (vi) production of wood, stone, glass, and clay products. Owlad et al. [69] reported the concentration of Cr(VI) in the wastewater of several industries (as in **Table 6**) and estimated the concentration of Cr(VI) in wastewater caused by industries between 0.1 and 200 mg/L.

#### *<IP_ADDRESS>.1.2 Speciation of Cr(VI)*

*Water Chemistry*

Carcinogenicity

Toxicity

**Table 4.**

Density (g/cm3

**Bioaccumulative character Chemical elements**

Non-carcinogenic Cr, Ni, Cd, Zn, Cu

Relatively toxic elements Pb, Ni, As, Cu, Cr(VI)

Atomic number 24 Atomic mass 51.996 Atomic radius (pm) 185 Main oxidation state(s) +2, +3, +6

Electronegativity (pauling) 1.66

Melting/boiling point (°C) 1.907/2.671 Isotopes 4 stable + 17 unstable Acid/base of oxide Strong acid State (st 27 °C, 1 atm) Solid Metallic character Metal Element group(s) Transition element Affinity Lithophile

Very bioaccumulative Pb, Zn, Cu Relatively bioaccumulative Hg, Ni, Cd Slightly bioaccumulative As, Cr

Strong carcinogenicity As, Pb

Group of highly toxic elements Cd, Hg

*Classification of metals according to their toxicity [62].*

**Physical and chemical properties of Cr**

Slightly toxic elements Cr(III), Zn

**52**

**Table 5.**

and carcinogenic in nature [68].

*Physical and chemical properties of Cr [67].*

*<IP_ADDRESS>.1.1 Chromium VI sources*

The oxidation states of Cr can go from −2 to +6; however, only the +6 and +3 oxidation states are commonly encountered in the environment. Cr(III) is highly insoluble, relatively immobile, and most thermodynamically stable [58], while Cr(VI) has high water mobility and solubility and can be easily reduced. Cr(VI) is recognized to be more poisonous (100 times more toxic than Cr(III)), mutagenic,

Ionic radius (pm) 87–94 (+2), 75.5 (+3), 55–69 (+4), 48.5–71 (+5), 40–58 (+6)

) 7.19

To date, naturally occurring Cr(VI) in groundwater has been detected in the following geologic environments: (i) chromite ore bodies, (ii) arid alluvial basins in the southwest United States, (iii) saline brines in evaporate basins, and (iv)

In aqueous solution, Cr(VI) may exist in a variety of oxospecies (viz., dichromates (Cr2O7 2−), chromates (CrO4 2−), H2CrO4, and acid chromates (HCrO4 −)) depending on pH of the solution, total Cr concentration, the presence of oxidizing and reducing compounds, the redox potential, and kinetics of the redox reactions [64, 70]. At pH < 1, the predominant species is H2CrO4, while as the pH is raised from pH 2 to 6, the HCrO4 − and Cr2O7 2− anions prevail.

According to Palmer and Puls [71] under pH 6.5, HCrO4 − dominates when the Cr(VI) concentrations are low (<30 mM), but Cr2O7 2− becomes significant when concentrations are greater than 1 mM, or it may even dominate when the total Cr(Vl) concentrations are greater than 30 mM. At a pH > 8 only the yellow ion CrO2 − exists. Cr2O7 2− dominates in acidic solution at Cr(VI) concentration above 0.01 M, while CrO4 2− dominates in basic solutions independently of Cr(VI) concentration, and H2CrO4 is a very strong acid [70]. The chemical equilibria which are involved for Cr(VI) dissociation are as in Eqs. (4)–(6):

$$\text{H}\_2\text{CrO}\_4^\* \leftrightarrow \text{H}^\* + \text{HCrO}\_4 \tag{4}$$

$$\rm{HCrO\_4^- \leftrightarrow H^+ + CrO\_4^{2-}}\tag{5}$$

$$2\,\text{HCrO}\_4^- \leftrightarrow \text{Cr}\_2\text{O}\_7^{2-} + \text{H}\_2\text{O} \tag{6}$$

#### *<IP_ADDRESS>.1.3 Cr(VI) effects on human health and environment*

Ingesting large quantity of Cr(VI), either by human or animal, can be corrosive to the skin and eyes and causes stomach upsets and ulcers, convulsions, kidney and liver damage [72, 73]. Allergic reactions consisting of severe redness and swelling of the skin have been noted. Some evidence on animal studies show that Cr(VI) compounds can cause cancer in various tissues due to the low water insolubility [35, 67, 72]. Indeed, due to the fact that Cr(VI) is unstable in the body, easy to diffuse across the membrane, and is reduced intracellularly, it can provide very reactive pentavalent Cr and trivalent Cr which can alter DNA. Ingestion of 1.0–5.0 g of Cr(VI) as chromate results in severe acute gastrointestinal disorders, hemorrhagic diathesis, and convulsions [74]. In 1998, Dartsch et al. [75] noticed that 5 mmol/L Cr(VI) resulted in 50% cell death.

Furthermore, Cr(VI) compounds at high concentration are extremely toxic to plants and retard their growth. Cr and Cr(VI) compounds can cause severe phytotoxicity which may result in reduction of seed germination, nutrient imbalance, degradation of pigments, decrease of antioxidant and enzyme concentration, and oxidative stress [76]. Indeed, Rout et al. [77] demonstrated a 25% reduction in seed germination in the presence of 200 μM Cr(VI). According to Davies et al. [78] Cr

#### **Table 6.**

*Industrial wastewater containing Cr(VI) [69].*

is mostly toxic to higher plants at 100 μg/kg dry weights. Sinha et al. [79] reported that Cr is toxic for most agronomic plants at a concentration of about 0.5–5.0 mg/L in nutrient media and of 5–100 mg/g under soil condition. The Cr toxicity in plants affects photosynthesis in terms of CO2 fixation, photophosphorylation, electron transport, and enzyme activities as reported by H. Oliveira [76, 80].


**3.4.3 Experimental work**

102 Selected Topics on Optical Amplifiers in Present Scenario

*D fL D fL H ( f ) cos LEF sin*

*f D fL j LEF sin f c*

Where D is the fibre dispersion parameter, L is the fibre length, f is the modulation frequency, c is the speed of light in vacuum, λ is the operating wavelength. For simplicity

The calculated RF responses of 400 km fibre for different chirp parameters of the optical transmitter are depicted in Fig.19. All the results have been calculated for an optical input power of 0 dBm in order to reduce the influence of the nonlinear effects in the fibre. By comparing the results to the reference case of a zero-chirp situation (*LEF*=0), for *LEF*<0, the

**Deep [dB]**

*c*

LEF=-0.5 LEF=-0.2 LEF=-0.1 LEF=0

Fig. 19. Calculated quality of MW/MMW transmission for different SOA and system

In real radio over fibre systems the radio frequency carrier is modulated by digital information. The above presented dispersion compensation technique affects the eye diagram, the BER (Bit Error Rate) and the EVM (Error Vector Magnitude), which are the important quality factors of signal transmission. If the subcarrier frequency is close to one of the frequency notches caused by dispersion the eye diagram closes, the BER and EVM decay, the communication deteriorates or lost. Applying the SOA compensator the eye diagram opens, low BER is obtained and EVM can greatly benefit from the improved link performance. The simulations were executed with different modulation schemes on radioover-fibre systems and VPI software was applied. Fig. 20 presents the quality of 4 QAM (quadrature amplitude modulation) radio signal transmission with different SOA chirp parameter, it shows the Symbol Error rate improvement applying SOA dispersion compensator. The EVM and SER increase versus chirp parameter. If the subcarrier frequency is close to the frequency notch, the communication lost. Applying a SOA dispersion compensator the constellation diagram can be detected, but the diagram rotates

(a) Transfer function with different chirp parameter of SOA, fiber length=100km (b) Rejection frequency and the deep versus chirp parameter of SOA, fiber length=1km

2 2 2 2

2 2

*c c*

(13)

**Rejection Frequency [GHz]**

**3.4.2 Calculation and simulation results**  The frequency transfer function of the optical link:

*link*

the linear loss and delay of the fibre are neglected.

012345 **Modulation Frequency [GHz]**

parameters, Optical wavelength=1550nm, D=16.8 ps/(nm·km)

because of the additional phase modulation generating the SOA chirp.

SOA

achievable bandwidth increases.

**Transmission [dB]**

The calculated and simulated results can be verified by experimental work. The SOA under test was driven by different bias (dc) currents. The polarization state of the incoming optical power was optimized by polarization controller. The harmful effect of the optical reflection was eliminated by optical isolators. The required optical power and wavelength were produced by a tuneable laser source. The intensity modulated optical signal was detected by a photodetector. The setup was controlled by a computer program, hence the measurement parameters were carefully set by the program and the measurement results were processed and stored.

The measured RF responses of 50km fibre for different operation points of SOA are depicted in Fig. 21. By comparing the results to the reference case of a zero-chirp situation (*LEF*=0), for *LEF*<0, the achievable bandwidth increases. As the SOA bias current (optical gain) increases the frequency notches of the RF response are reduced and shifted to higher modulation frequencies. Based on the results, we may conclude that the interplay of chirp generated by the saturated SOA and chromatic dispersion enables a significant reduction of the dispersion-induced effect.

Fig. 21. Simulated constellation diagrams (a) without SOA, (b) with SOA compensator, L=500m, fsubcarrier=60GHz, modulation= 2048Mbps, 4QAM

Multi-Functional SOAs in Microwave Photonic Systems 105

C. Arellano, J. Prat: Semiconductor Optical Amplifiers in Access Networks, in Proc. ICTON

E. Udvary, T. Berceli: Linearity and chirp investigations on SOA as an external modulator

E. Udvary, T. Berceli: Optical Subcarrier Label Swapping by Semiconductor Optical

E. Udvary, T. Berceli: Semiconductor Optical Amplifier for Detection Function in Radio over

Jesper Mork et al, "The modulation response of a Semiconductor Laser Amplifier". IEEE J. Sel. Topics. Quantum Electron., vol.5, no.3, pp.851-860, May/June, 1999. J. Marti, F. Ramos, J. Herrera, "Experimental reduction of dispersion-induced effects in

J-M. Kang, S-K. Han: A Hybrid WDM/SCM-PON Sharing Wavelength for Up- and Down-

Josep Prat, et al.: Optical Network Unit Based on a Bidirectional Reflective Semiconductor

L. Occhi, L. Schares, G. Guekos, "Phase modeling based on the α factor in bulk

M.Connelly, "Wideband Semiconductor Optical Amplifier Steady-State Numerical Model",

M.Gustavsson, A.Karlsson, L.Thylen: "Traveling wave semiconductor laser amplifier

R.Olshansky et al, "Subcarrier Multiplexed Lightwave Systems for Broadband Distribution",

Sang-Yun Lee, et al., "Reduction of chromatic dispersion effects and linearization of dual-

T. Berceli, E. Udvary: Transmission Challenges of Cascaded Semiconductor Optical Amplifiers, in Proc. MWP 2005, Seoul, Korea, 12-14 October 2005, pp. 129-132 T. Watanabe et al., "Transmission performance of chirp-controlled signal by using

drive Mach-Zehnder Modulator by using semiconductor optical amplifier in analog optical links" in Proc. ECOC 2002, September 8-12, 2002, Copenhagen,

semiconductor optical amplifier", IEEE Journal of Lightwave Technology, August

IEEE J. Quantum Electron., vol.37, no.3, pp.439-447, March, 2001.

detectors" J. of Lightwave Technology, vol.8., pp.610-617,1990

J. Lightw. Technol., vol.7, no.9, pp.1329-1342, Sept. 1989.

Govind P. Agrawal: Fiber-Optic Communication Systems, John Wiley & Sons, 1985. H.Ghafouri-Shiraz: "Laser Diode Amplifiers", John and Wiley, Chichester, 1996

in SCM systems, Proc. of the EUMA, Issue on MWP, vol. 3. pp. 217-222, Sept.

Amplifiers, J. Lightwave Technol., Special Issue on MWP, vol. 21. pp. 3221-3225,

Fiber Systems, *J. Lightwave Technol.*, Special Issue on MWP, vol. 26. pp. 2563-2570,

microwave optical links employing SOA boosters", Photonic Technology Letters,

Link Using Reflective Semiconductor Optical Amplifier, Photon. Technol. Letters,

Optical Amplifier for Fiber-to-the-Home Networks, Photon. Technology Letters,

semiconductor optical amplifiers", IEEE Journal of Selected Topics in Quantum

2005, Barcelona, Spain, June 2005, paper WeA1.4

Vol. 13, No. 9, Sept. 2001, pp.999-1001

Vol. 18, pp. 502-504, Feb. 2006

Electronics, 2003, pp. 788-797

vol. 17. Jan. 2005

Denmark

2000, pp. 1069-1077

2007

Dec. 2003

Aug. 2008

Fig. 22. Measured RF responses of the optical link for different SOA parameters Length of the optical fiber=50km, Length of the reference optical fiber=4m, Optical wavelength=1550nm, bias current of the SOA=400mA, 200ma, 125mA, 100mA, Optical Gain=15dB, 13.5dB, 4,5dB, -9.5dB


**4.2 Factors affecting transfection efficiency**

There are many factors that affect transfection efficiency. These include Mw, DDA, DNA complexes' charge ratio, pH and particle sizes, as well as the type of cell lines used.

### **4.2.1 Molecular weight (Mw)**

High Mw chitosan can bind DNA tightly, which is due to the high number of positive charge of amino groups, but binding DNA tightly may give low transfection efficiency, due to not releasing the DNA to the nucleus after endocytosis to the cell. The Mw of chitosan also influences the size of the chitosan–DNA complexes, as the higher sizes of chitosan-DNA complexes can affect the cellular uptake. These factors lead to transfection efficiency (see review of Mao et al., 2010).

If the N/P ratio, which is the molar ratio between the amino groups of chitosan and the phosphate groups of DNA, was fixed, then the higher the Mw , the larger the chitosan-DNA complexes diameter (MacLaughlin et al., 1998). However, there have been differing conclusions proffered between the Mw of chitosan and transfection efficiency. Some studies have reported of high transfection efficiency with high Mw chitosan (Huang et al., 2005; Kiang et al., 2004; MacLaughlin et al., 1998). Other studies have reported that low Mw chitosan has better transfection efficiency (Koping-Hoggard et al., 2004; Lavertu et al., 2006; Supaprutsakul et al., 2010).

MacLaughlin et.al. (1998) synthesised depolymerised chitosan oligomers with a Mw from 7 - 92, but the transfection efficiency was much lower than at the higher Mw of 102 and 230 kDa, respectively, and being about 1000 times lower in transfection efficiency compared to

Chitosan and Its Modifications: Are They Possible Vehicles for Gene Therapy? 445

Hydrophobic modifications of chitosan have been performed in many studies. The main objectives of these modifications were increasing transfection efficiency by modulating complex interactions with cells, especially in the complexes' adsorption on the cell surfaces and cell uptake (Kurisawa et al., 2000). Some hydrophobic units also help in the dissociation between the chitosan DNA complexes to release DNA to enter the nucleus after cellular uptake, as well as protecting it from enzymatic degradation and facilitating intra cellular pDNA (plasmid DNA) association, which can enhance transfection efficiency. These hydrophobic modifications included deoxycholic chitosan, N-alkylated chitosan, thiolated chitosan and hybrid chitosan (Mao et al., 2010). The combination of hydrophilic and hydrophobic modification of chitosan structure has been another interesting area that looks highly promising for the development of high transfection efficiency in a non-viral vector,

One of the significant applications of chitosan is in its application to gene therapy. It has a number of benefits. It has non-toxic, biodegradable and biocompatible with high cationic charge potential; protects DNA from degradation by nucleases; and has high yield transfection efficiency (Sui et al., 2006). Genetic material (DNA and RNA) has been explored for use as a treatment of genetic abnormalities or deficiencies, which is described as gene therapy. Gene therapy functions by transferring healthy genetic material or nucleic acid constructs, such as ribozymes, antisense molecules, decoy oligodeoxy nucleotides (ODNs), DNAzymes and siRNA, into diseased cells in an attempt to achieve a therapeutic effect that results in restoration of protein production, which was absent or deficient due to the preexisting genetic disorder (Tan et al., 2009). But using small nucleic acid, such as DNAzymes and siRNA, has some limitations, since they are rapidly degraded in plasma and cellular cytoplasm and cannot passively diffuse through cellular membrane, which is due to the strong anionic charge of the phosphate backbone and the consequent electrostatic repulsion from the anionic cell membrane surface as well as limited size of cellular entrance. So, these small nucleic acids encapsulated with chitosan nanoparticles can reduce the limitations of

The development of chitosan and its modification for non-viral gene delivery is also a target for gene therapy. This is because chitosan nanoparticles have a low toxicity and are taken up by endosomes allowing the DNA or nucleic acid to overcome the permeability barrier posed by epithelium and also to protect against enzymatic degradation. There are some studies that have attempted to use chitosan for cancer therapy. Chitosan itself was able to demonstrate growth inhibitory effects on cancer cells and has apoptosis effect on bladder tumour cells via caspase-3 activation (Tan et al., 2009). The various manufacturing processes for chitosan nano-/micro- particles/spheres (nanofabrication) has been described elsewhere

In current developments in chitosan for gene therapy, there is an attempt to develop siRNA loaded chitosan nanoparticles to silence the target gene. This method can silence the gene by means of RNA interfering (RNAi). SiRNAs, usually containing 20-25 base pairs (see Figure

using chitosan as a core structure.

**5. Chitosan and gene therapy**

these small nucleic acids.

(Masotti et al., 2009).

**5.1 Use of chitosan nanoparticles**

**5.2 Use of siRNA loaded chitosan nanoparticles**

lipofectamineTM. Haung et al. (2005) also found a decreased A549 cellular uptake with the decreasing Mw or DDA of chitosan and a N/P ratio of 6 was used in that study. But the study of Supaprutsahul et al. (2010) revealed much higher transfection efficiency with the depolymerised chitosan at Mw ~16 kDa (or Mn ~ 6.5). This may be because of the different chitosan/DNA ratio, as the previous study used low N/P ratio, while Supaprutsakul et al. (2010) used chitosan/plasmid at an N/P ratio of about 56:1, which meant than a much higher amount of chitosan was used for the lower Mw. This was consistent with the study of Romøren et al. (2003), who found that low Mw chitosan was beneficial at the higher charge ratio of the complexes.


**Preface to "Modelling the Deformation, Recrystallization and Microstructure-Related Properties in Metals"**

Experimental investigations of the thermomechanical processing (TMP) of metals clearly demonstrate that technological process tuning parameters have a great influence on both the microstructure and texture evolution, which determine the chemical, physical, or mechanical properties. During the processing chain, the behavior of polycrystalline material is correlated with the grain size, grain crystal structure, and crystallographic orientation. The mesoscopic transformations of polycrystalline aggregates, involving microstructural and crystallographic changes on the grain level, can be interpreted by a vast body of modelling approaches developed. Advances in modelling have created a solid platform for understanding the evolution of microstructural features in polycrystalline systems during particular processing steps and bring to light many hidden aspects of production as well as assist in revealing the behavior of materials under specific circumstances. Since mesoscopic changes in TMP are "genetically" connected, the modelling techniques enable tuning particular processing step to tailor the desired material performance for a given application.

In this Special Issue, we intend to provide a wide spectrum of articles dealing with the modelling of microstructural changes in various metallic systems. The major aim was to discuss different features of microstructure evolution such as morphological changes, crystallographic aspects, phase transformation as well as structure-property relationships in polycrystalline aggregates.

> **Jurij J. Sidor** *Editor*


*4.8. Extraction of Amino Acid Signature from the Alignments and Visualization of Respective Positions on the three-dimensional (3D) Model of S. cerevisiae Isomaltase IMA1*

Protein sequences of *S. cerevisiae* maltase MAL62 (UniProtKB: P07265), *S. cerevisiae* isomaltase IMA1 (UniProtKB: YGR287C), *O. polymorpha* maltase-isomaltase MAL1 (UniProtKB: Q9P8G8) and *B. adeninivorans* AG2 were aligned using Clustal Omega [72] and amino acids corresponding to IMA1 signature positions determining the substrate specificity [12,15] were extracted from the alignment. The *S. cerevisiae* IMA1 structure in complex with isomaltose (PDB: 3AXH) [29] was visualized with PyMol [30] and amino acid signature was designated on the structure.


**1. Introduction**

By August of 1975 [1], 277 patients had received an orthotopic heart transplantation and 49 were alive. The longest survivor lived 6.8 years.

Christian Barnard reported the heterotopic heart transplant (HHT) technique. In 1976 [1], Barnard noted the benefits of heterotopic heart transplantation to be that the donor heart acts as an assist device, assists during episodes of rejection, can be removed in case of severe graft rejection, and still the patient may receive a subsequent heart transplant. Barnard et al. [2] published a case report of a heterotopic heart transplant recipient that suffered acute rejection and was supported by the native heart while the heterotopic graft recovered.

At the time the paper was written, cyclosporine was not used for post-heart transplantation and the incidence of acute rejection was more common. The heterotopic heart transplant technique offered an extra layer of prevention and/or treatment during the pre-cyclosporine

era when death within 24 hours of the onset of rejection was common. In addition, to the benefits from treatment of acute rejection, the heterotopic heart transplantation technique allows selected recipients with pulmonary hypertension to receive a transplant.

to 13 mmHg), (2) donor to recipient weight ratio of less than 0.8, (3) anticipated ischemic time greater than 6 hours, and (4) a marginal donor heart. Marginal donors were described as those that required high inotropic support, had a history of a cardiac arrest or arrhythmia, wall motion abnormalities on the echocardiogram, and/or ischemic changes on the electrocardiogram (EKG). Fourteen of the donor hearts were marginal and had been declined by other

Heterotopic Heart Transplantation

125

http://dx.doi.org/10.5772/intechopen.74582

In the study of Newcomb et al. [5], the heterotopic heart transplant recipients were significantly older (mean 58 years vs. 47.1 years for OHT); the donors were also significantly older (mean age 45.2 years vs. 34.5 years for OHT). The ischemic time was also much higher for the HHT recipients; 366 minutes vs. 258 minutes for OHT. The intensive care unit and the total length of the hospital stay was higher for HHT recipients; though, not statistically significant. The study demonstrated lower survival for heterotopic heart transplant recipients compared to orthotopic heart transplant recipients in the same time period; though, the survival benefit for OHT recipients disappeared when they performed a subgroup analysis for the recipients who had elevated pulmonary artery pressures. The study demonstrates the successful use of the heterotopic heart transplant. The survival in HHT recipients were not as good as in those of OHT recipients because of the HHT technique was more often used in marginal donors and more high-risk recipients. Marginal donor hearts may not have performed as well in OHT recipients. Furthermore, high risk recipients have decreased survival expectations especially

Boffini et al. [6] described their single center experience with the heterotopic heart transplant; and, found the HHT to be comparable to OHT. HHT was used between in 1985–2003, in 12 patients [(1.7%) of the all the heart transplant performed during that time]. The 1-year and 5-year survival was 92 and 64% respectively. These results demonstrated when the HHT technique is used in the usual recipient risk patient, the outcomes can be effective and acceptable for recipients. The HHT technique was used for body size mismatch in 11 patients and

In addition to donor-recipient size mismatch, elevated pulmonary vascular resistance (PVR), and, fixed pulmonary hypertension are also indications for HHT. Vassileva et al. [7] reviewed 18 recipients with fixed pulmonary vascular resistance who received a HHT with the donor pulmonary artery anastomosed to the recipient right atrium. The indications were

(PA) systolic pressure > 60 mmHg. All of the recipients had some degree of pulmonary hypertension, and, 8 of the patients had a restrictive cardiomyopathy. Twelve of the patients were New York Heart Association class III or IV; the remaining six were in the hospital with continuous inotropic support, and, one was intubated. The mean aortic cross clamp time was 58 minutes and a mean ischemic time of 122 minutes. The follow-up right heart catheterizations demonstrated a progressive decrease in the pulmonary artery pressures after transplant with a mean systolic pulmonary artery pressure of 29 mmHg, a TPG of 10 mmHg, and, a

patients with elevated, and/or, fixed pulmonary artery pressures, and, elevated pulmonary

, (2) transpulmonary gradient (TPG) > 15 mmHg, or, (3) pulmonary artery

. The group concluded that the HHT technique was a valuable option for

centers. Most of the HHT recipients had more than one indication for an HHT.

with the use of a marginal donor heart.

1 recipients for a marginal donor heart.

(1) PVR > 6 units/m2

PVR of 3.7 units/m2

vascular resistance.